Clonal hematopoiesis (CH) is defined by the expansion of a lineage of genetically identical cells in blood. Genetic lesions that confer a fitness advantage, such as leukemogenic point mutations or mosaic chromosomal alterations (mCAs), are frequent mediators of CH. However, recent analyses of both single cell-derived colonies of hematopoietic cells and population sequencing cohorts have revealed CH frequently occurs in the absence of known driver genetic lesions. To characterize CH without known driver genetic lesions, we use 51,399 deeply sequenced whole genomes from the NHLBI TOPMed sequencing initiative to perform simultaneous germline and somatic mutation analyses among individuals without leukemogenic point mutations (LPM), which we term CH-LPMneg. We quantify CH by estimating the total mutation burden. Because estimating somatic mutation burden without a paired-tissue sample is challenging, we develop a novel statistical method, the Genomic and Epigenomic informed Mutation (GEM) rate, that uses external genomic and epigenomic data sources to distinguish artifactual signals from true somatic mutations. We perform a genome-wide association study of GEM to discover the germline determinants of CH-LPMneg. We identify seven genes associated with CH-LPMneg (TCL1A, TERT, SMC4, NRIP1, PRDM16, MSRA, SCARB1).Functional analyses of SMC4 and NRIP1 implicated altered hematopoietic stem cell self-renewal and proliferation as the primary mediator of mutation burden in blood. We then perform comprehensive multi-tissue transcriptomic analyses, finding that the expression levels of 404 genes are associated with GEM. Finally, we perform phenotypic association meta-analyses across four cohorts, finding that GEM is associated with increased white blood cell count, but is not significantly associated with incident stroke or coronary disease events. Overall, we develop GEM for quantifying mutation burden from WGS and use GEM to discover the genetic, genomic, and phenotypic correlates of CH-LPMneg.

Introduction: Abnormal levels of the coagulation factor VIII (FVIII) are a risk factor for venous thromboembolism (VTE). Genome-wide association studies have identified novel candidate gene associations that may regulate FVIII levels in humans, including CD36. Hypothesis: We hypothesized that CD36 regulates FVIII release from endothelial cells. Methods and Results: We identified a subset of human liver endothelial cells (HLEC) expressing FVIII and purified them using a differentially expressed cell surface protein CD32. CD32+ HLEC expressed FVIII, transported FVIII in intracellular vesicles, and secreted FVIII. We stimulated CD36 signaling or silenced CD36 to test the effect of CD36 on FVIII release. Oxidized LDL (oxLDL), a CD36 agonist, increased endothelial release of FVIII. Conversely, silencing CD36 decreased FVIII release. We searched for an intracellular signaling pathway that mediates CD36 stimulation of FVIII secretion and found that oxLDL increased p38 activity and a p38 inhibitor decreased FVIII release. Conclusions: Taken together, our data show that CD36 stimulates FVIII release through a p38 dependent pathway in specialized liver endothelial cells. These results validate genetic epidemiology data linking CD36 to FVIII levels in humans and provide new insights into the regulation of FVIII levels.

Introduction: Genome-wide association studies (GWAS) have identified multiple novel loci that contribute to CAD pathogenesis, but the mechanisms of these associations remain largely unknown. Research Question: This study aims to functionally characterize the association between the chromosomal GWAS locus for 4q12 and CAD. Methods: We combined computational methods with in vitro assays and mouse models to study REST. Results: A meta-analysis of expression quantitative trait locus (eQTL) data and multi-trait colocalization across atherosclerosis-relevant cell types revealed that the CAD-associated 4q12 locus regulates the expression of REST, a transcriptional repressor, in human aortic endothelial cells (HAEC). This analysis identified two enhancer variants as candidate causal variants for the GWAS association. CRISPR interference confirmed that the rs6853156 variant regulates REST expression in inducible human endothelial cells. Pathway analysis in HAEC after REST silencing indicated that epithelial-to-mesenchymal transition was the most upregulated pathway. Endothelial-to-mesenchymal transition (endMT) was validated in HAEC after REST silencing, as evidenced by rt-qPCR, western blot, and immunofluorescence. Consistently, REST silencing increased endothelial permeability and migration in vitro. CUT&Tag sequencing, jointly analyzed with RNA-sequencing, highlighted L1CAM and its interactors as the most significant gene-set directly affected by REST in the endothelium. L1CAM, a known endMT activator, was expressed in HAEC only upon REST silencing and direct binding of REST to its promoter was confirmed. Simultaneous silencing of L1CAM and REST in HAEC inhibited the upregulation of mesenchymal genes and the enhanced migration induced by REST silencing. Pcsk9-overexpressing mice with an endothelial-specific knockout of Rest exhibited increased atherosclerotic plaque formation in their aortas, with increased macrophage and lipid deposition within the plaque after 16 weeks of high-fat diet exposure compared to littermate controls. Conclusion: In summary, our data reveal the novel role of REST in atherosclerosis as a repressor that functions to constitutively inhibit endMT and protect against atherosclerosis.

