Pharmacological and biochemical studies on the contribution of NADPH oxidases to oxidative stress in the aorta of spontaneously hypertensive rats

Vascular oxidative stress is associated with a dysfunction of endothelium-dependent relaxation. Inactivation of nitric oxide by superoxide and other reactive oxygen species (ROS) appears to occur under conditions such as diabetes mellitus, hypercholesterolemia or arterial hypertension. NADPH oxidases have been identified as major source of ROS in blood vessels. The present study investigates the role of NADPH oxidases in aortae of 12-14 month old spontaneously hypertensive rats (SHR) in comparison to age-matched Wistar Kyoto rats (WKY). ROS production was largely increased in aortae of SHR as measured by in situ detection of DHE fluorescence. Suppression of ROS formation by the NADPH oxidase inhibitors, apocynin and the novel compound VAS2870, but not by the eNOS inhibitor L-NAME or the xanthine oxidase inhibitor oxypurinol, suggested NADPH oxidases as a major source of oxidative stress. In line with these findings, NADPH oxidase activity in aortic homogenates of SHR was significantly elevated. NADPH oxidases are multimeric complexes consisting of several cytosolic and two membranic subunits, namely p22phox and a catalytic Nox isoform. In rat aortae, the isoforms Nox1, Nox2 and Nox4 are expressed, which was confirmed by RT-PCR in aortic homogenates of SHR and WKY. Immunohistochemical analysis of the vessels demonstrated a predominant location of Nox1 in the aortic media. Nox2 was localized in the adventitia as well as the intima and Nox4 was located throughout the aortic wall. Quantitative Western blot analysis revealed increased levels of Nox1 and Nox2 protein in SHR aortic homogenates, while Nox4 expression was not significantly changed. Furthermore, Nox1 protein appeared to be upregulated especially in the intima of SHR aortae as indicated by colocalization of Nox1 immunofluorescence with the VSMC marker α-SM-actin and the endothelial cell marker RECA-1. These data suggest that Nox1 and Nox2-based NADPH oxidases are mainly responsible for oxidative stress in the aortic wall of aged SHR. Acetylcholine-induced endothelial-dependent relaxation was determined to investigate whether the NADPH oxidase-produced oxidative stress is linked to endothelial dysfunction. Vasorelaxation was impaired in aortic rings of SHR in comparison to the WKY controls. However, it was significantly improved in the presence of apocynin and VAS2870 in SHR and to a smaller extent in WKY, indicating the functional relevance of the enhanced NADPH oxidase activity. In conclusion, Nox1 and Nox2 comprising NADPH oxidases were identified as major sources of oxidative stress in SHR aortae and thus contributed significantly to endothelial dysfunction. Targeting these enzymes therefore promises to be of immense therapeutic relevance. The novel NADPH oxidase inhibitor VAS2870 was shown to effectively suppress the oxidative stress and to improve the endothelial function in SHR. In consequence, the present study highlights VAS2870 as an experimental tool in the pharmacology of NADPH oxidases and – furthermore - as an interesting model compound for the treatment of cardiovascular and other diseases associated with oxidative stress.