June 2021 Discover CircRes

This month on Episode 25 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the June 11th Compendium on Peripheral Vascular Disease, as well as discussing two original research articles from the May 28th issue of Circulation Research. This episode also features an in-depth conversation with Drs Eric Small and Ryan Burke from the University of Rochester Medical Center about their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin.   Article highlights:   Ghosh, et al. IAP Overexpression Attenuates Atherosclerosis   Dörr, et al. Etelcalcetide for Cardiac Hypertrophy   Compendium on Peripheral Vascular Disease   Cindy St. Hilaire:        Hi 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, and today I'm going to be highlighting articles presented in our May 28th and June 11th issues of Circ Res. I'm also going to speak with Drs Eric Small and Ryan Burke from the University of Rochester Medical Center about their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin. Cindy St. Hilaire:        The first article I want to share comes from the May 28th issue and is titled Over-Expression of Intestinal Alkaline Phosphatase Attenuates Atherosclerosis. The first author is Siddhartha Ghosh, and the corresponding author is Shobha Ghosh, and they're from VCU Medical Center. The Western diet is a colloquial term that is used to say a diet that is high in fats, sugars, refined grains, and red meat. A diet consisting of these foods can cause intestinal inflammation, which weakens the gut lining and facilitates transfer of the bacterial toxin lipopolysaccharide, or LPS. Once in the blood, LPS causes systemic inflammation. Cindy St. Hilaire:        Patients with diseases such as diabetes and atherosclerosis, in which inflammation is a major contributor, have increased levels of LPS in the blood. In the gut, the enzyme, intestinal alkaline phosphatase, or IAP, is a critical barrier for the intestine. It regulates the integrity of epithelial cell junctions and helps to detoxify LPS, both of which limit intestinal inflammation. Clinical trials of oral IAP have hinted at its potential to treat patients with ulcerative colitis. In this study, Dr Ghosh and colleagues investigated whether over-expression of IAP can reduce systemic LPS and help to prevent atherosclerosis. They fed atherosclerosis-prone mice engineered to over-express gut IAP, a Western diet for 16 weeks and found that the animals had improved gut integrity, reduced plasma levels of LPS, reduced gut lipid absorption, lower body weight, and decreased aortic plaque burden as compared to normal controls. Together, these results indicate that improving gut barrier integrity by boosting IAP, either by diet choices or pharmacologically, may help to slow atherosclerosis. Cindy St. Hilaire:        The second article I want to share is titled Randomized Trial of Etelcalcetide for Cardiac Hypertrophy and Hemodialysis. The first author is Katharina Dörr, and the corresponding author is Rainer Oberbauer, and they're from the Medical University of Vienna. In chronic kidney disease, or CKD, loss of renal function leads to systemic mineral imbalances. These imbalances trigger further physiological problems, such as the excess production of parathyroid hormone and growth factor, FGF23. The former can cause muscle and bone weakness, and the latter has been implicated in left ventricle hypertrophy. Hyperparathyroidism can be treated with calcimimetics or with vitamin D, but while both approaches lower parathyroid hormone levels, calcimimetics also lower FGF23. Cindy St. Hilaire:        This study investigated whether CKD patients treated with a calcimimetic, etelcalcitide, had any measurable improvements in left ventricle mass, as compared to patients given a vitamin D analog, alfacalcidol. In a single blind randomized study, 32 CKD patients were treated with etelcalcitide and 30 were treated with alfacalcidol for a year. At the end of the study, left ventricle mass measured by magnetic resonance imaging, was found to be significantly lower in the etelcalcitide group. FGF23 levels had also declined in this group, but had risen in the alfacalcidol group. The results indicate that calcimimetics reduce the risk of cardiac hypertrophy, as well as treating hyperthyroidism, and thus, might be a preferable treatment option in CKD. Cindy St. Hilaire:        The June 11th issue of Circulation Research is the Peripheral Vascular Disease Compendium, and in this compendium, we have 14 articles that are written by the leading experts who present an update on the state of the field of peripheral vascular disease research. They discuss current research and also current therapeutic options. Drs Nick Leeper and Naomi Hamburg serve