ABSTRACT:Coagulation factor VIII (FVIII) and its carrier protein von Willebrand factor (VWF) are critical to coagulation and platelet aggregation. We leveraged whole-genome sequence data from the Trans-Omics for Precision Medicine (TOPMed) program along with TOPMed-based imputation of genotypes in additional samples to identify genetic associations with circulating FVIII and VWF levels in a single-variant meta-analysis, including up to 45 289 participants. Gene-based aggregate tests were implemented in TOPMed. We identified 3 candidate causal genes and tested their functional effect on FVIII release from human liver endothelial cells (HLECs) and VWF release from human umbilical vein endothelial cells. Mendelian randomization was also performed to provide evidence for causal associations of FVIII and VWF with thrombotic outcomes. We identified associations (P < 5 × 10-9) at 7 new loci for FVIII (ST3GAL4, CLEC4M, B3GNT2, ASGR1, F12, KNG1, and TREM1/NCR2) and 1 for VWF (B3GNT2). VWF, ABO, and STAB2 were associated with FVIII and VWF in gene-based analyses. Multiphenotype analysis of FVIII and VWF identified another 3 new loci, including PDIA3. Silencing of B3GNT2 and the previously reported CD36 gene decreased release of FVIII by HLECs, whereas silencing of B3GNT2, CD36, and PDIA3 decreased release of VWF by HVECs. Mendelian randomization supports causal association of higher FVIII and VWF with increased risk of thrombotic outcomes. Seven new loci were identified for FVIII and 1 for VWF, with evidence supporting causal associations of FVIII and VWF with thrombotic outcomes. B3GNT2, CD36, and PDIA3 modulate the release of FVIII and/or VWF in vitro.

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 x 10-8; -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 P = 5.03 x 10-8). -8 ). We tested associations for 138 candidate variants and identified 2 that exceeded statistical significance (0.05/138 = 3.62 x 10-4): -4 ): F5 rs6025 (P P = 1.87 x 10-5; -5 ; SI, 1.29; previously observed) and F11 rs2036914 (P P = 2.0 x 10-4; -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.

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.

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.

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-β signaling. Deleting TGF-β 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.

Introduction: Despite the emergence of new lipid-lowering therapies, coronary artery disease (CAD) remains the leading cause of death worldwide. Genome-wide association studies (GWAS) for CAD have discovered multiple novel loci that contribute to CAD pathogenesis. However, limitations in the interpretability of those GWAS associations have hindered the clinical translation of those findings. Hypothesis: In this study, we sought to evaluate the mechanisms of the association between chromosomal locus 4q12 and CAD, replicated in multiple independent GWAS for CAD. Methods: We utilized a combination of computational methods and in vitro assays to achieve our objectives. Results: Meta-analysis of public expression quantitative trait locus (eQTL) data and multi-trait colocalization across atherosclerosis-relevant cell types revealed that the CAD 4q12 locus regulates the expression of REST, a transcriptional repressor, in human aortic endothelial cells (HAEC) and prioritized 2 enhancer variants as candidate causal variants for the GWAS association. CRISPR interference confirmed that the rs6853156 variant regulates REST expression in inducible human endothelial cells. Pathway enrichment analysis in HAEC after REST silencing showed that epithelial to mesenchymal transition was the most upregulated pathway suggesting a potential mechanism via which silencing REST contributes to atherosclerosis. Endothelial to mesenchymal transition (endMT) was confirmed in HAEC after REST silencing at the RNA and protein level by rt-qPCR, western blot, and immunofluorescence. Concordantly, REST silencing increased endothelial permeability and matrix metalloproteinase activity in endothelial cell supernatants in vitro. L1CAM, a known endMT activator was only expressed in HAEC upon REST silencing. Chromatin immunoprecipitation sequencing supports direct binding of REST to the L1CAM promoter. Simultaneous silencing of L1CAM and REST in HAEC inhibited the observed upregulation of mesenchymal genes induced by REST silencing. Conclusions: In summary, our data reveal a novel role of REST as a repressor that functions as a constitutive inhibitor of endMT in human arterial endothelial cells.

Key Points Question What is the effect of P2Y12 inhibition, a proposed therapeutic target and preventive strategy, on clinical outcomes in critically ill patients hospitalized for COVID-19? Findings In this randomized clinical trial that included 949 participants, use of a P2Y12 inhibitor did not result in a greater number of days alive and free of cardiovascular or respiratory organ support up to day 21 of the index hospitalization. Meaning These data do not support routine use of a P2Y12 inhibitor in critically ill patients hospitalized for COVID-19.

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 ERβ 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 ERβ (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.

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.

