Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing.

Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

Impact of the mitochondria-targeted antioxidant MitoQ on hypoxia-induced pulmonary hypertension.

Increased mitochondrial reactive oxygen species (ROS), particularly superoxide have been suggested to mediate hypoxic pulmonary vasoconstriction (HPV), chronic hypoxia-induced pulmonary hypertension (PH) and right ventricular (RV) remodelling.We determined ROS in acute, chronic hypoxia and investigated the effect of the mitochondria-targeted antioxidant MitoQ under these conditions.The effect of MitoQ or its inactive carrier substance, decyltriphenylphosphonium (TPP+), on acute HPV (1% O2 for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O2) or after banding of the main pulmonary artery (PAB) were treated with MitoQ or TPP+ (50 mg/kg/day).Total cellular superoxide and mitochondrial ROS levels were increased in pulmonary artery smooth muscle cells (PASMC), but decreased in pulmonary fibroblasts in acute hypoxia. MitoQ significantly inhibited HPV and acute hypoxia-induced rise in superoxide concentration. ROS was decreased in PASMC, while it increased in the RV after chronic hypoxia. Correspondingly, MitoQ did not affect the development of chronic hypoxia-induced PH, but attenuated RV remodelling after chronic hypoxia as well as after PAB.Increased mitochondrial ROS of PASMC mediate acute HPV, but not chronic hypoxia-induced PH. MitoQ may be beneficial under conditions of exaggerated acute HPV.

Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability.

Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1alpha on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1alpha enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.

Heme oxygenase-2 and large-conductance Ca2+-activated K+ channels: lung vascular effects of hypoxia.

RATIONALE: Hypoxic pulmonary vasoconstriction (HPV) is an important mechanism by which pulmonary gas exchange is optimized by the adaptation of blood flow to alveolar ventilation. In chronic hypoxia, in addition to HPV a vascular remodeling process leads to pulmonary hypertension. A complex of heme oxygenase-2 (HO-2) and the BK channel has been suggested as a universal oxygen sensor system. OBJECTIVES: We investigated whether this complex serves as an oxygen sensor for the vascular effects of alveolar hypoxia in the lung. METHODS: The investigations were performed in chronically hypoxic mice, in isolated perfused and ventilated lungs, and on the cellular level, including HO-2- and BK-channel deficient mice. MEASUREMENTS AND MAIN RESULTS: Immunohistochemical analysis of mouse lungs identified HO-2 mainly in pulmonary arteries, the bronchial epithelium, and alveolar epithelial cells. BK channel alpha-subunit (BKalpha) immunoreactivity was found primarily in the bronchial and vascular smooth muscle layer. Immunofluorescence staining and coimmunoprecipitation suggested only a weak complexation of HO-2 and BKalpha in pulmonary arterial smooth muscle cells. The strength of acute and sustained HPV, determined in isolated perfused and ventilated lungs, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. Exposure of mice to 3 weeks of chronic hypoxia resulted in a slight down-regulation of HO-2 and no alteration in BKalpha expression. The degree of pulmonary hypertension that developed, quantified on the basis of right ventricular pressure, right-heart hypertrophy, and the degree of muscularization of precapillary pulmonary arteries, was not different among wild-type, HO-2-deficient, and BKalpha-deficient mice. CONCLUSIONS: It is demonstrated that neither deletion of HO-2 nor BK channels affect acute, sustained, and chronic vascular responses to alveolar hypoxia in the lung.

Impact of mitochondria and NADPH oxidases on acute and sustained hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation to optimize pulmonary gas exchange. However, it remains unclear whether acute HPV (occurring within seconds) and the vasoconstrictor response to sustained alveolar hypoxia (developing over several hours) are triggered by identical mechanisms. We investigated the effect of mitochondrial and NADPH oxidase inhibitors on both phases of HPV in intact rabbit lungs. These studies revealed that the sustained HPV is largely dependent on mitochondrial complex I and totally dependent on complex IV, whereas NADPH oxidase dependence was only observed for acute HPV. These findings were reinforced by an alternative approach employing lungs from mice deficient in the NADPH oxidase subunit p 47(phox). In these mice (which lack a subunit suggested to be important for the function of most NADPH oxidase isoforms), but not in gp 91(phox)-deficient mice (which represent only one isoform of NADPH oxidases), acute HPV was significantly reduced, while non-hypoxia-induced vasoconstrictions elicited by the thromboxane mimetic U46619 were not affected. We concluded that the acute phase and the sustained phase of HPV are differentially regulated, with NADPH oxidase activity predominating in the acute phase, while a strong dependence on mitochondrial participation was observed for the second phase.

