Good morning, everyone. Please welcome our president of the Endocrine Society, Dr. Carol Wysham. ♪♪ -♪♪ Welcome to our plenary session on cardiovascular health in women. Today's presenters will be discussing all aspects of cardiovascular research and care from bench to bedside. This afternoon, we continue to recognize the 2022 Laureate Award winners by honoring three more of our esteemed colleagues. The International Excellence in Endocrinology Award is presented to an endocrinologist who has made exceptional contributions to the field of endocrinology in geographic areas with undeveloped resources for hormone health research, education, clinical practice, or administration. The recipient of Endocrine Society's 2022 International Excellence in Endocrinology Award is Dr. Lourdes Ibáñez, a pediatric endocrinologist at San Juan de Dubuque Barcelona Hospital and chairman of pediatrics, chair of clinical research in endocrinology at the University of Barcelona in Barcelona, Spain. Dr. Ibáñez is a worldwide leader in the field of PCOS who pioneered the use of combined low-dose insulin sensitization for the treatment of the disease, a novel therapeutic approach directed at the pathophysiology of the disorder. Dr. Ibáñez initiated the fellowship program in pediatric endocrinology and diabetes at the University of Barcelona back in 2002 and has mentored 25 fellows from Latin America. Dr. Ibáñez has previously served as a member of the editorial boards for the Journal of Clinical Endocrinology and Metabolism as well as the Journal of the Endocrine Society. Thank you to lead nominator Fred Cavallo with supporting letters from Selma Feldman-Witchell and Ana Claudia Latronica. And congratulations to Dr. Ibáñez for her commitment of contribution for improving care of adolescents and children in Latin America. Thank you. First, I would like to thank the Endocrine Society for honoring me with this award, and thank Ana Claudia Latronico, Selma Guitier, and Sharon Oberfield for nominating me. Many thanks go to my mentors, including Zvi Laron in Tel Aviv, Maria Nieu in New York, and Bill Crowley in Boston. Today's award honors Barcelona's fellowship program in pediatric endocrinology and provides a nice opportunity to thank my fellows from Latin America for their confidence. It has been a pleasure to train pediatricians from countries such as Argentina, Colombia, Costa Rica, Dominican Republic, Ecuador, Guatemala, and Venezuela. Many former fellows are now key pediatric endocrinologists in their own country. For me, it is a daily source of happiness to feel that I may have contributed indirectly to better patient care in those countries. Last but not least, many thanks go to my team in Barcelona, which did so much for our fellowship program. This award is also for them. Thank you very much. Our next award, the Outstanding Clinical Investigator Award, is presented to an internationally recognized clinical investigator who's contributed significantly to understanding the pathogenesis and therapy of endocrine and metabolic diseases. The recipient of the 2022 Outstanding Clinical Investigator Award is Dr. Karl Patzik, Chief of the Section of Medical Neuroendocrinology and Head of the Development of Endocrinology, Metabolism, Genetics, and Endocrine Oncology Affinity Group of the Eunice Kennedy Shriver National Institute of Child Health and Human Development at the Intramural NIH Research Program in Bethesda, Maryland. An extraordinary clinician whose translational research has provided a novel understanding of and treatments for patients with neuroendocrine tumors, especially pheochromocytoma and paraganglioma. Dr. Patzik established the International Symposia on Pheochromocytoma, the most internationally recognized meeting in this field. He was also part of the Endocrine Society's 2014 Pheochromocytoma Task Force and previously served on the editorial board of the Journal of Clinical Endocrinology and Metabolism. Thank you for nominators Jack Janowski and Lynette Nieman, and congratulations to Dr. Karl Patzik, who has dedicated his research career to improved patient care. Ladies and gentlemen, good afternoon. It is really a privilege and honor to be a recipient of the Outstanding Clinical Investigator Award. I would like to thank the Endocrine Society, the nominating committee, and those who nominated me, Dr. Jack Janowski and Dr. Lynette Nieman. I also would like to thank Dr. Weissman for very nice and kind words and introduction. Becoming a physician scientist is not an easy task. You find yourself constantly oscillating between the demanding clinical work and the laboratory experiments, which ultimate goal to improve the lives of patients. Although it is very challenging, it has provided me with a tremendous sense of purpose in my life. To shine light in moments of darkness and to provide hope in moments of despair. I would like to add that this award is not solely my own. Rather, it is the culmination of the sacrifice and very hard work of many colleagues, collaborators, clinical staff, and mentors. These individuals have supported and guided me through many years. I'm also grateful to my family, especially to my wife, Michaela, who is sitting here, my son, Tom, and parents from whom I learn what it means to care for others. I am truly blessed to receive this award. Thank you all from my heart. Thank you. The Gerald D. Auerbach Award for Outstanding Translational Research is presented annually in recognition of outstanding research that accelerates the transition of scientific discoveries to clinical applications. The 2022 recipient of the Gerald Auerbach Award is Dr. Terry Smith. The Frederick G. L. Hutwell Professor in Ophthalmology and Visual Sciences and Professor of Internal Medicine at the University of Michigan School of Medicine in Ann Arbor. Dr. Smith has studied Graves' disease, its ocular manifestations, and related autoimmune diseases for over 35 years. His laboratory group was the first to describe the unique molecular attributes of tissue surrounding the eye that make the orbit susceptible to immune activation and inflammation in Graves' disease. Dr. Smith and his colleagues have mapped the mechanisms involved in the tissue remodeling occurring in thyroid-associated ophthalmopathy, or TAO, a disfiguring and potentially blinding