Cover image of Discover CircRes

Discover CircRes

Each 15-minute podcast will provide an overview of the issue’s contents and relevant news in the field of basic/translational cardiovascular biology followed by an in-depth discussion of a featured article. This discussion will pull opinions from the podcast hosts, editorial team, research leaders and authors – both the corresponding authors as well as the trainee(s). We will provide lively discussions that give the listener a behind the scenes look at how science gets done and the implications of these fascinating discoveries.

Weekly hand curated podcast episodes for learning

Popular episodes

All episodes

The best episodes ranked using user listens.

Podcast cover

October 2019 Discover CircRes

This month on Episode 5 of the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from the September 27 and October 11, 2019 issues of Circulation Research and talks with Sarvesh Chelvanambi and Matthias Clauss  about their article HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients. Article highlights:   Stamatelopoulos, et al. Reactive Vasodilation in AL Amyloidosis Cao, et al. Miro2-Mediated Cardiac Mitochondrial Communication Georgakis, et al. Circulating MCP-1 Levels and Incident Stroke Sun, et al. Body Mass Index and DNA Methylation Tan, et al. Yy1 Suppresses DCM Through Bmp7 and Ctgf Transcript Cindy St. H:                       Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the September 27th and October 11th issues of Circulation Research.                                            We'll also have an in-depth conversation with Drs Matthias Clauss and Sarvesh Chelvanambi, who are the lead authors in one of the exciting discoveries from our October 11th issue.                                            The first article I want to share with you is titled, Reactive Vasodilation Predicts Mortality in Primary Systemic Light Chain Amyloidosis. The first authors are Drs Kimon Stamatelopoulos, Georgios Georgiopoulos, and the corresponding author is Dr Efstathios Kastritis. And the studies were conducted at the National Kapodistrian University of Athens School of Medicine in Athens, Greece.                                            So we hear about amyloids a lot in things like Alzheimer's, but amyloids are really just aggregates of protein that fold into shapes. And the nature of these shapes allows these individual protein molecules to bind and form many copies that form these fibers that are rather sticky. And the fibers then aggregate into larger and larger globules. And light chain amyloidosis is the most common type of amyloidosis. It's a rare but deadly disease, and it's caused by antibody-producing cells that are aberrantly churning out parts of antibodies called light chains. And it's these light chains that will aggregate and form sticky fibers.                                            So these fibers aggregate and form amyloid deposits, and these deposits build up and damage the organs and the tissue in which they're accumulating. And because it's dependent on where the aggregates are accumulating, AL amyloidosis can present with a wide variety of symptoms. However, symptoms of heart dysfunction and low blood pressure correlate with poor prognosis.                                            And because vascular dysfunction can contribute to hypotension or low blood pressure, this group decided to examine the vascular health of patients by conducting a measurement called flow-mediated vasodilation. And so this is a measurement where the diameter of the brachial artery, which is located in your arm, is measured before and then after a brief period of lower arm ischemia. And they formed a cohort of 115 newly diagnosed AL patients and another cohort of 115 matched controls. This study found that in AL patients, flow-mediated vasodilation was higher than in aged, sex, and cardiovascular risk factor-matched controls. The mean follow-up time for this study was 54 months, and in that time, the authors went on to find that high values of FMD in the amyloidosis patients was strongly predictive of mortality. In fact, high FMD values were more predictive of death than some measures of cardiovascular health. These results suggest that flow-mediated vasodilation may be a superior means of identifying AL patients most at risk and for assessing potential benefits of therapeutic interventions.                                            The next article I'd like to highlight is titled, Miro2 Regulates Inter-Mitochondrial Communication in the Heart and Protects Against TAC-Induced Cardiac Dysfunction. The first author is Yangpo Cao, and the corresponding author is Ming Zheng. And the work was conducted at Peking University, Beijing, China, Key Laboratory of Molecular Cardiovascular Science at the Ministry of Education, also in Beijing, China.                                            Beating heart cells have very high energy requirements, and thus they need lots of fully functioning mitochondria. And as we all know from our good old high school biology days, mitochondria are the powerhouse of the cell. Mitochondrial health and performance is directly dependent on the ability of individual mitochondria to be able to communicate with each other. In many cells, this mitochondrial communication occurs via the fusion of mitochondria into a giant network. However, in cardiomyocytes, the mitochondrial movement is much more constrained. In cardiomyocytes, mitochondria communicate by briefly connecting with neighboring mitochondria, which is often called kissing, mitochondrial kissing, or by nanotunneling, which is when the mitochondria create a sustained connection by means of long nanometer-sized tubular protrusions called nanotubes. And it's thought that the proper health of the cell is dependent on proper mitochondrial communication.                                            Miro2 is a Rho GTPase on the outer mitochondrial membrane and it harbors a calcium sensing domain. Miro2 can interact with transport proteins to promote mitochondrial transport along microtubules in a calcium-dependent manner. This group wanted to investigate whether Miro2 regulates cardiac inter- mitochondrial communication. To do this, they used transverse aortic constriction or TAC or they used an Ang II infusion model to induce hypertrophy in murine hearts. Using these two models, they found Miro2 expression was decreased via Parkin-mediated ubiquitination, and they also found that inter-mitochondrial communication was disrupted.                                            By contrast, transgenic mice over-expressing Miro2 were more resistant to hypertrophy, and they were able to do this by maintaining proper cardiac function than their wild type counterparts. Together these results reveal a novel role for Miro2 in mitochondrial communication and show that maintaining such communication may mitigate effects of hypertrophy.                                            The next paper I want to highlight is titled, Circulating Monocyte Chemoattractant Protein-1 or MCP-1 and the Risk of Stroke: A Meta-Analysis of Population-Based Studies Involving 17,180 individuals. That is a huge study. The first author is Marios Georgakis, and the corresponding author is Martin Dichgans. And they are from the University of Munich in Munich, Germany.                                            A major component of atherosclerosis is chronic inflammation and inhibiting the activity of proinflammatory cytokines has been identified as a potential therapeutic strategy to help slow the disease progression. One such cytokine under study is monocyte chemoattractant protein-1 or MCP-1, and animal studies have shown that blocking MCP-1 limits, or boosting MCP-1, accelerates atherosclerosis. However, large scale observational studies of MCP-1 in humans are lacking. To address this gap in knowledge, this group performed a meta-analysis of previously unpublished data from six population cohorts, which totaled over 17,000 individuals.                                            These individuals were followed for an average of 16 years, which when you think about it, this is an absolutely huge study. So in looking at this cohort of patients, the team identified a significant association between high baseline MCP-1 levels and the likelihood of suffering a future ischemic stroke. Interestingly, this effect was not seen with hemorrhagic stroke, which is typically not associated with atherosclerosis. These findings not only support the previous animal studies, but also support a recent study in humans in which a genetic predisposition for high levels of MCP-1 was associated with an increased risk of coronary artery disease and stroke. This study also suggests that future studies should explore the potential of lowering MCP-1 levels as a possible prevention strategy. Perhaps there could be another CANTOS-like trial where we use something to block MCP-1 signaling. Maybe that would have much broader effects. I guess we'll have to wait and see what the data says.                                            The next paper I want to highlight is titled, Body Mass Index Drives Changes in DNA Methylation, a Longitudinal Study. The first authors are Dianjianyi Sun, Tao Zhong and Shaoyong Su, and the corresponding authors are Shengxu Li and Wei Chen. And they're from the Children's Minnesota Research Institute, Children's Hospitals and Clinics of Minnesota in Minneapolis, Minnesota and The Peking University Health Science Center in Beijing, China, respectively.                                            So it's well appreciated that obesity is increasing worldwide. And obesity contributes to a whole host of cardiovascular morbidity, and ultimately contributes to mortality. It's also well known that environmental factors such as the food we eat and the air we breathe, as well as genetic factors, can influence a person's risk of obesity. And recently there have been studies that suggest that perhaps epigenetic factors also contribute to obesity. And just to remind you what epigenetics is, DNA is the genetic code, and mutations can happen on DNA that can alter either gene expression or maybe protein folding or whether a protein is made at all. But epigenetic factors are not as permanent as DNA mutations.                                            Epigenetic factors are alterable modifications that can happen to DNA itself or that can happen to the proteins on which the DNA is wrapped around. And epigenome-wide association studies have shown that DNA methylation at certain loci is linked to an increase in body mass index, or BMI. However, it's unknown whether these methylations are a cause or consequence of obesity.                                            So to get to the bottom of this, this group performed a large-scale longitudinal study. They examined thousands of DNA methylation sites in 995 white individuals and 490 black individuals. And they also determined the subjects' BMIs. They did this at a baseline measurement and then approximately six years later, they collected the same data in the same patient cohort. What they found was that only a handful of methylation sites were shared between the two ethnicities. And in both groups, however, there was a similar unidirectional link between BMI and methylation. Very interestingly, baseline BMI could predict methylation at a number of genetic loci. However, the team found that none of those baseline methylation sites could predict future BMI. From this data, the authors are able to conclude that it's obesity driving the methylation at certain genetic loci as opposed to certain genetic loci driving obesity, which I think is just extremely interesting. Really nice study.                                            The last article I want to highlight for you is a paper titled, Yin Yang 1 Suppresses Dilated Cardiomyopathy and Cardiac Fibrosis Through Regulation of Bmp7 and Ctgf. The first author is Chia Yee Tan, and the corresponding author is Jianming Jiang, and they're from the National University of Singapore.                                            Dilated cardiomyopathy or DCM is characterized by left ventricle enlargement and associated contractile dysfunction and fibrosis. Patients with DCM are at risk of arrhythmia and also of sudden death. And there's actually a huge number of genetic variants that have been linked to DCM, but the most common one or the most well-studied are mutations that affect the nuclear lamin gene or LMNA. So LMNA knockout mice are used to study the role of this gene in DCM, and these animals exhibit not only cardiac defects but also systemic defects. And those systemic defects include things like shorter lifespan, growth retardation, muscular dystrophy, neuropathy, and lipodystrophy.                                            Recently, LMNA-related dilated cardiomyopathy was linked to the deregulation of cardiac cell cycle. Meaning there was issues in how these cardiac cells are proliferating. So in this study, Tan and colleagues showed that boosting expression of a protein involved in cell cycle regulation, this protein is called Yin Yang 1, so boosting this gene's expression actually reversed the dilated cardiomyopathy symptoms in mice with heart-specific LMNA deficiency. Compared with untreated mice, mice receiving Yy1 via an adenoviral vector exhibited improved cardiac function and also reduced fibrosis after four weeks. The team then went on to show that Yy1 treatment prompted suppression of the extracellular matrix factor, Ctgf, and the upregulation of the growth factor, Bmp7.                                            Now, neither of these factors alone could rescue the symptoms of LMNA lacking mice. However, when both of these factors were manipulated together, they mimicked Yy1 treatment. These results highlight that Yin Yang 1 and its downstream targets Bmp7 and Ctgf are key players and potential therapeutic targets that can be harnessed for tackling LMNA-driven dilated cardiomyopathy.                                            Okay, so now we're going to have our interview with Drs Matthias Clauss and Sarvesh Chelvanambi. And they are from Indiana University School of Medicine in Indianapolis, Indiana. And their title of their paper is, HIV-Nef Protein Transfer to Endothelial Cells Requires Rac1 Activation and Leads to Endothelial Dysfunction: Implications for Statin Treatment in HIV Patients. So thank you both very much for joining me. Sarvesh C:                         Thank you so much, Cindy. Matthias C:                       Thanks for having us here. Cindy St. H:                       Could you both introduce yourselves and tell us a little bit about your background? Sarvesh C:                         My name is Sarvesh Chelvanambi. I grew up in Chennai, India. I did my undergraduate degree at Miami University in Oxford, Ohio. I got a Bachelor of Arts in Zoology with a minor in Finance. I then went to the Pennsylvania State University where I got my Masters in Biotechnology before coming over to Indiana University in 2014 to do my PhD work. And then I joined the lab of Dr Matthias Clauss, and in 2016, I got an American Heart Association predoctoral fellowship to study this project specifically. Cindy St. H:                       Wow! Congratulations. That's wonderful. Sarvesh C:                         Thank you so much. Cindy St. H:                       And now you completed the circle by publishing your AHA grant in Circulation Research. Sarvesh C:                         Exactly. Cindy St. H:                       And Matthias, how about you? Matthias C:                       I'm a Research Professor at IU School of Medicine, and my research interests focus in understanding how stressors connected with endothelium in this way contribute to vascular disease. These stressors include cigarette smoke and viral infections. Regarding viral agents, we are studying both acute infections and chronic infections and that is HIV. This HIV interest started actually 12 years ago in collaboration with Dr Samir Gupta who is also of course on this paper. We started off with a simple question, why are there so many cardiovascular events in patients, in HIV patients, with interrupted antiretroviral therapy? Cindy St. H:                       So it's not just the fact that they're HIV positive, it's that they were on therapy and then went off it? Matthias C:                       Yes. And this was part of this SMART study and this study was then actually halted because of the safety issues. Cindy St. H:                       So you're starting with the idea that patients with HIV who go off this antiviral therapy are more prone or get more cardiovascular events. So what did you start with, with this particular study? Matthias C:                       Well, our overarching idea was that the HIV virus could also do damage in the era of the combined antiretroviral therapy. And we started up with two questions, one was, is there an HIV protein which is persistent? And the other question, how is this HIV protein, if there's any one which is persistent, performing this? And this may be then leading over to your specific way to address these questions. Sarvesh C:                         That's kind of where we are starting with this project. Because back in 2016, the START trial came out saying, "We need to change the way we treat HIV patients," because initially the previous regimen of our drugs had a lot of metabolic side effects, but the current regimen of integrase inhibitors is actually really good and has very low metabolic effects. So there was a New England Journal Of Medicine paper that said, "Well, if a patient walks into the clinic, they're diagnosed as being HIV positive, put them on antiretroviral therapy right away."                                            But even in this era when everybody is on ART and there's almost no viral replication, you still see the persistence of a lot of comorbidities. And especially those associated with vascular events, whether it's peripheral arterial disease, coronary arterial disease, and a lot of other vascular diseases in the lung, or the kidney or the brain. So that kind of is what set us up, is there an element in the blood of these patients that is contributing towards vascular dysfunction? Cindy St. H:                       And so the protein that you are talking about in this paper is a protein called Nef, and is that where you come in,  Sarvash? Sarvesh C:                         Yes, because the project before I joined the lab, that's kind of where it led off, saying that Nef can get to the endothelium and it's very good at killing endothelial cells, but the mechanism through which it transfers into endothelial cells and the signaling pathways that Nef hijacks to induce this apoptosis was not clearly elucidated. A lot of work is done in Nef in monocytes and macrophages because as an HIV protein, it was studied in CD40 cells and the whole immune system as a whole, but we were the first to leverage all of those findings within an endothelial context and answer the questions, so what does Nef do and how does it get there? Cindy St. H:                       All right, so tell us a little bit what does it do and how does it get there? Sarvesh C:                         So we started doing some experiments with starting with HIV patient blood. So we took two fragments, we took the PBMC fraction, that Dr Clauss was talking about, which we knew had Nef within many of those cells. We also took the extracellular vesicle fraction, and we chose to look at this because there's a lot of literature out there saying that this fraction could not only disseminate particles throughout the body but also help signal through that. So in both of these fractions we added to the endothelial cells, we found increased apoptosis in HIV patients when compared to HIV negative patients.                                            And we were excited, but then we went and asked which of these patients had HIV Nef positivity in their blood, and then using that information when we stratified our apoptosis results, we made the surprising observation that the HIV positive, Nef positive patients were more prone to endothelial cell apoptosis. And this sparked a lot of conversation, so how do we target this and what is the signaling pathway it gets into? And that is kind of what led to most of the work in this paper, where you're showing that the transfer is mediated by extracellular, because this is such a nice tool, for HIV I guess, to spread itself into literally every cell type. Because while the HIV virus can only infect very few cell types, extracellular vesicles can be taken up by anything.                                            And the second observation we made was within endothelial cells, we found the signaling pathways that Nef was able to hijack to induce cell death. And that became the focus of this paper. Cindy St. H:                       That was one thing I wanted you to clarify, because I think what a really interesting aspect of this study is that it's the immune cells that are infected. The endothelial cells themselves are healthy and really they're getting this damage from the vesicles. That is,…wow! I don't know. It's just a really, really neat study. So can you tell us a little bit about the techniques you used in this paper? Sarvesh C:                         Yes, so we did a lot of assays to evaluate endothelial cell stress. So we started by looking at apoptosis, and a lot of those studies were done by looking at caspase-3 activity, which is a classic marker for cell death. We also did a lot of microscopy work where we took out extracellular vesicles out of those vesicles on the endothelial cells to show the uptake of Nef protein and thereby hammer that extracellular vesicles are indeed a mechanism of transfer for this protein in particular.                                            Now, one of the interesting experiments that we actually ended up doing, which was not a part of this paper really, was we wanted to see if chemotaxis was being affected by this. So we took an endothelial monolayer and separated T-cells that are expressing Nef using a Transwell membrane. And I had this huge problem where I couldn't read for a week because instead of using the 4-micron filters that allow T-cells to transfer, I was using 0.4 micron filters that T-cells cannot transfer through. But I still went about it and did my whole experiment because I didn't make that realization until a week later, because when I looked at the bottom of these chambers, there were no T-cells at all. But when I looked at the endothelial cells, I observed cytoplasmic transfer and Nef transfer, and we had a couple of conversations going, why is this happening? Did the T-cells all die or did they disappear?                                            And that's when we went back and looked in literature and found that Nef is very good at making virion particles. And those are the similar pathways that extracellular vesicle trafficking comes from. And so that was a huge shift in the way this project was designed and where we then started looking into the modes of transfer, the protein and the subsequent apoptosis that that transfer can cause. Cindy St. H:                       I love this story. So essentially your mistaken filter created this paper and this finding of the vesicles affecting the endothelial cells. Matthias C:                       Yeah, that's a typical finding for practitioner Chelvanambi, because he has this gift to turn negative things into positive things. So we have a lot of fun, and this mistake was really the beginning of a great study. Cindy St. H:                       That's wonderful. Really beautiful images, as well. So a little bit digging into, I guess, the next step. So first off, how were the endothelial cells getting damaged? They're getting damaged from these extracellular vesicles, but then what's Nef doing in the endothelial cell? What's happening there? Sarvesh C:                         So that was a very big question because if you look at it, Nef is a very small protein with almost no known enzymatic function. And yet it is able to interact with a lot of host proteins, which I guess makes it a very good viral protein. So when I went back and looked at literature, there were a host of studies in the 90s to show that Nef interacts with this kinase and that small GTPases, and there was a giant list for us to go after. And we were kind of left a bit fuddled, because we were like, which signaling pathway do we start with? Cindy St. H:                       Right. It's almost like there's too many. Sarvesh C:                         Exactly. And so what we ended up doing was we started looking into one of the various mutants of Nef that we had access to. And one of these mutants was a mutant that was incapable of PAK2 activation, and we showed that that doesn't have a lot of these stress damages. So we asked, "What is directly upstream of PAK2?" And that is where Rac1 came into the picture. And the small GTPase Rac1 is a nice candidate because it is also a master of many, many trades. Cindy St. H:                       I love this because it's such an interesting multidisciplinary approach to addressing the question, why are patients with HIV getting more cardiovascular events? What do you think evolutionarily is going on? Why would this be beneficial? Why would damaging the endothelium be beneficial? What are your thoughts on that? Sarvesh C:                         Personally, I think this is a side effect because HIV is never meant to exist in the era of ART. One of the analogies I always like to use is from Harry Potter, where HIV is Voldemort, which is the big bad villain. And what we have done is a really good job of banishing Voldemort. But what we have failed to do as a field is target its Death Eater, Nef. And I think with what we are suggesting, this paper with additional statins and other strategies that focus this, we can get to that point where we not only block HIV expansion but also the expansion of its minions, Nef. Cindy St. H:                       I love this analogy. I think you should redo your graphical abstract in a Harry Potter theme. Matthias C:                       Yeah, but I like your question. But also in this regard, I think it may be an example of a novel mechanism, how viral infections work in a different way than just infecting cells. And there's evidence from lots of viruses that they make toxic proteins, and why they are doing this, we don't know. But we noticed that the systemic effect of Nef may have some advantage for the infectious agent, because it makes T-cells more sticky, it makes them stick and transmigrate through the endothelium, and that is also shown in our paper. Cindy St. H:                       You have evidence that perhaps statins would be beneficial to give to these HIV patients on ART therapy. Can you tell us a little bit about that and how that would work? Sarvesh C:                         So based on what we did on our mouse studies that was a part of this paper, even after there is endothelial dysfunction, treatment with statins was able to restore endothelial function. Currently, there is a study going on called The Reprieve Trial where they're giving a statin called pitavastatin to HIV patients. The interesting part here is that these are HIV patients who don't have dyslipidemia. And the long-term goal is that statin treatment can help prevent the development of cardiovascular events. We're eagerly awaiting the results of this trial. Cindy St. H:                       Well done. Well thank you so much for speaking with me today. It was a pleasure to speak with you, Dr Chelvanambi and Dr Clauss. And congratulations again on this beautiful project, this beautiful story. And really, the implications for helping patients with HIV is really profound. HIV used to always be in the news and now that we have the ART therapy it's not talked about as much, but these patients are still in danger and I think your study is really doing a lot to highlight that and maybe even help them. So thank you very much and congratulations. Matthias C:                       Thank you. Sarvesh C:                         Thank you so much for the opportunity. Cindy St. H:                       So that's it for highlights from the September 27th and October 11th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams.                                            I'm your host, Dr Sidney St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


