Idiopathic pulmonary arterial hypertension (IPAH) is pathogenetically related to low levels of the vasodilator nitric oxide (NOcellular respiration ͉ nitric oxide ͉ oxygen consumption ͉ pulmonary hypertension ͉ mitochondrion I diopathic pulmonary arterial hypertension (IPAH) is a fatal disease of unknown etiology characterized by a progressive increase in pulmonary artery pressure and vascular growth (1, 2). Secondary forms of pulmonary arterial hypertension (PAH) are associated with known diseases, such as collagen vascular diseases or portal hypertension but in the absence of an identifiable etiology are classified as IPAH. Abnormalities in vasodilators, specifically nitric oxide (NO), have been implicated in the pathogenesis of pulmonary hypertension (1-5). NO is produced in the lung by NO synthases (NOS; EC 1.14.13.39) (6-8). There is conclusive evidence from animal models of pulmonary hypertension, mice genetically deficient in endothelial NOS (eNOS), and complementation studies with gene transfer of NOSs for the concept that NO is a critical determinant of pulmonary vascular tone (6, 7, 9). Furthermore, pulmonary and total body NO are lower in IPAH patients as compared with healthy controls (3,(10)(11)(12), and the decrease of NO has been linked to increased arginase II and decreased eNOS expression in IPAH pulmonary endothelial cells in vivo (10,13).In addition to effects on vascular tone, NO regulates cellular bioenergetics through effects on glycolysis, oxygen consumption by mitochondrion, and mitochondrial biogenesis (14-17). For example, eNOS-deficient mice, which have mild pulmonary hypertension under normoxia and an exaggerated pulmonary vasoconstrictive response to hypoxia (18), have reduced mitochondria content in a wide range of tissues in association with significantly lower oxygen consumption and ATP content (14-17). Mitochondria are essential to cellular energy production in all higher organisms adapted to an oxygen-containing environment, i.e., ATP produced through oxidative phosphorylation. The electrochemical gradient used by mitochondrial F 0 F 1 ATP synthase to synthesize ATP from ADP is generated by the proton pump action performed by Complexes I, III, and IV of the respiratory chain. The proton pumping is accompanied by electron shuttling, whereby Complexes I and II, along with the flavoprotein-ubiquinone oxidoreductase, transfer electrons from different sources to ubiquinone (coenzyme Q). The electrons are then transferred sequentially to Complex III, cytochrome c, Complex IV, and finally to molecular oxygen, the terminal electron acceptor. All multisubunit complexes of the respiratory chain (I-IV) are located in the mitochondrial inner membrane. Thus, mitochondria are the primary oxygen demand in the body, accounting for Ϸ90% of cellular oxygen consumption. Conversely, under limiting oxygen conditions, cells turn to glycolysis to generate energy. In endothelial cells, ATP is generated nearly equivalently by glycolysis and cellular respiration (19), accounting for a relative tolerance ...

Idiopathic pulmonary arterial hypertension (IPAH) is characterized by plexiform vascular lesions, which are hypothesized to arise from deregulated growth of pulmonary artery endothelial cells (PAEC). Here, functional and molecular differences among PAEC derived from IPAH and control human lungs were evaluated. Compared with control cells, IPAH PAEC had greater cell numbers in response to growth factors in culture due to increased proliferation as determined by bromodeoxyuridine incorporation and Ki67 nuclear antigen expression and decreased apoptosis as determined by caspase-3 activation and TdT-mediated dUTP nick end labeling assay. IPAH cells had greater migration than control cells but less organized tube formation in in vitro angiogenesis assay. Persistent activation of signal transducer and activator of transcription 3 (STAT3), a regulator of cell survival and angiogenesis, and increased expression of its downstream prosurvival target, Mcl-1, were identified in IPAH PAEC. A Janus kinase (JAK) selective inhibitor reduced STAT3 activation and blocked proliferation of IPAH cells. Phosphorylated STAT3 was detected in endothelial cells of IPAH lesions in vivo, suggesting that STAT3 activation plays a role in the proliferative pulmonary vascular lesions in IPAH lungs.

