November 2021 Discover CircRes - a podcast by Cynthia St. Hilaire, PhD & Milka Koupenova, PhD
from 2021-11-18T19:00
This month on Episode 30 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the October 29 and November 12 issues of Circulation Research. This episode also features a conversation with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.
Article highlights:
Subramani, et al. CMA of eNOS in Ischemia-Reperfusion
Liu, et al. Macrophage MST1 Regulates Cardiac Repair
Van Beusecum, et al. GAS6/Axl Signaling in Hypertension
Pati, et al. Exosomes Promote Efferocytosis and Cardiac Repair
Cindy St. Hilaire: Hi and welcome to Discover CircRes, the podcast of the American Heart Association's Journal Circulation Research. I'm your host, Dr Cindy St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh and today I'll be highlighting articles presented in our October 29th and November 12th issues of Circulation Research. I also will speak with Dr Elisa Klein from the University of Maryland about her study, Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.
Cindy St. Hilaire: The first article I want to share is titled, Chaperone-Mediated Autophagy of eNOS in Myocardial Ischemia Reperfusion Injury. The first author is Jaganathan Subramani and the corresponding author is Kumuda Das from Texas Tech University Health Sciences Center. Reestablishing blood flow to ischemic heart muscle after myocardial infarction is critical for restoring muscle function but the return of flow itself can cause damage, a so-called reperfusion injury. The generation of reactive oxygen species or ROS and loss of nitric oxide or NO both contribute to reperfusion injury.
Reperfusion injury is exacerbated when the NO producing enzyme, endothelial nitric oxide synthase or eNOS, produces damaging super oxide anions instead of NO. This switch in eNOS function is caused by glutathionylation of the enzyme, termed SG-eNOS. But how long this modification lasts and how it is fixed is unclear. This group used an in vitro model of ischemia reperfusion where human endothelial cells are exposed to several hours of hypoxia followed by reoxygenation. In this model, they found the level of SG-eNOS steadily increases for 16 hours and then sharply decreases. By blocking several different cellular degradation pathways, they discovered that this decrease in S-G eNOS was due to chaperone mediated autophagy with the chaperone protein, HSC70, being responsible for SG-eNOS destruction. Importantly, this team went on to show that pharmacological D-glutathionylation of eNOS in mice promoted NO production and reduced reperfusion injury, suggesting this approach may be of clinical benefit after myocardial infarction.
Cindy St. Hilaire: The second article I want to share is titled Macrophage MST1/2 Disruption Impairs Post-Infarction Cardiac Repair via LTB4. The first author is Mingming Liu and the corresponding author is Ding Ai and they're from Tianjin Medical University. Myocardial infarction injures the heart muscle. These cells are unable to regenerate and instead a non-contractile scar forms and that fibrotic scar can lead to heart failure.
Cardiomyocytes specific inhibition of the kinase MST1 can prevent infarction induced death of the cells and preserve the heart function, suggesting that it may have clinical utility. However, MST1 also has anti-inflammatory properties in macrophages. So inhibition of MST1 in macrophages may delay inflammation resolution after MI and impair proper healing. Thus, targeting this enzyme for therapy is not a straightforward process. This study examined mice lacking MST1 in macrophages and found that after myocardial infarction, the inflammatory mediator leukotriene B4 was upregulated in macrophages and the animal's heart function was reduced compared to that of wild type controls. Blocking the action of leukotriene B4 in mice reduced infarction injuries in the hearts of MST1-lacking animals and enhanced repair in the injured hearts of wild type animals given an MST1 inhibitor. The results suggest that if MST1 inhibition is used as a future post infarction regenerative therapy, then leukotriene B4 blockade may prevent its inflammatory side effects.
Cindy St. Hilaire: The next article I want to share is titled Growth Arrest Specific-6 and Axl Coordinate Inflammation and Hypertension. The first author is Justin Beusecum and the corresponding author is David Harrison and they're from Vanderbilt University. Inflammation contributes to hypertension pathology but the links of this relationship are unclear. It's thought one trigger of inflammation may be the hypertension-induced mechanical stretch of vascular endothelial cells. Mechanical stretch causes endothelial cells to release factors that convert circulating monocytes into inflammatory cells. And one such factor is the recently identified Axl and Siglec-6 positive dendritic cells, also called AS DCs.
