Effect of SHP on angiotensin II-stimulated PAI-1 mRNA expression and promoter activity. (A) Northern blot analysis of the effect of overexpression of SHP on PAI-1 mRNA expression. VSMCs were infected at the indicated multiplicity-of-infection (moi) with adenovirus expressing SHP (Ad-SHP), or green fluorescent protein (GFP) as a control, for 4 h. VSMCs were incubated in medium containing 0.5% FBS for 24 h, and then treated with angiotensin II (Ang II, 10 nmol/l) for 3 h before harvest. Data represent the means ± SEM. *P < 0.05, **P < 0.01, #P < 0.001 as compared to Ang II alone. (B and C) Luciferase gene reporter assay of the effect of SHP on PAI-1 promoter activity. (B) VSMCs were transfected with a reporter gene under the control of the PAI-1 promoter (-840 PAI-1 promoter Luc, 300 ng) together with the indicated amounts of an SHP expression vector (pcDNA3) for 5 h, and were then incubated in medium containing 10% FBS for 24h. Data represent the means ± SEM. *P < 0.01, **P < 0.001 were as compared with the control. (C) VSMCs were transfected with a reporter gene under the control of the PAI-1 promoter (-840 PAI-1 promoter Luc, 300 ng) together with the indicated amounts of an SHP expression vector (pcDNA3) for 5 h. Cells were incubated in medium containing 0.5% FBS for 24 h, and then treated with angiotensin II (Ang II, 10 nM) for 1 h before harvest. Data represent the means ± SEM. *P < 0.001 as compared to control. **P < 0.01, #P < 0.001 as compared to Ang II alone.