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 ...

Pulmonary arterial hypertension (PAH), a fatal disease of unknown etiology characterized by impaired regulation of pulmonary hemodynamics and vascular growth, is associated with low levels of pulmonary nitric oxide (NO). Based upon its critical role in mediating vasodilation and cell growth, decrease of NO has been implicated in the pathogenesis of PAH. We evaluated mechanisms for low NO and pulmonary hypertension, including NO synthases (NOS) and factors regulating NOS activity, i.e. the substrate arginine, arginase expression and activity, and endogenous inhibitors of NOS in patients with PAH and healthy controls. PAH lungs had normal NOS I-III expression, but substrate arginine levels were inversely related to pulmonary artery pressures. Activity of arginase, an enzyme that regulates NO biosynthesis through effects on arginine, was higher in PAH serum than in controls, with high-level arginase expression localized by immunostaining to pulmonary endothelial cells. Further, pulmonary artery endothelial cells derived from PAH lung had higher arginase II expression and produced lower NO than control cells in vitro. Thus, substrate availability affects NOS activity and vasodilation, implicating arginase II and alterations in arginine metabolic pathways in the pathophysiology of PAH.

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.

Severe pulmonary hypertension is irreversible and often fatal. Abnormal proliferation and resistance to apoptosis of endothelial cells (ECs) and hypertrophy of smooth muscle cells in this disease are linked to decreased mitochondria and preferential energy generation by glycolysis. We hypothesized this metabolic shift of pulmonary hypertensive ECs is due to greater hypoxia inducible-factor1␣ (HIF-1␣) expression caused by low levels of nitric oxide combined with low superoxide dismutase activity. We show that cultured ECs from patients with idiopathic pulmonary arterial hypertension (IPAH-ECs) have greater HIF-1␣ expression and transcriptional activity than controls under normoxia or hypoxia , and pulmonary arteries from affected patients have increased expression of HIF-1␣ and its target carbonic anhydrase IX. Severe pulmonary arterial hypertension is characterized by significant increases in pulmonary artery pressures to levels present in the systemic circulation. Pulmonary hypertension (PH) is a major determinant of morbidity and mortality in several pulmonary and heart diseases. The pathogenesis of severe pulmonary arterial hypertension has revolved around excessive vasoconstriction and/or abnormal pulmonary vascular remodeling. Recent experimental evidence has linked the pulmonary vascular disease in severe pulmonary arterial hypertension to an abnormal proliferative vascular cell phenotype, which is also characterized by resistance to endothelial and/or vascular smooth muscle cell apoptosis.

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Reactive oxygen species and reactive nitrogen species produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Detection of 3-nitrotyrosine in the asthmatic lung confirms the presence of increased reactive oxygen and nitrogen species, but the lack of identification of modified proteins has hindered an understanding of the potential mechanistic contributions of nitration/oxidation to airway inflammation. In this study, we applied a proteomic approach, using nitrotyrosine as a marker, to evaluate the oxidation of proteins in the allergen-induced murine model of asthma. Over 30 different proteins were targets of nitration following allergen challenge, including the antioxidant enzyme catalase. Oxidative modification and loss of catalase enzyme function were seen in this model. Subsequent investigation of human bronchoalveolar lavage fluid revealed that catalase activity was reduced in asthma by up to 50% relative to healthy controls. Analysis of catalase isolated from asthmatic airway epithelial cells revealed increased amounts of several protein oxidation markers, including chloro- and nitrotyrosine, linking oxidative modification to the reduced activity in vivo. Parallel in vitro studies using reactive chlorinating species revealed that catalase inactivation is accompanied by the oxidation of a specific cysteine (Cys(377)). Taken together, these studies provide evidence of multiple ongoing and profound oxidative reactions in asthmatic airways, with one early downstream consequence being catalase inactivation. Loss of catalase activity likely amplifies oxidative stress, contributing to the chronic inflammatory state of the asthmatic airway.

