January 2022 Discover CircRes - a podcast by Cynthia St. Hilaire, PhD & Milka Koupenova, PhD
from 2022-01-20T19:00
This month on Episode 32 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the January 7 and January 21 issues of Circulation Research. This episode also features a conversation with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.
Article highlights:
Carlson, et al. AKAP18δ Controls CaMKIIδ Activity
Gan, et al. sEV and Adipocyte ER Stress Following MI/R
Khan, et al. Long-term Risk Prediction of Heart Failure
Awan, et al. Wnt5a Is Essential for Cholesterol Homeostasis
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. Today, I'm going to be highlighting the articles from our January issues of Circulation Research. I'm also going to speak with Ms Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill about their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling.
Cindy St. Hilaire: The first article I want to share is titled AKAP18δ Anchors and Regulates CaMKII Activity at Phospholamban-SERCA2 and Ryanodine Receptors. The first and corresponding author for this article is Cathrine Carlson, and the study was conducted at University of Ohio. In cardiac muscle cells, calcium is continuously released and taken up by the sarcoplasmic reticulum to drive alternating contractions and relaxations. The kinase, CaMKII, regulates this calcium signaling via phosphorylation of the sarcoplasmic reticulum proteins ryanodine receptors also called RYR.
Cindy St. Hilaire: These receptors promote calcium release, and phospholamban promotes calcium uptake via the transporter SERCA, but how CaMKII localizes to and associates with these sarcoplasmic reticulum factors was unclear. Because AKAP18 delta enables phosphorylation of phospholamban and calcium uptake into the sarcoplasmic reticulum, this group suspected it might be involved. The team's immuno precipitation and functional experiments in rodent cardiomyocytes show that AKAP18 delta associates with CaMKII and phospholamban SERCA2 as well as with CaMKII and ryanodine receptors, and that these interactions are linked to CaMKII activity.
Cindy St. Hilaire: The team identified two separate CaMKII binding domains within the AKAP18 delta protein, one that inhibits the kinase and one that actuates it, suggesting they may somehow serve to fine tune CaMKII activity. While such regulatory details remain to be resolved, the isolated domains may be utilized as tools for studying calcium handling in cardiomyocytes, and for developing therapeutic CaMKII regulating reagents for treating arrhythmia.
Cindy St. Hilaire: The second article I want to share is titled Ischemic Heart-Derived Small Extracellular Vesicles Impair Adipocyte Function. The first author is Lu Gan, and the corresponding authors are Yajing Wang and Yu Cao from Thomas Jefferson University. While diabetes and obesity increase a person's risk of myocardial infarction, suffering a myocardial infarction itself can lead to metabolic dysfunction. One of the main regulators of systemic metabolic homeostasis is the body's adipose tissue, but whether and how an injured heart communicates with adipocytes was unclear.
Cindy St. Hilaire: The infarcted heart is known to release microRNA containing extracellular vesicles, also called EVs, and so this group hypothesized that these EVs might constitute a heart-to-fat communication system. They isolated circulating EVs before and after myocardial infarction in mice, and incubated these vesicles with cultured adipocytes. After 24 hours, differences in adipocyte gene and protein expression were apparent. Notably, a key cardioprotective metabolic factor called adiponectin was downregulated in cells treated with the extracellular vesicles from myocardial infarcted mice, while genes involved in endoplasmic reticulum stress were increased.
Cindy St. Hilaire: Analysis of the myocardial infarction extracellular vesicle content showed an increased abundance of specific microRNAs, and the team went on to show that inhibiting production of these microRNAs or the EVs themselves, prevented adipocyte ER stress and adiponectin production in mice after myocardial infarction. Together, these data hints that such microRNA inhibition may be a clinical strategy that can be used to prevent infarction-associated metabolic dysfunction in humans.
Cindy St. Hilaire: The next article I want to share is titled Development and Validation of A Long-Term Incident Heart Failure Risk Model. The first and corresponding author of this study is Sadiya Khan from Northwestern University. Heart failure contributes to approximately 1.2 million hospitalizations, and 300,000 deaths in the U.S. annually. Heart failure also has an estimated healthcare cost of over $10 billion. With both the incident rates and costs expected to rise in the future, a method for predicting an individual's heart failure risk would enable preventative interventions such as diet and blood pressure treatments to be initiated early, thus prolonging the number of healthy years.
