Urokinase plasminogen activator stimulates vascular smooth muscle cell proliferation via redox-dependent pathways.

OBJECTIVE: We showed previously that increased urokinase plasminogen activator (uPA) expression contributes to vascular smooth muscle cell (VSMC) proliferation and neointima formation after injury. Proliferation of cultured rat aortic VSMCs induced by uPA was inhibited by the antioxidant ebselen. Because increases in VSMC reactive oxygen species (ROS) contribute to VSMC proliferation, we hypothesized that uPA increases ROS generation by regulating expression or activity of cellular oxidases. METHODS AND RESULTS: uPA stimulated ROS production to levels equivalent to angiotensin II as measured by electron spin resonance and fluorescent redox indicators (dichlorofluorescein diacetate, lucigenin, and hydroethidine). The increase in ROS was biphasic, with the first peak at 30 minutes and the second peak at 4 hours. uPA increased expression of the NAD(P)H oxidases Nox1 and Nox4 as measured by RT-PCR and Western blot analysis. Knockdown of Nox1 and Nox4 expression with small interfering RNA showed that both isoforms (Nox1>Nox4) contributed significantly to uPA-stimulated ROS production and VSMC proliferation. Transfection of VSMCs with uPA cDNA to increase endogenous uPA expression enhanced ROS production dramatically, suggesting that autocrine uPA production may be an important mechanism for uPA-mediated VSMC events. CONCLUSIONS: These data show that uPA is an autocrine VSMC growth factor that increases ROS generated by both Nox1 and Nox4 oxidases.

Expression of adhesion molecule T-cadherin is increased during neointima formation in experimental restenosis.

Phenotypic modulation, migration and proliferation of vascular smooth muscle cells (SMCs) are major events in restenosis after percutaneous transluminal angioplasty. Surface cell adhesion molecules, essential to morphogenesis and maintenance of adult tissue architecture, are likely to be involved, but little is known about cell adhesion molecules expressed on SMCs. T-cadherin is a glycosyl phosphatidylinositol-anchored member of the cadherin superfamily of adhesion molecules. Although highly expressed in vascular and cardiac tissues, its function in these tissues is unknown. We previously reported increased expression of T-cadherin in intimal SMCs in atherosclerotic lesions and proposed a role for T-cadherin in phenotype control. Here we performed immunohistochemical analysis of spatial and temporal changes in vascular T-cadherin expression following balloon catheterisation of the rat carotid artery. T-cadherin expression in SMCs markedly increases in the media early (1-4 days) after injury, and later (day 7-28) in forming neointima, especially in its preluminal area. Staining for monocyte/macrophage antigen ED-1, proliferating cell nuclear antigen and smooth muscle alpha-actin revealed that spatial and temporal changes in T-cadherin level coincided with the peak in cell migration and proliferation activity during neointima formation. In colchicine-treated cultures of rat aortic SMCs T-cadherin expression is increased in dividing M-phase cells but decreased in non-dividing cells. Together the data support an association between T-cadherin expression and SMC phenotype.