Abstract 288: Nox1 is Required for Smooth Muscle Cell Activation of Matrix-Metalloproteinase-9 and Cell Migration in Response to Thrombin
Smooth muscle cell (SMC) degradation of the extracellular matrix and migration to the intima are fundamental processes in the vascular response to injury. NADPH oxidase-derived reactive oxygen species (ROS) are involved in development of vascular disease, however, the specific contribution of Nox1 and Nox4, the primary catalytic subunits of NADPH oxidase in SMC, is poorly understood. We hypothesized that Nox1-derived ROS mediate thrombin-dependent activation of matrix-metalloproteinase-9 (MMP-9) and migration of SMC. Studies were performed in SMC cultured from the aorta of Nox1 null and littermate control mice. Thrombin (2 U/ml) increased superoxide levels in control SMC, as measured by dihydroethidium, and this response was inhibited by the flavoenzyme inhibitor diphenylene iodonium (DPI, 10 μM). In contrast, thrombin failed to increase ROS in Nox1 null SMC. Previous studies have identified Src and mitogen activated protein kinases as key redox-dependent regulatory proteins in thrombin stimulated responses. Five minutes following thrombin stimulation, both Src and ERK1/2 phosphorylation were significantly decreased in Nox1 null SMC as compared to normal SMC (0.5±0.1 vs. 2.0 ± 0.4 for Src and 0.1 ± 0.1 vs. 2.9 ± 0.1 for ERK1/2, p<0.05), measured by densitometry of Western blots. Conditioned media was collected 24 hours after cells were treated with thrombin and MMP-9 activity measured by gelatin zymography. Thrombin increased MMP-9 more than 2-fold in control cells, however, thrombin failed to increase MMP-9 activity in Nox1 null cells. Using a wound-scratch assay, the number (7 ± 1 vs. 35 ± 3, p<0.05) and distance (15.2 ± 2.5 vs. 60.8 ± 4.9 μm, p<0.05) of cells migrating into the injured area was markedly reduced in SMC deficient in Nox1. In conclusion, the Nox1 subunit of NADPH oxidase is required by SMC for thrombin-dependent activation of MMP-9 and cell migration. In addition, Nox1 generation of ROS participates in phosphorylation of Src and of ERK1/2. These findings suggest that Nox1 may play an important role in the pathogenesis of vascular disease.