Cindy St. Hilaire: To develop such a prediction tool, this group studied decades of health data from over 24,000 individuals that was collected as part of five separate, long-running national heart, lung and blood institute studies. The individuals included in the model for development were at baseline aged between 20 and 59 years old, and had no cardiovascular disease diagnosis at that time. Analysis of their body mass indices, blood pressures, total cholesterol levels, high density lipoprotein levels, smoking statuses, diabetes diagnoses, and other cardiovascular health data over several decades enabled the team to develop an equation for predicting an individual's likelihood of developing heart failure in the next 30 years. The hope is such personalized risk assessments will help to guide patient-doctor discussions regarding cardiovascular health, lifestyle choices and medical interventions.
Cindy St. Hilaire: The last article I want to share is titled Wnt5a Promotes Lysosomal Cholesterol Egress and Protects Against Atherosclerosis. The first authors are Sarah Awan and Magalie Lambert, and the corresponding author is Philippe Boucher from the University of Strasbourg. The Wnt family of signaling proteins drives many developmental processes, such as cell fate determination, proliferation and migration. Recently, Wnt signaling has been implicated in lipid homeostasis. Mutations that impair Wnt signaling have been shown to cause hyperlipidemia in mice, and in humans, decreased Wnt signaling activity inversely correlates with atherosclerosis severity.
Cindy St. Hilaire: Because the protein Wnt5a in particular has been shown to inhibit cholesterol accumulation in cells, this group investigated the role of Wnt5a protein in mice and human cells. Mice whose vascular smooth muscle cells lacked Wnt5a developed more severe atherosclerosis compared to control animals, and human smooth muscle cells lacking Wnt5a accumulated far greater amounts of cholesterol in the lysosomes than did cells with normal levels of Wnt5a. The group then showed that Wnt5a normally associates with lysosomes, where it promotes the catabolism of lysosomal cholesterol via activating lysosomal lipase, and promoting cholesterol egress via the endoplasmic reticulum. In revealing how cholesterol efflux is trafficked by Wnt5a, these findings may help to inform future cholesterol regulating therapies.
Cindy St. Hilaire: Today, Natalie Harris and Dr Kathleen Caron from the University of North Carolina Chapel Hill are here with me to discuss their study, VE-Cadherin Is Required for Cardiac Lymphatic Maintenance and Signaling, which is featured in our January 7th issue of Circulation Research. Thank you both for joining me today.
Kathleen Caron: Thanks, Cindy, for having us. We're really honored and excited to talk with you.
Cindy St. Hilaire: I'm excited too, because I think this is my first lymphatic paper I'm talking about. That's where I'm going to start my questions. Your study is investigating cardiac lymphatics. But like I said, I haven't talked a lot about lymphatics here, so I was wondering if you could at least give a little bit of background about what the role is of the lymphatic system, especially because I feel like it's the unappreciated member of the circulation, and also give us a little bit of background on what cardiac lymphatics are.
Kathleen Caron: That's a really great question. We sometimes talk about lymphatic vessels as the third vascular system or the understudied vascular system. I'm hoping that that's not the case so much anymore, because the lymphatic field has really boomed in the past 15 years or so. I think where we are right now in the field is in early days, we and others had discovered key signaling molecules, and transcription factors, and growth factors that are important and specific to the lymphatic vasculature as compared to blood endothelial cells. Through those unique tools, now, the field has fast forwarded where we're starting to look into organ-specific functions of lymphatics.
Kathleen Caron: We're appreciating that perhaps a little unlike the blood vascular system, which has one main function of delivering blood, lymphatics actually have very different functions depending on the organ that they're in. Some of the more common ones that you'll read about in textbooks in about a paragraph in a medical textbook are that lymphatics are important for immune cell trafficking through the lymph nodes, so they're the major route of trafficking for immune cells and for their maturation. Lymphatics are also important for draining interstitial fluid, and maintaining the homeostasis of tissue fluid balance.
Kathleen Caron: A third really big one, which is sometimes underappreciated, is that lymphatics are the key vessels within the intestine that absorb lipid, and so all of our dietary lipids are absorbed through lymphatic vessels as opposed to the blood vasculature. Those three hallmark functions of lymphatics are the cornerstone of what they do throughout our body. But when you start to look into different organs and recognizing the different extrinsic and intrinsic forces that govern the function of these endothelial cells and different organs, you start to realize that they're even more complex, and that brings us to the heart.
