April 2021 Discover CircRes - a podcast by Cynthia St. Hilaire, PhD & Milka Koupenova, PhD

This month on Episode 23 of Discover CircRes, host Cindy St. Hilaire highlights the topics covered in the April 2nd Compendium on Hypertension issue, as well as discussing two articles from the April 16 issue of Circulation Research. This episode also features an in-depth conversation with Dr Kathryn Moore from the New York University School of Medicine, discussing her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques.

 

Article highlights:
 

Compendium on Hypertension

 

Mustroph, et al. CASK Regulates Excitation-Contraction Coupling

 

Ward, et al. NAA15 Haploinsufficiency and CHD

 
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.

Cindy St. Hilaire:         Today, I'm going to be highlighting the topics presented in our April 2nd Compendium on Hypertension, as well as two articles from the April 16th issue of Circ Res. I also will speak with Dr Kathryn Moore from New York University School of Medicine about her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques.

So the April 16th issue of Circulation Research is a compendium on hypertension. As introduced by Rhian Touyz and Ernesto Schiffrin, there are over 10,000 articles in PubMed related to hypertension. Hypertension is a major cause of morbidity and mortality worldwide, and data trends suggest that fewer and fewer patients are able to control their blood pressure medically. Further, the recent Sprint trial showed us that lowering blood pressure to levels below previously recommended values strongly correlated with significantly reduced rates of cardiovascular events and risk of death.

Cindy St. Hilaire:         As such, the April 2nd issue of Circ Res provides an extensive and expansive review on the current knowledge in the field. The series starts with an article on hypertension in low and middle-income countries by Aletta Schutte and colleagues. There they present the stark differences in the trajectory, healthcare, inequality, and established and emerging risks that are specific to low and middle-income countries.

Cindy St. Hilaire:         Robert Carey and colleagues present an evidence-based update in their article titled Guideline-Driven Management of Hypertension. In Pathophysiology of Hypertension, David Harrison and colleagues present the concept of the mosaic theory of hypertension originally proposed by Dr Irvine Page in the 1940s, which proposes that hypertension is the result of multiple factors that in some, raise blood pressure and induce end-organ damage. This article further refines this theory by incorporating what is known regarding the role of things like oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome in hypertension pathogenesis.

Cindy St. Hilaire:         Phil Chowienczyk and Jay Humphrey and colleagues cover the contribution of Arterial Stiffness and Cardiovascular Risk in Hypertension and identify steps required for making arterial stiffness measurements a keystone in hypertension management, and cardiovascular disease prevention as a whole. In Renin Cells, The Kidney, And Hypertension, Maria Luisa Sequeira Lopez and Ariel Gomez cover the major mechanisms that control the differentiation and fate of renin cells, the chromatin events that control the memory of the renin phenotype, and the major pathways that determine the cells’ plasticity.

Cindy St. Hilaire:         Meena Madhur and Annet Kirabo and colleagues penned the article, Hypertension: Do Inflammation and Immunity Hold the Key to Solving this Epidemic? In this Teview, they covered the emerging concepts of how environmental, genetic, and microbial-associated mechanisms promote both innate and adaptive immune cell activation and help lead to hypertension.

Cindy St. Hilaire:         In the article, The Gut Microbiome in Hypertension. Dominik N. Müller and colleagues present insights into the host-microbiome interaction and summarize the evidence of its importance in the regulation of blood pressure and provide recommendations for ongoing and future research.

Cindy St. Hilaire:         Paul Cohen, James Sowers, and colleagues cover Obesity, Adipose Tissue, and Vascular Dysfunction in which they discuss the abnormal remodeling of specific adipose tissue depots during obesity and how this contributes to the development of hypertension, endothelial dysfunction, and vascular stiffness.

Cindy St. Hilaire:         Clinton Webb, Satoru Eguchi, Rita Tostes, and colleagues cover Vascular Stress Signaling in Hypertension. In this Review, they discuss common adaptive signaling mechanisms against stresses, including the unfolded protein response, antioxidant response element signaling, autophagy, mitophagy, mitochondrial fission and fusion, STING-mediated responses, and activation of pattern recognized receptors. And how all of these responses contribute to vascular stress and ultimately hypertension.