17 Oct 2019

Rank #1

Podcast cover

August 2019 Issue

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Denisa Wagner and Nicoletta Sorvillo about their article on how PAD4 in blood promotes VWF strings and thrombosis. Article highlights: Goodyer et al: ScRNA-seq of the Cardiac Conduction System Xiong et al: Chemotaxis Mediated Second Heart Field Deployment Ranchoux et al: Pulmonary Hypertension and Metabolic Syndrome Rühl et al. Thrombin/APC Response in FVL and FII 20210G>A Mahmoud et al. LncRNA SMILR’s Mechanism and Therapeutic Potential   Transcript Cindy St. H:                         Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's Journal, Circulation Research. I'm your host, Cindy St. Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you some highlights from the recent articles published in the August 2nd and August 16th issues of Circulation Research. Cindy St. H:                         After I discuss some highlights, we'll also have an in-depth conversation with Drs. Denisa Wagner and Nicoletta Sorvillo, from Boston Children's Hospital and Harvard Medical School, who are the lead authors of one of the exciting discoveries from the August 16th issue. Cindy St. H:                         The first article I want to share with you today is titled Transcriptomic Profiling of the Developing Cardiac Conduction System at Single-Cell Resolution. The first author is William R. Goodyer, and the corresponding author is Sean Wu. They are both located at the Cardiovascular Institute and the Department of Pediatrics at Stanford University. Cindy St. H:                         Have you ever wondered how your heart beats, and why there's always this glub-glub pattern, and where did it come from? How is the heart able to initiate that pattern, from cells that don't contract to cells that contract? Well, the beating of the heart is regulated by what's called the cardiac conduction system, and this is an area in the heart of specialized cells, and these cells establish the rhythmic beating by coordinating the contraction of the chambers of the heart. Cindy St. H:                         There's several components to the CSS. The sinoatrial node acts as the pacemaker in the right atrium. The arterial ventricle node is the electrical relay that slows down the pulse from the SA node. A His bundle helps to transmit those impulses, and the Purkinjie fibers are the terminus of the electrical signal. Between all of these different components are a heterogeneous population of what are called transitional cells. There are several studies that have linked these somewhat amorphous or heterogeneous transitional cells to different arrhythmic disorders. Cindy St. H:                         For the normal function of the heart, all of these parts must come together, and when they don't, there's severe clinical manifestations such as arrhythmias, like I said, but also you can get decreased cardiac output and even sudden cardiac death. While important, the cells of the CSS are rather elusive, and that's because they're in a relatively small number compared to the rest of the cells in the heart, and there also aren't very clear markers to identify the cells in the CSS. Cindy St. H:                         To address this, Goodyer and colleagues harvested cells from embryonic mouse hearts and performed single-cell RNA sequencing on 22,000 individually barcoded cells. What they were looking for is learning what type of cells they are, but more importantly, they had the goal of identifying what these elusive transitional cells are, and can we find a marker for these cells to study them? And in some, yes. Together, the sequencing and spatial data provided gene expression atlas of the mouse CSS. Hopefully, this atlas will guide future studies into the essential electrical system that regulates the heartbeat. Cindy St. H:                         The next article I'd like to highlight is titled Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intra-Organ Crosstalk During Cardiogenesis. We're really going to hit you over the head with some single-cell transcriptomics in this month's podcast. The first authors of this article are Halqing Xiong, Yingjie Lou, Yanzhu Yue, Jiejie Zhang and the corresponding author is Aibin He and they're all from the Institute of Molecular Medicine, Beijing Key Laboratory of Cardiometabolic Molecular Medicine and the Peking-Tsinghua Center for Life Sciences, all at Peking University in Beijing, China. Cindy St. H:                         During development, the mammalian heart originates from two distinct areas in the early embryo and they're called the first heart field and the second heart field. Progenitor cells from these regions give rise to very different structures. From the first heart field comes the atria and the left ventricle, and the second heart field forms the right ventricle and the outflow tract. While we know the outcomes of these different developmental layers, a full understanding of how the first and second heart fields are regulated and how they actually interact with one another is actually lacking a lot of detail and we're not exactly sure how those structures can influence one another. Cindy St. H:                         So to learn more, Xiong and colleagues utilized two different murine models that were engineered to label cells coming from either the first or second heart fields red, and by labeling these cells red, it allows for their very pure isolation and then downstream studying at the single-cell level. So from each of these two models, they collected about 600 red-labeled cells and they collected these cells at four different time points, that were essentially at 12 hour intervals, and they did this starting at embryonic day 7.5, and that's because that's the time point in the mouse where these second and first heart fields are starting to develop. Cindy St. H:                         What they found, by using single-cell RNA sequencing, is that the first heart field cells differentiated into cardiomyocytes, in what they called a gradual, wave-like manner, while the second heart field cells differentiated in what they referred to as a more stepwise, defined pattern. The team also found high expression of migration factor MIF in first heart field cells and they found MIF's receptor CXCR2 in the second heart field progenitor cells. This suggests that perhaps the first heart field cells could regulate the migration of the second heart field cells. Sure enough, blocking MIF- CXCR2 interaction in cultured mouse embryos prevented second heart field cell migration and also prevented normal development of the right ventricular outflow tract structures. So together these results provide insight into both normal heart development and also suggest what might go awry in certain congenital heart malformations. Cindy St. H:                         The next paper I want to highlight is titled Metabolic Syndrome Exacerbates Pulmonary Hypertension due to Left Heart Disease. The first author is Benoit Ranchoux and the corresponding author is Francois Potus, and they are from the Pulmonary Hypertension Research Group at Laval University in Quebec City in Quebec, Canada. The disease pulmonary hypertension can arise from a number of causes, but one of the main drivers of what's called group two pulmonary hypertension is left heart disease. Left heart disease itself is caused by several conditions, such as diastolic dysfunction, aortic stenosis, which is a disease that I study, or mitral valve disease. All of these pathologies result in the left heart not beating efficiently or exerting too much energy. Cindy St. H:                         More than half of all group 2 PH patients also have metabolic syndrome, and metabolic syndrome is a condition that is ever increasing in the modern age, especially in America, and it's characterized by obesity coupled with pathology such as dyslipidemia, type 2 diabetes and high blood pressure. Metabolic syndrome is also marked by elevated levels of the inflammatory cytokine IL6. Rat studies have shown that IL6 can induce proliferation of the pulmonary artery smooth muscle cells and consequently, pulmonary hypertension. Cindy St. H:                         In this study Ranchoux and colleagues pulled together all these different pieces in a rat model and essentially want to test left heart disease coupled with metabolic syndrome coupled with does pulmonary hypertension happen or get worse? What they found was really interesting. Left heart disease was induced in a rat model using super coronary aortic banding and then metabolic syndrome was induced with a high fat diet feeding, or with treatment with Olanzapine, which is a second generation anti-psychotic agent, and it's known to induce metabolic syndrome not only in rats, but also in humans. The data from this paper show that inducing metabolic syndrome in rats coupled with left heart disease resulted in elevated IL6 levels and also greatly exacerbated pulmonary hypertension. Cindy St. H:                         Digging into this mechanism, they found that inhibition of IL6, using either an anti-IL6 antibody or by reducing IL6 secretion from macrophages, using the diabetes drug Metformin, ameliorated the pulmonary hypertension in the rats. They then went on and looked at human samples and they found that IL6 was higher in the lungs of pulmonary hypertension patients and that this IL6 could induce proliferation of human pulmonary artery smooth muscle cells. So together these data suggest that the observation in rats holds true for humans, but further goes on to suggest that perhaps Metformin, which is a well-known, well-used diabetic drug, could perhaps be used for the potential treatment of Group 2 pulmonary hypertension patients. Cindy St. H:                           In the August 16th issue, we have an article titled Increased Activated Protein C Response Rates Reduce the Thrombotic Risk of Factor V Leiden Carriers but not of Prothrombin 20210G>A Carriers. That is some title. The first authors are Heiko Rühl, and Christina Berens, and Dr Rühl is also the corresponding author, and they are at the Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, in Bonn, Germany. Genetic studies have found two mutations that convey particularly increased risk for venous thrombo-embolism, and VTE is also more commonly referred to as deep vein thrombosis. These mutations are called factor five Leiden mutations, or FVL, and the prothrombin 20210G>A mutation we're just going to call F2. Interestingly, the penetrance of these mutations, or how likely they are to exhibit a phenotype, is variable. Some individuals with mutations never experience deep vein thrombosis, while others experience multiple episodes. Cindy St. H:                         As a group, the FVL carriers produce a higher than normal level of an anticoagulation factor called APC, or activated protein c. They also produce high levels of the pro-coagulation factor thrombin, and the authors of this study wondered if it was the balance, or rather perhaps an imbalance, of these factors that could explain the phenotypic variations in the patients that harbor the same mutation. To test this, they collected 58 patients. 30 were FVL and 28 were F2 carriers, and they injected these patients with clotting factors and examined their response rates. In both of the groups, about half of the individuals had no history of deep vein thrombosis, while the other half had had at least one episode. Cindy St. H:                         The team found that while both types of mutations were associated with increased APC and thrombin levels after coagulant injection compared with a control group, in the FVL group lower APC levels correlated with a much higher risk of deep vein thrombosis. In other words, the FVL carriers who had never experienced deep vein thrombosis produced higher levels of APC. Translating this to the clinic, perhaps APC testing could help identify individuals who are carriers of the FVL mutation and determine which of them are at higher risk due to lower levels of APC. Cindy St. H:                         The last paper we're going to highlight before switching to our interview is titled The Human- and Smooth Muscle Cell Enriched lncRNA, SMILR, Promotes Proliferation by Regulating Mitotic CENPF mRNA and Drives Cell Cycle Progression Which Can Be Targeted to Limit Vascular Remodeling. Now that is a crazy title! We’ve got to limit these names here this is difficult. The first authors are Amira Mahmoud and Margaret Ballantyne and the corresponding author is Andrew Baker, and they're all from Queens Medical Research Institute, BHF Center for Cardiovascular Sciences at University of Edinburgh in Edinburgh, UK. Cindy St. H:                         Before we dive into this article, I think it's important that we give a quick explanation of what is a lncRNA? lncRNA, or L-N-C RNA, stands for long non-coding RNA, and these are described as being transcripts which are made into RNA that are in lengths exceeding 200 nucleotides. So that differs them from micro RNAs or peewee RNAs or snRNAs, and they are classically or, I guess originally, considered not to be translated into protein. However, I think now more and more studies are finding that perhaps they are made into peptide sequences. However it's not fully clear what the function of those sequences are. Similar to micro RNAs, they also harbor regulatory functions that can control cellular functions by helping to fine tune the regulation of gene transcription and translation. Cindy St. H:                         Largely speaking, vascular smooth muscle cells are quiescent, but they can be stimulated to proliferate and migrate following injury to the vessel wall. While such activation of smooth muscle cells is essential for wound healing, these same processes are operative in vascular disease or after a cardiovascular procedure. Often what happens is an excess of proliferation of the smooth muscle cell wall can lead to dangerous occlusion of the blood vessel. The long non-coding RNA, SMILR, was recently identified as a promoter of smooth muscle cell proliferation and now in this article, Mahmoud and colleagues have defined its mechanism of action. Through transcriptome analysis of human smooth muscle cells, in which the levels of SMILR were either modulated to be increased or suppressed, the team found that lncRNA regulated expression of several genes involved in mitosis, or cell division. Furthermore, RNA interaction experiments revealed that the messenger RNA encoding the mitotic centromere protein, CENPF, was a direct interaction partner of SMILR. So just like the suppression of SMILR, the inhibition of CENPF resulted in reduced mitosis of the smooth muscle cells. Cindy St. H:                         The team then went on to show the inhibition of SMILR via RNA interference could block the smooth muscle cell proliferation ex-vivo, and they did this using intact sections of human saphenous vein. These results suggest that targeting this lncRNA could be a potential clinical treatment in situations where vessel occlusion is at risk. Cindy St. H:                       Okay, so now we're going to switch and have our interview with Drs Denisa Wagner and Nicoletta Sorvillo, and we're going to discuss their paper entitled Plasma Peptidylarginine Deiminase IV Promotes VWF-Platelet String Formation and Accelerates Thrombosis after Vessel Injury. Thank you Drs. Wagner and Sorvillo for joining us today. I think a funny thing is that between Nicoletta in Switzerland, me and you on the East coast and my producer on the West coast, I think we're spanning about nine hours of time zones here. Thank you all for taking the time, whatever time of day it is, wherever you are. Dr Wagner:                         Thank you. Cindy St. H:                         I was wondering, Denisa, if you could please introduce yourself and tell us a little bit about your background. Dr Wagner:                         I am a vascular biologist. I was always interested in platelets, endothelial cells, and leukocyte. I started with a background of von Willebrand factor research. Von Willebrand factor is the most important adhesion molecule for platelets and it is stored in endothelial cells as we have found very early on, in an organelle called Weibel-Palade bodies. So my work on this paper is actually related to the first observation I ever made scientifically of showing that von Willebrand factor is released from endothelium. Cindy St. H:                         Wow, that's wonderful. And Nicoletta, could you please introduce yourself and tell us a little bit about your background? Nicoletta:                            I'm Italian, I studied in Italy and I did my PhD in the Netherlands, and I've always worked on inflammation and thrombosis during my PhD. One of the major proteins I was working on is ADAMTS13. That is again a protagonist of our paper. Then I moved to Boston, where I had the pleasure to be able to work in Denisa Wagner's lab, and there I continued working on inflammation and ADAMTS13 and now currently I moved here to Bern and I'm bringing my expertise here, but I moved a little bit towards ischemia and reperfusion injury and transplantation. Cindy St. H:                         Interesting. Wow. Denisa, I want to circle back to this factor being one of the first findings that you worked on. How does it feel to still be working on it? Is it still exciting? Dr Wagner:                         It is nice and it's refreshing to come back to it. I did a lot of stuff in between. We did a lot of adhesion molecule work, leukocyte rolling. We made the early knockouts like b-selectin, p-selectin, and von Willebrand factor knockout as well. So it's fun. And by the way, since Nicoletta said that she was Italian, I am originally Czech, from Prague. Cindy St. H:                         Interesting. I did not know that. And actually, Denisa, I don't know if you remember, but when I was a graduate student in Katya Ravid’s lab, we collaborated with you to use some of this intravital imaging on one of our JCI papers. Dr Wagner:                         Oh right, right. I was wondering where I knew your name from. That's funny. Cindy St. H:                         Yes. Yeah, yeah. So it's wonderful to speak to you again. Really I wanted to interview you because I loved this paper, not only because it was a really interesting mechanism that actually I wasn't very well aware of, this citrullination and also because of the beautiful intravital imaging you could do and then link it to patient disease states. Maybe you can start by telling me what's the clinical unmet need or the question that your paper was trying to address? Nicoletta:                            So Denisa Wagner's lab always has worked on neutrophils and NETs and it has been shown that these NETs are involved in thrombosis. So we were curious what happens when even the enzyme that is important to make these NETs, this extracellular DNA, does when it's in the circulation. And this enzyme is of course PAD4 and it is known that it can modify our [inaudible] residues on protein through this process of citrullination. So we went to see if it could modify plasma proteins and as Denisa already said, an important molecule that initiates thrombotic processes is vWF that can be released during inflammation or when there's a damage to the endothelium .  So we went to see what happens if the enzyme that is involved in removing this vWF that is ADAMTS13 happens if it gets modified by this enzyme path. So our question was more like what happens if you have the release of an enzyme that is normally intracellular? What would happen if it gets outside of the cell? Cindy St. H:                         Interesting. So before we get too deep in the weeds, what is citrullinization and why is it important? What do these modifications do? Nicoletta:                            It changes the charge of a protein. It goes and modifies arginine, and it transforms it into citrulline. It changes the charge of a protein and therefore you can imagine if you change a charge of protein it can change even the structure of a protein and if you change the structure then you can change the function. So this is what this modification can do. Cindy St. H:                         And that's what it's doing on the ADAMTS13? It's essentially altering or inhibiting its function? Nicoletta:                            Yes. What we saw is that we can find these citrullinated residues on ADAMTS13 and we identify them by mass spectrometry and then we saw that if it is modified by citrullination, it loses its activity so it doesn't function anymore. Cindy St. H:                         Interesting. Very neat. Could you talk a little bit about the process of where this is happening naturally and where it goes wrong in a diseased state such as either sepsis or aging or just general clotting? Dr Wagner:                         These neutrophil extracellular traps are generated often more during a disease state when there is either an infection or exacerbated inflammation that would be like in sepsis or for example, in a metabolic disorder like diabetes. So there is a lot more of them being generated. Also, for example, in diabetes, PAD4 is elevated inside the neutrophil four-fold. If it's released from diabetic neutrophils , then there would be really a lot more of it. And in aging also, then a NETosis becomes much more prominent. We have done this only with mice, but I believe that it will be also, unfortunately, the case with humans that old mice make a lot more NETs than young mice. Therefore this is relevant to look. Since thrombosis increases both with aging, the incidence of thrombosis, thrombosis increases with a disease like diabetes or in sepsis, you will have micro thrombosis. We thought it would be interesting to study those processes as well, then. Cindy St. H:                         That's really neat. One of the techniques that you utilize heavily in this paper and several of your papers that I'm familiar with is this intravital imaging or intravital microscopy. Just so people can get a sense of what it is you're actually doing, could you maybe describe what that experiment is? Maybe Nicoletta, you could describe that for us? Nicoletta:                            During intravital microscopy, we are able to image in vivo, a vessel in a live mouse. And in this case we use mice and we can label leukocytes and platelets and then look at them in the vessel in vivo and you can then look for a thrombus forming or you can look at the [inaudible 00:23:43] already had leukocyte rolling and you can see what is happening inside the vessel during a proper blood flow and you can damage the vessel in some cases. In our case, in our paper, we do a ferric chloride injury where we damaged the vessel with ferric chloride and therefor you initiate a thrombus development and you can visualize it in vivo and real time. Cindy St. H:                         Excellent. Yes. And hopefully our listeners will look and see the beautiful pictures because those are some serious clots you get forming in the vessels. Yeah. Yeah. And so the other thing that you did was confirming the modification on ADAMTS13, you use mass spectrometry. How difficult was it to confirm that what you thought was happening was happening using that technique? Nicoletta:                            It was very difficult and challenging, I have to say. Dr Wagner:                         See, I would love to hear more about it because you often read, Oh, then we did mass spec and we got this beautiful whatever. Could you tell us a little bit about the struggles? Nicoletta:                            It was quite a struggle. I mean I think trying to identify such a modification that is very, first of all, novel and it changes the math only of one thousandth it's very difficult. To identify you can confuse it with a deiminasion again because of the increase of mass is the same. And another problem was that ADAMTS13, our plasma protein, is low abundance in plasma compared to other plasma proteins like Fibrinogen, that is very, very much abundant. It was a challenge for this reason. So trying to pinpoint out a small, tiny modification already in a protein that is not so abundant in plasma and therefore we have to use this probe, this Biosyn PG program. And we did this in collaboration with Paul Thompson's lab and we were able to then fish out what was modified by the citrullination, but it was very challenging. We tried several different types of techniques that were different types of approaches before being able to show that in vivo. So in human samples we can find this modification. Dr Wagner:                         Nicoletta grew a lot of gray hair during that period. (laughs) Dr Wagner:                         It took us about a year to figure out how we could detect it in vivo because also some antibodies to ADAMTS13 don't work so well. It's a minor protein, but she figured it out. Cindy St. H:                         Wow. That's amazing. Well, congratulations on that. That's excellent. I guess what I'm wondering now is what are the next steps and what might your findings mean in terms of future potential therapeutic options or treatment strategies for different detrimental thrombotic events? Dr Wagner:                         I think what we have really verified that the PAD4 remains active when it circulates in circulation, when the release, and there are several diseases in which PAD4 levels were found to be elevated, like rheumatoid arthritis and what it means in general. That is PAD4 is actually causing havoc. It is citrullinating probably quite indiscriminately. Several proteins may be finding the exposed parts. Maybe it could have some binding sites, but I think it just affects proteins in general and for some of them like, ADAMTS13, this had a very detrimental effect. So in diseases where there is a lot of PAD4, one has to worry about the consequences of citrullinating things and perhaps spot for inhibitors should be used. What do you think, Nicolleta? Nicoletta:                            I totally agree with you. Yes, I totally agree. I mean PAD4 outside the cell could be dangerous, of course. However, we never know if there's something good that it can do that protects by citrullinating proteins so there's so much more to discover about extracellular PAD4 and its effect on the environment. Dr Wagner:                         However, Nicoletta when she wrote a paper at the end she decided to talk about ADAMTS13 as a therapeutic because both she and I, we are convinced that ADAMTS13 it's a possible future therapeutic and it's already given to patients who are lucky in ADAMTS13 and may be given to patients who have thrombotic events in the future, like stroke or myocardial infarction. And these situations are highly pro-inflammatory. Therefore, we would anticipate that in these situations, NETs, and we know NETs are released and therefore, what Nicolleta suggests at the end, is that introducing together with ADAMTS13 an inhibitor of citrullination would be a good thing so that the protein, the ADAMTS13, remains active in circulation. Cindy St. H:                         Wow. So a two-hit strategy. I mean I can think of a handful of potential diseases this would be good for. You know, patients with sickle cell, there's a lot of NETs released then thrombotic events or even stroke. I mean, do you see that this is a potential mechanism that's common to all thrombotic disease or just kind of specific subsets? Nicoletta:                            All is a big word I think, but I think that there are many disorders where together with a thrombotic event, you can find also low levels or low activity of ADAMTS13 and in many of these disorders, nobody knew really why you have a reduction of ADAMTS13 activity, what is happening? Why do you lose this ADAMTS13? What we believe, but of course further studies are needed, is that maybe in these disorders, what is causing the loss of ADAMTS13 is this release of PAD4 because in stroke or in some DIC sepsis, you can find patients or many patients who do have low levels of ADAMTS13 activity and we believe that it's due to maybe citrullination by PAD4. So in that case, I agree with you maybe then that this therapy can be used in different thrombotic events as you suggested. Cindy St. H:                         So what does PAD4 normally do when it's intracellular? What is its, I guess healthy role, in a cell, if it has one? Nicoletta:                            So what is known now is that it really regulates transcription. So that's very important because it citrullinates transcription factors to facilitate transcription. And what Denisa Wagner's lab has identified is that it's extremely important to form these NETs because it citrullinates histone and allows the unraveling of the chromatin and then the NET release. However, it's extremely interesting. We are very interested to understand what else does it do within the cell. Cindy St. H:                         Interesting. That is so neat. I love this story. Dr Sorvillo and Dr Wagner, thank you so much for joining us and congratulations again on a wonderful paper. Dr Wagner:                         Thank you. Nicoletta:                            Thank you for having us and inviting us. Thank you. Cindy St. H:                         So that's it for the highlights from our August issues of Circulation Research. Thank you for listening. This podcast is produced by Rebecca McTavish and edited by Melissa Stoner and supported by the editorial team of Circulation Research. Copy text for the highlighted articles is provided by Ruth Williams. I'm your host, Cindy St Hilaire and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


