Abstract
Objective: Vascular smooth muscle cell (VSMC) phenotype switch depends on extrinsic/intrinsic cues including NOX NADPH oxidase-linked redox signaling. Growth factor-triggered NOX1 expression/activity requires the chaperone oxidoreductase protein disulfide isomerase-A1 (PDI). Acute PDI overexpression induces agonist-independent NOX1 expression. PDI is required for VSMC migration and cytoskeleton organization, and extracellular PDI supports expansive vascular remodeling via cytoskeleton reshaping. Such PDI effects led us to hypothesize that PDI may orchestrate VSMC phenotypic alterations. Approach and Results: In primary VSMC, PDI silencing spontaneously decreased differentiation marker expression. Transfection with a doxycycline-inducible lentiviral vector encoding PDI showed that sustained PDI overexpression (72h) enhanced actin branching pattern vs. baseline (anisotropy index, 0.103±0.019 vs. 0.220±0.027, 72h vs. 0h, mean±SEM, N=5, P<0.05), increased cell length and induced expression of differentiation marker calponin (2-fold, 72h vs 0h, N=5, P<0.05), alpha-actin and smoothelin, which were abrogated upon catalase incubation. Intracellular superoxide enhanced upon 48h of PDI overexpression (2-hydroxyethidium levels, 0.998±0.102 vs. 2.887±0.227 AU, N=4, P<0.05) and was NOX1-dependent, based on inhibition with GKT136901 (by 48%) or NOXA1ds peptide (by 54%) (N=6, P<0.05). Increased NOX1 mRNA occurred early after PDI overexpression (74%, 24h vs. 0h, N=4, P<0.05), while NOX4 mRNA was upregulated only after long-term PDI induction (100%, 72h vs. 0h, N=4, P<0.05). In rabbit restenosis model exhibiting strong PDI upregulation (12-fold at day 14 after injury), ex vivo PDI silencing 7 or 14 days after injury reversed PCNA expression, while promoting increased NOX1 and decreased NOX4 mRNA levels (+54% and -45%, respectively, siPDI vs. siSCR, N=3, P<0.05 for both). Conclusions: While short-term PDI overexpression supports NOX1 activation/expression, sustained PDI overexpression drives NOX4 expression and VSMC differentiation. Effects on cytoskeleton, NOX1/4 activation and temporal control of NOX1/4 expression suggest a central role for PDI as a hub for redox-mediated VSMC phenotype regulation.
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