Redox regulation of vascular smooth muscle cell differentiation

Experiments were performed to determine the role of reactive oxygen species (ROS) in regulating vascular smooth muscle cell (VSMC) phenotype. After qu iescence, cultured human VSMCs increased their expression of differentiatio n proteins (alpha -actin, calponin, and SM1 and SM2 myosin), but not beta - actin. ROS activity, determined using the H2O2-sensitive probe dichlorodihy drofluorescein (DCF), remained high in quiescent cells and was inhibited by catalase (3000 U/mL) or by N-acetylcysteine (NAC, 2 to 20 mmol/L). A super oxide dismutase mimic (SOD; MnTMPyP, 25 mu mol/L) or SOD plus low concentra tions of NAC (SODNAC2, 2 mmol/L) increased DCF fluorescence, which was inhi bited by catalase or by NAC (10 to 20 mmol/L). Inhibition of ROS activity ( by catalase or NAG) decreased the baseline expression of differentiation pr oteins, whereas elevation of ROS (by SOD or SODNAC2) increased expression o f the differentiation markers. The latter effect was blocked by catalase or by NAC (10 to 20 mmol/L), None of the treatments altered p-actin expressio n. SODNAC2-treated cells demonstrated contractions to endothelin that were absent in proliferating cells. p38 Mitogen-activated protein kinase (MAPK) activity was decreased when ROS activity was reduced (NAC, 10 mmol/L) and w as augmented when ROS activity was increased (SODNAC2). Inhibition of p38 M APK with pyridyl imidazole compound (SB202190, 2 to 10 mu mol/L) reduced ex pression of differentiation proteins occurring under basal conditions and i n response to SODNAC2. Transduction of VSMCs with an adenovirus encoding co nstitutively active MKK6, an activator of p38 MAPK, increased expression of differentiation proteins, whereas transduction with an adenovirus encoding dominant-negative p38 MAPK decreased expression of the differentiation pro teins. These findings demonstrate that ROS can increase VSMC differentiatio n through a p38 MAPK-dependent pathway.