Genomic profiling of human vascular cells identifies TWIST1 as a causal gene for common vascular diseases

Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD

Low prevalence of connexin-40 gene variants in atrial tissues and blood from atrial fibrillation subjects

Abstract Background The atrial gap junction protein connexin-40 (Cx40) has been implicated to play an important role in atrial conduction and development of atrial fibrillation (AF). However, the frequency of Cx40 mutations in AF populations and their impact on Cx40 expression remains unclear. In this study, we sought to identify polymorphisms in the Cx40 gene GJA5, investigate the potential functional role of these polymorphisms, and determine their allelic frequencies. The prevalence of nonsynonymous Cx40 mutations in blood and atrial tissue was also compared to mutation frequencies reported in prior studies. Methods We conducted direct sequencing of the GJA5 coding and 3′ UTR regions in blood samples from 91 lone AF subjects and 67 atrial tissue-derived samples from a lone cohort, a mixed AF cohort, and several transplant donors. Reporter gene transfection and tissue allelic expression imbalance assays were used to assess the effects of a common insertion/deletion polymorphism on Cx40 mRNA stability and expression. Results We identified one novel synonymous SNP in blood-derived DNA from a lone AF subject. In atrial tissue-derived DNA from lone and mixed AF subjects, we observed one novel nonsynonymous SNP, one rare previously reported synonymous SNP, and one novel 3′ UTR SNP. A previously noted 25 bp insertion/deletion polymorphism in the 3′ UTR was found to be common (minor allele frequency = 0.45) but had no effect on Cx40 mRNA stability and expression. The observed prevalence of nonsynonymous Cx40 mutations in atrial tissues derived from lone AF subjects differed significantly (p = 0.03) from a prior atrial tissue study reporting a high mutation frequency in a group of highly selected young lone AF subjects. Conclusions Our results suggest that Cx40 coding SNPs are uncommon in AF populations, although rare mutations in this gene may certainly lead to AF pathogenesis. Furthermore, a common insertion/deletion polymorphism in the Cx40 3′ UTR does not appear to play a role in modulating Cx40 mRNA levels

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles

<div><p>Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous <i>Bin1</i> deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.</p></div

Genomic profiling of human vascular cells identifies TWIST1 as a causal gene for common vascular diseases

Genome-wide association studies have identified multiple novel genomic loci associated with vascular diseases. Many of these loci are common non-coding variants that affect the expression of disease-relevant genes within coronary vascular cells. To identify such genes on a genome-wide level, we performed deep transcriptomic analysis of genotyped primary human coronary artery smooth muscle cells (HCASMCs) and coronary endothelial cells (HCAECs) from the same subjects, including splicing Quantitative Trait Loci (sQTL), allele-specific expression (ASE), and colocalization analyses. We identified sQTLs for TARS2, YAP1, CFDP1, and STAT6 in HCASMCs and HCAECs, and 233 ASE genes, a subset of which are also GTEx eGenes in arterial tissues. Colocalization of GWAS association signals for coronary artery disease (CAD), migraine, stroke and abdominal aortic aneurysm with GTEx eGenes in aorta, coronary artery and tibial artery discovered novel candidate risk genes for these diseases. At the CAD and stroke locus tagged by rs2107595 we demonstrate colocalization with expression of the proximal gene TWIST1. We show that disrupting the rs2107595 locus alters TWIST1 expression and that the risk allele has increased binding of the NOTCH signaling protein RBPJ. Finally, we provide data that TWIST1 expression influences vascular SMC phenotypes, including proliferation and calcification, as a potential mechanism supporting a role for TWIST1 in CAD

Isolated cardiomyocytes release cardiac bridging integrator 1 (cBIN1) microparticles (MPs).

<p>(A) Western blot data of cBIN1 in cardiomyocyte lysates (positive control), MP pellets, and MP-free supernatant of culture medium. (B) Nanoparticle tracking analysis of resuspended MPs purified from culture medium. (C) Transmission electron microscopy (TEM) images (15,000x) of MPs prepared from cardiomyocyte culture medium after negative staining and immunogold labeling. Left, mouse immunoglobulin G (IgG) control; right, mouse anti-BIN1. Histogram data of cBIN1-MP sizes are also included. (D) Top: spinning-disc confocal images of cardiomyocyte-derived MPs colabeled with annexin V (green) and cBIN1 (red) or IgG control (scale bar, 10 μm). Frequency distribution of cBIN1-MP sizes measured by confocal imaging are shown on the right. Bottom: representative enlarged confocal images of annexin V/cBIN1-MPs (scale bar, 1 μm). Quantification of particle counts with different cBIN1 labeling patterns. (E) Representative stochastic optical reconstruction microscopy (STORM) images of annexin V/cBIN1-MPs (scale bar, 1 μm). Frequency distribution of cBIN1-MP sizes measured by STORM imaging are shown on the right. CM, cardiomyocyte; Conc., concentration; IB, immunoblotting.</p

Cartoon of cardiac bridging integrator 1 (cBIN1)-recruited charged multivesicular body protein 4B (CHMP4B) for microparticle (MP) release from cBIN1-microfolds to extracellular space, responsible for the blood availability of cBIN1.

