Background: HIF pathway is quickly activated during myocardial ischemia after myocardial infarction(MI), and cardiac microvascular leakage contributes to heart tissue damage. HIF2α isprofoundly expressed in cardiac endothelial cells (ECs) and the embryonic deletion of HIF2Aresults in increased vascular permeability and aberrant ECs behavior. However, the direct roleof endothelial cell-specific HIF2α (ecHIF2α) in ischemic heart disease is not known. Wehypothesized that ecHIF2α expression in response to myocardial infarction (MI) is protectiveagainst heart failure through the reduction of cardiac ECs apoptosis and inflammation. Methods and Results: To address our hypothesis, we generated EC-specific inducible-HIF2α knockout mice (ecHIF2α -/- ) by crossing Hif2a flox/flox mice with Cre ERT2 mice. To assess the functional role of HIF2α inischemic heart injury, we ligated the proximal left anterior descending coronary artery to induceMI using the same age and gender-matched ecHIF2α -/- and control ( Hif2a flox/flox ) mice. Cardiacfunction was determined by echocardiography after two and four weeks of ligation. Analysis ofechocardiography revealed worsened heart function, and Masson’s Trichrome stain displayedincreased fibrosis in ecHIF2α -/- mice. In vitro , ECIS analysis of isolated cardiac microvascularendothelial cells showed decreased endothelial barrier function in ecHIF2α -/- cells. In addition,hypoxic stimulation reduces the tube formation capacity in ecHIF2α-/- cells and is sensitive tohypoxia-induced early-stage apoptosis. Deletion of HIF2α, as well as its binding partner ARNT,increased the expression of several inflammatory genes, including IL-6. Interestingly,overexpression of ARNT alone abolishes the HIF2α deletion-induced inflammatory geneexpression. IL-6 protein levels in HIF2α deleted human aortic endothelial cells (HAoEC) show asignificant reduction (n=3-5, p<0.001) in ARNT overexpressed ECs. Conclusion: Collectively our data revealed an essential role of endothelial HIF2α/ARNT in maintaining cardiacfunctions by increasing endothelial barrier function and decreasing inflammation. Therefore,HIF2A/ARNT could provide a potential therapeutic target for the treatment of ischemic heartdisease.

Pharmacology Research & PerspectivesVolume 10, Issue 4 e00988 INVITED REVIEWOpen Access A practical treatment for COVID-19 and the next pandemic Jahar Bhattacharya, Jahar Bhattacharya Department of Physiology & Cellular Biophysics, Vagelos Columbia College of Physicians & Surgeons, New York, New York, USASearch for more papers by this authorRobert Booy, Robert Booy Department of Child and Adolescent Health, University of Sydney Medical School, Camperdown, New South Wales, AustraliaSearch for more papers by this authorArturo Casadevall, Arturo Casadevall Department of Molecular Biology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USASearch for more papers by this authorCharles Dela Cruz, Charles Dela Cruz Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USASearch for more papers by this authorDavid S. Fedson, Corresponding Author David S. Fedson davidsfedson@gmail.com orcid.org/0000-0001-9963-1934 Sergy Haut, France Correspondence David S. Fedson, 57 chemin du Lavoir, 01630 Sergy Haut, France. Email: davidsfedson@gmail.comSearch for more papers by this authorJoe G. N. Garcia, Joe G. N. Garcia Department of Medicine, University of Arizona College of Medicine, Tucson, Arizona, USASearch for more papers by this authorGary Grohmann, Gary Grohmann Director of Immunobiology, Therapeutic Goods Administration, Canberra, AustraliaSearch for more papers by this authorIvan F. N. Hung, Ivan F. N. Hung Department of Medicine, University of Hong Kong, Pokfulam, Hong KongSearch for more papers by this authorJeffrey R. Jacobson, Jeffrey R. Jacobson Department of Medicine, University of Illinois College of Medicine-Chicago, Chicago, Illinois, USASearch for more papers by this authorLance C. Jennings, Lance C. Jennings Department of Pathology & Biomedical Science, University of Otago, Dunedin, New ZealandSearch for more papers by this authorLester Kobzik, Lester Kobzik Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USASearch for more papers by this authorAleksandra Leligdowicz, Aleksandra Leligdowicz Toronto General Research Institute, University of Toronto, Toronto, CanadaSearch for more papers by this authorJames K. Liao, James K. Liao Department of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Illinois, USASearch for more papers by this authorJennifer H. Martin, Jennifer H. Martin Discipline of Clinical Pharmacology, University of Newcastle School of Medicine and Public Health, Callaghan, New South Wales, AustraliaSearch for more papers by this authorDaniel M. Musher, Daniel M. Musher Department of Medicine, Baylor College of Medicine, Houston, Texas, USASearch for more papers by this authorCharles