15 Aug 2019

Rank #2

Similar Podcasts

Podcast cover

September 2019 Issue

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from recent issues of Circulation Research and talks with Matthew Stratton, Rushita Bagchi, and Tim McKinsey about their article on Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation. Article highlights: Vincentz, et al. HAND1 Enhancer Variation Impacts Heart Conduction Zhuang, et al. EC-Klf2-Foxp1-Nlrp3 Regulates Atherogenesis Quintanilla, et al. Robust Targets for Persistent AF Ablation Lambert et al. Characterization of Kcnk3-Mutated Rats Myagmar et al. Gq Mediates Cardioprotection Transcript Cindy St. H:       Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St Hilaire, and I'm an assistant professor at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the August 30th and September 13th issues of Circulation Research. We'll also have an in-depth conversation with Drs. Matthew Stratton, Rushita Bagchi, and Tim McKinsey, who are the lead authors of one of the exciting discoveries presented in the September 13th issue. Cindy St. H:         The first article I want to share with you is titled, "Variation in a Left Ventricle–Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function." The first author is Joshua Vincentz and the corresponding author is Anthony Firulli, and this work was conducted in the Departments of Pediatrics, Anatomy, and Medical and Molecular Genetics at Indiana Medical School in Indianapolis, Indiana. Cindy St. H:         The heart's ventricular conduction system, or VCS, is composed of specialized muscle cells that propagate electrical signals through the working myocardium of the ventricles to coordinate the rhythmic contractions of the heart chambers. Disorders of the VCS can lead to certain types of arrhythmia. Genome-wide association studies have identified a number of single nucleotide polymorphisms, or SNPs, that appear to increase the risk of VCS-mediated arrhythmias. Two such SNPs are located in the upstream region of a gene encoding for Hand1. And Hand1 is a transcription factor that is involved in left ventricle development. Conditional cardiac Hand1 ablation during embryogenesis leads to ventricular septal defects and hyperplastic arterial ventricular valves, and a reduction in Hand1 expression could lead to morphological, and therefore functional defects. Vincentz and colleagues hypothesized that these SNPs might reside in an enhancer element, and that's a region of DNA and a promoter that allows for the increased expression of a gene. The region containing the SNPs is highly conserved from mammals to reptiles and includes two sequences that allow for the binding of GATA transcription factors. And GATA transcription factors are well known to drive cardiac development. So this team used CRISPR-Cas9 technology to show that the deletion of the enhancer impaired normal VCS morphology and therefore function. And they did this in a mouse model and in the in vitro electromobility shift assay (which frankly was one of my favorite love-to-hate experiments of my PhD). So this group did their own electromobility shift essay and showed that GATA-4 binds to these enhancer sites. And together, these results support a role for Hand1 in the formation and function of the VCS and offer insights to possible arrhythmia etiologies. And what I really love about this paper is that they could actually go from a SNP in a GWAS to a functional role of a protein, which is great. A lot of times with GWAS studies, you have no clue what the heck is going on. So this was a beautiful study where they actually could link a single nucleotide polymorphism to differential expression of a gene. Cindy St. H:         The next article I'd like to highlight is titled, "Endothelial Foxp1 Suppresses Atherosclerosis via Modulation of Nlrp3 Inflammasome Activation." The first authors are Tao Zhuang and Jie Liu, and the corresponding authors (there's three of them) are Zhongmin Liu, Muredach Reilly, and Yuzhen Zhang. The Liu and Zhang teams are from the Key Laboratory of Arrhythmias of the Ministry of Education of China, and the Research Center for Translational Medicine at Shanghai East Hospital, which is part of Tongji University School of Medicine in Shanghai. And the Reilly team is from the Cardiology Division in the Department of Medicine and the Irving Institute for Clinical and Translational Research at Columbia University in New York, New York. And I have to say my good friend Rob Bauer is also a coauthor on this article. So Rob, I hope you're listening. Cindy St. H:         Chronic inflammation contributes to atherosclerotic disease and is a major pathological mechanism contributing to the dysfunction of the vascular endothelium. So leukocytes, which are inflammatory cells that float around in your blood, leukocytes can adhere to the endothelial layer, and then they can migrate through the endothelial wall into the wall of the vasculature. And it's this activity, along with the uptake of oxidized LDL and the formation of a little fatty streak, that is the start of atherosclerosis. And now Zhuang and colleagues have identified that the transcription factor Foxp1 is a potential regulator of vascular endothelial health. So first they showed that while healthy arteries express Foxp1 robustly, atherosclerotic endothelium from both mice and humans exhibits reduced expression of this transcription factor. The team then generated atheroprone mice that either lacked Foxp1 or overexpressed Foxp1 specifically in the endothelium. The mice lacking Foxp1 were shown to have exacerbated athero with much larger plaque sizes and increased macrophage infiltration into the vessels, while overexpression of Foxp1 had largely the opposite effect. It actually curtailed progression of atherosclerotic disease. The team went on to examine the atherosclerosis-suppressing mechanism of Foxp1, showing that the factor suppressed expression of the inflammasome components in the endothelial cells. Cindy St. H:         So all together, these results highlight that Foxp1-mediated regulation of the inflammasome is a potential targetable pathway for atherosclerotic treatments, and having a new targetable pathway is important, as the CANTOS trial, which provides proof of concept of the inflammation hypothesis of atherosclerosis in humans, showed robust effects in only a small subset of the population tested. Thus, there is a need to identify other means, a plan B if you will, by which we can control the inflammation that contributes to atherosclerosis. Cindy St. H:         The next paper I want to highlight is titled, "Instantaneous Amplitude and Frequency Modulations Detect the Footprint of Rotational Activity and Reveal Stable Driver Regions as Targets for Persistent Atrial Fibrillation Ablation." The first author is Jorge Quintanilla, who is also a corresponding author alongside David Filgueiras-Rama, and they are from the National Center for Cardiovascular Research and the Center for Biomedical Research in Cardiovascular Diseases Network in Madrid, Spain. Uncoordinated contractions of the atria to the ventricles of the heart is called atrial fibrillation, or AFib, and AFib causes symptoms such as heart palpitations, dizziness, and taken to the extreme, AFib can actually cause death. To correct such rhythm problems, doctors can ablate certain regions of the heart suspected to be driving this misfiring. In an ablation procedure, a catheter is inserted through the blood vessels and into the heart. An electrophysiologist then identifies the locations of the heart that are sending abnormal electrical impulses, and with either delivery of tiny pulses of painless, low-level energy or using a catheter that has a cold tip to freeze the misfiring areas, the electrophysiologist can ablate and hopefully stop  AFib. The problem is that this approach often fails, and AFib still occurs or can reoccur after a length of time. So Quintanilla and colleagues wanted to develop a more personalized medicine approach to treating AFib. So to do this, they wanted to make it something simple, something affordable, and something that hospitals currently have access to. So they used the standard electroanatomical mapping system to track the amplitude and also the frequency modulations of the electrical signals from the hearts with AFib. And they found that regions with high and stable instantaneous frequency signals were the drivers of fibrillation in the hearts. When these regions were ablated in pigs with persistent AFib, the misfiring stopped in almost all cases and was sustained. The team went on to test the system in three patients with Afib, and two of the three remained arrhythmia-free without drugs for at least 16 months. So with further development and testing, this frequency mapping could potentially replace systems that are currently in use, and more importantly, this could provide a more accurate and patient-tailored way to find and ablate the drivers of AFib. Cindy St. H:       The next paper I want to highlight is titled, "Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension." Oh, now that was a nice title. That was nice and short. The first author is Mélanie Lambert, and the corresponding author is Fabrice Antigny, and they are from the INSERM Hôpital Marie Lannelongue in Le Plessis Robinson, France. Cindy St. H:       Pulmonary hypertension is a rare but life-threatening condition where the adverse remodeling of the pulmonary arteries causes an increase in the blood pressure that's needed to push the blood through the lungs, and this high blood pressure causes the heart to work harder, and it leads ultimately to right ventricular hypertrophy and heart failure. So genome-wide association studies have identified a number of mutations that have been linked to pulmonary hypertension and these include several loss-of-function mutations in the gene encoding for a potassium channel, and that's a protein that can release potassium from a cell to the extracellular environment. And the particular one that has been found to be mutated in pulmonary hypertension patients is Kcnk3. And this channel regulates the resting membrane potential of pulmonary artery smooth muscle cells. To date, it is not known how the loss of Kcnk3 contributes to pulmonary hypertension. To start to unravel this mystery, Lambert and colleagues created a full-body knockout of Kcnk3 in rats, and they used rats because that's a much more robust model for studying pulmonary hypertension than some of the murine models available. These knockout animals exhibited an increased pulmonary artery pressure. They also had faster heart rates and they were more susceptible than their wild-type counterparts to both pharmacological or hypoxia-induced pulmonary hypertension. These Kcnk3 knockout rats also had evidence of remodeled pulmonary vasculature, and this vasculature showed signs of endothelial dysfunction, altered vaso transcription, and altered neomuscularization. In in vitro studies, they used pulmonary artery smooth muscle cells that they isolated from these knockout rats, and these cells showed increased activation of proliferation markers, which is another signature of pulmonary hypertension. And this was also mirrored in human pulmonary artery smooth muscle cells that were treated with a Kcnk3 inhibitor. So together, this work starts to uncover the role of Kcnk3 in pulmonary hypertension pathogenesis. And it also provides the field with a novel model system from which people can learn more about the role of membrane potential of pulmonary artery smooth muscle cells in pulmonary hypertension. Cindy St. H:       The last paper I want to highlight before our interview is titled, "Coupling to Gq Signaling Is Required for Cardioprotection by an Alpha-1A-Adrenergic Receptor Agonist." The first author is Bat-Erdene Myagmar, and the corresponding author is Paul Simpson from the VA Medical Center in San Francisco, California. So like their name says, G-protein coupled receptors interact with G-protein subunits to propagate the signal when a ligand binds. The protein G alpha q has long been considered a key mediator of cardiac hypertrophy. And that's because in mice, when this Gq protein was overexpressed, it induced hypertrophy, myocardial apoptosis, and contractile failure. However, this sub unit Gq can interact with a multitude of G-protein coupled receptors that themselves bind a variety of ligands. So which receptor or which signaling pathway specifically is responsible for the hypertrophic phenotype? Recent studies by others had shown that stimulation of the alpha-1A adrenergic receptor prevents cardiotoxicity and heart failure. So Myagmar and colleagues asked whether this cardio-protective alpha-1A stimulation is dependent on the alpha q subunit. So using mice with a mutant version of alpha-1A that allows the binding of the ligand but does not couple with the Gq subunit, the team found that alpha-1A induced cardioprotection was absent. The mutant animals were more likely to die than their wild-type counterparts when hypertrophy was induced pharmacologically or surgically. And furthermore, in the mutant myocytes themselves, the group observed that alpha-1A induced ERK signaling, which is essential for the receptors cardioprotective activity, was impaired. So together these results showed that alpha-1A-induced cardioprotection is dependent on alpha q, and actually it showed that alpha q signaling is not always maladaptive. Cindy St. H:       Now we're going to move to our interview with Drs. Matthew Stratton, Rushita Bagchi and Tim McKinsey and we're going to talk about their great paper titled "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." Cindy St. H:       Okay, so now we're going to have our interview with Drs. Stratton, Bagchi, and McKinsey on their paper titled, "Dynamic Chromatin Targeting of BRD4 Stimulates Cardiac Fibroblast Activation." So welcome, everyone. Dr Tim M:          Thank you. Dr Rushita B:    Thank you. Dr Matt S:          Thank you. Cindy St. H:       I was wondering if you could just all maybe go around and introduce yourselves. Dr Tim M:          Sure. I'm Tim McKinsey. I'm a professor in the Division of Cardiology at the University of Colorado Anschutz Medical Campus. I also direct a newly formed fibrosis center on campus. It's called the CFReT, the Consortium for Fibrosis Research and Translation, and our goal is to understand new mechanisms that regulate fibrosis and develop new therapies to treat scarring, or fibrosis, in organs. Dr Rushita B:    I'm Rushita Bagchi. I'm currently a postdoctoral fellow in Dr McKinsey's lab. I grew up in India, and that's where I did my undergrad and master's degrees. Then I moved to Canada to do my PhD focusing on transcriptional regulation of cardiac fibrosis under the supervision of Dr Michael Czubryt. After that, I transitioned to Dr McKinsey's lab here in Denver to enhance or add to my expertise of transcription by studying epigenetics, and especially trying to find the underlying mechanisms that cause cardiovascular disease. The nice thing about this position for me has been that I have been able to constantly build up on my experience studying tissue fibrosis, but at the same time, Tim has been very generous and has let me develop projects of my own as well. Cindy St. H:       You're lucky. That's awesome. Thank you for joining us. And Dr Stratton. Dr Matt S:          I'm Matt Stratton. I'm an assistant professor in the Department of Physiology and Cell Biology at Ohio State University. I did my graduate training at Colorado State University in neurodevelopment and neuroendocrinology and then moved to Tim's lab for a postdoc and assumed my current position this past December. Cindy St. H:       Wow. How's it going? Dr Matt S:          It's going well. Starting a lab is a lot of fun and a lot of stuff going on. Cindy St. H:       Yeah, I'm four years in now and at the same time you feel brand new and excited and then, oh my God, what am I doing? So that's great. Well thank you all for joining me. So I really like this paper, mostly because I'm also a vascular biologist. I kind of focus more on the heart valves, but I have a real interest in cell phenotype transitioning and cell shifting, and so when you started to talk about chromatin remodeling and bromodomain protein, I was really interested and wanted to hear more. So maybe we can start by telling everyone what is the clinical need that your paper at base is trying to address? Dr Tim M:          Well before we get into that, could I start by saying that we're honored to have our work published in Circulation Research. We're really grateful for that. I also want to point out that this is the result of a very detailed collaborative effort involving at least six other labs, including the labs of Charles Lan at Baylor College of Medicine, Jun Qi at the Dana-Farber Cancer Institute, Kunhua Song and Maggie Lam here in Colorado, as well as Sap Haldar and Deepak Srivastava at the Gladstone Institutes in San Francisco. Without this collaborative effort, none of this would have been possible. Cindy St. H:       That's great to hear and I'm really happy you mentioned that. Team science is so important, and I feel like we almost can't get these big, groundbreaking papers unless we really work as a good team, so thank you for highlighting that. Dr Tim M:          So we're really interested in fibrosis, which is a hallmark of heart failure. Fibrosis can actually be a good thing for the heart. If you have a myocardial infarction, you need a strong scar to form to prevent the ventricle from rupturing. But in response to chronic stress like hypertension and other things, you can get this longstanding fibrosis that results in cardiac dysfunction. That's because fibrosis is essentially a scarring process and one of the things that that does is to create a stiff in the left ventricle that can't relax effectively. Unfortunately, despite the well-known roles of fibrosis in cardiac disease, there are no targeted anti-fibrotic therapies for the heart, and that's really our focus in the lab. We've had a long-standing interest in epigenetic regulation of heart failure and cardiac fibrosis, and we've known for some time that inhibitors have a family of epigenetic reader proteins called the bromodomain and extraterminal proteins, the BET proteins. Inhibitors of those BET proteins can block cardiac fibrosis in rodent models and improve cardiac function. What we knew going into this work is that systemic delivery of those compounds was efficacious. But as you know, the heart is made up of many different cell types. So we really wanted to understand if the efficacy of these compounds was related to effects in resident cardiac fibroblasts. Cindy St. H:       Excellent. So what is the role of a cardiac fibroblast in a healthy cell, and where does that go awry? Dr Matt S:            So in a undiseased heart, fibroblasts are necessary to provide structure, right? They lay down the extracellular matrix that really holds the heart together. Without them, you would not have a good pump function. Where they go awry, I mean, that's one of the things that we're trying to study, right? They become proliferative, they become contractile, and they secrete, or we call them super-secretors, of extracellular matrix. So TGF-beta is really a known signaling molecule that kicks the fibroblasts into this activated or myofibroblast state. We use that in the paper as a agonist for our cultured cells. Cindy St. H:         Great, thank you. So what was the hypothesis you were testing in this paper? Dr Matt S:            So what we wanted to know, if BRD4 and BET proteins are important for this activation of cardiac fibroblasts? So going from a quiescent fibroblast to a proliferative and super-secretor of extracellular matrix fibroblast in the heart. And those experiments hit right away. I mean, we did those experiments, and it was quite dramatic that if you use JQ1 to inhibit these BET proteins, you completely blocked this myofibroblast differentiation. We went in and did some siRNA and shRNA work to show that really BRD4 appears to be the main culprit of the BET protein families. Cindy St. H:         Rushita, could you tell us a little bit about what a bromodomain protein is and what maybe specifically BRD4 is in relation to the other bromodomain proteins? Dr Rushita B:      Sure. So when we talk about the chromatin, there are various players in there that are known as, in general, chromatin modifiers. So you have enzymes that add acetylation mark on lysine residues on histone tails, which is basically DNA is wound around these histones and those histones have lysine tails, but you have the big acetyl group sitting. Now when you have this acetyl group sitting, this makes it more accessible for the transcriptional machinery and allowing gene transcription to happen. Those enzymes are known as histone acetyltransferase, the ones that add the acetyl mark there. The ones that take it away, which is what our lab has been studying for a long time, and Tim is a known world expert in the field, those are known as histone deacetylases, or HDACs, which basically remove those acetyl marks and compact the chromatin, thereby suppressing gene expression. This BET proteins or bromodomains are transcriptional coactivators. So this bromodomain is actually in charge or takes up the duty of identifying these acetyl marks on the lysine residues and therefore, tells the transcription machinery to come in and allow gene transcription to happen. There are a few BET proteins. Of them, BRD4 has been studied extensively in cancer as well as in the heart. But as Tim mentioned, the role of BRD4 has been studied vastly in the heart in terms of the cardiac myocytes, but not so much in the non-myocyte population, which is where our work stands out really well and starts highlighting the role of this specific chromatin modifier protein in activation or control of profibrotic gene expression. Cindy St. H:         Yeah. So correct me if I'm wrong, but my understanding is, you're going to need a little bit of the cardiac fibroblasts remodeling in the early phase. But where it is really detrimental is when that overcompensates and overproliferates and throws down too much matrix and then is bad. So do you see your study as a way to kind of target that window of where a potential treatment might be applicable? Dr Tim M:            Yeah, we think that BRD4 is a nodal regulator of cardiac fibrosis and therefore, an excellent therapeutic target. The challenge will be developing selective BRD4 inhibitors that are safe, as well as effective. We know that BRD4 is not only expressed in cardiac fibroblasts-it's all over the body. But we think our work provides an entry point to the development of highly selective BRD4 inhibitors for fibrotic indications, including heart failure. Cindy St. H:         So that's one of the things I was wondering, how specific your drug is to BRD4 versus the other ones, but also you mentioned the myofibroblast versus the immune cells infiltrating the heart. Do we know what BRD4 is doing in those cells in this system? Dr Tim M:            BRD4 is definitely pro-inflammatory, and BET protein inhibitors like JQ1 are anti-inflammatory, that's for sure. Interestingly, there's a BET family inhibitor called Apabetalone, RVX-208, that's in Phase III clinical testing for people with atherosclerosis. So if that's successful, it will provide proof of concept that you can target this family of epigenetic readers to treat cardiovascular disease. I also wanted to point out that JQ1 was initially discovered by Jay Bradner's lab, in particular Jun Qi, who is a coauthor and collaborator on this paper. Cindy St. H:         Oh, very nice. Okay, good conflict of interest too, I guess. So maybe you guys can talk a little bit about how you managed to get this huge team of scientists together efficiently, and what were any hang-ups? Matt is laughing a bit, but you two are the lead authors, Matthew and Rushita. How did you two kind of lead the way on this and divvy up this huge project? Dr Matt S:            So it is definitely a project management-style approach I think you have to take. I mean, there's a lot of communication, really a lot of communication with bioinformatics, analysts, and getting the right sequencing done, and that was fun, but it took a lot of effort. And once you get this big data, how do you present it in an intelligible story and how do you pick things out that may lead to new discoveries, right? So we highlight Sertad4 in here as a gene that's very much BRD4-dependent. And I think this is a proof of concept for using this genomics, and particularly BRD4, as kind of a molecular string to pull on to unwind this puzzle. So that was a lot of fun. And you know, Rushita was super awesome in helping with this project. Dr Rushita B:      Yeah, I think having stared at cardiac fibroblasts for six years during my PhD definitely gave me the confidence that I could step up to the plate and deliver what was necessary. And like Matt said, there was a lot of omics-based stuff that we did in the paper. And that is actually one of the key highlights, because we see papers or manuscripts that are published that have RNA-Seq, ChIP-Seq and proteomics, but I believe the strength in our article is the combination of all three. So we were actually able to do overlapping ChIP-Seq and RNA-Seq experiments, and then there was proteomics involved. So we are looking at it at the genomic transcriptome and protium-wide changes that are happening all together, put in one manuscript. And the beauty of this work is it has now created data sets that people can mine and get more information out of. And this is something that will definitely continue to drive our future studies in the lab as well. Cindy St. H:         Can you maybe expand on that? Could you maybe describe briefly for the audience what ChIP-Seq is and what RNA-Seq is, and really the power that is created when you can couple those techniques with the same samples? Dr Matt S:            Sure. So BRD4 was the center point of the paper, right? So we did BRD4 ChIP-Seq and RNA Pol II ChIP-Seq in fibroblasts treated with TGF-beta or not. So in ChIP-Seq, you basically immunoprecipitated your target protein, and that brings with it, if it's bound to chromatin, that brings with it the DNA that it's bound to. And then you can sequence the DNA that comes out of your immunoprecipitation and map that to the genome, and you get a very nice picture of where is BRD4 enriched, and where does it go after stimulation like TGF-beta, when the fibroblast becomes a myofibroblast. So you can line all these up and you can pick out what gene changes we think are directly dependent on BRD4. That's something that we like, because we now know that BRD4 is a good target, right, so that kind of pulls it together. Cindy St. H:         Great. Thank you. What else do you want to bring up? Dr Matt S:            I think understanding how signals get translated to changes in gene expression is obviously something that the field is very much interested in. And because BRD4 is basically a step away from RNA polymerase II, it gives you a little bit more specificity in knowing that that's a disease-activated pathway, right? So trying to figure out what directs BRD4 to new locations in the chromatin and cause it to be removed from previous locations in the chromatin is really an interesting area of research. So we did a pathway screen basically using inhibitors, and we use Sertad4 as the readout, right. And we found that a p38 inhibitor was able to block the ability of TGF-beta to induce Sertad4. And we were able then to show that p38 had a role in targeting BRD4 specifically to the Sertad4 locus. Dr Tim M:            I wanted to say, you know, one of the challenges with this project is that fibroblasts are difficult to work with. You would think that they would be easier to work with than a myocyte. But when a fibroblast hits a plastic cell culture dish, it rapidly transforms into an activated cell, because that plastic has a very high tensile strength. So it took a lot of optimization to figure out methods to culture these cells to maintain them in a quiescent state. Cindy St. H:         What did you do? What was that trick? Dr Tim M:            I mean, it involves changing cell density, changing the constituents of the medium, and doing other things. Cindy St. H:         Science magic. Dr Tim M:            Yeah. Dr Rushita B:      And I'll just add to that. The nice thing about being able to contribute to a study like this is also that, like Tim said, fibroblasts, they change phenotype rapidly. You take them out of a biological system, whether it's a heart or any other tissue, you plate them out in cell culture, they start changing. The nice thing about the in vivo study, the RNA-Seq that was done using the in vivo study with JQ1, was that we used a very simple pressure overload model known as the TAC model, which is a very well-established and accepted model worldwide in the field of cardiovascular disease, treating animals with JQ1. So we isolated fibroblasts, but the time from the isolation of cells to the time an RNA was prepared was an hour or two. So we made sure that we minimally exposed them to culture conditions in the lab, so we retained their biology. So what we did on plastic dishes before, although they were plated on plastic, and we had RNA-Seq done on those cells, like Tim said, we did optimize the conditions. And then being able to similarly treat or use the cells that come from an animal directly and both of them contributing to a similar cohort of genes or pathways that we can look at, that has definitely given immense strength to this manuscript. Cindy St. H:         And that's why it's in Circ Research, so it's a beautiful paper. Very well done. So I can't imagine all these hearts that you had to isolate and get single cells of and culture. What kind of days were you pulling? What was the actual boots on the ground of getting this done? How did that work? Dr Tim M:            It wasn't uncommon for me to get emails from Matt and Rushita at very odd hours of the night or early in the morning. Dr Rushita B:      Yeah, it was like we had the animals being sacrificed, hearts taken, and running to the cell culture room to do everything under sterile conditions. Most important thing- I think what worked out really well is we made sure we had all the reagents prepared ahead of time, so that once the heart is out, it's weighed, because we were also looking at hypertrophy because of the TAC model. We weighed the heart and it goes into your BST right away. Cindy St. H:         I try to teach that to my lab. It's like the cooking idea of mise en place. I make them lay out everything in the cell culture hood ahead of time, and it's all in the order and you just boom, boom, boom, boom. Dr Rushita B:      And a lot of our experiments were done later in the evening, so the nice thing was we had access to multiple centrifuges, which is usually a huge plus. And I still remember Matt being on one side, I'd be on the other. And then we had help from members of the lab as well. They were running between the cell culture room and the centrifuge. So it was actually quite fun. It turned out really well. Cindy St. H:         I'm picturing like those old water brigades to put out a fire where like a bucket is just passed. Is that what this was? Dr Rushita B:      That was very similar to the situation you just talked about. Cindy St. H:         That's great. It sounds kind of painful, but also kind of fun. I guess lastly, maybe one of you can end with telling us what are the bigger picture results of this, and what are the next steps in terms of maybe possibly translating this to the clinic? Dr Tim M:            Well as I mentioned, one of the things we're trying to do is to selectively inhibit BRD4. We're also trying to inhibit it only in cardiac fibroblasts with the hope that we'll be able to improve the therapeutic index of BRD4 inhibition. So create a situation where patients can tolerate this anti-fibrotic therapy better than if it was delivered systemically. We're also looking at other regions of BRD4. BRD4 contains the bromodomains, and those are the targets of JQ1, but there are other interesting domains on BRD4 that we're actively pursuing. Cindy St. H:         Thank you. And Matthew, what are you doing in your new lab, or is it just set up right now? Dr Matt S:            Well I have a K Award from the National Institute of Aging. Cindy St. H:         Congratulations. Dr Matt S:            Thank you. To look at BRD4's role in the heart and cardiac aging. And I also have a couple projects based on some of the mining that we've done from these datasets. So hopefully those lead to good publications and follow-on grants. Cindy St. H:         Well, if this is a good start, I'm sure they will. And Rushita, what are your next plans? How long have you been with Tim? Dr Rushita B:      So I've been here with Tim for almost four years now, so I'm pretty much in the final leg of my postdoctoral training. So I'm still continuing to work on tissue fibrosis projects, including the heart. But I have been able to develop a new field of interest and something that Tim has entrusted me to carry on in the lab in the field of cardiometabolic disease, but definitely with an epigenetic focus. So hopefully in a year's time I see myself having an independent academic scientist position. My dream job will be to be at an academic institute where I can lead a research team which focuses on deciphering or trying to even find the most basic molecules that define the underlying mechanisms of tissue fibrosis and cardiometabolic disease. Cindy St. H:         That sounds like a great plan. Very best luck to you. Dr Rushita B:      Thank you. Cindy St. H:         Do you guys want to add anything else? Dr Tim M:            The field of cardiovascular epigenetics is in its infancy and we still have a lot to learn. Cindy St. H:         And I'm sure all of you will do your parts in moving that field forward. So with that, we're going to end our interview with Drs. Stratton, Bagchi, and McKinsey. Thank you all for joining me and thank you to the listeners for listening. Have a great day. Dr Tim M:            Thank you. Dr Rushita B:      Thank you. Dr Matt S:            Thank you. Cindy St. H:         That's it for highlights from the August 30th and September 13th issues of Circulation Research. Thank you for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Copy text for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


19 Sep 2019

Rank #3

Podcast cover

April 2020 CircRes

This month on Episode 111 of the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from the March 27 issue of Circulation Research and talks with Dr. Matthias Nahrendorf  and Dr. Maximilian Schloss about their article Modifiable Cardiovascular Risk, Hematopoiesis and Innate Immunity. Article highlights: Liu et al. Genetics of Transposition of the Great Arteries Park et al. Mild Lipid Abnormalities and ASCVD in the Young Yan, et al. Gut Flora Adjusts Blood Pressure By Corticosterone Transcript Cindy St. Hilaire:              Hello and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh. Today, I'm going to share with you articles selected from the March 27th issue of Circulation Research, as well as give you a hint of the cutting-edge ideas in the Compendium on atherosclerosis. We'll also have a discussion with Dr Maximilian Schloss and Matthias Nahrendorf about their article On Modifiable Cardiovascular Risk, Hematopoiesis And Innate Immunity. So, first the highlights.                                            The first article I'm sharing with you is titled Exome-Based Case Control Analysis Highlights the Pathogenic Role of Ciliary genes and Transposition of the Great Arteries Exome-Based Case-Control Analysis Highlights the Pathogenic Role of Ciliary Genes in Transposition of the Great Arteries. The first authors are Xuanyu Liu and Wen Chen and the corresponding author is Zhou Zhou from Peking Union Medical College in Beijing, China. In normal healthy hearts, the aorta develops from the left ventricle and the pulmonary arteries from the right ventricle, but in the common congenital heart malformation called transposition of the great arteries or TGA, the plumbing of these two major vessels is switched. Thus, the pulmonary arteries arise from the left ventricle and the aorta from the right.                                            This is a life-threatening condition, requires surgery in the earliest days of life and currently, the genetic etiology of this congenital disease is largely unknown. To identify the genetic drivers of transposition of the great arteries, the authors of this study performed whole exome sequencing of 249 TGA patients and, in 66 cases, they were actually able to do exome sequencing on their parents as well. The analysis identified 82 candidate genes in which the allele variant or mutation that was found in TGA patients was predicted to alter protein function.                                            Interestingly, a quarter of these mutations or variants were found to be in genes that are involved in cilia function. So, the cilium is an organelle that's found on all eukaryotic cells and is in the shape of a slender protuberance that projects from the much larger cell body. Recently, cilia have been identified as playing a central role in the pathogenesis of congenital heart diseases, and it has been suggested that congenital heart disease may be a new class of ciliopathy. Transposition of the great arteries has been hypothesized to arise from disturbances in the left right patterning during embryo development, and cilia are required cellular organelles and they are essential for left-right axis determination in early development. These findings add to the growing body of evidence that has identified a role of cilia genes and congenital heart disease and may lead to future prenatal diagnostic screenings.                                            The next article I want to highlight is titled Mildly Abnormal Lipid Levels, but Not High Lipid Variability, Are Associated with Increased Risk of Myocardial Infarction and Stroke in ‘Statin-Naive’ Young Population: A Nationwide Cohort Study. The first author is Jun-Bean Park and the corresponding author is Hyung-Kwan Kim from Seoul National University Hospital in Seoul in the Republic of Korea. High levels of lipids in the blood increase a person's risk of cardiovascular disease, and evidence suggests that this risk builds over lifetime. However, in young adults, and in this case, young adult means any individual between 20 and 39 years of age. In young adults, relatively little evidence is available that identifies individuals at high risk for atherosclerotic cardiovascular diseases, except for very high LDLC levels.                                            Variability in lipid levels has recently emerged as a predictor of adverse clinical outcomes and lipid level variability may be causally linked with the atherosclerotic cardiovascular disease risk. This is because theoretically, high lipid levels can induce fluctuations in the atherosclerotic plaque composition. These fluctuations result in plaque instability and rupture and ultimately, plaque related clinical events, such as myocardial infarction. However, high lipid level variability may merely reflect other risk factors or confounders for atherosclerotic cardiovascular diseases, including unhealthy lifestyle and unrecognized comorbidities. This study therefore examined health data of close to two million Korean individuals aged 20 to 39. None of them had ever been treated for high cholesterol with statins nor had any of them suffered any myocardial infarctions or stroke.                                            Over a four-year period, the subjects had undergone at least three lipid measurements as part of their general health assessments and then they were followed for a further four years or until death. The data showed that high baseline lipid levels was linked with an increased risk of adverse cardiovascular events, and in particular, myocardial infarctions. They also found that individuals who exhibited high lipid variability, so sometimes getting high readings, sometimes getting low readings, these individuals who exhibited high variability and lipid level measurements were not at any greater risk of such cardiovascular events. While such up and downs have previously been linked to cardiovascular disease, this study argues that perhaps statin use in other cohorts may have contributed to the variability and thus confounded research interpretation, an issue that was specifically avoided in this study. Together the results indicate that lipid in young adults can indeed indicate future cardiovascular risk and therefore suggest lipid-lowering strategies could be beneficial for this age group.                                            The next article I want to share with you is titled Intestinal Flora Modulates Blood Pressure by Regulating the Synthesis of Intestinal-Derived Corticosterone in High Salt-Induced Hypertension. The first author is Xuefang Yan and the corresponding authors are Zhe Wang and Qunye Zhang from Shandong University in China. Hypertension is highly prevalent in the adult population all over the world and it is a major risk factor for heart disease and stroke. A high salt diet can help to drive hypertension pathogenesis, but complete details about the mechanisms by which high salt intake shapes vascular pathology are lacking.                                            Recent studies show that fecal transfer from salt hypertensive to salt normotensive animals can lead to hypertension in the recipients, and this suggests that perhaps gut flora may play a role in hypertension. In the article by Yan and colleagues, they show that rats on a high salt diet have altered gut flora profiles and in particular that levels of the bacterium, Bacteroides fragilis, was reduced. Analysis of intestinal metabolites and substrates in high salt diet fed rats also showed that levels of arachidonic acid, which is produced by this bacterium, were low and levels of the stress hormone, corticosterone, which regulates blood pressure, were elevated.                                            The team went on to show that supernatants from this bacterial culture could prevent corticosterone production in the intestinal tissue of high salt fed mice as could direct treatment with arachidonic acid. Moreover, both B. fragilis and arachidonic acid were found to be lower in the feces of humans with hypertension compared to that of healthy controls. The results suggest B. fragilis and arachidonic acid normally curb corticosterone production and could therefore be novel targets for hypertension treatment strategies.                                            The last thing I want to mention before we switch to our interview is the Circulation Research Compendium on Atherosclerosis. The last compendium on this topic was back in 2016 and this new compendium provides the most cutting-edge ideas in the field. The topics highlighted in this compendium are polygenic scores to assess atherosclerotic risk, clinical perspectives, and basic implications, epigenetic reader proteins and cardiovascular transcriptional programs, sex as a biological variable in atherosclerosis, neutrophil extracellular traps in cardiovascular diseases, CD31 as a therapeutic target in athero, interleukin-1 and the inflammasome as therapeutic targets in cardiovascular disease, non-coding RNAs in vascular diseases, intracellular aspects of macrophage immunometabolism in atherosclerosis, single cell RNA sequencing in atherosclerosis, vaccination strategies and immune modulation in atherosclerosis and we have an update from the group leading the One Brave Idea. Please check out this compendium.                                            All right. So, now we're going to switch over to our interview portion of the podcast. I have with me today Dr. Matthias Nahrendorf, who is a professor at the Center of Systems Biology at Massachusetts General Hospital Research Institute and Harvard Medical School and his research fellow, Dr. Maximilian Schloss. Today, we're going to be discussing the article Modifiable Cardiovascular Risk, Hematopoiesis, Innate Immunity, which is part of our Compendium on Atherosclerosis. Circulation Research puts together two to three compendiums annually and this current one is the Compendium on Atherosclerosis. We will have two additional compendiums planned for 2020. One on Obesity, Metabolic Syndrome and Cardiovascular Disease and that should come out in late May and another on Atrial Fibrillation scheduled for June. So stay tuned.                                            So, thank you very much for being with me here today, Matthias and Maximilian. Matthias Nahrendorf:    Thanks for having us. Maximilian Schloss:        Thanks for having us. Cindy St. Hilaire:              So, I really enjoyed this review article. I actually learned a lot. I also really liked your cartoons at the end, so maybe we can talk about those a little bit later, but what it's on is essentially the role of inflammation and cardiovascular disease and years of study, which have recently culminated in the completion of the CANTOS trial, have showed us that reducing inflammation can help reduce cardiovascular events. When we look at the factors that we know drive cardiovascular disease, it's things like bad diet choices, lack of exercise, stress, and inadequate or disrupted sleep and in this article you make the more nuanced argument that these modifiable factors are in fact influenced by the innate immunity. So, before we dig too deep into what you are really discussing in this article, could you maybe give us a brief introduction to the role of innate immunity and cardiovascular disease initiation and progression? Matthias Nahrendorf:    Sure. Yeah. So, I think one very instructive experiment that had been done by more than one lab actually almost two decades ago, is stopping innate immune cells from migrating to atherosclerotic plaque by deleting the chemokine MCP-1 or the chemokine receptor CCR2 in mice that have a propensity to develop atherosclerosis. What became apparent is that these mice, despite having very high blood cholesterol levels, they don't really develop atherosclerosis. This really led the whole field now almost 20 years ago, to the insight that it's not only the hypercholesterolemia, it's also the immune system that contributes to the disease. So, innate immune cells, most numerous neutrophils and monocytes then in tissue also macrophages and they're meant to defend us against infections and they support healing. In this particular setting, they are probably doing more harm than good because they promote inflammation in areas where inflammation shouldn't be i.e., in the vessel wall. Maximilian Schloss:        Yeah, I would add that what Matthias said is that basically it's all about a balance between necessary inflammation and too much inflammation. If we take, for instance, myocardial infarction as an example, we need a certain amount of inflammation, local inflammation. We need a recruitment of innate immune cells like neutrophils and monocytes and eventually macrophages, to do their job. For instance, phagocytizing a dying cardiomyocytes or inducing fibrosis. So in this example, we need inflammation, but what we see in different models where we can manipulate inflammation being at reducing or increasing inflammation, we can see that if we do either/or then wound healing and scar formation is impaired. I think that's all we are interested in studying not only the mechanisms, how inflammation can be increased or decreased, but also what is actually the perfect balance in view also of finding ways of improving outcomes in cardiovascular patients. Cindy St. Hilaire:              One of the things in my research, so I focus on cardiovascular calcification, which is very hard to do in a mouse. They don't like to calcify similarly like they don't like to make plaque without a proper genetic background. Are there aspects of the mouse versus the human innate immune system that are very different? I mean I know specific receptors are slightly different, but in general, are they matched up pretty well or is there things that are quite different about them? Matthias Nahrendorf:    I think the answer is both and there are very important parallels and then there are very important differences. So, one important difference is just if you look at sheer numbers and the contribution of immune cells in the blood and, possibly also in the plaque, can be quite different. So, recent studies that use unbiased profiling in human plaques, for instance, say that there's quite a lot of lymphocytes and we still have to understand whether this is due to the retrieval or if it says species difference or the situation, but I think there are important differences. On the other hand, I think that it really make sense to study mice because a lot of the important discoveries about the immune system in the setting have translated to humans. Cindy St. Hilaire:              Like the IL-1 beta story. Matthias Nahrendorf:    That's right. Yeah. Cindy St. Hilaire:              So, actually one of the topics that you started out with in your article is on the role of hematopoiesis in cardiovascular disease. You had a beautiful paper years ago actually with my colleague at University of Pittsburgh, Partha Dutta, who's right down the hall from me, where you guys showed that myocardial infarction itself further exacerbates atherosclerotic plaques mid part through recruiting monocytes from the spleen and mobilizing the immune system. So, I'm wondering, what are the role of the cells when they get mobilized? You talk about these modifiable risk factors of stress and sleep interruption, unhealthy diet. So, how can these risk factors help or promote this mobilization of hematopoietic cells? Matthias Nahrendorf:    Yeah. So, I think that early on when we thought about going down this road and studying these risk factors, even before going there, you realize that the cells that we're interested in, innate immune cells are very short lived. So they live on the order of hours or days. So, they're really produced just in time. That's different to lymphocytes and resident macrophages, which have much longer lifespans. So, this really triggered the insight that we should look at production and release because it's a just in time supply situation. So, what we were wondering is whether in the setting of cardiovascular disease, whether production rates are increased and we now know and a number of labs have studied hematopoiesis in this setting including Fil Swirski, Alan Tall, and some others.                                            We now know that this is really the case, so hematopoiesis increases in chronic atherosclerosis. It increases in acute myocardial infarction and increases in heart failure. What we don't know is what mechanisms actually ramp up blood cell production and we're beginning to understand that the sympathetic nervous system is involved. But I think we only see the tip of the iceberg here. That's why we wanted to study modifiable risk factors, because if you look at others such as high cholesterol, once the insight was gained that lowering cholesterol is helpful, we had the statins which make a huge change. So, we hope to repeat that. Cindy St. Hilaire:              Maximilian, one of the things that you brought up is this balance. The inflammation's a little bit good and then it's a little bit bad or a lot bad. So, where is that good and bad spectrum in terms of mobilizing hematopoiesis or hematopoietic cells? Maximilian Schloss:        Yeah. I think that depends a bit on the disease type or we're talking about a chronic disease or an acute disease? For instance, to stay at the example of myocardial infarction, once cardiomyocytes become ischemic, they will release certain chemokines and cytokines into the blood, which then circulate to the bone marrow and tell the cells that leukocytes need to leave the bone marrow to enter the blood circulation system and then go to the heart to fulfill their very important functions there. Once the cells leave the bone marrow, the bone marrow need to reproduce themselves, then this process starts of hematopoiesis and there we can go back again to the concept of a balance. Of course, there is a certain beneficial physiological need of cell production, but one sees mechanisms so to say maybe go out of control and too many leukocytes are produced and released to the blood.                                            Then that again impairs patient outcome. There are very many papers, clinical papers, who have shown that leukocyte counts after myocardial infarction have a certain U shape relationship with the outcome. That I think is best described that if leukocyte counts are very high, that they actually negatively correlate with the outcome of MI patients. If you look at the bone marrow specifically, there are certain mechanisms, which we know, and what we are more closely looking at now, what are actually the modifier of this process, what are the signals which tell these cells to secrete more hematopoietic factors or quiescence factors? I think that's what also the Review is a little bit about. Cindy St. Hilaire:              Yeah, it's great. So, you were speaking about that kind of U-shaped curve in the release of these cells. Do we know based on some of the other things you spoke about, I guess I'm thinking about like diet or exercise or sleep in contributing to that release after an event like myocardial infarction. Is that known yet or has anyone looked into that? Matthias Nahrendorf:    Yeah. So, I think we're in the very beginning of understanding what's happening acutely. There's more knowledge on the chronic side and this is what we've been working on. Often the things that influenced the chronic situation can be quite different from what happens acutely. So I think in general, we're just beginning to understand what happens in acute myocardial infarction. Well, we know for instance is that exercise doesn't compromise the release and supply of leukocytes that's necessary in acute infection or acute myocardial infarction. So, if the mouse or the individual was exercising before the event, that may reduce overall leukocyte levels, but not to a degree that it's harmful. Cindy St. Hilaire:              Yeah. You can't exercise your way beyond a certain point. Matthias Nahrendorf:    Maybe that's also possible. If you run more than one marathon a day, I'm sure that's… Cindy St. Hilaire:              That will do something else. Matthias Nahrendorf:    Yeah. Cindy St. Hilaire:              Actually, so one of the interesting things that I saw in the article was when you were talking about diet and the role of diet in innate immunity, which is something I really never thought about, and you did bring up things like intermittent fasting. Can you discuss what's known at least scientifically about how that kind of diet timing can impact the immune system and therefore maybe cardiovascular disease? Matthias Nahrendorf:    So, that's a very emerging field. There's very little known about this. I think it's very interesting because very relevant and a lot of people are excited about it, but it's basically, from what I know, it's mostly two papers that were published, I think both in Cell, and they say that intermittent fasting leads to a decline of cells that are in circulation. So, that's a very exciting observation. I think it's similar insight as to discovering that immune cell levels circulate the circadian rhythms, which had been discovered a while ago. So, I think there's definitely an impact and we're just beginning to understand why this is and what regulates it. Cindy St. Hilaire:              Yeah, that segues nicely into the next thing I was wondering about and that is we all know not enough sleep, you get tired, your brain's not focused and stuff like that, but it really does impact the inflammatory system and also cardiovascular disease. So how is sleep involved in this innate immunity cardiovascular disease progression? Matthias Nahrendorf:    The way we approached this was actually thinking about lifestyle factors and their impact on cardiovascular disease. Maybe a decade ago, Fil, who's our middle author on this Review, and I started thinking about lifestyle factors and what struck us is that the association of some of these risk cardiovascular events is really high. So, if you look at sleep or if you look at psychosocial stress, psychosocial stress has an odds ratio of 2.4 for premature myocardial infarction. That is right on scale with all these powerful risk factors that everybody knows about like hypertension, but then what isn't really clear or maybe not entirely, is whether or not these risk factors also act via the innate immune system and that's where we were coming from.                                            I think at this point it's pretty clear that they do have an influence via the immune system. What I think what we've done is we uncovered a couple mechanisms that lead to the activation or dampening of inflammation depending on what you look at, but we don't really understand the broader network. I think there's a lot of work to be done looking into these pathways, which is exciting because I think that we can learn from nature what's dangerous and what's helpful. That this is how humans learn to fly. So, I think that observing what leads to cardiovascular disease, which behaviors are really harmful, will maybe lead us to new ways of mitigating it. Cindy St. Hilaire:              Yeah. Also, I think all of this, it's interesting. We all went after smoking for decades, stop smoking, reduce cardiovascular risk and maybe it's stress and sleep is the next smoking. Matthias Nahrendorf:    Smoking was so successful, right? I mean if you look 50 years back, it was promoted as this healthy thing that you should do. Then people really started to learn how bad it is and now we're at a time where smoking is declining and has declined and we see the results. Lung cancer is really on the decline. So, I think that's a good example how understanding health effects of behavior can be really helpful. Maximilian Schloss:        I think one thing I would like to add is when you ask more general question about innate immunity and when we talk about sleep and sleeping habits, I think what's generally quite interesting to know is that the immune system or these leukocyte numbers in circulation, they oscillate quite dramatically over the course of a day in a healthy human being and also in mouse models. I think one aspect also among others to consider is when we have unhealthy steeping habits, like for example, going to bed late or being a shift worker, drinking for example before going to bed. Then this will also confuse a system on the circadian entrainment, which then subsequently will lead to other problems.                                            I also think another thing is that what you were mentioning with the fasting is what we learned from this similar to these extreme circadian patterns seen when we fast or when a mouse is fasting, then monocyte levels drop into extreme low levels and these monocytes hone back into the bone marrow. I think this is interesting because it shows how dynamic actually a system like innate immune cells actually is. So, it's a very delicate system which responds to sleep disruption, exercise, diets in a very dramatic way. Cindy St. Hilaire:              All right, I'm going to bed early tonight and eating a good dinner. Well, this was a wonderful Review. I really enjoyed reading it. I really do think it's introducing the next targets that we have to go after in modifying cardiovascular disease. Thank you so much for taking the time to speak with me today. Matthias Nahrendorf:    Thank you. Maximilian Schloss:        Thank you so much. Cindy St. Hilaire:              That's it for our highlights from the March 27th and Compendium issue of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles was provided by Ruth Williams. Thank you to our guests, Max Schloss and Matthias Nahrendorf. I'm your host, Dr. Cindy St. Hilaire, and this is Discover CircRes, your on the go source for the most up-to-date and exciting discoveries in basic cardiovascular research.