Introduction: Human liver endothelial cells (HLEC) are a heterogeneous cell population. Single cell transcriptomics and immunostaining assays have categorized HLEC into two major groups according to their location within the liver lobules (zone 1 or periportal HLEC and zones 2/3 or central venous (CV) HLEC). Hypothesis: Cellular deconvolution of the bulk transcriptome of HLEC will highlight important properties of HLECs and their transcriptional regulators. Methods: We uniformly processed all bulk RNA-seq data from HLECs in Gene Expression Omnibus. We used CIBERSORTx with human liver single cell RNA-seq data to perform cell type deconvolution of bulk HLEC RNA-seq. We compared the transcriptome of HLEC subpopulations to that of HUVEC. We then applied the HOMER motif finding algorithm on the promoters of genes up/downregulated in HLEC compared to HUVEC. Results: We analyzed data from 10 HLEC samples and 107 HUVEC samples from 37 independent experiments. Principal component analysis revealed that 6 of the 10 HLEC samples have robust transcriptional differences compared to HUVEC. CIBERSORTx showed that those 6 HLEC samples correspond to CV HLECs and can be identified by FCGR2B expression. Differential gene expression analysis revealed enrichment of inflammatory and coagulation pathways in CV HLEC and enrichment of proliferation and mitosis pathways in HUVEC. CV HLEC showed increased expression of factors VIII, X, XII, Protein S and antithrombin III. We validated gene expression at the protein level by Western blot. A transcription factor (TF) prioritization approach using HOMER motif enrichment revealed a potential role for E2F, and RXR-RAR TF as primary determinants of the differences between HUVEC and CV HLEC. Conclusions: We identify and characterize CV HLEC as a distinct endothelial subtype relevant to inflammation and coagulation. We also identify downregulation of E2F and upregulation of RXR-RAR TF as potential key regulators of the CV HLEC transcriptome.

Introduction: Human liver endothelial cells (HLEC) are a heterogeneous cell population. Single cell transcriptomics and immunostaining assays have categorized HLEC into two major groups according to their location within the liver lobules (zone 1 or periportal HLEC and zones 2/3 or central venous (CV) HLEC). Hypothesis: Cellular deconvolution of the bulk transcriptome of HLEC will highlight important properties of HLECs and their transcriptional regulators. Methods: We uniformly processed all bulk RNA-seq data from HLECs in Gene Expression Omnibus. We used CIBERSORTx with human liver single cell RNA-seq data to perform cell type deconvolution of bulk HLEC RNA-seq. We compared the transcriptome of HLEC subpopulations to that of HUVEC. We then applied the HOMER motif finding algorithm on the promoters of genes up/downregulated in HLEC compared to HUVEC. Results: We analyzed data from 10 HLEC samples and 107 HUVEC samples from 37 independent experiments. Principal component analysis revealed that 6 of the 10 HLEC samples have robust transcriptional differences compared to HUVEC. CIBERSORTx showed that those 6 HLEC samples correspond to CV HLECs and can be identified by FCGR2B expression. Differential gene expression analysis revealed enrichment of inflammatory and coagulation pathways in CV HLEC and enrichment of proliferation and mitosis pathways in HUVEC. CV HLEC showed increased expression of factors VIII, X, XII, Protein S and antithrombin III. We validated gene expression at the protein level by Western blot. A transcription factor (TF) prioritization approach using HOMER motif enrichment revealed a potential role for E2F, and RXR-RAR TF as primary determinants of the differences between HUVEC and CV HLEC. Conclusions: We identify and characterize CV HLEC as a distinct endothelial subtype relevant to inflammation and coagulation. We also identify downregulation of E2F and upregulation of RXR-RAR TF as potential key regulators of the CV HLEC transcriptome.

Severe coronavirus disease-19 (COVID-19) is characterized by vascular inflammation and thrombosis. We and others have proposed that the inflammatory response to coronavirus infection activates endothelial cells, leading to endothelial release of pro-thrombotic proteins. These mediators can trigger obstruction of the pulmonary microvasculature, leading to worsening oxygenation, acute respiratory distress syndrome, and death. In the current study, we tested the hypothesis that higher levels of biomarkers released from endothelial cells are associated with worse oxygenation in patients with COVID-19. We studied 83 participants aged 18-84 years with COVID-19 admitted to a single center. The severity of pulmonary disease was classified by oxygen requirement, including no oxygen requirement, low-flow oxygen, high-flow nasal cannula oxygen, mechanical ventilation, and death. We measured plasma levels of two proteins released by activated endothelial cells, von Willebrand Factor (VWF) antigen and soluble P-Selectin (sP-Sel), and a biomarker of systemic thrombosis, D-dimer. Additionally, we explored the association of endothelial biomarker levels with the levels of pro-inflammatory cytokine and chemokines, and vascular inflammation biomarkers. We found that levels of VWF, sP-sel, and D-dimer were increased in individuals with more severe COVID-19 pulmonary disease. Biomarkers of endothelial cell activation were also correlated with proinflammatory cytokines and chemokines. Taken together, our data demonstrate increased levels of VWF and sP-selectin are linked to the severity of lung disease in COVID-19 and correlated with biomarkers of inflammation and vascular inflammation. Our data support the concept that COVID-19 is a vascular disease which involves endothelial injury in the context of an inflammatory state.