N. Serhan, Charles N. Serhan Department of Anesthesiology, Perioperative & Pain Medicine, Mass General Brigham-Harvard Medical School, Boston, Massachusetts, USASearch for more papers by this authorMasato Tashiro, Masato Tashiro National Institute of Infectious Diseases, Tokyo, JapanSearch for more papers by this author Jahar Bhattacharya, Jahar Bhattacharya Department of Physiology & Cellular Biophysics, Vagelos Columbia College of Physicians & Surgeons, New York, New York, USASearch for more papers by this authorRobert Booy, Robert Booy Department of Child and Adolescent Health, University of Sydney Medical School, Camperdown, New South Wales, AustraliaSearch for more papers by this authorArturo Casadevall, Arturo Casadevall Department of Molecular Biology & Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USASearch for more papers by this authorCharles Dela Cruz, Charles Dela Cruz Department of Medicine, Yale School of Medicine, New Haven, Connecticut, USASearch for more papers by this authorDavid S. Fedson, Corresponding Author David S. Fedson davidsfedson@gmail.com orcid.org/0000-0001-9963-1934 Sergy Haut, France Correspondence David S. Fedson, 57 chemin du Lavoir, 01630 Sergy Haut, France. Email: davidsfedson@gmail.comSearch for more papers by this authorJoe G. N. Garcia, Joe G. N. Garcia Department of Medicine, University of Arizona College of Medicine, Tucson, Arizona, USASearch for more papers by this authorGary Grohmann, Gary Grohmann Director of Immunobiology, Therapeutic Goods Administration, Canberra, AustraliaSearch for more papers by this authorIvan F. N. Hung, Ivan F. N. Hung Department of Medicine, University of Hong Kong, Pokfulam, Hong KongSearch for more papers by this authorJeffrey R. Jacobson, Jeffrey R. Jacobson Department of Medicine, University of Illinois College of Medicine-Chicago, Chicago, Illinois, USASearch for more papers by this authorLance C. Jennings, Lance C. Jennings Department of Pathology & Biomedical Science, University of Otago, Dunedin, New ZealandSearch for more papers by this authorLester Kobzik, Lester Kobzik Department of Environmental Health, Harvard T.H. Chan School of Public Health, Boston, Massachusetts, USASearch for more papers by this authorAleksandra Leligdowicz, Aleksandra Leligdowicz Toronto General Research Institute, University of Toronto, Toronto, CanadaSearch for more papers by this authorJames K. Liao, James K. Liao Department of Medicine, University of Chicago Pritzker School of Medicine, Chicago, Illinois, USASearch for more papers by this authorJennifer H. Martin, Jennifer H. Martin Discipline of Clinical Pharmacology, University of Newcastle School of Medicine and Public Health, Callaghan, New South Wales, AustraliaSearch for more papers by this authorDaniel M. Musher, Daniel M. Musher Department of Medicine, Baylor College of Medicine, Houston, Texas, USASearch for more papers by this authorCharles N. Serhan, Charles N. Serhan Department of Anesthesiology, Perioperative & Pain Medicine, Mass General Brigham-Harvard Medical School, Boston, Massachusetts, USASearch for more papers by this authorMasato Tashiro, Masato Tashiro National Institute of Infectious Diseases, Tokyo, JapanSearch for more papers by this author First published: 15 July 2022 https://doi.org/10.1002/prp2.988 Gary Grohmann, Lester Kobzik, and Masato Tashiro: Retired. AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Scientists are concerned about the origins of SARS-CoV-2, which has caused a devastating global pandemic. In a letter recently published in Science, 18 investigators called for more studies of its origins.1 In 2001 and in a different context, Malcom Gladwell wrote "it is a strange kind of public health policy that concerns itself more with the provenance of illness than with its consequences …".2 Studies on the origins of SARS-CoV-2 are unlikely to be undertaken, primarily for political reasons. As a result, investigators can only speculate on the origins of SARS-CoV-2 and its many variants: delta and more recently omicron.3, 4 Scientists and health officials believe we will eventually face another pandemic. Its impact on global mortality could be worse than the more than 20 million or more excess deaths we have experienced thus far.5 Highly effective COVID-19 vaccines have been developed, but vaccine nationalism and vaccine hesitancy have meant they have been used largely in developed countries. Scientists also hope to develop better antiviral drugs. Pharmaceutical companies have developed two new antivirals that appear to reduce the development of serious disease when given to patients when they first test positive for SARS-CoV-2. However, these drugs will be expensive for people living in developed countries and they will not be available in developing countries anytime soon. Moreover, antiviral resistance will always be a threat. For the next