February 2023 Discover CircRes

This month on Episode 45 of Discover CircRes, host Cynthia St. Hilaire highlights four original research articles featured in the February 3rd and February 17th issues of Circulation Research. This episode also features an interview with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation.   Article highlights:   Pi, et al. Metabolomic Signatures in PAH   Carnevale, et al. Thrombosis TLR4-Mediated in SARS-CoV-2 Infection   Cai, et al. Macrophage ADAR1 in AAA   Koide, et al. sEVs Accelerate Vascular Calcification in CKD   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 Cynthia St. Hilaire from the Vascular Medicine Institute at the University of Pittsburgh, and today I'm going to be highlighting the articles from our February 3rd and 17th issues of Circulation Research. I'm also going to have a chat with Dr Hind Lal and Dr Tousif Sultan from the University of Alabama at Birmingham about their study, Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. But before I get to the interviews, here are a few article highlights.   Cindy St. Hilaire:        The first article I want to highlight comes from the laboratory of Dr Peter Leary at the University of Washington, and the title is Metabolomic Signatures Associated With Pulmonary Arterial Hypertension Outcomes. Pulmonary Arterial Hypertension or PAH is a rare but life-threatening disease in which progressive thickening of the walls of the lung’s blood vessels causes increased blood pressure and that increased blood pressure ultimately damages the heart's right ventricle.   Interestingly, progression to heart failure varies considerably among patients, but the reasons why there is variability are not well understood. To find out, this group turned their attention to patient metabolomes, which differ significantly from those of healthy people and thus may also change with severity. Blood samples from 117 PAH patients were analyzed for more than a thousand metabolites by mass spectrometry and the patient's progress was followed for the next three years. 22 patients died within a three-year period and 27 developed significant right ventricle dilation. Other measures of severity included pulmonary vascular resistance, exercise capacity and levels of BNP, which is a metric of heart health. Two metabolic pathways, those relating to polyamine and histidine metabolism, were found to be linked with all measures of severity suggesting a key role for them in disease pathology. While determining how these pathways influence disease as a subject for further study, the current findings may nevertheless lead to new prognostic indicators to inform patient care.   Cindy St. Hilaire:        The next article I want to discuss is coming from our February 3rd issue of Circulation Research and this is coming from the laboratory of Dr Francisco Violi at the University of Rome and the title is Toll-Like Receptor 4-Dependent Platelet-Related Thrombosis in SARS-CoV-2 Infection. Thrombosis can be a complication of COVID-19 and it is associated with poor outcomes, including death. However, the exact mechanism by which the virus activates platelets, which are the cells that drive thrombosis, is not clear. For one thing, platelets do not appear to express the receptor for SARS-CoV-2. They do however, express the TLR4 receptor and that's a receptor that mediates entry of other viruses as part of the immune response. And TLR4 is ramped up in COVID-19 patient platelets. This group now confirms that, indeed, SARS-CoV-2 interacts with TLR4, which in turn triggers thrombosis.   The team analyzed platelets from 25 patients and 10 healthy controls and they found that the platelet activation and thrombic activity were both boosted in the patient samples and could not be blocked using a TLR4 inhibitor. Additionally, immunoprecipitation and immunofluorescent experiments further revealed colocalization between the virus protein and the TLR4 receptor on patient platelets. The team went on to show that the signaling pathway involved reactive oxygen species producing factors p47phox and Nox2, and that inhibition of phox 47, like that of the TLR4 receptor itsel,f could prevent platelet activation. As such, this study suggests that inhibiting either of these proteins may form the basis of an antithrombotic treatment for COVID-19.   Cindy St. Hilaire:        The third article I want to highlight is coming from the lab of Shi-You Chen at University of Missouri and the title of this article is ADAR1 Non-Editing Function in Macrophage Activation and Abdominal Aortic Aneurysm. Macrophage activation plays a critical role in abdominal aortic aneurysm development, or AAA development. Inflammation is a component of this pathology; however, the mechanisms controlling macrophage activation and vascular inflammation in AAA are largely unknown. The ADAR1 enzyme catalyzes the conversion of adenosine to inosine in RNA molecules and thus this conversion can serve as a rheostat to regulate RNA structure or the gene coding sequence of proteins. Several studies have explored the role of ADAR1 in inflammation, but its precise contribution is not fully understood, so the objective of this group was to study the role of ADAR1 in macrophage activation and AAA formation.   