Temporal expression and activation of matrix metalloproteinases-2, -9, and membrane type 1-matrix metalloproteinase following acute hindlimb ischemia.
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.
Muhs
Bart E
BE
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.
Plitas
George
G
Delgado
Yara
Y
Ianus
Ioana
I
Shaw
Jason P
JP
Adelman
Mark A
MA
Lamparello
Patrick
P
Shamamian
Peter
P
Gagne
Paul
P
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
C507096:Mmp14 protein, mouse; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D053511:Matrix Metalloproteinase 14