Podcasts about abca1

References Infect Immun. 2013 Dec; 81(12): 4478–4489 Cell Cycle. 2022; 21(11): 1121–1139. The Journal of Nutritional Biochem.2023.Volume 112, 109217 Lightfoot, G. 1964. "Early Morning Rain" https://youtu.be/ZFJ5Bj_put0?si=m1Ue7rFfk_ggSLR3 Simon ,P. 1968. "Old Friends" SImon and Garfunkle Bookends LP. https://youtu.be/7A76lTte8qE?si=rfk3Q6wTA6Nsiok7 Mozart, WA. 1782 Serenade in C. Minor K.388. https://youtu.be/R6iSGBNdqSw?si=S2eGPmcySUhw4uvU --- Send in a voice message: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/message Support this podcast: https://podcasters.spotify.com/pod/show/dr-daniel-j-guerra/support

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.07.24.550299v1?rss=1 Authors: Allende, L. G., Natali, L., Cragnolini, A. B., Musri, M. M., de Mendoza, D., Martin, M. G. Abstract: Cholesterol is crucial for the proper functioning of eukaryotic cells, especially neurons, which rely on cholesterol to maintain their complex structure and facilitate synaptic transmission. However, brain cells are isolated from peripheral cholesterol by the blood-brain barrier and mature neurons primarily uptake the cholesterol synthesized by astrocytes for proper function. This study aimed to investigate the effect of aging on cholesterol trafficking in astrocytes and its delivery to neurons. Using in vitro and in vivo models of aging, we found that aged astrocytes accumulated high levels of cholesterol in the lysosomal compartment, and this cholesterol buildup can be attributed to the simultaneous occurrence of two events: decreased levels of the ABCA1 transporter which impairs ApoE-cholesterol export from astrocytes, and reduced expression of NPC1, which hinders cholesterol release from lysosomes. We show that these two events are accompanied by increased microR33 in aged astrocytes, which is known to downregulate ABCA1 and NPC1. In addition, we demonstrate that the microR33 increase is triggered by oxidative stress, one of the hallmarks of aging. By co-culture experiments we also show that aging in vitro impairs the cholesterol delivery from astrocytes to neurons. Remarkably, we found that this altered transport of cholesterol could be alleviated through treatment with endocannabinoids as well as cannabidiol or CBD. Given that reduced neuronal cholesterol affects synaptic plasticity, the ability of cannabinoids to restore cholesterol transport from aged astrocytes to neurons holds significant implications in the field of aging. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

How does you unique genome increase the risk for Alzheimer's disease as you age? In todays episode—#7, I'll be giving a comprehensive overview on the APOE gene, the ApoE4 genetic variant, and other genes and their genetic variants linked to the risk for late-onset Alzheimer's Disease (LOAD), and how, when, and why Alzheimer's takes root in certain individuals that are carriers of these genes and the variants associated with them. While the ApoE4 genetic risk variant is widely recognized as a risk factor for LOAD, how ApoE4 contributes to the risk for Alzheimer's is not as so well recognized by most individuals. Indeed, the ApoE4 genetic variant is often highlighted as the most significant risk factor for LOAD, but how often have you run across the reasons why ApoE4 raises your risk for LOAD, and how other genetic variants may similarly and synergistically increase the risk for LOAD? Yes, many other risk variants add to the polygenic (more than one gene) disease profile of LOAD. The known functional and structural vulnerabilities linked to the ApoE4 variant are multifaceted, and I describe these functional and structural abnormalities that are linked to ApoE4 in my book, "The Diabetic Brain in Alzheimer's Disease". However, since the mechanisms that underly the link between ApoE4 in LOAD are a vast topic, I focus on two key points—cholesterol and fat binding and transport, and beta-amyloid deposition and clearance from the brain. ApoE4, APOJ, ABCA1 and ABCA7, and TREM2 variants greatly determine how these two key mechanisms—cholesterol and beta-amyloid metabolism are factored into the risk for LOAD. Additionally, I briefly describe another very common variant—MTHFR 677T, that is a critical risk variant in methylation and homocysteine metabolism—yet another pathway that links the importance of the heart-brain axis in the risk for Alzheimer's disease. Please listen in and get ready for about 35 minutes of a revealing overview on the genes and their variants that are widely available in genetic profiling tests, and are major risk factors in LOAD. Ralph Sanchez, MTCM, CNS, D.Hom www.TheAlzheimersSolution.com https://www.facebook.com/TheAlzheimersSolution https://www.linkedin.com/in/ralph-sanchez https://www.instagram.com/alzheimers_solution

This month on Episode 34 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the March 4 and March 18th issues of Circulation Research. This episode also features a conversation with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.   Article highlights:   Pauza, et al. GLP1R in CB Suppress Chemoreflex-Mediated SNA   Lim, et al. IL11 in Marfan Syndrome   Hohl, et al. Renal Denervation Prevents Atrial Remodeling in CKD   Liu, et al. Smooth Muscle Cell YAP Promotes Arterial Stiffness   Cindy St. Hilaire:        Hi and welcome to Discover CircRes, the podcast of the American Heart Association's journal, Circulation Research. I'm your host, Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting articles from our March issues of Circulation Research. I'm also going to speak with Dr Mireille Ouimet and Sabrina Robichaud from the University of Ottawa Heart Institute, and they're with me to discuss their study, Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis.   The first article I want to share is titled GLP1R Attenuates Sympathetic Response to High Glucose via Carotid Body Inhibition. The first author is Audrys Pauza, and the corresponding authors are Julian Paton and David Murphy at the University of Bristol.   Cindy St. Hilaire:        Hypertension and diabetes are risk factors for cardiovascular disease. And yet, for many patients with these two conditions, lowering blood pressure and blood sugar is insufficient for eliminating the risk. The carotid body is a cluster of sensory cells in the carotid artery, and it regulates sympathetic nerve activity. Because hypertension and diabetes are linked to increased sympathetic nerve activation, this group investigated the role of the carotid body in these disease states. They performed a transcriptome analysis of crowded body tissue, from rats with and without spontaneous hypertension. And they found among many differentially-expressed genes that the transcript encoding glucagon-like peptide-1 receptor or GLP1R, was considerably less abundant in hypertensive animals.   Cindy St. Hilaire:        This was of particular interest because the gut hormone GLP-1 promotes insulin secretion and tends to be suppressed in Type 2 diabetes. Moreover, GLP1R agonists are already used as diabetic treatments. This group showed that treating rat carotid body with GLP1R agonist suppresses sympathetic nerve activation and arterial blood pressure, suggesting that these drugs may provide benefits in more than one way. Perhaps the carotid body could be a novel target for lowering cardiovascular disease risk in metabolic syndrome.   Cindy St. Hilaire:        The second article I want to share is titled Inhibition of IL11 Signaling Reduces Aortic Pathology in Murine Marfan syndrome. The first author is Wei-Wen Lim, and the corresponding author is Stuart Cook and they're from the National Heart Center in Singapore. People with the genetic connective tissue disorder Marfan syndrome, are typically tall and thin with long limbs and are prone to skeletal, eye and cardiovascular problems, including a life-threatening weakening of the aorta. While Marfan syndrome patients commonly take blood pressure-lowering treatments to minimize risk of aortic aneurysm and dissection, there's currently no cure for Marfan syndrome or targeted therapy.   Cindy St. Hilaire:        The cytokine IL11 is strongly induced in vascular smooth muscle cells upon treatment with the growth factor TGF-beta, which is over activated in Marfan syndrome patients. And TGF-beta is also considered a key feature of the syndrome's molecular pathology. This study found that IL11 is strongly upregulated in the aortas of Marfan syndrome model mouse, and that genetically eliminating IL11 in these animals protected them against aortic dilation, fibrosis, inflammation, elastin degradation and loss of smooth muscle cells. Treating Marfan syndrome mice with anti-IL11 neutralizing antibodies exhibited the same beneficial effects. These results suggest that perhaps inhibiting IL11's activity could be a novel approach for protecting the aortas of Marfan syndrome patients.   Cindy St. Hilaire:        The next article I want to mention is titled Renal Denervation Prevents Atrial Arrhythmogenic Substrate Development in Chronic Kidney Disease. The first authors are, Mathias Hohl, Simina-Ramona Selejan and Jan Wintrich, and the corresponding authors also Mathias Hohl, and they're from Saarland University. People with chronic kidney disease have a two to three fold higher risk than the general population of developing atrial fibrillation, which is a common form of arrhythmia that can be life-threatening. Chronic kidney disease is associated with activation of the sympathetic nervous system, which can be damaging to the heart. Thus, this group examined myocardial tissues from atrial fibrillation patients with and without chronic kidney disease to see how they differ. They found that atrial fibrosis was more pronounced in patients with both conditions than in patients with atrial fibrillation alone, suggesting that chronic kidney disease perhaps exacerbates or even drives arterial remodeling.   Cindy St. Hilaire:        Sure enough, induction of chronic kidney disease in rats led to greater atrial fibrosis and incidence of atrial fibrillation than seen in the control animals. Renal denervation is a treatment in which the sympathetic nerves are ablated, and it's a medical procedure that's used for treating uncontrolled hypertension, and it has also been shown in animals to reduce atrial fibrillation. Performing renal denervation in the rats with chronic kidney disease reduced atrial fibrosis and atrial fibrillation susceptibility. This study not only shows that chronic kidney disease induces atrial fibrosis and in turn atrial fibrillation, but also suggests that renal denervation may be used in chronic kidney disease patients to break this pathological link and prevent potentially deadly arrhythmias.   