Aortic transplantation was conducted to determine the importance of nonvascular ADAR1 in AAA development and dissection and angiotensin II infusion of ApoE knockout mice combined with a macrophage specific knockout of ADAR1 was used to study the role of ADAR1 macrophage specific contributions to AAA formation and dissection. Allograft transplantation of wild type abdominal aortas to ADAR1 haploinsufficient recipient mice significantly attenuated AAA formation. ADAR1 deficiency in hematopoietic stem cells also decreased the prevalence and the severity of AAA and it also inhibited macrophage infiltration into the aortic wall. ADAR1 deletion blocked the classic macrophage activation pathway. It diminished NF-κB signaling and it enhanced the expression of a number of anti-inflammatory microRNAs. Reconstitution of ADAR1 deficient but not wild type human monocytes to immunodeficient mice blocked the aneurysm formation in transplanted human arteries. Together these results suggest that macrophage ADAR1 promotes aneurysm formation in both mouse and human arteries through a novel mechanism of editing the microRNAs that target NF-κB signaling, which ultimately promotes vascular inflammation in AAA.     Cindy St. Hilaire:        The last article I want to highlight is also from our February 17th issue of Circulation Research and it is coming from the lab of Shintaro Mandai at Tokyo Medical and Dental University and the title of the article is Circulating Extracellular Vesicle Propagated MicroRNA signatures as a Vascular Calcification Factor in Chronic Kidney Disease. Chronic Kidney Disease or CKD accelerates vascular calcification in part by promoting the phenotypic switching of vascular smooth muscle cells to osteoblast like cells. This study investigated the role of circulating small extracellular vesicles or SUVs from the kidneys in promoting this osteogenic switch. CKD was induced in rats and in mice by an adenine induced tubular interstitial fibrosis and serum from these animals induced calcification in in vitro cultures of A-10 embryonic rat smooth muscle cells. Intraperitoneal administration of a compound that prevents SEV biosynthesis and release inhibited thoracic aortic calcification in CKD mice under a high phosphorus diet. In Chronic Kidney Disease, the microRNA transcriptome of SUVs revealed a depletion of four microRNAs and the expression of the microRNAs inversely correlated with kidney function in CKD patients.   In vitro studies found that transected microRNA mimics prevented smooth muscle cell calcification in vitro. In silico analyses revealed that VEGF-A was a convergent target of all four microRNAs and leveraging this, the group used in vitro and in vivo models of calcification to show the inhibition of the VEGF-A, VEGFR-2 signaling pathway mitigated calcification. So in addition to identifying a new potential therapeutic target, these SUV propagated microRNAs are a potential biomarker that can be used for screening patients to determine the severity of CKD and possibly even vascular calcification.   Cindy St. Hilaire:        Today I have with me Dr Hind Lal who's an associate professor of medicine at the University of Alabama Birmingham and his post-doctoral fellow and the lead author of the study Dr Tousif Sultan. And their manuscript is titled Ponatinib Drives Cardiotoxicity by S100A8/A9-NLRP3-IL-1β Mediated Inflammation. And this article is in our February 3rd issue of Circulation Research. So thank you both so much for joining me today.   Tousif Sultan:              Thank you.   Hind Lal:                     Thank you for taking time.   Cindy St. Hilaire:        So ponatinib, it's a tyrosine kinase inhibitor and from my understanding it's the only treatment option for a specific group of patients who have chronic myelogenous leukemia and they have to harbor a specific mutation. And while this drug helps to keep these patients alive essentially, it's extremely cardiotoxic. So cardiotoxicity is somewhat of a new field. So Dr Lal, I was wondering how did you get into this line of research?    Hind Lal:                    So I was fortunate enough to be in the lab of Dr Tom Force and he was kind of father of this new area, now is very developed, it's called cardio-oncology. On those days there were basically everything started in cardio-oncology. So I just recall the first tyrosine kinase approved by FDA was in 2000 and that was... Imagine and our paper came in Nature Medicine 2005 and discovering there is... so to elaborate it a little bit, the cancer therapy broadly divided in two parts. One is called non-targeted therapy like chemotherapy, radiations, et cetera, and then there are cytotoxic drugs. So those cytotoxic drugs because they do not have any targeted name on it so they are, cardiotoxic are toxic to any organ was very obvious and understanding. When these targeted therapy came, which is mainly kinase inhibitor are monoclonal antibodies. So these are targeted to a specific pathway that is activated only in the cancer cells but not in any other cells in the body so they were proposed as like magic bullets that can take off the cancer without any cardiotoxity or minimal side effects. But even in the early phase like 2005 to 2010, these came out, these so-called targeted, they are not very targeted and they are not also the magic bullets and they have serious cardiotoxicity.   Cindy St. Hilaire:        And so what's the mechanism of action of ponatinib in the leukemia and how does that intersect with the cardiovascular system?   