Abstract 144: A Single Nucleotide Polymorphism of p27 Kip1 Associated with Vein Graft Patency Regulates Expression of p27 Kip1 in Both Venous Adventitial Cells and Smooth Muscle Cells but Selectively Regulates Proliferation of Adventitial Cells

Abstract

Introduction: p27 Kip1 (p27) is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (SNP) accounts for ~40% of the risk of peripheral vein graft failure. However, whether this SNP is functional has not been definitely established. Methods: To determine functionality, we investigated paired adventitial cells and smooth muscle cells (SMC) derived from fresh human saphenous veins (N≥7 lines per group). After growth arrest in 2% serum followed by stimulation with 10 ng/ml PDGF-BB, we measured p27 mRNA, p27 protein, and cell proliferation using qRT-PCR, Western blotting, and cell counts, respectively. Results: The SNP genotype was associated with 72 hour adventitial cell growth, but not SMC growth. Adventitial AA cells grew 33% slower than those with the CC genotype (Figure 1A, B, p=.004). We expected AA adventitial cells to produce more p27 mRNA, but paradoxically, p27 mRNA was lower in both cell types of the AA genotype (Figure 1C, D, P<.01). However, levels of p27 protein (a single ~27 kD band in both cell types) were ~2 fold higher in AA adventitial cells and SMCs compared to the CC cells at both 0 and 72 hours (Figure 1E, 72 hours; P<.001 for genotype; 0 h data not shown). Adventitial cells and SMCs made comparable amounts of p27 protein. Conclusion: The p27 Kip1 -838C>A SNP regulates levels of p27 in both venous adventitial cells and SMCs. There is an effect of genotype on the growth of adventitial cells, but not on SMCs. AA cells produce ~2 fold higher p27 protein than CC cells, despite AA cells having ~2 fold lower levels of p27 mRNA. These data demonstrate that the p27 SNP is functional, that different cell types within the vein wall respond uniquely to this genetic polymorphism, and suggest an important role in graft failure for the precursor of the cultured adventitial cell.