The novel coronary artery disease risk gene JCAD/KIAA1462 promotes endothelial dysfunction and atherosclerosis

Abstract

Aims: Recent genome-wide association studies (GWAS) have identified that the JCAD locus is associated with risk of coronary artery disease (CAD) and myocardial infarction (MI). However, the mechanisms whereby candidate gene JCAD confers disease risk remain unclear. We addressed whether and how JCAD affects the development of atherosclerosis, the common cause of CAD.

Methods and results: By mining data in the Genotype-Tissue Expression (GTEx) database, we found that CAD-associated risk variants at the JCAD locus are linked to increased JCAD gene expression in human arteries, implicating JCAD as a candidate causal CAD gene. We therefore generated global and endothelial cell (EC) specific-JCAD knockout mice, and observed that JCAD deficiency attenuated high fat diet-induced atherosclerosis in ApoE-deficient mice. JCAD-deficiency in mice also improved endothelium-dependent relaxation. Genome-wide transcriptional profiling of JCAD-depleted human coronary artery ECs showed that JCAD depletion inhibited the activation of YAP/TAZ pathway, and the expression of downstream pro-atherogenic genes, including CTGF and Cyr61. As a result, JCAD-deficient ECs attracted fewer monocytes in response to lipopolysaccharide (LPS) stimulation. Moreover, JCAD expression in ECs was decreased under unidirectional laminar flow in vitro and in vivo. Proteomics studies suggest that JCAD regulates YAP/TAZ activation by interacting with actin-binding protein TRIOBP, thereby stabilizing stress fiber formation. Finally, we observed that endothelial JCAD expression was increased in mouse and human atherosclerotic plaques.

Conclusion: The present study demonstrates that the GWAS-identified CAD risk gene JCAD promotes endothelial dysfunction and atherosclerosis, thus highlighting the possibility of new therapeutic strategies for CAD by targeting JCAD.

Keywords: Atherosclerosis; Coronary artery disease; Endothelial function; GWAS; JCAD/KIAA1462; Therapeutic target.

Figure 1

CAD-associated risk variants at JCAD locus are associated with increased JCAD gene expression in human aortic artery tissues. (A) Identification of lead single nucleotide polymorphism (SNP) rs9337951 in the CARDIoGRAMplusC4D and UK Biobank meta-analysis. (B) Box plots showing expression quantitative trait loci data from GTEx (v7) datasets for the lead genome-wide association studies SNP (rs9337951) associated with normalized JCAD gene expression in aortic artery tissue. Both genome-wide significant expression quantitative trait loci P-values and normalized effect size are shown for indicated SNPs. (C) Single-tissue expression quantitative trait loci normalized effect sizes for rs9337951 are shown for 48 different tissues in GTEx (v7), depicted as a color-coded forest plot with 95% confidence intervals. METASOFT based m-values representing the posterior probability of tissue-specific expression quantitative trait loci’s are also shown with their respective single-tissue expression quantitative trait loci P-values.

Figure 2

JCAD deficiency in mice improves endothelium-dependent vasorelaxation under high fat-diet feeding conditions. (A) Endothelium-dependent vasodilation was determined by relaxation of aortic rings pre-constricted with phenylephrine (10⁻⁶ mol/L). The dose-response curves of aortic rings to the vasodilator acetylcholine (10⁻⁹–10⁻⁵ mol/L) in JCAD^−/− mice (n = 6) and JCAD^+/+ (n = 9) mice under high fat diet conditions for 12 weeks. (B) The dose-response curves of aortic rings to NO donor sodium nitroprusside (10⁻⁹–10⁻⁵ mol/L) in JCAD^−/− mice (n = 6) and JCAD^−+/+ (n = 9) mice fed a high fat diet for 12 weeks.