Cindy St. Hilaire:         Rhian Touyz and colleagues then specifically dig into the topic of Oxidative Stress and Hypertension, focusing in on recent advances in delineating the primary and secondary sources of reactive oxygen species, the posttranslational oxidative stress modification ROS induces on protein targets important for redox signaling, their interplay between ROS and endogenous antioxidant systems, and the role of inflammation activation and endoplasmic reticular stress in the development of hypertension.

Cindy St. Hilaire:         Curt Sigmund and then colleagues cover the Role of the Peroxisome Proliferator Activated Receptors in Hypertension. In this Review, they discuss the tissue- and cell-specific molecular mechanisms by which PPARs in different organ systems modulate blood pressure and related phenotypes, such as endothelial cell dysfunction. Importantly, they also discuss the role of placental PPARs in preeclampsia which is a life-threatening form of hypertension that accompanies pregnancy.

Cindy St. Hilaire:         Daan van Dorst, Stephen Dobbin, and colleagues provide the Review, Hypertension and Prohypertensive Antineoplastic Therapies in Cancer Patients. Many cancer therapies have prohypertensive effects. And this Review covers some of the mechanisms by which these antineoplastic agents lead to hypertension and details the current gaps in knowledge that future clinical studies must investigate, to identify the exact pathophysiology and the optimal management of hypertension associated with anticancer therapy.

Cindy St. Hilaire:         In Hypertension, a Moving Target in COVID-19, Massimo Volpe, Reinhold Kreutz, and Carmine Savoia, review available data on the role of hypertension and its management in COVID-19.

Cindy St. Hilaire:         Melvin Lobo and colleagues review Device Therapy of Hypertension. In this Review, they discussed the newer technologies, which are predominantly aimed at neuromodulation of peripheral nervous system targets, and discuss the preclinical data that underpin their rationale and the human evidence that supports their use.

Cindy St. Hilaire:         Last but not least, in Artificial Intelligence in Hypertension: Seeing Through a Glass Darkly, Anna Dominiczak and colleagues cover a clinician-centric perspective on artificial intelligence and machine learning as applied to medicine and hypertension. In this Review, they focus on the main roadblocks impeding implementation of this technology in clinical care and describe efforts driving potential solutions.

Cindy St. Hilaire:         This is an expansive set of Reviews written by the leading experts in the field and provides an up-to-date assessment of all aspects of hypertension. The graphics, and the articles are absolutely beautiful. And I'm sure we will be seeing a lot of them in upcoming presentations. Hopefully at AHA and the other sub-meetings when we're all back in person.

Cindy St. Hilaire:         In the April 16th issue, I want to highlight the article, Loss of CASK Accelerates Heart Failure Development. The first author is Julian Mustroph, and the corresponding authors are Lars Maier and Stefan Wagner from the University Medical Center in Regensburg, Germany. Despite advances in cardiovascular medicine, heart failure takes the lives of tens of thousands of Americans each year. To develop novel treatments, a better understanding of the conditions of molecular pathology is needed. One contributing factor in heart failure is increased activity of the Ca/calmodulin-dependent kinase II (CaMKII).

Cindy St. Hilaire:         In this paper, the authors suggest a way to get CaMKII levels under control. Ca/CaM-dependent serine protein kinase or CASK, suppresses CaMKII neurons and the team showed that CASK is also expressed in human heart cells, where it associates with CaMKII. Next, they engineered mice to CASK specifically in cardiomyocytes, finding that when these animals are subjected to beta-adrenergic stimulation, cardiomyocyte like CaMKII activity was significantly greater than that seen in control animals. Calcium spark frequency and the propensity for arrhythmia were also increased. Furthermore, in a mouse model of heart failure, mice lacking CASK fared worse and had reduced survival compared to the wild type control animals while boosting CASK expression in wild type animals reduced the elevated CaMKII activity and calcium sparks associated with heart failure. The author suggests that increasing CASK activity might be a heart failure treatment strategy worthy of further study.