pandemic, as with the current COVID-19 pandemic, it is unlikely that people in resource-poor countries (where most deaths will occur) will ever get new vaccines and antivirals in time to significantly reduce mortality.6 Instead of counting on vaccines and treatments that target a newly emergent pandemic virus, we could also target the host response to infection and excessive inflammation using inexpensive repurposed generic drugs. This idea was suggested more than a decade ago.7 Generic drugs are inexpensive and most of them are familiar to practicing physicians everywhere. They would be available on the first pandemic day in any country with a basic healthcare system.6 Several generic drugs already meet these criteria. Severe COVID-19 is dominated by endothelial dysfunction,8, 9 which is associated with immunological dysfunction and often immunothrombosis.10, 11 For COVID-19, most of the attention on repurposed generic drugs for host response treatment has focused on statins, ACE inhibitors, and angiotensin receptor blockers (ARBs). These drugs help maintain or restore endothelial barrier integrity.12 They are safe to use in patients with acute critical illness. Dexamethasone is another inexpensive generic drug that reduces COVID-19 mortality in patients who require oxygen treatment with or without mechanical ventilation.13 It is not effective in those not receiving respiratory support. Early treatment with selective serotonin reuptake inhibitors (SSRIs) is also effective in reducing symptomatic COVID-19.14 It is uncertain whether supplies of SSRIs and physician familiarity with this group of drugs will ever be sufficient to allow them to be widely used in resource-poor countries. Most studies of statin treatment of COVID-19 are based on outpatient documentation, which does not account for statin withdrawal after hospital admission.15, 16 However, in all but one of 13 observational studies, inhospital statin treatment reduced COVID-19 mortality (Table 1).15, 17-19 Perhaps more important, inhospital treatment with a combination of a statin and either an ACE inhibitor or an ARB was associated with a threefold reduction in the risk of 28-day COVID-19 mortality.19 This is not surprising. In an earlier study, combination statin/ACE inhibitor treatment was associated with reduced inflammatory biomarkers in patients with coronary artery bypass surgery.20 TABLE 1. Observational studies of the reduction in 28–30-day COVID-19 mortality following inhospital statin treatment and statin withdrawal. Adapted from reference15 First author Adjusted (ref) Methods HR/OR 95% CI p value Zhang15 CCS, PSM (4:1) 0.58 0.43–0.80 .001 Rodriguez-Nava15 cohort, ICU 0.38 0.18–0.77 .008 Mallow15 Cohort 0.54 0.49–0.60 <.001 Saeed15 Diabetes mellitus, multivariate adjusted 0.51 0.43–0.61 .001 PM, IPTWa a The PS-matched IPTW cohort analysis included demographic and comorbidity factors, clinical and laboratory test values, and the use of ACE inhibitors and angiotensin receptor blockers. 0.88 0.84–0.91 <.001 Masana15 GM (1:1) 0.60 0.39–0.92 .020 Fan15 cohort, PSM 0.25 0.07–0.92 .037 Torres-Pena15 PSM, statins continued versus withdrawalb b Statin treatment continued after hospital admission versus statin withdrawal; conditional logistic regression. 0.67 0.54–0.84 <.001 Memel15 Marginal structural Cox model, IPTW 0.57 0.37–0.86 .008 Statins continued versus withdrawalc c Statin treatment continued after hospital admission versus statin withdrawal; marginal structural Cox model. 0.27 0.11–0.64 .003 Byttebier15, 19 CCS, PSM (1:1) 0.56 0.39–0.93 .020 Terlecki15 Logistic regression 0.54 0.33–0.84 .008 Lohia15 Cohort, PSM (1:1) 0.47 0.32–0.70 <.001 Choi17 Cox model, high-intensity statin 0.53 0.43–0.65 Not done Ayeh18 Cox proportional regression 0.92 0.53–1.59 Not significant Kuno19 Statins continued versus withdrawal, PSM (l:1) 0.53 0.41–0.62 <.001 Abbreviations: CCS, case–control study; CI, confidence interval; GM, genetic-matched; HR, hazard ratio; ICU, intensive care unit; IPTW, inverse probability treatment weighted; OR, odds ratio; PSM, propensity score-matched. a The PS-matched IPTW cohort analysis included demographic and comorbidity factors, clinical and laboratory test values, and the use of ACE inhibitors and angiotensin receptor blockers. b Statin treatment continued after hospital admission versus statin withdrawal; conditional logistic regression. c Statin treatment continued after hospital admission versus statin withdrawal; marginal structural Cox model. We (a group of 17 clinicians and investigators) believe that greater emphasis should be given to testing treatment with generic statins, ACE inhibitors, or ARBs (either by themselves or in combination) during the current COVID-19 pandemic. These studies could include randomized controlled trials,21 although they should not discount the value of observational studies.22 Because