disease. They identified the insulin-like growth factor 1 receptor as a therapeutic target, and their work has culminated in the creation of Tiprotumab, the first FDA-approved drug to treat thyroid eye disease. Thank you to nominator Jeffrey Sherman, with supporting letters from George Kahali and Samuel Refitoff. And congratulations to Dr. Smith, whose translational research is changing the treatment paradigm for this complex disease. Good afternoon to my esteemed colleagues and dear friends and family. I gratefully accept this award from the Endocrine Society and our awards committee on behalf of the many individuals who contributed to our work. I am also grateful to my mentors, including David Ramsden, Edward Siegel, Isidore Edelman, and Samuel Refitov. They taught me with grace, intellectual generosity, and great patience. The many trainees in my laboratory and clinic and my colleagues taught me more than I could ever teach them. Finally, I want to thank my wife, Linda, our son, Jeremy, and the rest of my dear family and extremely close friends, including Shelley Stoyer and Jeff Korf, whose love and support provided me strength and a sense of purpose. They have always made me better than I had any right to become. Thank you again for this award, which is certainly a highlight of my medical career. It's an honor to have C. Barry Mertz here to discuss her research into women's cardiovascular health. Dr. Barry Mertz is a director of the Barbara Streisand Women's Heart Center, the Linda Joy Paulin Women's Heart Health Program, the Erica J. Glaser Women's Heart Research Initiative, and the Preventive Cardiac Center at the Schmidt Cedars-Sinai Heart Institute. She is also a professor of medicine at Cedars-Sinai Medical Center. Dr. Barry Mertz's research interests include women and cardiovascular disease, mental stress and cardiovascular disease, the role of exercise and stress management in reversing disease, the role of cholesterol and nutrition management in heart disease, adverse pregnancy outcomes in cardiovascular disease, and precision medicine monitoring to predict unexpected cardiovascular events. She is chair of the National Heart, Lung, and Blood Institute-sponsored WISE Study or Women's Ischemic Syndrome Evaluation Initiative, which is investigating potential methods for more effective diagnosis and evaluation of ischemic heart disease in women. Dr. Barry Mertz has received investigational grants from the National Heart, Lung, and Blood Institute, the NIH National Center for Alternative and Complementary Medicine, the National Institutes of Aging, the Flight Attendance Medical Research Institute, the Pfeiffer Foundation, the Eli and Edith Broad Foundation, the Barbara Streisand Foundation, the Erica J. Glaser Women's Heart Research Institute, the Society for Women's Health Research, the Linda Joy Paulin Women's Heart Health Program, and the Congressionally-directed Medical Research Program of the Department of Defense. Please join me in welcoming Dr. Barry Mertz to the podium. Thank you. What an honor and a privilege to have a cardiovascular scientist invited to the Endocrine Society. It is very exciting also to see another large body of specialists and subspecialists interested in cardiovascular disease in women. What I hope to do now is share with you our journey of the last 30-plus years. These are my disclosures, dominantly, as you heard, grant support, and do not represent a conflict of interest, but we disclose, of course. Well, of course, we all know, as scientists and as physicians, that there are important differences between women and men, and they start with biology. And of course, our XX men are XY, and women have two copies of everything, so you wonder why we're built to last. This demonstrates, though, that there are important differences also related to hormones. And as I prepared this talk several weeks ago, I thought, what am I going to tell a lot of investigative and practicing endocrinologists about hormones? So you're going to see I don't say very much about hormones, because it turns out I don't know as much as you do. There's also important interrelationships between sex and gender. Sex, of course, is the biological sex, as we saw in the prior slide, where gender is really the gender constructs that we as society assign, girls and boys, women and men, for better or for worse, and it's obviously something of intense political interest currently. But in terms of our area, health science, there is an intersect between these sex differences as well as gendered and cultural attributes. So what I'm going to walk you through now is cardiovascular health in women, equity and inclusion. We'll talk about a preamble, 1984 through 97, some science and discovery, 1997 through 2015, policy changes, 2004 through 2019, and then, of course, going forward, because as we mountain climb, there's always a higher mountain when we get to the top. So this actually started for me with these two women. The gal on the right, of course, everybody knows well. That's Barbara Streisand in her Yentl movie. And if you remember the storyline between that, it was that as a young woman in Eastern Europe at the turn of the century, this young woman wanted to become literate. She wanted to have an education, and, of course, that was open only to men, rights and privileges. And then the Yentl, it turns into a love story, and, of course, Barbara sings a lot of songs. Watch it if you haven't seen it. The gal on your left you probably don't know, some of you know, that's Dr. Bernadine Healy now passed, unfortunately, but she was at the time the director of our National Institutes of Health, and she was a female investigative cardiologist, so a bit of a role model for me. Dr. Healy, after having watched the Yentl movie in 1991, wrote an editorial in the New England Journal that opined regarding this new data. And what we saw in the first time in 1984 was our death of cardiovascular disease in men, stratified in the black line, was actually continuing to go down where the death rate of cardiovascular disease in women, shown in the red line, was actually going up to the point where women had become the primary victims of cardiovascular disease. This was a new finding, pretty alarming. The Yentl movie came out. The Yentl syndrome, again, the