p27kip1 Knockout enhances collateralization in response to hindlimb ischemia

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Abstract

Objective:

The natural response to arterial occlusive disease is enlargement of collaterals; however, the molecular factors that control collateralization are not well understood. The gene p27Kip1 (p27) affects human response to arterial injury. Previous studies have shown that overexpression of p27 inhibits vascular endothelial and vascular smooth muscle cell (VSMC) proliferation and angiogenesis. To test the hypothesis that knockout of p27 would improve collateralization in reaction to ischemia, we performed in vivo and in vitro experiments using p27 knockout (p27−/−) and wild-type (wt) mice.

Methods:

Hindlimb ischemia was induced by left femoral artery ligation in p27−/− and wt (C57BL/6) female mice. The mice underwent weekly laser Doppler perfusion imaging of the footpads until sacrifice on postoperative day 28 followed by microcomputed tomography scanning of both hindlimbs. VSMCs were isolated from p27−/− and wt mice and used in migration and gel contraction assays in the absence and presence of the nonspecific matrix metalloproteinase (MMP) inhibitor BB94. MMP-2 and MMP-9 messenger RNA (mRNA) expression was measured by quantitative reverse transcription-polymerase chain reaction in p27−/− and wt VSMCs.

Results:

p27−/− mice reperfused more effectively than wt mice by laser Doppler starting from day 7 (ischemic/nonischemic ratio, 0.33 ± 0.02 vs 0.25 ± 0.02; P < .05) and continuing through day 28 (0.45 ± 0.04 vs 0.31 ± 0.04; P < .05). The gracilis collateral diameter was similar for the nonischemic hindlimbs of the p27−/− and wt mice, and this collateral pathway increased similarly after ischemia as assessed by microcomputed tomography. However, the p27−/− mice significantly enlarged a novel collateral pathway that bridged directly between the femoral artery proximal to the ligation site and the saphenous or popliteal artery distal to the ligation site more than wt mice (158 ± 18.3 vs 82 ± 22 μm; P < .001). p27−/− VSMCs migrated more (79% ± 5% vs 56% ± 6%; P < .05) and caused more gel contraction (18% ± 5% of the initial area vs 43% ± 4%; P < .05) than wt cells. Migration and collagen contraction were abolished in p27−/− and wt cells by MMP inhibition. p27−/− cells expressed significantly more MMP-2 mRNA than wt cells did.

Conclusions:

Knockout of p27 enhances arterial collateralization in response to hindlimb ischemia through enlargement of a new collateral pathway. In vitro, knockout of p27 increases collagen gel contraction in addition to stimulating VSMC migration. We speculate that p27 may affect collateralization through its role in regulating MMP-2 expression.

Clinical Relevance:

Atherosclerosis is the leading cause of mortality and morbidity in the United States. _ENREF_2 The adaptive response to the progressive occlusion of arteries is collateralization (arteriogenesis). As many patients with severe atherosclerosis are not good candidates for angioplasty or surgical bypass, therapies directed toward improving collateralization are needed. The molecular pathways controlling collateralization, however, are not well understood. The human response to arterial injury is affected by a genetic polymorphism in the gene CDKN1B (p27Kip1 or p27). We demonstrate that knockout of p27 improves collateralization. Study of the molecular partners of p27 will identify therapeutic candidates to enhance this process for patients.

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