Cynthia St. Hilaire, PhD & Milka Koupenova, PhD

November 2019 Discover CircRes

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.  

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