Cindy St. Hilaire:         The last article I want to share from the April 16th issue is titled, Mechanisms of Congenital Heart Disease Caused by NAA15 Haploinsufficiency. The first author is Tarsha Ward, and the co-senior authors are Kris Gevaert, Christine Seidman, and JG Seidman from Harvard University in Boston, Massachusetts. A number of genetic variants are associated with congenital heart disease, including loss of function variants of the gene encoding NAA15,  a sub N-terminal acetyltransferase complex called NatA, which acetylates a large portion of newly forming proteins. To find out how these variants contribute to defective heart development, the authors performed genome editing on human pluripotent stem cells to convert one or both copies of NAA15 gene into congenital heart disease linked to variants. The team then examined cardiomyocyte differentiation, protein acetylation, and protein expression in the edited and unedited cells.

Cindy St. Hilaire:         They found that while NAA15 haploinsufficiency cells were able to develop into cardiomyocytes seemingly normally, the cell's contractile ability was significantly impaired. Cells homozygous for NAA15 variants failed to differentiate and had poor viability. The team also found that while only a small number of proteins had reduced end terminal acetylation in NAA15 haploinsufficiency cells, over 500 proteins had altered expression levels, four of which were encoded by congenital heart disease-linked genes. This work provides the first insights into the effects of NAA15 variants in human cells and sets the stage for analyzing other congenital heart disease-linked variants in this manner.

Cindy St. Hilaire:         Today, Dr Kathryn Moore from NYU School of Medicine is with me to discuss her study, miR-33 Silencing Reprograms the Immune Cell Landscape in Atherosclerotic Plaques, which is in our April 16th issue of circulation research. So thank you so much for joining me today, Kathryn.

Kathryn Moore:          My pleasure.

Cindy St. Hilaire:         Atherosclerosis is the result of lipid-induced chronic inflammation, and while lipids are kind of thought to be an initial driver, therapies that target lipids alone, such as statins, they're not sufficient. They can obviously bring things down and improve things a lot, but a lot of research now is focused on uncovering the nuances of the inflammatory component of atherosclerosis to help identify new targets for therapies. One specific arm of this research has focused on resolving atherosclerotic inflammation. And my first question to you is, what exactly does resolving inflammation mean in the context of an atherosclerotic plaque? And maybe could you give us a little primer on some of those key cell types or processes involved in that.

Kathryn Moore:          I'm really fascinated by the resolution of inflammation and in particular, in the atherosclerotic plaques. So inflammation used to be thought of as an active process, almost a one-way process, which in order to resolve had to stop. But actually, the pro-inflammatory and anti-inflammatory responses are a continuum. And so inflammation resolution, we now recognize is an active process, and it's not just a matter stopping the influx of immune cells but these cells take on new phenotypes and different functions. And the immune cells themselves are required for resolution of inflammation and tissue repair. And so we're really interested in looking at what those pathways are, that tip the balance between pro-inflammatory responses and pro-resolving responses and how to incite them in the plaque so that you can start to remodel the plaque to be more stable or have a more favorable phenotype, or even to regress the plaque, to shrink the plaque in size.

Cindy St. Hilaire          This study specifically focused on microRNA-33, and I believe your lab was one of the very first to look at this specific, but also other micro RNAs in atherosclerosis. And the prior research that you and others have shown is that this microRNA modulates a variety of genes that control lipid metabolism. You found this in mice, but also in monkeys. And really by using anti-miRs against this microRNA, you can induce cholesterol efflux and that cholesterol will leave the liver and the macrophage cells, and it's incorporated into the protective HDL particles and excreted.

Cindy St. Hilaire:       And so it has this really nice protective effect. However, the effects seen in these animal studies were suggested that microRNA-33 had HDL independent action, which I think is where your story starts. So could you tell us some of the premises or the gaps in knowledge between those first initial findings of miR-33 that led you to conduct this study and then kind of what the design of the study was?

