Abstract
A variety of vascular pathologies, including hypertension, restenosis and atherosclerosis, are characterized by vascular smooth muscle cell (VSMC) hypertrophy and migration. NADPH oxidase 1 (Nox1) plays a pivotal role in these phenotypes via distinct downstream signaling. However, the mediators differentiating these distinct phenotypes and their precise role in vascular disease are still not clear. The present study was designed to identify novel targets of VSMC Nox1 signaling using 2D Differential In-Gel Electrophoresis and Mass Spectrometry (2D-DIGE/MS). VSMC treatment with scrambled (Scrmb) or Nox1 siRNA and incubation with the oxidant hydrogen peroxide (H2O2; 50 μM, 3 h) followed by 2D-DIGE/MS on cell lysates identified 10 target proteins. Among these proteins, actin-related protein 2/3 complex subunit 2 (ARPC2) with no previous link to Nox isozymes, H2O2, or other reactive oxygen species (ROS), was identified and postulated to play an intermediary role in VSMC migration. Western blot confirmed that Nox1 mediates H2O2-induced ARPC2 expression in VSMC. Treatment with a p38 MAPK inhibitor (SB203580) resulted in reduced ARPC2 expression in H2O2-treated VSMC. Additionally, wound-healing “scratch” assay confirmed that H2O2 stimulates VSMC migration via Nox1. Importantly, gene silencing of ARPC2 suppressed H2O2-stimulated VSMC migration. These results demonstrate for the first time that Nox1-mediated VSMC migration involves ARPC2 as a downstream signaling target.
Highlights
Vessel wall remodeling plays a key role in the development of vascular disorders, including systemic hypertension and atherosclerosis [1,2]
These results demonstrate for the first time that NADPH oxidase 1 (Nox1)-mediated vascular smooth muscle cells (VSMC)
Since p38 MAPK signaling is linked to NF-κB activation [25,26], we further investigated whether inhibition of the NF-κB pathway attenuates actin-related protein 2/3 complex subunit 2 (ARPC2) expression in response to H2O2
Summary
Vessel wall remodeling plays a key role in the development of vascular disorders, including systemic hypertension and atherosclerosis [1,2]. Nox primarily generates O2−, while Nox appears to constitutively produce H2O2 [8,9] These Nox isoforms serve distinct functions within cells, purportedly a consequence of their distinct intracellular compartmentalization, unique mechanism of regulation and activation, and the ROS they produce. Our laboratory has recently demonstrated that pathophysiologically-relevant concentrations of H2O2 stimulate Nox1-derived O2− generation in rat aortic VSMC, leading to activation of apoptosis signal-regulating kinase 1 (Ask). Our laboratory has recently demonstrated that pathophysiologically-relevant concentrations of H2O2 stimulate Nox1-derived O2− generation in rat aortic VSMC, leading to activation of apoptosis signal-regulating kinase 1 (Ask1) These data delineated a previously unknown molecular cascade by which feed-forward ROS signaling in the vascular wall is propagated [10]. ARPC2, shedding light on a new signaling pathway in vascular biology
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