these drugs target the host response, they could also be tested against everyday critical illness (sepsis, community-acquired pneumonia, and seasonal influenza). For COVID-19, they might even be used for patient treatment before this research is undertaken.23 We believe that treating patients with these repurposed generic drugs could help reduce mortality during the current and all future pandemics.6 Undertaking studies of this idea before the next pandemic might provide convincing evidence that these drugs could reduce its mortality. A few randomized controlled trials of these drugs have been undertaken in COVID-19 patients, but their results will not be reported for some time. (When the results of statin trials become available, investigators should be able to identify hyperinflammatory subphenotypes that might show improved survival with statin treatment.24, 25) In the meantime, thousands will continue to die of COVID-19 every day. This includes patients in high-income countries, some of whom have received three or more doses of COVID-19 vaccines.26 For patients who live in resource-poor countries, the social and economic consequences of the COVID-19 pandemic have been extraordinarily severe and might dwarf those of the disease itself.27 For patients and physicians in these countries, "… there is no guarantee that treating the host response will be effective, but we urgently need to find out. If a highly virulent and easily transmissible pandemic … virus emerges, it will spread rapidly throughout the world, overwhelming healthcare systems everywhere. In the absence of pandemic vaccines and effective antiviral treatments, the only way, physicians might reduce pandemic mortality will be to treat seriously ill patients with easily administered inexpensive generic drugs that are already available and that modify the host response to infection. … (T)he challenge of the next pandemic must make this the central element of preparedness planning".28 In 2001, Malcolm Gladwell also wrote that many threats to health and happiness "are the result of what, through simple indifference, we do to ourselves".2 We were unprepared for COVID-19 and are still not prepared to reduce global mortality during the next pandemic. The World Health Organization and non-governmental organizations and foundations have been disinterested in sponsoring laboratory and clinical studies of these generic drugs to treat the host response. This represents a failure of both scientific and political imagination.6 We believe this must change. ACKNOWLEDGMENTS This article contains no new data. All co-authors agree with its contents. They declare no conflicts of interest related to this article. REFERENCES 1Bloom JD, Chan YA, Baric RS, et al. Investigate the origins of COVID-19. Science. 2021; 372: 694. CrossrefPubMedWeb of Science®Google Scholar 2Gladwell M. The Scourge You Know. The New Yorker; 2001. Google Scholar 3Worobey M. Dissecting the early COVID-19 cases in Wuhan. Science. 2021; 374: 1202- 1204. CrossrefCASPubMedWeb of Science®Google Scholar 4Callaway E. Beyond omicron: what's next for COVID's viral evolution. Nature. 2021; 600: 2047- 2048. CrossrefWeb of Science®Google Scholar 5Adam D. The effort to count the pandemic's global death toll. Nature. 2022; 601: 312- 315. CrossrefCASPubMedWeb of Science®Google Scholar 6Fedson DS. COVID-19, host response treatment, and the need for political leadership. J Public Health Policy. 2021; 42: 6- 14. CrossrefPubMedWeb of Science®Google Scholar 7Fedson DS. Pandemic influenza: a potential role for statins in treatment and prophylaxis. Clin Infect Dis. 2006; 43: 199- 205. CrossrefCASPubMedWeb of Science®Google Scholar 8Libby P, Luscher T. COVID-19 is, in the end, an endothelial disease. Eur Heart J. 2020; 41: 3038- 3044. CrossrefCASPubMedWeb of Science®Google Scholar 9Nicosia RF, Ligresti G, Caporarello N, Akilesh S, Ribatti D. COVID-19 vasculopathy: mounting evidence for an indirect mechanism of endothelial injury. Am J Pathol. 2021; 191: 1374- 1384. CrossrefCASPubMedWeb of Science®Google Scholar 10Bonaventura A, Vecchie A, Dagna L, et al. Endothelial dysfunction and immunothrombosis as key pathogenic mechanisms in COVID-19. Nat Rev Immunol. 2021; 21: 319- 329. CrossrefCASPubMedWeb of Science®Google Scholar 11Gu SX, Tyagi T, Jain K, et al. Thrombocytopathy and endotheliopathy: crucial contributors to COVID-19 thromboinflammation. Nat Rev Cardiol. 2021; 18: 194- 209. CrossrefCASPubMedWeb of Science®Google Scholar 12Fedson DS. Treating emerging virus diseases: lessons learned from sepsis, pneumonia, influenza and Ebola. Ann Transl Med. 2016; 4: 421. CrossrefPubMedWeb of Science®Google Scholar 13 RECOVERY Collaborative Group, Horby P, Lim WS, et al. Dexamethasone in hospitalized patients with Covid-19. N Engl J Med. 2021; 384: 693- 704. CrossrefPubMedWeb of Science®Google Scholar 14Reis G, Moreira-Silva EADS, Silva DCM, et al. Effect of early treatment with fluvoxamine on risk of emergency care and hospitalisation among patients with COVID-19: the TOGETHER randomised, platform clinical trial. Lancet Glob Health. 