Cindy St. Hilaire:        The last article I want to highlight is titled YAP Targets the TGFβ Pathway to Mediate High-Fat/High-Sucrose Diet-Induced Arterial Stiffness. First author is Yanan Liu and the corresponding author is Ding Ai from Tianjin Medical University. Metabolic syndrome is characterized as a collection of conditions that increase the risk of cardiovascular diseases, such as obesity, hypertension and diabetes. Among the tissue pathologies associated with metabolic syndrome is arterial stiffness, which itself is a predictor of cardiovascular disease incidence and mortality. To specifically investigate how arterial stiffness develops in metabolic syndrome, this group fed mice a high-fat, high-sugar diet, which is known to induce metabolic syndrome and concomitant arterial stiffness.   Cindy St. Hilaire:        After two weeks on the diet, the animals' aorta has exhibited significant upregulation of TGF-beta signaling, which is a pathway known for its role in tissue fibrosis, and the aorta has also exhibited increased levels of yes-associated protein, or YAP, which has previously been implicated in vascular remodeling, collagen deposition and inflammation. YAP gain and loss of function experiments in transgenic mice revealed that while knockdown of protein in the animals' smooth muscle cells attenuated arterial stiffness, increased expression exacerbated the condition.   Cindy St. Hilaire:        The team went on to show that YAP interacted with and prevented the activation of PPM-1 B, which is a phosphatase that normally inhibits TGF-beta signaling and thus fibrosis. Together the results suggest that targeting the YAP, PPM-1 B pathway, could be a strategy for reducing arterial stiffness and associated cardiovascular disease risk in metabolic syndrome.   Cindy St. Hilaire:        Today, Sabrina Robichaud and Dr Mireille Ouimet from University of Ottawa Heart Institute are with me to discuss their study Autophagy is Differentially Regulated in Leukocyte and Non-Leukocyte Foam Cells During Atherosclerosis, which is in our March 18 issue of Circulation Research. So thank you both for joining me today.   Sabrina Robichaud:    Thank you so much for having us. It's a pleasure.   Mireille Ouimet:         Thank you for having us.   Cindy St. Hilaire:        Yeah, and congrats on the study. So we know that LDL particles contain cholesterol and fats, and these are the initiating factors in atherosclerosis. And it's also really now appreciated that inflammation in the vessel wall is a secondary consequence to this lipid accumulation. Macrophages are an immune cell that, in the context of the plaque, gobble up this cholesterol to the point that they become laden with lipids and exhibit this foamy appearance, which we now call foam cells. And these foam cells can exhibit atheroprotective properties, one of them called reverse cholesterol transport, and that's really one of the focuses of your paper. So before we dig into what your paper is all about, could you give us a little bit of background about what reverse cholesterol transport is in the context of the atherosclerotic plaque? And maybe introduce how it links to this cellular recycling program, autophagy, which is also a big feature of your study.   Mireille Ouimet:         Yes, so the reverse cholesterol transport pathway is a pathway that's very highly anti-atherogenic. It's linked to HDL function and the HDL protective effects, in that HDL can serve as a cholesterol acceptor for any excess cholesterol from arterial cells or other cells of the body and return this excess cholesterol to the liver for excretion into the feces. There is also trans-intestinal cholesterol efflux that can help eliminate any excess bodily cholesterol. Mireille Ouimet:         So reverse cholesterol transport is a way that we can eliminate excess cholesterol from foam cells in the vascular wall, and that's why we're really interested in the process. But the rate-limiting step of cholesterol efflux out of foam cells in plaques is actually, they have to be mobilized in the form of free cholesterol to be pumped out of the cells through the action of the ATP-binding cassette transporters. And so the rate-limiting step of the process is the hydrolysis of the cholesterol esters and the lipid droplets, because that's where the excess cholesterol is stored in foam cells.   Mireille Ouimet:         And so for years, people investigated the actions of cytosol like lipases in mobilizing free cholesterol from lipid droplets, although the identity of those lipases are not well-known and in macrophage themselves, but our recent work showed a role for autophagy in the catabolism of lipid droplets. And in fact, in macrophage foam cells, 50% of lipid droplet hydrolysis is attributable to autophagy while the other half is mediated by neutral lipases, which makes it really important to investigate the mechanisms of autophagy-mediated lipid droplet catabolism.   Cindy St. Hilaire:        That is so interesting. I guess I didn't realize it was that significant a component in that kind of rate-limiting step. That's so cool. So really, a lot of the cholesterol efflux studies, and maybe this is just limited to my knowledge of a lot of these cholesterol efflux studies, but to my knowledge, it's been really focused on the foam cell itself, the macrophage foam cell. However, there's been a lot of recent work that has now implicated vascular smooth muscle cells in this process. So could you share some of the research specific to smooth muscle cells and smooth muscle-derived foam cells that led you to want to investigate the contributions of smooth muscle cell-derived foam cells in cholesterol efflux?   Mireille Ouimet:         Yeah, so you're right in the sense that macrophages have always been the culprit foam cells in the atherosclerotic plaques but pioneering work from several groups, including Edward Fisher and Gordon Francis, they've shown that the smooth muscle cells can actually acquire a macrophage-like phenotype becoming lipid-loaded and foamy. And there's been work specifically looking at the ABC transporters, and their ability to efflux cholesterol from these vascular smooth muscle cell-derived foam cells, because as they trans-differentiate into macrophage-like cells, they acquire the expression of ABCA1, but this is to a lower extent, as compared to their macrophage counterparts.   Mireille Ouimet:         And the efflux is defective because there's an impairment in liposomal cholesterol processing of the lipoproteins that's really important to activate a like cell, and the expression of the ABC transporters, so vascular smooth muscle cell-derived foam cells are very poor effluxes.   Sabrina Robichaud:    There's very few studies that look at the vascular smooth muscle cell foam cells, and the very few that did look at it mostly focused on the ABCA1 transporters, and did show that they were poor effluxes. And as we all know, ABC1 is not the only cholesterol transporters that can transport cholesterol out of cells, there's also ABCG1 which is also one of our major findings in our paper.   Cindy St. Hilaire:        Can you tell us a little bit about the models you chose in the study and why you picked them? And also maybe a step back in terms of, what are the pros and cons of using mouse models in atherosclerotic studies?   Sabrina Robichaud:    So we chose to use the GFP-LC3 reporter mouse model because it allows us to track in lifestyle the movement of LC3, which is the main component of the autophagosome which is involved in pathology. So by using this reporter model, we could infer whether or not the cells had high autophagy or low autophagy. And to induce atherosclerosis in these mice, instead of backcrossing them to either an LDLR knockout or an ApoE knockout, we chose to do the adeno-associated virus that encode the gain of function PCSK9 instead to kind of minimize the time for breeding. It did have the effect that we needed in terms of raising plasma cholesterol to induce the atherosclerosis. So that was one of the models that we used in our paper.   Mireille Ouimet:          There's not very many good mouse models to study autophagy flux in vivo and GFP-LC3 is kind of the main one currently. We're working on developing some other tools to track lipophagy in vivo, but these things take time to put in place. So in the future, we hope to have some better tools to track lipophagy in real-time in vivo.   Cindy St. Hilaire:        How difficult is it to measure autophagy flux in vivo? I know there's certain part like LC3 or P62, a lot of people use a western blot and it's like, oh, it's high, it must be active, but it's a flux. So it's a little bit more... There's more subtleties to that, dynamic than that. So how difficult is it to really measure this flux in in vivo tissues?   Mireille Ouimet:         Yes, so now there are more recent mouse models that have been developed more recently to replace kind of the GFP-LC3 is the Rosella LC3. So it has both a red and a green tag, and so two LC3, so when autophagosomes are fused to lysosomes and are degraded, then there's preferential quenching of the GFP first, and then you have the red appearance that predominates so we know that then it's kind of like it a live flux measurements. Because we use the GFP-LC3 mouse, Sabrina treated her cells ex vivo. When we dissected out the aortic arches, digested the cells then we divided those into two components and added bafilomycin so that we can inhibit lysosome acidification to see the changes in the flux. And that's really to get the differences in untreated versus bafilomycin-treated.   Mireille Ouimet:         When we inhibit the lysosome, then we're sure that it is a functional flux or not. But it's kind of an indirect way of measuring it, and it reads very complex when we're talking about P62 and LC3 degradation with or without lysosome inhibition, but you really need that lysosomal inhibition, to show that if you block the degradation of the autophagosomes that fuse in with a lysosome, then you get an increase in the LC3 and the P62, and that's when you know that the flux is you intact.   Mireille Ouimet:         Because you could get an increase in LC3, that's just related to a defect in the breakdown of the autophagosome. But in our study, we've used phosphorylated ATG16L1, which is a now better marker of active autophagy. And I would recommend researchers to begin to use that rather than the combination of P62 and LC3 together with or without a lysosome inhibitors such as- Cindy St. Hilaire:        Oh, interesting. So let's repeat that, phosphorylated ATG-   Mireille Ouimet:         16L1, yes. So there's been an antibody that was developed by a colleague at the University of Ottawa, Dr Ryan Russell, and it's commercially available through cell signaling now, and it really has been a great tool to track active autophagy.   