editorial Dr. Bernadine Healy wrote. The Women's Ischemia Syndrome Evaluation, as you heard, I've had the honor to chair for now almost 30 years, the American Heart Association Heart Truth, these societies got to take action, 1984 through 1996, 12 years to recognize or take action. Of course, now we say if it had anything to do with erectile dysfunction, it would have gone a lot faster. But we were gratified that NHLBI dug in. So let's talk a little bit about the science. Of course, I'm going to have to give you a whirlwind tour, and we're going to hear a lot more about the science of endothelial function from our next speaker. But at that time, this cartoon really represented what we were dealing with. The male physician is seated and telling his female patient, we have studies of fruit flies, mice, hamsters, frogs, monkeys, and men with this condition. But as medical research using women as subjects just never occurred to anyone. It turned out that our study intensively, deeply phenotyping 1,000 women undergoing invasive coronary angiography for suspected coronary artery disease, we were able to describe a lot of very fundamental sex differences that then led to better understanding as well as clinical trials and improved outcomes. This is an example of one working with our uniformed health services pathologists, victims of sudden cardiac death related to myocardial infarction. We demonstrated for the first time that women erode their plaques where men explode. These heart attacks are very recognizable because there's a huge thrombus in the coronary artery. These heart attacks sometimes are hard to recognize, not as dramatic symptoms or EKG changes, and yet they distally embolize resulting eventually in microvascular dysfunction. We also demonstrated working with Cleveland Clinic for male controls that women and men deposited the fatty plaque in their coronary arteries in very sex-specific patterns. A male pattern is evidenced in the top, and this is, of course, what interventional cardiologists deal with every day. The luminogram is shown in red. The fatty plaque is shown in yellow. A second-year medical student could tell you where the blockage is and therefore, where you might place the stent. Women, on the other hand, in our study-wise, for the first time using intravascular ultrasound, demonstrated a very diffuse pattern of atherosclerotic fat. And yet, the perfusion was equally poor at the end of the artery. And again, this turns into what we now call microvascular coronary dysfunction. Now again, there probably are some hormonal and endocrine mechanistic pathways responsible for this. But if we think about where males deposit their visceral obesity, it is viscerally, where women are much more likely to have that cellulite very diffusely settled and underneath the skin. We culminated, again, in our first probably 10 years with describing some translational findings that because women, when they looked more like men, as Barbara does here in her Juntl movie, they were more likely to actually be treated because they were recognized. In guidelines therapy, aspirin, beta blocker, ACE, and statin would be deployed. Conversely, when women look like this microvascular dysfunction, obliteration of their capillaries, reactivity that is abnormal due to endothelial function, they more likely have this microvascular pattern, as Barbara looks here with her husband. And again, we and other large data sets could attribute the surplus of mortality observed since 1984 in women due to this lack of recognition and therefore lack of treatment. When you have diagnostic uncertainty, you will have therapeutic uncertainty as a treating physician. Moving on, and these, of course, is a whirlwind tour of the observations, we moved into policy pretty quickly. Again, we had an epidemic of death that was disproportionately impacting women. And clinical practice guidelines are very big in cardiovascular disease. Thankfully, it is the leading killer. Thankfully, we think we're doing something about it. Those identified in red here are those that we started to impact guidelines to the point where women would be either treated differently or treated appropriately. And indeed, we started seeing work of others looking in their registries. So here's an example of guideline implementation in acute coronary syndromes, threatened heart attacks or real heart attacks, and the sex survival gap. It's not particularly well-diagrammed here, so I've color-coded it for you. Following implementation of guidelines therapy, as we discussed, low-dose aspirin, statin, ACE, and beta blocker, the mortality improvement was seen exclusively in women. Very little mortality change in men. Why is that? Because they were being recognized already receiving guidelines therapy. The persistent sex gap suggests more work is needed to understand sex-specific pathophysiology to continue to improve outcomes in both women and men. And in fact, within this first decade and a half, we saw pretty dramatic turning of the tide from these American Heart Association and National Heart, Lung, and Blood stats. With the adoption of the Heart Truth Campaign, the American Heart Association, Red Dress, as well as these guideline campaigns, we saw a pretty dramatic 43% reduction in female mortality. Male mortality, of course, continued to decline. Well, do we have everything that we need to know at this point? Unfortunately not. The rates of awareness of heart disease in women, which initially doubled with these campaigns, clearly stalled. We have attention deficit disorder often with our public campaigns. We start to get distracted by something else, and we forget that heart disease is the leading killer of women. We continue to have important sex and gender gaps in our clinical trials. This is a review done by the Office of Research of Women's Health in the FDA, and you can see that those highlighted with the red bars, we continue to under-enroll women in diseases of the heart and vascular system that equally impact women and men, meaning this was prevalence corrected. Acute coronary syndrome, coronary artery syndrome, heart failure. So we have work to do. Here's one of my few endocrine slides. How about under-reporting of the sex of animals in basic science studies? Dominantly, a lot of science remains