Effect of the P-Selectin Inhibitor Crizanlizumab on Survival Free of Organ Support in Patients Hospitalized for COVID-19: A Randomized Controlled Trial.

BACKGROUND: COVID-19 has been associated with endothelial injury, resultant microvascular inflammation and thrombosis. Activated endothelial cells release and express P-selectin and von Willebrand factor, both of which are elevated in severe COVID-19 and may be implicated in the disease pathophysiology. We hypothesized that crizanlizumab, a humanized monoclonal antibody to P-selectin, would reduce morbidity and death in patients hospitalized for COVID-19. METHODS: An international, adaptive, randomized controlled platform trial, funded by the National Heart, Lung, and Blood Institute, randomly assigned 422 patients hospitalized with COVID-19 with moderate or severe illness to receive either a single infusion of the P-selectin inhibitor crizanlizumab (at a dose of 5 mg/kg) plus standard of care or standard of care alone in an open-label 1:1 ratio. The primary outcome was organ support-free days, evaluated on an ordinal scale consisting of the number of days alive free of organ support through the first 21 days after trial entry. RESULTS: The study was stopped for futility by the data safety monitoring committee. Among 421 randomized patients with known 21-day outcomes, 163 patients (77%) randomized to the crizanlizumab plus standard-of-care arm did not require any respiratory or cardiovascular organ support compared with 169 (80%) in the standard-of-care-alone arm. The adjusted odds ratio for the effect of crizanlizumab on organ support-free days was 0.70 (95% CI, 0.43-1.16), where an odds ratio >1 indicates treatment benefit, yielding a posterior probability of futility (odds ratio <1.2) of 98% and a posterior probability of inferiority (odds ratio <1.0) of 91%. Overall, there were 37 deaths (17.5%) in the crizanlizumab arm and 27 deaths (12.8%) in the standard-of-care arm (hazard ratio, 1.33 [95% CrI, 0.85-2.21]; [probability of hazard ratio>1] = 0.879). CONCLUSIONS: Crizanlizumab, a P-selectin inhibitor, did not result in improvement in organ support-free days in patients hospitalized with COVID-19. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT04505774.

Dyslipidemia.

Dyslipidemia is an important risk factor for coronary artery disease and stroke. All persons with dyslipidemia should be advised to focus on lifestyle interventions, including regular aerobic exercise, a healthy diet, maintenance of a healthy weight, and abstinence from smoking. In addition to lifestyle interventions, lipid-lowering therapy should be considered for persons at moderate to high risk for atherosclerotic cardiovascular disease based on validated risk equations. Statin therapy is the first-line medical treatment for dyslipidemia due to its effectiveness and favorable adverse effect profile, but newer treatments provide additional tools for clinicians to effectively treat dyslipidemia.

Myocardial Injury in Severe COVID-19 Compared With Non-COVID-19 Acute Respiratory Distress Syndrome.

BACKGROUND: Knowledge gaps remain in the epidemiology and clinical implications of myocardial injury in coronavirus disease 2019 (COVID-19). We aimed to determine the prevalence and outcomes of myocardial injury in severe COVID-19 compared with acute respiratory distress syndrome (ARDS) unrelated to COVID-19. METHODS: We included intubated patients with COVID-19 from 5 hospitals between March 15 and June 11, 2020, with troponin levels assessed. We compared them with patients from a cohort study of myocardial injury in ARDS and performed survival analysis with primary outcome of in-hospital death associated with myocardial injury. In addition, we performed linear regression to identify clinical factors associated with myocardial injury in COVID-19. RESULTS: Of 243 intubated patients with COVID-19, 51% had troponin levels above the upper limit of normal. Chronic kidney disease, lactate, ferritin, and fibrinogen were associated with myocardial injury. Mortality was 22.7% among patients with COVID-19 with troponin under the upper limit of normal and 61.5% for those with troponin levels >10 times the upper limit of normal (P<0.001). The association of myocardial injury with mortality was not statistically significant after adjusting for age, sex, and multisystem organ dysfunction. Compared with patients with ARDS without COVID-19, patients with COVID-19 were older and had higher creatinine levels and less favorable vital signs. After adjustment, COVID-19-related ARDS was associated with lower odds of myocardial injury compared with non-COVID-19-related ARDS (odds ratio, 0.55 [95% CI, 0.36-0.84]; P=0.005). CONCLUSIONS: Myocardial injury in severe COVID-19 is a function of baseline comorbidities, advanced age, and multisystem organ dysfunction, similar to traditional ARDS. The adverse prognosis of myocardial injury in COVID-19 relates largely to multisystem organ involvement and critical illness.

