Reactive oxygen species in pulmonary vascular remodeling

Abstract

The pathogenesis of pulmonary hypertension is a complex multifactorial process that involves the remodeling of pulmonary arteries. This remodeling process encompasses concentric medial thickening of small arterioles, neomuscularization of previously nonmuscular capillary-like vessels, and structural wall changes in larger pulmonary arteries. The pulmonary arterial muscularization is characterized by vascular smooth muscle cell hyperplasia and hypertrophy. In addition, in uncontrolled pulmonary hypertension, the clonal expansion of apoptosis-resistant endothelial cells leads to the formation of plexiform lesions. Based upon a large number of studies in animal models, the three major stimuli that drive the vascular remodeling process are inflammation, shear stress, and hypoxia. Although, the precise mechanisms by which these stimuli impair pulmonary vascular function and structure are unknown, reactive oxygen species (ROS)-mediated oxidative damage appears to play an important role. ROS are highly reactive due to their unpaired valence shell electron. Oxidative damage occurs when the production of ROS exceeds the quenching capacity of the antioxidant mechanisms of the cell. ROS can be produced from complexes in the cell membrane (nicotinamide adenine dinucleotide phosphate-oxidase), cellular organelles (peroxisomes and mitochondria), and in the cytoplasm (xanthine oxidase). Furthermore, low levels of tetrahydrobiopterin (BH4) and L-arginine the rate limiting cofactor and substrate for endothelial nitric oxide synthase (eNOS), can cause the uncoupling of eNOS, resulting in decreased NO production and increased ROS production. This review will focus on the ROS generation systems, scavenger antioxidants, and oxidative stress associated alterations in vascular remodeling in pulmonary hypertension.

Figure 1. Schematic view of the pathology of pulmonary hypertension

The figure summarizes the major pathological events that lead to the development of pulmonary hypertension (PH). In humans, PH is a complex and multifactorial, diffuse disorder of the pulmonary vasculature. However, for the purposes of research and inducing PH in animal models, the etiology of PH can be broadly distinguished into: (a) inflammatory; (b) high shear stress; and (c) hypoxia, even though none of these factors act alone during the progression of the disease. A common mechanism by which these insults provoke vascular dysfunction is by either increasing the production of reactive oxygen species (ROS) or by attenuating the ROS scavenging capability of the cells. Once produced, ROS can influence the growth and morphology of all three layers of the pulmonary vessels. In endothelial cells, ROS promote endothelial proliferation, decrease NO production, and increase the release of vasoactive mediators, leading to endothelial dysfunction. In smooth muscle cells (SMC), oxidative stress caused by ROS induces contraction and a switch to a synthetic phenotype. These SMC alterations are brought about by increasing the intracellular free Ca²⁺ and decreasing the expression of contractile phenotypic markers, while simultaneously enhancing the expression of proliferative phenotypic markers and growth factors. ROS can also augment the proliferation of fibroblasts in the adventitial layer and their expansion between the endothelium and the internal elastic lamina, known as the neointima (not depicted in the figure). Together, these changes result in vascular remodeling and the development of PH.

