Agmatine inhibits matrix metalloproteinase-9 via endothelial nitric oxide synthase in cerebral endothelial cells

Objectives: Matrix metalloproteinases (MMPs) are up-regulated by ischemic injury and degrade the basement membrane of brain vessels to promote cell death and tissue injury. We previously showed that agmatine has a neuroprotective effect on neurons against ischemic injury. In the present study, we investigated the effect of agmatine on the expression of MMPs and nitric oxide (NO) production in cerebral endothelial cells (CECs) after oxygen-glucose deprivation (OGD)-reperfusion injury and its potential association with endothelial nitric oxide synthase (eNOS). Methods: Primary cultured endothelial cells from murine brain and bEnd.3 cells were subjected to OGD-reperfusion injury. Protein and mRNA levels of both MMP-2 and MMP-9 were determined by immunocytochemical analysis, Western blot and RT-PCR. Protein levels of eNOS were evaluated by Western blot in the CECs. The production of NO was measured using the Griess reagent. Results: Agmatine attenuated the expression of MMP-2 and MMP-9 induced by ischemic injury at the protein and mRNA level, while agmatine increased the expression of eNOS directly. NO production was decreased in CECs after similar insult and was increased by agmatine treatment. In the presence of a nitric oxide synthase (NOS) inhibitor, N^ω-nitro-L-arginine methyl ester (L-NAME), the expression levels of MMP-2 were decreased, but the expression of MMP-9 was not decreased by agmatine administration. However, NO production was suppressed by a non-specific NOS inhibitor in the agmatine treatment group. Conclusion: Our study supports that the down-regulation of MMP-9 by agmatine runs parallel to the up-regulation of eNOS and the maintenance of functional NO release.