Abstract 366: Nox1-Derived Superoxide Anion Mediates Stretch-induced Phenotypic Switching in Smooth Muscle Cells

Abstract

Hemodynamic forces in the vessel wall have profound influences on function, structure and pathology of vascular cells. The predominant mechanical force influencing vascular smooth muscle cells (VSMC) structure and signaling is cyclic stretch (CS). Aberrant CS can direct VSMC to a synthetic phenotype (SP) characterized by increased VSMC migration, loss of contractility and matrix production. Some evidence suggests that CS is capable of inducing reactive oxygen species (ROS); however, the mechanisms by which stretch-derived ROS can induce a VSMC SP are poorly understood. Here we postulate that the NADPH oxidase isoform Nox1, which is an important source of ROS in vascular disease, is involved in a CS-mediated SP. In VSMC subjected to CS (10%, 1Hz) there was an increase in Nox1 mRNA levels (3.9 ± 0.14 fold of increase after 6 hr of CS), Nox1 protein levels (1.44 ± 0.04 fold increase after 24 hr CS) and Nox1-derived superoxide production (CS: 13.82±1.34 vs static: 6.61±0.67 pmol of superoxide/min/mg protein after 24 hr). Using our recently-developed specific peptide inhibitor of Nox1 (NoxA1ds), CS-induced superoxide production abated to baseline levels (6.61±0.67, 13.82±1.34 and 5.06±0.76 pmol of superoxide/min/mg protein; for static, CS and CS+NoxA1ds respectively). Furthermore, osteopontin protein levels, a VSMC SP marker, were increased by CS for 24 hrs (1.25±0.04 fold higher than static) and abolished by NoxA1ds pretreatment (0.96±0.19 fold of static). On the other hand, calponin protein levels, a VSMC contractile phenotype marker, were decreased by CS and restored by pretreatment with NoxA1ds (0.67±0.05- and 0.93±0.12-fold change from static respectively). Finally, we demonstrate that stretch-induced migration of smooth muscle cells is promoted by Nox1-derived superoxide (46.36±1.47, 56.16±1.74 and 40.98±2.46 % of scratch wound closure for static, CS and CS+NoxA1ds treatments respectively). These results suggest that Nox1-derived superoxide mediates a synthetic phenotype in vascular smooth muscle under CS, and identify Nox1 as a potential therapeutic target for the treatment of diseases such as atherosclerosis and hypertension which are characterized by VMSC SP.