Abstract 15673: CRISPR-Cas9 Gene Editing in Mice Reveals Novel Thrombotic Function of a Human SNP in STXBP5

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Qiuyu Zhu ◽  
Kyung Ae Ko ◽  
Sara Ture ◽  
Craig N Morrell ◽  
Joseph M Miano ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Gene Editing ◽  
Association Studies ◽  
Blood Stream ◽  
Genome Wide Association Studies ◽  
Wild Type ◽  
Human Endothelial Cells ◽  
Mesenteric Vessel ◽  
Prolonged Bleeding Time ◽  
Bleeding Score

Introduction: Endothelial cells responds to vascular injury by exocytosis, releasing von Willebrand factor (vWF) into the blood stream. However, the regulation of endothelial vWF release remains poorly understood. Recent genome-wide association studies (GWAS) have identified syntaxin-binding protein 5 (STXBP5) as a candidate gene linked to changes in vWF plasma levels. One top nonsynonymous single nucleotide polymorphism (SNP), rs1039084 (hg19 chr6:g.147635413A>G), encodes p. Asn436Ser substitution (STXBP5-N436S), and is associated with lower plasma vWF, higher bleeding score, and decreased venous thrombosis in humans. We recently discovered that STXBP5 inhibits endothelial vWF exocytosis and regulates thrombosis. However, the role of the STXBP5 genetic variants linked to vWF levels are not completely understood. Hypothesis: We hypothesized that STXBP5-N436S further inhibits endothelial exocytosis than wild type (STXBP5-WT). Methods: We overexpressed STXBP5-WT and STXBP5-N436S in cultured human endothelial cells and measured VWF release changes. Using CRISPR-Cas9 technique, we generated mice carrying the human rs1039084 SNP in Stxbp5 locus (Stxbp5-N437S mice). We conducted phenotypic analyses including endothelial exocytosis, hemostasis, and thrombosis in wild-type and Stxbp5-N437S mice. Results: In human endothelial cells, overexpression of STXBP5-N436S inhibits vWF exocytosis more potently than STXBP5-WT. Germline CRISPR-Cas9 gene editing efficiently and precisely knocked-in the human rs1039084 SNP in murine Stxbp5 locus, without causing detectable off-target genome cleavage. The baseline plasma vWF levels of Stxbp5-N437S mice are similar to WT mice, but Stxbp5-N437S mice showed impaired vWF exocytosis in response to epinephrine challenge. Moreover, Stxbp5-N437S mice have severe hemostasis defects displayed as prolonged bleeding time. Finally, Stxbp5-N437S mice have impaired mesenteric vessel thrombosis and carotid artery thrombosis. We are now studying the effects of the SNP upon STXBP5 structure and function. Conclusions: Our study validates the functional relevance of a candidate SNP identified by GWAS, and suggests that genetic variations within STXBP5 is a risk factor for thromboembolic disease.

scholarly journals Functional non-coding SNPs in human endothelial cells fine-map vascular trait associations

2021 ◽  
Author(s):  
Anu Toropainen ◽  
Lindsey K Stolze ◽  
Tiit Ord ◽  
Michael B Whalen ◽  
Paula Marta Torrell ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Disease ◽  
Complex Disease ◽  
Disease Risk ◽  
Association Studies ◽  
Chromatin Accessibility ◽  
Genome Wide Association Studies ◽  
Large Artery ◽  
Human Endothelial Cells ◽  
Common Complex Disease