Kathleen Caron: The heart just has this beautiful network of lymphatic vessels that begin in the subendocardial space, and then project out and cover the subepicardial surface of the heart. And because the heart is always pumping, and because lymphatic vessels don't have an intrinsic mechanism for the flow of fluid through them, they rely on the movement of the tissue that they're in to help propel the fluid. So, this really raises the question of how are lymphatics functioning physically within a myocardium that is pumping with a very strong extrinsic force, and what is the function of those vessels if the heart is a very dense, thick organ that is not necessarily prone to edema necessarily as maybe our peripheral tissue and our skin is?
Kathleen Caron: We've been studying this for many years now, and we've had several studies exploring genetic factors that are important for the growth and development of cardiac lymphatics. That's the focus of this paper today. They're quite unique and very different vessels.
Cindy St. Hilaire: Reading your paper, I definitely learned a lot about lymphatics in general. One of the things I was thinking about, obviously, you're looking at VE-Cadherin, which is an endothelial cell marker. When I think of VE-Cadherin, and when I think of endothelial cells, my mind goes primarily to those that are in arteries and veins. In those conduits, their role is to really keep a tight seal to keep things out. But in the lymphatic system, it's very different, so how exactly different are the endothelial cells in the lymphatic tissue, and are they different, say, in the cardiac lymphatics versus, like you said, the mesenteric lymphatic?
Kathleen Caron: Lymphatics are very different than the blood vasculature. First of all, the lymphatic vasculature has key differences in terms of its architecture and structure. The lymphatic endothelial cells themselves, as they exist in vessels, don't put down a basement membrane, and in general, the dermal capillaries or the initial collector lymphatics that are the ones that are taking in fluid also don't have smooth muscle cells surrounding them like our typical vasculature does. All of this is guided and precedented by the differences in gene expression patterns of these very specialized endothelial cells.
Kathleen Caron: They also have very different cell-cell junctions. So when we think of a blood endothelial cell, we typically think of these tight junctions that bring them together, but the lymphatic endothelial cells have oak leaf shaped overlapping junctions. They're really beautiful to see on an EM, and they're very different than the blood vasculature, because, Cindy, as you mentioned, the function is very different. You're supposed to let things leak out, and big things too, right, like immune cells and large proteins.
Cindy St. Hilaire: One of the neat things that really made your study possible is this really nice PROX1 inducible CRE that you crossed with the flox-cadherin5 gene. I was wondering a little bit about that protein. Is that one of these, I guess, markers that allows lymphatic EC to be a lymphatic EC, and how specific is that protein for those specific ECs?
Natalie Harris: The PROX1 CRE that we use is based off of the PROX1 transcription factor, which we consider to be one of the master transcription factors of lymphatics. In fact, that was one of the very first lymphatic specific transcription factors that help maintain the lymphatic identity. So in this case, PROX1 turns on from blood endothelial cells, because many lymphatics are of venous origin, so actually, PROX1 turning on is a hallmark of them becoming a lymphatic endothelial cell.
Natalie Harris: Those are really great CRE specifically to look at lymphatics in this case, and it actually is a perfect model system because VE-Cadherin itself is only expressed in lymphatics and blood vessels, and then we have PROX1 as our free driver. Therefore, it will only be lymphatic, so it's a very specific lymphatic knockout of VE-Cadherin.
Cindy St. Hilaire: That's so wonderful when we discover things that are so specific like that. So using this really nice model that's also Tamoxifen inducible, you then have control to look at things temporally. One of the neat things that you did was you looked at this in terms of an embryonic level knockout, but then another one postnatally, and then another one, it was an adult mouse, which not a lot of people do that intricate, temporal spacing of things. So I was wondering if you could just share with us what you were thinking behind doing that, and then really importantly, what those different models actually taught you about the cardiac lymphatics?
Kathleen Caron: That's a great question, Cindy. It would take me 20 minutes to answer. It really represents work by all of the co-authors. Really, it's the first effort to look at the different stages. That's because the growth and development of lymphatics, particularly within the myocardium, differs a lot during embryogenesis, and then the vessels themselves are quiescent in an adult animal. Then of course, we were interested in seeing what might happen in an injured myocardium, and that was also part of the study.
Kathleen Caron: We felt that it was important to address the changing and dynamic role of this protein in a developing lymphatic, because it's growing and forming these nascent vessels, and then as it's starting to remodel an early life, and then in adulthood when it's in a quiescence state. That was the rationale for looking at this. It was also... Sometimes, science just takes you where it takes you, and it was a co-author of ours, and collaborator of ours, who had noted a phenotype in the hearts of these animals that he generated and suggested that maybe it would be a good idea to look early in development. Then as one thing leads to another, you start looking later in development and so on and so forth, so the science just kind of…
Cindy St. Hilaire: Sometimes tells you where to go on its own.
