Summary

In this study we aimed to test the hypothesis that in human vascularsmooth muscle cells (VSMC) homocysteine influencessynthesis and release of matrix metalloproteinase-2 (MMP-2),which is deeply involved in vascular remodeling and atheroscleroticplaque instabilization. Experiments were carried out in culturedhuman VSMC exposed to 50–500 µ mol/l homocysteineafter a 24-hour culture with MEM containing 0.1% BSA. Both insupernatants and cell lysates we evaluated MMP-2 activity (gelatinzimography), MMP-2 and TIMP-2 protein synthesis (Westernimmunoblotting). Homocysteine effects were investigated alsoafter cell exposure to i) specific MEK inhibitor PD98059 (30µ mol/l) to evaluate the involvement of Mitogen-Activated ProteinKinase (MAPK) and ii) specific phosphatidylinositol 3-kinase(PI3-K) inhibitor LY294002 (100 µ mol/l) to evaluate the involvement of PI3-K pathway. Gelatin zimography evidenced thatMMP-2 activity is increased both in conditioned media and in celllysates starting from 8-hour incubation with 100 µ mol/l homocysteine.Western blot analysis evidenced increased MMP-2levels in both conditioned media and cell lysates. Cell exposureto PD98059 and LY294002 prevented homocysteine effects onMMP-2 synthesis. Homocysteine, at concentrations associatedwith increased risk of cardiovascular events, increases MMP-2activity, synthesis and secretion in VSMC through a mechanisminvolving the activation of MAPK and PI3-K pathways.These datasuggest that homocysteine is directly involved in mechanismsleading to remodelling and instabilization of atheroscleroticplaques.