Kathryn Moore:          So, as you mentioned, we discovered miR-33 as an inhibitor of cholesterol efflux and the pathways that lead to the generation of HDL, the so-called good cholesterol. And when you inhibit miR-33 in mice and monkeys, you can raise plasma levels of HDL. But we also saw that in mice that had been fed a Western diet continuously, we saw favorable changes in the atherosclerotic plaque under conditions where we didn't see the increase in HDL. So if the mice are on a Western diet, the levels of miR-33 in the liver are very low, and inhibiting it doesn't cause the increase in HDL cholesterol. But we still saw this 25% regression in atherosclerotic plaques. And that got us thinking about the other things that miR-33 could be doing and around the same time, I was also very interested in immunometabolism and how the metabolic state of macrophages influences their function.

Kathryn Moore:          And Mihail Memet, who is a former postdoc in my lab made the discovery that miR-33 could inhibit fatty acid oxidation in macrophages and that this polarized the cells to a more inflammatory phenotype. So when we give the miR-33 inhibitors, we're raising a level of fatty acid oxidation in the macrophages and they become more tissue reparative. And so we suspected that could be the mechanism going on in the plaque but those studies, those initial studies were done over five years ago. And that was before the advent of single-cell technologies, which have really revolutionized how we're studying the atherosclerotic plaque. So in this study, we were able to apply some of these more high dimensional analyses of all of the immune cells in the plaque. And really look at how inhibiting miR-33 was altering their transcriptome and their phenotype.

Cindy St. Hilaire:         Yeah, so that is a perfect segue to my next question, which is you're doing this single-cell RNA-sequencing on tissue, but it's not just any tissue. It's not like a nice spleen that you can kind of pop open and all the cells fall out nicely and you can fax them or whatever. This is from an aorta, which itself is fibrous and tough on top of the atherosclerotic plaque, which is also difficult. So can you discuss maybe some of the challenges regarding doing this exact kind of analysis with this tissue and maybe some of the limitations or controls that you used to help really refine your result?

Kathryn Moore:           It is a little bit challenging to learn how to digest the aorta to release the immune cells, so to isolate the CD45+ immune cellsthat then go on to the sequence that takes some trial and error to get the right conditions. But actually, once you've done that a couple of times, it's not as difficult as it seems but I think that one of the challenges of doing these types of studies is integrating the results that we get from the single-cell RNA-sequencing with the other technologies that we've used in the past to analyze atherosclerosis.

Kathryn Moore:          So, previously when we were analyzing atherosclerotic plaque size or immune cell content, we are doing this through histology and immunostaining. And single-cell RNA-sequencing has identified all these new immune cell subsets based on transcriptomic signatures. And they don't really match up nicely with the protein signatures that we've used in the past.

Cindy St. Hilaire:         Yeah.

Kathryn Moore:          I saw this as a great opportunity to try to integrate all these techniques. And see if we could come to some middle ground. To understand how maybe the new subsets that we're identifying with single-cell RNA-seq from the aortic immune cells matched some of the things that we were able to do by looking at histology and tracing monocytes and macrophage entry and retention in the plaque.

Cindy St. Hilaire:         How did it line up? What's the nice Venn diagram of this study and what we've all been doing previously?

Kathryn Moore:          Well, it's a challenge, but what I thought was really really fascinating was we did monocyte-macrophage tracing experiments. Because one of the things we find when we inhibit miR-33 is we have a 50% decrease in the macrophage content of the plaque, but how is that happening? And what we found was there was an increase in the recruitment of monocytes into the plaque which may sound surprising if the plaque is shrinking, but they are the cells that are needed. They're the cleanup crew that are being introduced. But we saw a decrease in retention of macrophages and a decrease in proliferation and an increase in macrophage death and clearance of the apoptosis cells. And then through the single-cell RNA-sequencing, we were able to look at the different macrophage subsets. We had resident macrophages, Trem2hi metabolic macrophages, and MHCIIhi inflammatory macrophages.