Cindy St. Hilaire:        That's great. I remember my lab was looking at that at one point, and I was trying to explain the flux as... I don't know if people are going to remember this, but there's this amazing, I Love Lucy skit, where her and Ethel are working on a chocolate factory conveyor belt, and it picks up speed. And because she can't get it all done quick, she starts stuffing them in her mouth. And it's like, if you just took a snapshot of that, you would not know whether it's going too fast, or not functioning properly. And so I equate the flux experiments to that. Which are probably aging myself a lot on so.   Cindy St. Hilaire:        All right, so sticking to kind of the autophagy angle, what were the differences you found in autophagy in early and late atherosclerotic plaques? Because I know you looked at those two time points, but also, importantly, between the macrophage foam cells and the smooth muscle cell-derived foam cells?   Sabrina Robichaud:    So surprisingly, there weren't that big of a difference between each time point when we were looking at the individual cell type by themselves. Surprisingly, we did find that the macrophages did have a functional autophagy flux, even at the later stages of atherosclerosis, which was kind of interesting in itself. But when we looked at the vascular smooth muscle cell foam cells, though, that was a whole other story, and we found that these were actually defective at a very early stage and stayed defective up until the very late stage of atherosclerosis.   Cindy St. Hilaire:        And what is the very early stage like? What's that definition with the smooth muscle cell?   Sabrina Robichaud:    So we did a six-week time points in terms of our atherosclerosis study, and then a 25-week time point. So there are far apart, which shows like the very early, early stage and what would be considered the most effective autophagy at that point with the necrotic core and everything. So surprisingly, the two phenotype were quite similar at early and both late stages for both cell types, but were functional in the macrophages but dysfunctional in the smooth muscle cells.   Cindy St. Hilaire:        So you mentioned at one point in the discussion that you observed inconsistent lipid loading of the smooth muscle cells, and you mentioned that a lipase, which is excreted from the foam cells can then be internalized by, I assume kind of neighboring or in the vicinity, smooth muscle cells. And so the question I had it's kind of one of those chicken-and-egg question, and it's, is the smooth muscle cell-derived foam cell an independent process? Does it happen alone or de novo as a function of a smooth muscle-mediated process? Or is it really dependent first on this macrophage foam cell providing this lipid that is efflux that is then internalized by a smooth muscle cell that kind of goes on to become a foam cells. It's kind of a question of like the continuum of an atherosclerotic plaque and what do you think is happening, either based on your data or just kind of a hunch?   Mireille Ouimet:         That's an excellent question. And there's no doubt that macrophages really drive the initiating events of atherosclerosis. So I don't think that without the macrophage there would ever be a vascular smooth muscle cell, or there would be minimal vascular smooth muscle cell-derived foam cells. Definitely the inconsistencies that we observed in our study, were if we added like aggregated LDL on its own to a primary mouse vascular smooth muscle cell, we would get poor lipid loading and a very low percentage of those cells that would become foamy, relative to treating them with cyclodextrin complex cholesterol, for instance.   Mireille Ouimet:         So free cholesterol, that's cell permeable, will go into the vascular smooth muscle cell, no problem, and generate the foaminess and then allow that cell to acquire the macrophage-like phenotype. But aggregated LDL on its own in our hands, just gave very poor loading. And when we treated the vascular smooth muscle cells with aggregated LDL along with macrophage-derived condition media, we got some improvements, but it was still kind of inconsistent. But then we thought if we treat the vascular smooth muscle cells with aggregated LDL in the presence of conditioned media from macrophage foam cells that were preloaded with the aggregated LDL, would that promote their foaminess to a greater extent? And it did.   Mireille Ouimet:         So, there have been studies from Gordon Francis's lab that showed that adding recombinant lysosomal acid lipase to vascular smooth muscle cells that contained aggregated LDL, promoted the lysosomal hydrolysis of the aggregated LDL and to generate the foamy macrophages and allow the lysosomal processing. So we know that that vascular smooth muscle cells take up lysosomal acid lipase, and we know that macrophages undergo lysosome exocytosis and they can secrete lysosome acid lipase and acidify the extracellular milieu.   Mireille Ouimet:         So work from Fred Maxfield group has shown the presence of these cell surface connected compartments that are acidified, containing macrophage-derived lysosomal acid lipase, that even hydrolyze extra cellularly-aggregated LDL for macrophages. So we're not sure whether there's probably a local production of free cholesterol in the plaque by macrophages, this free cholesterol could be taken up by the vascular smooth muscle cell. And also the vascular smooth muscle cells do express some scavenger receptors, whether the expression of these scavenger receptors like LRP or CD36 even goes up when they've taken up a little bit of the free cholesterol. And then that allows the aggregated LDL to come in and then there would be some lysosomal acid lipase secreted by the macrophage foam cells that would promote the lysosomal processing of this aggregated LDL. All of those are very complex questions that will require some addressing in vivo models.     Cindy St. Hilaire:        You also mentioned in the paper that studies... There's a handful of them now. Studies have shown that between 30% and 70% of the cells that are staining positively for macrophage markers, meaning they're foam cells, are of the smooth muscle cell lineage. And so I believe people have seen that in mouse plaques with lineage tracing, but they've also used newer techniques to really see this also in human atherosclerotic plaques. So we know it's not just from a mouse, we know that smooth muscle cells can turn into a macrophage-like foam cell, and it's 30% to 70%, which is a huge range. Cindy St. Hilaire:        So do we know the factors that dictate whether a specific plaque is going to have more or less smooth muscle cell derived foam cells? And I guess more important to what you found in your paper is, how important would it be to know whether a plaque is on the 30% end or on the 70% end in terms of therapeutic strategies?   Sabrina Robichaud:    Yeah, most of these studies, the range can be attributed to the different time points at which these studies have been collected early on will be a little bit more macrophage understanding would be at a later time point. Now of course in terms of therapeutics, as we saw in our paper, metformin actually will positively increase cholesterol efflux in the vascular smooth muscle cell foam cells, but not in the macrophages. So obviously, being able to know at which point there's a majority of macrophages versus vascular smooth muscle cells, definitely going to determine which therapeutic we're going to be able to use.   Sabrina Robichaud:    Ideally, we would be able to find a therapeutic that would work in both foam cell, but from what we've seen, the mechanistic behind the autophagy dysfunction between both cell types are so different, that I'm not entirely sure that that would be possible, we would need some sort of combination therapy. But again, we need to be a little bit more targeted depending on the percentage of the foam cells that are comprising the plaque at that particular moment in time.   Cindy St. Hilaire:        Yeah, so you mentioned there's a function of time there. If you look earlier, there's more macrophage, if you look later, the percent of smooth muscle cell-derived foam cell increases. Is there a point in a very advanced atherosclerotic plaque where it's just mostly smooth muscle cells? Or do those macrophage foam cells stay, and it's just the increasing number of smooth muscle cell-derived foam cells? Do we know?   Mireille Ouimet:         This is an excellent question, and I was going to bring up the topic of clonal expansion of the vascular smooth muscle cells. So it's a very heterogeneous population and understanding that might be some of the differences that we see in different studies. It could be the model has one type of a smooth muscle cell that's expanding more than another, what are the factors that govern that? Does one clone take over at the later stages versus the earlier stages? We don't know.   Mireille Ouimet:         But we were surprised in our studies to see that the macrophages that are present at least on the lumen of the plaques were very active in autophagy. They had the highest staining for the phospho-ATG16L1 in that late stage. So we're not sure if it's newly-recruited macrophages that come in, that are more active and in autophagy, and then have good lysosomal capacity that keeps degrading the lipid present in the plaque and tries to ingest it, but also as a consequence keeps releasing some of the degraded cholesterol into the milieu where the smooth muscle cells that are proliferating are internalizing it and becoming more foamy. So these are really great open questions that need to be addressed in the field.   Cindy St. Hilaire:        So drug-eluting stents are coated with rapamycin or the various chemical compositions that are derived from rapamycin. And rapamycin itself induces autophagy. So while the thought behind using this coating on stents was to prevent smooth muscle cell proliferation, and thus restenosis or ingrowing of the stent, your study suggests that this could also help to promote autophagy in the cells underlying the stent. So has anyone gone in and looked at plaques that have been stented and either failed or not, and investigated the foam cell content or markers for autophagy activity?   Mireille Ouimet:         Not to my knowledge, and this has been something we've definitely... We think that this is what's happening. Some of the protective effects of these drug-eluting stents that have everolimus or sirolimus or the rapamycin or rapamycin analogs, we do believe that some of their protective effect can be attributed to autophagy activation, but this remains to be demonstrated. We think that autophagy activation locally would promote reverse cholesterol transport and would be one of the processes that prevents restenosis because we can promote the efflux of cholesterol out.   