2021; 10: e42- e51. CrossrefPubMedWeb of Science®Google Scholar 15Fedson DS. 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Validation of inflammopathic, adaptive, and coagulopathic sepsis endotypes in coronavirus disease 2019. Crit Care Med. 2021; 49: e170- e178. CrossrefCASPubMedWeb of Science®Google Scholar 25Calfee CS, DeLucchi KL, Sinha P, et al. Acute respiratory distress syndrome subphenotypes and differential response to simvastatin: secondary analysis of a randomised controlled trial. Lancet Respir Med. 2018; 6: 691- 698. CrossrefCASPubMedWeb of Science®Google Scholar 26Barda N, Dagan N, Cohen C, et al. Effectiveness of a third dose of the BNT162b2 mRNA COVID-19 vaccine for preventing severe outcomes in Israel: an observational study. Lancet. 2021; 398: 2093- 2100. CrossrefCASPubMedWeb of Science®Google Scholar 27Appiah KA. A tale of two pandemics: the true cost of Covid in the global south. The Guardian. 2021. Accessed December 3, 2021. https://www.theguardian.com/world/2021/nov/23/a-tale-of-two-pandemics-the-true-cost-of-covid-in-the-global-southGoogle Scholar 28Fedson DS. Influenza pandemic preparedness: a special challenge for India. Ind J Med Res. 2019; 150: 217- 220. CrossrefPubMedWeb of Science®Google Scholar Volume10, Issue4August 2022e00988 ReferencesRelatedInformation

Over the past four decades, no class of drugs has had more impact on cardiovascular health than the HMC-CoA reductase inhibitors or statins. Developed as potent lipid-lowering agents, statins were shown to reduce mortality and morbidity of patients who are at risk for cardiovascular disease. However, retrospective analyses of some of these clinical trials have uncovered some aspects of their clinical benefits that may be additional to their lipid-lowering effects. In cell culture and animal studies, these effects alter the expression of endothelial nitric oxide synthase, the stability of atherosclerotic plaques, the production of pro-inflammatory cytokines and reactive oxygen species, the reactivity of platelets, and the development of cardiac hypertrophy and fibrosis. Such cholesterol-independent or "pleiotropic" effects of statins generated intense interest as to their potential mechanism and created debate over their relative contribution to cardiovascular risk reduction. One potential mechanism for statin pleiotropy is through inhibition of isoprenoid synthesis and protein prenylation. In particular, the prenylation of Rho GTPases such as Rho, Rac, and Cdc42 is critical to their cellular localization and function. Thus, inhibition of Rho and its downstream effector, Rho kinase, by statins may constitute an important pleiotropic mechanism that could be exploited therapeutically for non-lipid conditions beyond cardiovascular disease.

BACKGROUND:The blood-brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of microvascular endothelial cells (BMVECs), which form the lining of cerebral blood vessels and are linked via tight junctions (TJs). The BBB is regulated by components of the extracellular matrix (ECM), and matrix metalloproteinase 3 (MMP3) remodels the ECM's basal lamina, which forms part of the BBB. Oxidative stress is implicated in activation of MMPs and impaired BBB. Thus, we investigated whether MMP3 modulates BBB permeability.METHODS:Experiments included in vivo assessments of isoflurane anesthesia and dye extravasation from brain in wild-type (WT) and MMP3-deficient (MMP3-KO) mice, as well as in vitro assessments of the integrity of monolayers of WT and MMP3-KO BMVECs and the expression of junction proteins.RESULTS:Compared to WT mice, measurements of isoflurane usage and anesthesia induction time were higher in MMP3-KO mice and lower in WT that had been treated with MMP3 (WT+MMP3), while anesthesia emergence times were shorter in MMP3-KO mice and longer in WT+MMP3 mice than in WT. Extravasation of systemically administered dyes was also lower in MMP3-KO mouse brains and higher in WT+MMP3 mouse brains, than in the brains of WT mice. The results from both TEER and Transwell assays indicated that MMP3 deficiency (or inhibition) increased, while MMP3 upregulation reduced barrier integrity in either BMVEC or the coculture. MMP3 deficiency also increased the abundance of TJs and VE-cadherin proteins in BMVECs, and the protein abundance declined when MMP3 activity was upregulated in BMVECs, but not when the cells were treated with an inhibitor of extracellular signal related-kinase (ERK).CONCLUSION:MMP3 increases BBB permeability following the administration of isoflurane by upregulating the ERK signaling pathway, which subsequently reduces TJ and VE-cadherin proteins in BMVECs.

