MEF2B-Nox1 signaling is critical for stretch-induced phenotypic modulation of vascular smooth muscle cells

Abstract

Objective: Blood vessel hemodynamics have profound influences on function and structure of vascular cells. One of the main mechanical forces influencing vascular smooth muscle cells (VSMC) is cyclic stretch (CS). Increased CS stimulates reactive oxygen species (ROS) production in VSMC, leading to their dedifferentiation, yet the mechanisms involved are poorly understood. This study was designed to test the hypothesis that pathological CS stimulates NADPH oxidase isoform 1 (Nox1)-derived ROS via MEF2B, leading to VSMC dysfunction via a switch from a contractile to a synthetic phenotype.

Approach and results: Using a newly developed isoform-specific Nox1 inhibitor and gene silencing technology, we demonstrate that a novel pathway, including MEF2B-Nox1-ROS, is upregulated under pathological stretch conditions, and this pathway promotes a VSMC phenotypic switch from a contractile to a synthetic phenotype. We observed that CS (10% at 1 Hz) mimicking systemic hypertension in humans increased Nox1 mRNA, protein levels, and enzymatic activity in a time-dependent manner, and this upregulation was mediated by MEF2B. Furthermore, we show that stretch-induced Nox1-derived ROS upregulated a specific marker for synthetic phenotype (osteopontin), whereas it downregulated classical markers for contractile phenotype (calponin1 and smoothelin B). In addition, our data demonstrated that stretch-induced Nox1 activation decreases actin fiber density and augments matrix metalloproteinase 9 activity, VSMC migration, and vectorial alignment.

Conclusions: These results suggest that CS initiates a signal through MEF2B that potentiates Nox1-mediated ROS production and causes VSMC to switch to a synthetic phenotype. The data also characterize a new Nox1 inhibitor as a potential therapy for treatment of vascular dysfunction in hypertension.

Keywords: MEF2B; Nox1; oxidative stress; vascular remodeling.

Figure 1

Uniaxial cyclic stretch induces Nox1 expression and activity. RASMCs were subjected to a time course of cyclic stretch. A) Nox1 mRNA was measured by qPCR (n = 5). B) Nox1 protein was measured by Western blot (n = 6). C) RASMC were incubated with NoxA1ds or scrambled (Scr) peptide (10 μM) and subjected to 24 hr cyclic stretch. SOD-inhibitable O₂^.- was measured by cytochrome c reduction (n = 9). Data are expressed as means + SEM. *P<0.05 versus static control.

Figure 2

Uniaxial cyclic stretch induces MEF2B promoter activity, MEF2B protein expression, and MEF2B-dependent Nox1 expression. A) RASMCs were subjected to a time course of cyclic stretch and MEF2B promoter reported was measured by dual firefly luciferase reporter assay, using MEF2F firefly luciferase reporter and a control reporter expressing Renilla luciferase (n = 6). B) RASMCs were subjected to a time course of cyclic stretch and MEF2B protein expression was investigated by Western blot (n = 4). C) RASMCs were pretreated with MEF2B siRNA or scrambled (Scr) siRNA, subjected to 24 hr cyclic stretch. Nox1 protein expression was measured by Western blot (n = 6). Data are expressed as means + SEM. *P<0.05 versus vehicle or static condition.

Figure 3

Nox1 mediates a decreased contractile phenotype in response to uniaxial cyclic stretch. RASMCs were subjected for 24 hr to stretch or static conditions. A and B) CNN1 protein expression measured by Western blot (n = 4). C and D) Smoothelin B protein expression measured by Western blot (n = 4). E) Cell nuclear perpendicular alignment versus the direction of the stretch vector represented by radial histograms (n = 5, over 130 cells). F) Summary of the average alignment angles (n = 5 over 130 cells). In figures A, C, E, and F, cells were pretreated with NoxA1ds or scrambled peptide (10 μM). In figures B and D, cells were pretreated with Nox1 siRNA or scrambled (Scr) siRNA. Data are expressed as means + SEM. *P<0.05 versus static control.

Figure 4

Nox1-mediated increase in synthetic phenotype in response to uniaxial cyclic stretch. RASMC were subjected for 24 hr to stretch or static conditions. A and B) OPN was measured by Western blot (n = 4). C, Extracellular MMP9 activity was measured by zymography (n = 5). D and E) Cell migration was measured by wound healing assay (n = 8). F) Uniaxial CS increases actin fiber density via Nox1. Fluorescence microscopy images of phalloidin-stained RASMC. Fiber density was assessed using ImageJ software and with the help of the Hessian matrix plug-in. The graph depicts mean values of 8 cells from 4 independent experiments. The green bars represent 20 μm. In figures A, C, D, E and F cells were treated with NoxA1ds or scrambled (Scr) peptide (10 μM). In B) cells were pretreated with Nox1 siRNA or scrambled siRNA (5 pmol/ml) for 48 hr. Data are expressed as means + SEM. *P<0.05 versus static control. ^#P<0.05 vs. stretch scrmb.