Functional analyses of coronary artery disease associated variation on chromosome 9p21 in vascular smooth muscle cells

Abstract

Variation on chromosome 9p21 is associated with risk of coronary artery disease (CAD). This genomic region contains the CDKN2A and CDKN2B genes which encode the cell cycle regulators p16(INK4a), p14(ARF) and p15(INK4b) and the ANRIL gene which encodes a non-coding RNA. Vascular smooth muscle cell (VSMC) proliferation plays an important role in the pathogenesis of atherosclerosis which causes CAD. We ascertained whether 9p21 genotype had an influence on CDKN2A/CDKN2B/ANRIL expression levels in VSMCs, VSMC proliferation and VSMC content in atherosclerotic plaques. Immunohistochemical examination showed that VSMCs in atherosclerotic lesions expressed p16(INK4a), p14(ARF) and p15(INK4b). Analyses of primary cultures of VSMCs showed that the 9p21 risk genotype was associated with reduced expression of p16(INK4a), p15(INK4b) and ANRIL (P = 1.2 × 10(-5), 1.4 × 10(-2) and 3.1 × 10(-9)) and with increased VSMC proliferation (P = 1.6 × 10(-2)). Immunohistochemical analyses of atherosclerotic plaques revealed an association of the risk genotype with reduced p15(INK4b) levels in VSMCs (P = 3.7 × 10(-2)) and higher VSMC content (P = 5.6 × 10(-4)) in plaques. The results of this study indicate that the 9p21 variation has an impact on CDKN2A and CDKN2B expression in VSMCs and influences VMSC proliferation, which likely represents an important mechanism for the association between this genetic locus and susceptibility to CAD.

Figure 1.

Smooth muscle cells in atherosclerotic lesions express p16^INK4a, p14^ARF and p15^INK4b. Sections of formaldehyde-fixed paraffin-embedded tissue blocks of atherosclerotic plaques were subjected to double immunostaining of SMA together with p16^INK4a (A and D), p14^ARF (C and E) or p15^INK4b (E and F). In (A–C), Fast red staining indicates expression of the VSMC marker SMA, and dark brown colour (DAB) indicates expression of p16^INK4a (A), p14^ARF (B) or p15^INK4b (C). Arrows indicate cell co-expressing SMA with p16^INK4a (A), p14^ARF (B) or p15^INK4b (C). No nuclear counterstaining was used in (A–C). In (D–F), brown staining with DAB indicates expression of the VSMC marker SMA, blue colour (haematoxylin) indicates nuclei and Fast red staining indicates expression of p16^INK4a (D), p14^ARF (E) or p15^INK4b (F). Arrows indicate expression of p16^INK4a (D), p14^ARF (E) or p15^INK4b (F), in the nuclei of smooth muscle cells. The haematoxylin nuclear counterstain has partially obscured Fast red nuclear staining of p16^INK4a, p14^ARF and p15^INK4b, in (D–F). ×200 magnification.

Figure 2.

Relationship of 9p21 variation with p16^INK4a and p15^INK4b expression levels in cultured VSMCs. Primary cultures of VSMCs from different individuals (n = 69) were genotyped for SNP rs1333049 and subjected to quantitative reverse transcriptase–polymerase chain reaction and western blot analyses. (A–C) Relative fold differences in p16^INK4a, p14^ARF and p15^INK4b mRNA levels in VSMCs of the G/C or G/G genotype for SNP rs1333049 compared with mRNA levels of the corresponding genes in VSMCs of the C/C genotype. (D) Representative images acquired from western blot analyses of p16^INK4a and p15^INK4b and the gel load reference protein β-actin. (E and F) Relative fold differences in p16^INK4a and p15^INK4b protein levels in VSMCs of the G/C or G/G genotype compared with levels of the corresponding proteins in VSMCs of the C/C genotype. Data shown in (A–C, E and F) are mean ± standard deviation of mean.

Figure 3.

Relationships of ANRIL with 9p21 variation, p16^INK4a, p14^ARFand p15^INK4b in cultured VSMCs. Primary cultures of VSMCs from different individuals (n = 69) were genotyped for SNP rs1333049 and subjected to quantitative reverse transcriptase–polymerase chain reaction analysis of ANRIL, p16^INK4a, p14^ARF and p15^INK4b. (A) Relative fold differences in the ANRIL level in VSMCs of the G/G or G/C genotype for SNP rs1333049 compared with the ANRIL level in VSMCs of the C/C genotype. Data shown are mean ± standard deviation of mean. (B–D) Pair-wise correlation of ANRIL with p16^INK4a, p14^ARF and p15^INK4b, respectively.

Figure 4.

Relationships of p16^INK4a, p15^INK4b and 9p21 variation with the cell proliferation marker PCNA in cultured VSMCs. Primary cultures of VSMCs from different individuals (n = 69) were genotyped for SNP rs1333049 and subjected to quantitative reverse transcriptase–polymerase chain reaction analysis of PCNA, p16^INK4a and p15^INK4b. (A and B) Correlation of PCNA with p16^INK4a and p15^INK4b, respectively. (C) Relative fold differences in the PCNA mRNA level in VSMCs of the G/C or G/G genotype for SNP rs1333049 compared with the PCNA level in VSMCs of the C/C genotype. Data shown are mean ± standard deviation of mean.

Figure 5.

Association between 9p21 variation and VSMC proliferation in culture. Primary cultures of VSMCs from different individuals (n = 73) were genotyped for SNP rs1333049 and subjected to cell proliferation assay by the BrdU labelling and detection method. The amount of BrdU incorporated into cellular DNA was detected using the enzyme-linked immunosorbent assay with the use of a 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit (Roche). Presented in the graph are relative fold differences in absorbance of samples from VSMCs of the G/C or G/G genotype for SNP rs1333049 compared with the absorbance of samples from VSMCs of the C/C genotype. Data shown are mean ± standard deviation of mean.

Figure 6.

Relationship of 9p21 variation with p16^INK4a and p15^INK4b protein levels in atherosclerotic plaques. Atherosclerotic coronary arteries from different individuals (n = 41) were genotyped for SNP rs1333049 and subjected to double immunostaining of SMA together with p16^INK4a or p15^INK4b. Immunostaining images were analysed using Image-Pro software. Shown in the graphs are percentages of p16^INK4a stain area in VSMC stain area (A) and percentage of p15^INK4b stain area in VSMC stain area (B) in plaques of the C/C, C/G and G/G genotypes for SNP rs1333049. Data shown are mean ± standard deviation of mean.

Figure 7.

Association of 9p21 variation with smooth muscle cell content in atherosclerotic plaques. Atherosclerotic coronary arteries from different individuals (n = 52) were genotyped for SNP rs1333049 and subjected to immunostaining of SMA. Immunostaining images were analysed using Image-Pro software. (A) A representative image of immunostaining, purple colour indicates SMA stains, ×40 magnification. (B) Percentages of SMA stain area over total plaque area in plaques of the C/C, C/G and G/G genotypes for SNP rs1333049. Data shown are mean ± standard deviation of mean.