Estrogen activates endothelial exocytosis.

Estrogen therapy is used to treat patients with post-menopausal symptoms, such as hot flashes and dyspareunia. Estrogen therapy also decreases the risk of fractures from osteoporosis in post-menopausal women. However, estrogen increases the risk of venous thromboembolic events, such as pulmonary embolism, but the pathways through which estrogen increase the risk of thromboembolism is unknown. Here, we show that estrogen elicits endothelial exocytosis, the key step in vascular thrombosis and inflammation. Exogenous 17ß-estradiol (E2) stimulated endothelial exocytosis of Weibel-Palade bodies (WPBs), releasing von Willebrand factor (vWF) and interleukin-8 (IL-8). Conversely, the estrogen antagonist ICI-182,780 interfered with E2-induced endothelial exocytosis. The ERα agonist propyl pyrazole triol (PPT) but not the ERß agonist diarylpropionitrile (DPN) induced vWF release, while ERα silencing counteracted vWF release by E2, suggesting that ERα mediates this effect. Exocytosis triggered by E2 occurred rapidly within 15 min and was not inhibited by either actinomycin D or cycloheximide. On the contrary, it was inhibited by the pre-treatment of U0126 or SB203580, an ERK or a p38 inhibitor, respectively, suggesting that E2-induced endothelial exocytosis is non-genomically mediated by the MAP kinase pathway. Finally, E2 treatment enhanced platelet adhesion to endothelial cells ex vivo, which was interfered with the pre-treatment of ICI-182,780 or U0126. Taken together, our data show that estrogen activates endothelial exocytosis non-genomically through the ERα-MAP kinase pathway. Our data suggest that adverse cardiovascular effects such as vascular inflammation and thrombosis should be considered in patients before menopausal hormone treatment.

Acetylcholine Inhibits Platelet Activation.

Platelets are key mediators of thrombosis. Many agonists of platelet activation are known, but fewer endogenous inhibitors of platelets, such as prostacyclin and nitric oxide (NO), have been identified. Acetylcholinesterase inhibitors, such as donepezil, can cause bleeding in patients, but the underlying mechanisms are not well understood. We hypothesized that acetylcholine is an endogenous inhibitor of platelets. We measured the effect of acetylcholine or analogs of acetylcholine on human platelet activation ex vivo. Acetylcholine and analogs of acetylcholine inhibited platelet activation, as measured by P-selectin translocation and glycoprotein IIb IIIa conformational changes. Conversely, we found that antagonists of the acetylcholine receptor, such as pancuronium, enhance platelet activation. Furthermore, drugs inhibiting acetylcholinesterase, such as donepezil, also inhibit platelet activation, suggesting that platelets release acetylcholine. We found that NO mediates acetylcholine inhibition of platelets. Our data suggest that acetylcholine is an endogenous inhibitor of platelet activation. The cholinergic system may be a novel target for antithrombotic therapies.

Novel Thrombotic Function of a Human SNP in STXBP5 Revealed by CRISPR/Cas9 Gene Editing in Mice.

OBJECTIVE: To identify and characterize the effect of a SNP (single-nucleotide polymorphism) in the STXBP5 locus that is associated with altered thrombosis in humans. GWAS (genome-wide association studies) have identified numerous SNPs associated with human thrombotic phenotypes, but determining the functional significance of an individual candidate SNP can be challenging, particularly when in vivo modeling is required. Recent GWAS led to the discovery of STXBP5 as a regulator of platelet secretion in humans. Further clinical studies have identified genetic variants of STXBP5 that are linked to altered plasma von Willebrand factor levels and thrombosis in humans, but the functional significance of these variants in STXBP5 is not understood. APPROACH AND RESULTS: We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) techniques to produce a precise mouse model carrying a human coding SNP rs1039084 (encoding human p. N436S) in the STXBP5 locus associated with decreased thrombosis. Mice carrying the orthologous human mutation (encoding p. N437S in mouse STXBP5) have lower plasma von Willebrand factor levels, decreased thrombosis, and decreased platelet secretion compared with wild-type mice. This thrombosis phenotype recapitulates the phenotype of humans carrying the minor allele of rs1039084. Decreased plasma von Willebrand factor and platelet activation may partially explain the decreased thrombotic phenotype in mutant mice. CONCLUSIONS: Using precise mammalian genome editing, we have identified a human nonsynonymous SNP rs1039084 in the STXBP5 locus as a causal variant for a decreased thrombotic phenotype. CRISPR/Cas9 genetic editing facilitates the rapid and efficient generation of animals to study the function of human genetic variation in vascular diseases.

A CRISPR Path to Engineering New Genetic Mouse Models for Cardiovascular Research.

Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologically informative sequences required complex vector design and a series of arduous steps only a handful of laboratories could master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) method has now superseded traditional means of genome modification such that virtually any laboratory can quickly assemble reagents for developing new mouse models for cardiovascular research. Here, we briefly review the history of CRISPR in prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom. Core components of CRISPR technology are reviewed and updated. Practical pointers for 2-component and 3-component CRISPR editing are summarized with many applications in mice including frameshift mutations, deletion of enhancers and noncoding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sites for conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of genetic mosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressed as the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previously intractable questions.

Associations between ambient wood smoke and other particulate pollutants and biomarkers of systemic inflammation, coagulation and thrombosis in cardiac patients.

BACKGROUND: Increased particulate air pollution has been associated with both an increased risk of myocardial infarction (MI) and adverse changes in cardiac biomarkers. Up to 30% of ambient wintertime fine particles (PM2.5) in Rochester, NY are from wood burning. Our study examined associations between ambient levels of a marker of wood smoke (Delta-C) and other particulate air pollutants and biomarkers of inflammation, coagulation and thrombosis. METHODS: We measured blood concentrations of C-reactive protein (CRP), D-dimer, fibrinogen, P-selectin, platelet factor 4 (PF-4), von Willebrand factor (vWF), and myeloperoxidase (MPO) of 135 patients undergoing cardiac catheterization during the winters of 2011-2013. We coupled these data with hourly ambient concentrations of Delta-C, black carbon (BC; marker of traffic pollution), and ultrafine (10-100nm; UFP), accumulation mode (100-500nm; AMP), and fine particles (<2.5µm; PM2.5). Using linear regression models, we estimated the change in each biomarker associated with increased pollutant concentrations at intervals between 1 and 96h preceding blood collection. RESULTS: Each 0.13µg/m3 increase in Delta-C concentration in the prior 12h was associated with a 0.91% increase in fibrinogen levels (95% CI=0.23%, 1.59%), but unexpectedly in the prior 48h, each 0.17µg/m3 increase in Delta-C concentration was associated with a 2.75% decrease in MPO levels (95% CI=-5.13%,-0.37%). We did not see associations between Delta-C concentrations and any other biomarkers. Interquartile range (IQR) increases in PM2.5, BC, UFP, and AMP concentrations were generally associated with increased CRP and fibrinogen, but not PF4, D-dimer, vWF, or P-selectin. CONCLUSIONS: In a population of cardiac patients, we noted adverse changes in fibrinogen associated with increased concentrations of a marker of wood smoke. Increases in PM2.5, BC, AMP, and UFP concentrations in the previous 96h were also associated with adverse changes in markers of systemic inflammation and coagulation, but not with markers of endothelial cell dysfunction or platelet activation.

Enhanced assay of endothelial exocytosis using extracellular matrix components.

Vascular inflammation plays a key role in the pathogenesis of atherosclerosis. The first step in vascular inflammation is endothelial exocytosis, in which endothelial granules fuse with the plasma membrane, releasing prothrombotic and proinflammatory messenger molecules. The development of cell culture models to study endothelial exocytosis has been challenging because the factors that modulate exocytosis in vitro are not well understood. Here we report a method for studying endothelial exocytosis that optimizes extracellular matrix components, cell density, and duration of culture. Human umbilical vein endothelial cells plated on collagen I-coated plates and cultured in the confluent state for 7-12 days in low-serum medium showed robust secretion of von Willebrand factor when stimulated with various agonists. This exocytosis assay is rapid and applicable to high-throughput screening.

Genome-wide investigation of exogenous female hormones, genetic variation, and venous thromboembolism risk.

BACKGROUND: Increased risk of venous thromboembolism (VTE) is a life-threatening side effect for users of oral contraceptives (OCs) or hormone therapy (HT). OBJECTIVES: To investigate the potential for genetic predisposition to VTE in OC or HT users, we conducted a gene-by-environment case-only meta-analysis of genome-wide association studies (GWAS). METHODS: Use or nonuse of OCs (7 studies) or HT (8 studies) at the time of the VTE event was determined by pharmacy records or self-report. A synergy index (SI) was modeled for each variant in each study and submultiplicative/supramultiplicative gene-by-environment interactions were estimated. The SI parameters were first meta-analyzed across OC and HT studies and subsequently meta-analyzed to obtain an overall estimate. The primary analysis was agnostic GWAS and interrogated all imputed genotypes using a P value threshold of <5.0 × 10-8; secondary analyses were candidate-based. RESULTS: The VTE case-only OC meta-analysis included 2895 OC users and 6607 nonusers; the case-only HT meta-analysis included 2434 HT users and 12 793 nonusers. In primary GWAS meta-analyses, no variant reached genome-wide significance, but the smallest P value approached statistical significance: rs9386463 (P = 5.03 × 10-8). We tested associations for 138 candidate variants and identified 2 that exceeded statistical significance (0.05/138 = 3.62 × 10-4): F5 rs6025 (P = 1.87 × 10-5; SI, 1.29; previously observed) and F11 rs2036914 (P = 2.0 × 10-4; SI, 0.91; new observation). CONCLUSION: The candidate variant approach to identify submultiplictive/supramultiplicative associations between genetic variation and OC and HT use identified a new association with common genetic variation in F11, while the agnostic interrogations did not yield new discoveries.