Functional consequences of genetic variation in the non-coding human genome are difficult to ascertain despite demonstrated associations to common, complex disease traits. To elucidate properties of functional non-coding SNPs with effects in human endothelial cells (EC), we utilized molecular Quantitative Trait Locus (molQTL) analysis for transcription factor binding, chromatin accessibility, and H3K27 acetylation to nominate a set of likely functional non-coding SNPs. Together with information from genome-wide association studies for vascular disease traits, we tested the ability of 34,344 variants to perturb enhancer function in ECs using the highly multiplexed STARR-seq assay. Of these, 5,592 variants validated, whose enriched attributes included: 1) mutations to TF binding motifs for ETS or AP1 that are regulators of EC state, 2) location in accessible and H3K27ac-marked EC chromatin, and 3) molQTLs associations whereby alleles associate with differences in chromatin accessibility and TF binding across genetically diverse ECs. Next, using pro-inflammatory IL1B as an activator of cell state, we observed robust evidence (>50%) of context-specific SNP effects, underscoring the prevalence of non-coding gene-by-environment (GxE) effects. Lastly, using these cumulative data, we fine-mapped vascular disease loci and highlight evidence suggesting mechanisms by which non-coding SNPs at two loci affect risk for Pulse Pressure/Large Artery Stroke, and Abdominal Aortic Aneurysm through respective effects on transcriptional regulation of POU4F1 and LDAH. Together, we highlight the attributes and context dependence of functional non-coding SNPs, and provide new mechanisms underlying vascular disease risk.

Impaired adhesion of neutrophils expressing Slc44a2/HNA-3b to VWF protects against NETosis under venous shear rate

Blood ◽  
2021 ◽  
Author(s):  
Gaia Zirka ◽  
Philippe Robert ◽  
Julia Tilburg ◽  
Victoria Tishkova ◽  
Chrissta X Maracle ◽  
...  
Keyword(s):  
Transmembrane Protein ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Wild Type ◽  
Shear Rates ◽  
Extracellular Traps ◽  
Genome Wide ◽  
Healthy Donors ◽  
Von Willebrand ◽  
Willebrand Factor

Genome wide association studies linked expression of the human neutrophil antigen 3b (HNA-3b) epitope on the Slc44a2 protein with a 30% decreased risk of venous thrombosis (VT) in humans. Slc44a2 is a ubiquitous transmembrane protein identified as a receptor for Von Willebrand factor (VWF). To explain the link between Slc44a2 and VT we wanted to determine how Slc44a2 expressing either HNA-3a or HNA-3b on neutrophils could modulate their adhesion and activation on VWF under flow. Transfected HEK293T cells or neutrophils homozygous for the HNA-3a- or the HNA-3b-coding allele were purified from healthy donors and perfused in flow chambers coated with VWF at venous shear rates (100s-1). HNA-3a expression was required for Slc44a2-mediated neutrophil adhesion to VWF at 100s-1. This adhesion could occur independently of β2 integrin and was enhanced when neutrophils are preactivated with lipopolysaccharide (LPS). Moreover, specific shear conditions with high neutrophil concentration could act as a "second hit", inducing the formation of neutrophil extracellular traps. Neutrophil mobilization was also measured by intravital microscopy in venules from SLC44A2-knockout and wild-type mice after histamine-induced endothelial degranulation. Mice lacking Slc44a2 showed a massive reduction in neutrophil recruitment in inflamed mesenteric venules. Our results show that Slc44a2/HNA-3a is important for the adhesion and activation of neutrophils in veins under inflammation and when submitted to specific shears. Neutrophils expressing Slc44a2/HNA-3b not being associated with these observations, these results could thus explain the association between HNA-3b and a reduced risk for VT in humans.

scholarly journals Functionalization of the TMEM175 p.M393T variant as a risk factor for Parkinson disease

2019 ◽  
Vol 28 (19) ◽  
pp. 3244-3254 ◽  
Author(s):  
Sarah Jinn ◽  
Cornelis Blauwendraat ◽  
Dawn Toolan ◽  
Cheryl A Gretzula ◽  
Robert E Drolet ◽  
...  
Keyword(s):  
Parkinson Disease ◽  
Therapeutic Strategy ◽  
Association Studies ◽  
Causal Variant ◽  
Genome Wide Association Studies ◽  
Wild Type ◽  
Lysosomal Ph ◽  
Genome Wide ◽  
Attractive Candidate ◽  
Lysosomal Gene