Airway hyperresponsiveness and remodeling are defining features of asthma. We hypothesized that impaired superoxide dismutase (SOD) antioxidant defense is a primary event in the pathophysiology of hyperresponsiveness and remodeling that induces apoptosis and shedding of airway epithelial cells. Mechanisms leading to apoptosis were studied in vivo and in vitro. Asthmatic lungs had increased apoptotic epithelial cells compared to controls as determined by terminal dUTP nick-end labeling-positive cells. Apoptosis was confirmed by the finding that caspase-9 and -3 and poly (ADP-ribose) polymerase were cleaved. On the basis that SOD inactivation triggers cell death and low SOD levels occur in asthma, we tested whether SOD inactivation plays a role in airway epithelial cell death. SOD inhibition increased cell death and cleavage/activation of caspases in bronchial epithelial cells in vitro. Asthma is commonly diagnosed using physiological measures, but alterations in airway structure are the defining features of asthma. Damage to airway epithelium, eosinophil infiltration, smooth muscle hyperplasia, thickening and aberrant collagen, and protein composition of the basement membrane are well established elements of the asthmatic airway. 1,2 The injury to the bronchial epithelium in asthma is marked by loss of columnar epithelial cells. Extensive loss of cells and denuded basement membrane with few basal cells remaining on the airway surface are noted in severe asthma, but shedding of airway epithelium is present even in clinically mild asthma. 2,3 Physical loss of epithelial lining cells is considered one proximate cause of the airway hyperresponsiveness to inhaled mediators, and has been speculated to contribute to asthmatic airway remodeling, in particular abnormal collagen synthesis. Evidence from organ culture systems supports the concept of an epithelial-mesenchymal unit in which loss of epithelium leads to mucosal myofibroblast and fibroblast proliferation, and collagen deposition. 2,4 -6 Thus, if the epithelial injury and loss could be understood and prevented in asthma, the clinical symptoms of airway hyperresponsiveness and long-term progressive sequelae in the airways, which contribute to fixed airflow limitation, might be prevented.Several reports have proposed that loss of epithelial cells is because of apoptosis based on immunostaining for the proteins that regulate apoptosis, or by detection of DNA strand breaks by immunostaining with the terminal dUTP nick-end labeling (TUNEL) assay. 7-11 However, not all reports have confirmed increased TUNEL positivity in airways. 9 Furthermore, if airway epithelial cells are undergoing increased cell death, it is unclear whether this is because of an inherent cell defect or a response to the asthmatic airway environment. Although nonspecific events related to increased levels of reactive oxygen and

Pulmonary arterial hypertension (PAH) is a proliferative vasculopathy characterized by high circulating CD34 ؉ CD133 ؉ proangiogenic progenitors, and endothelial cells that have pathologic expression of hypoxia-inducible factor 1 ␣ (HIF-1␣). Here, CD34 ؉ CD133 ؉ progenitor cell numbers are shown to be higher in PAH bone marrow, blood, and pulmonary arteries than in healthy controls. The HIF-inducible myeloid-activating factors erythropoietin, stem cell factor (SCF), and hepatocyte growth factor (HGF) are also present at higher than normal levels in PAH blood, and related to disease severity. Primary endothelial cells harvested from human PAH lungs produce greater HGF and progenitor recruitment factor stromal-derived factor 1 ␣ (SDF-1␣) than control lung endothelial cells, and thus may contribute to bone marrow activation. Even though PAH patients had normal numbers of circulating blood elements, hematopoietic alterations in myeloid and erythroid lineages and reticulin fibrosis identified a subclinical myeloproliferative process. Unexpectedly, evaluation of bone marrow progenitors and reticulin in nonaffected family members of patients with familial PAH revealed similar myeloid abnormalities. Altogether, the results show that PAH is linked to myeloid abnormalities, some of which may be related to increased production of HIF-inducible factors by diseased pulmonary vasculature, but findings in nonaffected family suggest myeloid abnormalities may be intrinsic to the disease process. (Blood. 2011;117(13):3485-3493) IntroductionPulmonary arterial hypertension (PAH) is a vasculopathy of the pulmonary circulation characterized by arterial obliteration secondary to unchecked pathologic angiogenic processes. 1-3 An abundance of studies over the past decade provide evidence for the paradigm of lifelong interdependence between angiogenesis and hematopoiesis. [4][5][6] The concept of a common hematopoieticendothelial stem cell, that is, hemangioblast, with bidirectional, reversible gene transcription and persistence is well established in developmental biology. 7 In postnatal life to adulthood, hemangioblasts are readily identifiable in the bone marrow by the CD133-selective expression on a small subpopulation of CD34-positive hematopoietic stem cells. 8 Hemangioblasts give rise to all blood cellular components, but whether these cells give rise to endothelium during postnatal neovascularization is uncertain. 9,10 In contrast, studies clearly substantiate that CD34 ϩ CD133 ϩ progenitors are vital contributors to angiogenesis via proangiogenic effects on endothelial cells in vessels. [11][12][13][14][15][16][17][18] Our and other studies identify that CD34 ϩ CD133 ϩ progenitors are present at higher than normal levels in the circulation of PAH patients and are more proliferative than circulating progenitors of healthy controls. 19,20 The relationship of numbers of circulating CD34 ϩ CD133 ϩ cells to severity of PAH suggest that these cells may promote the angioproliferative vascular remodeling. 20 However, whether the source ...