23 Apr 2020

Rank #4

Most Popular Podcasts

Podcast cover

February 2020 Discover CircRes

This month on Episode 9 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the January 31 and February 14, 2020 issues of Circulation Research and talks with Dr Joe Miano and DrThomas Quertermous about their article Coronary Disease-Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. Article highlights:   Wang, et al. Multi-Omics Integration Study of AF Heianza, et al. Antibiotics and Risk of Mortality Dikalova, et al. Sirt3 Reduces Hypertension and Vascular Dysfunction Hu, et al. Lipid Overload Acetylates Drp1 in the Heart Transcript Dr St. Hilaire: Hi, welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm from the Vascular Medicine Institute at the University of Pittsburgh. Today I'm going to share with you four articles that we selected from the January 31st and February 14th issues of Circulation Research. I'm also going to have a discussion with corresponding authors, Drs. Joe Miano and Thomas Quertermous about their study on the role of TCF21 and smooth muscle cell lineage specificity in coronary artery disease. So first, the highlights. The first article I'm sharing with you is titled, Integrative Omics Approach to Identifying Genes Associated with Atrial Fibrillation. First author is Biqi Wang, and the corresponding author is Honghuang Lin from Boston University School of Medicine in Boston, Massachusetts. Atrial fibrillation, or Afib, is the most common form of heart arrhythmia and in the US alone there's somewhere between three and six million individuals with this condition. AFib can be either idiopathic or inherited, and genome-wide association studies, or GWAS studies, have identified hundreds of genetic loci that are linked to AFib. However, these loci explained only a small percentage of inherited cases. This suggests that there are many more AF related genes yet to be discovered. To try and identify these elusive a-fibrillated loci, this study integrated data from previously performed transcriptome, epigenome and GWAS studies. The TWAS and EWAS, as the transcriptome and epigenome-wide studies are short-handedly called, was collected from more than 150 Afib patients, and over 2,000 control individuals. While existing GWAS data, that's genomic data, was collected from tens of thousands of AFib and control participants. By combining and analyzing the data from these TWAS, EWAS, AND GWAS studies, the team was able to identify an additional 1700 genes that were associated with AFib. Now this is compared to the original 206 loci that were identified by the GWAS studies alone. Many of these new genes are involved in cardiac development as well as the regulation of the heart and the muscle cells. The additional gene hunting power afforded by co-analyzing multiple Omics data is not only helpful for approaching AFib but is really setting a platform upon which future studies might be done to provide novel insights for numerous other diseases of complex ideology. The second article I will highlight is titled, Duration and Life-Stage of Antibiotic Use and Risks of All-Cause and Cause-Specific Mortality, a Prospective Cohort Study. The first author is Yoriko Heianza, and the corresponding author is Lu Qi from Tulane University in New Orleans, Louisiana. So microbiome is a word that is used to describe all of the microbes; the bacteria, the fungi, the protozoa, and the viruses that live on and also live inside the human body. And how our microbiome influences human health as well as disease state is a new and hot research topic. Alterations to the gut microbiome have been suggested to influence the risk of developing certain chronic diseases, including cancer and cardiovascular disease. There are many factors that influence the constituents of the gut microbiome; things such as your diet, your environment, your stress level, but another factor that can significantly alter the gut microbiome is the use of antibiotics. So there's preliminary evidence that suggests long-term antibiotic use may be linked to increased mortality in adult women, and now this study defined that link in more detail. The authors performed a large-scale population study of antibiotic use in middle aged and older women with a follow up period of 10 years. Over 37,000 women who were in middle age or in late age at the start of the study show that long durations of antibiotic use, which was defined as using antibiotics over two or more months, was associated with increased risk of all-cause mortality and of cardiovascular disease-related mortality in late adulthood, even after adjusting for risk factors such as age, lifestyle, diet and obesity. While no such association was apparent in middle-aged women, the risk for older women was more pronounced if they had also used antibiotics during middle life. And middle life is defined as between the age of 40 and 59 years of age. This suggests that risk of mortality due to antibiotic use may be cumulative. While antibiotics are unquestionably beneficial for saving lives, the link is not necessary causative, and the results indicate a potential risk may exist that could be factored into prescription decisions. Obviously, there's much more details that need to be worked out, but this is quite a provocative study. While antibiotics unquestionably saved lives and the link is not necessarily causative, the results indicate a potential risk may exist that could be factored into prescription decisions. Moving to a metabolism theme, the next article I want to share with you is titled, Mitochondrial Deacetylase Sirt3 Reduces Vascular Dysfunction and Hypertension While Sirt3 Depletion in Essential Hypertension Is Linked to Vascular Inflammation and Oxidative Stress. The first author is Anna Dikalova and the corresponding author is Sergey Dikalov, and the work was completed at Vanderbilt University. Hypertension affects about a third of the global adult population. That's a huge number of individuals. It's a risk factor for stroke, myocardial infarction and heart failure. Although blood pressure lowering treatments are widely available, hypertension remains uncontrolled in about 30% of patients who are on those treatments. A thorough understanding of the complex pathophysiology of the condition would facilitate the search for much needed alternate treatments for this third of patients with hypertension. To that end, these investigators studied the role of Sirt3, which is an enzyme that tends to be at the lower than usual levels in blood vessels of patients with hypertension. Sirt3 regulates metabolic and antioxidant functions, and alterations in either of these functions can contribute to cardiovascular disease and vascular dysfunction. The team showed that mice genetically engineered to over express Sirt3 had healthier blood vessels and lower blood pressure than control animals who were subjected to experimentally induced hypertension. By contrast, Sirt3 depletion was shown to cause vascular inflammation and increased signs of vascular aging in mice. The team also confirmed that humans with hypertension exhibit low levels of Sirt3; however, the mechanism causing Sirt3 to be low in certain people is not clear. These data suggest that boosting Sirt3 may be potential therapy for hypertension; however, of course, more studies must be conducted to thoroughly investigate this. The last article I want to share with you before we switch to our interview is titled, Increased Drp1 Acetylation by Lipid Overload Induces Cardiomyocyte Death and Heart Dysfunction. The first author is Qingxun Hu and the corresponding author is Wang from the University of Washington School of Medicine in Seattle, Washington. In the heart, fat molecules are the main energy source. However, excessive lipids caused from diet induced dyslipidemia, AKA eating too much fat, can lead to cardiomyocyte dysfunction. It's known that lipid overload in the heart can cause increased activity of dynamin-related protein one, or Drp1. Drp1 is an enzyme that regulates mitochondrial fission, but exactly how Drp1 becomes activated due to lipid overload is entirely unclear. The authors of this paper confirmed that Drp1 activity and mitochondrial fission are abnormally increased in the hearts of mice fed a high-fat diet, and these mice also exhibit signs of heart dysfunction. They show similar effects in monkeys who were fed a high-fat diet. Interestingly, Drp1 mRNA was not altered in the hearts of mice. However, Drp1 protein acetylation was increased. So this suggests post-translational modifications are regulating its activity in dyslipidemia. The team went on to perform experiments on cultured rat cardiomyocytes, and they found that incubation with saturated fatty acid palmitate led to the acetylation of Drp1, and thus its activation. And this activation resulted in an excess of mitochondrial fission, which reduced cell viability. By contrast, mutation of Drp1 to prevent its acetylation protected the cells. Together, the results reveal the mechanism of how dyslipidemia can contribute to heart cell dysfunction. Further, this data suggests that Drp1 activity or acetylation state could be novel targets for treating obesity-related heart disease. Okay, we're now going to switch over to the interview portion of the podcast. I have with me Dr Thomas Quertermous, the William G Erwin Professor of Medicine and the Director of Research in the division of cardiovascular medicine at Stanford University. And Dr Joe Miano, Professor and Jay Harold Harrison Distinguished University Chair in vascular biology at the Medical College of Georgia at Augusta University. And today we're going to be discussing their manuscript titled Coronary Disease Associated Gene TCF21 Inhibits Smooth Muscle Cell Differentiation by Blocking the Myocardin-Serum Response Factor Pathway. So welcome to both of you. Thank you for joining me. Dr Miano: Thank you. Dr Quertermous: Thank you. Dr St. Hilaire: So I'm going to start with you, Dr Quertermous. You've been taking a genomics approach to identify factors that contribute to coronary artery disease. And I'm wondering if you could just give us a brief summary of your work thus far and how it brought you to this current study? Dr Quertermous: Well, as you know, the classical risk factors for coronary artery disease and vascular disease in general really only contribute about 30% of the total risk and the remainder has not been studied, and not been investigated, and can't currently be targeted by therapeutics. So the goal is to try and better understand what are the molecular mechanisms in the blood vessel wall that must contribute the remainder of the risk. And so with the advent of genome-wide association studies and the identification of genes and loci, we've been able to begin to uncover the signaling pathways and mechanisms of disease risk. Dr St. Hilaire: And so the one we're most interested in today, this TCF21, you pulled that up out of one of your GWA studies, or how did we get to this? Dr Quertermous: Well, it's an interesting story. I first cloned that gene about 15 years ago when I was trying to understand vascular development, and it's a basic helix loop helix factor, and I was, well, a number of labs at that point in time were cloning this class of transcription factor to try and better understand developmental processes, and so it was one of a number of bHLH proteins that we cloned at the time. I did some work on it and then named it Pod-1 at that point in time, and then lost interest, and went away from it. And then I was involved in the cardiogram genome-wide association study for coronary artery disease, and I was sitting at my desk one night, and I was watching the hits coming in, you know, as we were doing the association, as we were doing the analysis, and I saw this gene, TCF21, and I thought, "Well, I don't really know what the heck that gene is." And so I was going back and forth between our data and a spreadsheet on the web, and I saw that I had published on this gene, and I was like, "Wow, I didn't even know that I had written a paper about this gene." And then it became clear that it was the bHLH factor I'd cloned some time ago. And then knowing what I knew about the development, that this gene is involved in early processes that lead to the formation of the coronary artery, and in particular the development of smooth muscle cells, then I became super interested, and I said, "Okay, my gene, I'm coming back to you. You and me are going to have a great future together." And that was really how I got started. Dr St. Hilaire: It re-found you. Dr Quertermous: It found me, I guess in this case, yes, and so I began then to work very seriously, because it's hard to try and understand mechanisms. And so we had a good starting point. We had a transcription factor so I could quickly identify the targets downstream of that, and I can link it into some cell biology that I already had some insights into. Dr St. Hilaire: That's a really neat story. I like that. It's kind of penicillin-esque. Dr Quertermous: Thank you. Dr St. Hilaire: Dr Miano, those of us familiar with smooth muscle cells appreciate that they are plastic, that they have this ability to kind of switch their phenotypes per se, and those of us familiar with that also then know about the myocardin and serum response factor pathway. But for our listeners who are less familiar with that, could you maybe give a brief background about what myocardin SRF pathway is and what smooth muscle cell phenotype modulation is? Dr Miano: Sure. I wish I could say, as my colleague said, that I cloned one of those factors, but I didn't. I've been interested in SRF since I was a graduate student actually. Actually went to Eric Olson's lab to look for what we affectionately called back then SmyoD, which stands for smooth muscle myo D. So at that time, we didn't understand what the factors were, even the signaling, that directed cells to become differentiated smooth muscle cells. So I went to Eric's lab looking for SmyoD. Of course I didn't find it. I found some other things. Worked a little bit on SRF, but it was actually Daiju Wong in 2000 or 2001 who in a Cell paper described an elegant a way of finding myocardin, what he called myocardin. So SRF myocardin is a transcriptional switch that is necessary and sufficient to make just about any cell a smooth muscle cell. So when myocardin is not present, then smooth muscle cells lose their differentiated state and they become another cell type, depending on who SRF talks to. And so how does a factor that binds a very discrete element like the CArG box, how does it confer cell identity or specific cell states? And it does so through its interaction with these cofactors, one of which is myocardin. And as this paper describes so elegantly, what Tom did in his lab, is that this TCF1 transcription factor, which is DNA binding, unlike myocardin, it does a similar thing in that it competes for SRF binding with myocardin, so it binds myocardin, prevents myocardin's ability to bind SRF, and thereby directs a new program of gene expression. Dr St. Hilaire: Interesting. So it's kind of helping to fine tune that transcriptional regulation. So I always think of smooth muscle cells, they're kind of always in a contractile state when they're healthy, and it's when they're in either unhealthy, or diseased, or a stress state that they're in that more proliferative-like state. And Dr Quertermous, your previous studies have shown that TCF21 is required for the De-differentiation, and proliferation, and migration of smooth muscle cells. However, there was one sentence in the paper that I was slightly confused on and I'm hoping that you can expand about the bigger role of TCF21. And what it said was that TCF21 expression is protective towards human coronary artery disease. And so the data in the paper show that TCF21 inhibits smooth muscle cell contractility. So can you maybe reconcile the bigger mutations or things you identified in the GWAS with the functional activities you're seeing that you presented in the paper, and maybe talk about the timeline in the continuum of atherosclerosis where TCF is maybe good or maybe bad? Dr Quertermous: So this paper is one of a duo of papers, honestly, that the other paper being published in Nature Medicine almost exactly the same time, and so that paper sort of described some of the aspects of TCF21 at a population level and shows that if you look at all of the single base pairs in the genome that regulate disease risk at 6q23.2 and also regulate expression, you can gain an idea of what's the directionality of the expression of TCF21. And those data suggests that the more TCF21 you have, the less your risk of developing coronary artery disease. And Joe and other scientists have worked for a long, long time to characterize this process and characterize the plasticity of this cell type. And note that one can switch the cell back and forth between being a contractile0differentiated cell to a de-differentiated cell, and elegant work by Gary Owens and a number of investigators have profiled the phenotype of the cells that the smooth muscle cell can become if it undergoes this differentiation process. It's not been able to know though up until this point in time whether that's a good process or a bad process. I mean, 15, 10 years ago we thought smooth muscle cells are proliferating, they're creating a space-occupying lesion, they're decreasing the lumen of the blood vessel, and that's got to be a bad thing. And in honesty, I think over the past three or four years, it's been increasingly clear that perhaps the smooth muscle cells are actually doing a good thing. They are stabilizing the lesion, they're creating the fibrous cap, and there's been some nice work correlating the number of smooth muscle cells in the plaque to the risk that that plaque is going to rupture. Dr St. Hilaire: Yeah, that was kind of my next question. Do you think there's more nuance to it's not just contractile, and synthetic? There's much more broader scope and it's not so much a good or a bad smooth muscle cell. Dr Quertermous: I think it depends on the circumstances I guess, but it's important that the smooth muscle cell be able to migrate into the plaque, and begin to produce matrix components which stabilize the plaque, and to form the fibrous cap, and I think if the smooth muscle cell remains in the media as a contractile cell, it's really not able to do those things, right? And so the human genetics data, looking at the directionality, the expression, the different alleles and their expression patterns, and what is the risk allele at? In this region of the genome, it's pretty clear that more TCF21 is good, and what TCF21 does is to promote this phenotypic modulation. And so that suggests that the process as a whole is good. Not just the gene, but what it does. It's really not possible that TCF21 does anything else in the blood vessel wall. It's primarily restricted to the smooth muscle cell. It's not expressed in macrophages, or endothelial cells, or the other cell types that we think are important in the pathophysiology of the disease process. So putting everything together, it looks for the most part, like this is a positive force in the blood vessel wall, this gene and this process. Dr St. Hilaire: Interesting. And speaking to the vessel wall, I thought one of the very cool and really key experiments in the paper was taking your mechanistic in vitro studies into the mouse, and Dr Miano, maybe you could tell us a little bit about how you were able to do that and mutate these smooth muscle specific CArG boxes in a mouse model. Dr Miano: Well, that's a really good question. Again, it's a history. We've wanted to edit CArG boxes, well, mutate back then, for a long, long time, but it wasn't until the CRISPR craze took a foothold that we really recognize now the power of harnessing that and doing the experiments we wanted to do for so long. And so we've previously published on a CArG box in the first intron of the calponin locus, and found that, to our surprise, that a subtle mutation in that element completely abolished expression of calponin into the smooth muscle. So Tom and I were working in parallel and unbeknownst to me, Tom was working on this SRF enhancer in the second intron, and we've known for quite a while that SRF is auto regulated by itself, and there's CArG boxes in it 5-prime promoter, and there's CArG boxes in the interior of the locus as well, including the one in the second intron that Tom describes in the paper here. And so what we've been doing is using CRISPR in the mouse to make these subtle edits in these CArG boxes around the SRF locus. And unlike the affirmation calponin model I just described, if we mutate the two proximal CArG boxes of SRF, we don't see a lot of change in SRF expression. That was really surprising to us, because studies from Bob Schwartz' lab in Houston two decades ago showed those were important, at least in an artificial reporter assay for the autoregulatory loop that he first described. So we moved interior to this CArG box that's really the focus of this paper, highly conserved, much more so than other CArG boxes, and we first deleted the region, which we often do with CRISPR, and found there was a decrease. But we'd like to do more subtle things with CRISPR, which is really the power of this new editing technology. And so we went in and made just, I think it was like four or five base substitutions to create a novel restriction cipher ease in genotyping. And we reported in the paper, you can see that, to compliment Tom's group's data, that in vivo, indeed, that CArG box by itself, nothing else altered within the locus, did cause a, I would call, a substantial decrease in expression of this important regulator. And so that was really our main contribution to this paper. Dr St. Hilaire: Yeah. I know the opportunities are endless, but also complicated and expensive, and I thought this was a beautiful addition to really confirming those mechanistic studies. So I think my next big question is, if TCF21 is,  so important and protective, and perhaps it's upregulation is beneficial, what is regulating it, and do we know how we can potentially modulate this? Dr Quertermous: That's a great question. That's a great question. And so we know a few things; we certainly know that platelet-derived growth factor stimulation of smooth muscle cells will upregulate TCF21, which is sort of surprising. I mean, it's not so surprising, I guess. So we've spent a little time working on that, and there's a micro RNA which regulates the expression level of TCF21, but we haven't spent a lot more time than that, honestly. We spent a lot more time downstream trying to figure out what's the mechanism by which TCF21 works to suppress the smooth muscle contractile phenotype and activate this more de-differentiated migratory phenotype that the smooth muscle cell adopts. So we've not gone upstream, but your question's a really, really good one. We certainly mapped where TCF21 binds across the genome and we've mapped the variation that regulates its expression, and so we've made progress in that direction, and as I said, identified things which are downstream. But we definitely need to spend more time upstream, and I think that's the area of this intersection of molecular science and genomic science, that there are not many groups that really spend much time up above the gene trying to understand. And so we've not spent enough time doing that, and I think that as a community we've not spent enough time doing that, because I think that's where the big payoff can come in terms of therapeutics. Dr St. Hilaire: To that end, I think I'll end with that question. What do you think is the best way that we could leverage your findings in the clinic? Would it be to focus more on the downstream or to try to identify these more upstream factors in TCF21? Dr Quertermous: Well, I think both open up opportunities, right? If we can understand how TCF21 works and what's downstream, and we can activate those processes and activities, then that's good. If we can figure out what's above TCF21, that would be good as well. The danger there is that TCF21 does a lot of things in a lot of different cells in the body. Dr St. Hilaire: So it'd be a little bit harder to focus onto a smooth muscle cell in a plaque than perhaps some of the downstream effects of TCF21? Dr Quertermous: Correct. Right. That's my worry. It's sort of like thinking about TGF beta and you wouldn't really want to try and manipulate TGF beta. Dr St. Hilaire: That's a whole another can of worms. Dr Quertermous: Yeah, it gets you into a lot of difficulties, I think. So we're really pretty focused downstream now and thinking that we can find specific opportunities there that are resident in that smooth muscle cell in the blood vessel that may not be active in other cell types. So that's really our thinking and that's the way we're going. Dr St. Hilaire: Wonderful. Well, thank you so much to both of you for joining me today. I learned a lot and I really thought this was a beautiful, complex, but well-done study, so thank you very much. Dr Miano: Thank you, Cindy. Dr Quertermous: Thank you so much for calming us down, I guess. Dr St. Hilaire: Well, that's it for our highlights from the January 31st and February 14th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the Editorial team of Circulation Research. Some of the copy texts for the highlighted articles was provided by Ruth Williams. Thank you to our guests, Drs Thomas Quertermous and Joseph Miano. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