Abstract Multiple genome-wide association studies (GWAS) in Parkinson disease (PD) have identified a signal at chromosome 4p16.3; however, the causal variant has not been established for this locus. Deep investigation of the region resulted in one identified variant, the rs34311866 missense SNP (p.M393T) in TMEM175, which is 20 orders of magnitude more significant than any other SNP in the region. Because TMEM175 is a lysosomal gene that has been shown to influence α-synuclein phosphorylation and autophagy, the p.M393T variant is an attractive candidate, and we have examined its effect on TMEM175 protein and PD-related biology. After knocking down each of the genes located under the GWAS peak via multiple shRNAs, only TMEM175 was found to consistently influence accumulation of phosphorylated α-synuclein (p-α-syn). Examination of the p.M393T variant showed effects on TMEM175 function that were intermediate between the wild-type (WT) and knockout phenotypes, with reduced regulation of lysosomal pH in response to starvation and minor changes in clearance of autophagy substrates, reduced lysosomal localization, and increased accumulation of p-α-syn. Finally, overexpression of WT TMEM175 protein reduced p-α-syn, while overexpression of the p.M393T variant resulted in no change in α-synuclein phosphorylation. These results suggest that the main signal in the chromosome 4p16.3 PD risk locus is driven by the TMEM175 p.M393T variant. Modulation of TMEM175 may impact α-synuclein biology and therefore may be a rational therapeutic strategy for PD.

58 Differential binding of recombinant wild type and variant t-PA to human endothelial cells

1988 ◽  
Vol 2 ◽  
pp. 28 ◽  
Author(s):  
E.S. Barnathan ◽  
D.B. Cines ◽  
K. Barone ◽  
A. Kuo ◽  
G.R. Larsen
Keyword(s):  
Endothelial Cells ◽  
Wild Type ◽  
Human Endothelial Cells ◽  
Differential Binding

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus MicroRNA Mutants Modulate Cancer Hallmark Phenotypic Differences in Human Endothelial Cells

2021 ◽  
Vol 95 (7) ◽  
Author(s):  
Lauren A. Gay ◽  
Daniel Stribling ◽  
Peter C. Turner ◽  
Rolf Renne
Keyword(s):  
Endothelial Cells ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Wild Type ◽  
Human Endothelial Cells ◽  
Tubule Formation ◽  
Phenotypic Changes ◽  
Individual Mirnas ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi’s sarcoma (KS) results from the transformation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells. The contribution of the KSHV microRNAs (miRNAs) to the process of oncogenesis in endothelial cells has not been fully elucidated. To better understand the contributions of individual miRNAs to oncogenesis-related cellular phenotypes, we used KSHV miRNA knockout mutants, each lacking one of the 12 miRNA genes. An additional mutant lacked all miRNAs. Since KSHV infection causes a variety of phenotypic changes in endothelial cells, we tested the mutants for their ability to effect such changes in telomerase-immortalized vein endothelial (TIVE) cells infected with each of the mutant viruses. Wild type- and mutant-infected as well as uninfected cells were evaluated for perturbations to proliferation, migration, tubule formation, and glycolysis. We found broad variation between the different viruses in these aspects. With respect to proliferation rate, ΔmiR-K12-3, ΔmiR-K12-8, and ΔmiR-K12-11 showed significant impairment. Cells infected with ΔmiR-K12-11 had reduced migration. In tubule formation, the ΔmiR-K12-5, -6, and -7 viruses were deficient. At the same time, cells infected with the ΔmiR-K12-10 virus showed dysregulated glycolysis. By combining these observations with previously published KSHV miRNA targetome lists from ribonomics data, we were able to functionally validate a number of new miRNA targets in specific pathways. As proof of concept, miR-K12-3 was shown to target cathepsin D, a strong promoter of apoptosis. Taken together, the results demonstrate that KSHV miRNAs play different roles in inducing the phenotypic changes that are characteristic of transformed cells. IMPORTANCE Kaposi’s sarcoma-associated herpesvirus (KSHV) causes Kaposi’s sarcoma (KS). The contribution of KSHV microRNAs (miRNAs) to oncogenesis is not fully understood. This is particularly true for human endothelial cells, the cell type from which KS tumors are derived. Here, we used a panel of KSHV miRNA knockout viruses to shed light on the roles of individual miRNAs in the process of transformation. Latently infected endothelial cells were studied for phenotypic changes related to cancer, including proliferation, migration, angiogenesis, glycolysis, and apoptosis. The mutant-infected cell lines displayed a wide range of phenotypes in these selected measures of oncogenesis, which differed from those of wild-type-infected cells and from each other. These results indicate that KSHV miRNAs contribute to different aspects of oncogenesis and that each one has a unique role to play.

