Abstract 891: Nox2-Deficient Mice are Protected against Cigarette Smoke-Induced Inhibition of Neovascularization: Role of Reactive Oxygen Species, Nitric Oxide and Endothelial Progenitor Cells
Background: We have previously shown that cigarette smoke exposure is associated with impaired neovascularization following tissue ischemia and that this is at least partly due to increased generation of reactive oxygen species (ROS). Because NADPH oxidase is a major source of ROS in the vasculature, we investigated its role in the modulation of neovascular-ization by cigarette smoke.
Methods and results: Mice deficient for the Nox2-containing NADPH oxidase (Nox2−/−) and control (Nox2+/+) mice were exposed to cigarette smoke for a total of 4 weeks. After two weeks, hindlimb ischemia was surgically induced by femoral artery removal. We found that Nox2−/− mice had a significantly faster rate of blood flow recovery when compared to Nox2+/+ mice, as assessed by Doppler Flow Ratios (DFR) between the ischemic and normal hindlimbs at day 14 after surgery (0.89 ± 0.04 vs. 0.61 ± 0.02, p < 0.001). At the microvascular level, Nox2−/− mice showed a statistically significant higher capillary density in ischemic muscles (535.20 ± 20.75 vs. 279.63 ± 16.11 capillaries per mm2, p < 0.001). We found that Nox2−/− mice displayed reduced oxidative stress in serum (antioxidant levels) and ischemic tissues (nitrotyrosine immunostaining) compared to controls. This was associated with an increased plasma nitrite concentration (Griess reaction) and a preserved expression of the angiogenic factor VEGF in ischemic tissues (Western blot). In addition, we found that the number of peripheral endothelial progenitor cells (EPCs) isolated from the spleen (FACS analysis) was significantly increased in Nox2−/− vs. control mice (0.77 ± 0.07% vs. 0.45 ± 0.05%). In vitro, endothelial cells isolated from the aorta of Nox2−/− mice or HUVECs treated with the NADPH oxidase inhibitor apocynin exhibit reduced superoxide formation (DHE staining) together with preserved migration capacity (Boyden chamber) and angiogenic activities (matrigel assay) when exposed to cigarette smoke extracts.
Conclusions: NADPH oxidase deficiency protects against cigarette smoke-induced impairment of neovascularization. The potential mechanisms involved include a reduction of ROS formation, a preserved expression of the angiogenic factors VEGF and NO, and an increased number of endothelial progenitor cells.