AS DCs produce a large amount of inflammatory cytokines but little is known about the role of AS DCs or their cytokines in hypertension. This group found elevated levels of AS DCs in hypertensive people compared to normal tensive individuals. Mechanical stretch of human endothelial cells promoted the release of GAS6, which is an activator of the AS DC cell surface kinase, Axl. This stretch induced GAS6 release also promoted conversion of co-cultured monocytes to AS DCs. Inhibition of GAS6 or Axl in the co-cultured system prevented conversion of monocytes to AS DCs. This team went on to show that hypertensive humans and mice have elevated levels of plasma GAS6 and that blocking Axl activity in mice attenuated experimentally induced hypertension and the associated inflammation. This work highlights a new signaling pathway, driving hypertension associated inflammation and identifies possible targets to treat it.
Cindy St. Hilaire: The last article I want to share is titled Novel Mechanisms of Exosome-
Mediated Phagocytosis of Dead Cells in Injured Heart. The first author is Mallikarjun Patil and Sherin Saheera and the corresponding author is Prasanna Krishnamurthy from the University of Alabama, Birmingham. After myocardial infarction inflammation must quickly be attenuated to avoid excessive scarring and loss of muscle function. Macrophage mediated efferocytosis of dead cells is a critical part of this so-called inflammation resolution process. And resolution depends in part on the protein. MFGE8. MFGE8 helps macrophages engage with eat me signals on the dead cells and loss of macrophage MFGE8 delays inflammation resolution in mice. Because stem cell-derived exosomes promote cardiac repair after infarction and are anti-inflammatory and express MFGE8, this group hypothesized that perhaps part of a stem-cell derived exosomes proresolven activity may be due to boosting macrophage efferocytosis.
They showed that stem cell derived exosomes did indeed boost efferocytosis of apoptotic cardiomyocytes in vitro and in vivo. An in vitro experiments showed that if exosomes lacked MFGE8 then efferocytosis by macrophages was reduced. Furthermore, after myocardial infarction in mice, treatment with MFGE8 deficient exosomes did not reduce infarct size and did not improve heart function compared to control exosomes. These results suggest MFGE8 is important for the cardioprotective effects of stem cell-derived exosomes. And that this protein may be of interest for boosting efferocytosis after myocardial infarction and in other pathologies where inflammation is not readily resolved.
Cindy St. Hilaire So today, Dr Elisa Klein from the Department of Biomedical Engineering at the University of Maryland is with me to discuss her study Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity and this article is in our November 12th issue of Circulation Research. So Dr Klein, thank you so much for joining me today.
Elisa Klein: Thank you for having me.
Cindy St. Hilaire: Yeah. So broadly your study is investigating how blood flow patterns specifically, kind of, laminar and oscillatory flow, how those blood flow patterns impact protein modifications and activity. So before we, kind of, get to the details of the paper, I was wondering if you could just introduce for us the concept of blood flow patterns, how they change in the body naturally but then how they might influence or contribute to disease pathogenesis in the vessels?
Elisa Klein: Sure. So obviously we have blood flow through all of our vessels and since we are complex human beings, we have complex vascular beds that turn and that split or bifurcate. And so every place we get one of these bifurcations or a turn in a vessel, the blood flow can't quite make that turn or split perfectly. So you get a little area where the flow is a oscillatory or what we call disturbed. There's lots of different kinds of disturbed flow. And the reason why that's important is because you tend to develop atherosclerotic plaques at locations where the blood flow is disturbed. So in my lab, we look a lot at what it is about that disturbed flow that makes the endothelial cells there dysfunctional and that leads to the atherosclerotic plaque development.
Cindy St. Hilaire: That is so interesting. So I can picture how this is happening in a mouse at the bifurcation of different arteries but how are you able to model this in vitro? Can you describe the setup and then also how that setup can mirror the physiological parameters?
Elisa Klein: Sure. So we have a couple of different systems we can use to model this and they all have their advantages and disadvantages, right? So a few years ago we made a system that's a parallel plate flow chamber. So you basically have your cells that you see that on a microscope slide and you use a gasket that's a given shape and that either drives the flow… Usually it drives the flow straight across the cells. So that's a nice laminar steady flow. And we see that the cells align and they produce nitric oxide in that type of flow which are measures that they are responding to the flow in vitro. So, a few years ago we made a device that actually makes the flow zigzag as it goes across the endothelial cells. And that creates these little pockets of disturbed flow and we did that in our parallel plate flow chamber.