Basic features of hypoxic pulmonary vasoconstriction in mice.

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation which tends to optimize pulmonary gas exchange. Investigations using genetically engineered mice represent a promising approach to understand the underlying mechanisms. Our goal was to characterize basic features of HPV in the isolated buffer-perfused and ventilated mouse lung system. HPV was reproducible for several hours when ventilating the lungs with 1% O2 (10 min) alternated with normoxic ventilation periods (21% O2, 15 min). HPV was well elicitable and most constant using Krebs-Henseleit buffer with the addition of hydroxyethylamylopectin as an oncotic agent. Inhibition of both lung NO and prostanoid formation amplified HPV in an over-additive fashion. HPV was higher in BALB/c mive as compared to C57BL/6 mice, and was approximately threefold enhanced under positive pressure ventilation as compared to negative pressure ventilation. A three hour hypoxic ventilation period resulted in a biphasic vasoconstrictor response with loss of posthypoxic vasodilatation. In summary, we have characterised HPV and established an experimental set-up optimized for investigation of the basic mechanisms of HPV in mice.

Prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits.

Bleomycin is a well known fibrogenic agent, provoking an initial adult respiratory distress syndrome-like injury with subsequent strong fibroproliferative response. Severe abnormalities of the alveolar surfactant system, which may be linked to the appearance of alveolar fibrin deposition, have been implicated in the pathogenetic sequence of events. Using a model of standardized aerosol delivery of 1.8 U bleomycin/kg body weight in rabbits, we investigated the influence of repetitive nebulization of heparin or urokinase-type plasminogen activator (u-PA) on the development of lung fibrosis. In an "early" (Days 2-12 postbleomycin) or "late" (Days 14-24 post-bleomycin) treatment protocol, approximately 3,500 U heparin or approximately 6,500 U u-PA was delivered to the bronchoalveolar space. Within four weeks, the bleomycin challenge provoked severe pulmonary fibrosis with reduction of lung compliance, marked increase in soluble collagen (bronchoalveolar lavage fluid) and hydroxyproline content (lung tissue), a typical reticular fibrosis pattern on high-resolution computed tomography, and typical histologic findings. Therapeutic intervention resulted in a far-reaching normalization of compliance, suppression of soluble collagen and hydroxyproline accumulation, and virtual abrogation of the computed tomography scan and histologic features of lung fibrosis, with most prominent effects seen in the early heparin and late u-PA administration. No bleeding complications occurred. These findings strongly support the concept that alveolar fibrin generation is an important event in the development of postbleomycin lung fibrosis. "Compartmentalized" anticoagulation and/or fibrinolysis via inhalational deposition of interventional agents in the alveolar compartment may thus offer a new therapeutic strategy for prevention of fibrosis.

Downregulation of hypoxic vasoconstriction by chronic hypoxia in rabbits: effects of nitric oxide.

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion to ventilation for optimizing pulmonary gas exchange. Chronic alveolar hypoxia results in vascular remodeling and pulmonary hypertension. Previous studies have reported conflicting results of the effect of chronic alveolar hypoxia on pulmonary vasoreactivity and the contribution of nitric oxide (NO), which may be related to species and strain differences as well as to the duration of chronic hypoxia. Therefore, we investigated the impact of chronic hypoxia on HPV in rabbits, with a focus on lung NO synthesis. After exposure of the animals to normobaric hypoxia (10% O(2)) for 1 day to 10 wk, vascular reactivity was investigated in ex vivo perfused normoxic ventilated lungs. Chronic hypoxia induced right heart hypertrophy and increased normoxic vascular tone within weeks. The vasoconstrictor response to an acute hypoxic challenge was strongly downregulated within 5 days, whereas the vasoconstrictor response to the thromboxane mimetic U-46619 was maintained. The rapid downregulation of HPV was apparently not linked to changes in the lung vascular NO system, detectable in the exhaled gas and by pharmacological blockage of NO synthesis. Treatment of the animals with long-term inhaled NO reduced right heart hypertrophy and partially maintained the reactivity to acute hypoxia, without any impact on the endogenous NO system being noted. We conclude that chronic hypoxia causes rapid downregulation of acute HPV as a specific event, preceding the development of major pulmonary hypertension and being independent of the lung vascular NO system. Long-term NO inhalation partially maintains the strength of the hypoxic vasoconstrictor response.