being done in male animals. Physiology, pharmacology, and endocrinology continue to have these gaps. And then a minority number of articles reporting data on women by year. Is this appropriate? Female data is scarce, as you can see, and this was all CAD, the leading killer of women and men. Seventeen percent of the articles compared treatment strategies. Now, the NIH also disproportionately uses a share of its resources to disease that primarily impact men at the expense of those that affect primarily women. This is data from a Lancet article that we put together most recently. And with these gaps, so you can see things that disproportionately impact women, those that disproportionately impact men, you can see, moving over to our NIH expenditure, that female-predominant diseases, exclusive or just more prevalent, are below the line in terms of prevalence funding, where things that predominantly impact men are more above the line, meaning more often funded. And this is an important issue. These are our federal tax dollars, and so recognition and understanding and then trying to make a difference on a policy standpoint. So going forward, we have identified science and we have advocacy as well as philanthropy campaigns. You probably heard some of my funding, particularly important for young investigators, is seed funding from women who care. We also work on policy, and we have a separate think tank. In addition to our research and publications, we work and target policies that will do a better job with precision medicine. And of course, as we know, education is key. Putting sex and gender into our medical curriculum will help eliminate disparities. And use of technology, as we've seen during this COVID pandemic, is likely a way to additionally address these knowledge gaps as well as barriers to care. This is a champion who has continued with us. She endowed our center for these missions and goals. I want to share with you just a bit of science that we try to make sure that everyone is aware and available. These are published methods that have gone out not only to our cardiovascular community, but our larger community of investigators increasingly interested in issues of sex and gender. You'll recognize that a lot of these are endocrine. And I would like to think that investigators and other physicians will reach out to you and ask you about these issues as they go forward with their science and their treating of patients with translational science. We clearly identify relatively simple steps. When you talk to the average cardiologist and you ask, do they have any idea about how the menstrual cycle might be impacting the care of their patient, their eyes glaze over and they look like they're about to faint. It's not that hard. And again, we provide these kinds of tools. The United States NIH has, to some extent, listened to some of this advocacy. Dr. Healy, of course, was responsible for clinical trials, including women and men. This was a mandate from Congress. The Women's Health Initiative was also started by Dr. Healy. These centers of excellence were funded under the Clinton administration. Many of them were defunded under the G.W. Bush administration. The Obama administration initiated our National Institute of Minority Health, as well as Precision Medicine. And again, under our director, Francis Collins, who recently stepped down, basic science studies must include male and female animals. Again, a congressional decree. So we're gonna see if this has important differences. We also, in our grant review criteria now for NIH, sex is a biological variable, S-A-B-Z-B, is now a mandatory inclusion criteria, and you must address these issues. Is testing sex differences a goal? Yes, you'll do interaction analyses, more highly powered. Are conclusions by sex without comparing a goal? Yes, you can sex stratify the data. If you're not interested in any of those, it's adjusting for confounding, which is most often seen in our journals. And hopefully, we'll be seeing better sex differences as we go forward. We're also working to educate physicians. Here's a national poll done by a professional group from the Women's Heart Alliance, and you can see that by polling primary care OBGYNs and cardiologists, only 40% included a heart risk check. 39% made cardiovascular disease a priority. Only 22 and 42% fell well-prepared. And so, these are important issues in physician education that the ACGME now is undertaking. We've also worked with this concept that advancing women's health through leadership in endowed chairs could make a difference. Women frequently drop out of academic medicine and are less likely to be in leadership positions. And we've published in Academic Medicine that across these initiatives to assess the current status of endowed chairs in women's health, we found that the number of chairs is growing and the consensus is that organized efforts to further increasing this number are warranted. We saw recently if an award has a woman's name on it, it is more likely to actually be awarded to a woman. This is our circle of translational care and again, from bench to bedside, we have the research concept, which would be the knowledge gap. We then develop the evidence base, which is needed for clinical guidelines. Again, guidelines make good doctors, great doctors, good hospitals, great hospitals. This then forms a clinical practice, outcomes are measured and we identify again as looking at the next mountain, the next knowledge gap. Well, with this bench to bedside approach than cardiovascular disease mortality trends, the most recent update or the one before the most recent update demonstrated as of 2013 that CBD mortality had approached parity in women and men. The question is, is this optimal for women? We recognize that pre-1984 women were less likely to die of cardiovascular disease than men. And so perhaps a lower threshold might be what we should be anticipated given the important knowledge of sex differences between women and men. And so here's actually our up-to-date numbers. And unfortunately, pre-pandemic mortality was rising in cardiovascular disease in both women and men and the pandemic deferred and delayed care that has been observed and well-described is expected to disproportionately impact cardiovascular disease