And that parallel plate flow chamber is really good for visualizing the cells. So you can stick it on a microscope. You can see what's happening, we can label for specific markers but it's not good for doing the things that we did in this Circ Research paper, where we want it to measure metabolism, because you need a lot more cells to measure metabolism and we needed a better media to cell ratio, so less media and more cells. So for this one, we designed and built a cone-and-plate device. So what it is, it's a cone and you spin that cone on top of a dish of endothelial cells and that cone produces flow. So it's going around in a circle. And if we just make it go around in a circle, it'll produce a steady laminar flow but if we oscillated it, so basically we kind of turn it back and forth, it'll make this oscillating disturbed flow. And then we have our dish of cells.
We do this in a 60-millimeter dish and then we have a small amount of media in there and a lot of cells. And we can culture the cells in there for a while.
Cindy St. Hilaire: That is so neat. And so I'm assuming that then your cone system is very tuneable. You could either speed it up, slow it down or change that oscillatory rate with different, I guess, shifts of it?
Elisa Klein: Yeah, that's exactly right. So we can do all those things. It's programmable with a motor and so we can run whatever type of flow we want.
Cindy St. Hilaire: That's great. So before your study, what was known regarding this link between hemodynamics and endothelial cell dysfunction and also endothelial cell metabolism? Because I feel like that's a really interesting space that a lot of people look at, kind of, metabolism and EC dysfunction or they just look at shear stress and EC dysfunction and you're, kind of, combining the three. So what was kind of the knowledge gap that you were hoping to investigate?
Elisa Klein: Yeah, so we're really interested in macrovascular endothelial cell dysfunction. So this pro atherosclerotic phenotype that you can get in endothelial cells. And most of the work on endothelial cell metabolism had actually been done in the context of angiogenesis. So how much energy and how do cells get their energy to make new blood vessels? And that's more of a microvascular thing. So there was a study that came out before ours, actually, before we started this study, that was looking at how steady laminar flow could decrease endothelial cell glycolysis. And so that was after 72 hours of flow and they showed some gene expression changes at that time. Our study is shorter than that and we were still able to see a decrease in glycolysis in our cells in laminar flow. Before we started this study, no one had really looked at disturbed flow. So in the meantime, there are a few other papers that came out showing that the cells don't decrease glycolysis when they're in disturbed flow but not so much connecting them back to this function of making nitric oxide.
Cindy St. Hilaire: So we were kind of dancing to the topic of O linked N acetylglucosamine or how do you say it?
Elisa Klein: GlcNAC.
Cindy St. Hilaire: GlcNAC? O- GlcNAC. So, O- GlcNAC is a sugar drive modification and I think it's added to Syrian and three Indian residues and proteins.
Elisa Klein: Yup, that's right.
Cindy St. Hilaire: Okay, good. And that modification, it does help dictate a protein's function. And you were investigating the role of this moiety on endothelial nitric oxide synthase or eNOS and so what exactly does this GlcNAC do for eNOS’ function and under what conditions or disease states is this modification operative?
Elisa Klein: Yeah. So there's some really important studies from a little bit ago that showed that eNOS gets GlcNAcylated in animals with diabetes, right? So if you have constantly high sugar levels, you get this modification of eNOS. The thought was that eNOS gets GlcNAcylated at the same site where it gets phosphorylated. But a more recent study came out and said, well, maybe that's not the case but it definitely gets GlcNAcylated somewhere where it affects this phosphorylation site. So it may be near it and prevent the folding or prevent the phosphorylation site availability. So if the eNOS gets GlcNAcylated, the thought is that it can't get phosphorylated and therefore it can't make nitric oxide.
Cindy St. Hilaire: And so an interesting thing about this GlcNAcylation, which is probably the hardest thing I've ever said on this podcast, is that it's integrated with lots of different things. Obviously you need glycolysis and the substrates from the breakdown of sugars to make that substrate but also the enzymes that make that substrate are required. And so what's known about that balance in endothelial cells? Is there much known regarding the metabolic rate of the cells and this N-Glcynation?
Elisa Klein: Yeah. So endothelial cells are thought to be highly glycolytic in terms of how they use glucose but they definitely take up glutamine to fuel the tricarboxylic acid or TCA cycle. And another paper came out a few years ago showing that quiescent and endothelial cells metabolize a lot of fatty acids. So they're fueling their energy needs that way. So there wasn't a lot known about GlcNAcylation in endothelial cells.