Abstract Background The blood–brain barrier (BBB) regulates the exchange of molecules between the brain and peripheral blood and is composed primarily of microvascular endothelial cells (BMVECs), which form the lining of cerebral blood vessels and are linked via tight junctions (TJs). The BBB is regulated by components of the extracellular matrix (ECM), and matrix metalloproteinase 3 (MMP3) remodels the basal lamina of the ECM, which forms part of the BBB. Thus, we investigated whether MMP3 modulates BBB permeability. Methods Experiments included in-vivo assessments of isoflurane anesthesia and dye extravasation from brain in wild-type (WT) and MMP3-deficient (MMP3-KO) mice, as well as in-vitro assessments of the integrity of monolayers of WT and MMP3-KO BMVECs (via measurements of transendothelial electrical resistance [TEER] and transwell assays in a co-culture of BMVECs with astrocytes and the expression of junction proteins. Results Compared to assessments in WT mice, measurements of isoflurane usage and anesthesia induction time were higher in MMP3-KO mice and lower in WT mice that had been treated with MMP3 (WT + MMP3), while anesthesia emergence times were shorter in MMP3-KO mice and longer in WT + MMP3 mice than in WT mice. Extravasation of systemically administered dyes was also lower in MMP3-KO mouse brains, and higher in WT + MMP3 mouse brains, than in the brains of WT mice, and the results from both TEER and transwell assays indicated that MMP3 deficiency (or inhibition) increased, while MMP3 upregulation reduced, barrier integrity in either BMVEC monolayers or the co-culture. MMP3 deficiency also increased the abundance of TJ and VE-cadherin proteins in BMVECs, and the protein abundance declined when MMP3 activity was upregulated in BMVECs, but not when the cells were treated with an inhibitor of extracellular signal related kinase (ERK). Conclusion MMP3 increases BBB permeability by upregulating the ERK signaling pathway, which subsequently reduces TJ and VE-cadherin proteins abundance in BMVECs. Collectively, these observations suggest that MMP3 could be therapeutically targeted to manipulate BBB permeability and treat neurological disease.

Abstract Background and introduction The difference in cardiovascular disease risk between age-matched women and men narrows as transition through menopause in observational studies. Estrogen exerts complex physiological effects via its non-nuclear and nuclear actions. Experimental studies have shown that endothelial estrogen receptors mediate vasoprotection via endothelial nitric oxide production, reendothelialization, and atherosclerosis. Prior studies in vitro addressed estrogen's effects on endothelial cells and vascular smooth muscle cells, leading to vasoprotection. However, the in vivo evidences are lacking for beneficial effects of endothelium non-nuclear ERα signaling on vascular remodelling in response to injury. Purpose This study aims to clarify the impact of endothelial ERα non-nuclear signaling in the vasoprotection, using a novel mouse model lacking tissue-specific ERα non-nuclear signaling. Methods We identified the amino acids of ERα which were responsible for its binding to p85α subunit of phosphatidylinositol 3-kinase in vitro. We generated a novel mouse model in which non-nuclear signaling of ERα was ablated in endothelial cells by crossing Tie2-Cre transgenic mice with floxed ERα mutants (RR259/260AA) in which p85α and ERα interaction was disrupted. Results In endothelial cells isolated from ERαKI/KITie2 cre/+ animals, E2 failed to induce phosphorylation of Akt, confirming the absence of ERα non-nuclear signaling. Baseline characteristics at 8 to 12 weeks of age were undistinguishable between the genotypes, including body weight, systolic blood pressure, uterine weight and echocardiographic fractional shortening. We then assessed how vascular remodelling process was impacted in a carotid artery wire injury model. Histological analyses with Elastica van Gieson staining two weeks after injury revealed that estrogen dependent suppression of remodelling response (intima to medial ratio) was abolished in ERαki/kiTie2cre/+mice (P=0.0004). Masson's Trichrome staining showed that in the presence of E2 fibrosis was significantly higher in ERαki/kiTie2cre/+ mice than ERαki/kiTie2cre/− mice (P=0.0015). Conclusions We generated a novel mouse model for tissue-specific ablation of ERα non-nuclear signaling by interfering ERα-PI3K interaction. Our results demonstrate that the pivotal role for ERα non-nuclear signaling of endothelial cells in carotid arterial protection following injury with its minimal impact on baseline cardiovascular phenotype. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Japan Heart Foundation Research Grant, SENSHIN Medical Research Foundation

