Rationale Acute pulmonary oxygen sensing is essential to avoid life-threatening hypoxemia via hypoxic pulmonary vasoconstriction (HPV) which matches perfusion to ventilation. Hypoxia-induced mitochondrial superoxide release has been suggested as critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing remains unresolved. Objectives To investigate the role of the pulmonary specific isoform 2 of subunit 4 of mitochondrial complex IV (Cox4i2) and the subsequent mediators superoxide and hydrogen peroxide for pulmonary oxygen sensing and signaling. Methods and Results Isolated ventilated and perfused lungs from Cox4i2−/− mice lacked acute HPV. In parallel, pulmonary arterial smooth muscle cells (PASMCs) from Cox4i2−/− mice showed no hypoxia-induced increase of intracellular calcium. Hypoxia-induced superoxide release which was detected by electron spin resonance spectroscopy in wild type (WT) PASMCs was absent in Cox4i2−/− PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in WT but not Cox4i2−/− PASMCs. Downstream signaling determined by patch clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2−/− PASMCs compared to WT PASMCs, which could be normalized by application of hydrogen peroxide. In contrast, chronic hypoxia-induced pulmonary hypertension and pulmonary vascular remodeling were not or only slightly affected by Cox4i2 deficiency, respectively. Conclusion Cox4i2 is essential for acute but not chronic pulmonary oxygen sensing by triggering mitochondrial hyperpolarization and release of mitochondrial superoxide which, after conversion to hydrogen peroxide, contributes to cellular membrane depolarization and HPV. These findings provide a new model for oxygen sensing processes in the lung and possibly also in other organs.

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% O for 10 minutes) was investigated in isolated blood-free perfused mouse lungs. Mice exposed for 4 weeks to chronic hypoxia (10% O) 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.

Background and Purpose: Chronic obstructive pulmonary disease, encompassing chronic airway obstruction and lung emphysema, is a major worldwide health problem and a severe socio-economic burden. Evidence previously provided by our group has shown that inhibition of inducible NOS (iNOS) prevents development of mild emphysema in a mouse model of chronic tobacco smoke exposure and can even trigger lung regeneration. Moreover, we could demonstrate that pulmonary hypertension is not only abolished in cigarette smoke-exposed iNOS −/− mice but also precedes emphysema development. Possible regenerative effects of pharmacological iNOS inhibition in more severe models of emphysema not dependent on tobacco smoke, however, are hitherto unknown. Experimental Approach:We have established a mouse model using a single dose of porcine pancreatic elastase or saline, intratracheally instilled in C57BL/6J mice.Emphysema, as well as pulmonary hypertension development was determined by both structural and functional measurements.Key Results: Our data revealed that (i) emphysema is fully established after 21 days, with the same degree of emphysema after 21 and 28 days post instillation, (ii) emphysema is stable for at least 12 weeks and (iii) pulmonary hypertension is evident, in contrast to smoke models, only after emphysema development. Oral treatment with the iNOS inhibitor N(6)-(1-iminoethyl)-L-lysine (L-NIL) was started after Abbreviations: COPD, chronic obstructive pulmonary disease; GOLD, global initiative for chronic obstructive lung disease; iNOS, inducible NOS; L-NIL, N(6)-(1-iminoethyl)-L-lysine; LV, left ventricle; MLI, mean linear intercept; ONOO-, peroxynitrite; PBGD, porphobilinogen deaminase; PH, pulmonary hypertension; PPE, porcine pancreatic elastase; RV, right ventricle; RVSP, right ventricular systolic pressure; S, Septum. Athanasios Fysikopoulos, Michael Seimetz, Norbert Weissmann and Simone Kraut have equal contribution.Br J Pharmacol. 2020;1-20.wileyonlinelibrary.com/journal/bph 1 emphysema establishment and continued for 12 weeks. This resulted in significant lung regeneration, evident in the improvement of emphysema and reversal of pulmonary hypertension. Conclusion and Implications:Our data indicate that iNOS is a potential new therapeutic target to treat severe emphysema and associated pulmonary hypertension.