Effects of Mycophenolic Acid, Rapamycin, and Carvedilol on the PDGF-induced Fibronectin Secretion by Rat Vascular Smooth Muscle Cells: Implication of MAP Kinase / 대한이식학회지

ABSTRACT

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation and extra-cellular matrix (ECM) protein accumulation play important roles in transplant vascular sclerosis and re- stenosis after balloon vascular injury. Mycophenolic acid (MPA), rapamycin (RPM), and carvedilol (CA) were proven to inhibit the proliferation of VSMC. Fibronectin is a multifunctional ECM protein and induces tissue fibrosis. Since mitogen-activated protein kinases (MAPK) are upstream signaling molecules of VSMC proliferation and fibronectin production, this study examined the effects of MPA, RPM, and CA on the fibronectin secretion and MAPK activation in rat VSMC stimulated by platelet derived growth factor (PDGF). METHODS: VSMC was isolated from the aorta of male Sprague-Dawley rat, weighing 200-250 g and cultured with EMEM containing 10% fetal bovine serum and insulin/transferrin supplement. Near confluent VSMC were incubated with serum-free media for 48 hours to arrest and synchronize the cell growth. Test drugs were administered 15 minutes before the addition of PDGF 10 ng/mL. Cell proliferation, fibronectin secretion, and MAPK activation in VSMCs were measured by Western blot analysis. RESULTS: PDGF induced cell proliferation, fibronectin secretion, and extracellular- regulatary protein kinase 1/2 (ERK 1/2) and p38 MAPK activation by 1.7-, 1.5-, 3.3-, 3.9-fold, respectively, compared to control. MPA (>1 microM), CA (>100 nM), PD98059 (>30 microM), and p38 MAPK inhibitor (>10 nM) effectively inhibited PDGF-induced proliferation and fibronectin secretion. RPM, up to 100 nM, effectively inhibited cell proliferation, but did not inhibit fibronectin secretion. MPA and CA, but not RPM, inhibited PDGF-induced ERK 1/2 and p38 MAPK activation. CONCLUSION: The present study demonstrates that MPA and CA inhibit both cell proliferation and fibronectin secretion in rat VSMC stimulated by PDGF. Inhibition of both ERK 1/2 and p38 molecules are significantly associated with these events. Even though, it has a significant anti-proliferative effect on the rat VSMC, RPM neither affected the phosphorylation of ERK 1/2 and p38 nor secretion of fibronectin. These data suggest that ERK 1/2- and p38 MAPK-independent, more proximal pathway may exist for PDGF-induced proliferation of rat VSMC.