Nitric Oxide in Vascular Endothelial Growth Factor-Induced Focal Angiogenesis and Matrix Metalloproteinase-9 Activity in the Mouse Brain

Background and Purpose-Vascular endothelial growth factor (VEGF) can induce matrix metalloproteinase (MMP)-9 activities and focal angiogenesis. We hypothesized that VEGF activation of cerebral MMP-9 would require nitric oxide participation. Methods-We compared the in vivo effects of: (1) N-G-monomethyl-L-arginine, a nonspecific nitric oxide synthase inhibitor; (2) L-N-6-(1-iminoethyl)lysine, an inducible nitric oxide synthase selective inhibitor; and (3) doxycycline, a known nonspecific inhibitor of MMP in the mouse brain, using in situ zymography and endothelial marker CD31. 3-nitrotyrosine was used as a surrogate for nitric oxide activity. Inflammatory cell markers CD68 and MPO were used to confirm leukocyte infiltration. Results-VEGF-stimulated MMP-9 activity expressed primarily around cerebral microvessels. N-G-monomethyl-L-arginine suppressed cerebral angiogenesis (P<0.05), especially those microvessels associated with MMP-9 activation (P<0.02) induced by VEGF, comparable to the effect of doxycycline. L-N-6-(1-iminoethyl)lysine showed similar inhibitory effects. 3-nitrotyrosine confirmed nitric oxide levels in the brain. Compared with the lacZ control, VEGF increased inflammatory cell infiltration, especially macrophages, in the induced brain angiogenic focuses. Conclusions-Inhibition of nitric oxide production decreased MMP-9 activity and focal angiogenesis in the VEGF-stimulated brain. Both specific and nonspecific inhibition of nitric oxide synthase resulted in similar reductions, suggesting that VEGF-stimulated cerebral MMP activity and angiogenesis are predominantly mediated through inducible nitric oxide synthase, a specific nitric oxide synthase isoform mediating inflammatory responses. (Stroke. 2009; 40: 2879-2881.)