Cindy St. Hilaire:        Great. So I guess stemming from my question on the stents, what are the other translational implications of the findings of your study? And what would you like to see come out of this?   Mireille Ouimet:         So one of the things is, as Sabrina mentioned, would be to target both foam cell populations because it seems as though the vascular smooth muscle cell foam cells are very much defective in their autophagy capacity, and they're very poor effluxes, but we could potentially restore autophagy in the cell population to promote reverse cholesterol transport.   And looking at prevention of atherosclerosis is a bit different than looking at regression, because regression is at a later stage where the plaques are more advanced. And if they're mostly vascular smooth muscle cell-derived, maybe then those drugs that we're considering that protect against the development of atherosclerosis are effective on the macrophage themselves early on, but might not be mimicking what we would see in the clinic where the patients that present are older.   Cindy St. Hilaire:        Yeah, it's kind of really reminiscent of like the CANTOS trial and like, where do we want to target the therapy? It's going to be very different if it's an early smaller plaque, versus a late-stage possibly pro close to rupturing type of plaque. Well, Sabrina Robichaud and Dr Ouimet, thank you so much for joining me today. Congratulations again on a wonderful study, and I'm really looking forward to hearing more about this from your group.   Sabrina Robichaud:    Thank you.   Mireille Ouimet:         Thank you very much. And we also want to thank all the co-authors on the study, specifically also Adil Rasheed, who is co-first author on the work and Katey Rayner's group for all the support and involvement in this study.   Cindy St. Hilaire:        That's it for the highlights from the March 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, Sabrina Robichaud and Dr Mireille Ouimet Sabrina. 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 was provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire and this is Discover CircRes, you're 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 2022, The opinions expressed by speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit ahajournals.org.

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.    

Dr. Carolyn Lam:               Welcome to Circulation on the Run, your weekly podcast summary and backstage pass to the journal and its editors. I'm Dr. Carolyn Lam, associate editor from the National Heart Center and Duke National University of Singapore. What is the association between fetal congenital heart defects and maternal risk of hypertensive disorders of pregnancy? We will be discussing new data in this area in just a moment, following these summaries.                                                 The first paper describes the effect of long-term metformin and lifestyle measures on coronary artery calcium. This is a paper from Dr. Goldberg of George Washington University Biostatistics Center and colleagues of the Diabetes Prevention Program Research Group. The Diabetes Prevention Program and its outcome study is a long-term intervention study in subjects with prediabetes, which showed reduced diabetes risk with lifestyle and metformin compared to placebo.                                                 In the current study, the authors looked at subclinical atherosclerosis, which was assessed in 2,029 participants using coronary artery calcium measurements after 14 years of average follow-up. They found that men but not women with prediabetes treated with metformin for an average duration of 14 years had lower coronary calcium scores than their placebo counterparts. No difference in coronary calcium scores was observed in the group receiving a lifestyle intervention as compared to the placebo group.                                                 These findings provide the first evidence that metformin may protect against coronary atherosclerosis in men with prediabetes, although demonstration that metformin reduces cardiovascular disease events in these subjects is still needed before firm therapeutic implications of these findings can be made. The reason for an absence of an effect in women is unclear and deserves further study.                                                 The next study provides insights on the physiology of angina from invasive catheter laboratory measurements during exercise. Dr. Asrress of Royal North Shore Hospital in Sydney, Australia, and colleagues, studied 40 patients with exertional angina and coronary artery disease who underwent cardiac catheterization via radial axis and performed incremental exercise using a supine cycle ergometer. As they developed limiting angina, sublingual GTN was administered to half the patients and all patients continued to exercise for two minutes at the same workload. Throughout exercise, distal coronary pressure and flow velocity, and central aortic pressure were recorded using sensor wires.                                                 Using this novel invasive approach, the authors showed that administration of GTN ameliorated angina by reducing myocardial oxygen demand as well as increasing supply with a key component being the reversal of exercise-induced coronary lesion vasoconstriction. This was evidenced by the fact that there was a relationship between the diastolic velocity pressure gradient with significant increase in relative stenosis severity. In keeping with exercise-induced vasoconstriction of stenosed epicardial segments and dilation of normal segments, with trends towards reversal with GTN.                                                 Thus, this study describes the development of a paradigm where patients with coronary artery disease can exercise while simultaneously having coronary and central aortic hemodynamics measured invasively, and has shown that this provides a unique opportunity to study mechanisms underlying the physiology of angina. In treating patients with exercise-induced angina, the results highlight the importance of after-load reduction and the use of agents that reduce arterial wave reflection and promote coronary artery vasodilation.                                                 The next study provides mechanistic insights into reverse cholesterol transport, where excess cholesterol is removed from macrophage-derived foam cells in atherosclerotic plaques. It suggests that melanocortin receptor-1, or MC1-R, may play a role. As background, the melanocortin system, consisting of melanocyte-stimulating hormones and their receptors, regulate a variety of physiological functions, ranging from skin pigmentation to centrally-mediated energy balance control. At the cellular level, the biological actions are mediated by G protein-coupled melanocortin receptors, such as MC1-R. MC1-R not only affects melanogenesis in the skin but also has immunomodulatory effects through its wide expression in the cells of the immune system.                                                 In the current study from Dr. Rinne of University of Turku in Finland, and colleagues, human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was further assessed in apolipoprotein E deficient mice. Their findings identified a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R conferred protection against macrophage foam cell formation through a dual mechanism. It prevented cholesterol uptake while it concomitantly promoted reverse cholesterol transport by increasing the expression of ATP-binding cassette transporters, ABCA1 and ABCG1.                                                 Thus, the identification of MC1-R in lesional macrophages, demonstration of its role in regulating reverse cholesterol transport, combined with its established anti-inflammatory effects, suggests that MC1-R could be a novel new therapeutic target for preventing atherosclerosis.                                                 The next study suggests that obesity-related heart failure with preserved ejection fraction, or HFpEF, is a genuine form of cardiac failure and a clinically relevant phenotype that may require specific treatments. First author, Dr. Obokata, corresponding author, Dr. Borlaug, and colleagues from Mayo Clinic Rochester and Minnesota studied 99 patients with obese HFpEF with a BMI above 35, with 96 non-obese HFpEF with a BMI less than 30, and 71 non-obese controls without heart failure. All subjects underwent detailed clinical assessment, echocardiography, and invasive hemodynamic exercise testing.                                                 The authors found that, compared to non-obese HFpEF, obese HFpEF patients displayed greater volume overload, more biventricular remodeling, greater right ventricular dysfunction, worse exercise capacity, more impaired pulmonary vasodilation, and more profound hemodynamic arrangements, despite a lower NT-proBNP level. Obese HFpEF patients displayed other important contributors to high left ventricular filling pressures, including greater dependence on plasma volume expansion, increased pericardial restraint, and enhanced ventricular interaction, which was exaggerated as pulmonary pressure load increased.                                                 These data provide compelling evidence that patients with the obese HFpEF phenotype have real heart failure and display several pathophysiological mechanisms that differ from non-obese patients with HFpEF. These and other issues are discussed in an accompanying editorial by Dr. Dalane Kitzman and myself. We hope you enjoy it.                                                 The final study identifies a novel long noncoding RNA that regulates angiogenesis. As background, although we know that the mammalian genome is pervasively transcribed, a large proportion of the transcripts do not encode a protein, and are thus regarded as noncoding RNAs. Based on their length, they can be divided into small or long noncoding RNAs, long being described as more than 200 nucleotides. Although their function is not fully understood, long noncoding RNAs have been increasingly reported to mediate the expression of other genes, affect the organization of the nucleus, and modify other RNAs.                                                 