Experimental hindlimb ischemia leads to neutrophil-mediated increases in gastrocnemius MMP-2 and -9 activity: A potential mechanism for ischemia induced MMP activation

Introduction Matrix metalloproteinases (MMP)-2 and -9 are Type 4 collagenases instrumental in basement membrane degradation, a process necessary for angiogenesis to occur. Polymorphonuclear leukocytes (PMNs) contain MMP-9, and in the presence of both PMN-derived serine protease and membrane type 1 (MT1)-MMP, are able to activate pro-MMP-2 following hindlimb ischemia. We hypothesized that neutrophil depletion (ND) of animals prior to hindlimb ischemia (HI) would abrogate the activation of pro-MMP-2 and decrease the level of MMP-9 Materials and methods 12 FVB/N Tie2/LacZ-182 SATO female mice were randomly divided into four blinded groups; HI + PBS, HI + anti-PMN antibody (GR-1), HI + isotype matched control antibody (IgG_2b,K), and no HI + PBS. PMN depletion was achieved prior to the time of ischemia and maintained until sacrifice. HI was achieved by unilateral femoral artery ligation. Three days postligation the animals were sacrificed and the gastrocnemius muscle from each hindlimb was harvested. MMP-2 and -9 (gelatin zymography) and MT1-MMP (Western blot) expression and activation were quantified by densitometry and NIH Image Analysis software. MMP values were expressed as a ratio of ischemic-to-nonischemic hindlimbs and compared between groups. Statistical significance was determined with analysis of variance (ANOVA) Results Zymograms revealed a greater than 10-fold increase in active MMP-9 and greater than 4-fold increase in active MMP-2 from HI + PBS compared to no HI + PBS (P < 0.05). HI + anti-PMN antibody demonstrated reduction of both active MMP-2 and -9 levels to that of the nonischemic group. Pro-MMP-2 was constitutively expressed in all four groups with no significant differences between any group (P = NS). There was no difference between the HI + isotype-matched antibody group and the HI + PBS group throughout the experiments (P = NS). ND did not affect MT1-MMP activation or expression Conclusions Limb ischemia causes activation of MMP-2 and -9, which is eliminated by ND. ND animals undergoing hindlimb ischemia exhibit identical levels of active MMP-2 and -9 as animals that did not have hindlimb ischemia. Neutrophils may be an important activator of MMP-2 and the suppliers of MMP-9 in the ischemic hindlimb and may be essential for tissue remodeling, basement membrane degradation, and angiogenesis in ischemic limbs