as the guest editors of this compendium. Drs Derek Klarin, Phil Tsao, and Scott Damrauer discuss the genetic determinants of peripheral artery disease. Drs Kunihiro Matsushita and Aaron Aday present a Review on the epidemiology of peripheral artery disease and polyvascular disease. Cindy St. Hilaire:        The potential of leveraging machine learning and artificial intelligence to improve peripheral artery disease detection, treatment, and outcomes is covered by Drs Alyssa Flores, Falen Demsas, Nicholas Leeper, and Elsie Ross. The benefits of walking as exercise therapy and its benefits on lower extremity skeletal muscle is presented by Drs Mary McDermott, Sudarshan Dayanidhi, Kate Kosmac, Sunil Saini, Josh Slysz, Christiaan Leeuwenburgh, Lisa Hartnell, Robert Sufit, and Luigi Ferrucci. Drs Marc Bonaca, Naomi Hamburg, and Mark Creager discuss medical therapies currently available to improve outcomes in patients with PAD. In a similar vein, Drs Joshua Beckman, Peter Schneider, and Michael Conte cover the recent advances in revascularization for peripheral artery disease. Cindy St. Hilaire:        Racial and ethnic disparities in PAD is discussed by Drs Eddie Hackler, Naomi Hamburg, and Khendi White Solaru. Drs Tom Alsaigh, Belinda Di Bartolo, Jocelyne Mulangala, Gemma Figtree, and Nicholas Leeper present their thoughts on optimizing the translational pipeline for patients with peripheral artery disease. New directions and therapeutic angiogenesis and arteriogenesis in PAD is covered by Drs Brian Annex and John Cooke. Drs Esther Kim, Jacqueline Saw, Daniella Kadian-Dodov, Melissa Wood, and Santhi Ganesh review sex-biased arterial diseases with clinical and genetic pleiotropy, focusing in on multi-focal fibromuscular dysplasia and spontaneous coronary artery dissection, which have a much higher prevalence in women. Cindy St. Hilaire:        Drs Matthew Fleming, Ling Shao, Klarissa Jackson, Joshua Beckman, Anna Burke, and Javid Moslehi cover the vascular impact of cancer therapies and focus on how cardiac and vascular sequelae of novel targeted cancer therapies can provide insights into cardiovascular biology. Epidemiology and genetics of venous thrombosis and chronic venous diseases is presented by Drs Richard Baylis, Nicholas Smith, Derek Klarin, and Eri Fukaya. Dr Stanley Rockson reviews advances in our understanding of lymphedema and the compendium concludes with an article by Drs Yogendra Kanthi, Meaghan E. Colling, and Benjamin Tourdot, which reviews, inflammation, infection, and venous thromboembolism. This comprehensive compendium on peripheral vascular disease is found in our June 11th issue. Cindy St. Hilaire:        So today, Drs Eric Small and Ryan Burke from the University of Rochester Medical Center are with me to discuss their study Prevention of Fibrosis and Pathological Cardiac Remodeling by Salinomycin. This article is in our May 28th issue of Circ Res. So thank you both for joining me today. Eric Small:                  Thanks Cindy, for having us. Excited to talk about our research with you. Ryan Burke:                Yeah, thank you very much for having us. Cindy St. Hilaire:        Absolutely. So fibrosis, it's essentially a wound healing mechanism, it's where connective tissue replaces the innate tissue of the organ system that it's happening in. It's really a component of many disease states. As far as I know, treatment options are pretty limited or really non-existent except in a couple rare cases, and in particular, your study, as it's in Circ Research, is focused on cardiomyopathy and the fibrosis related to that. But before we dig into your findings, which is really focused on a great therapeutic angle, I really want to take a step back and ask about what we know about fibrosis or the fibrotic process itself, maybe in the context of the heart, and despite why it's relatively common, it's been so difficult to target in terms of either therapies or really just understanding some of the basic processes. Eric Small:                  Sure, I'd be happy to discuss this. So as you know, and you alluded to already, pathological fibrosis contributes to progression of many debilitating human diseases. So in injury response in many tissues or organs, including the heart, kidneys, lungs, even the skin, leads to a wound healing process and that wound healing process is meant to repair the tissue and that includes an inflammatory response and secretion of extracellular matrix that fortifies the structural integrity of the tissue. But you can imagine in the context of a heart, that has to beat 60 plus times per minute, any alterations to the biomechanical properties of that tissue can alter the function. Eric Small:                  So extracellular matrix, which is