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Discover CircRes


Sep 17, 2020

This month on Episode 16 of the Discover CircRes podcast, host Cindy St. Hilaire highlights four featured articles from the August 28 and September 11 issues of Circulation Research. This episode features an in-depth conversation with Drs Andrew Murphy and Michelle Flynn from The Baker Heart and Diabetes Institute at Monash University in Melbourne, Australia regarding their study Transient Intermittent Hyperglycemia Accelerates Atherosclerosis By Promoting Myelopoiesis.

 

Article highlights:
 

Fish, et al. KRAS Mutations Cause Arteriovenous Malformations

 

Ehling, et al. B55a in Vascular Biology

 

Barrett, et al.  Platelet Activity and Vascular Health in COVID-19

 

Furmanik, et al. Nox5 in VSMC Phenotype and Calcification

 
 

Cindy St. Hilaire:  Hi. Welcome to Discover CircRes, the podcast to the American Heart Association's journal, Circulation Research. I'm your host, Dr Cindy St. Hilaire, from the Vascular Medicine Institute at the University of Pittsburgh. And today I'm going to share with you four articles selected from our late August and early September issues of Circulation Research. I'm also going to speak with Drs Andrew Murphy and Michelle Flynn from The Baker Heart and Diabetes Institute at Monash University in Melbourne, Australia regarding their study Transient Intermittent Hyperglycemia Accelerates Atherosclerosis By Promoting Myelopoiesis. So first, the highlights.

The first article I'm sharing with you is titled Somatic Gain of KRAS Function in the Endothelium is Sufficient to Cause Vascular Malformations that Require MEK but not PI 3-Kinase Signaling. First authors are Jason Fish, Carlos Perfecto Flores-Suarez, and Emily Boudreau. And the corresponding authors are Jason Fish and Joshua Wythe, and they're from University of Toronto and Baylor College of Medicine.

Arterial venous malformations, or AVMs, are tangles of blood vessels in which the arteries are directly connected to the veins without going through the capillary bed. These are thought to be present from birth and when they occur in the brain, they can cause an array of symptoms such as headaches or seizures, but they are also the leading cause of hemorrhagic stroke in children and young adults. This is because the venous system is not muscularized to respond to the pressure forces that are exerted on arteries.

These pressure forces cause distension and eventual leakage at the site of AVMs. Vessel tissue recovered from patients undergoing AVM repair has been shown to contain sematic gain of function mutations in the protein RAS GTPase, which is encoded by the gene, KRAS. However, whether these gain of function mutations directly cause AVMs has not been established. This study now shows that endothelial cells with constitutive expression of gain of function KRAS mutants in mice and zebra fish causes vascular malformations and cranial hemorrhages. Inhibiting a MEK kinase, which is a downstream mediator of RAS signaling, prevented hemorrhages in the mutant KRAS carrying fish. In vitro studies also showed that overactive RAS GTPase protein caused excessive angiogenic behavior of endothelial cells. Together, this work confirms the link between gain of function KRAS mutations and brain AVMs, and suggests that MEK inhibition could be a potential strategy for nonsurgical treatment.

The second article I want to share with you is titled B55a/PP2A Limits Endothelial Cell Apoptosis During Vascular Remodeling: A Complimentary Approach To Kill Pathological Vessels. The first author is Manuel Ehling and the corresponding author is Massimiliano Mazzone. And the work was completed at Leuven Center for Cancer Biology in Belgium. Building a mammalian vascular system is a dynamic process that is dependent on both growth of new vessels, as well as the pruning of unwanted ones. But while much is known about molecular mechanisms underlying angiogenesis, comparatively little is understood about the mechanisms regulating vascular pruning. This study discovered that suppression of the protein phosphatase 2 subunit, B55A, is a key protein regulating the pruning process. They found that in mouse vascular development, B55a is widely expressed. However, in adult mice expression is restricted only to sites of active angiogenesis.