Background: Dual antiplatelet therapy (aspirin plus clopidogrel/ticagrelor - DAPT) is a mainstay treatment for NSTEMI. However, studies suggest that early administration of clopidogrel may not confer significant benefit over later administration. As the time to peak plasma concentration is much shorter for ticagrelor (1-2 hours) vs. clopidogrel (8-12 hours), we hypothesized that earlier administration of ticagrelor may confer survival benefit over later administration. Methods: Patients treated for NSTEMI at our center using DAPT, January 2012 through May 2017, between 18 and 90 years old, were retrospectively identified. Patients who left the hospital against medical advice, were discharged to hospice, or were already treated with DAPT or an anticoagulant at the time of presentation were excluded. Patients who received ticagrelor ≤ 2 hours from arrival were matched with similar patients who received ticagrelor > 2 hours from arrival using 30 clinical and demographic variables. Patients who received clopidogrel were matched similarly. We then compared survival for 500 days using a log rank test. Results: 349 patients met the inclusion criteria. Of these, 18 received ticagrelor within 2 hours and 41 received clopidogrel within 2 hours. These were matched with 18 and 41 control patients, respectively, who received ticagrelor/clopidogrel > 2 hours from arrival. As shown in Figure 1a, patients who received ticagrelor ≤ 2 hours from arrival had significantly lower 500-day mortality than patients who received ticagrelor > 2 hours from arrival (0.0% vs. 27.7%; p=0.017). There was no difference in mortality between the early and late clopidogrel groups (Figure 1b). Conclusion: In the ticagrelor era, early administration of DAPT with ticagrelor (within 2 hours of arrival) may be associated with improved mortality in patients with NSTEMI, an effect not seen with clopidogrel. Larger studies investigating the impact of door to DAPT time in patients with NSTEMI are needed.

BACKGROUND:Rho kinases (ROCKs) contribute to allergic airways disease. ROCKs also play a role in lymphocyte proliferation and migration.OBJECTIVE:To determine the role of ROCK2 acting within CD4+ cells in allergic airways responses.METHODS:ROCK2-haploinsufficient (ROCK2+/- ) and wild-type mice were sensitized with ovalbumin (OVA). ROCK2+/- mice then received either CD4+ cells from ROCK2-sufficient OVA TCR transgenic (OT-II) mice or saline i.v. 48 h before challenge with aerosolized OVA. Wild-type mice received saline before challenge. Allergic airways responses were measured 48 h after the last challenge. Allergic airways responses were also assessed in mice lacking ROCK2 only in CD4+ cells (ROCK2CD4Cre mice) vs. control (CD4-Cre and ROCK2flox/flox ) mice.RESULTS:OVA-induced increases in bronchoalveolar lavage lymphocytes, eosinophils, IL-13, IL-5, and eotaxin were reduced in ROCK2+/- vs. wild-type mice, as were airway hyperresponsiveness and mucous hypersecretion. In ROCK2+/- mice, adoptive transfer with CD4+ cells from OT-II mice restored effects of OVA on lymphocytes, eosinophils, IL-13, IL-5, and mucous hypersecretion to wild-type levels, whereas eotaxin and airway hyperresponsiveness were not affected. ROCK2 inhibitors reduced IL-13-induced release of eotaxin from airway smooth muscle (ASM), similar to effects of these inhibitors on ASM contractility. Despite the ability of adoptive transfer to restore allergic airways inflammation in ROCK2-insufficient mice, allergic inflammation was not different in ROCK2CD4Cre vs. control mice.CONCLUSION:ROCK2 contributes to allergic airways responses likely via effects within ASM cells and within non-lymphocyte cells involved in lymphocyte activation and migration into the airways.

Background: Recent studies have suggested that inherent process delays associated with a universal reperfusion strategy of inter-hospital transfer of ST elevation myocardial infarction (STEMI) patients (pts) for primary PCI (pPCI) may adversely affect clinical outcomes compared to pts directly admitted to a pPCI facility. However, long-term clinical outcomes associated with inter-hospital STEMI reperfusion strategies incorporating pPCI and selective use of pharmacologic reperfusion (FIB) is poorly described. Methods: In this prospective, observational study we define the 10-year mortality among 3,117 consecutive STEMI pts directly admitted (n=1,188; 38%) to 3 pPCI facilities or undergoing inter-hospital transfer (n=1,929; 62%) within an integrated program from 31 rural STEMI-referral hospitals in Central and Southern Illinois between 1/2005 and 12/2014. The inter-hospital transfer STEMI program used a pre-defined reperfusion protocol with emergent transfer for pPCI or use of targeted pre-transfer FIB if...

