The mechanisms by which NADPH oxidase complex is activated are still poorly unkown, although its importance for understanding of the redox cell signaling pathways. Recently we described that loss-of-function of protein disulfide isomerase (PDI), a chaperone superfamily of thioredoxin, decreased Nox1 NADPH oxidase isoform expression as well as reactive oxygen species (ROS) production induced by angiotensin II stimulus in VSMC (Janiszewski et al, 2005; Fernandes et al, 2009). In addition, PDI overexpression (~ 2.3 fold) spontaneously increased basal Nox1 (mRNA) expression and NADPH oxidase activity; so that transfected VSMC were not able to increase the generation of ROS after angiotensin II stimulus, suggesting a preemptive activation of Nox1 (Fernandes et al, 2009). This increased spontaneous ROS production in VSMC overexpressing PDI was prevented by AT1 receptor inhibitors, losartan and candesartan (and not by ACE inhibitors, captopril and enalapril). Finally, PDI strongly co-localized with AT1R in VSMC stimulated with AngII or transfected with PDI. Thus, this project aimed to investigate the mechanism of AT1R activation by PDI overexpression in vascular smooth muscle cells. Thus, specific objectives are to investigate: i) ROS producton mediated by PDI overexpression dependence on PDI thiols and/or AT1R disulfide bridge Cys18-Cys274, ii) whether PDI overexpression causes constitutive activation of the AT1R and/or transactivates EGF receptor, and iii) Nox1 involvement during spontaneous VSMC migration after PDI overexpression. These data should provide mechanistic information relevant to understanding the pathways by which PDI regulates the activation of AT1R/NADPH oxidase and angiotensin II-mediated signaling in VSMC.
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