scholarly journals The Human Cytomegalovirus Ribonucleotide Reductase Homolog UL45 Is Dispensable for Growth in Endothelial Cells, as Determined by a BAC-Cloned Clinical Isolate of Human Cytomegalovirus with Preserved Wild-Type Characteristics

2002 ◽  
Vol 76 (18) ◽  
pp. 9551-9555 ◽  
Author(s):  
Gabriele Hahn ◽  
Hanna Khan ◽  
Fausto Baldanti ◽  
Ulrich H. Koszinowski ◽  
M. Grazia Revello ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Clinical Isolate ◽  
Human Cytomegalovirus ◽  
Ribonucleotide Reductase ◽  
Human Fibroblasts ◽  
Artificial Chromosome ◽  
Cell Types ◽  
Factor X ◽  
Wild Type ◽  
Human Endothelial Cells

ABSTRACT An endothelial cell-tropic and leukotropic human cytomegalovirus (HCMV) clinical isolate was cloned as a fusion-inducing factor X-bacterial artificial chromosome in Escherichia coli, and the ribonucleotide reductase homolog UL45 was deleted. Reconstituted virus RVFIX and RVΔUL45 grew equally well in human fibroblasts and human endothelial cells. Thus, UL45 is dispensable for growth of HCMV in both cell types.

scholarly journals A hypertension patient-derived iPSC model demonstrates a role for G protein-coupled estrogen receptor in hypertension risk and development

2020 ◽  
Vol 319 (5) ◽  
pp. C825-C838 ◽  
Author(s):  
Natalie C. Fredette ◽  
Eliyah Malik ◽  
Marah L. Mukhtar ◽  
Eric R. Prossnitz ◽  
Naohiro Terada
Keyword(s):  
Endothelial Cells ◽  
Estrogen Receptor ◽  
G Protein ◽  
Vascular Endothelial Cells ◽  
Association Studies ◽  
No Production ◽  
Genome Wide Association Studies ◽  
Nucleotide Polymorphisms ◽  
Increased Risk ◽  
G Protein Coupled

Hypertension (HTN) is a polyfactorial disease that can manifest severe cardiovascular pathologies such as heart failure or stroke. Genome-wide association studies (GWAS) of HTN indicate that single-nucleotide polymorphisms (SNPs) contribute to increased risk for HTN and resistance to some HTN drug regimens (Hiltunen TP et al., J Am Heart Assoc 4: e001521, 2015; Le MT et al., PLoS One 8: e52062, 2013; McDonough CW et al., J Hypertens 31: 698–704, 2013; Vandell AG et al., Hypertension 60: 957–964, 2012). However, cellular mechanistic insights of such SNPs remain largely unknown. Using a bank of induced pluripotent stem cells (iPSCs) derived from patients with HTN and CRISPR/Cas9-mediated gene-editing approach, we investigated the effects of a female HTN risk-associated SNP (rs1154431) of the G protein-coupled estrogen receptor (GPER) (Bassuk SS, Manson JE., Clin Chem 60: 68–77, 2014) in vascular endothelial cells. Although GPER1 deletion reduced endothelial nitric oxide synthase (eNOS) activation in iPSC-derived endothelial cells (iECs), the polymorphism itself did not significantly affect eNOS and NO production in a comparison of isogenic hemizygous iECs expressing either normal (P16) or HTN-associated (L16) GPER. Interestingly, we demonstrate for the first time that GPER plays a role in regulation of adhesion molecule expression and monocyte adhesion to iECs. Moreover, the L16 iECs had higher expression of inflammation genes than P16 iECs, implying that the risk variant may affect carrier individuals through increased inflammatory activity. This study further indicates that iPSCs are a useful platform for exploring mechanistic insights underlying hypertension GWAS endeavors.

scholarly journals Progerin Expression Induces Inflammation, Oxidative Stress and Senescence in Human Coronary Endothelial Cells

Cells ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 1201 ◽  
Author(s):  
Guillaume Bidault ◽  
Marie Garcia ◽  
Jacqueline Capeau ◽  
Romain Morichon ◽  
Corinne Vigouroux ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Premature Aging ◽  
Lamin A ◽  
Wild Type ◽  
Human Endothelial Cells ◽  
Endothelial Cell Function ◽  
Age Related

