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 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.
Doronzo
Gabriella
G
Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy.
Russo
Isabella
I
Mattiello
Luigi
L
Trovati
Mariella
M
Anfossi
Giovanni
G
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
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