20 Feb 2020

Rank #5

Podcast cover

January 2020 Discover CircRes

This month on Episode 8 of the Discover CircRes podcast, host Cindy St. Hilaire  speaks with Nikki Purcell and  Sean Wu, the chair and vice-chair of the BCVS Early Career Committee. The episode also features an interview with the 2019 BCVS Early Career Finalists, Dr Luigi Adamo, Dr Swati Dey, and Dr Jihoon Nah. In addition, we highlight three featured articles from the January 3 and January 17, 2020 issues of Circulation Research. Article highlights: Souza, et al. Upregulation of Plasma SPM by Enriched Marine Oils Paredes, et al. Metabolic Control of VSMC Phenotype Ritterhoff, et al.  ACC2 Deletion Prevents Aspartate Synthesis Transcript Dr St. Hilaire: Hi. Welcome to Discover CircRes, the podcast of the American Heart Association Journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm an assistant professor at the University of Pittsburgh. The goal of this podcast is to share with you highlights from recent articles published in the Circulation Research Journal. But today, we're going to have a special edition focused on early career. The American Heart Association has 16 different councils. One of which is basic cardiovascular sciences,  or BCVS. The BCVS Scientific Sessions is held annually and brings together basic and translational cardiovascular scientists from around the world. It has become the go-to meeting for intra and interdisciplinary cross fertilization of ideas in basic cardiovascular research. The overarching goal is to integrate molecular, and cellular, and physiological approaches to address problems relating to functional genomics, cell signaling, myocardial biology, circulatory physiology, pathophysiology, and peripheral vascular disease. In addition to highlighting new approaches and discoveries from the general scientific community, the BCVS Council also plays a pivotal role in training the next generation of junior scientists and trainees. At the recent meeting in Boston, I had the opportunity to interview the chair and vice chair of the BCVS Early Career Committee, Dr Nicole Purcell from UCSD, and Dr Sean Wu from Stanford University, as well as the three finalists of the Outstanding Early Career Investigator Award competition, Dr Luigi Adamo, Dr Swati Dey, and Jihoon Nah. Hope you enjoy. Before we get to our interviews with the BCVS early career committee chairs, and the finalists of the BCVS Outstanding Early Career Investigator Award, I want to give you a few highlights from three articles that were published in the January 3rd and January 17th issues of Circulation Research. The first article I'd like to highlight is titled Enriched Marine Oil Supplements Increase Peripheral Blood Specialized Pro-Resolving Mediators Concentrations and Reprogram Host Immune Responses: A Randomized Double-Blind Placebo-Controlled Study. The first authors are Patricia Souza and Raquel Marques, and the corresponding author is Jesmond Dalli, and they are from Queens Mary University of London. So, this study is attempting to answer the longstanding question of whether I should or should not take fish oil supplements. Once ingested into the body, essential fatty acids, which is a group that includes those that are found in fish oils--those fatty acids are converted into molecules called specialized pro resolving mediators, or SPMs. SPMs can reduce inflammation and can also promote a process called phagocytosis where immune cells can essentially eat up dead cell debris and also micro-organisms like bacteria. While these actions are beneficial, whether ingesting fish oils translates to beneficial cardiovascular effects in humans, is unclear. Some studies show the oils reduce inflammation, while others have shown no effect. And part of the lack of clarity on this is that in previous studies there was no impartial measure of the clinical efficacy of the supplements. So to combat this, Souza and colleagues performed a double blind, placebo-controlled crossover study to determine the effects of marine oil supplementation, which by the way was tested at three different concentrations on both SPM levels and on immune cell function. Now for those of you who are unfamiliar with the clinical trial lingo, that means that both the patients, and the scientists, and the doctors did not know who got what pill, whether they got the placebo or the fish oil, and each patient was tested with both the placebo and with the fish oil so that they could be somewhat used as their own control at the end of the study. The scientists then obtain the blood from the patients before and then during the experiment at several time points. Blood samples from the subjects revealed a dose dependent increase in SPMs that was significant in the two high dose groups. Meaning the more fish oil they took, the more beneficial effects we're seeing. These effects peaked a few hours after they ingested the fish oil. Further, they found that high dose blood samples, monocytes and neutrophils, so those are inflammatory cells, those cells had increased phagocytic activity, while leukocyte activation, which is a sign of inflammation, was decreased. These beneficial effects persisted after SPM levels returned to baseline. These results suggest that SPMs are not only likely mediators of fatty acid-induced immune effects, but perhaps they could be useful efficacy indicators for future trials. The second article I want to share with you is titled Mitochondrial Protein Poldip2 Controls VSMC Differentiated Phenotype by O-Linked GlcNAc Transferase-Dependent Inhibition of a Ubiquitin Proteasome System. The first author is Felipe Paredes and the corresponding author is Alejandra San Martin, and they are located at Emory University in Atlanta, Georgia. So, I've said it before and I'll say it again, we all learned in elementary school that the mitochondria are the powerhouse of the cell. The nuclear encoded protein, Preliminaries Interacting Protein Two or Poldip2, is required for the activity of the TCA cycle, which is called the tricarboxylic acid cycle. It's also known as the citric acid cycle, and it's also known as the Krebs cycle. So interestingly, this nuclear protein, Poldip2, has effects on mitochondria, which as we also know have their own DNA encoding some of their proteins. So, Poldip2 deficiency reduces the activity of the citric acid cycle, which then induces a shift in the metabolic reprogramming, which leads to a lower rate of oxidative metabolism, and a higher glycolytic rate. So previously, this group found in mice that were heterozygous knock-outs for Poldip2, that these mice exhibited no avert phenotypes at baseline, but after an injury they actually showed protective effects. This was done with a wire induced injury that causes neointima formation, and also in an experimental aneurysm model. In normal healthy vessels, smooth muscle cells reside in a quiescent state. However upon injury, they can be induced to lose their fully differentiated smooth muscle cell phenotype, and acquire a more plastic, undifferentiated state. And in that state, these cells can migrate, proliferate, and in some cases even transdifferentiate where they acquire non-smooth muscle cell-like markers. The investigators, because of these phenotypes in response to injury, decided to look at the role of Poldip2 in metabolism in the phenotypic switching of smooth muscle cells. They found that reduced levels of Poldip2 in vascular smooth muscle cells in vitro induced the expression of transcription factors that are necessary for the expression of smooth muscle specific markers. And further, repressed the transcription factor KLF4, known to promote the loss of the smooth muscle contractile phenotype. Poldip2 deficient mouse aortas expressed high levels of contractile proteins, and more significantly did not de-differentiate or acquire macrophage-like characteristics when exposed to known stimuli, cholesterol, or PDGF. These effects are caused by inducing the enzymes that perform protein glycosylation, which helps to stabilize smooth muscle specific transcription factors yet repress the differentiation factor KLF4. Altogether, this work suggests that mitochondria metabolism and mitochondria-induced signaling plays a main role in the maintenance and phenotypic switching of vascular smooth muscle cells, and that this access could be targeted to modulate smooth muscle phenotype during vascular diseases. Sticking with the metabolism theme, the last article I want to share with you before our interviews is titled Metabolic Remodeling Promotes Cardiac Hypertrophy by Directing Glucose to Aspartate Biosynthesis. The first author is Julia Ritterhoff, and the corresponding author is Rong Tian, and the work was completed at the University of Washington School of Medicine in Seattle, Washington. Cells of hypertrophied hearts switch from using fatty acids for energy production to glucose. This is less efficient. It has been shown that preserving fatty acid oxidation prevents the pathological shift of substrate preference, which then preserves cardiac function, and improves energetics, and reduces cardiomyocyte hypertrophy. While the effects of preserving fatty acid oxidation are well known, it remains unclear whether substrate metabolism regulates cardiomyocyte hypertrophy directly, or whether this metabolic shift is a secondary effect of improving cardiac energetics. So to that end, the investigator sought to determine the mechanisms of how preservation of fatty acid oxidation prevents the hypertrophic growth of cardiomyocytes. They went about this in two ways, in vitro and in vivo. And so for in vitro, they took some cultured adult rat cardiomyocytes, and they grew them in a medium that contained glucose and mixed chain fatty acids and induced pathological hypertrophy by supplementing these cells with Phenylephrine. This hypertrophy in a dish caused increase glucose consumption and higher intracellular aspartate. Interestingly, adding aspartate alone was enough to promote hypertrophy. In vivo, they found that fatty acid oxidation prevented the metabolic shift to anabolic energy production, which further prevented cardiac hypertrophy, and overall improved myocardial energetics. Together their data shed new light on the contribution of intermediary metabolism, specifically aspartate to the hypertrophic growth of the heart. They found that aspartate synthesis is a rate limiting step, and they found that specific mechanisms for aspartate production support growth and proliferation of postmitotic cells, and these studies now provide potential therapeutic strategies to target for reducing cardiac hypertrophy. Okay, well I'm here with the BCVS Vice Chair and Chair of the Early Career Committee, Nikki Purcell and Sean Wu. Thank you for coming. Nikki Purcell: Thank you for having us. Sean Wu: Yeah, thank you. Dr St. Hilaire: Yeah. I was wonder if you could just introduce yourselves and maybe a little bit about where you are in your career. Sean Wu: I'm Sean Wu. I'm at Stanford University. I'm part of the Stanford Cardiovascular Institute. I do both clinical work as a general cardiologist, and I also work on research in stem cell and developmental biology. Dr St. Hilaire: And how long have you had your lab? Sean Wu: I have had my lab for about 10 years now. Dr St. Hilaire: That's good. Success story. How about you, Nikki? Nikki Purcell: All right. I am at the University of California, San Diego, in the Department of Pharmacology. I'm an Associate Professor there. My research deals with phosphatases in the heart and their role in cardiac hypertrophy and heart failure. And I've gotten my position as a professor for about eight and a half years now. Dr St. Hilaire: Excellent. So, my lab just had its fourth birthday, so we're still on the, okay, we're moving up. We're moving up. Nikki Purcell: Congrats. Dr St. Hilaire: Yeah. Sean Wu: Congrats, yeah. Fantastic. Dr St. Hilaire: Yeah. Thank you. So, what is the role of the BCVS Early Career Committee? Nikki Purcell: So, the real role of the Early Career Committee is to support early career investigators. And our role in the AHA is, one, to keep the early career investigators engaged in the AHA and BCVS, but also to help them with their questions as they transition from either their predoc to their postdoc, or getting their first faculty position. So, we try to make events where they can network, and get the mentorship they need, and help them along the way. Sean Wu: And also, the Early Career Committee serve as a connector between the early career and the bigger AHA with all the different activities that we have. But it also connects the early career member with each other because we feel like having an opportunity for them to be able to meet people in their similar stages of training, they can share stories with each other, and build a network of scientists themselves as they grow up into being full faculty. Dr St. Hilaire: Great. So, what kind of events are these that you use to either help bring them together or help them move along? Nikki Purcell: We've done multiple along the years. One of the ones that is really popular is probably our mentor lunches where we actually have established PIs from all different fields come in, and the one that's worked we've done the speed dating, but that's hard to get everybody in. And the last one we did was we had 12 to 13 people around a table with two PIs and that worked amazing. And they had that chance because a, early career sometimes don't like to talk as much- Dr St. Hilaire: You're a little shy. Nikki Purcell: ... but other people are going to ask questions they want to hear. So, it was a great opportunity. And for network we had a mentoring talk. So, that's one of the things we do is to try to connect people with other people in their field that maybe they are afraid to normally just go up to. But it's a much more relaxed setting I would say. Sean Wu: We also have some of the mainstream session, so having well known people come up and talk about their career, like the ones that we just now have with Litsa Kranias describing her long and distinguished career as a scientist with some of the lessons that she's learned along the way. But we also like to offer people opportunity to experience what the careers of people who have not necessarily done in academic, but into industry, in the legal world, or into the venture world to give the trainees some ideas of what they could do with their career. So, that's one of the sessions that we have this time called, Oh, The Places That You'll Go, so that they can actually see what the opportunities that they have as they complete their training in science and looking forward to the next stage of their career. Dr St. Hilaire: That's so important because I mean, what is it, maybe 10% to 20% of people actually stay and go on in academia? Nikki Purcell: And we get a lot of questions on mentors, and how do we find our mentors. And so, the other session that we're having is our Lunch and Learn really is to show new faculty with their chairs, postdocs with their mentor, and graduate students with their mentor, and really get it that the early careers can ask them questions. Especially if you're leaving the lab, how are you taking your project? How did you work that out with your mentor? You're a new faculty, how did you negotiate? All those questions that they don't teach you maybe so much along the way. Dr St. Hilaire: No. They don't. Nikki Purcell: So, we're having the panel up there so that they can throw questions at them- Dr St. Hilaire: That's great. Nikki Purcell: ... and really ask from Chair down to the graduate student. Dr St. Hilaire: And so, we're at the BCVS conference right now recording this, but you have events also at the AHA Scientific Sessions in the fall? Nikki Purcell: Yes. Sean Wu: Mm-hmm (affirmative). Dr St. Hilaire: Is there any non-conference related things? Do people ever kind of cold call the ECC saying, "I need advice," or is that a thing? Nikki Purcell: We've gotten emails, yeah. Sean Wu: Yeah. Right. Nikki Purcell: Yeah. We're really accessible. Yeah. Sean Wu: We've had inquiries, too. Dr St. Hilaire: Yeah. Sean Wu: Yeah. Besides things that we do at the meetings, we just now have this brand-new mentoring program with the BCVS where a young investigator, if they like to be able to interact with certain established investigator, can now make a request and apply to be able to make a travel visit. Dr St. Hilaire: Very nice. Sean Wu: And so, we have a full list of mentors who are willing to either sponsor the person, or both sponsor and provide some financial support for the training to come. Dr St. Hilaire: Wow, so you've already gotten buy-in from these senior mentors. Sean Wu: Yeah. Right. Nikki Purcell: Yeah. Dr St. Hilaire: That's wonderful. Nikki Purcell: We have over 30 mentors who have said yes, and we're always looking for more. So, if there's faculty out there. And what's great is also it gives a chance for let's say you want to transition to a different field, if you're a grad student postdoc it gives you that chance to meet that lab in that new field that you're interested in. And it's also for young PI, so if you're just starting out your lab, and you want that little help in that field, so it's open to that, too. So, we're really excited about that new program. Sean Wu: And I think it provides them a lot of opportunity to get exposure. Because that's one thing that the young scientists always tells us about, it's hard for them to really get to meet people or get other people to know who they are and what they do. And the Early Career Committee is one that really helped facilitate giving people opportunity, and put them in front of other people to help getting leadership training, and getting exposure. Nikki Purcell: And this year, we're actually having the poster competition, which we've never had before. So again, three established judges will definitely come to all these posters and speak to them, and that also gets them exposure. So, we're giving out five awards to both the early career trainees as well as postdoctoral. Dr St. Hilaire: That's wonderful. Nikki Purcell: Yeah. And so, we'll have some honorable mentions, too. The more people we can recognize, the more names we can get out there. Dr St. Hilaire: Absolutely. I remember one of my very first conferences as a postdoc, we just kind of made this discovery of a new disease, and I had written down the names of five big names that I was like, "I know they're going to be at that meeting. I really want to meet them." And I just had business cards at the ready, "Please come to my poster. Please come to my poster." That's the biggest fear as a poster presenter, what if nobody comes? What if I can't share this? So, I think that's an amazing avenue of interactions you've created. Nikki Purcell: And we have over 200 that have applied for our award, but there's 600 posters. And we know that a large proportion of those are Early Career. Sean Wu: Yeah, yeah. Dr St. Hilaire: That's great. If there's one thing a trainee, or a postdoc, or even junior faculty could do at a conference, of this size at BCVS we're about I think 500 to 1,000 people. Nikki Purcell:1,000 this year. Dr St. Hilaire: 1,000 this year. Amazing. Nikki Purcell: 1,000. Biggest conference ever. Dr St. Hilaire: That's wonderful. Oh, that's great. Sean Wu: Yeah. That's right. The first time. Dr St. Hilaire: So, 1,000 at BCVS versus, I don't know, the tens of… Nikki Purcell: Yeah. 13,000. Dr St. Hilaire: 13,000 at Scientific Sessions. What can someone who just feels lost in this little sea of all these big people do? What would you recommend as advice to a more junior person to interact? Nikki Purcell: I would say one, find us, because if we know your name and we know what you do, we promote the early careers, and we try to, and I would introduce who I'm with. In our social event that we have at Tuesday night brings together not only early career but the established investigators. It's everyone can come in, and we try to welcome that. My biggest thing, and when I met with a small group the other day of postdocs and students, is just grab people. You see people walk past your poster, start saying, "Are you interested in seeing what I'm doing?" Engage them. People are going to look at your poster and keep walking, but if you engage and are excited about what you do, they're going to be excited to say, "Oh, okay. I'm going to stop and listen." So, my big thing is get out of your comfort zone and just engage. Sean Wu: I do think that it is a little daunting. If you were just starting out as a new graduate student, and you have never been to an AHA meeting, and especially if you go to the annual session in November, it does feel a little bit like you're just overwhelmed by too many people, too many sessions, too much- Nikki Purcell: It's too much walking. Sean Wu: Right. Too much going on. And I do think that the Early Career Committee is what gives you a little bit more of a home for you as a trainee because by, at least, starting out meeting other people in the specific sessions that we create for them, now they can start building their network from a much more cozier, comfortable environment of the Early Career Committee activity. And before eventually moving out to get to know more people in other councils and the bigger AHA. Hopefully that also allows people to get excited about taking on more of a leadership role in the AHA down the row in the future as they become more familiar. Dr St. Hilaire: Well, thank you so much Dr Sean Wu and Dr Nikki Purcell. Everyone come to BCVS and meet them in person. Nikki Purcell: Thank you. Sean Wu: All right, thank you. Dr St. Hilaire: Yes. Thank you. Nikki Purcell: Thank you so much. Dr St. Hilaire: All right, so now we're going to talk with the three finalists of the outstanding Early Career Investigator Competition that's held annually at the BCVS Conference. With me today is Dr Luigi Adamo, Dr Swati Dey, and Dr Jihoon Nah. So, welcome all of you. Jihoon Nah: Thank you. Thank you for inviting us. Dr St. Hilaire: Yeah, thank you. Swati Dey: Yeah. Nice to meet you. Dr St. Hilaire: Congratulations again on becoming the finalists. I don't know how many submitted, but I know it's always very steep competition. Jihoon Nah: Well, thank you. It's an honor to be here. Dr St. Hilaire: Maybe we can all take a turn, and you can just give a quick introduction. Say where you're at, and what stage you're in in your career. Jihoon Nah: Yes. So, I'm Jihoon Nah in Rutgers University, and I'm now a post doctorate fellow in Dr Sadoshima’s lab. Dr St. Hilaire: How long have you been there? Jihoon Nah: I came to USA three years ago from South Korea. Dr St. Hilaire: Oh, goodness. How do you like it? Jihoon Nah: Yeah, very good. Dr St. Hilaire: Very Good? Jihoon Nah: Yeah. Dr St. Hilaire: Good. Luigi Adamo: So, hi, my name is Luigi Adamo. I'm a physician scientist. I'm a cardiologist at Washington University in St. Louis, and I just concluded this long training as a physician scientist doing residency fellowship, advanced fellowship, postdoc final, and faculty. I'm an instructor of medicine there, and I'm looking for starting my own lab. So, it's a transition phase. Dr St. Hilaire: Ah, so maybe we can use the podcast to promote you. Luigi Adamo: Oh, anybody want a motivated physician scientist in cardio immunology? Please send me an email. Dr St. Hilaire: Perfect. Okay. Wonderful. Swati Dey: So, my name is Swati Dey. I am a new PI. I started my lab at Vanderbilt University Medical Center a couple of months ago. So, it's a really new experience for me right now. I did my postdoctoral training from Johns Hopkins for about four years with Dr Brian O’Rourke, and then for two years I was a junior faculty. And like I said, I recently transitioned. Dr St. Hilaire: Yeah, yeah. Excellent. So, we got kind of three different spots in the career. I'm also relatively new faculty. I got my lab in 2015, so we just had our fourth birthday, and I have at the same time feelings of I've been here forever and I'm still brand new. Hopefully we can share some of the early career ups and downs with everybody who's listening. So, thanks again for coming. Dr St. Hilaire: I was actually wondering, how did you guys ... We're all in the cardiovascular field, and at this conference in a little bit more cardiology than the rest of it, so how did you get into this field? What was your path? Swati Dey: So for me, it was I don't know how I got there. I have been very lucky. Dr St. Hilaire: It just happened. Swati Dey: No. So, my PhD was from Ohio State. I got a PhD in microbiology. Dr St. Hilaire: Interesting. Swati Dey: And then, I was looking for postdocs. So when I was young, my mother, she passed away due to a cardiac condition. So, when I got an opportunity to start a postdoc in a cardiovascular lab studying cardiovascular diseases, I just couldn't believe that this would just fall in front of me. I just went with the flow. It was a little hard in the beginning because the learning curve is essentially longer. Dr St. Hilaire: I'm sure. Swati Dey: I had no background in cardiac physiology, electrophysiology, anything related to cardiac. But it was also fun. I was learning so many new things. Everything I did was just brand new, and it made my postdoctoral training period so exciting, and I was just feeling proud of myself, everything I accomplished. Dr St. Hilaire: Well, the title of your talk, I'm not going to read it because it's very long, but essentially, you're looking at the neurocardiac access, and how that can be manipulated to help with myocardial infarction treatments. You've gone from teeny tiny bugs to ... Swati Dey: Absolutely. Yeah. It's been great. So yeah, this work essentially focuses on nonischemic heart failure. So sudden cardiac death, like if you have been to my talk, it talks about it happens in patients with even before the signs and symptoms of heart failure appear. Like very early stages of heart failure. I could be doing late stage of heart failure, too. So, there's no signs and symptoms for sudden cardiac death. Swati Dey: And to prevent that, there are very few treatment options available. There are ICDs, the defibrillators, but they are expensive, and if you go running they might just shock you,. So there are very appropriate shocks, but there are also inappropriate shocks. So, the quality of life for these patients are very poor. And I remember when I started working on this project, we got some human samples of patients who were my age, and they had defibrillators, and they were in such poor health that they had to go through this new surgical treatment. And it was so exciting to study the underlying mechanisms because I could see it translate into the real world. Dr St. Hilaire: And in people young enough to really benefit from it. That's wonderful. That's a great story. Luigi Adamo: Great story. Dr St. Hilaire: Yeah. I started in microbiology, too, actually with a fungal genetic lab. Swati Dey: Oh, wow. Dr St. Hilaire: Yeah. Now I do valve research, so it's funny where it takes you. Swati Dey: Yeah, exactly. Dr St. Hilaire: Luigi, how'd you get in this field? Luigi Adamo: So today, I presented work about B-cells in the heart. I think God has a good sense of humor, and also kind of serendipitous stories. So, I've always wanted to do cardiology. My dad is a cardiologist. But so, I did a PhD in hemodynamics, and then when I joined the physician scientist, phew, I actually ended up doing cardiac inflammation and heart failure just because I was so strong there. And I picked a mentor, Dr Doug Mann, a giant in cardio inflammation, but he didn't want to study B-cells, I didn't want to study B-cells. So, I started doing a different experiment, and then a friend was teaching me how to do a flow cytometer and heart samples gave me as a control an anti B-cell antibody. Luigi Adamo: And then, I remember telling her, "Why do you give me this?" It was like, "Whatever. We need a control in that color." And then in my model, I saw this dramatic effect on B-cells, and then I kept doing my experiments, and all my hypotheses were wrong, and there was always a signal on B-cells- Dr St. Hilaire: Oh, that is so funny. Luigi Adamo: ... and it was you know what? I think I need to study B-cells. Dr St. Hilaire: I think there's a B-cell there. Luigi Adamo: And it was a blessing because I might be one of the very few people in the world studying B-cells in the heart, and that's what I think sometime make people interested in my work because there's very little known about it. Dr St. Hilaire: Yeah. And how about you? What's your story? Jihoon Nah: So when I was a PhD student, I was working on some molecular biology in neurons. But it was a little bit basic in the field, and I just focusing on some cells, some intracellular cells And after I got my PhD, I want to move my field from basic to more clinical field. And I have a lot of interest in autophagy and mitophagy, and I tried to find some post doctorate position in more some clinical field which focusing on autophagy. And fortunately, I can join Dr Sadoshima’s laboratory, focusing on autophagy and mitophagy in cardiovascular disease. That's what I can do in this field. And I think autophagy is a very important role, especially in non-dividing cell like a cardiomyocyte. And I can allow many news and knowledge [inaudible 00:26:54] in this cardiovascular field, and I knew there are many things that are known about the role of autophagy or mitophagy in cardiovascular disease. And I think I want to stay this field, yeah, to find- Dr St. Hilaire: Yeah? You don't think you'll switch? Maybe like the microbiology, that hard switch. Jihoon Nah: Yeah. I really like this field. Dr St. Hilaire: Well, you say that. We're going to come back in five years and we'll see. Dr St. Hilaire: So, everyone's a little bit early career. So, what's really been a hard hurdle for you to overcome? Whether it was in a particular experiment, or paper, or just in the career itself. Swati Dey: It wasn't a hurdle, but one thing which took a long time to learn and was very hard was grant writing. So as a postdoc, so Hopkins is a great institution, but also you're competing with people who are equally, or well, actually smarter than you. Dr St. Hilaire: Well and also, if it's English as a first language versus English as a foreign language, I can't imagine. Swati Dey: Exactly. So, the tools and techniques to write your science on paper is not easy to communicate such that I understand my science, but to make another person convinced that my science is good, it's not easy. So, I think grant writing has been the hardest thing I had to learn. Because as a graduate student, you don't write. So, other things I knew I could do, it was in my control, but grant writing required so much rigorous training. And I'm happy I had colleagues and my mentor who took the time out to actually make my entire grant red. It came back with all tracking and comments, and- Dr St. Hilaire: Oh, yeah. I know. It's so disheartening. Swati Dey: It's actually so helpful. Dr St. Hilaire: Yeah, yeah. Swati Dey: But yeah, they took the time out. They thought that they were invested in my success, and my colleagues, and my mentor. Dr St. Hilaire: So essentially, you kind of formed a mentoring team that really helped train you or teach you how to properly write a grant. Swati Dey: Yeah. Dr St. Hilaire: That's really good advice for people to have. Swati Dey: And it doesn't really have to be someone who is very senior. Maybe someone who is recent. You have to find people who have been successful in that particular phase of science. Let's say grant writing, or maybe networking, which is also very important, or doing certain techniques. Reach out to different people, seek help, and learn from them, and never think that you are just too good or someone is going to judge you. That's the problem more that we think that if we go to someone with our weaknesses, they're going to judge that, "Oh, you're not good enough," but we all are not good enough in some parts. Dr St. Hilaire: Yeah. We're all weak at some point. Exactly. Swati Dey: Yeah. Dr St. Hilaire: Oh, that's really great advice. Luigi Adamo: I agree with all that. It sounds like very wise and very thoughtful. I am a physician scientist, as I mentioned. For me, one thing that has been very hard has been the length of the path because when you try to do both things, and you have the passion for both, it just takes a long time. And then, there are people that ... people who did their PhD with me, and they did it well as I did, or maybe a little bit more, I don't know, but then they were already an established investigator, and I was a trainee back to the bottom. And then, I was doing medicine, and then my classmates were leaving and getting big jobs, and I was back in the lab. And it just it takes a very supportive family, a lot of dedication. I think if you have that fire and passion for science and for medicine, it just keeps you afloat. But sometimes it's hard. Dr St. Hilaire: How about you? Any big hurdles yet or not? Jihoon Nah: No. So fortunately, I think with the experiment. I think I have a very small some problems, and I can overcome every time. But in my case, it always feels a little bit difficult to grant-writing because using English is very difficult to me. And also, in my case, I came USA around the two and half years later after I got my PhD degree. And many of fellowship, or first doctor fellowship, or grant has some time limitation within four years of the PhD degree. So, it's very tough to me to get a grant. Dr St. Hilaire: No, that's a really good point. I don't think enough graduate students fully understand all the different requirements of getting the postdoc, and then knowing if you want to write a K grant you need to have a project at year two because you'd have to submit it about twice, and you need the timeframe to resubmit it. And that's the thing that's really important that I don't think we spend enough time teaching trainees on. Swati Dey: My PhD was not in cardiovascular sciences, so I did not know what a K grant was. Dr St. Hilaire: Sure. Swati Dey: So when I came in, after two years I started seeing, okay, people are talking about this Dr St. Hilaire: What is this K99? Swati Dey: What is it? And apparently, a lot of my colleagues they had this in their life plan. They planned this out when they were still in their, probably med school, or in the graduate school, and I had no clue. Dr St. Hilaire: I was similar. I'm actually the first one in my family to go to college, let alone get a PhD and become a professor. And I can remember at every step of the way always finding people who I was like, "How did you already know this? How did you know what this award was, or to apply for this grant, or to go to a conference?" And I think that's partly why we want to have these conversations and get them out there is so that people in similar situations at least hear what these are in time, and have those opportunities. Dr St. Hilaire: All right. So, we'll end with a question that hopefully can help everybody else. If you could go back and give yourself any piece of advice, or any bit of nugget of knowledge that would help you had you known it then, what would you say? What would be one little thing you'd want to tell your past self? Swati Dey: Meet with your PI very early on, and sketch out your career plan. Luigi Adamo: I actually…I was about to say the same thing. I've been blessed with great mentors, but I think it's very important to have open communication about expectation and goals because very appropriate goals could be different in the minds of people. Then, there are personal needs, there are personal feelings about things. And in the end, a good mentor, if you set a goal, will help you work toward that goal. Dr St. Hilaire: How about you? You're still early, early, but that's okay. What would you tell your grad school self? Jihoon Nah: I'd say it's very interesting question. So, when I was a PhD student, I didn't want to learn some new techniques, and I think I was a little afraid to learn new techniques. And also, I feel it's very difficult and I frustrated my PI to set up new things in the laboratory. I think if I can beg, I want to say I just want to learn new techniques, and I just want to try to push my PI to set up new things because it’s very important to support our hypotheses. Swati Dey: No, he said something very important. I noticed this. Some of my fresh PhD friends, I asked them to apply to a good lab for postdoc. They're like, "Oh no, I am molecular biology. I can only do what will ... If I switch fields, I can't learn anything new." Dr St. Hilaire: Yeah. I can't do that. Swati Dey: Yeah. That's not true. You can switch fields. Go from engineering, to science, to English, to anything if you want to. Dr St. Hilaire: Absolutely. Jihoon Nah: And one more thing then. In my case, I came USA a little bit late after I got PhD degree. I just want to recommend to somebody who want to go some overseas to study, I think it's better to decide earlier, and try to just go to overseas as soon as possible. Dr St. Hilaire: Yeah. Do you guys want to add anything else? Luigi Adamo: I would say thank you. Jihoon Nah: Yeah, I'm very happy. Swati Dey: Yeah, me too. Dr St. Hilaire: Thank you. This has been really fun. Thank you for coming, and we'll talk again soon. Jihoon Nah: Thank you. Luigi Adamo: Thank you. Dr St. Hilaire: That's it for this special early career focused edition of Discover CircRes. Thank you for listening. Dr St. Hilaire: This podcast is produced by Rebecca McTavish, and edited by Melissa Stoner, and sponsored by the editorial team of Circulation Research. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your source for the most up to date and exciting discoveries in basic cardiovascular research.