Non-surgical bleeding (NSB) due to angiodysplasia is common in patients with LVADs. We reported that thrombin-induced Angiopoietin-2 (Ang-2) expression in LVAD patients drives altered angiogenesis and is associated with decreased Angiopoietin-1 (Ang-1) expression. However, the mechanism for decreased Ang-1, which is made by pericytes, is not known and the etiology of thrombin activation in LVAD patients is not clear. We aimed to assess if high levels of Tumor Necrosis Factor-α (TNF-α) in patients with LVADs induce pericyte apoptosis, promote Tissue Factor (TF) expression, and promote vascular instability.

Background Rac1 is a small GTPase, which plays a critical role in cell movement, spreading, proliferation, transformation, and production of reactive oxygen species (ROS). Because smooth muscle cell (SMC) migration and proliferation are important for vascular remodeling after injury, we hypothesized that SMC Rac1 may play an important role in neointima formation. Methods and Results To investigate the role of SMC Rac1 in neointimal formation, we used global haploinsufficient Rac1 knock out mice (Rac1+/−) and also generated SMC‐specific Rac1+/− (smRac1+/−) mice by crossing smMHC‐Cre mice with Rac1 flox/flox mice. Four weeks after carotid artery ligation, neointimal formation in Rac1+/− and smRac1+/− mice was substantially reduced compared to wild‐type (WT) or Rac1 flox/+ (control) mice (intima area, WT vs. Rac1+/−: 59.59±12.43 vs. 27.72±5.97 10 3 μm 2 ; control vs. smRac1+/−: 50.69±9.75 vs. 14.32±2.43 10 3 μm 2 ). Aortic SMC isolated from smRac1+/− mice exhibited normal cytoskeletal architecture, and similar migratory and adherent function as WT SMCs. However, smRac1+/− SMCs showed decreased chemotaxis, and proliferation compared with WT SMCs. Furthermore, SMCs from smRac1+/− mice showed decreased phosphorylation of PAK1 and ERK, and reduced ROS production in response to mechanical stretch compared to that of SMCs from WT mice. Inhibition of Rac1 in vitro using NSC23766 lead to decreases in vascular SMC migration and proliferation and significantly reduced SMC glycolytic metabolism measured via changes in ECAR and lactate production. The effects of Rac1 inhibition on migration and proliferation could be rescued via supplementation with the glycolytic end products lactate or pyruvate. Conclusion These findings indicate that Rac1 regulation of SMC migration and proliferation is mediated in part via control of glycolytic metabolism in these cells. These findings illustrate the critical role of Rac1 and glycolysis in neointimal formation after vascular injury. Therapeutic modalities, which target Rac1 in SMCs, therefore, may be beneficial in preventing vascular proliferative diseases. Support or Funding Information Project funded by NIH R01 HL052233 to James K Liao

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.

Introduction: Cardiac hypertrophy and fibrosis can often lead to heart failure. Presently, the pathophysiological mechanism underlying the development of cardiac fibrosis is not known. We hypothesized that Rho-associated coiled-coil containing kinase (ROCK)-2 in cardiac fibroblasts (CFs) may be involved in the pathogenesis of cardiac hypertrophy and fibrosis. Methods and Results: We developed CF-specific ROCK2-deficient (ROCK2 Postn-/- ) and CF-specific constitutively active ROCK knockin (caROCK Postn-/- ) mice by using fibroblast-specific promoter (Postn) with the Cre/loxP system. Despite comparable increases in systemic blood pressure, angiotensin II (Ang II) infusion (1000ng/kg/min, 28d)-induced cardiac hypertrophy and fibrosis were substantially reduced in ROCK2 Postn-/- mice and were enhanced in caROCK Postn-/- mice compared to littermate controls (n=5-10, P Postn-/- mice and were developed in caROCK Postn-/- mice compared to littermate controls (n=8-10, P Postn-/- mice was substantially decreased compared to littermate controls (n=5 each, P Conclusions: These findings indicate that ROCK2 in CF is an important mediator of cardiac hypertrophy and fibrosis through the upregulation of CTGF and FGF2. Inhibition of ROCK2 may prevent the deleterious cardiac remodeling associated with elevated Ang II and TGF-β1 levels.

Background: Hospitalization for Acute Decompensated Heart Failure (ADHF) carries a high rate of All-Cause Readmission (ACR). While isolated interventions may reduce HF-related readmission, methods ...