Kathryn Moore:          We were able to look at their transcriptomes and say, "Which of these subsets are most likely to be performing those functions that we saw before?" And that was fun because that was like piecing together a puzzle. And what we saw, what it leads us to believe is that the Trem2hi metabolic macrophages are the ones that are undergoing aptosis. They have an increase in aptosis genes and eat-me signals and the MHCIIhi, having an increase in athoscoertic genes like mirTK that will help them clear the dying cells and the MHCIIhi macrophages also have decreased markers of proliferation. So although we used to think about macrophages as this one big pool, now we're able to say that these different subsets are performing different functions. And to me that's really exciting.

Cindy St. Hilaire:        Oh, that is exciting. And it's also extremely complicated because I was having enough trouble with just the two types of macrophages of a couple of years ago. The study showed that inhibiting this miR-33 using these anti-miR-33 oligos, and you're just kind of injecting oligos against it. And you're doing this in mice with established atherosclerosis. This helped to alter these monocyte and macrophage populations in the plaque itself.

Cindy St. Hilaire:        Do you think a function of the success of this study and essentially this therapy in the mouse is really dependent on the fact that it's targeting these circulating cells that are then going to the plaque? And I guess part of that question is, do you think part of this is because it's a circulating cell that can take it up, and then change and be delivered to the location it's going to, as opposed to that oligo targeting the plaque itself and the cells that are already residing there. Do you have any sense of that?

Kathryn Moore:          So it's interesting because one of the things that we did with our single-cell RNA-seq was to look at all immune cells in the plaque and say, "How many miR-33 target genes are changing in the ones from the treated mice?" And in the monocytes, you see very little change in miR-33 target genes. And that's consistent with what we know from Regulus Therapeutics who designed the anti-miR-33 antisense oligonucleotides. So we don't think that the ASO are being taken up in the circulation. I think they're actually being taken up by the macrophages in the plaque. And one of the great things about trying to target macrophages is they're very phagocytic. So they're going to be the ones that take up these ASOs, and the single-cell really allowed us to see whether it was just macrophages that were being affected or whether there were other immune cell populations that also seemed to have miR-33 induce changes. And of course it's hard from the single-cell to infer whether this is direct or indirect.

Cindy St. Hilaire:         Yeah.

Kathryn Moore:          But it seemed as if T-cells also were targeted by the anti-miR-33, definitely macrophages. We saw some changes in dendritic cells, very little changes in K cells, for example. And no changes in monocytes. And so it also begins to tell us how many different cell types are being affected and who's driving the bus when it comes to these changes. But by far the most miR-33 target genes change were the macrophage populations. And I think that's really due to their phagocytic ability.

Cindy St. Hilaire:         So I know there's a great divergence generally in microRNAs between mice and humans or really any species, but there are homologs to this in humans. What is the same and what is different between, I guess, this particular targeting micro RNA or what we know about it in mice and humans?

Kathryn Moore:          So mice have only one copy of miR-33, whereas humans and monkeys have two copies but those two copies are very similar in sequence. They differ only by two nucleotides. So you can use the same antisense oligonucleotides to target in mice and in non-human primates, for example. It's never been tried in humans.

Cindy St. Hilaire:         Yeah, of course. Not yet.

Kathryn Moore:          But it has been tried in monkeys, and we were able to effectively inhibit both miR-33a and miR-33b in the non-human primates. But the different variants of miR-33 have different transcriptional regulation. So they're induced under different conditions. And I think that's one way that mice and humans will really differ-the conditions where you'd have high levels of miR-33 will be different.

Cindy St. Hilaire:         Got it. Yeah. And the mice has that in the SREBP gene and humans.

Kathryn Moore:          And miR-33a is an SREBP-2 gene, which is SREBF2. And in humans there's an additional copy, which is SREBF. So it's in both of the SREBP genes in humans.

Cindy St. Hilaire:         Interesting. So I wonder, we need to ask the evolutionary biologist. Did they segregate together? I mean, I guess they must have. That's really interesting. That's cool.

Kathryn Moore:          One of the things that I love about miR-33 is that the SREBP-2 gene is turned on when cholesterol levels are low and it acts to increase the pathways involved in cholesterol synthesis and uptake. And miR-33 is transcribed at the same time. And what it does is it blocks the exits for cholesterol from the cell and from the body. And so it's just this hidden gem in the locus that sort of boosts SREBP-2 function.

