Genetic variant at coronary artery disease and ischemic stroke locus 1p32.2 regulates endothelial responses to hemodynamics

Abstract

Biomechanical cues dynamically control major cellular processes, but whether genetic variants actively participate in mechanosensing mechanisms remains unexplored. Vascular homeostasis is tightly regulated by hemodynamics. Exposure to disturbed blood flow at arterial sites of branching and bifurcation causes constitutive activation of vascular endothelium contributing to atherosclerosis, the major cause of coronary artery disease (CAD) and ischemic stroke (IS). Conversely, unidirectional flow promotes quiescent endothelium. Genome-wide association studies (GWAS) have identified chromosome 1p32.2 as strongly associated with CAD/IS; however, the causal mechanism related to this locus remains unknown. Using statistical analyses, assay of transposase accessible chromatin with whole-genome sequencing (ATAC-seq), H3K27ac/H3K4me2 ChIP with whole-genome sequencing (ChIP-seq), and CRISPR interference in human aortic endothelial cells (HAECs), our results demonstrate that rs17114036, a common noncoding polymorphism at 1p32.2, is located in an endothelial enhancer dynamically regulated by hemodynamics. CRISPR-Cas9-based genome editing shows that rs17114036-containing region promotes endothelial quiescence under unidirectional shear stress by regulating phospholipid phosphatase 3 (PLPP3). Chromatin accessibility quantitative trait locus (caQTL) mapping using HAECs from 56 donors, allelic imbalance assay from 7 donors, and luciferase assays demonstrate that CAD/IS-protective allele at rs17114036 in PLPP3 intron 5 confers increased endothelial enhancer activity. ChIP-PCR and luciferase assays show that CAD/IS-protective allele at rs17114036 creates a binding site for transcription factor Krüppel-like factor 2 (KLF2), which increases the enhancer activity under unidirectional flow. These results demonstrate that a human SNP contributes to critical endothelial mechanotransduction mechanisms and suggest that human haplotypes and related cis-regulatory elements provide a previously unappreciated layer of regulatory control in cellular mechanosensing mechanisms.

Keywords: GWAS; coronary artery disease; endothelial cells; mechanotransduction; shear stress.

Fig. 1.

CAD-associated SNP rs17114036 is located in an enhancer element (chr1:56962213–56963412) in HAECs. (A) In silico prediction of causal SNPs in the CAD locus 1p32.2. Diagrams demonstrate the association (−log₁₀P) and LD pattern of a total of 45 common SNPs in the 1p32.2 locus. Green circles indicate possible causal SNPs predicted by the Bayesian Statistical Approach. Red circles indicate possible causal SNPs predicted by the Approximate Conditional and Joint Association Analysis. Purple circles indicate putative causal SNPs predicted by both statistical analyses. (B) Chromatin accessibility and canonical enhancer marks in chr1:56962213–56963412 region enclosing rs17114036 in HAEC. ATAC-seq and H3K27ac/H3K4me2 ChIP-seq collectively identified an enhancer-like region in chr1:56962213–56963412 in HAECs. All sequencing experiments were performed in duplicate, and the merged tracks are shown. (C) Enhancer activity of chr1:56962213–56963412 in vascular endothelium. DNA sequences of chr1:56962213–56963412 were cloned into luciferase reporters (firefly luciferase construct pGL4) that contain a minimal promoter or human PLPP3 promoter. The red asterisks denote the relative position of rs17114036 in the luciferase construct. Dual luciferase reporter assays were conducted in HAECs 24 h after the electroporation-based transfection (using pRL-TK plasmid carrying Renilla luciferase as transfection controls) in HAECs, detecting increased firefly luciferase as the result of insertion of chr1:56962213–56963412. Data represent mean ± SEM. ***P < 0.0005 as determined by Student’s t test.

Fig. 2.

CRISPR-based approaches identify rs17114036-containing genomic locus as a cis-regulatory element regulating endothelial PLPP3 expression. (A) RNA RNP complex that contains recombinant S. pyogenes Cas9 and two sgRNAs flanking rs17114036. (B) Experimental overview of CRISPR-Cas9–mediated genomic deletion in HAECs. TeloHAECs were treated with RNP complex four times before single-cell sorting and isogenic clone selection. (C) Reduced PLPP3 expression, (D) elevated E-selectin expression, (E) increased leukocyte adhesion, and (F) higher monolayer permeability in teloHAECs with genomic deletion at rs17114036-containing region. (G) Reduced PLPP3 expression in HAECs treated with CRISPR interference targeting rs17114036-containing region. n = 3–8. Data represent mean ± SEM. *P < 0.05 as determined by Student’s t test; **P < 0.005 as determined by Student’s t test; ***P < 0.0005 as determined by Student’s t test.

