title: 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.
abstract: 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 micromol/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 andTIMP-2 protein synthesis (Western immunoblotting). Homocysteine effects were investigated also after cell exposure to i) specific MEK inhibitor PD98059 (30 micromol/l) to evaluate the involvement of Mitogen-Activated Protein Kinase (MAPK) and ii) specific phosphatidylinositol 3-kinase (P13-K) inhibitor LY294002 (100 micromol/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 micromol/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.
authors.affiliation: Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Doronzo
authors.forename: Isabella
authors.initials: I
authors.lastname: Russo
authors.forename: Luigi
authors.initials: L
authors.lastname: Mattiello
authors.forename: Mariella
authors.initials: M
authors.lastname: Trovati
authors.forename: Giovanni
authors.initials: G
authors.lastname: Anfossi
mesh_terms: D001011:Aorta / Q000166:cytology / Q000201:enzymology; D002465:Cell Movement; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003609:Dactinomycin / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005419:Flavonoids / Q000494:pharmacology; D006710:Homocysteine / Q000494:pharmacology*; D006801:Humans; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009025:Morpholines / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D032389:Myocytes, Smooth Muscle / Q000187:drug effects / Q000201:enzymology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors
chemical_list: D002867:Chromones; D005419:Flavonoids; D009025:Morpholines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011500:Protein Synthesis Inhibitors; D006710:Homocysteine; D003609:Dactinomycin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D020928:Mitogen-Activated Protein Kinases; D020778:Matrix Metalloproteinase 2; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one