Decreased Apoptosis and Increased Proangiogenic Gene Expression in p27Kip1-Deficient Mouse Collaterals After Hindlimb Ischemia

Abstract

Previous studies showed that the gene p27Kip1 (p27) affects the human response to arterial injury as well as collateralization after hindlimb ischemia in mice. The exact mechanism through which p27 works is not yet known. We hypothesized that there would be less apoptosis (lower Bax/Bcl2 ratio) in p27−/− (knockout [ko]) mice collateral arteries after hindlimb ischemia and more expression of proangiogenic genes (Mmp2, Foxo3, Tgf-β, and Tnf-α) than in p27+/+ (wild type [wt]) collateral arteries. The ko and wt mice had their femoral and collateral arteries harvested 7 days after hindlimb ischemia for RNA isolation and quantitative reverse transcription polymerase chain reaction. Small interfering RNA (siRNA) knockdown of p27 was performed on vascular smooth muscle cells isolated from wt aortae, and Mmp2 messenger RNA (mRNA) expression level was measured using quantitative reverse transcription polymerase chain reaction. Movat pentachrome staining was performed on wt and ko hindlimb sections 28 days after hindlimb ischemia. Bax mRNA expression increased significantly more in wt collaterals after hindlimb ischemia than in ko collaterals. There was no significant difference in the Bcl2 mRNA expression for the ischemic collaterals between the two groups. Therefore, the ratio of Bax/Bcl2 mRNA expression was significantly lower in ko gracilis collaterals compared with wt collaterals after hindlimb ischemia. Expression of Mmp2, Foxo3, Tgf-β, and Tnf-α mRNA increased significantly more in ko collaterals after ischemia than in wt collaterals. We also found that Mmp2 mRNA expression increased by 237% after 89% siRNA knockdown of p27 in wt vascular smooth muscle cells. Movat staining showed that the collateral wall thickness was 133% ± 27% higher in ko mice compared with wt mice by 28 days after hindlimb ischemia (P < .02). The ko collaterals had significantly less apoptosis than wt collaterals and significantly higher mRNA expression of proangiogenic genes. Mmp2 mRNA expression increased significantly more in p27 ko collaterals after hindlimb ischemia. These results may explain why p27 ko mice revascularize better than wt mice after hindlimb ischemia and suggest that p27 may have additional effects other than regulating Mmp2 expression in the collateralization response.