Figure 3

JCAD deficiency reduces atherosclerosis in ApoE^−/− mice fed a western-type diet. (A-B) En face Oil Red O–staining of whole aorta from male (A, n = 15–16) and female (B, n = 7–12) JCAD^+/+; ApoE^−/− and JCAD^−/−; ApoE^−/− mice after 12 weeks of western-type diet feeding. Quantification of en face lesion area. Data were represented as percent Oil Red O positive area of the entire aorta measured by Image J. Images presented were from a composite of 2–3 images from the same aorta. (C) Representative Oil Red O–stained aortic sinus cryosections from male JCAD^+/+; ApoE^−/− (n = 6) and JCAD^−/−; ApoE^−/− (n = 5) mice. Quantification of aortic sinus lesion area between two groups. Data were represented as percent Oil Red O positive area of the entire sinus measured by Image J. (D–F), Analysis of plaque composition in aortic sinus. The contents of macrophages (CD68 positive, green, D), smooth muscle cells (α-SMA positive, red, E) were analyzed by immunostaining-based quantification of the positive signal (mean fluorescence intensity in arbitrary units) was provided in right panels, n = 6 per each group, scale bar = 100 μm. Collagen content (blue, F) was determined by Masson’s trichrome staining. Quantification data were presented as % lesion area in the right panel, n = 8 for JCAD^+/+; ApoE^−/− and n = 7 for JCAD^−/−; ApoE^−/− group. α-SMA, alpha-smooth muscle actin.

Figure 4

Endothelial cell-specific JCAD deficiency reduces atherosclerosis in ApoE^−/− mice fed a western-type diet. (A) Confocal microscopy shows the colocalization of JCAD (red) with adherens junction protein VE-cadherin (green) in HUVECs. DAPI was used for counterstaining of cell nuclei. Enlarged images with arrows showing both membrane and perinuclear vesicular localization of JCAD. Bar = 40 µm, n = 4. (B) JCAD gene expression in mouse vascular cells. Relative expression of JCAD gene was compared in primary mouse lung endothelial cells (EC), mouse aortic smooth muscle cells (SMC), and mouse peritoneal macrophages (MPM), n = 3. (C) JCAD gene expression in human vascular cells. Relative expression of JCAD gene was compared in human coronary artery endothelial cells (HCAEC), human coronary artery smooth muscle cells (HCASMC), and human THP1-derived macrophages (THP1 monocytes stimulated with 100 nM PMA for 48 h), n = 2. (D-E) En face Oil Red O–staining of whole aorta from male JCAD^ecWT; ApoE^−/− (n = 7) and JCAD^ecKO; ApoE^−/− (n = 7) mice after 12 weeks of western-type diet feeding. Quantification of en face lesion area. Data were represented as percent Oil Red O-positive area of the entire aorta measured by Image J. Images presented were from a composite of 2–3 images from the same aorta. DAPI, 4′,6-diamidino-2-phenylindole; HUVECs, human umbilical vein endothelial cells; PMA, phorbol 12-myristate 13-acetate.

Figure 5

JCAD deficiency attenuates vascular inflammation by acquiring an atheroprotective transcriptional program. (A–B) Gene ontology (A) and KEGG2016 pathway (B) analysis of differentially expressed genes in JCAD depleted HCAECs by RNA-seq. RNA-seq data were deposited in NCBI Gene Omnibus (http://www.ncbi.nlm.nih.gov/geo/) with the accession number of GEO: GSE102498. (C) Selected atherosclerosis-relevant genes regulated by JCAD revealed by RNA-seq. * indicates established downstream target genes of YAP/TAZ. For a whole list of differentially expressed genes, please refer to Supplementary material online, Table S7. (D) Validation of RNA-seq data by real time PCR. Select downregulated and upregulated genes were presented, n = 4. (E–F) JCAD deficiency attenuates LPS-induced vascular inflammation. JCAD^+/+ (WT) and JCAD^−/− (KO) primary mouse lung endothelial cells were stimulated with LPS for 6 h before monocyte (THP1) adhesion assay was performed (E, n = 6). In parallel, whole cell lysates were collected for Western blot analysis to detect protein expression of VCAM1 and ICAM1 (F, n = 4), ***P < 0.001 vs. WT, ^##P < 0.01 vs WT + LPS. Log2FC, Log2 fold change; siNC, scramble control siRNA; siJCAD, JCAD siRNA; LPS, lipopolysaccharide.