Kathleen Caron: Exactly. It was a long project.
Natalie Harris: Part of the reason too is that the cardiac lymphatics have been shown to have a little bit of a different development and maintenance and pruning cycle than some of the other lymphatics. Some other lymphatics are totally fully formed in embryonic development, but the cardiac lymphatics have been shown to develop through birth and a little bit postnatally as well. That makes them a little bit unique in the sense that their maturation is very prolonged, so that's part of the reason as well we wanted to look both in embryonic development as well as that postnatal period.
Cindy St. Hilaire: That's so interesting. There are a lot of little nuggets that my antennas would perk up as I read your paper, really neat observations. One of them was that I think it was the postnatal and the adults. There was lymphatic endothelial cells in the cardiac tissue were disrupted. They were discontinuous and fragmented, yet there was no cardiac edema. I thought that was interesting because normally, you'd think about any of these mice with lymphatic issues. You think of edema. You think of swelling, and yet it wasn't happening in the heart. What do you think that means either about the lymphatic system in the heart or in lymphatics as a whole?
Kathleen Caron: That's a really great question, and one that we think about all the time. I think it goes back to the first question or the first comment about the really remarkable differences in the functions of lymphatics and different tissues, right? And within the myocardium, because it is continuously moving and pumping with great force, the extrinsic forces within that tissue will help to mitigate the formation of edema. This is not to say that you can't get myocardial edema, and we've actually developed surgical models in our lab to form myocardial edema in mice.
Kathleen Caron: It is a very common clinical condition in humans as well, but the lymphatics themselves being fully invested within this myocardium probably are being regulated differently in their function in draining fluid than, for example, the lymphatics that you might have in the skin or in your thigh or in other organs in your body. The fact that there wasn't edema, even though you had leaky vessels, didn't alarm us too much because we knew and sensed that with this constant pressure and pumping of the myocardium, that in itself helps to keep the tissue fluid balanced.
Natalie Harris: That might be another reason why we're not seeing such extremes in edema, and then going back to what Kathrine said, again, because lymphatics have multiple functions, perhaps it's more in the immune cell realm or even other functions we haven't uncovered yet.
Cindy St. Hilaire: One of the other neat observations you had was that you were doing a myocardial infarction model on the adult animals, and you noticed that the infarct size and the fibrosis was indeed larger in the knockouts, but the cardiac function wasn't exactly affected. What does this mean, and were you surprised by this?
Kathleen Caron: Yeah, we were surprised. We absolutely were surprised, and we think that's actually one of the key big reveals for the field. To balance this, to counterbalance the absence of a phenotype, that was really remarkable to us, and I hope to many others as well, is that other studies including work from our lab and Paul Riley's lab and Eva Brackinham’s lab have very convincingly shown in multiple different ways that if you stimulate lymphangiogenesis after injury, if you have a model, either genetic or induced, where there are more lymphatics for whatever reason, that's a beneficial thing. That's a great thing, and having more lymphatics is positive and beneficial to improving heart repair, and mitigating heart injury, and helping in the context of myocardial infarction.
Kathleen Caron: Of course, it was really surprising that now we have a mouse model where we essentially have little to no lymphatics with very little to no function, and yet the ejection fractional shorting of the heart was doing just fine. I think that was a big moment and a big discovery for us, but very convincing. Then I think it leads us to really asking while more might be better, what really could be the critical function of the lymphatics in an injured myocardium? As Natalie just mentioned previously, it might be related to immune cell trafficking. Paul Riley's group has made some really seminal discoveries in that regard.
Natalie Harris: It's just very interesting, because it's really against everything that you would expect from, again, all the previous studies. It just goes to show again that the lymphatics are so heterogeneous in their organ level function that that's really worth exploring more, because maybe if you can figure out strategies to selectively target certain beds, you can really do a treat on the disease by disease, organ by organ basis. That makes the lymphatics just really cool in my opinion, because they are so different, but it's all the same system, so it's just a very interesting organ, in my opinion.
Kathleen Caron: I should also say serendipitously or right about a few months ago... Shout out to Mark Kahn's lab at University of Pennsylvania. They had a recent paper, I believe, in JCI that had a similar finding to ours. It's always gratifying when another lab says, "Oh, wow, really?" Their study was very different than ours and on a different series of signaling molecules, but similarly, they ablated or reduced cardiac lymphatics through different mechanisms, and then had an injury model. Also, were rather surprised to see that it didn't have this negative effect.