meant to improve the structural integrity of an injury, even in the heart, ultimately can lead to reduced cardiac function. So this extracellular matrix, and in the context of disease, this extracellular matrix is called fibrosis, can reduce the contractility and the relaxation of the heart. The relaxation of the heart is actually an important aspect in insufficient relaxation called diastolic dysfunction, is becoming a more prevalent disease phenotype and it is called heart failure with preserved ejection fraction. What we're finding and what some investigators are alluding to is that fibrosis is a major component of this disease, and so understanding how extracellular matrix is secreted, why it is deposited in the context of injury, especially in the context of the heart, why does that process not stop sufficiently and revert once the injury is repaired, is a really important basic science and clinical question. Cindy St. Hilaire:        So why, specifically, has fibrosis or cardiac fibrosis been so difficult to target therapeutically? Eric Small:                  From my point of view, one of the reasons that fibrosis, organ fibrosis in general, and especially within the heart, is hard to target is because I think we're understanding now that one of the major cellular sources of extracellular matrix in disease is the fibroblast. This cell type has been sort of underappreciated for many years and is coming to the forefront now of biomedical research. So fibroblasts until maybe 10 or 15 years ago were thought to be more of a structural component. Of course, they contribute to wound healing, but it was thought that they contribute mostly to structural integrity and homeostasis of the injury. It's becoming more apparent now that resident cardiac fibroblasts contribute to extracellular matrix deposition in disease. But these cell types are really plastic, phenotypically plastic cell, they respond to a lot of biomechanical stimuli, especially that are induced in the context of tissue injury or disease, and so they respond to mechanical stretch or cellular deformation, and they can respond to many secreted factors, especially the canonical factor that has been studied extensively, TGF-beta. Cindy St. Hilaire:        Which itself is extremely complicated, to say the least. Eric Small:                  Absolutely, and so it does so much, and they respond to factors that are really high up on this hierarchy, that do so many things that I think obviously targeting TGF-beta is not going to be really an efficacious therapeutic option. So understanding what's more downstream and much more specifically related to the fibroblast, I think is really important to come up with new therapeutics. Cindy St. Hilaire:        So in your quest to identify novel therapeutics, or even really understanding that below the surface signaling you just talked about, you developed a high-throughput screen. I think this is a term that we often use, but we don't really know the details of that term, like what does high-throughput actually mean when you're doing it with cells and disease models? Dr Eric Small:             Sure. So I think in our case, we really let the science lead the way when it came to the high-throughput screen. So I'm not a chemical biologist, I have never, before now, developed a high-throughput screen and the science just pointed me in this direction. So the basic science research related to fibroblast plasticity and what induces fibroblasts to secrete extracellular matrix in the context of disease, all culminated in this one reporter that I thought would be good for the assay. So maybe as a way of a little bit of background, one difficulty in understanding fibrosis and fibroblast plasticity is that there are no really unique specific markers for an activated fibroblast. So most of the markers that people say are myofibroblast markers, which is the term for an activated ECM-secreting fibroblast, are expressed in other tissues or cells. Probably the most used and best characterized marker of a myofibroblast, is the smooth muscle alpha-actin gene, which encodes the smooth muscle actin protein, which is highly up-regulated in myofibroblasts, but obviously is expressed in a lot of other cell types, including smooth muscle cells. Eric Small:                  So it is a good marker of a myofibroblast, but it's not unique to myofibroblasts. But, this smooth muscle alpha-actin gene allowed us to make inroads into better understanding how fibroblasts respond to different stimuli. So what we did was, in the lab, one of the earlier things that we did when I set up my lab as an independent investigator, was to try to develop a stable cell line that expressed this reporter in a way that we could easily