P53-induced microRNA-107 inhibits HIF-1 and tumor angiogenesis.

The pathway involving the tumor suppressor gene TP53 can regulate tumor angiogenesis by unclear mechanisms. Here we show that p53 regulates hypoxic signaling through the transcriptional regulation of microRNA-107 (miR-107). We found that miR-107 is a microRNA expressed by human colon cancer specimens and regulated by p53. miR-107 decreases hypoxia signaling by suppressing expression of hypoxia inducible factor-1beta (HIF-1beta). Knockdown of endogenous miR-107 enhances HIF-1beta expression and hypoxic signaling in human colon cancer cells. Conversely, overexpression of miR-107 inhibits HIF-1beta expression and hypoxic signaling. Furthermore, overexpression of miR-107 in tumor cells suppresses tumor angiogenesis, tumor growth, and tumor VEGF expression in mice. Finally, in human colon cancer specimens, expression of miR-107 is inversely associated with expression of HIF-1beta. Taken together these data suggest that miR-107 can mediate p53 regulation of hypoxic signaling and tumor angiogenesis.

High risk oral contraceptive hormones do not directly enhance endothelial cell procoagulant activity in vitro.

BACKGROUND: Oral contraceptive (OC) use increases venous thromboembolism risk 2-5-fold. Procoagulant changes can be detected in plasma from OC users even without thrombosis, but cellular mechanisms that provoke thrombosis have not been identified. Endothelial cell (EC) dysfunction is thought to initiate venous thromboembolism. It is unknown whether OC hormones provoke aberrant procoagulant activity in ECs. OBJECTIVE: Characterize the effect of high-risk OC hormones (ethinyl estradiol [EE] and drospirenone) on EC procoagulant activity and the potential interplay with nuclear estrogen receptors ERα and ERß and inflammatory processes. METHODS: Human umbilical vein and dermal microvascular ECs (HUVEC and HDMVEC, respectively) were treated with EE and/or drospirenone. Genes encoding the estrogen receptors ERα and ERß (ESR1 and ESR2, respectively) were overexpressed in HUVEC and HDMVEC via lentiviral vectors. EC gene expression was assessed by RT-qPCR. The ability of ECs to support thrombin generation and fibrin formation was measured by calibrated automated thrombography and spectrophotometry, respectively. RESULTS: Neither EE nor drospirenone, alone or together, changed expression of genes encoding anti- or procoagulant proteins (TFPI, THBD, F3), integrins (ITGAV, ITGB3), or fibrinolytic mediators (SERPINE1, PLAT). EE and/or drospirenone did not increase EC-supported thrombin generation or fibrin formation, either. Our analyses indicated a subset of individuals express ESR1 and ESR2 transcripts in human aortic ECs. However, overexpression of ESR1 and/or ESR2 in HUVEC and HDMVEC did not facilitate the ability of OC-treated ECs to support procoagulant activity, even in the presence of a pro-inflammatory stimulus. CONCLUSIONS: The OC hormones EE and drospirenone do not directly enhance thrombin generation potential of primary ECs in vitro.

Cardiac pericytes mediate the remodeling response to myocardial infarction.

Despite the prevalence of pericytes in the microvasculature of the heart, their role during ischemia-induced remodeling remains unclear. We used multiple lineage-tracing mouse models and found that pericytes migrated to the injury site and expressed profibrotic genes, coinciding with increased vessel leakage after myocardial infarction (MI). Single-cell RNA-Seq of cardiac pericytes at various time points after MI revealed the temporally regulated induction of genes related to vascular permeability, extracellular matrix production, basement membrane degradation, and TGF-ß signaling. Deleting TGF-ß receptor 1 in chondroitin sulfate proteoglycan 4-expressing (Cspg4-expressing) cells reduced fibrosis following MI, leading to a transient improvement in the cardiac ejection fraction. Furthermore, genetic ablation of Cspg4-expressing cells resulted in excessive vascular permeability, a decline in cardiac function, and increased mortality in the second week after MI. These data reveal an essential role for cardiac pericytes in the control of vascular homeostasis and the fibrotic response after acute ischemic injury, information that will help guide the development of novel strategies to preserve vascular integrity and attenuate pathological cardiac remodeling.