mortality in women. Women were more likely to be fired or drop out of the workforce losing their insurance and Kaiser Family Foundation data demonstrate clearly that women's risk factors have risen disproportionately compared to men during the pandemic. So I'll close with that. I hope you feel invigorated about taking care of those cardiovascular disease patients in your practice as well as getting back to the bench or the clinical research area. I thank my Barbara Streisand Women's Heart Center, my Smith Heart Institute. And as you can see here, we gather annually. This was pre-pandemic. Our patients, volunteers are in the middle who participate in our cohort registries as well as our clinical trials, our faculty and our research team. Thank you for your attention. Thank you. Thank you, Dr. Barry Mertz. This afternoon's second plenary presentation will be by Dr. Philip Schall. Philip Schall is a professor of pediatrics and vice chair for research in Department of Pediatrics at UT Southwestern. He is a physician scientist whose laboratory is focused on endothelial cell biology in the context of cardiovascular and metabolic health and disease. His clinical expertise is in neonatal perinatal medicine, and his clinical activities entailed attending in one of the largest neonatal intensive care units in the US. His research has received longstanding support from the NIH, the American Heart Association, and numerous other organizations. Dr. Schall is the director for the Center of Pulmonary and Vascular Biology in the Department of Pediatrics. He's a co-program director of the NIH T32 training program in lung biology and disease and director of the Physician Scientist Training Program in Pediatrics. In addition, he leads Southwestern Medical School's Scholarly Activity Program. Dr. Schall is a holder of the Associates First Capital Corporation Distinguished Chair in Pediatrics, and he's a member of the Genetics Development and Disease Graduate Program. Dr. Schall was president of the Society for Pediatric Research in 2005 and 2006. Please join me in welcoming Dr. Schall as he discusses endothelial estrogen receptor signaling in cardiometabolic health and disease. Thank you so much, Dr. Wysham. And I'd like to definitely thank the organizers as well. It's absolutely a pleasure to have this opportunity to talk about the attempts in my laboratory to better understand hormone biology, particularly related to estrogen and actions of hormone receptors, particularly the estrogen receptor, and how they impact cardiovascular and metabolic health and disease. And the center of our attention today will be on the estrogen receptor in endothelial cells. And what I'd like to do is like to tell you two new stories. In the spirit of a meeting like this, these are entirely new stories. So when we consider the classical functions of estrogen receptors, we're thinking about estrogen binding to the receptor, the receptor binds to DNA and regulates gene expression. Well, for now quite a number of years, we've realized that there are some non-classical aspects of this biology. The first is that there are important actions of estrogen receptors outside the nucleus. And that's particularly the case in endothelial cells. And then secondly, there is a second ligand endogenously that we have to consider. And that is the abundant cholesterol metabolite, 27-hydroxycholesterol, or 27HC. So the two stories I'd like to tell you about today have to do with both of these non-classical, if you will, aspects of the biology. So the first story that I'd like to tell you relates to the endothelial receptor in metabolism. And at the outside, we were going to study adiposity. And the receptor in endothelium is known to promote angiogenesis and mediate anti-inflammatory processes. And if that happens in the adipose tissue, that would most certainly be considered to afford protection from adiposity. Well, not every hypothesis is correct. And it was not the case. The endothelial estrogen receptor did not impact adiposity. And I should mention, all these studies are in preclinical models. However, we found that it affords protection from insulin resistance. So the surprise about insulin resistance really came from studies that we first did in males. And these are mice that either have normal amounts of estrogen receptor alpha in the endothelium, so those are the phlox mice, or they have a deficiency selectively in the endothelial cells. And those are the delta EC. And when we simply measured fasting glucose, we found that the males without the receptor in the endothelium were hyperglycemic. And when doing glucose tolerance tests, the curve was shifted upward. And so they were glucose intolerant. And then when we evaluated their sensitivity to insulin, be it by quantifying HOMA-IR or by glucose infusion rates during clamps, we found that the mice were also insulin resistant. We certainly, importantly, did studies in females as well. We over-activized the mice and put them on high-fat diets and then treated them with either vehicle or estradiol. And what we found in the GTT, the glucose tolerance test, was quite dramatic. Now, it's well known in control mice, which are the phlox mice, that if they receive estrogen replacement, there is a dramatic improvement in their glucose tolerance. However, in the mice that lack the receptor in endothelium, there was a total loss of the antidiabetic actions of estrogen. And the same was the case in insulin tolerance tests. In the control mice, if they are given estradiol, there is an improvement in insulin sensitivity. It doesn't happen when the mice lack the receptor solely in the endothelium. So that was curious. So we had to then figure out, well, why? Why would that be the case? And it ends up that the answer is in the skeletal muscle. And the key defect in both groups of mice was actually an impairment in glucose disposal in the muscle. So here in the males, there's a decrease in glucose disposal in the mice lacking the receptor in the endothelium. And in the four groups of mice that were female, in the control mice, there is an enhancement of glucose disposal with estradiol treatment. But that simply is absent in the mice lacking the receptor. So that's curious. We're manipulating a mechanism in the endothelium, yet it's affecting actions