Natascha Sommer

Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms

Hypoxic pulmonary vasoconstriction (HPV), also known as the von Euler–Liljestrand mechanism, is a physiological response to alveolar hypoxia which distributes pulmonary capillary blood flow to alveolar areas of high oxygen partial pressure. Impairment of this mechanism may result in hypoxaemia. Under conditions of chronic hypoxia generalised vasoconstriction of the pulmonary vasculature in concert with hypoxia-induced vascular remodelling leads to pulmonary hypertension. Although the principle of HPV was recognised decades ago, its exact pathway still remains elusive. Neither the oxygen sensing process nor the exact pathway underlying HPV is fully deciphered yet. The effector pathway is suggested to include L-type calcium channels, nonspecific cation channels and voltage-dependent potassium channels, whereas mitochondria and nicotinamide adenine dinucleotide phosphate oxidases are discussed as oxygen sensors. Reactive oxygen species, redox couples and adenosine monophosphate-activated kinases are under investigation as mediators of hypoxic pulmonary vasoconstriction. Moreover, the role of calcium sensitisation, intracellular calcium stores and direction of change of reactive oxygen species is still under debate. In this context the present article focuses on the basic mechanisms of hypoxic pulmonary vasoconstriction and also outlines differences in current concepts that have been suggested for the regulation of hypoxic pulmonary vasoconstriction.

Elevated CO2 Levels Cause Mitochondrial Dysfunction and Impair Cell Proliferation

Background: Cells are exposed to elevated levels of CO2 (hypercapnia) in many diseases. Results: Hypercapnia decreased cell proliferation, which was prevented with α-ketoglutarate, IDH2 overexpression, and microRNA-183 inhibition. Conclusion: Hypercapnia causes mitochondrial dysfunction by up-regulation of microRNA-183, which decreases the levels of IDH2. Significance: Hypercapnia causes mitochondrial dysfunction, which is relevant for patients with lung diseases. Elevated CO2 concentrations (hypercapnia) occur in patients with severe lung diseases. Here, we provide evidence that high CO2 levels decrease O2 consumption and ATP production and impair cell proliferation independently of acidosis and hypoxia in fibroblasts (N12) and alveolar epithelial cells (A549). Cells exposed to elevated CO2 died in galactose medium as well as when glucose-6-phosphate isomerase was knocked down, suggesting mitochondrial dysfunction. High CO2 levels led to increased levels of microRNA-183 (miR-183), which in turn decreased expression of IDH2 (isocitrate dehydrogenase 2). The high CO2-induced decrease in cell proliferation was rescued by α-ketoglutarate and overexpression of IDH2, whereas proliferation decreased in normocapnic cells transfected with siRNA for IDH2. Also, overexpression of miR-183 decreased IDH2 (mRNA and protein) as well as cell proliferation under normocapnic conditions, whereas inhibition of miR-183 rescued the normal proliferation phenotype in cells exposed to elevated levels of CO2. Accordingly, we provide evidence that high CO2 induces miR-183, which down-regulates IDH2, thus impairing mitochondrial function and cell proliferation. These results are of relevance to patients with hypercapnia such as those with chronic obstructive pulmonary disease, asthma, cystic fibrosis, bronchopulmonary dysplasia, and muscular dystrophies.

Oxygen-dependent expression of cytochrome c oxidase subunit 4-2 gene expression is mediated by transcription factors RBPJ, CXXC5 and CHCHD2

Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded by both mitochondrial and nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 and the tissue-specific (predominantly lung) isoform 2 (COX4I2). COX4I2 renders lung COX about 2-fold more active compared with liver COX, which lacks COX4I2. We previously identified a highly conserved 13-bp sequence in the proximal promoter of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at a 4% oxygen concentration. Here, we have identified three transcription factors that bind this conserved ORE, namely recombination signal sequence–binding protein Jκ (RBPJ), coiled-coil-helix-coiled-coil-helix domain 2 (CHCHD2) and CXXC finger protein 5 (CXXC5). We demonstrate that RBPJ and CHCHD2 function towards activating the ORE at 4% oxygen, whereas CXXC5 functions as an inhibitor. To validate results derived from cultured cells, we show using RNA interference a similar effect of these transcription factors in the gene regulation of COX4I2 in primary pulmonary arterial smooth muscle cells. Depending on the oxygen tension, a concerted action of the three transcription factors regulates the expression of COX4I2 that, as we discuss, could augment both COX activity and its ability to cope with altered cellular energy requirements.

