PMID- 19766294 OWN - NLM STAT- MEDLINE DCOM- 20100513 LR - 20131121 IS - 1879-2472 (Electronic) IS - 0049-3848 (Linking) VI - 125 IP - 2 DP - 2010 Feb TI - 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. PG - e23-32 LID - 10.1016/j.thromres.2009.08.015 [doi] AB - 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 micromol/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 micromol/L) or L-glycine (10 micromol/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. CI - Copyright 2009 Elsevier Ltd. All rights reserved. FAU - Doronzo, Gabriella AU - Doronzo G AD - Metabolic Disease and Diabetes Unit, Department of Clinical and Biological Sciences of the University of Turin, San Luigi Gonzaga Hospital, Orbassano (Turin), Italy. gabriella.doronzo@unito.it FAU - Russo, Isabella AU - Russo I FAU - Del Mese, Paola AU - Del Mese P FAU - Viretto, Michela AU - Viretto M FAU - Mattiello, Luigi AU - Mattiello L FAU - Trovati, Mariella AU - Trovati M FAU - Anfossi, Giovanni AU - Anfossi G LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20090918 PL - United States TA - Thromb Res JT - Thrombosis research JID - 0326377 RN - 0 (Receptors, N-Methyl-D-Aspartate) RN - 0LVT1QZ0BA (Homocysteine) RN - EC 2.7.1.- (Phosphatidylinositol 3-Kinases) RN - EC 2.7.11.24 (Mitogen-Activated Protein Kinases) SB - IM MH - Cells, Cultured MH - Enzyme Activation/drug effects MH - Homocysteine/*pharmacology MH - Humans MH - Mitogen-Activated Protein Kinases/*metabolism MH - Muscle, Smooth, Vascular/*drug effects MH - Phosphatidylinositol 3-Kinases/*metabolism MH - Receptors, N-Methyl-D-Aspartate/*metabolism MH - Time Factors EDAT- 2009/09/22 06:00 MHDA- 2010/05/14 06:00 CRDT- 2009/09/22 06:00 PHST- 2009/03/03 00:00 [received] PHST- 2009/06/26 00:00 [revised] PHST- 2009/08/05 00:00 [accepted] PHST- 2009/09/22 06:00 [entrez] PHST- 2009/09/22 06:00 [pubmed] PHST- 2010/05/14 06:00 [medline] AID - S0049-3848(09)00386-7 [pii] AID - 10.1016/j.thromres.2009.08.015 [doi] PST - ppublish SO - Thromb Res. 2010 Feb;125(2):e23-32. doi: 10.1016/j.thromres.2009.08.015. Epub 2009 Sep 18.