<p>TT, transverse-tubule.</p

Cardiac bridging integrator 1 (cBIN1) is present in human plasma and reduced in heart failure.

<p>(A) Western blot (WB) analysis of protein lysates and immunoprecipitation products from normal human heart lysates and plasma. (B) Mass spectrometry analysis of a cBIN1 protein band from human heart lysate immunoprecipitation (IP). (C) Hypotonic shock induced by double-distilled water increases plasma cBIN1 measured by a cBIN1-specific enzyme-linked immunosorbent assay (ELISA), allowing detection of full blood content. Left: final plasma cBIN1 measured by ELISA following increasing amount of water dilution. Red arrow: Maximal concentration was reached at 25% dilution of plasma (1 volume of plasma with 3 volumes of water). Right: final plasma cBIN1 concentration in 25% plasma diluted in physiological buffered saline (PBS), triton detergent buffer (TB), and double-distilled water. (D) Plasma cBIN1 quantified in healthy control patients and patients with heart failure (HF) (<i>n</i> = 10). *** indicates <i>p</i> < 0.001 using a Student <i>t</i> test. Con., control; IgG, immunoglobulin G; SH3, SRC homology 3.</p

Large-Scale Single-Cell RNA-Seq Reveals Molecular Signatures of Heterogeneous Populations of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells

RATIONALE: Human-induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) have risen as a useful tool in cardiovascular research, offering a wide gamut of translational and clinical applications. However, inefficiency of the currently available iPSC-EC differentiation protocol and underlying heterogeneity of derived iPSC-ECs remain as major limitations of iPSC-EC technology. OBJECTIVE: Here, we performed droplet-based single-cell RNA sequencing (scRNA-seq) of the human iPSCs after iPSC-EC differentiation. Droplet-based scRNA-seq enables analysis of thousands of cells in parallel, allowing comprehensive analysis of transcriptional heterogeneity. METHODS AND RESULTS: Bona fide iPSC-EC cluster was identified by scRNA-seq, which expressed high levels of endothelial-specific genes. iPSC-ECs, sorted by CD144 antibody-conjugated magnetic sorting, exhibited standard endothelial morphology and function including tube formation, response to inflammatory signals, and production of NO. Nonendothelial cell populations resulting from the differentiation protocol were identified, which included immature cardiomyocytes, hepatic-like cells, and vascular smooth muscle cells. Furthermore, scRNA-seq analysis of purified iPSC-ECs revealed transcriptional heterogeneity with 4 major subpopulations, marked by robust enrichment of CLDN5, APLNR, GJA5, and ESM1 genes, respectively. CONCLUSIONS: Massively parallel, droplet-based scRNA-seq allowed meticulous analysis of thousands of human iPSCs subjected to iPSC-EC differentiation. Results showed inefficiency of the differentiation technique, which can be improved with further studies based on identification of molecular signatures that inhibit expansion of nonendothelial cell types. Subtypes of bona fide human iPSC-ECs were also identified, allowing us to sort for iPSC-ECs with specific biological function and identity

Small interfering RNA (siRNA) mediated charged multivesicular body protein 4B (CHMP4B) knockdown reduces cardiac bridging integrator 1 (cBIN1) microparticle (MP) formation and release.

<p>(A) HeLa cells overexpressing cBIN1–green fluorescent protein (GFP) release cBIN1-MPs. Live-cell imaging of HeLa cells expressing cBIN1-GFP with surface labeling of annexin V-Alexa 647. Left, a maximum projection image of a time series of images (every 5 seconds for 2 minutes) of a HeLa cell expressing cBIN1-GFP and surface labeled with annexin V. Right, time-lapse frame images of the boxed area in the left image at the indicated time points. Western blot also identified cBIN1-GFP in MPs from HeLa cell culture medium. (B–D) The effect of CHMP4B siRNA on cBIN1-MP formation and release. (B) Western blot of CHMP4B in HeLa cells treated with CHMP4B siRNA or nontargeting control siRNA. (C) Representative images and quantification of surface annexin V particles in control or CHMP4B siRNA pretreated HeLa cells overexpressing cBIN1-GFP. (D) Western blot results of cBIN1-GFP in cell lysates and medium MP fraction from HeLa cells treated with control or CHMP4B siRNA. Cells are from 3 independent experimental repeats. ** and *** indicate <i>p</i> < 0.05 and <i>p</i> < 0.001, respectively, using an unpaired Student <i>t</i> test. ESCRT, endosomal sorting complexes required for transport; GFP, green fluorescent protein.</p