Thioredoxin increases exocytosis by denitrosylating N-ethylmaleimide-sensitive factor.

Exocytosis involves membrane fusion between granules and the plasma membrane. Nitric oxide (NO) inhibits exocytosis by chemically modifying N-ethylmaleimide-sensitive factor (NSF), a key component of the exocytic machinery. However, cells recover the ability to release messenger molecules within hours of exposure to NO through unknown mechanisms. We now identify thioredoxin (TRX1) as a denitrosylase that reverses NO inhibition of exocytosis. Endogenously synthesized NO increases S-nitrosylated NSF levels, but S-nitrosylated NSF levels decrease within 3 h after exposure to NO. We found that NO increases the interaction between TRX1 and NSF, and endogenous TRX1 removes NO from S-nitrosylated NSF. Knockdown of TRX1 increases the level of S-nitrosylated NSF, prolongs the inhibition of exocytosis, and suppresses leukocyte adhesion. Taken together, these data show that TRX1 promotes exocytosis by denitrosylating NSF. Our findings suggest that TRX1 might regulate exocytosis in a variety of physiological settings, such as vascular inflammation, thrombosis, and insulin release.

Increased endothelial exocytosis and generation of endothelin-1 contributes to constriction of aged arteries.

RATIONALE: Circulating levels of endothelin (ET)-1 and endogenous ET(A)-mediated constriction are increased in human aging. The mechanisms responsible are not known. OBJECTIVE: Investigate the storage, release, and activity of ET-1 system in arteries from young and aged Fischer-344 rats. METHODS AND RESULTS: After NO synthase inhibition (L-NAME), thrombin contracted aged arteries, which was inhibited by endothelial denudation, ET(A) receptor antagonism (BQ123), and ECE inhibition (phosphoramidon, SM19712) or by inhibiting exocytosis (TAT-NSF, N-ethylmaleimide-sensitive factor inhibitor). Thrombin did not cause endothelium-dependent contraction of young arteries. In aged but not young arteries, thrombin rapidly increased ET-1 release, which was abolished by endothelium denudation or TAT-NSF. L-NAME did not affect ET-1 release. ET-1 immunofluorescent staining was punctate and distinct from von Willebrand factor (VWF). VWF and ET-1 immunofluorescent intensity was similar in young and aged quiescent arteries. Thrombin rapidly increased ET-1 staining and decreased VWF staining in aged but had no effect in young aortas. After L-NAME, thrombin decreased VWF staining in young aortas. NO donor DEA-NONOate (1 to 100 nmol/L) reversed thrombin-induced exocytosis in young (VWF) but not aged L-NAME-treated aortas (VWF, ET-1). Expression of preproET-1 mRNA and ECE-1 mRNA were increased in aged compared to young endothelium. BigET-1 levels and contraction to exogenous BigET-1 (but not ET-1) were also increased in aged compared to young arteries. CONCLUSIONS: The stimulated exocytotic release of ET-1 is dramatically increased in aged endothelium. This reflects increased reactivity of exocytosis, increased expression and storage of ET-1 precursor peptides, and increased expression of ECE-1. Altered endothelial exocytosis of ET-1 and other mediators may contribute to cardiovascular pathology in aging.

Aldosterone activates endothelial exocytosis.

Although elevated levels of aldosterone are associated with vascular inflammation, the proinflammatory pathways of aldosterone are not completely defined. We now show that aldosterone triggers endothelial cell exocytosis, the first step in leukocyte trafficking. Exogenous aldosterone stimulates endothelial exocytosis of Weibel-Palade bodies, externalizing P-selectin and releasing von Willebrand factor. Spironolactone, a nonselective mineralocorticoid receptor (MR) blocker, antagonizes aldosterone-induced endothelial exocytosis. Knockdown of the MR also decreases exocytosis, suggesting that the MR mediates exocytosis. Aldosterone triggers exocytosis within minutes, and this effect is not inhibited by actinomycin D, suggesting a nongenomic effect of aldosterone. Aldosterone treatment of endothelial cells increases leukocyte adherence to endothelial cells in culture. Taken together, our data suggest that aldosterone activates vascular inflammation in part through nongenomic, MR-mediated pathways. Aldosterone antagonism may decrease vascular inflammation and cardiac fibrosis in part by blocking endothelial exocytosis.