Rationale: A cellular prooxidant state promotes cells to neoplastic growth, in part because of modification of proteins and their functions. Reactive nitrogen species formed from nitric oxide (NO) or its metabolites, can lead to protein tyrosine nitration, which is elevated in lung cancer. Objective: To determine the alteration in these NO derivatives and the role they may play in contributing to lung carcinogenesis. Methods: We analyzed levels of NO, nitrite (NO 2 Ϫ ), nitrate (NO 3 Ϫ ), and the location of the protein nitration and identified the proteins that are modified. Measurements and Main Results: Although exhaled NO and NO 2 Ϫ were increased, endothelial NO synthase or inducible NO synthase expression was similar in the tumor and tumor-free regions. However, immunohistochemistry showed that nitrotyrosine was increased in the tumor relative to non-tumor-bearing sections. We used proteomics to identify the modified proteins (two-dimensional polyacrylamide gel electrophoresis; mass spectrometry). Both the degree of nitration and the protein nitration profile were altered. We identified more than 25 nitrated proteins, including metabolic enzymes, structural proteins, and proteins involved in prevention of oxidative damage. Alterations of the biology of NO metabolites and nitration of proteins may contribute to the mutagenic processes and promote carcinogenesis. Conclusions: This study provides evidence in favor of a role for reactive nitrogen and oxygen species in lung cancer.Keywords: lung cancer; nitric oxide; nitrotyrosine; protein nitration; proteomics Nitric oxide (NO) plays a variety of regulatory roles in vivo, including control of vascular tone and host defense. However, recent data have suggested that NO metabolites, such as nitrite or NO-modified peptides or proteins, may also play important physiologic roles. Enzymatic generation of NO in mammals is by three isoforms of NO synthase (NOS), inducible NOS (iNOS), endothelial NOS (eNOS), and neuronal NOS (nNOS) (1). The function and regulation of these proteins reflect the role they play in normal physiology. Excessive or inappropriate production of endogenous or exogenous reactive oxygen species (ROS) and NO are implicated in the pathogenesis of cancer (2, 3). For example, cigarette smoke, a major source of exogenous oxidants, is associated with the development of lung cancer (2, 4). Furthermore, cigarette smoking leads to chronic airway inflammation (Received in original form November 15, 2004; accepted in final form May 18, 2005) Supported in part by grants HL60917, AI70649, HL076491, CA53914, and HL04265 from the National Institutes of Health; Debartolo Endowed Funds; American Heart Association 0325313B; a Betsy DeWindt American Cancer Society fellowship, GCRC M01RR018390; and Cafaro Endowed Funds. with accumulation and activation of leukocytes, which produce high levels of ROS and NO. Both these processes lead to oxidative damage (5). Although it is generally accepted that the prooxidant state promotes cells to neoplastic growth through DNA ...

Idiopathic pulmonary arterial hypertension (IPAH) is associated with lower levels of the pulmonary vasodilator nitric oxide (NO) and its biochemical reaction products (nitrite , nitrate ), in part, due to the reduction in pulmonary endothelial NO synthesis. However, NO levels are also determined by consumptive reactions, such as with superoxide to form peroxynitrite, which subsequently may generate stable products of nitrotyrosine (Tyr-NO 2 ) and/or NO 3 -. In this context, superoxide dismutase (SOD) preserves NO in vivo by scavenging superoxide and preventing the consumptive reactions. Here, we hypothesized that reactive oxygen species (ROS) consumption of NO may contribute to the low NO level and development of pulmonary hypertension. To test this, nitrotyrosine and antioxidants glutathione (GSH), glutathione peroxidase (GPx), catalase, and SOD were evaluated in IPAH patients and healthy controls. SOD and GPx activities were decreased in IPAH lungs (all p < 0.05), while catalase and GSH activities were similar among the groups (all p > 0.2). SOD activity was directly related to exhaled NO (eNO) (R 2 = 0.72, p = 0.002), and inversely related to bronchoalveolar lavage (BAL) NO 3 -(R 2 = -0.73, p = 0.04). Pulmonary artery pressure (PAP) could be predicted by a regression model incorporating SOD, GPx, and NO 3 values (R 2 = 0.96, p = 0.01). These findings suggest that SOD and GPx are associated with alterations in NO and PAP in IPAH.