title: Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1-matrix metalloproteinase following acute hindlimb ischemia.
abstract: OBJECTIVE
  Matrix metalloproteinase (MMP) activity is essential for remodeling of ischemic tissue. The murine hindlimb ischemia model exhibits tissue remodeling including revascularization in part due to angiogenesis. MMP-2 and -9 are type IV collagenases necessary for basement membrane degradation as a part of extracellular matrix remodeling and angiogenesis. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of membrane type 1 (MT1)-MMP, are able to activate proMMP-2 in vitro. Activation of MMP-2 and -9 may be essential in ischemic limbs both for tissue remodeling and revascularization via angiogenesis. We hypothesized that MMP-2 and -9 would be activated following acute hindlimb ischemia (HI), and this activation would be temporally related to PMN infiltration.
  
  
  METHODS
  HI was achieved by unilateral femoral artery ligation in 20 FVB/N mice. Five mice underwent sham operation without hindlimb ischemia. Gastrocnemius muscle was harvested from both hindlimbs at 1, 3, 14, and 30 days following ligation and assayed for MMP-2, -9 (gelatin zymography), and MT1-MMP (Western blotting). MMP-2 and -9 expression and activation were analyzed by gelatin zymography and quantified by densitometry with NIH Image Analysis software. Neutrophils per high power field were counted. The results were expressed as a ratio of ischemic to nonischemic limbs and compared at each time point using ANOVA.
  
  
  RESULTS
  Zymographic analysis revealed a 212% increase in active MMP-2 3 days postligation (P <.05). Active MMP-9 reached its maximum level (800% over baseline) on postoperative day 3 and continued to be elevated on day 14 (737% over baseline) (P <.05). The increase in active MMP-2 and -9 levels paralleled PMN infiltration that also peaked 3 days postligation (1184% over baseline) (P <.05). PMN count, MMP-2, and -9 all returned to baseline levels by postoperative Day 30. MT1-MMP was present in tissue samples from all time points as confirmed by Western blot.
  
  
  CONCLUSIONS
  Limb ischemia causes an early activation of MMP-2 and -9 in temporal relation to PMN infiltration. HI may prime PMNs, leading to their sequestration in ischemic tissue. Primed PMNs, along with constitutively expressed MT1-MMP, may activate MMPs-2 and -9 and enable tissue remodeling essential for limb revascularization and angiogenesis.
authors.affiliation: S.A. Localio Laboratory for Surgical Research, New York Harbor Healthcare System, New York University School of Medicine, 530 First Ave., Suite 6F, New York, New York 10016, USA.
authors.forename: Bart E
authors.initials: BE
authors.lastname: Muhs
authors.forename: George
authors.initials: G
authors.lastname: Plitas
authors.forename: Yara
authors.initials: Y
authors.lastname: Delgado
authors.forename: Ioana
authors.initials: I
authors.lastname: Ianus
authors.forename: Jason P
authors.initials: JP
authors.lastname: Shaw
authors.forename: Mark A
authors.initials: MA
authors.lastname: Adelman
authors.forename: Patrick
authors.initials: P
authors.lastname: Lamparello
authors.forename: Peter
authors.initials: P
authors.lastname: Shamamian
authors.forename: Paul
authors.initials: P
authors.lastname: Gagne
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004789:Enzyme Activation; D005263:Femoral Artery / Q000601:surgery; D006614:Hindlimb / Q000098:blood supply*; D007511:Ischemia / Q000201:enzymology* / Q000473:pathology; D007700:Kinetics; D007958:Leukocyte Count; D008026:Ligation; D053511:Matrix Metalloproteinase 14; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000378:metabolism*; D051379:Mice; D018919:Neovascularization, Physiologic; D009504:Neutrophils / Q000201:enzymology / Q000473:pathology
chemical_list: C507096:Mmp14 protein, mouse; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053511:Matrix Metalloproteinase 14