Deletion of B55a in mice caused death in mid to late stages of embryogenesis as a result of vascular problems that appeared to be due to excessive vessel pruning. Switching off B55a in adult mice when the vascular development is for the most part complete did not cause any apparent problems. They did find though, that inhibition of B55a significantly delayed growth of tumors that form from the injection of cancerous cells. Inhibition of B55a produced tumors with less dense vasculature and reduced metastatic potential. Thus, the author suggests that ramping up blood vessel pruning, be it inhibition of B55a, could be a novel strategy for limiting tumor growth.

The next article I want to share is titled Platelet and Vascular Biomarkers Associated With Thrombosis and Death in COVID-19. The first author is Tessa Barrett and the corresponding author is Jeffrey Berger, and they're from New York University. Our knowledge of the complications of COVID-19 is evolving every day. Laboratory testing done to date suggests that approximately 30% of hospitalized COVID-19 patients go on to develop thrombotic events. Platelets are central characters in both arterial and venous thrombosis, and it is known that virus platelet interactions can stimulate a pro-coagulant and inflammatory state during a viral infection. Further, recent studies have reported COVID-19 patients have hyperactive platelets and autopsies of COVID-19 patients exhibit micro and macro thrombi across vascular beds, even in patients without clinical thrombosis.

This group then hypothesized that biomarkers of platelet activation are associated with incident thrombosis or death in COVID-19 patients. To test this, they randomly selected 100 COVID-19 positive patients and analyzed banked samples collected on the day of the COVID-19 diagnosis to investigate in vivo platelet activity, as well as vascular health biomarkers. They show for the first time that biomarkers of platelet activation at the time of diagnosis are associated with thrombosis or death in patients hospitalized with COVID-19. Their findings suggest platelet activation mechanisms may contribute to adverse events and highlight the potential role of antiplatelet therapy in this disease.

The last article I want to share with you before we switch to our interview is titled Reactive Oxygen-Forming Nox5 Links Vascular Smooth Muscle Cell Phenotypic Switching and Extracellular Vesicle-Mediated Vascular Calcification. The first authors are Malgorzata Furmanik and Martijn Chatrou. And the corresponding author is Leon Schurgers from Maastricht University in The Netherlands. Vascular calcification is an active process regulated by several mechanisms, including vascular smooth muscle cell apoptosis, osteochondral genic transdifferentiation, extracellular vesicle release, and cellular senescence. In healthy adult arteries, smooth muscle cells maintain a contractile phenotype. However, various insults such as oxidative or mechanical stress, can induce smooth muscle cells to lose their contractility and this process of de-differentiation is termed phenotypic switching. And phenotypic switching is thought to precede the development of vascular disease. Patients with conditions such as chronic kidney disease have mineral imbalances in their circulation and also exhibit higher levels of vascular calcification.

However, the mechanisms behind these observations are not well defined. This group found that extracellular calcium can enter the smooth muscle cells via extracellular vesicles and this increased cytosolic calcium concentration. Increased calcium induces expression and activity of Nox5 in NADPH oxidase. Activation of Nox5 increased production of reactive oxygen species, which in turn decreased contractile marker expression, and also promoted calcification in vitro. Intracellular calcium signaling also further enhanced extracellular vesicle secretion, and decreased extracellular vesicle uptake. This then promoted the accumulation of extracellular vesicles in the extracellular matrix, which is a mechanism that promotes calcification. Together, these data suggest that mineral imbalances, such as those seen in chronic kidney disease patients, contribute to loss of smooth muscle cell contractility, which promotes osteochondral genic transdifferentiation.

For the interview portion today, I have with me Drs Andrew Murphy and Michelle Flynn from the Baker Heart and Diabetes Institute and Monash University in Melbourne Australia. And we're going to be discussing their manuscript titled Transient Intermittent Hyperglycemia Accelerates Atherosclerosis by Promoting Myelopoiesis. But really I like the running title, which is Hyperglycemic Spikes Accelerate Atherosclerosis. So thank you both very much for joining me today.

Michelle Flynn: Thanks for having us.

Cindy St. Hilaire: So before we start to ‘stalk a bit about what the details of this study is, could you maybe give us a little primer on what you've done that led up to this study?

Andrew Murphy: Yeah, so this really was a continuation of a study that began actually when I was in my postdoc in Allan Tall lab and working with Ira Goldberg’s lab with the postdoc  Prabhakara R Nagareddy there. We've shown along with Ed Fisher’s group at NYU, that mice that had established atherosclerotic lesions that were then made diabetic, failed to have lesion regression compared to those that were non-diabetic with normalized plasma cholesterol levels. We showed that if we gave an SGLT-2 inhibitor to normalize glucose that regression then started to occur. And then we found that this was primarily driven by myelopoiesis, suddenly increased production of monocytes, which through that entered the plaque. And so from that, that was in the hyperglycemic model which is sort of a very rare patient group these days, because most people are on well-controlled glucosteroid drugs. And really the SGLT-2 inhibitors have been a game changer in that scenario. And what we were trying to do with this study was bring it into a more clinically relevant setting that might show the potential importance of glucose on a much larger population.

Cindy St. Hilaire: Excellent. Maybe you could give us an introduction to the link between what's known about diabetes and cardiovascular disease and the interplay?

Michelle Flynn: So diabetic and pre-diabetic patients actually account for 65% of all cardiovascular deaths, which really indicates that diabetes itself plays a major factor alongside other things like obesity and hypercholesterolemia. And so we've previously shown that hyperglycemia was actually driving atherosclerosis in a chronic hyperglycemic setting. So given that kind of vascular disease actually affects both diabetic and pre-diabetic patients, we suspected that it may not just be chronic hyperglycemia or really intense hyperglycemia that could be driving this issue. And so what we were actually looking at in this paper was how more transient levels of hyperglycemia, which actually occur quite often in both diabetic patients and pre-diabetic patients, how much this can contribute to cardiovascular disease.

Andrew Murphy: I guess this link between poor glucose control and cardiovascular disease is obviously very well established. The interesting part is that HbA1c only predicts part of the risk. If you look at fasting blood glucose, again, that's only partially responsible, but if you look at postprandial or two hour glucose loads, you'll see that that is more predictive of cardiovascular events than the other two measures. And it seems to be a continuum. So even if you are a healthy or non-diabetic individual, you obviously still have those postprandial events and depending how high that goes up is thought to be a predictive of future cardiovascular outcomes. And so obviously that's worse than people with pre-diabetes and then again worse with people that have actual, full blown diabetes.

Cindy St. Hilaire: And what is a transient hyperglycemic event? What would do that in maybe you and me who don't have diabetes versus someone who has diabetes or is pre-diabetic?

Michelle Flynn: So essentially what we're modeling with this transient hyperglycemia is that postprandial increase in glucose after you have a meal, which in people who have impaired glucose tolerance is going to be more pronounced than in someone who has a normal glucose tolerance.

Cindy St. Hilaire: Got it. And so how did you test this in the mice?

Michelle Flynn: We did this by developing a novel model of transient hyperglycemia. So we used ordinary wild type mice that weren't diabetic, and we injected them with glucose intraperitoneally, which then increased blood glucose levels in the plasma after about 15 minutes up to about 15 to 20 millimolar. And then after about two hours, this decreased back down to baseline levels. So this was very similar to what you actually see in a postprandial event. And by doing this four times throughout the day, we were able to mimic what you might see in a patient who has had several meals across the day who has impaired glucose tolerance.

Andrew Murphy: One other advantage with the model that we used was that we were trying to really isolate the effects of glucose. And so by injecting glucose intraperitoneally in otherwise healthy mice, it bypasses the incretin response, which we know loses efficacy, I guess, in people that are diabetic. And so we were just really mimicking acute glucose rises that would occur after a meal. And then obviously in this wild type mouse the insulin response would then kick in to clear the glucose so it really tests that glucose hypothesis.

Cindy St. Hilaire: So it's really digging in deeply on the actual sugar component, not just eating in general or other aspects. So in some of your experiments, or I guess in actually most of them, you show that the injection of glucose, it increased the plaque size in these mice, but it didn't alter the cholesterol levels. So can you explain a bit what's going on there? A little bit about the mechanism you discovered and kind of specifically introducing RAGE and the S100A8 and A9 axis?

Michelle Flynn: Yeah, so what we showed was that regardless of cholesterol levels, we were seeing an increase in clot plaque size, and this was actually driven by the monocytes and neutrophils which were increased in the circulation of these mice. And then these are able to infiltrate into the plaque where they promote plaque progression. And what we found was that the increase in monocytes and neutrophils was due to an increase in their production within the bone marrow.