In the current study by first author, Dr. Leisegang, corresponding author, Dr. Brandes, and colleagues of Goethe University in Frankfurt, Germany, epigenetically controlled long noncoding RNAs in human umbilical vein endothelial cells were searched by axon array analysis following knockdown of the histone demethylase JARID1B. The authors discovered a novel noncoding RNA named MANTIS to be strongly upregulated. MANTIS is located in the antisense strand of an intronic region of the gene for annexin A4, calcium- and phospholipid-binding protein. MANTIS is a nuclear long noncoding RNA that is enriched in endothelial cells but also expressed in other cell types. Reducing MANTIS levels led to impaired endothelial sprouting, tube formation, attenuated endothelial migration, and inhibition of the alignment of endothelial cells in response to shear stress.                                                 Brahma-like gene 1, or BRG-1, was identified as a direct interaction partner of MANTIS, implying a role of MANTIS in the formation of the switch/sucrose non-fermentable chromatin remodeling complex. MANTIS binding to BRG-1 was shown to stabilize the BRG-1 interaction, hence by inducing an open chromatin conformation, MANTIS was proposed to maintain the endothelial angiogenic potential. The implications of these findings are discussed in an accompanying editorial by Dr. Zampetaki and Mayr from Kings College London.                                                 That brings us to the end of our summaries. Now for our feature discussion.                                                 Today, we are going to be discussing the association between fetal congenital heart defects and maternal risk of hypertensive disorders of pregnancy. To discuss this, I have the first and corresponding author of our feature paper, Dr. Heather Boyd, from Statens Serum Institut in Copenhagen, and our familiar Dr. Sharon Reimold, content editor for special populations from UT Southwestern. Welcome, Heather and Sharon. Dr. Heather Boyd:            Thank you. Dr. Sharon Reimold:        Thank you. Dr. Carolyn Lam:               Heather, it's a topic that I can't say I'm very familiar with, association between fetal congenital heart defects and maternal risk of hypertensive disorders of pregnancy. Could you start by sharing why would we think there would be a link? What was the hypothesis you were testing? Dr. Heather Boyd:            A couple years ago, there was a paper published in the European Heart Journal that reported evidence of angiogenic imbalance in women with fetuses with major congenital heart defects, so women who were pregnant with babies that had heart defects, and then in fetuses that were terminated because of this kind of defect. My research group focuses a lot of attention on preeclampsia. In the last decade or so, angiogenic imbalance in preeclampsia has been a really hot topic. Women with preeclampsia, particularly women with early-onset preeclampsia, have big angiogenic imbalances. When we saw the European Heart Journal paper, we immediately thought, "What's the connection between preeclampsia and heart defects in the offspring?" Dr. Carolyn Lam:               Oh! Dr. Heather Boyd:            Exactly. That was our entry point to it, was the term "angiogenic imbalance" in that paper sort of was a flag for us. It wasn't a completely new idea, but we in Denmark have one big advantage when considering research questions that involve either rare exposures and/or rare outcomes, and that's our National Health Registry. We have the ability to assemble these huge cohorts and study conditions like heart defects with good power, so we decided just to go for it. Dr. Carolyn Lam:               That makes a lot of sense now. Please, tell us what you did and what you found. Dr. Heather Boyd:            The first thing we did was look at the association between carrying a baby with a heart defect and then whether the mom had preeclampsia later in the same pregnancy. We had information on almost 2 million pregnancies for this part of the study. We found that women carrying a baby with a heart defect were seven times as likely as women with structurally normal babies to develop early preterm preeclampsia. We defined that as preeclampsia where the baby has to be delivered before 34 weeks, so the really severe form of preeclampsia. Then, women carrying a baby with a heart defect were almost three times as likely to develop late preterm preeclampsia as well. That's where they managed to carry it until 34 weeks but it has to be delivered some time before 37 weeks.                                                 These findings were similar to those of other studies, but we were able to go a step further and look at individual heart defect subtypes. What we found there waws that these strong associations were similar across defect categories. Then we decided to see if we could shed any light on the origin of the problem, whether it was coming from the mom's side or the baby's side. To do this, we looked at women with at least two pregnancies in our study period to see whether preeclampsia in one pregnancy had any bearing on the chance of having a baby with a heart defect in another pregnancy or vice versa.                                                 This part of the study included 700,000 women. We found very similar findings. We found that women with early preterm preeclampsia in one pregnancy had eight times the risk of having a baby with a heart defect in a subsequent pregnancy. Late-term preeclampsia in one pregnancy was associated with almost three times the risk of offspring heart defects in later pregnancies. Then, we found that it worked the other way around too. Women who had a baby with a heart defect were twice as likely to have preterm preeclampsia in subsequent pregnancies.                                                 Those results were really, really exciting, because whatever mechanisms underlie the associations between preterm preeclampsia in moms and heart defects in the babies, they operate across pregnancies. Therefore, that pointed towards something maternal in origin. Dr. Carolyn Lam:               That is so fascinating. Sharon, please, share some of the thoughts, your own as well as those of the editors when we saw this paper. Dr. Sharon Reimold:        I think that there's a growing data about the links between hypertensive disorders of pregnancy and preeclampsia with subsequent abnormal maternal outcome. But this paper, I think, has implications for how we look at moms who are going to have offspring with congenital heart defects as well as those with preeclampsia. For instance, I would look at a patient now that has preeclampsia, especially in more than one pregnancy, to identify that they may be at risk to have offspring with congenital defects in the future if they have additional children. But the mom is also at risk based on other data for developing other cardiovascular risk factors and disease as she gets older. It was really the link going back and forth with the hypertensive disorders and the congenital defects that we found the most interesting. Dr. Carolyn Lam:               That struck me too, especially when you can look at multiple pregnancies and outcomes. That's amazing. You know what, Heather, could you share a little bit about what it's like working with these huge Danish databases? I think there must be a lot more than meets the eye. Dr. Heather Boyd:            It's an interesting question, because I'm a Canadian and I was trained in the US. I did my PhD in epidemiology at Emery, and then I moved to Copenhagen. When I first got here, I was absolutely floored at the possibility of doing studies with millions of women in them. It opens some amazing possibilities, like I said earlier, for certain outcomes and certain exposures. You just need to have a question where the information you want is registered. Dr. Carolyn Lam:               Yeah. But I think what I also want to put across is, having worked with big databases, and certainly not as big as that one, it's actually a lot of work. People might think, "Oh, it's just all sitting there." But, for example, how long did it take you to come to these observations and conclusions? Dr. Heather Boyd:            I have a fabulous statistician. I think she's the second author there, Saima Basit. She spends a lot of her time pulling together data from different registers. But yes, you're right. The data don't always just mesh nicely. The statisticians we have working with us are real pros at this sort of data slinging. Dr. Carolyn Lam:               Could I just pose one last question to both of you. What do you think are the remaining gaps? Dr. Sharon Reimold:        I think that this is a clinical link. Then, going back to figure more about what's going on biologically to set up this difference? Because right now there's really no intervention that's going to make a difference, it's just a risk going forward. This is sort of like medicine done backwards, that there's this association and now we need to figure out exactly why. Dr. Heather Boyd:            I can piggyback on what Sharon said a little bit, because I think one of the things we need to remember is that not all women with preeclampsia have babies with heart defects. Not by a long shot. What we need to do now is to figure out what distinguishes the women who do get this double whammy from the vast majority who don't.                                                 One of the things that Denmark does really nicely is that there are large bio banks. One of the things we want to do is go back to bank first trimester maternal blood samples and see if we can identify biomarkers that are unique to the women with both preterm preeclampsia and babies with heart defects. That's one of the things we're thinking about to address this gap. Because, as Sharon says, we've got to figure out what the mechanism is.                                                 The other thing we want to do is to see whether the association between preeclampsia and heart defects extends, for example, to other things, to cardiac functional deficits, for example, because it's probably not just severe structural defects. If there's an association, it's probably on a continuum. Are babies born to preeclamptic moms, do their cardiac outputs differ? Do their electrical parameters differ? Do they just have different hearts?                                                 We're really lucky because right now the Copenhagen Baby Heart Study is offering to scan the hearts of all infants born at one of the three major university hospitals in the Copenhagen area. We're about to have echocardiography data on 30,000 newborn hearts to help us look at this. I'm really excited about that possibility. Dr. Carolyn Lam:               I've learnt so much from this conversation. I'm sure the listeners will agree with me. Thank you both very, very much.

