Homocysteine rapidly increases matrix metalloproteinase-2 expression and activity in cultured human vascular smooth muscle cells - Role of phosphatidyl inositol 3-kinase and mitogen activated protein kinase pathways

In this study we aimed to test the hypothesis that in human vascular smooth muscle cells (VSMC) homocysteine influences synthesis and release of matrix metalloproteinase-2 (MMP-2), which is deeply involved in vascular remodeling and atherosclerotic plaque instabilization. Experiments were carried out in cultured human VSMC exposed to 50-500 mu mol/l homocysteine after a 24-hour culture with MEM containing 0.1 % BSA. Both in supernatants and cell lysates we evaluated MMP-2 activity (gelatin zimography), MMP-2 and TIMP-2 protein synthesis (Western immunoblotting). Homocysteine effects were investigated also after cell exposure to i) specific MEK inhibitor PD98059 (30 pmol/l) to evaluate the involvement of Mitogen-Activated Protein Kinase (MAPK) and ii) specific phosphatidylinositol 3-kinase (P13-K) inhibitor LY294002 (100 mu mol/l) to evaluate the involvement of P13-K pathway. Gelatin zimography evidenced that MMP-2 activity is increased both in conditioned media and in cell lysates starting from 8-hour incubation with 100 mu mol/l homocysteine. Western blot analysis evidenced increased MMP-2 levels in both conditioned media and cell lysates. Cell exposure to PD98059 and LY294002 prevented homocysteine effects on MMP-2 synthesis. Homocysteine, at concentrations associated with increased risk of cardiovascular events, increases MMP-2 activity, synthesis and secretion in VSMC through a mechanism involving the activation of MAPK and P13-K pathways.These data suggest that homocysteine is directly involved in mechanisms leading to remodelling and instabilization of atherosclerotic plaques.