Figure 2. Reactive oxygen species generation in pulmonary hypertension

The major sources of ROS in the vasculature include uncoupled eNOS, mitochondrial dysfunction, NADPH oxidase, and XO. In PH, all of these sources contribute to the development of oxidative stress. Endothelial NOS uncoupling is mediated by limited L-arginine availability, as a result of increased degradation by arginase upregulation and attenuated synthesis by the downregulation of ASS and ASL, the enzymes responsible for the conversion of L-citrulline to L-arginine. Moreover, a sustained increase in ADMA levels, due to a decrease in DDAH2 activity, competes with L-arginine for binding to eNOS. In addition, in PH, eNOS function is impaired by a decrease in BH₄, an essential co-factor for NO generation. GCH1, the rate-limiting enzyme in BH₄ biosynthesis, is ubiquinated and targeted for degradation by Hsp70/CHIP. Therefore, the low GCH1 levels, limit the production of BH₄. Finally, The disruption of the zinc tetrathiolate (ZnS₄) cluster by oxidative attack disrupts the eNOS dimer, which is accompanied by decreased NO generation and increased ROS production. Further, in PH, several markers of mitochondrial dysfunction are observed, including increased levels of uncoupling protein-2 (UCP-2), decreased levels of the mitochondrial antioxidant, superoxide dismutase-2 (SOD2), and the impaired function of complexes I, II, and III of the respiratory chain. Interestingly, ADMA appears to promote these changes in the mitochondria, and also augment mitochondrial ROS generation and decrease ATP production. In addition, several subunits of NADPH oxidase, including p47^phox, p67phox, gp91phox, and Rac1, are upregulated in PH, increasing ROS production in the vasculature. Increased levels and activity of XO also contribute to oxidative stress and vascular dysfunction in the early stages of PH.

Figure 3. Reactive oxygen species signaling in pulmonary hypertension

The vasoactive mediators that contribute to the development of pulmonary vascular remodeling and PH can be broadly divided into two major categories: factors affecting vascular tone and factors influencing vessel wall thickening. ROS influence the generation of several of these factors. In PH, ROS decrease the expression and/or function of redox sensitive voltage gated K⁺ channels (Kv1.5, Kv2.1), leading to elevations in intracellular K⁺ and Ca²⁺ influx through the activation of voltage-gated L-type Ca²⁺ channels. In turn, Ca²⁺ induces SMC contraction through the calmodulin dependent activation of myosin light chain kinase (MLCK) and the subsequent phosphorylation of the myosin light chain (MLC). ROS also promote the activation of the small GTPase, RhoA, and its downstream effector, Rho kinase, which increase the phosphorylation of MLC through the inhibition of MLC phosphatase. In addition, ROS promote pulmonary vessel constriction by enhancing the production of endothelin-1 (ET-1) and thromboxane A2 (TXA2), while attenuating the levels of vasodilators, such as prostacyclin and peroxisome proliferator-activated receptors-γ (PPAR-γ). The role of ROS in mediating pulmonary SMC proliferation and vessel thickening is more complex. Oxidative stress induces the expression of several growth factors, such as transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), and platelet-derived growth factor (PDGF). In addition, ROS also activate p38MAPK and ERK1/2, which promote proliferation. The MAPK signaling pathway is also activated by pp60^Src, through ROS mediated activation. Further, ROS stimulate Akt1, which promotes SMC proliferation through the upregulation of PGC-1α. Together, the dysregulation of these constrictive and proliferative factors by ROS promotes the vascular remodeling seen in PH.

Figure 4. Reactive oxygen species scavenging in pulmonary hypertension

ROS are scavenged by antioxidant systems to preserve the cellular redox homeostasis and to prevent oxidative damage to cellular proteins, lipids, and DNA. The major vascular enzymatic antioxidants are SOD, catalase, and GPx. There are three types of SOD: cytoplasmic Cu/ZnSOD (SOD1), mitochondrial MnSOD (SOD2), and extracellular EC-SOD (SOD3) that catalyze the rapid conversion of O₂⁻ into H₂O₂. In PH, there is a decrease in the expression and/or activity of all three SOD isoforms, increasing O₂⁻ levels. The H₂O₂ that is produced by SOD can be enzymatically reduced in the cytoplasm by catalase and GPx. Catalase decomposes H₂O₂ to O₂ and H₂O, while GPx requires two molecules of GSH to reduce one molecule of H₂O₂ to two molecules of H₂O and in the process GSH is oxidized to GSSG. Catalase activity/expression is altered in PH, while the expression/activity of GPx is decreased in many forms of PH. The result of reduced ROS quenching by these antioxidants adds to the oxidative stress observed in PH.