assay. So we could do it with GFP or a luciferase reporter or something like that. We made a luciferase reporter of this smooth muscle actin myofibroblast, alpha-actin gene. So one important aspect of a screen is, especially in our screen, which we were looking for chemicals that would inhibit our reporter, that we would hope would be anti-fibrotic Eric Small:                  Our hope was that this reporter would actually, in some cases, lead to an anti-fibrotic compound, but an important aspect of this screen, which was, I think the original question, was to not come up with factors that would just kill fibroblasts, but come up with factors that would specifically inhibit smooth muscle actin and myofibroblast activation without being too toxic. We don't want to inject a toxic chemical into a person; we want to inject a chemical that would be specific to an activated myofibroblast. So that was the first consideration, is to make sure that these were not toxic compounds, but were acting specifically on the smooth muscle actin report. Cindy St. Hilaire:        So with this system, you were able to screen over 2000 compounds, it was like 2300 or something like that. From that 2000 compounds screen, you zeroed in on salinomycin and two other compounds that are in the same family, I think, of chemicals like polyether ionophores they were called, I think it was the top three were all this similar class. So that's probably unsurprising that similarly-structured chemicals have a similar function or phenotype, but it's also intriguing. So I'm wondering, what's known if anything, about this class of chemicals, have they been used in therapy or is there some kind of naturopathic history to salinomycin or these other compounds that maybe if we read more carefully, we would have got a hint before? Ryan Burke:                Salinomycin has a pretty storied history in the literature, but it's an odd history. It's a veterinary antibiotic. So it's actually used primarily in livestock management and it had really no approach in human science at all. Then it was discovered that salinomycin, its earliest contribution, was that it is a compound that is actually very selectively targeting cancer stem cells. So salinomycin has a very extensive literature in cancer. It affects a lot of relevant signaling pathways, it's actually where we got a lot of our insight as to what we should be evaluating in fibroblasts. Both, in terms of ... This is probably going to be a charged statement; but there's a lot of similarities in how ... Cancer cells, when they're metastasizing and activating and moving around, there's a lot of EMT involved in that, there's a lot of things that are very analogous to how fibroblasts activate in heart failure. Ryan Burke:                I'm not saying they're the same, that's the charge portion of it, but the pathways are often conserved. What we found is that salinomycin had been studied extensively in various models of both solid and blood tumors, and it was found that it was affecting a whole ton of signaling pathways and sparing others, which was actually some of the insight that we had about AKT signaling. In the heart, it seemed very easy to just apply that and say, "Well, activation of fibroblasts is largely dependent on signaling pathways like SMADs and p38 signaling, so let's see what salinomycin does to these pathways in fibroblasts," and it turned out that that wound up being a very fruitful avenue for exploration, because it does behave very similarly in fibroblasts to the way it behaves in cancer cells. We didn't really find a lot of discordance in those results. Ryan Burke:                This study was very iterative, right? So do the high-throughput screen, find the drug, then try a preclinical model in animals. Then when it worked quite well in the angiotensin, hypertension-induced remodeling, that's a pretty mild model, right? Give the mouse an MI, see if it works in that, because that's a much more serious remodeling and when it performed well there, it's like, "Wow, you really actually probably have something here." Cindy St. Hilaire:        Yeah, and that's a perfect segue for my next question really, was I wanted to ask about these different murine models. Like you identified this compound, now you want to test it. Could you maybe give us a little brief background on why you chose the models you did and the treatment regimens that you also tried? Ryan Burke:                Sure. When we began, we began with angiotensin infusion because it's a fairly mild remodeling. You get some hypertrophic remodeling of the heart, you get some proliferation and some mild fibrosis in the mouse model. We figured this would give us the best chance to see a signal versus