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.

Bronchopulmonary dysplasia (BPD) is a neonatal lung disease developing in premature babies characterized by arrested alveologenesis and associated with decreased Fibroblast growth factor 10 (FGF10) expression. One-week hyperoxia (HYX) exposure of newborn mice leads to a permanent arrest in alveologenesis. To test the role of Fgf10 signaling to promote de novo alveologenesis following hyperoxia, we used transgenic mice allowing inducible expression of Fgf10 and recombinant FGF10 (rFGF10) protein delivered intraperitoneally. We carried out morphometry analysis, and IF on day 45. Alveolospheres assays were performed co-culturing AT2s from normoxia (NOX) with FACS-isolated Sca1 Pos resident mesenchymal cells (rMC) from animals exposed to NOX, HYX-PBS, or HYX-FGF10. scRNAseq between rMC-Sca1 Pos isolated from NOX and HYX-PBS were also carried out. Transgenic overexpression of Fgf10 and rFGF10 administration rescued the alveologenesis defects following HYX. Alveolosphere assays indicate that the activity of rMC-Sca1 Pos is negatively impacted by HYX and partially rescued by rFGF10 treatment. Analysis by IF demonstrates a significant impact of rFGF10 on the activity of resident mesenchymal cells. scRNAseq results identified clusters expressing Fgf10, Fgf7, Pdgfra, and Axin2, which could represent the rMC niche cells for the AT2 stem cells. In conclusion, we demonstrate that rFGF10 administration is able to induce de novo alveologenesis in a BPD mouse model and identified subpopulations of rMC-Sca1 Pos niche cells potentially representing its cellular target.

Acute and sustained hypoxic pulmonary vasoconstriction (HPV), as well as chronic pulmonary hypertension (PH), is modulated by nitric oxide (NO). NO synthesis can be decreased by asymmetric dimethylarginine (ADMA), which is degraded by dimethylarginine dimethylaminohydrolase-1 (DDAH1). We investigated the effects of DDAH1 overexpression (DDAH1 tg ) on HPV and chronic hypoxia-induced PH. HPV was measured during acute (10 min) and sustained (3 h) hypoxia in isolated mouse lungs. Chronic PH was induced by the exposure of mice to 4 weeks of hypoxia. ADMA and cyclic 39,59-guanosine monophosphate (cGMP) were determined by ELISA, and NO generation was determined by chemiluminescence. DDAH1 overexpression exerted no effects on acute HPV. However, DDAH1 tg mice showed decreased sustained HPV compared with wild-type (WT) mice. Concomitantly, ADMA was decreased, and concentrations of NO and cGMP were significantly increased in DDAH1quinoxalin-1-one potentiated sustained HPV and partly abolished the differences in sustained HPV between WT and DDAH1 tg mice. The overexpression of DDAH1 exerted no effect on the development of chronic hypoxia-induced PH. DDAH1 overexpression selectively decreased the sustained phase of HPV, partly via activation of the NO-cGMP pathway. Thus, increased ADMA concentrations modulate sustained HPV, but not acute HPV or chronic hypoxia-induced PH.Keywords: asymmetric dimethylarginine; cyclic guanosine monophosphate; hypoxia-induced pulmonary hypertension; hypoxic pulmonary vasoconstriction; nitric oxide Exposure of the lung vasculature to hypoxia induces vasoconstriction of the precapillary vessels (hypoxic pulmonary vasoconstriction [HPV]) and, if chronic, vascular remodeling develops, fixing pulmonary hypertension (PH). HPV can be divided into two phases inducible by alveolar hypoxia, lasting seconds to minutes (acute HPV), or minutes to hours (sustained HPV). Such a biphasic response of the lung vasculature to hypoxia has been reported for isolated pulmonary arteries, as well as for isolated lungs (1, 2). Both phases of HPV play an important role in matching local perfusion to ventilation, thus optimizing pulmonary gas exchange. Acute HPV is finally triggered by an intracellular calcium increase, whereas sustained HPV additionally depends on calcium-sensitizing factors released by the endothelium, resulting in the search for an "endothelium-derived vasoconstrictive factor" (3, 4). In contrast to acute HPV, the increase of pulmonary arterial pressure (PAP) during sustained HPV is not spontaneously reversible upon reexposure to normoxia, and has been suggested to facilitate the development of PH. PH is characterized by vascular remodeling and vasoconstriction, resulting in an increased workload for the right heart. Vascular alterations in PH may also be triggered by endothelial dysfunction, resulting in smooth muscle cell proliferation, platelet aggregation, and fibroblast proliferation.Nitric oxide (NO) is one of the major endothelium-derived vasoactive mediators controlling a diverse range of pul...

