Abstract 17096: CDKN2B Regulates Cell Fate Decisions in Human Vascular Smooth Muscle Cells
Objectives: Genome wide association (GWA) studies have identified single nucleotide polymorphisms (SNPs) at chromosome 9p21.3 that are highly associated with vascular disease in man. It is unclear, however, how these noncoding SNPs control gene expression and what their effects are on constituents of the vessel wall. We aimed to define expression SNPs (eSNPs) present at 9p21.3 and employ in vitro vascular models to identify causal genes and disease-related mechanisms.
Methods: Simultaneous genotyping and mRNA microarray analysis of biologically relevant tissues was performed on 698 subjects. The expression and regulation of the 9p21 cyclin dependent kinase (CDK) inhibitor genes was assessed in human arterial SMC (smooth muscle cells). In vitro modulation of these genes was performed via siRNA knockdown and transient overexpression experiments. The effect of the CDK inhibitors was then queried with Boyden chamber (chemotaxis), MTT (proliferation and survival), annexin staining (apoptosis), and SMC gene expression (differentiation) assays.
Results: The disease-associated variant rs4977574 was found to be an eSNP and to correlate with variation in CDKN2B expression- but not other 9p21.3 genes — in visceral fat biopsies. CDKN2B, but not CDKN2A, ANRIL or ARF, expression was significantly upregulated during cellular differentiation. Cells deficient in CDKN2B had a significantly impaired capacity to undergo phenotype switching in response to serum starvation (4.2–6.9 fold reduction in SMC-specific gene expression, p<0.02). Similarly, SMC deficient in CDKN2B also displayed accelerated proliferation (0.028 +/− 0.011 vs. 0.019 +/− 0.009 relative proliferation units, p = 0.007), and cellular migration (72.5 +/− 35.6 vs. 56.9 +/− 22.1 cells/hpf, p <0.05). H2O2- induced apoptosis was significantly reduced in CDKN2B-overexpressing cells relative to control (2.97 +/− 1.0 vs. 6.41 +/− 2.2 %, p < 0.02).
Conclusions: The noncoding 9p21.3 polymorphism, rs4977574, correlates with the expression of the distant tumor suppressor gene, CDKN2B, likely via a long-range cis-acting mechanism. CDKN2B is modulated during the differentiation of vascular SMCs and regulates several pathways relevant to atherosclerogenesis and aneurysm formation.
- © 2010 by American Heart Association, Inc.