16 Jan 2020

Rank #6

Podcast cover

December 2019 Discover CircRes

This month on Episode 7 of the Discover CircRes podcast, host Cindy St. Hilaire highlights two featured articles from the December 6, 2019 issue of Circulation Research and talks with Roy Silverstein and Yiliang Chen about their article, Mitochondrial Metabolic Reprogramming by CD36 Signaling Drives Macrophage Inflammatory Responses. Article highlights: McArdle, et al, et al. Migratory and Dancing Atherosclerotic Macrophages Skaria, et al. Cardioprotection with Endogenous αCGRP Transcript Dr Cindy St. Hilaire: Hi, welcome to Discover CircRes the monthly podcast of the American Heart Association journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, and I'm an Assistant Professor at the University of Pittsburgh. In this episode I'm going to share with you highlights from recent articles published in the December 6 issue of Circulation Research. We're also going to have an in-depth conversation with Drs Roy Silverstein and Yiliang Chen about their recent article on how macrophage CD36 modulates immunometabolism. Also, the American Heart Association Scientific Sessions were recently held in Philadelphia, PA and in this edition of Discover CircRes, we're going to feature a conversation with the editors in chief of Circulation Research and Circulation, Drs Jane Friedman and Joe Hill. The first article I'd like to highlight is titled Migratory Dancing Atherosclerotic Macrophages. The first author is Sarah McCardell and the corresponding author is Klaus Ley and the work was conducted at the La Jolla Institute of Immunology in La Jolla, California. A major component of atherosclerosis is the inflammatory response and atherosclerotic plaques contain a mix of macrophages. Some macrophages arise from proliferation of resident cells, while other macrophages can infiltrate in from the blood. And a few studies have shown that smooth muscle cells can acquire some macrophage-like markers. Some macrophages are anti-inflammatory while others are more pro-inflammatory. These variations have largely been determined using techniques that examine the cell surface marker expression, the transcription profiles, or by mass spectrometry. But how all these different types of macrophagia cells look and function in vivo has not been clearly defined nor visualized. McCardell and colleagues have now observed fluorescently-labeled macrophages in the atherosclerotic plaques of live mice. First, using single cell RNA sequencing, they identified key markers of macrophage subsets. These markers are Cx3cr1 and CD11c. They then generated Apoe knockout mice that could then express green fluorescent protein under the direction of the Cx3cr1 promoter and yellow fluorescent protein under the direction of CD11c. These fluorescent proteins could be expressed individually, they could be expressed together, or they could be expressed not at all. And then in these mice they used intravital microscopy to look at the carotid artery plaques and they found while green cells and double positive cells, so that is, cells expressing Cx3cr1or both Cx3cr1 and CD11c--these cells tended to stay in one place, but they could extrude these protrusions akin to dancing, while the yellow cells or the cells that were expressing CD11c alone were more spherical and migratory. RNA analysis revealed that migratory genes were indeed upregulated in the yellow cells as compared to the green cells. The work provides preliminary insights into plaque macrophage dynamics and presents a technical resource for investigating how such behaviors may influence disease progression and I highly recommend you check this article out online. They have included several videos in the supplementary data and they're really beautiful. You can actually see the macrophages moving around and dancing and moving through the tissue and it's really neat to think about maybe how people are going to use this in the future to study the role of macrophages and maybe even other inflammatory cells in atherosclerotic disease progression. The next paper I want to highlight is titled Blood Pressure Normalization-Independent Cardioprotective Effects of Endogenous, Physical Activity-Induced Alpha Calcitonin Gene-Related Peptide (αCGRP) in Chronic Hypertensive Mice. The first author is Tom Skaria and the corresponding author is Johannes Vogel and they are from the University of Zurich in Zurich, Switzerland. So chronic hypertension affects a ton of people, over a billion worldwide, and it is a main driver of cardiovascular mortality and morbidity and it's a leading risk for heart failure. The way chronic hypertension can contribute to heart failure is by increasing the sarcomere gene expression in cardiomyocytes. And this gene expression helps to promote cellular hypertrophy or the swelling of cells, the enlarging of cells. High blood pressure can also promote interstitial fibrosis. And this fibrosis, which is happening in between the cardiomyocytes, impairs the contractile function of those cardiomyocytes. And while there are some medications available to help treat hypertension, many patients are unresponsive to these anti-hypertensive therapies. Interestingly, we all know exercise is good for us, and we all know exercise is good for specifically our heart, but exercise itself also induces cardiac hypertrophy, but it does so without impairing cardiac contractility. So how does this do this? How does exercise cause hypertrophy, but do it without impairing contractility? One of the proteins thought to be involved is called alpha calcitonin gene related peptide or alpha CGRP and mice that have been deleted of alpha CGRP, when they exercise, they exhibit hearts that look like hypertensive hearts. So this group hypothesized that exercise-induced endogenous alpha CGRP suppresses hypertension induced pathological cardiac remodeling and they tested this hypothesis in a murine model of chronic hypertension. What they found was interesting--they found that endogenous alpha CGRP suppresses pathological cardiac remodeling and it helps to preserve the heart function and it also mediates the cardioprotective effects of regular exercise in the setting of chronic hypertension. A really interesting thing that this article highlights is that alpha CGRP is currently approved for the treatment of migraines. So what might that mean? That might mean that someone who is taking this long-term for migraines may actually carry the risk of cardiac impairment if they have chronic hypertension. Mid-November is when the annual American Heart Association Scientific Sessions are held. This year's scientific sessions were in Philadelphia, PA and the Editors-In-Chief of Circulation and Circulation Research, Drs Joe Hill and Jane Friedman, had the chance to sit down for a chat and I'm going to share with you what they discussed, and I hope you enjoy it. Here they are. Amit Khera: I'm Amit Khera. I'm Digital Strategies Editor for Circulation and I'm standing in this week for Carolyn Lam and Greg Hunley. And I'm also doing the Circ on the Run Podcast as well as Discover CircRes podcast with our two Editors-In-Chief. This is Jane Friedman, who recently took over as editor-in-chief of Circulation Research and Joseph Hill, who is the editor-in-chief of Circulation. So welcome to you both. We’re excited to do this. Dr Joseph Hill:Thank you. Dr Jane Freedman: Thank you. Amit Khera: The idea behind this, there's a session here at Sessions where we're running a little bit about Circulation Research and Circulation, pulling back the cover, if you will, and seeing behind the cloak as to what happens in the journal. So Dr Freedman, I'll start with you. Tell me a little bit about as the incoming editor of Circulation Research, some of your vision for the journal, what you're excited about. Dr Jane Freedman: Well, I'm thrilled to be the new editor of Circulation Research and I've assembled a fabulous team of associate editors, deputy editors and other staff and support that are going to continue to grow what's already a wonderful journal, to be the preeminent and primary journal for basic and translational cardiovascular sciences and also support and interact with the other AHA family of journals. Amit Khera: So obviously that starts with a great team and it sounds like you've assembled that. Anything new that you're thinking about and the redesign of Circ Research in your term? Dr Jane Freedman: So we're hoping to expand the original scientific content, so we can have a larger number of articles in original science and we can have the pages to be able to handle other areas of basic cardiovascular science to include new areas, emerging areas, things like that. We're also increasing some of our early career initiatives, so that's very important to us as well. Amit Khera: Fantastic. And you talk about expanding for science and Joe, that that leads to you. In the session tomorrow, one of the goals is when people submit their science, it really goes into a black box and people don't know what happens on the Editorial level. Can you maybe enlighten us a little what happens? Dr Joseph Hill: Jane and I had been friends for 20 or more years and we now have established a bidirectional mutually synergistic collaboration where we send papers each way. We have distinct missions but yet with significant overlap, and I think it's an incredibly exciting time for the entire portfolio of AHA journals. So as you say, most people that you hit send and you wait four to six weeks and either get a happy note or an unhappy note. And what happens at both our journals is we have a strategy of multiple touches on every paper. The paper that first comes in is first touched by a senior editor, either myself or James de Lemos and two or three others. And we will reject without review about 50% of the papers at that point. We publish six papers a week, but we get 110 a week, so we don't need to review 50 of them to pick the top six. Out of respect to our authors to save them time, out of respect to our reviewers who devote tremendous effort to reviewing papers, we don't send them papers that we don't think have a shot. That said, if a paper makes it past that first stage, there's about a 50% chance it'll get published either in our journal or in one of the subspecialty journals. Probably a 50/50 chance it'll be published somewhere in an AHA family journal. So if it makes it past that stage, we send it to an Associate Editor, of which you are one, and we have about 50 of them. A third are in Dallas, another third is in the US outside of Dallas, and another third are in countries around the world, 17 different countries. And that person will probably reject without review another 5% or 10% maybe, but he or she will dig into that paper and, in parallel send it out to two or sometimes three reviewers, who are trusted and valued advisors. They help that associate editor make a strong recommendation. He or she makes a decision to bring to the larger group that is informed by those reviewers. So already that paper has been touched by five different investigators. Typically, that associate editor will reach out electronically within his or her affinity group. We have an affinity group in epidemiology, heart failure intervention, basic science… asking other AEs, "Could you take a look at this paper? One reviewer said this, one said that, I'm sort of thinking this…" And then we'll have a conversation on our weekly video conference and then a decision goes out to the authors. So every paper is touched by at least five and sometimes 10 different editors and reviewers, which we have found has been a powerful way to really dig into and identify things that one or two people might've missed. Amit Khera: You know, one thing I note here is how many people touch these articles, yet how efficient and how fast this process is. And that's a testament to the goals of the journal to be really responsive and rapid for our authors. One big part of that, and I'll come back to Dr Freedman, is peer review, right? So associate editors have a lot of work and we're affinity groups and so forth, but really critical is these peer reviewers, and in the modern era we're all so busy. Tell us a little bit about the value of peer review and how we enhance the value to the peer reviewers themselves. Dr Jane Freedman: Just as you said, the peer reviewers are absolutely central, valued, and vital parts of making the journal run correctly and we, like Circulation, our associate editors send them out to three different peer reviewers and they have a very fixed amount of time to review the articles and they provide these wonderful comments. We also very heavily rely on our Editorial Board. They know the drill that it needs to be back within a fixed amount of time and for the most part they do it. It's an interesting question. What's the value to them? I've been a reviewer too. It's part of your payback. It's part of educating yourself about what's new and interesting. There's a lot of reasons for doing it. People enjoy being on the Editorial Board and interacting with the journal. But fundamentally, as an editor, you're incredibly grateful to your reviewers. They are the unsung heroes of making a journal work. Amit Khera: And you mentioned sending out to three. When you have disparate reviews, it's amazing when some people love it and some people hate it. Dr Jane Freedman: Yeah. Amit Khera: How do you handle that? Dr Jane Freedman: Yeah. Well, sometimes it's apparent from the reviews why that happened. Someone may have focused on something that the editorial group thinks is less important or they focused on something that's addressable. The other thing we do similar to Joe, is we have a video conference call every single week on Wednesdays, and that's a period where people can vet any concerns or questions. And then my editors, my associate and deputy editors, know we have an open communication at all times. So I very frequently, when they have questions about reviews and how to reconcile disparate reviews, we'll have an ongoing conversation about that. Amit Khera: It sounds like of course you're actively engaged in how this is a dynamic process. I mentioned one thing as digital strategies editor, and I know both at Circ Research and Circulation, I was thinking how do we bring these articles to life? How do we have the most people read them or engage with them? And one is traditional social media, so Twitter and Facebook, which is incredibly important. Podcast, we have a monthly podcast, we have a weekly podcast, and really hope that people listen to them because they're really full of important information. And finally, I think what people don't appreciate is the media. So we work with AHA media. Some of our top stories get over a million media impressions, go all around the world and there's Professional Heart Daily. So there's so many ways that we're bringing articles to life. Joe, I'm going to finish with you. This is a Circ family. The value of having a family of journals and how we keep cohesion and for authors when they're submitting to serve a family of journals. What's the value and how does that add? Dr Joseph Hill: Well, there has been complete turnover of all the Editors-In-Chief in the entire family of journals, of which there are 12. And we are all quite similar in our personalities, in our perspectives on the importance, the ultimate importance of validity. The first question we ask is this true? If it's not, it's gone. It doesn't get referred. We reject it. Even if it's going to be on the front page of the New York Times and cited 10,000 times. And all of us hold ourselves to that same standard. So our vectors are all pointed in the same direction. We also care about impact, not impact factor, but does it change the way you think? Does it matter? Is it incremental or does it really move the needle? So we are now in a situation, I think, a wonderful situation where we all sink or swim together. We send papers all around, as you know very well. We send papers to the sub-specialty journals, we send 20 or 30 a week on an extraordinarily regular basis, and we send papers horizontally to Circ Research or Hypertension or Stroke and so forth. So it is a syncytium now I would say of a family of journals where we are all looking out for each other. Jane cares about our journal and we care about her journal and that's a really a wonderful situation to be in. Amit Khera: Well thanks. That family and how this fluidity of articles and thought and exchanges is really part of the value and ultimately the goal is for a great paper to find a great home and I think in the Circ family we do that. Dr Cindy St. Hilaire: Great. So I'm here with Drs Roy Silverstein and Yiliang Chen and today we're going to be discussing their paper titled Mitochondrial Metabolic Reprogramming by CD36 Signaling Drives Macrophage Inflammatory Responses. And this article is in the December 6th, 2019 edition of Circulation Research. So thank you both for joining me today. I'm really looking forward to learning more about the study, but before we really dig into it, could you please introduce yourselves and maybe give us a little bit about your background? Dr Roy Silverstein: Hi, I'm Roy Silverstein. I am a physician scientist, chair of the department of medicine at Medical College of Wisconsin in Milwaukee and also a senior investigator at the Blood Research Institute, which is part of what is now called Versiti Blood Center of Wisconsin. I'm a hematologist. Dr Yiliang Chen: Hi, my name is a Yiliang Chen. I graduate a PhD from University of Toledo, Ohio State. Then I chose to join Roy Silverstein's lab because I'm fascinated with this macrophage biology and immune functions in a disease called atherosclerosis, which is well-known inflammatory diseases. Dr Roy Silverstein: Can I make a little note that Dr Chen is currently supported by a scientist development grant from the American Heart Association, which is I think a nice tie-in? Dr Cindy St. Hilaire: Yeah. Dr Yiliang Chen: Yeah. I want to take this opportunity really saying American Heart Association to support our research. Dr Cindy St. Hilaire: Well that's wonderful. And now we get to publish this beautiful story. So it's come full circle. So you stated the objective of this paper was to investigate the mechanisms by which dyslipidemia, oxidative stress, and macrophage activation are linked in athero. And you focused on immunometabolism and you also focused on a protein called CD36. So before we get too deep in the weeds, can you give us a short little primer on what is immunometabolism in the context of athero, and also maybe a little bit about the molecule CD36? Dr Roy Silverstein: Well let me take the CD36 piece and then I'll let Dr Chen take the immunometabolism piece. So CD36 is a protein that's expressed on quite a few different cell types. We think that on muscle and fat its main purpose is to translocate free fatty acids from the external environment into the cell. In the case of fat, for storage, and in the case of muscle, for beta oxidation and energy, but in macrophages and immune cells and platelets, it has a different role. It serves as a scavenger receptor, part of the innate immune system, and it recognizes structures that we call DAMPs-danger associated molecular patterns. And the specific DAMPs that are recognized include oxidized low-density lipoprotein or what we call Ox-LDL. Dr Cindy St. Hilaire: Okay, so now could you give us a little bit about immunometabolism? Dr Yiliang Chen: Sure. So for metabolism, especially the process related to ATP or energy production, normally we call it bioenergetics and it is import. For so many years, people understand for the immune cell to get activated, they may produce proteins or somehow sometimes they need to proliferate. So there's a lot of energy is required during this whole process, right? But the old dogma is that the metabolism is only activated just to support the production of energy, especially the ATP. Right. But the emerging evidence has shown that, actually it's not that simple. For example, if you use LPS or bacterial product to activate to M1 status, the cells mainly use the glycolysis. They don't use the mitochondria Ox-LDL so TCA cycle to produce ADP. While the M2 activation is total different story. They switch it to the mitochondria ATP production and not using the glycolysis and it seems the metabolism is the underlying mechanism that driving these immune activations of the macrophages. So we want to ask, what kind of metabolism is going on in those ox-LDL-stimulated macrophages and is it related to atherosclerosis? So finally we figured out, okay, everything is focusing on a mitochondria function, which is interesting. But in our situation, very interesting, we find when the cells treat with oxidized LDL, actually they largely shut down the mitochondria OXPHOS. Then the Mito can switch ROS production of reactive oxygen species in shall we call it ROS. So that makes things quite interesting because it is well known oxidative stress is commonly observed during atherosclerosis. And also the mitochondria dysfunction actually, they are also commonly observed in the human patients with cardiovascular diseases. That kind of thing, everything together. Dr Cindy St. Hilaire: So you found that fatty acid metabolism, which is induced by this oxidized LDL, leads to the metabolic shift in the mitochondria, this switch you just described. And that shift leads to an accumulation of long chain fatty acids, but you also noticed independent of the metabolism in the mitochondria, you notice dysfunctions in what you call the mitochondrial network, and I'm wondering is it the accumulation of these long chain fatty acids that drives alterations in the mitochondrial network, or is it the other way around? I guess what I'm curious about is the interplay between that metabolic shift and just the baseline function of the mitochondria. Is one causing the other? Is it bi-directional? Dr Yiliang Chen: It sounds like a chicken and egg question. Dr Cindy St. Hilaire: Exactly. Dr Yiliang Chen: I think it's not that simple. For me, I was saying initially the cell try to adapt to this oxo LDL microenvironment. They try to stimulate the fatty acid trafficking into the mitochondria. But a side effect, what we think is, when you shut down a fatty acid oxidation while you're trafficking them there, that naturally will lead to accumulation of fatty acid. Those lipids may very well insert into the inner membrane of mitochondria then leads to the defects and that pretty much explained the EN images we show in our paper. Yeah. Exactly. Dr Cindy St. Hilaire: So the fact that the cell can't break down these long chain fatty acids, they're accumulating and potentially disrupting the mitochondrial membrane integrity. Dr Roy Silverstein: It's a form of lipodystrophy, not lipodystrophy, but lipotoxicity. Dr Yiliang Chen: Essentially, I think this is a defect in metabolism that leads to chronic inflammation. Dr Cindy St. Hilaire: Yeah. So your study focused on macrophages and atherosclerotic plaques and there's a huge body of evidence that shows inflammation and macrophage contribute to atherosclerotic disease progression pretty much throughout the whole plaque development. But there's also a body of evidence that shows smooth muscle cells can transdifferentiate and acquire macrophage-like phenotypes and they can also express CD36. It's one of the markers that people look for in that. And so I'm wondering, do you think this metabolic shift is operative in the smooth muscle or the macrophage-like smooth muscle cells? I guess that's the better thing to call them. Do you think that metabolic shift is operative and is contributing more so to the plaque or do you think this is innate to the macrophage from the immune system? Dr Roy Silverstein: I think that's a very provocative question. Thank you for it. Our in vitro experiments would not answer that question yet. We'd start with macrophages in those experiments, but it would be very interesting to look at smooth muscle cells that have been pushed towards that phenotype. We do, however, have some in vivo data that suggests that the cells that we call macrophages are behaving this way. And we can't say for certain that those are haematopoietically-derived cells versus smooth muscle derived cells. Dr Cindy St. Hilaire: So your study focused on the role of CD36 on macrophages, however, you mentioned in your introduction at the beginning that CD36 has also, it's in the GI track, it's on the muscle cells, it's on adipose tissue. It's also on the skin. And I'm wondering if you think these findings are specific only to the resident macrophages in the plaques or is this a broader function in macrophages? And I guess I'm thinking of this in the context of your study because it used the Apoe CD36 double knockout. So these are full body knockouts missing Apoe and functional CD36. And so I'm wondering, I guess, what would happen if CD36 was only removed from the macrophage cells itself? And I guess I'm thinking about this in context of something like metabolic syndrome. Could this be operative in adipose cells expressing CD36 or muscle cells or something like that? Kind of a more speculative question. Dr Roy Silverstein: That's great. You're helping us write our next grant. Dr Yiliang Chen: Yeah. Great question. Dr Cindy St. Hilaire: Give me 10%. Give me a little bit. Dr Roy Silverstein: I think one of the things that we've found over the years is that CD36 signaling in response to DAMPs, and even in response to fatty acid, involves a generalizable pattern that involves recruitment of what you might call a signalosome inside the cell. That signalosome typically would include members of the SARC family kinases, specific map kinases, a guanine nucleotide exchange factor called VAV, and other downstream signaling complexes. So we believe that that creates some opportunity for context specific signaling, but it does seem that a common theme is the generation of intracellular reactive oxygen species. Dr Cindy St. Hilaire: So I guess the bigger question after all of this, after your great findings, is what is the potential to leverage these findings in terms of developing therapies? Is there a novel pathway we can start to target or to think about targeting and how would you kind of go about that? Dr Yiliang Chen: Yeah. For that, that is our ongoing investigation. So based on the story in this paper, we are saying the mitochondria dysfunction and ROS production will activate and be pathway and drive this chronic inflammation, right? So if this is true, the particular question we are asking now is can we find a way to suppress mitochondrial ROS production or find a way to correct this fatty acid defect? Dr Cindy St. Hilaire: Do you think your findings on the metabolic shift of macrophage and how those contribute to atherosclerosis, how do you think those findings inform what the Cantos trial showed and the Cantos trial, which people may not be familiar with, used immunomodulation. Essentially, it was an antibody to block IO1 beta signaling and it had mediocre affects. The MI numbers were down, but the death rates were the same. Do you think that targeting the metabolism of the immune cells specifically as opposed to targeting the inflammation pathway outside of the cells is a more targeted and therefore maybe more precise approach? Dr Roy Silverstein: Yeah. I think that's a good observation. In my mind, what the Cantos study really showed us is that blocking inflammation in the broad sense, in a very upstream sense, can have an impact on human atherosclerotic heart disease. And I think that's really important observation and it validates the concept of inflammation as a target for atherosclerosis. Dr Cindy St. Hilaire: All of us breathed a sigh of relief once we're okay. It is an inflammation disease. Dr Roy Silverstein: Yeah. And it also decreased cancer, right? So it's double victory. Dr Cindy St. Hilaire: Yes. It did. Exactly. Yeah. Dr Roy Silverstein: But my thought is that we could maybe get a little bit upstream of that in a specific way. And perhaps the most translatable discovery here is the importance of mitochondrial reactive oxygen species as the source. Most people have looked at the NOX pathways, the NADP H oxidase pathways or broad spectrum. Dr Cindy St. Hilaire: I looked at that in my graduate studies. Dr Roy Silverstein: Yeah. So, you know that literature, and using broad heavy-handed approaches to create a “antioxidant effect” and most of those clinical trials have been extremely disappointing. But they haven't really targeted specific reactive oxygen species or specific sources. And we have this inhibitor that Dr Chen used in his experiment called mito-TEMPO which targets the mitochondria through a molecular mechanism and had a significant impact on the downstream production of pro-inflammatory product. So we think, or I think at least, that that is potentially an interesting target to basically prevent that reversal of mitochondrial function. Dr Cindy St. Hilaire: Thank you so much for taking the time to speak with me today. It's been wonderful, and I look forward to reading more of your papers in the future. Dr Roy Silverstein: Thanks. Dr Yiliang Chen: Thank you. Dr Cindy St. Hilaire: That's it for highlights from the December 6th issue of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. Thank you to our guests, Dr Roy Silverstein and Dr Yiliang Chen and Drs Jane Friedman and Joe Hill for sharing their discussion with us. I'm your host, Dr Cindy St Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


12 Dec 2019

Rank #7

Podcast cover

November 2019 Discover CircRes

This month on Episode 6 of the Discover CircRes podcast, host Cindy St. Hilaire highlights five featured articles from the October 25 and November 8, 2019 issues of Circulation Research and talks with Coleen McNamara and Aditi Upadhye about their article, Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked To Human Coronary Atherosclerosis. Article highlights: Omura, et al. ADAMTS8 in Pulmonary Hypertension. Rödel, et al. Blood Flow Suppresses CCM Phenotypes in Zebrafish Cai, et al. Proteomics Assessment of hPSC-CM Maturation Shin, et al. Leptin Causes Hypertension Via Carotid Body Trpm7 Lin , et al. Cellular Heterogeneity in Elastin Deposition Transcript Dr Cindy St. Hilaire:          Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire and I'm an assistant professor of medicine at the University of Pittsburgh. In this episode I'm going to share with you some highlights from recent articles that were published in the October 25th and our November 8th issues of Circulation Research. We're also going to have an in-depth conversation with doctors Coleen McNamara and Aditi Upadhye, who are the lead authors in one of the exciting discoveries from our October 25th issue. The first article I want to share with you is titled ADAMTS8 promotes the development of pulmonary arterial hypertension and right ventricular failure, a possible novel therapeutic target. The first author is Junichi Omura and the corresponding author is Hiroaki Shimokawa, and the work was conducted at Tohoku University, Sendai, Japan. Pulmonary hypertension is caused from the excessive proliferation of the vasculature in the lungs. It has contributions from smooth muscle cells, endothelial cells, inflammatory cells, and these cells proliferate and occlude the small vessels in the lungs. And this occlusion leads ultimately to failure of the right heart ventricle. Current therapies only treat the symptoms, not the underlying pathology. So there really is a big push right now to try to discover novel therapeutic targets. The authors of this study performed a gene expression screen, and in this screen, they compared pulmonary artery smooth muscle cells from pulmonary hypertension patients to those same cells from healthy controls. The research has found numerous differentially-expressed genes. However, they chose to focus on one called ADAMTS8. And they focused on this because the protein is expressed specifically in the lungs and heart tissues, and it was significantly upregulated in the patient's cells. So ADAMTS8 is a secreted zinc dependent protease, and this protease function makes it potentially a druggable target. So similar to human cells, ADAMTS8 was also found to be upregulated in the lungs of mice with pulmonary hypertension and a lack of vascular ADAMTS8 attenuated the disease symptoms. Conversely, overexpression of ADAMTS8 in pulmonary artery smooth muscle cells from both mice and humans prompted increased proliferation. They performed a high throughput screen to try and identify compounds that would suppress ADAMTS8 and pulmonary artery smooth muscle cell proliferation. And in this screen, they found mebendazole, which is a drug that is already in clinical use for parasitic worm infections. Thus, the study not only pins ADAMTS8 as a driver of pulmonary hypertension, but also suggests a potential existing drug might be useful for treating it. The next manuscript I want to share with you is titled Blood Flow Suppresses Vascular Anomalies In a Zebrafish Model of Cerebral Cavernous Malformations. The first author is Claudia Jasmin Rödel, and the corresponding author is Salim Abdelilah-Seyfried, and they are from the University of Potsdam in Potsdam, Germany. Vessel diameter and geometry as well as blood velocity and flow speed, all affect how the flow of blood impacts biomechanical forces that are received by the endothelial cells that line the lumen of vessels. Pathological changes in biomechanical signaling pathways or abnormal patterns of blood flow have been implicated in the etiology of various vascular diseases, and this manuscript is focusing on one: cerebral cavernous malformations, or CCMs. There are various genetic causes of CCMs, and this combined with several lines of evidence, point to a role for blood flow in CCM lesion development. Specifically, patients typically develop CCM lesions only in low perfused venous capillaries. Those are slow flow vessels. Rarely are high flow vessels affected. The authors want to answer the question, why do CCMs develop in low flow areas and more broadly, what is the role of hemodynamic forces in CCM pathology? To explore the role of blood flow and vascular remodeling, they use a zebrafish model. This is a great model to study this specific type of malformation, because the zebrafish itself is transparent and you can do an amazing way of imaging and I highly recommend that you go online and check out some of the videos that are supplemental figures for this paper. They're beautiful, they're neat, and you can really see the blood flow in these zebrafish models that they use. Okay, so which models did they use? They used ones that had normal levels of blood flow or normal speeds of blood flow, and then a zebrafish that is actually absent of any blood flow. Which is crazy that it can live for any amount of time. And so they use these zebrafish and looked at the lateral dorsal aorta, which is a high shear stress vascular bed. They found that blood flow induces a protective response in endothelial cells. This finding helps to explain why CCM patients never suffer from vascular anomalies within highly perfused blood vessels since these vessels are protected by the flow itself. The next paper I want to highlight is titled An Unbiased Proteomics Method to Assess the Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes. The first author is Wenxuan Cai and the corresponding author is Ying Ge, and they are from the University of Wisconsin Madison in Madison, Wisconsin. Cardiomyocytes are the beating cells of the heart and they're very difficult to work with in culture as they don't proliferate very well. As such, scientists are moving to use human induced pluripotent STEM cells as means to create cardiomyocytes. So cardiomyocytes derived from human pluripotent stem cells are a valuable resource for drug discovery and screening and disease modeling. While useful, these pluripotent stem cell-derived cardiomyocytes remain immature compared to their natural adult counterparts, and this immaturity slightly reduces their utility. So there are now several methods that people use to promote maturation of cardiomyocytes, but currently there's no consensus on the best way to assess cardiomyocyte maturity, or rather, IPS cardiomyocyte maturity. In this manuscript, Cai and colleagues have established a straightforward yet comprehensive mass spectrometry approach to ensure cardiomyocyte maturity. This method combines analysis of a subset of intact proteins with an unbiased screen of digested peptide fragments. The team used the method to examine early and late stage maturation of cardiomyocytes derived from embryonic, as well as human induced pluripotent stem cell sources, validating their findings against cells from mouse hearts. For the intact protein analysis, sarcomeres were isolated from cell samples which enabled the identification of the major sarcomeric proteins, as well as any post-translational modifications on these proteins that can fine tune our assessment of maturity. The unbiased screen further identified both known and novel maturation markers. This study not only provides a handy tool for assessing IPS-derived cardiomyocyte maturity, but it also defines a set of maturity markers for cross reference in future studies. The next paper I want to discuss is titled Leptin Induces Hypertension Acting on Transient Receptor Potential Melastatin 7, Trpm7, Channel In the Carotid Body. The first author is Mi-Kyung Shin, and the corresponding author is Vsevolod Polotsky, and they are from the Johns Hopkins University in Baltimore, Maryland. Leptin is a hormone that is secreted from fatty tissue, and it's secreted in response to eating something fatty and delicious. Leptin signaling increases metabolism and blood pressure, and it also helps to reduce appetite. That is, if you don't eat the fatty food too fast. So, obese individuals can exhibit high levels of leptin, yet their metabolism and appetite may be unaltered, while hypertension may still develop. Leptin's effects on appetite metabolism are mediated via signaling in the brain, while its effects on blood pressure are thought to be mediated elsewhere. In this manuscript, the authors suspected that the carotid body has something to do with this. The carotid body is a cluster of cells in the neck that detect blood levels of oxygen and other substrates, and the carotid body cells can communicate the information to the brain via the carotid sinus nerve. The carotid body has abundant expression of leptin receptor, and moreover, leptin has been shown to increase carotid sinus nerve firing. So in this manuscript, the authors now show that infusions of leptin into mice increased hypertension in the animals only when the carotid sinus nerve was intact. They also showed that hypertension in these mice was dependent on the iron channel Trpm7, which is very abundant in the carotid body. Inhibition of Trpm7 prevented the leptin-induced hypertension. Together, these results begin to explain why obese individuals' leptin still induces hypertension when the hormone's other effects on appetite and metabolism are diminished. They suggest that inhibition of Trpm7 could perhaps be a way to treat the hypertension seen in obese individuals. The last paper I want to highlight before we move over to our interview is titled Heterogeneous Cellular Contributions to Elastic Laminae Formation and Arterial Wall Development. The first author is Chien-Jung Lin, and the corresponding author is Jessica Wagenseil from the Washington University in St. Louis. Elastin is the extracellular matrix protein that provides structure to both large and small arteries. Vascular smooth muscle cells are known to produce the layered elastic laminae found in elastic arteries. However, they synthesize very little elastin in more muscular arteries. Muscular arteries also have well-defined internal elastic laminae that separates the smooth muscle cells from the endothelial cells, but the source of the elastin in these muscular arteries is not well-defined. The goal of this study was to define the extent to which endothelial cells can contribute to elastin in the eternal elastic laminae of various arteries. To address this question, they created several new strains of mice in which elastin is deleted specifically in a smooth muscle or an endothelial cell. They found that smooth muscle cells and endothelial cells can both independently form an internal elastic lamina in elastic arteries. In muscular and resistance arteries, however, endothelial cells are the major source of elastin. Further, in the ascending aorta, it was noted that ill-formed internal elastic laminae was associated with neointimal formation, confirming that the internal elastic laminae is a critical physical barrier for smooth muscle cells and endothelial cells in large elastic arteries. This study provides new information about how smooth muscle cells and endothelial cells contribute to elastin production in the artery wall, and also how local elastic laminae defects may contribute to cardiovascular disease. I'm here with Dr Coleen McNamara and Aditi Upadhye, and we'll be discussing their paper titled Diversification and CXCR4-Dependent Establishment of the Bone Marrow B-1a Cell Pool Governs Atheroprotective IgM Production Linked to Human Coronary Atherosclerosis. And this paper is was published in our October 25th edition of the journal. So thank you both so much for joining me today. Dr Coleen McNamara:    Thank you for having us. Dr Aditi Upadhye:            Thank you. Dr Cindy St. Hilaire:          I'm really looking forward to learning more about this paper. First, I'm wondering if you could just please introduce yourselves and give us a little bit about your background. Dr Coleen McNamara:    Well, I'm Coleen McNamara. I'm a physician scientist in the Cardiovascular Research Center at the University of Virginia in cardiovascular medicine. And my laboratory studies B cells and atherosclerosis predominantly. And that's the topic of Aditi's paper. Dr Aditi Upadhye:            And I'm Aditi Upadhye. I'm a PhD student in Coleen's lab and my project in Coleen's lab has focused on the role of CXCR4 in B-1 cell IgM production in atherosclerosis. Dr Cindy St. Hilaire:          If this is your project, you must be nearing the end of graduate school then. Dr Aditi Upadhye:            Yes, very close. Dr Cindy St. Hilaire:          Excellent. And congratulations on a beautiful paper. Dr Aditi Upadhye:            Thank you so much. Dr Cindy St. Hilaire:          So I was just reading the paper and I did see that you stated the objective of the paper, was that you wanted to characterize bone marrow IgM repertoire and determine whether CXCR4 regulated the B-1 cell production of this atheroprotective IgM. Could you maybe just give us a quick primer on what all those words mean? What is a B-1 cell, what is IgM, and what is this atheroprotectiveness, and why is this important to research? Dr Aditi Upadhye:            Sure. So research over the past few decades has shown that the role of B cells in atherosclerosis is subset specific. So in mice there are two broad categories of B cells. B-1 and B-2. And B-2 cells are the ones you typically learn about in immunology classes. They're the ones that produce really high affinity class-switched antibodies in a T-cell dependent manner. And there is evidence that B-2 cells are atherogenic. So either through their ability to modulate T-cells through cytokine production, or through their production of IgG and IgE antibodies, they may have atherogenic capability. B-1 cells are very, very different. They produce what are called these natural IgM antibodies. So they're present even in germ-free mice that don't have any prior antigen exposure, exogenous antigen exposure. And the kind of paradigm in the field thus far had been that B-1 cells produce germline-encoded antibodies. So they don't acquire quite as much diversity as their B-2 cell counterparts do. And really importantly, it has been shown that B-1 cells are an atheroprotective cell subset, primarily through their ability to produce IgM. So our coauthor, Dr Joseph Witztum, previously demonstrated that B-1 cells produced IgM antibodies against oxidation specific epitopes that arise on oxidized LDL in atherosclerosis. But really the mechanisms that regulate IgM production and what these IgMs are targeted against is less known. And that's something that we were trying to get at with this paper. Dr Cindy St. Hilaire:          One of the things you talked about in one of the earlier figures in your paper was that there's differences in the chemokine expression between these B cells that are in the spleen versus when they're in the bone marrow. And these differences are apparent at baseline, but also under hyperlipidemic conditions. Is there a cause or a consequence angle to asking this question about B-1 cells and atheroprotectiveness? Dr Aditi Upadhye:            Yeah, I think so. One of the points of this paper is that B-1 cells are very heterogeneous and so they may be going to multiple locations, not just the bone marrow, which we focus on in our paper, but also the spleen, also the perivascular adipose tissue, are sites that we're also interested in looking at. So the fact that there is different chemokine ligand expression level on these different sites might guide them to these different places and might help with their function. Dr Cindy St. Hilaire:          Yeah, and I guess that's a perfect segue for my next question. And it seems that the CXCR4 expression on the cells is really key to their proper migration and then the subsequent secretion of the IgM. Do we know what's happening to CXCR4 expression either as we age or as atherosclerosis progresses? Is there any evidence of environmental or behavioral or genetic angles that might predispose an individual to having more or less CXCR4 on their B cells? Dr Aditi Upadhye:            That's a great question. So there are a lot of things that regulate chemokine receptor expression, including expression of the ligands too. I don't know that much about how CXCR4 expression changes with age or with atherosclerosis. At least in mice, it seems that CXCR4 doesn't change during hyperlipidemia. So for example, in C57 black 6 mice versus a ApoE knockout mice, either child fed or Western diet fed, CXCR4 doesn't seem to change on the one cell subsets. Dr Cindy St. Hilaire:          Interesting. Maybe a future project then. Dr Aditi Upadhye:            Yeah. Yeah. Dr Cindy St. Hilaire:          So I found it really interesting, I think it was in figure five, I always like to try to pick out my favorite figures. My favorite figures of this paper are three, five, and seven. And so... Are those your favorite? But so I guess one of the things that I thought was interesting is that you made a mouse, a multiple knockout mouse that had ApoE knockout and it also was not able to make IgM antibodies. Is that correct? So when you took that mouse and then you looked at it, I think at 80 weeks of age, you could see differences in the atherosclerosis on these mice, but then you couldn't see it on kind of the standard model of what probably most atherosclerosis labs use. And that is a younger mouse that's put on a high fat diet for a shorter window of time. And so could you maybe talk about what that difference means, what your study shows, and then how do we move forward with studying the role of inflammation and atherosclerosis in younger versus older mice? Dr Aditi Upadhye:            Yeah, that's a great question. And I think that every model has its caveats that, and that's something that we ran into when we were trying to show whether B-1 cell CXCR4 is important in atheroprotection. But I think what our findings suggest is that there is a very delicate balance between the amount of IgM that you have and the lipid burden that you have. And in any given model, these might be factors to consider when it comes to studying atherosclerosis. Just taking those factors into consideration when you're analyzing your atherosclerosis results. Dr Cindy St. Hilaire:          Absolutely. Dr Coleen McNamara:    One of the reasons we liked this 100-week-old, or the 80-week-old mouse is the one where if it doesn't have IgM, there's significantly more atherosclerosis even on a chow diet. And so that's a cholesterol of about 300 or 400, and then an 80 to a 100-week old mouse is about the equivalent of a 70-year-old person, which is sort of more akin to the human situation. And so in that setting, the IgMs matter, whereas it didn't look like the IgMs as Aditi said were really capable of blocking the oxidized lipids that were generated in younger mice that had cholesterols well over a thousand. So we felt like that that was really relevant, which is why we use that same model for when we did the single cell sorting and sequenced the antibody repertoire. Thinking that that would give us more insight into the role of age and modest hyperlipidemia, which is more the clinical scenario. Dr Cindy St. Hilaire:          Do you think that has implications for how humans with familial hypercholesterolemia are treated versus someone with just a lower level but still elevated lipid profile? Dr Coleen McNamara:    Yeah, I do, and I think that's really an important point, Cindy, because the vast majority of people that suffer from cardiovascular disease, have heart attacks, die of cardiovascular disease, are typically older people with modest cholesterol levels. Familial hypercholesterolemia, obviously in those patients, they get significant cardiovascular disease at young ages. But that's certainly in a relative sense, a much less common occurrence. So I think that the model and the mechanisms that we were looking at are more applicable than garden variety atherosclerosis. Dr Cindy St. Hilaire:          Interesting. That's something I haven't really thought about. We always just kind of use these mice to model athero and try to do it in the quickest way possible to get the papers out. But it's really interesting. Dr Coleen McNamara:    We use a lot of mouse models and we use young models with hypercholesterolemia in our laboratory as well. So I think that there's a real role for doing that. And a lot of people have really advanced the field with those types of models as well because they allow you to ask mechanistic questions. Dr Cindy St. Hilaire:          One of the things you mentioned in the paper was the variability of the IgMs produced, that there's not just one IgM, there's different flavors, I guess is a way to put it. Can you maybe just talk about that a little bit, what that might mean? And then I have another follow-up question after that. Dr Aditi Upadhye:            Sure. So B-1 cells, the B cell receptor, it's different on different B cells. And so that is made through a process called VDJ recombination. And the B cell receptor determines what your antibody is going to be specific for. There's a lot of different IgMs present within a given B cell repertoire because the differential combination of all these genes makes up the repertoire. Dr Cindy St. Hilaire:          What is it about the IgM that makes it atheroprotective, what's it actually targeting? Dr Aditi Upadhye:            That's a great question. So Dr Witztum and colleagues and others have shown that these IgMs target oxidation specific epitopes. And for example, one of them that we focus on in this paper is malondialdehyde-modified LDL. And so these IgMs can recognize MDA and either facilitate its clearance or prevent it from being bound to macrophages and prevent inflammatory processes within those macrophages downstream. Dr Cindy St. Hilaire:          So essentially this IgM is kind of working to prevent foam cell formation? Dr Aditi Upadhye:            Yes. Dr Cindy St. Hilaire:          Excellent. Dr Coleen McNamara:    So these are, these modified lipids are danger associated molecular patterns, as you've heard about before. So not only are these modified lipids taken up into the macrophage by scavenger receptors, which we know is an atherogenic, a process that leads to atherosclerosis, but they can also activate inflammatory pathways through toll-like receptors. Dr Cindy St. Hilaire:          So in light of the variability, I guess what I'm wondering is, is there more variability in these IgMs based on atherosclerotic state or in humans, healthy or control, and then also how are these heterogeneous populations of cells, how does your finding coincide with the recent studies on clonal hematopoiesis? And I was wondering if you could talk a little bit about that. Actually, for people who don't know, the idea... I guess I should explain the idea of clonal hematopoiesis. So essentially there was a recent paper in Science by Ken Walsh, who's actually at UVA now, where they found that there's acquired mutations in hematopoietic stem cells, and as we age, those mutations can become enriched and therefore somewhat clonal, hence the term clonal hematopoiesis. So how does the variability of the B cell population kind of work with this clonal hematopoiesis theory? Dr Coleen McNamara:    Well, it's interesting that you ask that because that's actually another direction within the lab. So we're collaborating with Dr Walsh and Jose Fuster, who was the first author on that Science paper. And we think, and in particular related to Aditi's work, that this particular subset of B cells has quite a propensity for clonality. And what she was actually able to show is, in terms of the B-1a cells within the peritoneal cavity, when their complementarity determining region three was sequenced, which is the main region responsible for recognizing the antigen-in 70% of the single cells that were sequenced, it was identical. So that actually is quite clonal. Dr Cindy St. Hilaire:          Yeah. So essentially if it was random, you would expect those numbers to be much lower. Much more variable. Dr Coleen McNamara:    Absolutely. But yet in the bone marrow, we saw much less of any given sequence being overrepresented. And in addition, there was evidence that there was modification in the antibody repertoire in adult life. Sort of suggesting and getting back to your earlier questions, that it actually may be atherogenic stimuli or hyperlipidemia that could be stimulating selection of other B cell clones. Dr Cindy St. Hilaire:          Interesting. So we have a lot of chicken and egg questions to ask for the future. Dr Coleen McNamara:    Yeah, exactly. And we're really getting into that space because we do think that the subtype of immune cell lends itself to clonal expansion. Dr Cindy St. Hilaire:          I guess I want to end with one question about the translatability of some of your findings. So the last figure, figure seven, you show an inverse relationship between the level of CXCR4 on these B-1 cells with increasing plaque burden. And essentially I think the analysis you did suggests that it was actually very predictive, even more so than lipid levels. So is there base for this as a biomarker of sorts do you think moving forward? Dr Aditi Upadhye:            Yeah, I think that's how we'd like to move forward in the lab is to look at how CXCR4 might be atheroprotective on these B-1 cells. And if we can find a good preclinical model to test that and see how it's atheroprotective in a more mechanistic way, that would be great. I think also that our ability to show that increasing CXCR4 on mouse B-1 cells and getting them to increase their localization to the bone marrow and increase IgM production, that also indicates that this could be feasible. But whether or not that can be atheroprotective is a question for the future. Dr Cindy St. Hilaire:          That's great. Well thank you so much for taking the time speaking with me today. This was an amazing story with very cool implications for the future, and Aditi, I look forward to following your bright career in the future. Dr Aditi Upadhye:            Thank you so much for the opportunity. Dr Coleen McNamara:    Thank you. Dr Cindy St. Hilaire:          Thank you. Well, that's it for our highlights from the October 25th and November 8th issues of Circulation Research. Thank you so much for listening. This podcast is produced by Rebecca McTavish, edited by Melissa Stoner, and supported by the Editorial team of Circulation Research. Some of the copy texts for the highlighted articles was provided by Ruth Williams, and I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your source for the most up to date and exciting discoveries in basic cardiovascular research.


14 Nov 2019

Rank #8

Podcast cover

July 2019 Discover CircRes

This month on the Discover CircRes podcast, host Cindy St. Hilaire highlights three featured articles from recent issues of Circulation Research and talks with Steve Lim and James M. Murphy about their article on nuclear FAK regulation of smooth muscle cell proliferation. Article highlights: Li et al: Histone Turnover in Adult Heart Kurosawa et al: Celastramycin Ameliorates Pulmonary Hypertension Urban et al: NOS3 Gene Polymorphism and Coronary Heart Disease   Transcript Cindy S.:                               Hi, welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal, Circulation Research. I'm your host Cindy St. Hilaire, and I'm an assistant professor of medicine and bioengineering at the University of Pittsburgh. My goal as host of this podcast is to share with you highlights from recent articles published in the July 5th and July 19th issues of the Circulation Research Journal. We'll also have an in-depth conversation with Drs. Steve Limb and James Murphy, from the University of South Alabama College of Medicine, who are the lead authors in one of the exciting discoveries presented in the July 5th issue. Cindy S.:                               The first article I want to share with you is titled, Replication-Independent Histone Turnover Underlines the Epigenetic Homeostasis in the Adult Heart. The co-first authors are Yumei Li, Shanshan Ai, Xianhong Yu, and the corresponding author is Aibin He. This research was conducted at the Institute of Molecular Medicine Beijing Key Laboratory of Cardiometabolic Molecular Medicine and the Peking-Tsinghua Center for Life Sciences. Both of which are part of the Peking University in Beijing, China. Cindy S.:                               In the nucleus of cells, DNA is packaged into a structure called chromatin. Chromatin can reside in an open state that is permissive to gene transcription, or closed state where transcription is inhibited. The core units of chromatin are called nucleosomes. A nucleosome consists of DNA that is wrapped around proteins called histones. It's the position of these nucleosomes that determines whether the chromatin allows for DNA transcription or not. There is a large body of research that is focused on understanding the epigenetic processes that promote or repress transcription. Most of this research focuses on the processes that read, write, and erase covalent histone modifications. But, histones are proteins, and proteins, as we all know, have finite half-lives. Cindy S.:                               Far less research has been conducted to understand the dynamics of histone assembly and disassembly on specific regions of DNA. In this study, the authors took a novel approach of using a GFP-tagged histone H2B protein to track in vivo the rate at which nucleosomes are replaced in cardiac chromatin, and to what extent this rate varies across the genome of those cells. This is particularly interesting, and a particularly good cell type to study, as cardiomyocytes rarely divide or proliferate in the adult heart. What they found was intriguing. Nucleosome recycling is not even across the epigenome of cardiac cells. Instead, gene promoters, enhancer, and other regulatory regions that are known to promote gene transcription all exhibited a higher histone turnover rate than regions of the epigenome that are not occupied by these permissive remarks. Cindy S.:                               Further, they found greater histone turnover at loci for cardiac specific transcription factors as compared to loci for pluripotency transcription factors. This implies preferential access to these regions. Digging further into the mechanism, they discovered that the repressive chromatin regulator, EED, promoted this histone turnover. The epigenetic signature is what helps to define the identity and function of a fully differentiated cell. This study suggests that loss of histone turnover may promote loss of the proper epigenetic signature of a fully differentiated cell. These exciting findings suggest replication independent histone turnover is a requirement in maintaining both epigenetic and functional homeostasis in the adult heart. From this, one may hypothesize that perhaps aberrations in histone turnover contribute to age related diseases in the cardiac tissue, as well as possibly other tissues. Cindy S.:                               The next article I'd like to highlight is titled, Identification of Celastramycin as a Novel Therapeutic Agent for Pulmonary Arterial Hypertension-High-throughput Screening of 5,562 Compounds. The first author is Ryo Kurosawa, and the corresponding author is Hiroaki Shimokawa, both from the department of cardiovascular medicine at Tohoku University Graduate School of Medicine in Sendai, Japan. This article is focusing on the disease pulmonary arterial hypertension. Cindy S.:                               Pulmonary arterial hypertension, or PAH, is a disease that stems from the increased proliferation of arterial smooth muscle cells in the lungs. This proliferation leads to a progressive occlusion of the pulmonary arteries. This occlusion also causes increased pressure in the right heart ventricle. That can lead to heart failure, and ultimately death. Basal dilatory drugs are currently used as therapy in PAH, as they help to open the blood vessels, which can alleviate some of the symptoms. However, these drugs do not target the underlying cause of the symptoms, which is the hyperproliferation of the smooth muscle cell. Cindy S.:                               To identify novel compounds that inhibit smooth muscle cell proliferation, Kurosawa and colleagues used a high-throughput approach. They isolated cells from patients with pulmonary arterial hypertension and used these cells in a high-throughput approach to test 5,562 novel molecules on their ability to inhibit the proliferation of these cells. This unbiased approach yielded several potential compounds that potentially reduced smooth muscle cell proliferation from these patients, and also had very minimal deleterious effects on healthy control smooth muscle cells. From there, the team tinkered with the structure of the drug Celastramycin to try to increase its efficacy, and with that tinkering they found in vitro, that their new molecule could reduce both the inflammatory signal that helps to drive the proliferation of the smooth muscle cells, as well as reactive oxygen species, which helps to drive the inflammatory signaling. Cindy S.:                               Moving forward to in vivo studies, the team found that their new treatment also reduced right ventricle systolic pressure and hypertrophy in three different rodent models of pulmonary arterial hypertension. This treatment improved exercise capacity in one of the models. Together, these exciting results indicate that Celastramycin could be developed as a potential therapy for pulmonary arterial hypertension. Cindy S.:                               The last paper we're going to talk about before switching to our interview with Drs. Steve Limb and James Murphy, is a paper titled, 15-Deoxy-Δ12,14-Prostaglandin J2 Reinforces the Anti-Inflammatory Capacity of Endothelial Cells With a Genetically Determined Nitric Oxide Deficit. The co-first authors are Ivelina Urban, Martin Turinsky, Sviatlana Gehrmann, and the corresponding author is Marcus Hecker, all from the department of cardiovascular physiology at Heidelberg University in Heidelberg, Germany. Cindy S.:                               Nitric oxide is a vasodilatory and anti-inflammatory molecule, and thus, beneficial to cardiovascular health. Homozygosity of a single nucleotide polymorphism, or SNP, is a gene nitric oxide synthase results in reduced ability of endothelial cells to produce nitric oxide, specifically in response to fluid share stress. Decreased bioavailability of nitric oxide in the vessel wall helps to promote atherosclerosis. The SNP that we're referring to in this paper is called T-786C, where TT homozygosity is considered the control, or healthy genotype, and CC homozygosity is the disease associated. CC homozygosity of this SNP is predictive of atherosclerotic related diseases, and consequently, individuals with CC homozygosity have an increased risk for coronary heart disease. Cindy S.:                               Now, despite this detrimental evidence, homozygous patients do not develop atherosclerosis at an accelerated rate. This suggests that there's a compensatory mechanism at play. To identify how CC homozygous cells compensate for reduced nitric oxide synthase activity, the authors utilized human umbilical vein endothelial cells, that are also called huvecs, that harbored either the TT or the CC version of this SNP. They also used these in combination with a monocytic cell line. Cindy S.:                               Urban and colleagues found that under fluid share stress conditions, human endothelial cells homozygous with for the CC variant, had increased production of an anti-inflammatory prostaglandin called 15d-PGJ2. Signaling, via this prostaglandin, helps to compensate in part for the reduced endo production. This prostaglandin suppressed monocyte activation by reducing expression of pro-inflammatory genes such as aisle 1 beta, and decreased monocyte transmigration through endothelial cells. The team also found that patients with coronary heart disease were more likely to have the CC homozygous variant than age match controls. Thus, not only did they identify a partial compensatory mechanism, the authors suggest that 15d-PGJ2 could be a useful biomarker for the diagnosis of coronary heart disease. Cindy S.:                               So that's it for the highlights of the July issues of Circulation Research. Thank you very much to Ruth Williams, who writes the In This Issue copy for the journal, as well as the editorial team at the journal and at the podcast. Cindy S.:                               Okay, so now we're going to talk to our team of first author and last author. Today's paper that we're talking about is Nuclear Focal Adhesion Kinase Controls Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia Through GATA4-Mediated Cyclin D1 Transcription. The first authors of this papers are Kyuho Jeong, Jung-Hyun Kim, and James M. Murphy, and the corresponding author is Steve Lim. Today, we're going to be speaking with James and Steve about this paper. So thank you, both of you, for joining us today. Steve Lim:                           Thanks for having us today. Cindy S.:                               Great. Congratulations on your beautiful paper. I was wondering if maybe we could just start by both of you introducing yourselves, telling us your current position, and maybe about how you came into this field. Steve Lim:                           Hi, Cindy. We appreciate the opportunity to discuss our paper. I'm Steve Lim, an associate professor in the department of biochemistry and molecular biology and medicine at the University of South Alabama. I received my PhD from University of Alabama at Birmingham. I did my post-doctoral training studying the law of FAK in cancer biology at UC San Diego Moores Cancer Center. In 2012 I started my own lab here at South Alabama, where I decided to focus on vascular biology using some pharmacy data I generated at the end of my post-doctoral study. James Murphy:                 I'm James Murphy, I'm a post-doctoral fellow in Dr. Lim's lab at the University of South Alabama. My path to science was a little different than most. I got an undergraduate and graduate degree in mathematics before I joined the PhD program here at South Alabama. Due to a family history of cardiovascular related deaths, I decided to join Dr. Lim's lab due to his interest in studying vascular disease to find new therapeutic targets. Cindy S.:                               Interesting, a math major. James Murphy:                 Yeah. Cindy S.:                               Has that been able to help with any of your basic science studies? James Murphy:                 I'm pretty good at doing concentrations. Cindy S.:                               You're the expert in lab math. James Murphy:                 Yeah. I think the logic skills and critical thinking skills that I picked up in math really help out here in science. Cindy S.:                               Oh, I bet, that's wonderful. You're the dream PhD student who can hit the ground running with M1V1 equals M2V2. Great. Well, thank you so much. I really liked this paper because I love the mechanosensing and how does a cell read what's outside, and how does that message get brought to the inside. Really, that's what you're finding in this paper, specifically looking at how FAK is mediating transcriptional regulation. Maybe you can start by just telling us, what was your overarching question when you started this study? Steve Lim:                           Sure. It is very well-known fact that promotes cell proliferation and migration through interior receptors and gross factor receptor signaling. Both of which are key components in the smooth muscle cell hyperplasia. So naturally, we asked ourselves a simple question, "Is FAK activity important for smooth muscle cell proliferation, and leading into hyperplasia?" Cindy S.:                               So when you say FAK activity I think one thing that's interesting in your paper is, FAK really has kind of two different functions, and one is the kinase function. A kinase is when it can phosphorylate another protein, so it itself is an enzyme. But, then it has another function, so can you maybe tell us about those different functions of FAK? Steve Lim:                           Right. So FAK can function as a kinase, as well as a kind of independent scaffold, which can recruit different proteins. In the paper, we specifically described a kinase independent function as a nuclear function, nuclear FAK function. Cindy S.:                               Interesting. So what premise, or what gaps and knowledge were present before your study, that you were trying to address? Steve Lim:                           Actually, a study showed that the knocking off FAK in the smooth muscle cells prevented neointimal hyperplasia. As just you asked question, FAK has two different functions. Since FAK has both kind of dependent and independent [inaudible 00:14:48], this study lets the unanswered question, which of these two different functions of FAK plays a larger role in dealing with hyperplasia. We aimed to inhibit FAK activity to distinguish between FAK kind of dependent and independent roles in dealing with hyperplasia. Cindy S.:                               Interesting. How exactly were you able to do that? How could you take and dissect apart the two different functions of this protein? Steve Lim:                           We started off with a small pile of experiment to test if a small molecule FAK inhibitor could block neointimal hyperplasia, and we were very surprised at the degree to FAK inhibition actually prevented neointimal hyperplasia following vascular injury. Cindy S.:                               Yeah, and that's in figure one. I was looking at that, it's quite striking. Steve Lim:                           Actually, to distinguish these two different functions we generated new genetic FAK–Kinase-Dead mouse model in conjunction with a FAK inhibitor model, and that would allow us to study a lot of FAK activity in smooth muscle cells. Cindy S.:                               Great. James, could you tell us about the mouse model that you developed for this study, and the specific mutations that you created and what you were allowed to test with those models. James Murphy:                 So the FAK–Kinase-Dead knock-in model was actually generated during Dr. Lim's post-doctoral studies. Cindy S.:                               Is that the exciting data? James Murphy:                 The mutation is just a simple lysine to arginine mutation of amino acid 454. What they found was that, actually, homozygous kinase-dead embryos was lethal. So you need FAK activity to actually develop a full grown organism. We kind of had to cross a hetero wild-type kinase-dead mouse with a phlox FAK mouse, which eventually, if you cross with tissue-specific Cres, what you end up with is a phlox wild-type or a phlox kinase-dead mouse. Then, when you treat Tamoxifen in your Cre mouse, then you delete one copy of wild-type FAK and you're left with either a single copy of wild-type FAK, or a single copy of kinase-dead FAK. Cindy S.:                               Very nice. So for your study, you used, if I recall correctly, the myosin-11, Tamoxifen-inducible Cre model. Can you maybe talk about why you chose that model and why not the SM22 Cre or a non-inducible model? What was your strategy? James Murphy:                 As I mentioned, FAK activity is important for embryo genesis, so we thought we had to use an inducible model, so as to make sure we had an adult mouse at the time of the experiment. We originally actually had the SMA Cre model, however, some grant reviewers had told us that we should kind of shift to the more myosin-11 mouse to be more specific to the vascular. One downside to that, as we mentioned in the paper, is that that's actually only on the Y chromosome, so you can only use male mice. Cindy S.:                               Yes. But, at least it's in only the smooth muscle cells. Is that kind of the pros and cons of that model? James Murphy:                 Yes, and the MYH-11 Cre is kind of the most accepted model when you're doing smooth muscle studies. Cindy S.:                               Great. So can both of you go over some of the key findings of your paper? If we're going to say this in a tweet, what would we say? James Murphy:                 In a tweet. So I think, as we talked about, FAK can go to the nucleus. It's kind of constantly shuttling between the nucleus and the cytoplasm, at least what we've been able to observe in vitro. However, kind of a its localization in vivo still kind of was up in the air at the time. However, our immunostaining data actually rebuild that healthy uninjured arteries primarily showed FAK was in the nucleus. Suggesting that FAK was inactive, and maybe somehow suppressing smooth muscle cell proliferation by staying in the nucleus. But, after wire injury, FAK not only increased its activation, but also shifted to be primarily within the cytoplasm, and eventually we showed that that increase of GABA4 protein stability leading to proliferation. Cindy S.:                               Very interesting. That's great. So what was the hardest part of this whole study? James Murphy:                 Dr. Lim did the preliminary FAK inhibitor studies, but he had people when he started his own lab, he had to teach us how to do the wire injury. At first, learning gets kind of technical, you have to get used to using the microscope. Cindy S.:                               Could you describe the wire injury model for us? James Murphy:                 Yes. What you do is you anesthetize the mouse and you actually locate the femoral artery, and you want to kind of reveal the muscular branch. What you do is you add suture proximal and distal to the muscular branch of the femoral artery to stop blood flow. Then, you're going to cut a small incision in the muscular branch, and you insert a small wire through the branch up into the femoral artery towards the iliac branch. What this does is denude the endothelial layer and kind of causes an extension of the artery, damaging the smooth muscle layer. Once you remove it and suture off the muscular branch, then after a couple weeks you start to see hyperplasia. Cindy S.:                               Interesting. So what does this model clinically? James Murphy:                 This model kind of mimics angioplasty procedures that one may have if they have an occluded artery. There's multiple angioplasty procedures. There's a physical dislodging and opening of the artery. Then, there's some other methods such as using a stent to keep it open. Cindy S.:                               Great. Very interesting. What do you think would happen in maybe, I don't know, an LDLR knockout that was crossed with your FAK kinase deficient mutant? What do you think would happen in an athero model? James Murphy:                 We're actually- Cindy S.:                               Or is that the next paper? We don't have to talk about it if it's the next paper. James Murphy:                 We're actually currently testing that right now. Cindy S.:                               Oh, okay. James Murphy:                 So that's kind of our next step is to test this in atherosclerotic models to see what happens. Cindy S.:                               So, what might this mean for potential therapeutic target? How could we leverage this data to possibly translate it to the clinical setting, even if it's far off? What might we want to do moving forward? Steve Lim:                           Speaking of translational potential, currently most of the treatment options for narrow vessels rely on thrombolytic stents, that provides local delivery of anti-proliferative drugs. However, DES comes with several disadvantages, including location, work, size of these affected vessels. In fact, inhibitors are under cancer clinical development, have never been used in the vascular diseases. Our study, I think, at least to show the potential for using this type of FAK inhibitors in treating hyperplasia, which was not possible before. Cindy S.:                               That's interesting. So essentially, there's already potential, therapy's already available that would just have to be tested in this new ... in this new vascular realm, essentially. Steve Lim:                           Yeah. I was thinking about effication of these type of drugs. I think it could be, as you said, PAH could be one of the targets, because they're not really useful drugs available now. In the future, what we actually, we started already, but it's known, these moments of proliferation plays key role in the arthrosclerosis progression. Studies targeting neointimal hyperplasia and atherosclerosis, it's not existing. I think in the future probably, we would like to test whether in fact inhibition and the smooth muscle cells reduce its atherosclerity in animal models, and hopefully in humans. Cindy S.:                               Yeah, yeah, hopefully in humans, always. Yeah, and in those mouse models, there's always interesting studies where you can block things from the beginning. But, I think one of the beautiful things about the mouse model that you created, the fact that it's Tamoxifen inducible, you could essentially let that atherosclerotic plaque build up for a bit and then knock it out and see if it can reverse it. So the model you created is a really wonderful tool to use for a whole bunch of studies. So congratulations. Steve Lim:                           Thank you. Cindy S.:                               Yeah, I thought the most interesting aspect of this paper was really the fact that it could link this FAK protein, this integrin signal mediating protein to the transcription factor GABA4. So could you possibly tell us a little bit about that interaction, and exactly what GAB is doing in the smooth muscle cell? Steve Lim:                           I actually think that the identifying GABA4 factor actually was one of the difficulties, because normal cells do not express GABA4, that's what is known. I think it's because, based on our finding, the smooth muscle cells in vivo, you could package more predominantly localized in vivo, the nuclear FAK is predominant. So that nuclear FAK finds GABA4 and reduces ability through the process on degradation. But, actually, not changing ... Nuclear factor is not changed. GABA4 mRNA are the expression, so GABA4 is always expressed in smooth muscle cells. But, we never see in healthy, or very freshly isolated smooth muscle cells. We never see Gaba4. That was the most difficult part actually. Cindy S.:                               So the mRNA is always there, it's just never making it to a protein that accumulates in any measurable quantity. Steve Lim:                           So you become a protein, but FAK, nuclear FAK kills all GABA4 in the nucleus. Cindy S.:                               That's the proteasome mediated degradation? Steve Lim:                           Right. Then, GABA4 actually promotes cycling D1 transcription. So no GABA4, no cycling the new one, and smooth muscle cells do not cycle. Cindy S.:                               Interesting. So can you maybe close the loop and tell us essentially what's in figure nine, like this. Could you talk us through that? Steve Lim:                           It summarizes in figure nine, I think it would be best, we can put two different situations. In healthy R3, FAK is in the nucleus, and GABA4 is reduced, cycling D1 is not expressed, and smooth muscle cells become high acid. They don't proliferate. But, in injured, actually, FAK localization is it's the vaso injury promotes FAK localization, vaso injury shifts FAK nuclear localization to cytoplasm. Actually, FAK is activated. Now, GABA4, that increases cycling T1 expression. So that causes intimal hyperplasia. That could be a kind of summary. Cindy S.:                               No, that's perfect. Congratulations on a very nice paper. I thoroughly enjoyed reading it, and I enjoyed even more speaking with the two of you. So thank you very much. Steve Lim:                           Well, thank you so much. Cindy S.:                               Thank you for listening. I'm your host Cindy St. Hilaire, and this is Discover CircRes, your source for the most up to date and exciting discoveries in basic cardiovascular research.