ABCA3 ist als Phospholipidtransporter der ABC-Transporterfamilie wesentlich an der Synthese von Lungensurfactant beteiligt. Diese wichtige Rolle von ABCA3 in der Lunge ist mittlerweile bekannt und wird weiterhin näher erforscht. Des Weiteren wird ABCA3 auch in anderen Geweben exprimiert, darunter Leber, Niere, Gehirn [Stahlman et al. 2007]. ABCA3 wurde daneben auch in humanem Milchdrüsengewebe entdeckt und stellt in Mammakarzinomen einen Marker für eine gute Prognose dar [Schimanski 2010]. In der murinen Milchdrüse ist die Abca3-Expression molekularbiologisch auf Ebene der mRNA nachgewiesen worden [Hammel 2007]. Des Weiteren ist ABCA3 in immunhistochemischen Färbungen von humaner und muriner Milch darstellbar. Dort ist es auf der Außenseite der Milchfetttröpfchen-Membran lokalisiert [bislang nicht veröffentlichte Daten der eigenen Arbeitsgruppe]. Milchfetttröpfchen werden in Milchdrüsen-Epithelzellen gebildet, indem Lipidtransporter (unter anderem ABC-Transporter der ABCA-Subklasse wie ABCA1 oder ABCA7) Lipide importieren. In der Milchdrüse ist über den Mechanismus, mit dem ABCA3 an der Milchsekretion beteiligt sein könnte, nicht viel bekannt. Es kann aber analog zu der Rolle in der Lunge davon ausgegangen werden, dass ABCA3 auch in der Milchdrüse Phospholipide transportiert. Phospholipide verfügen über wichtige Eigenschaften in der Milch als Emulgatoren und als protektive Faktoren für das Neugeborene. Es ist nachgewiesen, dass ein Einschnitt in der Phospholipid-Sekretion und eine veränderte Zusammensetzung der Phospholipide in der Milch zu nachteiligen Auswirkungen auf das Neugeborene führen [Isaacs 2005]. Es stellt sich die Frage, ob und inwieweit ABCA3 in der Brustdrüse an der Milchbildung und – sekretion beteiligt ist. Dazu wurde ein Mausmodell mit spezifischer Deletion des ABCA3 Gens in der Milchdrüse generiert. Eine Mauslinie mit Cre-Expression unter dem während der Laktation spezifisch im Mammagewebe aktiven Lactoglobulin-Promotor wurde mit einer Mauslinie gekreuzt, welche im ABCA3-Gen LoxP Stellen enthielt. Es kommt zur Cre-getriggerten Deletion von ABCA3 im Mammagewebe. Das andere Allel wurde durch Einkreuzen einer klassischen Knockout-Linie gänzlich inaktiviert. Die Genotypisierung erfolgte mittels PCR. Zur quantitativen Bestimmung der Expression von Abca3 im Mammagewebe wurde die Methode der quantitativen RT-PCR angewendet. Der Melkvorgang erfolgte mittels einer eigens konzipierten Melkvorrichtung. Diese beinhaltet eine Flüssigkeitsfalle, wodurch ein besseres Handling beim Melkvorgang erreicht wurde. So konnte unter anderem eine höhere Milchmenge pro Maus gewonnen werden mit weniger technisch bedingten Schwankungen. Die Milchproben an Tag 5, 10 und 15 der Laktation wurden massenspektrometrisch auf den Gehalt der einzelnen Phospholipide analysiert. Es ergab sich eine Reduktion des Abca3-Gens im Milchdrüsengewebe der gefloxten Mäuse von 95% im Vergleich zum Wildtyp. Die Analyse der Phospholipide zeigte eine Verminderung von Phosphatidylethanolamin, Phosphatidylserin und Phosphatidylcholin, während innerhalb der Phosphatidylcholin-Spezies kurzkettige Moleküle (PC30:0, PC32:0) signifikant vermindert waren. Die Milchmenge der ABCA3 defizienten Linie an Tag 15 der Laktation war signifikant vermindert im Vergleich zur Wildtyp Gruppe (p = 0,005). Der Nachweis von ABCA3 in Milchfetttröpfchen und die Verminderung der Milchmenge bei dessen Fehlen weist auf eine Rolle dieses Transporters für die Milchsekretion hin. Da Phospholipide nur einen geringen Anteil der Milchfette darstellen, aber vor allem in der Membran der Milchfetttröpfchen enthalten sind, könnte ABCA3 möglicherweise durch Bereitstellung von Phospholipiden für die Bildung der Membran von Milchfetttröpfchen an der Milchbildung beteiligt sein. Am Höhepunkt der Laktation kann das Phospholipid-Defizit in den Milchfetttröpfchen nicht meht kompensiert werden und die Milchmenge nimmt durch die verminderte Bildung von Milchfetttröpfchen signifikant ab. Dabei ändert sich die Gesamtzusammensetzung der Milch nur unbedeutend, da jeweils das gesamte Organell „Milchfetttröpfchen“ mitsamt Inhalt fehlt. Dies muss im Rahmen dieser Studie allerdings hypothetisch bleiben.

Phytosterole, die Sterole von Pflanzen, unterscheiden sich von Cholesterol nur durch eine Alkylsubstitution an C24 und eventuell eine Doppelbindung in der Sterol-seitenkette. Trotz vergleichbarer Zufuhr von 200 - 600 mg/d und der großen strukturellen Ähnlichkeit werden von Phytosterolen nur 0,6 - 7%, von Cholesterol jedoch etwa 60% systemisch absorbiert. Phytosterole senken in hohen Dosen (> 2g/d) die Cholesterolabsorption um etwa 30% und die LDL-Cholesterol-Spiegel um bis zu 15% und werden deshalb zunehmend in „funktionellen Lebensmitteln“ vermarktet. Als Mechanismus wurde lange eine rein luminale, physico-chemische Interferenz mit der mizellären Emulgierung von Cholesterol postuliert. Spätestens seit der molekularen Aufklärung der Phytosterolspeicherkrankheit Sitosterolämie als dysfunktionelle Mutationen der apikalen Steroltansportproteine ABCG5/G8 stand fest, dass Phytosterole sehr wohl in Enterozyten aufgenommen, aber durch ABCG5/G8 effektiv ins Darmlumen resezerniert werden. Da ABCG5/8 auch Cholesterol transportieren kann, blieb die intestinale Sterol-Selektivität und -Interaktion weiterhin unklar. In allen Zellen wird ein Cholesterolüberschuss über bestimmte Oxycholesterole signalisiert, die den nukleären Transkriptionsfaktor LXRα aktivieren und so u.a. die Expression des zellulären Cholesterolexporters ABCA1 stimulieren. Dies ließ eine Rolle von Oxycholesterolen oder analogen regulatorischen Oxyphytosterolen bei der enterozytären Sterol-Interaktion und -Selektivität vermuten. Deshalb wurde am humanen Enterozytenmodell Caco-2 das Handling und die Metabolisierung von Phytosterolen und Cholesterol allein und in Kombination verglichen. Sitosterol wurde eindeutig, wenn auch langsamer als Cholesterol, von Enteroyzten akkumuliert, reduzierte aber bei Kombination die Cholesterolabsorption. Dies war teilweise durch Hemmung der apikalen Aufnahme, aber überwiegend der basolateralen Cholesterolsekretion bedingt, unabhängig von der Mizellenbildung, und nicht durch Sättigung einer limitierten Transportkapazität erklärbar. Im humanen Enterozyten und vergleichend in Hepatozyten und Makrophagen wurde deshalb nach potentiell regulatorischen Oxysterolen gesucht. Aus allen Sterolen wurden in diesen Zellen nur die 27-Hydroxy- und 27-Carboxy-Metaboliten gebildet, andere LXR-agonistische Oxysterole waren nicht nachweisbar. Der Umsatz war für Sitosterol und Campesterol abhängig von der Länge der C24-Alkylsubstitution deutlich geringer als für Cholesterol. In Ko-Inkubationen hemmten Phytosterole konzentrations- und C24-alkyl-abhängig die Bildung von 27-OH-Cholesterol. Diese kompetitive Hemmung und die geringe 27-Hydroxylierung von Phytosterolen selbst wurde auch in Präparationen des katalysierenden Enzyms, der an der inneren Mitochondrienwand lokalisierten Cytochrom P450 Oxidase CYP27, direkt gezeigt. Die Bioaktivität der 27-OH-Sterole als LXRα-Agonisten