title: p27(kip1) Knockout enhances collateralization in response to hindlimb ischemia.
abstract: The natural response to arterial occlusive disease is enlargement of collaterals; however, the molecular factors that control collateralization are not well understood. The gene p27(Kip1) (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.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Division of Vascular Surgery, University of Washington, Seattle, Wash.
authors.forename: Galit
authors.initials: G
authors.lastname: Ankri-Eliahoo
authors.affiliation: Division of Vascular Surgery, University of Washington, Seattle, Wash.
authors.forename: Kevin
authors.initials: K
authors.lastname: Weitz
authors.affiliation: Department of Pediatrics, University of Washington, and Center for Developmental Biology and Regenerative Medicine, Seattle Children's Research Institute, Seattle, Wash.
authors.forename: Timothy C
authors.initials: TC
authors.lastname: Cox
authors.affiliation: Division of Vascular Surgery, University of Washington, Seattle, Wash; Division of Vascular Surgery, Department of Surgery, VA Puget Sound Health Care System, Seattle, Wash. Electronic address: gtang@uw.edu.
authors.forename: Gale L
authors.initials: GL
authors.lastname: Tang
mesh_terms: D020533:Angiogenesis Inhibitors / Q000494:pharmacology; D000818:Animals; D001783:Blood Flow Velocity; D002465:Cell Movement; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D003097:Collateral Circulation / Q000187:drug effects; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000172:deficiency* / Q000235:genetics; D004195:Disease Models, Animal; D005260:Female; D005838:Genotype; D006614:Hindlimb; D007511:Ischemia / Q000235:genetics / Q000378:metabolism / Q000503:physiopathology*; D017078:Laser-Doppler Flowmetry; D020778:Matrix Metalloproteinase 2 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors / Q000494:pharmacology; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000378:metabolism / Q000503:physiopathology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010641:Phenotype; D012039:Regional Blood Flow; D013997:Time Factors; D055114:X-Ray Microtomography
chemical_list: D020533:Angiogenesis Inhibitors; C493961:Cdkn1b protein, mouse; D061965:Matrix Metalloproteinase Inhibitors; D050760:Cyclin-Dependent Kinase Inhibitor p27; D003094:Collagen; D020778:Matrix Metalloproteinase 2; C522362:Mmp2 protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse