Objective-Growth factor-and reactive oxygen species (ROS)-induced activation of VSMCs is involved in vascular disease. This study investigates whether inhibitory oxidation of protein tyrosine phosphatases (PTPs) contributes to signaling in VSMCs in vitro and in vivo, and analyzes whether ROS-and growth factor-dependent vascular smooth muscle cell (VSMC) signaling is blunted by antioxidants that are able to activate oxidized PTPs. Methods and Results-Signaling induced by H 2 O 2 and platelet-derived growth factor (PDGF) was analyzed in VSMCs with or without the antioxidants N-acetyl-cysteine (NAC) and tempol. Effects of antioxidants on PDGF-stimulated chemotaxis and proliferation were determined. In vivo effects of antioxidants were analyzed in the rat carotid balloon-injury model, by analyzing neointima formation, cell proliferation, PDGF -receptor status, and PTP expression and activity. NAC treatment prevented H 2 O 2 -induced PTP inhibition, and reduced H 2 O 2 -and ligand-induced PDGF -receptor phosphorylation, PDGF-induced proliferation, and chemotaxis of VSMCs. Antioxidants inhibited neointima formation and reduced PDGF receptor phosphorylation in the neointima and also increased PTP activity. Key Words: restenosis Ⅲ VSMC Ⅲ protein tyrosine phosphatase Ⅲ platelet-derived growth factor Ⅲ neointima formation V ascular injury induces oxidative stress and elevated production of reactive oxygen species (ROS) in the vessel wall. 1,2 Moreover, ROS are produced and act as second messengers as part of the signaling of receptor tyrosine kinases (RTKs) (reviewed in references 3,4 ), which are activated after vascular injury. The most important ROS for pathological conditions are superoxide (O2 Ϫ ) and hydrogen peroxide (H 2 O 2 ). Inhibition of ROS reduce vessel remodeling and restenosis. 5 The underlying mechanisms remain incompletely understood. Conclusion-PTP-inhibitionPDGF -receptor activation contributes significantly to vascular smooth muscle cell (VSMC) proliferation and migration, which is a hallmark of vascular diseases such as atherosclerosis and restenosis. PDGF ligands and receptors are significantly upregulated in atherosclerotic plaques. Moreover, PDGF -receptor antagonists inhibit atherogenesis and restenosis in various models. 6 PDGF -receptors and other tyrosine kinases involved in VSMC proliferation and migration are regulated by protein tyrosine phosphatases (PTPs). 7 PTPs which have been identified as negative regulators of PDGF -receptors include the receptor-like PTP DEP-1, and the cytosolic phosphatases TC-PTP and PTP-1B (reviewed in reference 8 ). However, there are also indications that some PTPs, such as SHP-2, act as positive mediators of PDGF -receptor signaling. The expression pattern of PTPs in VSMCs remains incompletely characterized. However, expression of PDGF receptor-antagonizing PTPs such as DEP-1, TC-PTP, and PTP-1B has been confirmed in previous studies. 9 PTPs themselves are subject to multiple regulatory mechanisms. Reversible oxidation of the active site cysteine residu...
Growth factor-dependent tissue remodeling, such as restenosis, is believed to be predominantly regulated by changes in expression of receptor-tyrosine-kinases (RTKs) and their ligands. As endogenous antagonists of RTKs, protein-tyrosine-phosphatases (PTPs) are additional candidate regulators of these processes. Using laser-capture-microdissection and quantitative RT-polymerase chain reaction (qRT-PCR), we investigated the layer-specific expression of the four platelet-derived growth factor (PDGF) isoforms, the PDGF-alpha and beta receptors, and five PTPs implied in control of PDGF-receptor signaling 8 and 14 days after balloon injury of the rat carotid. Results were correlated with analyses of PDGF-beta receptor phosphorylation and vascular smooth muscle cell (VSMC) proliferation in vivo. The expression levels of all components, as well as receptor activation and VSMC proliferation, showed specific changes, which varied between media and neointima. Interestingly, PTP expression--particularly, DEP-1 levels--appeared to be the dominating factor determining receptor-phosphorylation and VSMC proliferation. In support of these findings, cultured DEP-1(-/-) cells displayed increased PDGF-dependent cell signaling. Hyperactivation of PDGF-induced signaling was also observed after siRNA-down-regulation of DEP-1 in VSMCs. The results indicate a previously unrecognized role of PDGF-receptor-targeting PTPs in controlling neointima formation. In more general terms, the observations indicate transcriptional regulation of PTPs as an important mechanism for controlling onset and termination of RTK-dependent tissue remodeling.
Estrogens are known to display significant vasoprotective effects in premenopausal women. PDGF is an important mediator of vascular smooth muscle cell (VSMC) migration and proliferation, and thus atherogenesis. We analyzed the effects of 17beta-estradiol (E2) on beta-PDGF receptor (beta-PDGFR) expression/activation and PDGF-dependent VSMC proliferation, migration, and downstream signaling events. Pretreatment of VSMCs with E2 (0.3 microM-0.1 mM) for 24 h concentration-dependently inhibited PDGF-induced proliferation and migration up to 85.5 +/- 15.8% and 79.4 +/- 9.8%, respectively (both P < 0.05). These effects were prevented by coincubation with the ER antagonist ICI-182780. E2 did not alter beta-PDGFR expression, nor did it impair the ligand-induced tyrosine phosphorylation of the beta-PDGFR and consecutive binding of the receptor-associated signaling molecules Src homology region 2-containing phosphatase-2, PLC-gamma, phosphatidylinositol 3-kinase, and RasGAP. Thus estrogens inhibited PDGF-induced cellular responses at the postreceptor level. Although stimulation of VSMCs with PDGF-BB led to a transient increase of rac-1 activity, pretreatment with E2 for 24 h concentration-dependently inhibited PDGF-induced rac-1 activation. Furthermore, inhibition of rac-1 by Clostridium sordellii lethal toxin or overexpression of dominant-negative rac-1 (rac-N17) significantly inhibited PDGF-induced VSMC migration, indicating that rac-1 activity is essential for PDGF-dependent cellular responses. E2 did not further reduce PDGF-induced migration in rac-N17-overexpressing cells, suggesting that it diminishes VSMC migration by altering rac-1 activity. We conclude that E2 attenuates PDGF-dependent cellular functions of VSMCs downstream of the beta-PDGFR via inhibition of rac-1. These observations offer a molecular explanation for the vasoprotective effects of estrogens.
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