Objective: The pathophysiology underlying the chromosome (Chr) 9p21 locus of atherosclerosis susceptibility is presently unknown. Here, we sought to determine whether protein coding genes in the Chr9p21 region, i.e. cyclin-dependent kinase inhibitors CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)) and methylthioadenosine phosphorylase (MTAP) were expressed in human atherosclerotic lesions and whether expression was correlated with lesion composition.
Methods and results: Protein expression of p15(INK4b), p16(INK4a), p14(ARF) and MTAP was demonstrated by immunostaining in normal and atherosclerotic coronary arteries and co-localized with CD68 and smooth muscle alpha-actin positive cells. Quantitative RT-PCR in human endarteryectomy specimens (n = 57) revealed increased p16(INK4a) and decreased MTAP expression in macrophage-rich lesions (P<0.001 and P = 0.007, respectively). Functional studies suggest that decreased MTAP expression in macrophage-rich lesions might be mediated through down-regulation by TNF-alpha. No clear association of p15(INK4b), p16(INK4a), p14(ARF), and MTAP expression in plaque tissue with Chr9p21 haplotypes was found. The latter finding was corroborated by the lack of correlation of RNA expression of 9p21-regulated transcripts EU741058 and NR_003529 of antisense non-coding RNA in the INK4 locus (ANRIL) with mRNA expression of these genes. In contrast, ANRIL DQ485454 which is not genetically determined by the 9p21 genotype was significantly correlated with MTAP expression (P = 0.01).
Conclusion: CDKN2B (p15(INK4b)), CDKN2A (p16(INK4a), p14(ARF)), and MTAP are abundantly expressed in atherosclerotic lesions. While expression levels showed no clear association with Chr9p21 genotype, association of high p16(INK4a) and low MTAP expression with a less stable plaque phenotype suggests a more general role of these proteins in atherogenesis.
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