FURIN Expression in Vascular Endothelial Cells Is Modulated by a Coronary Artery Disease-Associated Genetic Variant and Influences Monocyte Transendothelial Migration

Background: Genome-wide association studies have shown an association between the single-nucleotide polymorphism rs17514846 on chromosome 15q26.1 and coronary artery disease susceptibility. The underlying biological mechanism is, however, not fully understood. rs17514846 is located in the FES Upstream Region (FURIN) gene, which is expressed in vascular endothelial cells (ECs). We investigated whether rs17514846 has an influence on FURIN expression in ECs and whether FURIN affects EC behavior. Methods and Results: Quantitative reverse transcription–polymerase chain reaction analysis showed that cultured vascular ECs from individuals carrying the coronary artery disease risk allele of rs17514846 had higher FURIN expression than cells from noncarriers. In support, luciferase reporter analyses in ECs indicated that the risk allele had higher transcriptional activity than the nonrisk allele. Electrophoretic mobility shift assays using EC nuclear protein extracts detected a DNA-protein complex with allele-specific differential binding of a nuclear protein. Knockdown of FURIN in ECs reduced endothelin-1 secretion, nuclear factor-κB activity, vascular cell adhesion molecule-1, and MCP1 (monocyte chemotactic protein-1) expression and monocyte-endothelial adhesion and transmigration. A population-based study showed an association of the rs17514846 risk allele with higher circulating MCP1 levels and greater carotid intima-media thickness. Conclusions: The coronary artery disease risk variant at the 15q26.1 locus modulates FURIN expression in vascular ECs. FURIN levels in ECs affect monocyte-endothelial adhesion and migration

Coronary-Heart-Disease-Associated Genetic Variant at the COL4A1/COL4A2 Locus Affects COL4A1/COL4A2 Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction

Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD risk

Association between SNP rs4773144 and MI incidence/prevalence in CHD patients.

<p>Association between SNP rs4773144 and MI incidence/prevalence in CHD patients.</p

Influence of SNP rs4773144 genotype on <i>COL4A2</i> and <i>COL4A1</i> expression levels.

<p>Primary cultures of vascular SMCs and ECs from different individuals were genotyped for rs4773144 and subjected to quantitative reverse transcriptase–polymerase chain reaction analysis of <i>COL4A2</i> and <i>COL4A1</i>. Showed in the graphs are relative fold differences in <i>COL4A2</i> (<b>A</b> and <b>B</b>) and <i>COL4A1</i> (<b>C</b> and <b>D</b>) RNA levels in SMCs (<b>A</b> and <b>C</b>) and ECs (<b>B</b> and <b>D</b>), respectively. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Association of SNP rs4773144 with collagen IV level in atherosclerotic plaques and plaque cap thickness.

<p>Atherosclerotic coronary arteries from different individuals were genotyped for SNP rs4773144 and subjected to histopathological analyses using Image-Pro software. <b>(A)</b> A representative image of immunostaining, blue color indicates COL4A2 staining, and brown smooth muscle alpha-actin. ×400 original magnification. <b>(B)</b> Percentages of COL4A2 immunostain positive areas in atherosclerotic plaques in different genotype groups. <b>(C)</b> Atherosclerotic plaque cap thickness in different genotype groups. <b>(D)</b> Atherosclerotic plaque cap/intima ratios in different genotype groups. Columns and error bars in <b>B</b>-<b>D</b> are mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Influence of SNP rs4773144 genotype on apoptosis.

<p><b>(A)</b> Primary cultures of SMCs from different individuals were genotyped for rs4773144 and subjected to apoptosis assay. Shown in the graph are relative differences in cell apoptotic rate in different genotype groups. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model. <b>(B)</b> Primary cultures of ECs of different genotypes for rs4773144 were subjected to immunoblot analysis of the anti-apoptotic protein BCL2. Shown on the left are representative immunoblot images and on the right a graphic presentation of BCL2 band intensities standardized against band intensities of the loading control HSC70. Columns and error bars represent mean and SEM values; <i>p</i>-values shown are for an additive genetic model.</p

Influence of SNP rs4773144 genotype on gene transcription.

<p>Primary cultures of vascular SMCs (A) and ECs (B) of the A/G genotype for SNP rs4773144 from different individuals were subjected to chromatin immunoprecipitation using an antibody against Pol II, followed by an allelic imbalance analysis of SNP rs4773144, with the use of the TaqMan method to determine the ratio of Ct value of the A allele (detected by a VIC fluorescein-labeled probe) versus the Ct value of the G allele (detected by a FAM fluorescent dye-labeled probe). Upper panels show amplification curves of the A allele (red) and G allele (green). Column charts in the lower panels show the relative fold difference in G to A ratio of Ct values between input DNA and anti-Pol II antibody precipitated chromatin DNA. Columns and error bars represent mean and SEM values.</p

Effect of SNP rs4773144 on transcription modulating activity of a DNA sequence encompassing the SNP and on nuclear protein interaction with the DNA sequence.

<p><b>(A)</b> Vascular SMCs were transfected with a firefly luciferase reporter gene plasmid (pGL3-promoter vector) with an insert corresponding to a DNA sequence encompassing the rs4773144 site of the A or G allele, or with the empty plasmid (control), together with a plasmid (pRL-TK) containing a <i>renilla</i> luciferase gene to serve as a transfection efficiency reference. At 48 hour after transfection, activities of firefly luciferase and <i>renilla</i> luciferase were measured. Columns and error bars represent mean and SEM values of the ratio of firefly luciferase activity to <i>renilla</i> luciferase activity. <b>(B)</b> A representative image of electrophoretic mobility shift assays. Nuclear protein extracts from vascular ECs were incubated with biotin-labeled probes corresponding to the A allele (lanes 1–7) or the G allele (lanes 8–14) of SNP rs4773144 in the absence or presence of competitors in 20-fold or 50-fold molar excess, as indicated underneath the image.</p