noise. It turned out that the results were really striking. Even the mice that were given the condition that we expected to see nothing in, is the drug with a saline infusion, even that had effects that were consistent with the effects that were seen. Consistent in direction in terms of the overall morphology and function of the heart, consistent with what you were seeing with the normalization of that hypertrophic remodeling in the angiotensin model that also got the drug. So that was really interesting to us. It was just consistent all the way through. Ryan Burke:                We wound up having a meeting about it and we were like, "All right, we've done the preventative regimen. We've preloaded them and then run them through with the drug. Now let's see if we can reverse established remodeling." So we did that study and when that worked out okay, there was yet another discussion where it was like, "All right, are we doing this?" And it was a myocardial infarction study. Myocardial infarctions, that's really extensive remodeling with huge changes, both the macro and microstructures of the heart. There's a lot more of an inflammatory component involved in that. Ryan Burke:                So we weren't sure how this would perform and it turns out that it performs exactly as it performs in pressure overload. You see normalization in physiology. I think that's part of the power of this study is that you're looking at non-ischemic and ischemic heart failure models, you're looking at preventative and interventional regimens, and it's just consistently performing at a level. We wanted to check all of our boxes, really, with this. Cindy St. Hilaire:        Sure. Yeah, maybe salinomycin's going to be the new aspirin we pop when we're over 50. Ryan Burke:                I doubt it, it's worth $7 a kilogram. I very highly doubt anyone's licensing that. Eric Small:                  But I think it's interesting you say that because understanding the mechanism after you understand that it's efficacious is sort of a similar idea here. We don't necessarily know precisely what it's targeting to act as an anti-fibrotic in this case, and so there's a lot of work to be done on this compound. I'd like to reiterate something that Ryan actually said is that really interesting, at least in cells and in the animal models, that salinomycin doesn't have a huge impact on the heart or on cultured fibroblasts in the absence of, for example, TGF-beta stimulation or a disease mechanism. It's really when we have a disease that salinomycin blocks the activation of the myofibroblasts and prevents that from contributing to the disease. Cindy St. Hilaire:        Interesting. So that can really, at least in the case of maybe cardiomyopathy, would help target it to the heart. Eric Small:                  That would be the hope, yeah. Cindy St. Hilaire:        Yeah, that's great. Wow. Speaking of the heart, and you mentioned this in that first answer that you had about the fibroblast being kind of the forgotten child of the heart and the focus is really more the cardiomyocyte, but did this drug have any impact on the cardiomyocytes itself that are also probably exposed to this TGF-beta signaling, in the context of an injury? Eric Small:                  So this is where we have some interesting, but not anticipated, results. So we obviously performed a screen in fibroblasts to look for specific anti-fibrotic compounds and when we put this into animals into ischemic or non-ischemic models, especially in the ischemic model, we found a much better outcome than we would have expected from simply an anti-fibrotic. So for example, we saw that pretreatment of mice with salinomycin prior to myocardial infarction, almost completely abrogated, not completely, but highly significantly abrogated necrotic tissue formation. So when Ryan went back and looked at the percentage of heart that became necrotic, or ischemic, after myocardial infarction, it actually reduced the necrotic core significantly. So we do think it's acting on cell types other than the fibroblasts in the context of ischemic remodeling, and it does seem to induce potentially protective signaling pathways in cultured myocytes. So that's definitely an area that we'd be interested in pursuing in more detail. Ryan Burke:                So of course the question there is, and this is a totally fair question for people to evaluate, we're looking at an organ in which all the cell types are talking to each other. We know we've affected the fibroblasts in a certain way, and we know to a certain extent, from what we found, what we've done for the fibroblasts, and we know what that looked like as a result in myocytes, but who initiated that, right? Did we affect the myocyte