in the muscle. So we decided we're going to have to learn more about insulin action in this scenario. And we looked at insulin signaling in the muscle. And one way to do that is to give the mice a dose of a vehicle or insulin, and then look at insulin signaling by evaluating the activation of AKT by evaluating its phosphorylation. And in the control mouse, that happens quite avidly. But that's blunted in the mice that have a loss of the receptor in their endothelium. And that, again, was unclear why that would happen. Because again, our manipulation of the receptor is in the endothelial cells, not in the myocytes. Well, it ends up that the answer is in insulin delivery to the muscle. And so instead of in the control mouse where you administer insulin it very rapidly goes to the muscle, that is dramatically blunted in the mice lacking the receptor. So it's a delivery issue. Now what regulates insulin delivery to the muscle? Well there are really two main aspects of that. The first is that insulin itself actually promotes capillary recruitment in the muscle and that enhances insulin delivery to the muscle and ensues an improvement in glucose disposal. And then there's a second mechanism which is the fact that the insulin that is in the microvasculature of the muscle has to make a journey across the endothelial cell layer to get to the myocytes. And that's a very regulated journey. So we did studies in mice where we can evaluate capillary recruitment in the muscle using ultrasonography and there was no difference between the two groups. So what about this insulin transport? Does estrogen possibly affect that? Well one of the best ways to look at that truly visually is to take cultured endothelial cells, be they from the mouse skeletal muscle or from other vascular sites, and take fluorescent tagged insulin and look at its uptake, the first step of transport across the endothelium. And what we found was that there was a dramatic enhancement of this insulin uptake with estradiol treatment, about a tenfold increase. The other journey that we can follow is the completion of that movement across the endothelium and that process is known as transcytosis and we can evaluate this in tight monolayers of endothelial cells. And we found that estradiol very avidly drives the insulin across the endothelium and usually either with genetic manipulation of the ester receptor alpha in the endothelium or with a pharmacological inhibitor, we see an attenuation in that effect of estradiol. So with a non-genomic centric view of the world, the first question that came to mind to us was whether this mechanism might actually involve non-nuclear actions of estrogen receptors. And one major function that occurs within endothelial cells is to activate the kinase, PI3 kinase. And so we used pharmacological inhibitor of that kinase and found that that completely prevented the estradiol promotion of the insulin transport. So the next question was, okay, it looks like this non-nuclear signaling is required. Is it sufficient? And so the sufficient question was answered by comparing the effects of estradiol versus the estrogen-dendrimer conjugate, which is a molecule that only selectively activates the non-nuclear receptors. And the EDC had no impact on insulin transport, certainly in contrast to estradiol. And so when we put this data together, we thought that this would suggest that it both involves non-nuclear as well as nuclear actions of the estroceptor for this promotion of the transport of insulin. So we certainly want to now learn, well, how could that possibly happen? And we decided that we're going to have to interrogate both nuclear mechanisms as well as non-nuclear mechanisms and do it in a very open fashion. So the first thing we tried to do was to look at the estrogen response of translatome in the endothelial cells in the muscle and actually do it in vivo. And so what we did is we generated mice that have a ribosomal protein that is tagged and expressed only in the endothelial cells. And then we took female mice and over-rectomized them, treated them with vehicle estradiol, and then isolated the muscle. And then from the muscle, we can immunoprecipitate the ribosomes only from the endothelial cells, and then we performed RNA-seq. And what we found is that in response to estradiol treatment of the mice, there were a large number of genes whose translation was increased, and there was actually a large number where it was decreased. Now what we're going to focus in on is the responses that were positive in response to estradiol treatment, again in vivo. Then the other two data sets that we wanted to bring to this were to look at the proteins that interact with the ester receptor in endothelial cells in response to estradiol treatment. And that was done by taking cultured endothelial cells, treating them with vehicle or estradiol, immunoprecipitating the receptor, and then using mass spectrometry to identify the proteins that are interacting with the receptor. And the green circle represents a large number of proteins that were recruited to the ester receptor in culture in response to E2. And then the third data set that we evaluated was to take cells in culture, treat them with vehicle or estradiol, and look at the changes in messenger RNA that occur. And so we put those three different data sets together, and it ends up that the Venn diagram gave us five genes or gene products that are upregulated in their expression as well as recruited to the estrogen receptor in the setting of estradiol treatment. And then what really caught our attention was a protein called SORTIN-NEXIN-5. And on the next slide, what I'd like to do is summarize what we learned about SORTIN-NEXIN-5 and its role in this process. And it caught our attention because SORTIN-NEXIN-5 is a vesicular trafficking regulator in other contexts. And so what we found is that the underpinnings of this promotion of insulin delivery to the muscle involves both the nuclear effects of estradiol through estroreceptor alpha to upregulate the expression of the SORTIN-NEXIN-5 and activation of the non-nuclear population of receptors to also recruit the SORTIN-NEXIN-5 to the plasma membrane. And the combination of those mechanisms then drives the uptake of the insulin into the endothelial cell and