Effects of hypercapnia with and without acidosis on hypoxic pulmonary vasoconstriction.

Acute respiratory disorders and permissive hypercapnic strategy may lead to alveolar hypoxia and hypercapnic acidosis. However, the effects of hypercapnia with or without acidosis on hypoxic pulmonary vasoconstriction (HPV) and oxygen diffusion capacity of the lung are controversial. We investigated the effects of hypercapnic acidosis and hypercapnia with normal pH (pH corrected with sodium bicarbonate) on HPV, capillary permeability, gas exchange, and ventilation-perfusion matching in the isolated ventilated-perfused rabbit lung. No alteration in vascular tone was noted during normoxic hypercapnia with or without acidosis compared with normoxic normocapnia. Hypercapnia with normal pH resulted in a transient increase in HPV during the course of consecutive ventilation maneuvers, whereas hypercapnic acidosis increased HPV over time. Hypercapnic acidosis decreased exhaled NO during hypoxia more than hypercapnia with normal pH and normocapnia, whereas intravascular NO release was unchanged. However, inhibition of NO synthesis by nitro-L-arginine (L-NNA) resulted in a loss of the increased HPV caused by hypercapnic acidosis but not that caused by hypercapnia with normal pH. Furthermore, capillary permeability increased during hypoxic hypercapnia with normal pH but not hypoxic hypercapnic acidosis. This effect was NO-dependent because it disappeared during L-NNA administration. Ventilation-perfusion matching and arterial PO2 were improved according to the strength of HPV in hypercapnia compared with normocapnia during Tween nebulization-induced lung injury. In conclusion, the increased HPV during hypercapnic acidosis is beneficial to lung gas exchange by improving ventilation-perfusion matching and preserving the capillary barrier function. These effects seem to be linked to NO-mediated pathways.

Redox signaling and reactive oxygen species in hypoxic pulmonary vasoconstriction

Hypoxic pulmonary vasoconstriction (HPV) is an essential physiological mechanism of the lung that matches blood perfusion with alveolar ventilation to optimize gas exchange. Perturbations of HPV, as may occur in pneumonia or adult respiratory distress syndrome, can cause life-threatening hypoxemia. Despite intensive research for decades, the molecular mechanisms of HPV have not been fully elucidated. Reactive oxygen species (ROS) and changes in the cellular redox state are proposed to link O2 sensing and pulmonary arterial smooth muscle cell contraction underlying HPV. In this regard, mitochondria and NAD(P)H oxidases are discussed as sources of ROS. However, there is controversy whether ROS levels decrease or increase during hypoxia. With this background we summarize the current knowledge on the role of ROS and redox state in HPV.

Mitochondrial Hyperpolarization in Pulmonary Vascular Remodeling. Mitochondrial Uncoupling Protein Deficiency as Disease Model

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.

Cytochrome c oxidase subunit 4 isoform 2-knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology

Cytochrome c oxidase (COX) is the terminal enzyme of the mitochondrial electron transport chain. The purpose of this study was to analyze the function of lung‐specific cytochrome c oxidase subunit 4 isoform 2 (COX4i2) in vitro and in COX4i2‐knockout mice in vivo. COX was isolated from cow lung and liver as control and functionally analyzed. COX4i2‐knockout mice were generated and the effect of the gene knockout was determined, including COX activity, tissue energy levels, noninvasive and invasive lung function, and lung pathology. These studies were complemented by a comprehensive functional screen performed at the German Mouse Clinic (Neuherberg, Germany). We show that isolated cow lung COX containing COX4i2 is about twice as active (88 and 102% increased activity in the presence of allosteric activator ADP and inhibitor ATP, respectively) as liver COX, which lacks COX4i2. In COX4i2‐knockout mice, lung COX activity and cellular ATP levels were significantly reduced (—50 and — 29%, respectively). Knockout mice showed decreased airway responsiveness (60% reduced Penh and 58% reduced airway resistance upon challenge with 25 and 100 mg methacholine, respectively), and they developed a lung pathology deteriorating with age that included the appearance of Charcot‐Leyden crystals. In addition, there was an interesting sex‐specific phenotype, in which the knockout females showed reduced lean mass (—12%), reduced total oxygen consumption rate (—8%), improved glucose tolerance, and reduced grip force (—14%) compared to wild‐type females. Our data suggest that high activity lung COX is a central determinant of airway function and is required for maximal airway responsiveness and healthy lung function. Since airway constriction requires energy, we propose a model in which reduced tissue ATP levels explain protection from airway hyperresponsiveness, i.e., absence of COX4i2 leads to reduced lung COX activity and ATP levels, which results in impaired airway constriction and thus reduced airway responsiveness; long‐term lung pathology develops in the knockout mice due to impairment of energy‐costly lung maintenance processes; and therefore, we propose mitochondrial oxidative phosphorylation as a novel target for the treatment of respiratory diseases, such as asthma.—Hüttemann, M., Lee, I., Gao, X., Pecina, P., Pecinova, A., Liu, J., Aras, S., Sommer, N., Sanderson, T. H., Tost, M., Neff, F., Aguilar‐Pimentel, J. A., Becker, L., Naton, B., Rathkolb, B., Rozman, J., Favor, J., Hans, W., Prehn, C., Puk, O., Schrewe, A., Sun, M., Höfler, H., Adamski, J., Bekeredjian, R., Graw, J., Adler, T., Busch, D. H., Klingenspor, M., Klopstock, T., Ollert, M., Wolf, E., Fuchs, H., Gailus‐Durner, V., Hrabě de Angelis, M., Weissmann, N., Doan, J. W., Bassett, D. J. P., Grossman, L. I. Cytochrome c oxidase subunit 4 isoform 2‐knockout mice show reduced enzyme activity, airway hyporeactivity, and lung pathology. FASEB J. 26, 3916–3930 (2012). www.fasebj.org