And this was in turn due to the signaling by a protein heterodimer of S100A8 and A9, which signals via the receptor RAGE in the bone marrow on the progenitors of these cells, which induces their proliferation and differentiation. And then that produces an increase in the production of those immune cells, which promote plaque progression.

Cindy St. Hilaire: Interesting. So it's really independent of kind of the basic thing that everyone thinks about, or I guess as non-scientists think about, is cholesterol. The public really focus on cholesterol, but what your study's showing is there's this whole other glucose mediated immune arm to it. What else does this S100A8-A9 regulate?

Andrew Murphy: So S100A8 and A9 has some intracellular roles, which may direct the development of the model itself, but really a lot of its extracellular roles and so on is promoting sterile inflammation, chemotaxis, so activation of local immune cells. And in the context of diabetes and obesity, many of other diseases, it can signal via RAGE, as Michelle said, but it can also signal by TLR4. And so it seems as though in those diseases driven mainly by glucose, such as the modeling of postprandial hyperglycemia or all kinase in general, it will signal via RAGE, but we've also shown in the setting of obesity that it will signal via TLR4 to stimulate things like interleukin 1 beta. We've also had a paper just recently in Circulation  with Prabhakara Nagareddy’s group where we've shown post myocardial infarction that prime neutrophils in the heart to eventually release IL1-beta and cause myelopoiesis in that way.

Cindy St. Hilaire: Wow, so this is really kind of an early activator of a much bigger immune response, whether it's in atherosclerosis or MI or probably, I don't know, a handful other things, I guess, right?

Andrew Murphy: It seems to be really important when neutrophils are involved. So in a setting of an MI, we know that they come into the heart very early and become activated and it really makes them about 40% of the cytosol proteins of the cells. So when it degranulates or lyses, they are kind of neutral, at least in the predominant protein.

Cindy St. Hilaire: Okay. So this is released in NETs in NETosis then?

Andrew Murphy: That's what we're sort of discovering so far. So I guess all I can say is, stay tuned, this is a story for another day.

Cindy St. Hilaire: Okay. That's really interesting though.

Andrew Murphy: We haven’t looked in gglucose driven events yet.

Cindy St. Hilaire: Yeah and actually one of the interesting things I've learned from your study, I had known about GLUT1 and that GLUT1 was I guess the constituently active of the glucose transporters, but I didn't realize it was so high on neutrophils and that neutrophils were so dependent metabolically on glucose. Can you maybe tell a little bit more about that story?

Michelle Flynn: Yes. So the neutrophil itself is actually very highly dependent on glycolysis because it doesn't actually have many mitochondria. So compared to most cells, they have very few mitochondria so they can't really rely upon the oxidative phosphorylation for their general metabolism. And so they predominantly rely on glucose coming into the cell and then being shuttled through glycolysis to generate their energy. And yeah this does seem to be predominantly due to uptake of glucose through GLUT1.

Cindy St. Hilaire: And then that excess glucose, the byproduct, is reactive oxygen species and upregulation and this cascade of-

Michelle Flynn: Yes, yes that's correct.

Cindy St. Hilaire: Okay, great. So currently we use HbA1c as a biomarker for overall kind of glucose regulation in diabetic patients. And based on your studies and perhaps the studies of others, would neutrophil numbers or even S100A8 or A9 be a better metric to figure out where a pre-diabetic or even a healthy patient is in terms of their glucose tolerability?

Michelle Flynn: Yeah. That could actually be an interesting marker to look at. Given that neutrophils and S100 are also associated with obesity and diabetes in general, and as well as the risk for cardiovascular disease. So with the progression of diabetes, you could expect that the levels of these would increase as well.

Andrew Murphy: We've shown previously when we first discovered that the S100 was important in diabetes, that in the Pittsburgh study with Trevor Orchard's group, he had followed people with type one diabetes for 20 years, that those that did develop a cardiovascular event had a higher S100A8 and A9 levels and that correlated with neutrophils. And so it certainly seems to be a marker of predictive outcomes. And so those that do have poorer glycemic control will have higher neutrophils. That's well known. And so perhaps you're right that probably in combination with HbA1c or things like two hour post glucose challenges, S100A8 and A9 and perhaps neutrophil counts would also be a nice predictive measure of potential cardiovascular outcomes of that person.