its ultimate journey all the way across. And this then promotes the delivery of the insulin to the skeletal muscle myocytes and therefore promotes glucose disposal. And we would suggest that this is now an important anti-diabetic action, if you will, of estrogens that we have to recognize. Okay. Now I'd like to turn to the second story. And this happens to do with that other ligand that I mentioned at the beginning, the cholesterol metabolite 27-hydroxycholesterol or 27HC. And then the common thread is the endothelial ER alpha. So this story really began with an interest in the production of the 27HC in macrophages. And in macrophages, they receive cholesterol from LDL. And then there's an enzyme, CYP27A1, that converts the cholesterol to 27HC. And preclinical, excuse me, in vitro studies had suggested that this conversion of cholesterol to 27HC is going to allow cholesterol to leave the macrophage and therefore it would be anti-athrogenic. So that was our initial hypothesis. This conversion of cholesterol to 27HC, again, an estroceptor ligand, by the enzyme CYP27A1 in the macrophages would promote the cholesterol removal and therefore be anti-athrogenic. Well, I hate to admit it, but this is the second time we were wrong. And that was not the case. The surprise was that the cholesterol conversion to 27HC in the macrophage is actually pro-athrogenic. So the first studies that showed us that are studies where we generated mice that either had the normal amount of CYP27A enzyme in their macrophages, those are the floxed mice, or we generated mice that lack the enzyme only in the macrophages, the Delta MAC, put them on a hypercholesterolemic background and fed them a hypercholesterolemic diet and looked at their atherogenesis. And what I'm showing here are images of the degree of lesion formation at the aortic root. I think you can see the leaflets of the aortic valves here. And again, the initial surprise was that simply by deleting this enzyme from the macrophage, there was actually less severe atherosclerosis. Now why is that? Well, it ends up that the main reason for that is that there's a dramatic impact on the accumulation of macrophages in the artery wall. And I think you will all remember that really the main processes of atherogenesis are two. You have to have hypercholesterolemia, and the cholesterol has to make its way into the artery wall. And then monocytes have to be recruited there to become macrophages that take up the cholesterol. Everybody becomes foam cells, and it drives the disease. Well the main impact of deleting this enzyme from the macrophage and monocyte is to have a dramatic impact on this accumulation of the macrophages in the lesion. And I think we could interpret this to say that actually the majority of recruitment of the macrophage into the artery wall is actually driven by this process. So we then had to, of course, try to understand, well, why could that happen? And this recruitment of the macrophage into the artery wall involves the activation of the endothelium. And what activates the endothelium in the setting of hypercholesterolemia? It's really not well understood. So we decided to look at the effect of this deletion of the macrophage enzyme on vascular inflammation by performing intravital microscopy, where we're going to visualize fluorescent tag leukocytes and their adhesion in a microvascular setting. And so I'm going to go ahead and show you a video here. And we're looking at fluorescent tag leukocytes that are adherent to this small vessel wall. And I should mention these mice are hypercholesterolemic, and they have a normal amount of this enzyme in their macrophages. And I think you can appreciate that a large number of macrophages will adhere. And then if they do adhere, they roll quite slowly. And then in contrast, let's take a look at the video for the mouse that is otherwise the same but lacks CYP27A1 in the macrophages. I think you can appreciate there's far less adhesion. And those leukocytes that are adhering are rolling much faster. So this enzyme in the macrophages is activating the endothelium to promote vascular inflammation. OK, well, how is this happening? Well, we decided we're going to have to test the possibility that this may actually involve the actions in the endothelium that are mediated by estrogen receptor alpha. And is there actually a crosstalk between the macrophage and the endothelial cell? And the messenger might possibly be 27HC. And so we performed bone marrow transplant experiments where the donor mouse either had normal CYP27A1 in the macrophages or lacked the enzyme in the macrophages. And then the recipient mouse was either going to have the estrogen receptor alpha in the endothelium or not. So we're going to end up with four conditions. We have mice that don't have the enzyme in the macrophage, and they don't have the receptor in the endothelium. Or they have one or the other. Or they have both. And this is really the wild type mouse, if you will, at the bottom. And then we looked at their severity of atherosclerosis. And if the starting point is mice that don't have any enzyme in the macrophage and no receptor in the endothelium, if we provide back one or the other of those components, there's no enhancement of atherosclerosis. It's only when we have both. So it's only when the enzyme can produce 27HC in a mouse that has the receptor in the endothelium that the atherosclerosis is driven. Now what about that major mechanism of accumulation of the macrophages? Well that showed the same pattern. It was really in mice that had both the enzyme producing the 27HC from the macrophage and endothelium that had estrogen receptor alpha that showed an enhancement in the macrophage accumulation in the artery wall. So we think that we found a crosstalk between the macrophages and the endothelium that is promoting the vascular inflammation component of atherosclerosis. Now how does that happen? Well we decided we're going to have to look at partner proteins possibly of the estrogen receptor in the endothelium. And so we performed an experiment where we did this interactome analysis once again. And in this case we took endothelial cells and treated them either with estradiol or with 27HC and we saw a large number of proteins that were either recruited or disassociated