Myocardial infarctions, ischemic strokes, and peripheral artery disease (PAD) are the most common causes of mortality and morbidity among citizens of the United States. p27^Kip1 (p27) is a cell cycle regulator that affects the human physiological response to acute arterial injury such as coronary angioplasty or arterial bypass vein grafts. Overexpression of the p27 gene inhibits angiogenesis in mice. Conversely mice knocked out for p27 have improved angiogenesis and arteriogenesis. Our preliminary data shows that p27^-/- (knockout) mice have increased vessel diameters after hind-limb ischemia, but the molecular pathways controlling this collateralization result are not well understood. Our lab is interested in conducting in-vitro assays upon p27^-/- and wild-type (WT) vascular smooth muscle cells (VSMCs) to study the role of the p27 gene in VSMCs under hypoxic conditions as opposed to normoxic conditions. We hypothesize that cell proliferation and migration of p27^-/-, p27^+/- (HET) and WT VSMCs under hypoxic conditions will better mimic the ischemic in-vivo model than VSMC under normoxic conditions, and provide better insight about the molecular basis of collateralization via genetic profiling. I am currently performing experiments to further develop a hypoxic experimental apparatus and conducting normoxic in-vitro cell proliferation and cell migration assays to compare with the hypoxic experimental model. Preliminary results reveal that p27^-/- VSMC proliferate and migrate more than WT VSMC in normoxia. We expect this difference to be magnified in hypoxia. The results of this project will enable us to establish the hypoxia in-vitro model in the laboratory, and better study gene expression changes in hypoxia between the two genotypes.

Collateral artery development is the body's natural adaptive response to bypass major artery blockage. However, this process restores about 30% of the original blood flow, which is insufficient to avoid end-organ ischemia. Therefore, there is a great need for better understanding of the angiogenesis process. Overexpression of the p27kip1 (p27 or cdkn1b) gene inhibits angiogenesis in mice, and p27 knock-out mice (p27-/- or KO) have improved angiogenesis and arteriogenesis. We previously showed that p27-/- mouse vascular smooth muscle cells (VSMC) have increased matrix metalloproteinase 2 (MMP2) mRNA and migrate more effectively than wild type (WT) VSMC. We wanted to better understand the link between p27, MMP2, and cell repair capability. We hypothesized that decreasing the p27 genetic dose would result in increased MMP2 expression. Experiments were performed on p27+/+ (WT), p27+/- (HET), and KO VSMC isolated from 8-10 wk old female mouse aortae. All groups were tested for MMP2 mRNA and protein expression level using qRT-PCR and Western blot respectively. A cytoplasmic fraction was used for protein analysis. MMP2 mRNA expression in HET VSMC was significantly higher than in WT (194±9 vs. 101±3%, p<0.001), and significantly lower than in KO VSMC (280±38%, p<0.001). TIMP1 and TIMP2 mRNA expression was significantly lower in the KO VSMC than in the HET and WT cells (p<0.01). In the cytoplasmic fraction there was no significant difference between MMP2 protein levels between KO and HET VSMC, but both were significantly higher than the level in WT (2.55±0.27 vs. 2.15±0.22 and 1.14±0.3 respectively, p<0.05). Decreasing genetic dose of p27 in VSMC leads to higher levels of MMP2 mRNA and protein levels. This suggests that p27’s effect on VSMC migration in vitro and possibly its effect on angiogenesis and arteriogenesis is through regulation of MMP2 expression. TIMP1 and TIMP2 may also play a role in this process.