and then fibroblasts changed? Or did we affect fibroblasts and myocytes changed? But those are important type questions. We've shown the changes, but how do we show the connections? I think that's the really interesting work that we're still doing. We even extended it a little bit to endothelial cells in the heart, because we were showing that there was sort of a preservation of vascularization in the MI model that was associated with salinomycin, and we wouldn't rule out that we were affecting endothelial cells as well. I mean, I think this is a subject for discussion in the field, in the future. Groundwork is there, it's time to move forward. Cindy St. Hilaire:        Yeah, that is so exciting, and it's also I guess the classic chicken and egg question of science. What's causing what? That's excellent. So what's next for this project? I mean, you just highlighted some other angles, endothelial cell, but is there plans to translate it to a clinical setting, especially because it's already used in humans, so there's all that safety data out there? What's the plan? Eric Small:                  So that's a really interesting question. So our collaborators here at the University, Colin Woeller, Patricia Sime, Rick Phipps, they have been involved in the study with us and they are interested in fibrosis in other aspects as well. So they're interested in lung fibrosis, idiopathic pulmonary fibrosis, ocular eye fibrosis, and they've found that in other situations, salinomycin can inhibit fibrotic disease remodeling, for example, in the eye and in the skin. So branching out into other animal models of fibrotic disease is one area that we'd like to pursue. One area that I'm really interested in looking at salinomycin would be, for example, in models of HFpEF to see whether salinomycin might be efficacious in limiting the progression of animal model HFpEF. These are now becoming more prevalent and so it'd be great to test that there. Eric Small:                  So I think probably with some of these small animal studies, it would lay the groundwork for larger animal studies as collaborations or with other investigators. Absolutely, I think that's where this could definitely go next. Ryan Burke:                Also, it's a high-throughput screen, right? It wasn't the only hit and so extending the screen outwards both ... So the screen was designed to pick up both anti and pro-fibrotic drugs. So pro-fibrotic drugs have applications in wound healing. It also gives us a hint as to if a drug has some unexpected side effects in large clinical populations, then we can look at that and say, "Oh, maybe we have mechanistic understanding of why this might be the case." I think you'll see some future explorations down that path as well in that study. Cindy St. Hilaire:        Well, I look forward to seeing all of them. This was a wonderful study. I'm more vascular biologist, but obviously being on Circ Res, I'm learning so much more about the heart, but this one, I just particularly love that you started with this crazy complex question of what the heck is going on and this high-throughput screen was just designed in such a way that it really narrowed down what was a huge amount of options to start with. So it was really elegantly done and I just love the story, so congrats to you both and I look forward to future publications. Eric Small:                  Thank you. I'm especially proud of this one because as a basic scientist and as a trained in graduate school as a developmental biologist, I was following the science and when this opportunity arose to try to make this high-throughput screen work, I mean, this was as clinically relevant as I could ever have imagined my lab becoming. I'm really proud that we're able to do that. Cindy St. Hilaire:        Absolutely. I know, it's something we always talk about, and this research can be translated to humans eventually, and you're almost there. That's great. Well, congrats again. Thank you both for taking the time today and I look forward to your future studies. Eric Small:                  Thank you, Cindy. Ryan Burke:                Yeah, thanks Cindy. Cindy St. Hilaire:        That's it for the highlights from the May 28th and June 11th issues of Circulation Research. Thank you for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Eric Small and Ryan Burke. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the editorial team of Circulation Research. Some of the copy text for highlighted articles is provided by Ruth Williams. 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. This program is copyright of the American Heart Association, 2021. The opinions expressed by the speakers of this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.