Hutchinson–Gilford progeria syndrome (HGPS) is a rare premature aging disorder notably characterized by precocious and deadly atherosclerosis. Almost 90% of HGPS patients carry a LMNA p.G608G splice variant that leads to the expression of a permanently farnesylated abnormal form of prelamin-A, referred to as progerin. Endothelial dysfunction is a key determinant of atherosclerosis, notably during aging. Previous studies have shown that progerin accumulates in HGPS patients’ endothelial cells but also during vascular physiological aging. However, whether progerin expression in human endothelial cells can recapitulate features of endothelial dysfunction is currently unknown. Herein, we evaluated the direct impact of exogenously expressed progerin and wild-type lamin-A on human endothelial cell function and senescence. Our data demonstrate that progerin, but not wild-type lamin-A, overexpression induces endothelial cell dysfunction, characterized by increased inflammation and oxidative stress together with persistent DNA damage, increased cell cycle arrest protein expression and cellular senescence. Inhibition of progerin prenylation using a pravastatin–zoledronate combination partly prevents these defects. Our data suggest a direct proatherogenic role of progerin in human endothelial cells, which could contribute to HGPS-associated early atherosclerosis and also potentially be involved in physiological endothelial aging participating to age-related cardiometabolic diseases.

Abstract 056: Integrative Genomic Analysis Unravels Novel Pathways And Key Regulatory Networks In Blood Pressure

Hypertension ◽  
2014 ◽  
Vol 64 (suppl_1) ◽  
Author(s):  
Yuqi Zhao ◽  
Xingyi Shi ◽  
Ville-Petteri Mäkinen ◽  
Qingying Meng ◽  
Xia Yang
Keyword(s):  
Blood Pressure ◽  
Endothelial Cells ◽  
Cardiovascular Diseases ◽  
Regulatory Networks ◽  
Association Studies ◽  
Data Driven ◽  
Genome Wide Association Studies ◽  
Tissue Specific ◽  
Aortic Endothelial Cells ◽  
Aortic Endothelial

Blood pressure (BP) is a highly heritable trait and hypertension is a major risk factor for cardiovascular diseases. Recent genome-wide association studies (GWAS) have implicated a number of susceptibility loci for systolic (SBP) and diastolic (DBP) blood pressure. However, these loci together only explain 1% of the BP variability and the underlying mechanisms remain elusive. In this study, we utilized an integrative genomics approach that leveraged multiple genetic and genomic datasets including 1) GWAS from CHARGE (The Cohorts for Heart and Aging Research in Genomic Epidemiology) Consortium for DBP and SBP, 2) expression quantitative trait loci (eQTLs) from genetics of gene expression studies of human tissues related to hypertension (such as peripheral blood, whole blood, human aortic endothelial cells, and adipose tissue), 3) knowledge-driven biological pathways, and 4) data-driven regulatory gene networks. The integration of these diverse data sources enabled tissue-specific investigations on whether the genetic variants associated with BP concentrated on specific parts of gene regulatory networks, termed as “subnetworks”, and whether novel key regulators in the subnetworks could be identified based on data-driven network topology. We identified 10 and 8 subnetworks for DBP and SBP respectively. Among these, subnetworks associated with ion homeostasis, ALK in cardiac myocytes, B cell receptor signaling, and Regulation of Insulin Secretion, were shared between DBP and SBP. More interestingly, we detected tissue-specific pathways, for example, L1CAM interactions in aortic endothelial cells and Leukocyte transendothelial migration in adipose. Among the trait-specific subnetworks, those involved in megakaryocyte development and cytoskeleton remodeling were found to be SBP-specific while GAB1 signalosome subnetwork was DBP-specific. Finally, by utilizing the gene-gene relationships revealed by the network architecture, we detected key regulator genes, both known (e.g. COL1A1, KL, and OSR1) and novel (e.g. EFEMP1, and CRABP2), in these blood pressure subnetworks. Our results shed lights on the complex mechanisms underlying blood pressure and highlight potential novel targets for hypertension and cardiovascular diseases.

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