wurde direkt im LXRE-Transaktivierungs-Assay nachgewiesen und die stimulierte Expression von CYP27 und des in Enterozyten nur basolateral lokalisierten Cholesteroltransporters ABCA1 gezeigt. Dementsprechend steigerte 27-OH-Cholesterol auch selektiv die basolaterale, systemische Cholesterolsekretion, während der apikal exprimierte Sterolexporter ABCG8 und die apikale Sterolresekretion unverändert blieben. Umgekehrt hob in Ko- Inkubationen mit Phytosterolen die exogene Substitution eines synthetischen LXRα- Agonisten als Ersatz für das reduzierte endogene 27-OH-Cholesterol die Hemmung der Cholesterolabsorption durch Phytosterole komplett auf und überfuhr die Sterolselektivität. Auch in Tracer-Experimenten mit nanomolaren Phytosterol- und Cholesterolkonzentrationen, die die Aktivierung von LXRα nicht beeinflussen können, konnte keine direkte Sterolselektivität der ABCG5/8 und ABCA1-Transporter nachgewiesen werden. Neben physico-chemischen mizellären Effekten und der allenfalls limitierten direkten Cholesterolpräferenz der Steroltransporter NPC1L1, ABCG5/G8 und ABCA1 wurde für ACAT2, die apikal einströmendes Cholesterol zu >35% verestert und in die ApoBabhängige basolaterale Chylomikronensekretion einschleust, bereits eine relative Sterolselektivität beschrieben. Bei den eigenen Untersuchungen wurde ein neuer Mechanismus auf der regulatorischen Ebene der LXRα-Aktivierung im Enterozyten gefunden: Im Zentrum steht die kompetitive Hemmung des „Cholesterol-Sensors“ CYP27 durch Phytosterole und die nur geringe 27-Hydroxylierung der Phytosterole selbst. Dadurch wird bei gleichzeitigem Einstrom von Phytosterolen und Cholesterol in Enterozyten die Bildung des dominierenden LXRα-Agonisten 27-OH-Cholesterol verhindert. Normaler-weise vermittelt dies über LXR-Aktivierung und Induktion von CYP27, LXRα und ABCA1 eine selbstinduzierbare Komponente der Cholesterolabsorption auf dem ApoA-abhängigen Weg. Der lokale LXRα-Antagonismus von Phytosterolen blockt diese Selbstbahnung, lenkt Cholesterol vermehrt um zur luminalen Resekretion durch die konstitutiv apikal exprimierten ABCG5/8 und trägt auch zur Sterolselektivität bei. In peripheren Makrophagen könnten Phytosterole über Hemmung von CYP27, LXRα und ABCA1 durch Sterol-„Trapping“ die frühe Atherosklerose trotz eher niedriger Cholesterolspiegel bei Sitosterolämie erklären. Ob auch bei ABCG5/G8-Gesunden die unter pharmakologischen Phytosteroldosen erhöhten Plasmaspiegel langfristig zur Phytosterol- und paradoxen Cholesterol-Akkumulation in peripheren Zellen führen können, ist gegenwärtig unklar.

Thu, 12 Feb 2009 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/9753/ https://edoc.ub.uni-muenchen.de/9753/1/Hrusovar_Natalie.pdf Hrusovar, Natalie

Ein bedeutender Mechanismus zur Prävention und Regression von atherosklerotischen Läsionen ist die Abräumung von akkumulierten extrazellulären Lipiden in der Gefäßwand und deren Einschleusung in den reversen Cholesterintransport durch Makrophagen. Wichtigste molekulare Effektoren sind dabei Scavenger Rezeptoren wie CD36 und Cholesterin-Exporter wie ABCA1 und ABCG1. Deren Expression wird durch spezifische oxidierte Sterole, die die nukleären Transkriptionsfaktoren wie PPARgamma und LXRalpha aktivieren, induziert. Da hochdosierte lipidlösliche Antioxidantien diese regulatorischen Oxylipide beeinflussen könnten, war es Ziel dieser Arbeit am Makrophagen-Modell die Wirkung von hochdosiertem alpha-Tocopherol auf Signalwege und Schlüsselrezeptoren der Cholesterin-Homöostase zu untersuchen. Der Einfluss von alpha-Tocopherol und teilweise auch von gamma-Tocopherol wurde auf regulatorischer, transkriptioneller, translationeller und funktioneller Ebene mittels Realtime RT-PCR, Reportergen-Assays, FACS, Immunoblot und Lipidaufnahme- und Lipidefflux-Assays analysiert. Der LDL-R wurde durch hochdosiertes alpha-Tocopherol nicht beeinflusst, während die Expression des Scavenger Rezeptors CD36, konzentrationsabhängig sowohl auf mRNA-Ebene als auch auf Protein-Ebene durch alpha-Tocopherol beeinträchtigt wurde. Auf funktioneller Ebene verringerte alpha-Tocopherol die Aufnahme von [H³]-Cholesterin markiertem oxLDL durch Makrophagen. Der Effekt konnte ebenso mikroskopisch dargestellt werden. Die verminderte Expression von CD36 durch alpha-Tocopherol konnte zumindest teilweise durch eine dosisabhängige Verminderung der mRNA-Transkription von PPARγ und eine verminderte Aktivierung von PPARgamma im PPRE-Luziferase-Assay auch durch exogene Stimuli erklärt werden. gamma-Tocopherol hatte keinen vergleichbaren Effekt auf die CD36- und PPARgamma-spezifische mRNA, weswegen bereits auch ein direkter transkriptioneller Effekt von alpha-Tocopherol postuliert wurde. Die vermehrte zelluläre Aufnahme von oxidiertem LDL über Scavenger Rezeptoren wie CD36 induziert normalerweise auch eine vermehrte Einschleusung von Cholesterin in den reversen Cholesterintransport durch ABC-Exporter wie ABCA1 und ABCG1, wodurch die Schaumzellbildung zumindest verzögern werden kann. Diese Induktion der Cholesterin-Exporter wird durch oxidierte Sterole vermittelt, die LXRalpha aktivieren. Deshalb wurde ebenfalls eine mögliche Interferenz von hochdosiertem alpha-Tocopherol mit dem zellulären Cholesterin-Export untersucht. In der Tat wurde der Cholesterin-Efflux von Makrophagen auf delipidiertes HDL durch alpha-Tocopherol beeinträchtigt, wodurch der zelluläre Cholesterin-Bestand anstieg. Dieser Effekt zeigte auch mikroskopisch vermehrte Lipidgranula. Die Aktivierung des LXR-Response Elements im Luziferase-Assay durch exogene Stimuli wie 22-OHC oder oxidiertes LDL wurde durch alpha-Tocopherol ebenfalls negativ beeinflusst. Dadurch könnte die Reduktion der Expression von ABCA1 und ABCG1 auf mRNA-Ebene und von ABCA1 auf Proteinebene zumindest teilweise erklärt werden. Mit gamma-Tocopherol konnte nur eine geringe Reduktion auf mRNA Ebene, sowohl für ABCA1 als auch LXRalpha festgestellt werden. Bei der verminderten Expression von ABCA1 und ABCG1 durch hochdosiertes alpha-Tocopherol handelt es sich also wahrscheinlich um einen spezifischen, teilweise durch LXRalpha vermittelten Prozess. Es scheinen aber weitere Signalwege beteiligt zu sein: Unerwarteterweise wurde die Transkription und die Aktivierung von LXRalpha auch durch delipidiertes HDL stimuliert, was durch hochdosiertes alpha-Tocopherol ebenfalls dosisabhängig reduziert werden konnte. Nichtsdestotrotz war ABCA1 in Makrophagen nach Cholesterinverarmung durch delipidiertes HDL supprimiert. Die gefundenen Effekte von alpha-Tocopherol auf Schlüsselrezeptoren der Cholesterin-Homöostase in Makrophagen können zur Erklärung der enttäuschenden Resultate der Preventionsstudien mit hochdosiertem alpha-Tocopherol beitragen: Durch Hemmung des Scavenger Rezeptors CD36 reduziert alpha-Tocopherol zwar einerseits den ersten Schritt zur Schaumzellbildung um den Preis einer verzögerten Abräumung extrazellulärer Lipiddepots, alpha-Tocopherol verlangsamt aber auch durch Hemmung von ABCA1 und ABCG1, den endgültigen Abtransport von Cholesterin aus der Gefäßwand durch den reversen Cholesterin-Transport.

