NAD(P)H oxidase is a major source of superoxide (O_{2^{· -}} ) and thus oxidative stress in VSMC. We recently described functional/spatial interaction between NAD(P)H oxidase and the endoplasmic reticulum oxidoreductase PDI in VSMC. We hypothesized that such pathway connects oxidative stress to UPR as part of the recently proposed integrated stress response (Harding et al, 2003). Western analysis showed that the UPR inducer tunicamycin (Tn) (2m g/ml) led to eIF2a phosphorylation at 2hs and overexpression of KDEL proteins (GRP78/GRP94/calreticulin) at 12 hs, detected in VSMC homogenates. At this time, onset of apoptosis was shown by marked decrease in procaspase 12 levels and cell loss after 48h. HPLC analysis of dihydroethidium (DHE) oxidation products showed Tn-induced increase (ca. 35% vs. control) in VSMC O_{2^{· -}} levels, starting after 4 hs, and significant decrease in GSH/GSSG ratio (HPLC- electrochemical detection), indicating oxidative stress, both at levels ³ the known oxidase agonist angiotensin-II (100 nM). Superoxide dismutase activity decreased at 4 hs, reaching 50% loss by 14 hs. Also, UPR induction by a novel TAP-directed peptide bearing N-glycosylation motifs strongly increased superoxide levels (by 60%) after 2hs and GRP78/GRP94 overexpression after 12 hs. UPR disruption by overexpression of GADD34 cDNA promoting eIF2a dephosphorylation reduced Tn-mediated O_{2^{· -}} production, thus confirming that UPR triggers VSMC oxidative stress. Tn induced concentration and time-dependent increase in VSMC membrane NADPH oxidase activity, which was totally prevented by Brefeldin-A. Such a role for protein traffic in oxidase activation was supported by Tn-induced PDI shift to membranes. VSMC membrane incubation with PDI inhibitors (DTNB, Bacitracin and neutralizing Ab-PDI) strongly decreased NADPH oxidase activity. Moreover, the mediator role of PDI in O_{2^{· -}} production during UPR was further supported by gain and loss-of-function experiments after transfection with sense or antisense PDI cDNA. In vivo pathophysiological relevance of such an integrated stress response was studied in the neointima after vascular balloon injury, a condition known to trigger NAD(P)H oxidase overexpression and oxidative stress. The neointima also co-overexpressed PDI and UPR markers and showed strongly increased level of procaspase-12 cleavage. Thus, PDI/NADPH oxidase interaction couples UPR and oxidative stress in VSMC. Such an integrative stress pathway may control apoptosis and act as a regulator of vascular repair. (Supported by FAPESP, CNPq Milênio Redoxoma)