Figure 6

JCAD promotes YAP/TAZ activation in endothelial cells. (A) JCAD depletion decreased YAP/TAZ/TEAD luciferase activity (8XGTIIc-lux). Human umbilical vein endothelial cells (HUVECs) were transfected with control siRNA (siNC, 100 nM) or JCAD siRNA (siJCAD, 100 nM) for 48 h before transfection with 8XGTIIc-lux for 24 h, n = 3. YAP/TAZ siRNA (siYAP/TAZ, 20 nM) transfection was used as the positive control. (B) JCAD depletion inhibits YAP/TAZ nuclear staining in HUVECs (left panel). Quantification of YAP/TAZ subcellular localization in siNC (196 cells) and siJCAD (207 cells) treated HUVECs (right panel). C, cytoplasm; N, nucleus. Mean % from four independent experiments were used for quantification. (C) JCAD depletion decreased YAP/TAZ downstream protein expression (CTGF and Cyr61) in HUVECs, n = 3. (D) JCAD depletion promotes YAP phosphorylation at Ser127 in HUVECs, n = 3. (E) JCAD depletion decreased F-actin stress fiber formation in HUVECs. Mean fluorescence intensity was calculated by dividing total fluorescent intensity by cell numbers, n = 4. (F) JCAD interacting proteins identified by immunoprecipitation-based mass spectrometry. Averaged number of peptides from three independent experiments were presented. For a complete list of JCAD interacting proteins, please refer to Supplementary material online, Table S8. Abbreviations: ARMC8: Armadillo repeat-containing protein 8; FLNC: Filamin-C; CKAP4: Cytoskeleton-associated protein 4; PPP2R1A: Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A alpha; TRIOBP: TRIO and F-actin-binding protein; WDR26: WD repeat-containing protein 26. (G) Validation of JCAD interaction with TRIOBP in HUVECs. HUVECs were infected with adenoviral HA-tagged (Ad-HA) JCAD for 48 h before whole cell lysate was collected for HA-beads pulldown assay, n = 3.

Figure 7

JCAD expression is decreased by unidirectional laminar flow in vitro and in vivo. (A) Scheme of a cone-plate viscometer to generate unidirectional laminar flow (UF) or disturbed flow (DF). Image was manually drawn using powerpoint. (B) JCAD gene expression is decreased by unidirectional laminar flow. HUVECs were exposed to unidirectional laminar flow for 24 h before RNA was collected for real time PCR analysis. KLF2 was used the positive control, n=4. (C) JCAD protein expression is decreased by unidirectional laminar flow. HUVECs were exposed to unidirectional laminar flow for 24 h before confocal microscopy was performed, n = 4, bar = 60 μm. (D) JCAD gene expression is increased in response to disturbed flow. VCAM1 was used as the positive control, n = 3. (E) JCAD gene expression in intimal RNA lysate isolated from aortic arch (AA) and thoracic aorta (TA) of ApoE^−/− mice fed a normal diet, n = 12. (F) JCAD expression in C57BL/6J mice undergoing partial ligation of left carotid artery (LCA). Intimal RNA lysate was collected for real-time PCR analysis, n = 4. HUVECs, human umbilical vein endothelial cells; RCA, right carotid artery.

Figure 8

JCAD expression is increased in mouse and human atherosclerotic plaques. (A) JCAD expression is increased in aortic endothelium of ApoE^−/− mice fed a high fat diet (HFD) vs. normal diet (ND), n = 5. (B) JCAD expression is increased in advanced human atherosclerotic plaques, n = 9. (C) Intimal endothelial JCAD expression is increased in human atherosclerotic plaques, n = 4. A, atheroma; I, intima; L, lumen; M, media.

Take home figure

JCAD promotes endothelial dysfunction and atherosclerosis. Junctional protein JCAD promotes the activation of YAP/TAZ/TEAD by interacting with TRIOBP and thus stabilizing F-actin stress fiber. By doing so, JCAD trigger the expression of inflammatory genes in endothelial cells, driving an inflammatory process via the recruitment of monocytes and resulting in the formation of atherosclerotic plaques.