Background: Curcumin induces apoptosis in many cancer cells and it reduces xenograft growth and the formation of lung metastases in nude mice. Moreover, the plant derived polyphenol has been reported to be able to overcome drug resistance to classical chemotherapy. These features render the drug a promising candidate for tumor therapy especially for cancers known for their high rates concerning therapy resistance like melanoma. Results: We show here that the melanoma cell line M14 is resistant to Curcumin induced apoptosis, which correlates with the absence of any effect on NF kappa B signaling. We show that CXCL1 a chemokine that is down regulated in breast cancer cells by Curcumin in an NF kappa B dependant manner is expressed at variable levels in human melanomas. Yet in M14 cells, CXCL1 expression did not change upon Curcumin treatment. Following the hypothesis that Curcumin is rapidly removed from the resistant cells, we analyzed expression of known multi drug resistance genes and cellular transporters in M14 melanoma cells and in the Curcumin sensitive breast cancer cell line MDA-MB-231. ATP-binding cassette transporter ABCA1, a gene involved in the cellular lipid removal pathway is over-expressed in resistant M14 melanoma as compared to the sensitive MDA-MB-231 breast cancer cells. Gene silencing of ABCA1 by siRNA sensitizes M14 cells to the apoptotic effect of Curcumin most likely as a result of reduced basal levels of active NF kappa B. Moreover, ABCA1 silencing alone also induces apoptosis and reduces p65 expression. Conclusion: Resistance to Curcumin thus follows classical pathways and ABCA1 expression should be considered as response marker.

In dieser Arbeit wurde die zelluläre Cholesterin-Homöostase in menschlichen Zellen von Patienten mit der seltenen Tangier-Erbkrankheit (TD) untersucht. Diese Patienten besitzen verschiedene Mutationen im ABCA1-Gen, welches eine zentrale Rolle im Cholesterin-Export spielt, und haben aufgrund des niedrigen bzw. fehlenden HDL im Plasma ein erhöhtes Risiko, kardiovaskuläre Erkrankungen auszuprägen. Es ist jedoch unklar, wieso es trotz der relativ einheitlichen HDL-Defizienz zu völlig unterschiedlichen Identitätsmustern der Arteriosklerose kommt. Ziel der vorgelegten Arbeit war es, Auswirkungen des Funktionsverlustes von ABCA1 auf die Regulation der zellulären Cholesterin-Homöostase zu untersuchen. Dazu wurden Telomerase-immortalisierte Fibroblasten zweier Tangier-Patienten mit verschiedenen ABCA1-Mutationen und unterschiedlich ausgeprägter klinischer Manifestation der Arteriosklerose (TD1 und TD2) mit Fibroblasten eines gesunden Spenders verglichen. Der Cholesterin-Gehalt in TD-Fibroblasten im Vergleich zu Kontrollzellen war 1,4 bzw. 1,5-fach erhöht. Diese zelluläre Cholesterin-Akkumulation führte zur verminderten Expression der an der Cholesterin-Synthese und -Aufnahme beteiligten Gene, HMG-CoA-Reduktase und des LDL-Rezeptors. Daher war die endogene Cholesterin-Biosynthese im Vergleich zur Kontrolle um 27 % (TD1) bzw. 58 % (TD2) reduziert. Die Anreicherung von Cholesterin in den TD-Fibroblasten ging mit der verminderten Expression der Gene einher, die an der Regulation der Cholesterin-Homöostase bzw. dem Cholesterin-Export (ABCA1, ABCG1 und SREBP1c) beteiligt sind. Diese Störung war auch an einem entsprechend gegenläufigen Gehalt an Oxysterolen erkennbar (ein geringer Cholesterin-Export bewirkte einen höheren Oxysterol-Spiegel). Diese Untersuchungsergebnisse deckten jedoch gleichzeitig die Tatsache auf, dass keine strikte Korrelation zwischen einer verminderten Expression des defekten Cholesterin-Export-Gens (ABCA1) und der intrazellulären Cholesterin-Akkumulation existiert. Da die Expression von ABCA1, ABCG1 und SREBP1c laut den Ergebnissen anderer Arbeitsgruppen durch den Transkriptionsfaktor LXR reguliert wird, muß es –auf der Basis der hier vorgelegten Ergebnisse– LXR-unabhängige, aber wichtige Regulationsmechanismen des Cholesterin-Stoffwechsels geben. Die komplexe und unerwartete Regulation der LXR-Zielgene könnte erklären, warum Patient TD2 eine schwere Arteriosklerose aufweist, während Patient TD1 keinen klinischen Befund hat. Weitere Studien sind jedoch notwendig, um diese unbekannten und von Oxysterolen unabhängigen Regulationsmechanismen aufzuklären.

In der vorliegenden Arbeit wurden Effekte von IL-10 auf monocytäre Lipidrezeptoren und -Transporter untersucht, die zusätzlich zu den anti-inflammatorischen Wirkungen von IL-10 für dessen anti-atherosklerotische Wirkung von Bedeutung sein könnten. Es konnte gezeigt werden, dass IL-10 die Expression des quantitativ wichtigsten Scavenger Rezeptors, CD36, in Monocyten/Makrophagen sowohl auf RNA- als auch auf Proteinebene hemmt. Diese Hemmung ging einher mit einer verringerten zellulären Aufnahme von oxidiertem LDL. Durch PPARgamma-FACS und -Western Blot in Cytosol-Kern-Fraktionen sowie Koinkubationen mit IL-10 und den PPARgamma-Agonisten 15d-PGJ2 (15-deoxy-Δ12,14-Prostaglandin J2) und Indomethacin konnte gezeigt werden, dass die IL-10 Hemmung von CD36 über die Hemmung des Transkriptionsfaktors PPARgamma vermittelt wird. Im Gegensatz dazu stimulierte IL-10 den Cholesterinexporter ABCA1 und dessen wichtigsten Transkriptionsfaktor LXRalpha auf RNA- und Proteinebene. Die Induktion von ABCA1 hatte funktionell einen verstärkten zellulären Cholesterinefflux zur Folge, welcher die Monocyten und Makrophagen vor Lipidakkumulationen schützte und das vermehrte Einschleusen von Cholesterin in den reversen Cholesterintransport ermöglicht. IL-10 stimulierte ABCA1 dabei trotz der Hemmung des Transkriptionsfaktor PPARgamma. Die IL-10 Stimulation von ABCA1 war durch Piceatannol, einem Inhibitor der proximalen, STAT3-vermittelten Signalübertragung des IL-10 Rezeptors, hemmbar. Mittels LXRalpha "knock down" Zellen konnte weiter gezeigt werden, dass für die IL-10-stimulierte ABCA1 Expression ein intakter LXRalpha-Signaltransduktionsweg nötig ist. Koinkubationen mit Fenofibrat, 9-cis RA (9-cis retinoic acid) und 22-OHC (22-Hydroxycholesterol) ergaben, dass IL-10 auch die Induktion von ABCA1 durch die beiden Transkriptionsfaktoren, PPARalpha und LXRalpha, verstärkte. Durch Hemmung der Proteinkinase A (PKA) und Messung der zellulären cAMP-Spiegel konnte des Weiteren ein distaler cross-talk des IL-10-Signalweges mit dem cAMP/PKA-Weg für die ABCA1 Stimulation nachgewiesen werden. Dagegen hatte IL-10 keinen anhaltenden Einfluss auf die Transkription von SR-BI. Der Scavenger Rezeptor BI (SR-BI) kann je nach Konzentrationsgradient sowohl die zelluläre Cholesterinaufnahme wie die Cholesterinabgabe vermitteln. Des Weiteren reduzierte IL-10 die LPS-induzierte ICAM-1 Expression, was auf die Attenuierung der NFkappaB- und PPARgamma-vermittelten ICAM-1 Expression zurückgeführt werden konnte. Insgesamt befördert IL-10 somit den ABCA1-initiierten peripheren Cholesterinabtransport aus Monocyten/Makrophagen durch HDL. Die gezeigten Effekte von IL-10 erklären tierexperimentelle Befunde eines deutlich reduzierten Lipidgehalts in atherosklerotischen Plaques unter IL-10 Behandlung. Sie erklären auch die niedrigen HDL-Spiegel bei IL-10 knock out (IL-10-/-) Mäusen, die durch exogene IL-10 Substitution korrigiert werden können. Demnach sollte IL-10 nicht nur durch seine anti-inflammatorischen Mechanismen, sondern auch durch seine Effekte auf das Cholesterinhandling von Monocyten/Makrophagen in der Gefäßwand die Entstehung und Progression atherosklerotischer Plaques reduzieren.