Cindy St. Hilaire: Wow. That'd be really great because you could then maybe kind of more fine tune and predict which patients might be more or less susceptible to cardiovascular events.

Andrew Murphy: That's right. Yeah. I think one other important aspect would be if HbA1c is deemed to be relatively well under control, yet you still have a high level of S100A8 and A9, that perhaps those transient spikes are contributing. You're not picking that up in the HbA1c, which looks like the average over approximately a month. And so that could be a nice way to add value onto that score.

Cindy St. Hilaire: Interesting. I didn't realize it was that stable about over a month. All right. So I'm relatively healthy. I'm not pre-diabetic, but if I eat a whole bunch of cake or a whole bunch of ice cream or drink a lot of beer, does that create un me a transient hyperglycemic event that is of the same range we're talking about and what do your findings suggest for people who are relatively healthy and things we should be aware about regarding eating habits and things like that?

Andrew Murphy: Yeah. I think it's a really good question. And it's sort of hard to give you an exact answer to that right now. We need to look at that in people, model these sort of same spikes in people, but what we I guess don't know yet, even in the preclinical models is how high and how long does that glucose have to be? And I think that's one of the most important questions first. So is there a danger zone where these neutrophils start be the innate senses of hyperglycemia that start to then release S100A8 and A9 to cause these downstream events? But what our data does show is that if you're doing this, having a binge night or a binge day once a week for your life, then that's probably not going to be a great thing.

Cindy St. Hilaire: Yeah. All right. So you need to figure out is one scoop of ice cream okay, but two not so great.

Andrew Murphy: Maybe if it's two different flavors it'll be okay.

Cindy St. Hilaire: Maybe, right? That's great. So, I mean, is there a way we could potentially therapeutically target this signaling axis or is it too ubiquitous in terms of what it regulates? Is there a way to harness what you've found potentially in the clinic?

Michelle Flynn: Yeah, so there's an inhibitor of S100A8 and A9 that prevents its binding to RAGE. It's currently approved as an Orphan Drug for systemic sclerosis in both the US and the UK. And that drug itself, we tested in our preclinical mouse model, and we found that it was in fact able to prevent their production of these immune cells, as well as prevent the accelerated atherosclerosis in response to these transient hypoglycemic spikes.

Andrew Murphy: So another sort of line of thinking that we're exploring is that we could actually target neutrophil metabolism itself. And so now we're sort of understanding, are there certain proteins that are more abundantly expressed in neutrophils and not other cells in the body that would regulate glycolysis? I know that might sound a bit of a pie in the sky sort of idea, because glycolytic pathway's quite regulated, but there we have found some proteins that are rich in neutrophils and not other cells that may be responsible for the early steps of glycolysis. And so whether that can be harnessed or not, we'll have to see in the future, but it might be a way of more directly targeting neutrophils rather than approaching that's important in sterile inflammation.

Cindy St. Hilaire: That makes sense. That is such a cool idea and this is really such a beautiful story. It's one of those papers that you just read it and it's just such a logical progression, but it's also really interesting and I really appreciated all those bone marrow transplants. I did those in grad school, so well done. It's a beautiful story. And then I'm just really happy that you published it with us. So thank you so much for joining me today.

Andrew Murphy: Yeah thanks for having us.

Michelle Flynn: Thank you.

Cindy St. Hilaire: That's it for highlights from the late August and early September issues of Circulation Research. Thank you so much for listening. Please check out the Circulation Research Facebook page and follow us on Twitter and Instagram with the handle @CircRes and #DiscoverCircRes. Thank you to our guests, Drs Andrew Murphy and Michelle Flynn. This podcast is produced by Rebecca McTavish and Ashara Ratnayaka, edited by Melissa Stoner, and supported by the Editorial Team of Circulation Research. Some of the copy text for the highlighted articles is provided by Ruth Williams. 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.