A lot of this work has been done in cancer cells, which are also highly glycolytic but their metabolism actually seems like it's maybe more diverse than people have thought for a long time. So the weird thing about GlcNAcylation, which if you're used to working with phosphorylation there's a thousand different enzymes that can phosphorolate right. But with GlcNAcylation there's one enzyme that's known to put the GlcNAC on and one enzyme that's known to take it off. And so they're global, right? So in our studies, if we say, okay, we're going to knock down that enzyme, you're effecting every single protein in the cell that's GlcNAcylated. And obviously ourselves in particular, we're not a big fan of that. Especially once you put them in flow, they were, like, nope, we're not going to make it.
Cindy St. Hilaire: Well, and that's a perfect segue to my next question because your results show that this flow really did not alter the expression of these enzymes that either add or subtract to the moiety. And rather it was the Hexosamine Biosynthetic Pathway that was decreased itself. So can you maybe give us a quick primer on what that is exactly and how that pathway feeds into the glycosylation... I think you wrote in the paper of over 4,000 proteins? So how would that fit in and why eNOS then?
Elisa Klein: Yeah, so the Hexosamine Biosynthetic Pathway is one of these branch pathways that comes off glycolysis and there are these numbers sometimes there are these pathways out there and people say for the HBP in particular, 2% to 5% of the glucose that's going down through glycolysis gets shunted off into the HBP. We've done a lot of looking to try and figure out exactly where that 2% to 5%-
Cindy St. Hilaire: Yeah, what exact percentage?
Elisa Klein: Yeah, but some percentage of it comes down and we really thought there were going to be changes in these enzymes that do the GlcNacylation, we thought there might be changes in the localization of the proteins and it's possible that those things do occur. We just couldn't detect them in our cells. And in the end, what we showed was the main thing was that when you have cells and steady laminar flow, you just decreased glycolysis. And therefore, that 2% to 5% goes down. So you seem to make less of this UDP- GlcNAC, which is the substrate that gets put on to eNOS in this case. The really strange thing that we could not explain despite a lot of work and obviously we don't get to put all of our experiments that didn't work in the paper-
Cindy St. Hilaire: The blood, sweat and tears gets left out. So-
Elisa Klein: Exactly. So we tried really hard to figure out why it was eNOS specifically, right? Because in steady laminar flow, you see a lot of these like GlcNAcylated proteins and a lot of them didn't change but eNOS changed hugely, essentially this GlcNAcylation just went away for the cells and steady laminar flow. So we couldn't quite answer that. We're still working on that part of the question and looking at some of the other proteins that maybe get GlcNAcylated more in this case and trying to figure out what they are.
Cindy St. Hilaire: I thought one of the cool results in your paper was one of the last ones. It was the one in healthy mice. In that you looked at healthy mice, just normal C57 black 6 mice that were 10 weeks old. So they just, kind of, reached maturity but you looked at their kind of these bifurcations and you looked at the inner aortic arch where there is more disturbed flow and you saw, similar to your in vitro studies, that there was this higher level of O-GlcNAcylation compared to the outer arch in the descending order. So my question is, these are healthy mice that are relatively young, they're not even full adults yet. That takes a couple more months. And so what are your thoughts about the role of this O-GlcNAcylation specifically on eNOS in driving atherogenesis. Where do you think this is happening in the disease process? It appears if it's in these wild type mice, it's already happening early. So where do you think this is most operative in the disease pathogenesis?
Elisa Klein: I mean, I think it's very early, the effects of disturbed flow on endothelial cells. I can't imagine that there's a time when it's not having an effect on the cells. So I teach college students and I tell them all the time you think you're invincible now but these choices you're making today are going to affect your cardiovascular future in 50 years, which is very hard to accept. So I think it's very early in the process and I think it's only made worse by the things that we eat, in particular, that changed our blood sugar and our blood fatty acids and things like that. And our lab is looking into this more to try and see how when you change your blood metabolites then how does that then also affect this GlcNAcylation and the endothelial cell metabolism and then how does that affect endothelial cell function?
Cindy St. Hilaire: Yeah. And it's funny, it's really making me think of those, kinds of, extreme diets like keto diets and things like that where you're just like depleting sugar. And obviously there's lots of controversy in that field, but if you just think about the sugar aspect what is that doing to those EC cells? Why do you think endothelial cells have this response? Meaning why do you think it is that they've adapted to induce a metabolic shift in response to disturbed flow? Because, obviously it's not going to be perfect laminar flow everywhere. So what do you think it is that provides some sort of advantage in the shift?