Fig. 3.

Unidirectional flow (UF) increases enhancer activity at chr1:56962213–56963412 that transcriptionally activates PLPP3 in human aortic endothelium. (A) Increased chromatin accessibility and H3K27ac mark at chr1:56962213–56963412 in HAECs subjected to 24-h atheroprotective UF compared with cells under 24-h atherosusceptible disturbed flow. The PLPP3 locus is shown and zoomed in to demonstrate details around the enhancer region of interest, with rs17114036 highlighted with a vertical blue line. (B) Reduced UF-induced PLPP3 expression in HAECs with genomic deletion at rs17114036-containing genomic locus. Control and genome-edited (∼66-bp deletion) HAECs were subjected to 24-h UF. The y axis represents the fold change of PLPP3 mRNA quantities between the static conditions and UF for each individual clone. Nondeletion clones, n = 3; deletion clones, n = 4. (C) H3K27ac ChIP-PCR performed in two CRISPR clones; one nondeletion clone and one biallelic deletion clone harboring the 66-bp deletion near rs17114036. The cells were subjected to static or UF conditions before cross-linking and ChIP. PCR primers were designed to amplify a region within the enhancer but not overlapping the deleted region. Data are shown as fold change of the UF-treated samples compared with static conditions. Data represent mean ± SEM. *P < 0.05 as determined by Student’s t test; ***P < 0.0005 as determined by Student’s t test.

Fig. 4.

CAD-protective allele C at rs17114036 confers higher enhancer activity of chr1:56962213–56963412. (A) Increased ATAC-seq reads in chr1:56962213–56963412 region from HAECs isolated from people heterogeneous (T/C) at rs17114036 compared with HAECs from people heterozygous (T/T) at rs17114036. (B) A positive correlation (R = 0.6, P value = 6.23e-06) between ATAC-seq reads at chr1:56962213–56963412 and PLPP3 mRNA detected by RNA-seq in 56 HAEC lines. (C) Increased ATAC-seq reads at rs17114036-containing genomic locus from C (rs17114036)-containing chromosome compared with T-containing chromosome in HAEC lines heterozygous (T/C) at rs17114036. (D) Increased enhancer activity of chr1:56962213–56963412 with C allele at rs17114036 compared with T allele. Dual luciferase reporter assays were conducted in teloHAEC. The red and green asterisks denote the relative position of rs17114036 polymorphisms in the luciferase construct. n = 4–6. Data represent mean ± SEM. ***P < 0.0005 as determined by Student’s t test.

Fig. 5.

CAD-protective C allele at rs17114036 promotes flow-induced, KLF2-mediated enhancer activity of chr1:56962213–56963412. (A) Increased enhancer activity of chr1:56962213–56963412 (with C allele at rs17114036) under unidirectional flow (UF) but not disturbed flow (DF). Experiment was performed in biological triplicate and technical triplicate. Data represent mean ± SEM. *P < 0.05 as determined by two-way ANOVA; **P < 0.005 as determined by two-way ANOVA. The green asterisk denotes the relative position of rs17114036 in the luciferase construct. (B) Increased ATAC-seq reads in rs17114036-containing region in HAECs under UF compared with DF. ATAC-seq was conducted in four HAEC lines heterozygous at rs17114036 under 24-h atherorelevant flows, detecting increased ATAC reads in cells under UF and higher reads from the C allele-containing chromosome compared with T allele. (C) KLF2 affinity to CACC sites in human PLPP3 promoter and intron 5. ChIP-qPCR was performed with either a control IgG antibody or the antibody against HA followed by qPCR using primers detecting CACC sites in PLPP3 promoter or rs17114036-enclosing region from control HAECs (Ctrl) or HAECs transfected with KLF2 transcripts with HA tag. Primers that detect a site ∼200 bp from the CACC at rs17114036 were used as a negative control. n = 4. (D) Increased enhancer activity of chr1:56962213–56963412 by KLF2 overexpression. Dual luciferase reporter assays were conducted in HAECx transfected with luciferase constructs containing the PLPP3 promoter and enhancer with either the major (T) or minor (C) allele at s17114036 along with KLF2-overexpressing or control plasmids. KLF2 overexpression resulted in a 2.9-fold increased luciferase activity in HAECs transfected with T allele-containing construct and a 4.7-fold increase in cells transfected with C allele-containing construct. n = 3. Data represent mean ± SEM. *P < 0.05 as determined by Student’s t test. (E) The interplay between hemodynamic forces, chromatin landscapes at PLPP3 intron 5, and rs17114036 at the molecular level in regulating endothelial PLPP3 expression and vascular functions.