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. DESIGN OF STUDY: 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.