Hind Lal:                     Yeah, so this is very good question I must say. So what we believe at this point because, so leukemia if you know is driven by the famous Philadelphia chromosome, which is a translicational gene, one part of human chromosome nine and one part of human chromosome 22 and they translocate make a new gene which is BCR-ABL gene. And because it was discovered in Philadelphia UPENN, is named that Philadelphia chromosome, which is very established mechanism, that's how CML is driven. But what we have discovered that the cardiotoxicity driven by totally, totally different from the ponatinib is one of the inflammatory So it's kind of goodening. So this question is so good. One kind of toxicity is called on-target, when toxicity is mediated by the same mechanism, what is the mechanism of the drug to cure the cancer? So in that case your absolute is minimal because if you manipulate that, the drug's ability to cure the cancer will be affected but if the toxicity and the efficacy is driven by two different mechanism, then as in case of ponatinib seems like it's NLRP3 and inflammasome related mechanism. So this can be managed by manipulating this pathway without hampering the drug efficacy on the cancer.   Cindy St. Hilaire:        So what exactly is cardiotoxicity and how does it present itself in these patients?   Hind Lal:                     So these drugs like ponatinib, they call broader CVD effects. So it's not just cardiac, so they also in hypertensives and atherosclerosis and thrombosis, those kind of thing. But our lab is primarily focused on the heart. So that's why in this paper we have given impresses on the heart. So what we believe at this point that ponatinib lead to this proinflammatory pathway described in this paper, which is just 108A9-NLRP3-IL-1β and this inflammatory pathway lead to a cytokine storm very much like in the COVID-19 and these cytokine storms lead to excessive myocarditis and then finally cardiac dysfunction.   Cindy St. Hilaire:        Is the cytokine storm just local in the cardiac tissue or is it also systemic in the patients? Is cardiotoxicity localized only or is it a more systemic problem?   Tousif Sultan:              I would like to add in this paper we have included that we look this cytokine things and explain blood circulation, bone marrow. So the effect is everywhere, it's not local. So we didn't check other organs, maybe other organs also being affected with the ponatinib treatment.   Cindy St. Hilaire:        And what's the initial phenotype of a patient has when they start to get cardiotoxicity, what's kind of like a telltale symptom?   Hind Lal:                     So good thing that in recent years cardio-oncology developed. So initially the patient that were going for cancer treatment, they were not monitored very closely. So they only end up in cardiology clinic when they are having some cardiac events already. So thanks to the lot of development and growth in the cardio-oncology field, now most patients who going for a long-term cancer treatment, they are closely monitored by cardiology clinics.   Cindy St. Hilaire:        Got it. So they can often catch it before a symptom or an event. That's wonderful.   Hind Lal:                     Yeah, so there's a lot of development in monitoring.   Cindy St. Hilaire:        Wonderful. So you were really interested in figuring out why ponatinib induces cardiotoxicity and you mentioned that really up until now it's been very difficult to study and that's because of the limitation of available murine models. If you just inject a wild type mouse with ponatinib, nothing happens really. So what was your approach to finding relatively good murine models? How did you go about that?   Hind Lal:                     So this is the top scientific question you can ask. So like science, the field is try and try again. So initially this is the first paper with the ponatinib toxicity using the real in vivo models. Any paper before this including ours studies published, they were done on the cellular model in hiPSC, that isolated cardiomyocytes. So you directly putting the ponatinib directly the isolated cells. So this is first case when we were trying to do in vivo, maybe other attempt in vivo but at least not published. So first we also treated the animals with ponatinib and that failed, we don't see any cardiotoxic effect. And then when we going back to the literature, the clinical data is very, very clear from pharmacovigilance that ponatinib is cardiotoxic in humans. So when we're not able to see any phenotype in mouse, we realize that we are not mimicking what's happening in the humans.   So we certainly missing something. Now once again I quote this COVID-19, so many people get infected with COVID-19 but people are having preexisting conditions are on high risk to developing CVD. So there was some literature on that line. So we use this very, very same concept that if there is preexisting conditions, so likely who'd have developing future cardiac event will be more. So we use two model in this paper one atherosclerosis model which is APoE null mice mice, another is tag branding which is pressure overload model for the heart and as soon as we start using what we call comorbidity model like patient is having some preexisting conditions and we very clearly see the robust defect of ponatinib on cardiac dysfunction.   Cindy St. Hilaire:        Yeah, it's really, really well done and I really like that you use kind of two different models of this. Do you think it's also going to be operative in maybe like the diabetic mirroring models? Do you think if we expand to other comorbidities, you might also recapitulate the cardiotoxicity?   Hind Lal:                     So you got all the best questions.   Cindy St. Hilaire:        Thank you. I try.   Hind Lal:                     So because this is CML drug and lot of the risk factor for cardiovascular and cancer are common and even metabolic disease. So most of the time these patients are elderly patients and they're having metabolic conditions and most of the time they have blood pressure or something CVD risk factors. So I agree with you, it'll be very relevant to expand this to the diabetes or metabolic models, but these were the first study, we put all our focus to get this one out so news is there then we can expand the field adding additional models et cetera. But I agree with you that will be very logical next step to do.   Cindy St. Hilaire:        Yeah. And so I guess going back to what you know from the human study or the clinical trials or the human observations, are different populations of patients with CML more predisposed to cardio toxicity than others or is that not known yet?   Hind Lal:                     So one other area called pharmacovigilance. So what pharmacovigilance does patient all over the world taking these drugs. So WHO have their own vigilance system and FDA have their own, so it's called BG-Base for the WHO and it's called the FAERS for the FDA. So one can go back in those data sets and see if X patient taking this Y drug and what kind of symptoms or adverse effect they are seeing and if these symptoms are associated with something else. So there is data that if patients having CVD risk factor, they are more prone to develop ponatinib induced cardiac events. But it needs more polish like you asked the just previous question, diabetes versus maybe blood pressure means hypertension, atherosclerosis, or thrombosis. So it has not been delineated further but in a one big bucket if patients are having CVD risk factor before they are more prone and more likely to develop the cardiac events.   Cindy St. Hilaire:        So after you established that these two murine models could pretty robustly recapitulate the human phenotype, what did you do next? How did you come upon the S100A8/A9-NLRP3-IL-1β signaling circuit? How did you get to that?   Hind Lal:                     So in basic science work, whenever we do mouse is called until we get there is cardiac dysfunction, it's called phenotype, right? So mouse had a cardiac phenotype. So next step is, "Why? What is leading to that phenotype?" That's what we call mechanism. So there the best idea to fit the mechanism is using one of the unbiased approaches like you do unbiased proteomics, unbiased RNC analysis, something like this that will analyze the entire transcript like RNC and say, "Okay, these pathway are," then you can do further analysis that will indicate these pathway are different, are altered. So in this case we used RNC analysis and it came out that this yes A8 and yes A9, 100A8 and nine, they were the most upregulated in this whole set. And thereafter we were very lucky. So we started this study at Vanderbilt, where my lab was and thereafter we very lucky to move here and found Sultan who had a lot of experience with this inflammation and immune system and then Sultan may add something on this so he'll be the better person to say something on this.   Tousif Sultan:              So after our RNC analysis, so we got this S100A8 and nine as top hit with the ponatinib treatment. So then we validated this finding with our flow cytometric, qRT PCR aand then we started which pathway is going to release cytokine and all that. So we found that is NLRP3 inflammasome.   Cindy St. Hilaire:        Yeah and well and I guess maybe step back, what is S100A8/A9? What are those? Tousif Sultan:              Yeah, S10A8/A9 is a calcium binding protein. So that's also called alarmin and they basically binds with the pathogen associated pattern and other TLR2 like receptors and then start inflammatory pathway to release cytokine and all that and it's stable in heterodimer form. So S100A8 heterodimer with A9 and then bind with TLR and a start in this inflammatory pathway.   Cindy St. Hilaire:        And what type of cell is that happening in? Is that happening in the immune cells only or is it also in the cardiomyocyte, or...?   Tousif Sultan:              Yeah, we have included all this data. So from where this alarmin is coming with ponatinib treatment, so literature also suggested that neutrophils and monocytes, those cells are the potential to release the alarmin. So here we also found these two type of cells, neutrophils and monocytes. They release huge alarmin with the treatment of ponatinib.   Cindy St. Hilaire:        And so really taking this really neat mechanism to the next level, you then tried attenuating it by using broad anti-inflammatory steroid dexamethasone but also by targeting these specific components, the NLRP and the S100A specific inhibitors and they worked well. It worked really nicely. Does your data show that any of these therapies work better than the other and then are these viable options to use in humans?   Hind Lal:                     Yeah, we have some data in the paper. Are very broad which help a lot in COVID patients, far very acute infections. So in this case, situation is very different cause most of CML patients will going to take ponatinib for lifelong, there is no remission, right? So in those case, its certainly not a very attractive option. We have shown data in the paper that dexamethasone help with the heart but lead to some metabolic changes. So we have compared those with the NLRP3 inhibitors, those metabolic alterations, dexa versus the NLRP3 inhibitors, CY-09. And we demonstrated that targeting is specifically with paquinimod, our NLRP3 inhibitor CY-09, feel better. It can still rescue the cardiac phenotype without having those adverse effect on metabolic parameters.   