Acute respiratory distress syndrome (ARDS) is a serious complication of severe systemic or local pulmonary inflammation, such as caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. ARDS is characterised by diffuse alveolar damage that leads to protein-rich pulmonary oedema, local alveolar hypoventilation and atelectasis. Inadequate perfusion of these areas is the main cause of hypoxaemia in ARDS. High perfusion in relation to ventilation (V/Q<1) and shunting (V/Q=0) is not only caused by impaired hypoxic pulmonary vasoconstriction but also redistribution of perfusion from obstructed lung vessels. Rebalancing the pulmonary vascular tone is a therapeutic challenge. Previous clinical trials on inhaled vasodilators (nitric oxide and prostacyclin) to enhance perfusion to high V/Q areas showed beneficial effects on hypoxaemia but not on mortality. However, specific patient populations with pulmonary hypertension may profit from treatment with inhaled vasodilators. Novel treatment targets to decrease perfusion in low V/Q areas include epoxyeicosatrienoic acids and specific leukotriene receptors. Still, lung protective ventilation and prone positioning are the best available standard of care. This review focuses on disturbed perfusion in ARDS and aims to provide basic scientists and clinicians with an overview of the vascular alterations and mechanisms of V/Q mismatch, current therapeutic strategies, and experimental approaches.

In addition to its renowned poisonous effects, carbon monoxide (CO) is being recognized for its beneficial actions on inflammatory and vasoregulatory pathways, particularly when applied at low concentrations via CO-releasing molecules (CO-RMs). In the lung, CO gas and CO-RMs are suggested to decrease pulmonary vascular tone and hypoxic pulmonary vasoconstriction (HPV). However, the direct effect of CO-RMs on the pulmonary vasoreactivity in isolated lungs has not yet been investigated. We assessed the effect of CORM-2 and CORM-3 on the pulmonary vasculature during normoxia and acute hypoxia (1% oxygen for 10 min) in isolated ventilated and perfused mouse lungs. The effects were compared with those of inhaled CO gas (10%). The interaction of CORM-2 or CO with cytochrome P-450 (CYP) was measured simultaneously by tissue spectrophotometry. Inhaled CO decreased HPV and vasoconstriction induced by the thromboxane mimetic U-46619 but did not alter KCl-induced vasoconstriction. In contrast, concentrations of CORM-2 and CORM-3 used to elicit beneficial effects on the systemic circulation did not affect pulmonary vascular tone. High concentration of CO-RMs or long-term application induced a continuous increase in normoxic pressure. Inhaled CO showed spectral alterations correlating with the inhibition of CYP. In contrast, during application of CORM-2 spectrophotometric signs of interaction with CYP could not be detected. Application of CO-RMs in therapeutic doses in isolated lungs neither decreases pulmonary vascular tone and HPV nor does it induce spectral alterations that are characteristic of CO-inhibited CYP. High doses, however, may cause pulmonary vasoconstriction.

Aims The pulmonary vascular tone and hypoxia-induced alterations of the pulmonary vasculature may be regulated by the mitochondrial membrane permeability transition pore (mPTP) that controls mitochondrial calcium load and apoptosis. We thus investigated, if the mitochondrial proteins p66shc and cyclophilin D (CypD) that regulate mPTP opening affect the pulmonary vascular tone. Methods and Results Mice deficient for p66shc (p66shc-/-), CypD (CypD-/-), or both proteins (p66shc/CypD-/-) exhibited decreased pulmonary vascular resistance (PVR) compared to wild-type mice determined in isolated lungs and in vivo. In contrast, systemic arterial pressure was only lower in CypD-/- mice. As cardiac function and pulmonary vascular remodelling did not differ between genotypes, we determined alterations of vascular contractility in isolated lungs and calcium handling in pulmonary arterial smooth muscle cells (PASMC) as underlying reason for decreased PVR. Potassium chloride (KCl)-induced pulmonary vasoconstriction and KCl-induced cytosolic calcium increase determined by Fura-2 were attenuated in all gene-deficient mice. In contrast, KCl-induced mitochondrial calcium increase determined by the genetically encoded Mito-Car-GECO and calcium retention capacity were increased only in CypD-/- and p66shc/CypD-/- mitochondria indicating that decreased mPTP opening affected KCl-induced intracellular calcium peaks in these cells. All mouse strains showed a similar pulmonary vascular response to chronic hypoxia, while acute hypoxic pulmonary vasoconstriction was decreased in gene-deficient mice indicating that CypD and p66shc regulates vascular contractility but not remodelling. Conclusions We conclude that p66shc specifically regulates the pulmonary vascular tone, while CypD also affects systemic pressure. However, only CypD acts via regulation of mPTP opening and mitochondrial calcium regulation. Translational Perspective Pulmonary hypertension is a progressive disease of the pulmonary vasculature ultimately resulting in right heart failure. Thus, therapeutic options targeting specifically the pulmonary vasculature are urgently needed. Our study describes for the first time the role of the proteins p66shc and CypD in the regulation of the pulmonary vascular tone. As the effect of p66shc-/- was specific for the pulmonary vasculature, it is an interesting target for future research on therapies for pulmonary vascular diseases like pulmonary hypertension.