Elisa Klein: That's a really good question. I haven't thought about the advantage that it might provide. There are a lot of things that are going on in this area of disturbed flow. So there is the shear stress, the differential shear stress that the cells are experiencing. There's also transport issues, right? So if you have this area of disturbed flow, you have blood and the contents of the blood, including the white blood cells and the red blood cells, everything else that's, kind of, sitting around in that area and not getting washed downstream as quickly. So it is possible that maintaining glycolysis provides energy for repair or for protecting the endothelial cell from some sort of inflammatory insult or something like that, that's happening in the area of disturbed flow. And I feel like I just read something recently, it was in a different genre but... if they stopped the increased glycolysis or stop the metabolic shifts, it actually was worse.
Right? So I also believe that we treat humans for a single metabolic change, right? So if you have diabetes, I'm going to give you this drug and if you have high triglycerides, I'm going to give you this drug. But it's possible that if you have this metabolic abnormality, your body shifts the rest of your metabolism to protect the cells because of that metabolic abnormality. And so part of what we do as engineers is try and build computational models or we can take into account some of this complexity. So that's a really interesting question and my guess is that there are some protective aspects of this maintenance of high glycolysis and disturbed flow.
Cindy St. Hilaire: Yeah, maybe it would be perfectly fine until we get athero and then it all goes awry. So in terms of... obviously it's early days and I know you're a bioengineer but in terms of translational potential, what do you think your findings suggest about future potential therapies or future targets for which we can use to develop therapies? Is modulating this O-GlcNAcylation itself, a viable option?
Elisa Klein: I don't think that modulating it is a super viable option, right? Because as I said, when we tried to change those enzymes ourselves did not enjoy going through flow or anything else. So it's very hard to change it overall. What I think is these things that are coming out about how metabolism may shift for endothelial cells when they're activated versus when they're quiescent, right? So when laminar flow or cells are quiescent, they decrease glycolysis, they increase fatty acid oxidation. Those things are important to take into consideration when you are treating a person who has a metabolic disorder. So that's the biggest translational piece that I think is, how do we give therapies that modify the metabolism of a cell holistically instead of trying to hit one pathway in particular.
We have done some studies where we tried to give endothelial cells something to inhibit a specific metabolic pathway and you see the cell shifts its entire metabolism to account for that. So we're starting to look at some of these other drugs like statins or metformin that do change endothelial cell metabolism, possibly even the SGLT2 inhibitors and trying to see not just how they change glycolysis but how they change metabolism as a whole and how that then affects endothelial cell function.
Cindy St. Hilaire: So what are you going to do next on this project?
Elisa Klein: So on this project, so we have some stuff in the works like I said on statins and how statins work together. And one of our big goals is to sort of build a comprehensive metabolic model of the endothelial cell. So this study really focused on glucose but there are other things that endothelial cells metabolize, glutamine, and fatty acids, and trying to look at some of those and then seeing how changes in the glycolytic pathway may affect some of those other pathways. We also have some really nice mass spec data part of which is in this paper but part of which is going to go into our next work, which is looking at how laminar flow impacts some of the other side branch pathways that are in metabolism and coming off of glycolysis as well as the TCA cycle, right? So we don't think of endothelial cells as being big mitochondrial energy producers but they do use their mitochondria. And so we think it's really interesting and part of our goal of building an endothelial cell model and then hopefully a model of the complexity of the whole vascular wall.
Cindy St. Hilaire: Wow. That would be amazing. Well, Dr Elisa Klein from the University of Maryland, thank you so much for joining me today. This is an amazing study and I'm looking forward to seeing hopefully more of your future work.
Elisa Klein: Thank you so much. It was a pleasure.
Cindy St. Hilaire: That's it for the highlights the from October 29th and November 12th 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 or #DiscoverCircRes. Thank you to our guest, Dr Elisa Klein. This podcast is produced by Asahara Ratnayaka, edited by Melissa Stoner and supported by the editorial team of Circulation Research. Some of the copy texts for highlighted articles is provided by Ruth Williams. I'm your host, Dr Cindy St. Hilaire, and this is Discover CircRes, your on-the-go source for the most exciting discoveries and basic cardiovascular research. This program is copyright of the American Heart Association, 2021. The opinions expressed by speakers on this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, visit AHAjournals.org.
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