Ang II–Stimulated Superoxide Production Is Mediated via Phospholipase D in Human Vascular Smooth Muscle Cells

Abstract

Abstract-Intracellular signaling events that mediate the long-term effects of Ang II in vascular smooth muscle cells are unclear, but oxidative stress may play an important role. This study examined the ability of Ang II to generate reactive oxygen species and investigated the putative role of phospholipase D (PLD)-dependent signaling pathways for its production in human vascular smooth muscle cells. In addition, we assessed whether redox-sensitive pathways influence Ang II-stimulated cell growth. Primary and low-passage cells (passages 1 to 4) derived from resistance arteries of subcutaneous gluteal biopsies from healthy subjects were studied. Key Words: oxidative stress Ⅲ superoxide anions Ⅲ intracellular signaling Ⅲ vascular hypertrophy T he multiple actions of Ang II are mediated via highly complex intracellular signaling pathways that are stimulated after binding of the peptide to its cell-surface receptors. 1 The signaling processes are multiphasic, with distinct temporal characteristics. 2 Ang II-induced activation of phospholipase C characteristically occurs within seconds, resulting in increased intracellular free Ca 2ϩ concentration and rapid vascular contraction, 3,4 whereas activation of signaling pathways mediating protein synthesis and cell growth are delayed, occurring within minutes or hours after Ang II stimulation. 2,5 Although the intracellular signaling events underlying Ang II-induced growth are not completely understood, there is increasing evidence suggesting that generation of reactive oxygen species (ROS), such as superoxide anion (⅐O 2 Ϫ ), hydrogen peroxide (H 2 O 2 ), and the reactive hydroxyl radical (⅐OH), may be fundamental in the mitogenic response to this peptide. 6Ϫ8 The major source of ⅐O 2 Ϫ in cardiovascular cells is NADH/ NADPH oxidase, 9Ϫ12 which transfers electrons from NADH or NADPH to molecular oxygen, producing ⅐O 2 Ϫ . The ⅐O 2 Ϫ that is generated by NADH/NADPH oxidase is converted by superoxide dismutase to H 2 O 2 , which is scavenged by catalase or by peroxidases. 13 ⅐O 2 Ϫ and H 2 O 2 stimulate vascular smooth muscle cell (VSMC) hyperplasia and hypertrophy. 6,9,14 These effects are associated with growth-related events such as intracellular alkalinization, increased intracellular free Ca 2ϩ concentration, MAP kinase activation, and induction of proto-oncogene expression. 6,15Ϫ17 Furthermore, antioxidants inhibit cell growth and trigger apoptosis, which implies that a basal level of oxidant stress is necessary for normal cell growth. 18,19 Vascular tissue is constantly exposed to endogenous and exogenous oxidants, which, if unscavenged, may lead to cellular proliferation. ROS concentrations are increased in atherosclerosis, neointimal formation, and hypertension. 20Ϫ22 In Ang II-induced but not catecholamineinduced hypertension, aortic superoxide is increased, which suggests that Ang II-induced effects in hypertension are mediated in part through oxidative stress. 23,24

Angiotensin II and Oxidative Stress

“…Impaired endothelium-dependent vasodilatation as well as increased markers of oxidative stress (urinary 8-hydroxy-2'-deoxyguanosine, malondialdehyde-modified LDL) were found before angioplasty and were abrogated by the intervention. This finding is in line with experimental studies in vascular smooth muscle cell (VSMC) cultures (13), including human VSMC (14), and studies in the kidney of ren2 rats, which overexpress human renin and overexpress the NADPH oxidase constituents Nox 1 and Nox 4 (15) The hypertensinogenic effect of reactive oxygen species (ROS) extends beyond the AngII infusion model. For instance in the kidney of the SHR rat, all the main components of the NADPH oxidase are overexpressed even before the onset of hypertension, particularly in the vasculature, the macula densa, and the distal nephron (16).…”

Spironolactone for poorly controlled hypertension in type 2 diabetes: conflicting effects on blood pressure, endothelial function, glycaemic control and hormonal profiles

“…Furthermore, in the EPHESUS study, aldosterone blockade significantly reduced hypoglycaemia by 43%, which again could be consistent with a worsening of glycaemic control and the attendant negative impact on endothelial function [37][38][39]. Second, spironolactone doubled plasma levels of angiotensin II, which could be an additional factor in worsening endothelial function [40,41]. The increase in angiotensin II in patients treated with spironolactone and ACE inhibitors may have been due to non-ACE pathways that mediate the production of angiotensin II [42,43].…”

Angiotensin II Induces Connective Tissue Growth Factor Gene Expression via Calcineurin-Dependent Pathways

“…46 The mechanisms whereby Ang II induces NADPH oxidase are not completely understood. Previous studies by Touyz et al 47,48 have provided evidence that Ang II-induced activation of NADPH oxidase is linked to activation of phospholipase D and protein kinase C, phosphorylation of p47phox, translocation of cytosolic subunits, and de novo protein synthesis. Interestingly, Ang II alone, even at micromolar concentrations, has little if any effect on CTGF mRNA level in cardiac fibroblasts.…”