18 Jul 2019

Rank #9

Podcast cover

Discover CircRes Intro Podcast

Cindy S.H.:                         Hi. Welcome to Discover CircRes, the monthly podcast of the American Heart Association's journal Circulation Research. I'm your host, Cindy St. Hilaire, and my goal is to bring you highlights of articles published in the Circ Research Journal as well as have in-depth conversations with senior scientists and the junior trainees who have led the most exciting discoveries in our current issues. Today is our premier episode, so I want to take some time to introduce myself, give you a little bit of background about the history of the journal, and then have a conversation with our new editor in chief, Dr. Jane Freedman, and my social media editor partner in crime, Dr. Milka Koupenova. Cindy S.H.:                         First, a little bit about me. I'm an assistant professor of medicine and bioengineering at the University of Pittsburgh. My lab is part of the division of cardiology and we're also a member of the Pittsburgh Heart, Lung and Blood Vascular Medicine Institute. I'm still a relatively new PI. I'm still learning as I go. One of the strengths of being a new PI in the current time is the amazing network we have through social media, whether it's through listening to podcasts or through Twitter or through select groups like one of my favorites, New PI Slack. Really one of my personal goals of starting this podcast for Circ Research is to have a career development angle. Because career development is so fresh in my mind and it's really something I want to incorporate into this podcast, we're hoping we can reach out to more junior trainees through these mediums. Really that's the impetus for Dr. Freedman wanting to have specific social media editors at the Circulation Research Journal. Cindy S.H.:                         I'm very honored to be the first host of this podcast and I'm very excited for this opportunity. As a team, Milka and I hope to expose the larger community to not only the most current and exciting discoveries in cardiovascular research but also a behind-the-scenes look of what it takes to get high-impact research done and published and planned and funded, and also talk about some of the maybe the non-bench aspects of this job, the networking, the behind-the-scenes look that really you learn on the fly as you go. Hopefully we can expose more people to these on-the-fly things in a slightly more rigorous manner. Cindy S.H.:                         Before I go into the articles summarized in this week's podcast, I want to give a very big thank you to Ruth Williams. Ruth is the person who writes the content of the In This Issue which is featured in every issue of the journal Circulation Research, and that content is extremely helpful in deciding which articles we're going to focus on in this podcast and also for helping me form the conversations and discussions. Thank you, Ruth, for all your hard work. Cindy S.H.:                         Now I'm going to highlight three articles that were featured in the June 21st issue of Circulation Research. The first is entitled Relationship Between Serum Alpha-Tocopherol and Overall and Cause-Specific Mortality: A 30-Year Prospective Cohort Analysis. The first author is Jiaqi Huang and the corresponding author is Demetrius Albanes , who are both at the Division of Cancer Epidemiology and Genetics at the National Cancer Institute, which is at the NIH in Bethesda, Maryland. Alpha-tocopherol is the more formal name for vitamin E, and vitamin E is an essential fat-soluble vitamin. By essential, that means that while your body absolutely needs it, it does not produce it itself. Therefore we need to consume products containing vitamin E. We do that by eating vegetable oils, nuts, seeds, whole grains and certain fruits and vegetables. Previously, population-based studies have shown inconsistent associations between circulating vitamin E and risk of overall death or death due to specific diseases such as cancer and cardiovascular disease. Cindy S.H.:                         To look more closely at cause-specific mortality, Huang and colleagues studied a cohort of close to 30,000 Finnish men, which is a huge study. Added to that, these men were in their 50s and 60s at the start of the study and then continued for the next 30 years of their life to be in this study. It's frankly an amazing achievement to keep that many individuals enrolled. From approximately 24,000 deaths, so about 80% of the original cohort, the authors adjusted for factors such as age and confounding things like smoking. They found that vitamin E levels were inversely associated with the risk of death from a variety of causes. What that means is that higher levels of vitamin E associated with lower risk of death. All of those causes of death that they found were cardiovascular disease, heart disease, stroke, cancer, and respiratory disease. This large prospective cohort analysis provides very strong evidence that higher vitamin E levels means greater protection. Cindy S.H.:                         It's really interesting to note though that this data did not seem to associate with a reduced risk of death by diabetes or, for that matter, injury and accidents, which I guess kind of makes sense. The authors say these results indicate that vitamin E may influence longevity, but they also highlight the need for further studies, specifically in more ethnically diverse populations and of course in women, because we all know a major limiting factor of a majority of cardiovascular studies is the fact that often there are just not enough women in these studies. But really that's a push now to include not only women but more ethnically and geographically diverse populations. Cindy S.H.:                         The second article I want to highlight is titled Mitochondria Are a subset of Extracellular Vesicles Released by Activated Monocytes and Induce Type I IFN and TNF Responses in Endothelial Cells . The first authors are Florian Puhm and Taras Afonyushkin , and the senior author is Christopher Binder. All three are in the Department of Laboratory Medicine, the Medical University of Vienna, in Vienna, Austria. This group is also part of the Research Center of Molecular Medicine of the Austrian Academy of Sciences. Cindy S.H.:                         I want to talk about this paper because I found that title extremely provocative. Extracellular vesicles or microvesicles are small particles that can be released from cells. These particles can act as cell-cell communicators. They can hold a variety of substances such as proteins and micro RNAs and minerals and all sorts of things that are derived from inside the cell. The matrix vesicle is then budded off. Matrix vesicles released from monocytes after bacterial LPS stimulation, so a stimulus that induces an inflammatory response, these matrix vesicles have been shown to contain mitochondrial proteins. Mitochondrial DNA-containing matrix vesicles have been reported in the mouse model of inflammation. From this premise, from these prior studies, Dr. Puhm and colleagues hypothesized that the mitochondrial content of matrix vesicles might actively contribute to pro-inflammatory effects. Cindy S.H.:                         What they then did was show that monocytic cells release free mitochondria and also matrix vesicles that contain mitochondria within them. These free and matrix vesicle-encapsulated mitochondria were shown to drive enothelial cells to induce inflammatory cytokines such as TNF-alpha and interferon. These circulating matrix vesicles were collected also in human volunteers that were injected with this same inflammatory substance, LPS. These circulating matrix vesicles isolated from humans also induced endothelial cell cytokine production. Very interestingly, inhibition of the mitochondrial activity drastically reduced the pro-inflammatory capacity of these matrix vesicles. Cindy S.H.:                         Together, this result suggests that the released mitochondria, whether it's free or whether it's encapsulated in a matrix vesicle, may be a key player in certain inflammatory diseases. This study shows that in addition to their central role in cellular metabolism, mitochondria, whether encapsulated or free, can actively participate in an inflammatory response in a cell other than the cell it was native in, which is just intriguing to think about. This work provides new insight to the contribution of mitochondria to the content and biological activity of extracellular vesicles. It also might suggest that perhaps targeting mitochondria and their release may represent a novel point for therapeutic intervention in inflammatory pathologies. Cindy S.H.:                         The last article I want to highlight is titled Macrophage Smad3 Protects the Infarcted Heart, Stimulating Phagocytosis and Regulating Inflammation . The first author is Bijun Chen and the senior author is Nikolaos Frangogiannis . When tissues are injured, there is localized increase in the cytokine TGF-beta. However, depending on conditions, this TGF-beta can function to stimulate macrophages to adopt either pro-inflammatory or anti-inflammatory phenotypes. To complicate matters more, the signaling pathway for both the pro- and anti-inflammatory phenotypes involves activation of the intracellular signaling protein Smad3. Inflammation, whether too much or too little, can influence the outcome of injuries, including injuries such as myocardial infarctions. An infarction, for those of you unfamiliar with the term, is a localized area of dead tissue and that results from a lack of blood supply. In this case, an infarction, a myocardial infarction, is essentially a heart attack that stops blood flow through the coronaries and causes death in the cardiac tissue and cells. Cindy S.H.:                         The authors hypothesized that in the infarcted myocardium, activation of TGF-beta and Smad signaling and macrophages may regulate repair and remodeling. They had a very specific question about a very specific cell type in the context of the whole heart. To address the role of Smad3, they utilized mice that were engineered to lack Smad3 in the myeloid lineage which produces macrophage cells. They found that these mice with myeloid cell-specific deletion of Smad3 had reduced survival compared to control mice. Additionally, the hearts from the animals with the myeloid cell-specific deletion of Smad3 exhibited increased adverse remodeling and greater impairment of function. That's a really interesting finding. The heart tissue itself was the same. All that was different were the cells of the myeloid lineage. Then to dig after what cells were mediating this effect, the investigators moved on to in vitro studies. They found that Smad3-lacking cells themselves showed reduced phagocytic activity, sustained expression of pro-inflammatory genes, and reduced production of anti-inflammatory mediators when compared with control macrophages. Cindy S.H.:                         In summary, these results suggest Smad3 is necessary for macrophages in the area of the infarction to transition to an anti-inflammatory phagocytic phenotype that protects against excess remodeling. However, we cannot go after global inhibition of Smad3 as a potential therapy post myocardial infarction, and that's because inhibition of Smad3 in cardiomyocytes is actually protective against the infarction. Inhibition in a macrophage is bad, but inhibition in a cardiomyocyte is good. Any potential Smad3-modifying therapies really needs to be designed to be cell type-specific and be able to be deployed to activate that cell type. Cindy S.H.:                         In addition to science, I love history. I thought I would take this opportunity of the first podcast to share with you a little bit of history about the Journal of Circulation Research. Circulation Research is now in its 66th year, but its origins can be traced to 1944. That was when the AHA established a council that was attempting to organize its research arm and its professional program arms. The AHA journal Circulation was already in existence, but in 1951 the executive committee decided to launch a basic research supplement, and it was called just that: Circulation Basic Research Supplement. But a few years later, Circulation Research was to be its own publication because of the interest and the excitement around the basic research supplements. The quote that I'm going to read is from that first executive committee meeting and there they wanted Circulation Research to be the authoritative new journal for investigators of basic sciences as they apply to the heart and circulation. Cindy S.H.:                         It's a fun little subgroup that they list after that. They list in anatomy, biology, biochemistry, morphology, which I just think is so neat to think about, pathology, physics, pharmacology, and others. It's interesting to think about what that would be today if we were now finding this journal. Biochemistry, genetics, molecular biology. It's fun to think about how much science has changed since they began this journal. Really the broader goal was to integrate and disseminate new knowledge. Leading that was Dr. Carl Wiggers, who was the first editor in chief of Circ Research. At the time, he was the head of physiology at Western Reserve University, and he's often referred to as the dean of physiology, as his research really provided much of the fundamental knowledge regarding the pressures in the heart and the vessels of the body and how they interact. Cindy S.H.:                         I actually went back and looked at some of the first titles in Volume One, Issue One, of Circ Research. It's really kind of neat. Some of them could be completely relevant today. I'm just going to read a few. Nucleotide Metabolism and Cardiac Activity, Fundamental Differences in the Reactivity of Blood Vessels in Skin Compared to Those in the Muscle. That was at the VRIC the other day. Haemodynamic Studies of Tricuspid Stenosis of Rheumatic Origin. Reading these for the first time I actually got chills because my two themes of my lab are both in that first Volume One, Issue One, of that journal. I study the extracellular nucleotide aCD73 and its impact on vascular homeostasis. I also study calcific aortic valve disease and are hugely curious about the role of inflammation and things like rheumatic heart disease in the progression of the disease. It's amazing how much science has changed, but yet how so much has stayed the same. Cindy S.H.:                         Dr. Wiggers wrote a few gems, a few quotes in his biography that I want to share with you. I find them inspiring and also humbling. The first is, "Research is a gamble in which the laws of chance favor the loser. The loser must remain a good sport," which I think is perfect to think about in science. I really wish I had read that after my first RO1 was triaged. The next two are more about the science writing and I think they're great not only for when we're thinking about papers but also grants. The first is, "Readers are greatly influenced in their judgment of a research project by literary style. A poor presentation can easily damage the best investigation," which is so true. No matter how good your science is, if you can't communicate it, it doesn't matter. And lastly, "A good paper, like a good glass of beer, should be neither largely foam nor flat. It should have just the right amount of head of foam to make it palatable." Cindy S.H.:                         With these nuggets of wisdom, we're now going to talk with Drs. Jane Freedman, who's now the editor in chief of Circ Research, and Dr. Milka Koupenova, who is the social media editor. Before I really introduce Jane, I want to recognize all of the former editors in chief of Circ Research, Dr. Carl Wiggers, Dr. Carl Schmidt, Dr. Eugene Landis, Dr. Julius Comroe, Dr. Robert Berne, Dr. Brian Hoffman, Dr. Francis Abboud, Dr. Harry Fozzard, Dr. Stephen Vatner, Dr. Eduardo Marbán, Dr. Roberto Bolli, and now Dr. Jane Freedman. Welcome, Jane. Thank you so much for this opportunity and congratulations on your new position. Dr. Freedman:                   Thank you very much. Cindy S.H.:                         I was wondering if you could just introduce yourself to the listeners and give us a little bit about your background. Dr. Freedman:                   Sure. I am the Budnitz Professor of Medicine at the University of Massachusetts, and I originally became interested in a scientific career while attending Yale University where I was both an architecture and geology major. Cindy S.H.:                         Interesting. Dr. Freedman:                   Yes, very interesting. Then, not exactly knowing what I wanted to do, I worked for a year as a research assistant for my later-to-be mentor Dr. Joe Loscalzo at Brigham and Women's Hospital. There one day he sent me up to the intensive care unit and said we need to get a tube of blood from someone who was in the throes of having a myocardial infarction. Really at that point I became hooked. Why was that person having a heart attack, and using their blood how could I figure out whether they would live, die, do well, not do well, or yield new things that might help us cure or diagnose people with heart attacks later on? After that. I went to Tufts Medical School. I did my residency and cardiology fellowship at Brigham and Women's Hospital and the Massachusetts General Hospital. After working at several different places, I have wound up at the University of Massachusetts where I am in the Division of Cardiology and where my laboratory currently resides. Cindy S.H.:                         Excellent. As the new editor in chief, what do you see as your vision for the journal? Dr. Freedman:                   I'm in a very fortunate position to be taking over a wonderful journal from an incredibly dedicated group of editors and associate editors and other supportive editors. Scientific pursuits and reporting and publications are really evolving at a rapid clip, so we hope to have several things happen over the next few years to survive and thrive. The first thing is we hope to define and expand Circulation Research's scientific identity. We want to extend its already outstanding portfolio of science that really demonstrates how elegant basic and translational mechanisms and pathways are part of a greater web of cardiovascular disease and stroke. This will include an increasingly diverse group of basic and translational sciences and they'll touch on both fundamental studies as well as how they translate to human disease. We also want to continue to pursue the excellence that Circulation Research already epitomizes and we want to extend its brand both to an increasingly diverse group of members, both nationally and internationally. Dr. Freedman:                   Circulation Research already has really wonderful publication metrics such as turnaround time, time to review, and we hope to maintain that so as to be a journal of choice for an increasingly growing number of investigators. We would also very much like to have greater interface with the American Heart Association. A lot of the research on our pages is funded by the American Heart Association, and the majority of science that the American Heart Association currently funds is basic cardiovascular science. We hope to have greater interface and help our users of the journal understand what the American Heart Association can do for them and for their scientific pursuits. Dr. Freedman:                   Last and very importantly, we really want to attract early and mid-career investigators to the journal. We already have some really nice programs that the previous editorship has started, such as Meet The First Author, but we would also like to be a site for education of how you can review papers, have a junior editor program and other types of programs that will help early and mid-career investigators in their future. One of the ways we're going to be doing that is to have enhanced social media programs. Cindy S.H.:                         Great. I really like that idea of having the junior editors because I think the best learning experience I had about how to write a grant did not happen until I actually served on a study section, because it was there you actually can understand all of those comments you got on your first grant that was triaged and why they were said. I think that is a key and really important aspect. Dr. Freedman:                   That's a perfect analogy because you want to remove the black box that people think is happening when they send their manuscripts in. There's so many reasons why manuscripts succeed and don't succeed, and we really do want to be as transparent as possible and we do want to educate investigators as much as possible about the process. Cindy S.H.:                         Actually, could you maybe tell us a little bit about that process? I made all my figures, I formatted my paper according to the instructions, I hit submit. Black box. What happens? What's the next step? Dr. Freedman:                   What's the next step? Cindy S.H.:                         What do you do? What does an editor in chief actually do? Dr. Freedman:                   I do have to say that none of this would happen, especially in the incredibly quick turnaround time, if we didn't have amazing support and help in our office that happens to be in Baltimore. The people there are just incredible. They make sure that papers move through. It's really 24/7. Our group has not been at it for very long, but I know Dr. Bolli's group as well as our group, people are handling manuscripts as fast as they really come in. We see the manuscript, they get quality checked. We try not to be too onerous with the first steps. Then typically they go to one of the associate or deputy editors who will handle them to send out for review. Cindy S.H.:                         Is that based on keywords or the title or how is that decided? Dr. Freedman:                   Sometimes it's based on keywords, so careful with your keywords. A lot of times, because each of the associate editors has an area of expertise that hopefully covers what your science is interested in, they will know experts in the field. We very heavily rely on our editorial board. We have an amazing editorial board at Circulation Research, and amazing contributions from the BCBS council. These individuals have over the years and currently provided just tireless and unsung, devoted help to making the journal run smoothly. It's a pretty quick turnaround time. Then the decision made based on the reviews of the article. Occasionally articles come in and they're not suitable for the journal because they're not what we perceive as what our readers would be interested in. Sometimes those articles don't go up for review. We don't want to keep them caught up, so we send them back right away. Dr. Freedman:                   When the articles come back in with the reviews, we're going to be discussing them at a weekly meeting. Other viewpoints will weigh in, and then we make a decision whether it's an accept, whether it's a revise, whether it needs a lot more science. That's called a de novo. Sometimes we think it's more suitable for one of the other 11 American Heart Journals and we might suggest that you consider sending it to that journal and we consult with that journal's editor. Cindy S.H.:                         Interesting. All that happens with about 14 days. Dr. Freedman:                   That's supposed to happen with 14 days. Cindy S.H.:                         It does pretty regularly based on the stats. That's amazing. One of the initiatives you mentioned was really the role of social media. Now I would like to introduce Dr Milka Koupenova, who is the co social media editor alongside me. Before I let Milka talk, I really have to be honest and say that my graduate school days were some of the best of my life. It was in part because Milka I were both in the same lab. We overlapped by a couple of years under the amazing mentorship of Dr. Katya Ravid. Every time we get together, all we'd talk about was how can we be like Katya? Maybe someday we'll actually have a podcast where we can get Katya in here and actually record all her nuggets of wisdom. Dr. Koupenova:                 I think the same thing about Katya. Cindy S.H.:                         How can it be more like Katya? But for now, Milka, welcome. Thank you. If you could just introduce yourself and give us a little bit about your background. Dr. Koupenova:                 Hi, everybody. My name is Milka Koupenova. I am an assistant professor at University of Massachusetts Medical School. Briefly about me, as Cindy mentioned, I did my PhD at Boston University and I studied at that time metabolism in atherosclerosis. Then I had this great opportunity to join this lab in thrombosis that studied these little cell fragments called platelets, which I knew something but not that much about. I joined Dr. Freedman lab as a postdoctoral fellow, and actually my interest evolved to be very much in platelet immunobiology and how platelets may contribute to thrombotic disease during viral infections. Luckily for me, I had two angels that I wanted to be. One of them was Katya Ravid, as you mentioned, and the other one was Dr. Freedman. Both set up a great example of scientists and how to do science in life. Cindy S.H.:                         Wonderful. Excellent. Thank you. I won't lie. I don't know if you feel this way. I definitely feel a little nervous about being a social media editor. I'm talking in a room to a box with a microphone on me and I don't know who's going to be listening. That's also exciting for me too. I get to disseminate all this cool knowledge and share our basic research with this huge audience. What are you most nervous about and excited about? Dr. Koupenova:                 You're doing the podcast, so I don't have to worry about that, that that particular part. I am quite excited actually about everything that's going to surround popularizing the science at Circulation Research. I think in the time that we live in and when social media is a huge part of our life, we definitely need to engage the community, scientific or lay, and communicate our ideas. I'm super excited about the creative part behind how we are going to achieve this via various social medias. Cindy S.H.:                         Can you talk about the platforms that you plan on using? Dr. Koupenova:                 We currently are using Twitter and Facebook. Please follow us on Twitter and Facebook. And we are going to launch Instagram. Find us, follow us, engage us. That will be great. You can always send us messages and like us, retweet whatever you decide. Cindy S.H.:                         Give podcast feedback on Twitter. Nice comments only. Dr. Koupenova:                 We'd like to hear your comments and we'd like to hear what you envision in certain cases when it comes to your Circulation Research, because this is your journal as much as it is ours. We're here for you. In addition to popularize and advertise the wonderful science that we're publishing in Circ Research, we want you to be engaged. We want you to be able to advertise in your own work and to think of it as something that you own and something you need to communicate to the rest of the world. That is one of the things that we want to do. Dr. Koupenova:                 Finally I'm going to echo on what Dr. Freedman said, is we want to attract truly early career and young investigators and help them be involved, help them own their science and help them communicate their ideas. That's pretty much what our social media platform is and we are going to evolve with you. That is perhaps one of the challenges. Cindy S.H.:                         I think one of the most interesting aspects, at least in academia as I see it, is really the role of self-promotion. It's something you're never taught and it's something that you don't really appreciate until you go to that conference. I remember my first conference as a new PI, I was standing there and I'm just like, "Okay, these are all other PIs. How are they all in groups? How does everybody know each other? Why are they all friends already?" It takes a lot of guts and you have to inject yourself. "Hi. I'm Cindy St. Hilaire and I'm new. Please be my friend," essentially, essentially. But it's important and I really liked the fact that when your journal is published you have that little button, share on Twitter, share on Facebook. I think that's really important. It helps you practice that self-promotion and can help really allow you to embrace your extrovert when you know how to. Dr. Koupenova:                 That's exactly what I was going to point out. Scientists or physician scientists, or physician scientists perhaps are a bit better. But as scientists we're very much introverted. But social media gives you a platform that it's not cheesy to popularize and communicate. Then you see those people on conferences and then you have your little group without- Cindy S.H.:                         It's amazing how many Twitter friends I have. "Oh, I met you on Twitter. It's so nice to meet you in real life." Dr. Koupenova:                 It's a new generation. We at Circ Research want to evolve with it. Is that correct, Dr. Freedman? Dr. Freedman:                   That is correct. Thank you very much. Cindy S.H.:                         It's exciting times. I guess maybe this is a question for all of us to talk about, but how do you think we can, number one, attract people to science, attract diverse people to science, and then really keep them in science and how do you think we can use Circ Research and also the social media aspects of Circ Research to do that? Dr. Freedman:                   I think, first of all, people have to see themselves in the journal. The journal, I think the first point I talked about, about being inclusive, inclusive types of people, way people consume science, types of science. We really want people to feel like Circ Research isn't just a journal that puts out scientific papers, but is a forum. It's a forum for them to exchange ideas and it's a forum for them to understand better about their scientific careers. Cindy S.H.:                         Great. Thank you. This has been an amazing first podcast. I'm so happy to share it with the two of you and I'm super excited for this opportunity. Again, Jane, I want to congratulate you on your new position as editor in chief and I can't help but mention as the first female editor in chief. That's a wonderful, wonderful thing. Cindy S.H.:                         You can find us on Twitter. The handle is @CircRes, at C-I-R-C-R-E-S. We're also on Instagram using the same name, C-I-R-C-R-E-S. We hope to hear from you there. Cindy S.H.:                         Thank you for listening. I'm your host, Cindy St. Hilaire, and this is Discover CircRes, your source for the most up-to-date and exciting discoveries in basic cardiovascular research.


20 Jun 2019

Rank #10