from the receptor in response to the two different ligands. And once again there was a protein that really grabbed our fancy and that is septin 11. And this protein is actually recruited to estrogen receptors when they're bound by estradiol but it's disassociated from the receptor when the ligand is 27HC. So we then embarked on cell culture studies to evaluate the requirement for this protein in the ability of 27HC to promote an activation of the endothelium to make it pro-inflammatory and that was indeed the case. And with that in hand we decided we need to go ahead and test this in vivo and we generated mice that lack the septin 11 only in their endothelium and did the bone marrow transplant experiment once again. So we have mice that don't have the enzyme in the macrophage or they don't have the partner protein in the endothelium or they have only one of those components or they have both. And what we found is that it takes both. There's got to be the enzyme making 27HC and there's got to be the partner protein in the endothelium for there to be an enhancement of androgenesis, atherogenesis, excuse me. And then what about that key aspect, that being the macrophage accumulation? Well really that is driven when you have the enzyme being produced in the macrophage and you have the partner protein in the endothelium. So we believe that we have indeed found an interesting crosstalk between the monocyte macrophage and the endothelium and actually a unique mode of NF kappa B activation as well. So in this schematic here I'm going to summarize some experiments that we did with purified proteins and other biochemical approaches. And what we found is that when 27HC binds to the non-nuclear ester receptors in an endothelial cell, the septin-11 is disassociated to be recruited to a kinase called MKK7 and that causes the disassociation of an inhibitor protein called GAD45 beta. That results in the activation of the MKK7 that inactivates junk and then junk leads to the activation of NF kappa B which very avidly will upregulate the expression of key adhesion molecules such as ICAM1 and VCAM1. And so we think we've figured out how hypercholesterolemia actually causes the vascular inflammation that is required to partner with the hypercholesterolemia in order to drive the disease. So is there anything we can do about it? Well, we decided to look at the effect of inhibiting this enzyme, CYP27A1, pharmacologically in mice that are going to be developing atherosclerosis. And we took a pharmacologic inhibitor that's abbreviated GW and even partway through the development of atherosclerosis we treated mice either with the inhibitor or with vehicle and we found that the inhibitor treatment even without having any impact on circulating lipids decreased the atherosclerosis development. And we even did another experiment where we had three groups of mice, vehicle treated, inhibitor treated, and then inhibitor treated in a group in which we provided exogenous 27HC back to the mice. And with that provision back of 27HC we found an increase in the lesion severity back to the baseline. And this sort of helps strengthen the fact that the effect of the inhibitor was actually through its impact on the production of 27HC. And then what about that key mechanism that I think is relevant to this set of processes? The macrophage accumulation in the lesion is really what is blunted by the inhibitor by about 50%. Now admittedly we're very excited about this because there is a big drive these days to try to come up with anti-inflammatory interventions to try to take care of the considerable residual in cardiovascular disease risk that still is present despite lipid lowering therapies. And I would suggest instead of hitting some major pathway of immune response, let's go ahead and inhibit the process that hypercholesterolemia itself is causing to cause the vascular inflammation that needs to partner with the hypercholesterolemia to drive the disease. So let me finish by just summarizing what admittedly were two surprises. The first in the context really of insulin sensitivity and type 2 diabetes is that through both non-genomic and genomic processes that endothelial ER alpha actually promotes peripheral insulin sensitivity and it does so by enhancing insulin delivery to the skeletal muscle. And then in the setting of atherosclerosis, it's primarily through non-genomic processes that that 27HC derived from the macrophage and endothelial ER alpha partner in a crosstalk between those two cell types and that we would suggest that that underlies how hypercholesterolemia promotes the vascular inflammation that together partner to drive atherosclerosis. So lastly and most importantly, I want to recognize individuals that I'm blessed to work with every day and who are responsible for this work. My scientific partner at UT Southwestern is Dr. Cheiko Mineo. The studies of glucose homeostasis were driven by Anastasia Sacradoo and Ken Chambliss and other members of the lab. And then the studies of atherosclerosis were done by Li-Ming Yu and Lin-Zhang Huang. And I want to highlight contributions really to both stories by Jun Peng. And I want to give a plug for Jun's oral presentation midday tomorrow where Jun is going to tell you about an entirely new major mechanism that impacts glucose homeostasis. So please look up Jun Peng and join him at his presentation. I have fantastic collaborators at UT Southwestern as well as a number of other institutions. And I want to thank you for listening. I look forward to your questions after the session, I imagine. And if anybody wants to learn more about how we try to learn more about how endocrinology impacts the health of both men and women, both cardiovascular and metabolic health, please visit us. Check us out. Take advantage of the code down here at the bottom. Thank you very much. Thank you. Oh, thank you, Dr. Schall. I'm so appreciative to today's amazing speakers and to all of you who joined this plenary. Please enjoy the rest of your day at ENDO 2022. And please join us for tomorrow morning's plenaries. Have a good day.

Dr. Carol Wysham

cardiovascular health

women

Endocrine Society

2022 laureate award winners

Dr. Lourdes Ibáñez

International Excellence in Endocrinology Award

polycystic ovary syndrome

adolescents

children