eNOS uncoupling in pulmonary hypertension.

Pulmonary arterial hypertension (PAH) is characterized by high blood pressure and vascular remodelling in pulmonary arteries and subsequently right ventricular hypertrophy. Although several studies in animal models of pulmonary hypertension (PH) and pathological examinations of lung tissue obtained from PAH patients show occlusion of pulmonary arteries, the exact molecular mechanisms for vascular abnormalities remain unclear. Endothelial dysfunction seems to be one of the earliest events in PAH and is characterized by reduced levels of vasodilators nitric oxide (NO) and prostacyclin and increased vasoconstrictor agents, such as endothelin-1 and prostanoids. NO formed from L-arginine by endothelial nitric oxide synthase (eNOS) plays an important protective role in vascular haemostasis. Activin receptor-like kinase 1 (Alk1) is a type I receptor of transforming growth factor-beta family proteins or bone morphogenic proteins and is mainly expressed in endothelial cells, regulating proliferation and migration in vitro and angiogenesis in vivo. Mutation of the Alk1 gene causes hereditary haemorrhagic telangiectasia type II in patients, an autosomal dominant vascular dysplasia that results in abnormal blood vessel formation in the skin, mucous membranes, and often in organs such as the lungs, liver, and brain, suggesting that Alk1 may play an important role during vascular development. Jerkic et al. showed that deletion of one copy of the Alk1 gene (Alk1+/2) caused PAH in adult mice as characterized by increased right ventricular systolic pressure, vascular remodelling, decreased vascular density, and endothelial dysfunction of pulmonary arteries. In contrast, no signs of endothelial dysfunction and PAH were detected in young Alk1+/2 mice. The authors identified increased production of oxygen-derived species (ROS) including superoxide anion and hydrogen peroxide (H2O2) in the lungs of adult but not newborn Alk1+/2 mice. Remarkably, endothelium-dependent relaxation to acetylcholine was enhanced in pulmonary arteries of adult Alk1+/2 mice despite reduced production of NO. Dysfunction of eNOS in endothelial cells can be an important source for production of H2O2, which is a potent vasodilator that activates soluble guanylate cyclase and elevates levels of cyclic guanosine monophosphate in vascular smooth muscle cells. Studies with an NOS inhibitor suggested that eNOS-catalysed formation of superoxide anion and subsequent formation of H2O2 may represent an important mechanism underlying endothelial dysfunction described in a number of vascular diseases. Furthermore, the authors elegantly showed that in vivo treatment with the cell-permeable superoxide dismutase mimetic tempol completely prevented characteristic signs of PAH in Alk1+/2 mice despite increased levels of H2O2, indicating that superoxide anion is a mediator of PH. The role of superoxide anion in several forms of PAH has been demonstrated in previous studies. There are several sources of superoxide anion in vascular endothelium, for example, increased activity of NADPH oxidase, xanthine oxidase, and/or cyclooxygenase. In addition, reduced antioxidant defence capacity and/or impaired enzymatic activity of eNOS may contribute to elevation of superoxide anion concentration and subsequent endothelial dysfunction. In the present study, however, no differences in protein expression of enzymes involved in ROS generation and scavenging were found between wild-type and Alk1+/2 mice. Since phosphorylation of eNOS is a key posttranslational modification that ensures optimal production of NO, increased basal phosphorylation of eNOS at Ser and uncoupling of eNOS as a source of superoxide anion were identified in the lungs of Alk1+/2 mice. It is well established that tetrahydrobiopterin (BH4) is an essential cofactor for allosteric and redox activation of eNOS and that BH4 is mainly produced in the vascular endothelium. 12 Several biochemical studies have demonstrated that suboptimal concentrations of BH4 result in the generation of superoxide anion by eNOS. 13,14