Cindy St. Hilaire:        That's wonderful. Do you think though that because you have to take ponatinib for life, that long-term NLRP inhibition would also cause problems or...?   Hind Lal:                     So because not every patient who taking ponatinib would develop the cardiac phenotype, right? Which is like a 10%, 12%, patient developing cardiac dysfunction. So I think someone like I strongly believe paquinimod, which is inhibitor of S100A9, will be really good option or at least we have enough data that make us nail for at least a small clinical trial. And we quickly moving on that. At UAB we have our clinical cardio-oncology program and we are already in touch with the director for the clinical cardio-oncology program. So what we trying to do in that small trial is if one of the standard therapy for heart like beta blocker or ARBs inhibitor, is there any preference like one work better than the other in the standard care? So first we doing that project, then we obviously looking forward if one small clinical trial can be done with paquinimod. I strongly believe it should be helpful.   Cindy St. Hilaire:        That is wonderful. And so do you think... There's other chemotherapeutic agents or probably even other non-cancer drugs that cause cardiotoxicity, do you think this mechanism, this pathway, this S100A-NLRP-IL-1β axis is operative in all cardiotoxicities or do you think it's going to be very specific to the ponatinib?   Hind Lal:                     So it's certainly not all, but it'll be certainly more than ponatinib. So in our lab we are using another kinase inhibitor, which is osimertinib and it's not published yet, but now we know that it's also cardiotoxic because it's taking metabolic root or energetics disruption but not this pro-inflammatory part, but we're doing another project which is strep pneumonia induced cardiac dysfunction, which is called pneumonia. So strep pneumoniae, which leads to the pneumonia ,and lot patient die because of the failing heart we see here in the hospitals and we see these pathways operational over there and we gearing up to do clinical trial on that aspect as well, but it's not generalized like all kind of heart will have the same mechanism.   Cindy St. Hilaire:        It's wonderful to see you're already taking those next steps towards really kind of bringing this to a translational/clinical study. So what was the most challenging aspect of this study?   Tousif Sultan:              The challenging aspect, ponatinib is a kinase inhibitor and that was surprising for us how it's activating immune cells. Generally kinase inhibitors, inhibits all the cells like that. So that was challenging. So we repeated it many times did in vitro experiment to confirm that. So we just added, just treated in vitro immune cells with the ponatinib and confirmed it. So that was little challenging.   Cindy St. Hilaire:        So what's next? You mentioned you're going to try some clinical trials, early stage clinical trials. What's next mechanistically, what do you want to go after?   Hind Lal:                     So what we are doing next and we are very, very eagerly trying to do that. So what it was done, we used the cardiac comorbidity models, but as you know, anybody who will take ponatinib will have cancer, right? So we strongly believe that we miss one factor. There was no cancer on these. So that is very logical next step. What that will allow us to do, what rescue experiment we'll have done in this paper. So we saw, "Okay, this rescue the cardiac phenotype, which is taken care of now," but very same time, we not able to demonstrate that this is happening without hurting the cancer efficacy. So if we have the dual comorbid mouse, which have CML a real thing and we have cardiac thing, then that will allow us to demonstrate, "Okay, we got something that can take care of the cardiac problem without hurting the efficacy on the cancer." And it will be best if you also help little bit to more potentiate the cancer efficacy.   Cindy St. Hilaire:        Yes. Excellent. Well, congratulations on a beautiful study, really exciting findings. Dr Lal and Dr Sultan, thank you so much for taking the time to talk with me today.   Tousif Sultan:              Thank you so much.   Hind Lal:                     Well thank you, Cynthia. We really appreciate your time. Thank you for having us.   Cindy St. Hilaire:        Yeah, it was great.   Cindy St. Hilaire:        That's it for our highlights from the February 3rd and February 17th 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, Dr Hind Lal and Dr Tousif Sultan. This podcast is produced by Ishara Ratnayake, edited by Melissa Stoner and supported by the editorial team at Circulation Research. Some of the copy text for the highlighted articles was provided by Ruth Williams. I'm your host, Dr Cynthia St. Hilaire, and this is Discover CircRes, you're on-the-go source for most exciting discoveries in basic cardiovascular research. This program is copyright of the American Heart Association 2023. And the opinions expressed by the speakers in this podcast are their own and not necessarily those of the editors or of the American Heart Association. For more information, please visit ahajournals.org.