Role of NMDA receptor in homocysteine-induced activation of Mitogen-Activated Protein Kinase and Phosphatidyl Inositol 3-Kinase pathways in cultured human vascular smooth muscle cells

INTRODUCTION: Exposure of vascular smooth muscle cells (VSMC) to homocysteine, at concentrations associated with an increased risk of cardiovascular events, enhances synthesis and secretion of Matrix Metalloproteinase-2 (MMP-2), which is involved in atherosclerotic plaque instabilization. This effect was prevented by inhibitors of Mitogen Activated Protein Kinase (MAPK) and Phosphatidylinositol 3-Kinase (PI3-K) pathways, allowing to hypothesize that homocysteine activates both these pathways, likely via a receptor-mediated mechanism. One possible receptor is N-methyl-D-aspartate receptor (NMDAr), which is expressed in VSMC and is involved in homocysteine effects in other cell types. MATERIALS AND METHODS: VSMC exposed to DL-homocysteine or NMDA (100 mumol/L for both; 5 min-8 hours), were investigated by measuring: i) phosphorylation of ERK1/2, p38MAPK (signaling molecules of MAPK pathway) and Akt and p70S6K (signaling molecules of PI3-K pathway) by western blot; ii) synthesis and secretion of MMP-2 (western blot); iii) activation of MMP-2 (gelatin zimography). To evaluate NMDAr involvement in the homocysteine effects, the experiments were repeated in the presence of a non-competitive NMDAr-antagonist MK-801 (50 mumol/L) or L-glycine (10 mumol/L), which inhibits NMDAr function by promoting its internalization. RESULTS: DL-homocysteine and NMDA time-dependently increased: i) the phosphorylation of ERK1/2, p38 MAPK, Akt and p70S6K (ANOVA, p<0.0001); ii) the synthesis, secretion and activation of MMP-2. DL-homocysteine and NMDA effects were prevented by VSMC pre-incubation with MK-801 or high L-glycine concentrations. CONCLUSIONS: In human VSMC homocysteine-at concentrations associated with increased cardiovascular risk- activates MAPK and PI3-K pathways and MMP-2 synthesis and secretion through NMDA receptor, a potential mechanism involved in intracellular signaling in response to homocysteine in VSMC.