Cynthia St. Hilaire, PhD & Milka Koupenova, PhD

December 2019 Discover CircRes

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.  

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