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.

Oxygen sensing and signal transduction in hypoxic pulmonary vasoconstriction.

Hypoxic pulmonary vasoconstriction (HPV), also known as the von Euler–Liljestrand mechanism, is an essential response of the pulmonary vasculature to acute and sustained alveolar hypoxia. During local alveolar hypoxia, HPV matches perfusion to ventilation to maintain optimal arterial oxygenation. In contrast, during global alveolar hypoxia, HPV leads to pulmonary hypertension. The oxygen sensing and signal transduction machinery is located in the pulmonary arterial smooth muscle cells (PASMCs) of the pre-capillary vessels, albeit the physiological response may be modulated in vivo by the endothelium. While factors such as nitric oxide modulate HPV, reactive oxygen species (ROS) have been suggested to act as essential mediators in HPV. ROS may originate from mitochondria and/or NADPH oxidases but the exact oxygen sensing mechanisms, as well as the question of whether increased or decreased ROS cause HPV, are under debate. ROS may induce intracellular calcium increase and subsequent contraction of PASMCs via direct or indirect interactions with protein kinases, phospholipases, sarcoplasmic calcium channels, transient receptor potential channels, voltage-dependent potassium channels and L-type calcium channels, whose relevance may vary under different experimental conditions. Successful identification of factors regulating HPV may allow development of novel therapeutic approaches for conditions of disturbed HPV. ROS originating from mitochondria and/or NADPH oxidases may initiate HPV but exact signalling mechanisms are unclear http://ow.ly/SkaGC

Mitochondrial cytochrome redox states and respiration in acute pulmonary oxygen sensing

Hypoxic pulmonary vasoconstriction (HPV) is an essential mechanism to optimise lung gas exchange. We aimed to decipher the proposed oxygen sensing mechanism of mitochondria in HPV. Cytochrome redox state was assessed by remission spectrophotometry in intact lungs and isolated pulmonary artery smooth muscle cells (PASMC). Mitochondrial respiration was quantified by high-resolution respirometry. Alterations were compared with HPV and hypoxia-induced functional and molecular readouts on the cellular level. Aortic and renal arterial smooth muscle cells (ASMC and RASMC, respectively) served as controls. The hypoxia-induced decrease of mitochondrial respiration paralleled HPV in isolated lungs. In PASMC, reduction of respiration and mitochondrial cytochrome c and aa3 (complex IV), but not of cytochrome b (complex III) matched an increase in matrix superoxide levels as well as mitochondrial membrane hyperpolarisation with subsequent cytosolic calcium increase. In contrast to PASMC, RASMC displayed a lower decrease in respiration and no rise in superoxide, membrane potential or intracellular calcium. Pharmacological inhibition of mitochondria revealed analogous kinetics of cytochrome redox state and strength of HPV. Our data suggest inhibition of complex IV as an essential step in mitochondrial oxygen sensing of HPV. Concomitantly, increased superoxide release from complex III and mitochondrial membrane hyperpolarisation may initiate the cytosolic calcium increase underlying HPV.