Cindy St. Hilaire: It's so neat. The whole observations that you saw with these knockouts was a paper in itself, but the next half of the paper, you dig into the mechanism, which is also interesting. Can you share a little bit about the links that you found between VE-Cadherin and the VEGF receptor signaling, and is your mechanism you think specific to all lymphatic ECs or even all ECs, or is it specific just to the cardiac lymphatic ECs?
Kathleen Caron: Yes, the mechanism, I find one of the funnest parts of this paper, because I think it really synergizes a lot of the key signaling molecules within our field. Also, I think it bridges together a G-protein-coupled-receptor signaling pathway that my lab has been interested in for decades now, and that is a pathway with the VEGFR3 signaling pathway. I think that's been a big open question in the field. How do these two critical requisite signaling paradigms for lymphatics converge together to maintain lymphatic function development?
Kathleen Caron: I think we've really made some really great inroads in the study, and VE-Cadherin is central to that because it forms a structural scaffold to keep a GPCR signaling pathway in register with the receptor tyrosine kinase signaling pathway, and basically allow for the transactivation of these two really powerful pathways. The mechanism really is gratifying to be able to finally pull how these molecules all interface together and regulate one another.
Natalie Harris: It's very interesting in the fact that VE-Cadherin, it's not necessarily like a lymphatic-specific molecule, but a lot of work in terms of VE-Cad has been more in studying mechanosensing and mechanotransductions. That's where a lot of little nuggets about maybe our mechanism has occurred that we know from really just on protein level studies that VE-Cadherin does interact with VEGFR2 and VEGFR3 by the transmembrane domain interaction. That was clue number one, and then clue number two is that we know that a lot of different mechanical signals that might affect VEGFR3 happened in the presence of VE-Cad.
Natalie Harris: So in a sense, this particular paper is just piecing together a lot of these nuggets of information, and it all makes sense. One thing that you were saying in terms of maybe specific to the heart, going back to some of the earlier studies on these papers on these mice, that we found very vessel-bed-specific effects. One of the vessel beds that is really impacted is the lacteals and the mesentery, so the gut lymphatics. We do know that these lymphatic beds are very sensitive to VEGFC. In fact, they require constant VEGFC signaling. So if you're not having VEGFR3 stable at the membrane to receive these signals, it makes sense if you would have really extreme effects. That might be, again, some of the case in the heart as well. We do know after a cardiac injury, we do see an increase in things like adrenomedullin, and an increase in VEGFC has been shown to increase lymphangiogenesis, so perhaps also the heart, the gut lymphatics also has a special requirement for VEFGR3 signaling.
Cindy St. Hilaire: So in terms of, I guess, the future of this line of research and maybe thinking about translation, what do you see as maybe a role for this in terms of developing therapeutic strategies or even preventative measures, I guess, specifically in the cardiac lymphatic area?
Natalie Harris: Like we mentioned earlier, there's been a lot of studies in mice that have looked at increasing lymphangiogenesis post-injury, so it would be interesting to see more when those hit the clinical end, and if you're seeing similar effects. Then the other thing that's interesting about lymphatics, you can think of them as both a target and also as a drug delivery route. There's a huge, huge field totally dedicated to using the lymphatics to deliver drugs like nanoparticles. That's very big in the cancer realm, and pretty much for any kind of drug delivery, if you can imagine using that as a super highway to deliver drugs as well.
Natalie Harris: That could be a potential avenue in terms of the heart as well, getting a more specific administration of cardiovascular drugs to the heart. So whether or not we're thinking of them as being modulated by disease, we can also use them to modulate the disease itself by delivering drugs as well, so it's interesting. You can think of the lymphatics as a therapeutic target and as a therapeutic administrator. That's going to be really interesting to see where the field goes.
Cindy St. Hilaire: I like that, a new super highway to deliver drugs. Thank you so much, soon to be Dr Harris and Dr Caron from UNC Chapel Hill. This was a wonderful conversation and a beautiful paper. Congratulations on all the hard work.
Kathleen Caron: Well, thanks so much, Cindy, and to the whole Circ Research team. We really appreciate your advocacy for our work and giving us this wonderful opportunity.
Natalie Harris: Thank you so much.
Cindy St. Hilaire: That's it for the highlights from our January 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, Natalie Harris and Dr Kathleen Caron. This podcast was 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, your on-the-go source for the most up-to-date and exciting discoveries in basic cardiovascular research.
Cindy St. Hilaire: This program is copyright of the American Heart Association, 2022. 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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