Cindy St. Hilaire:         Its amazing stuff works out like that. I love it. So if we were going to leverage this inflammation resolution as atherosclerotic therapy, wherein the continuum of the disease, should we target? You know, we have obviously atherosclerotic plaque does not happen overnight. Teenagers can even have evidence of a fatty streak. If we were going to leverage antisense oligos as therapy, especially specifically against miR-33, where do you think would be a good place to target? And do we know, or have the kind of imaging capabilities to maybe identify that window right now in patients?

Kathryn Moore:          That's an interesting question. So lipid-lowering therapies will remain the first line of treatment for atherosclerosis, but lipid-lowering alone is insufficient to regress the plaque. It can stabilize plaques, but it doesn't really cause them to shrink. And when you think about the patient population that presents with cardiovascular disease, it's adults, for the most part. These are people in their fifties and sixties, and we've missed the chance to stop the early events. And so those are the majority of the people that are being treated. And I think there is room there to treat inflammation at the same time in the hopes of tipping that balance between pro-inflammatory events and then inflammation resolution. So we know surprisingly little about that tipping point. And now I think when miR-33 inhibition is fascinating in that it can affect both lipid metabolism and inflammation. And so I think that as an add-on therapy with lipid-lowering, it would be interesting, but of course, I'm not ready.

Cindy St. Hilaire:         We're not there yet.

Cindy St. Hilaire:         So I guess what's next for this line of research? What are kind of the next questions that the single-cell RNA-seq discovered for you? Was there anything kind of surprising or really exciting that you want to pursue next?

Kathryn Moore:          One of the things that I thought was really interesting was that the different macrophage subpopulations had different miR-33 target genes being de repressed. And that's probably not surprising, but I didn't initially think that would happen, but of course, the subpopulations are identified based on their unique transcriptomes. So they're not all the same, which means that they'll have different levels of miR-33, and they'll have different levels of the miR-33 target genes. And so Abca1, which we think about all the time as a miR-33 target gene that's involved in cholesterol efflux, it went up in Trem2hi macrophages and the resident macrophage population, but not in the MHCIIhi. The target genes and the MHCIIhi were different than the other two populations. And I think this now gives us a chance to sort that out.

Kathryn Moore:          And some of the targets in the MHCIIhi macrophages were ones that are involved in chromatin reorganization-

Cindy St. Hilaire:         Oh, interesting.

Kathryn Moore:          ... and inscriptional regulation. And when I looked across the other subsets, I could see that common pattern in T-cells and B-cells that were changing. And I think that's one way that miR-33 could have a broad impact. MiR-33 is a little bit of a unique microRNA. It has a very potent impact on these pathways. Other microRNAs often can change gene expression by 10 to 20%, but miR-33, when we inhibit it, we see really powerful effects. And I think that if it is involved in targeting genes that mediate chromogenic reorganization or transcriptional complex formation, that gives us a hint of how it could be having additional impact.

Cindy St. Hilaire:         That's really cool. And this was an absolutely beautiful story, not only in kind of dissecting out the mechanisms at play, but you know, those beautiful tisney plots and the nice graphics of the single-cell stuff.

Kathryn Moore:          The first author of the paper, Milessa Afonso, is a postdoc that just left the lab, and she worked so hard on this and did such a beautiful job.

Cindy St. Hilaire:         Well, it's a wonderful story and I'm really happy we were able to publish it. So, Dr Moore, thank you so much for joining me today.

Kathryn Moore:                      My pleasure. Thank you.

Cindy St. Hilaire:        That's it for the highlights from the April 2nd and 16th issues of Circulation Research. Thank you so much for listening. Please check out the CircRes Facebook page and follow us on Twitter and Instagram with the handle @circres and hashtag DiscoverCircRes. Thank you to our guest, Dr Kathryn Moore. This podcast is produced by Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Copy text for the highlighted articles was 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 exciting discoveries in basic cardiovascular research.

 

 

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