Oleg Pak

The effects of hypoxia on the cells of the pulmonary vasculature

Pulmonary hypertension is associated with remodelling of pulmonary vessels. Chronic hypoxia is a common cause of pulmonary hypertension and pulmonary vascular remodelling. Vascular remodelling is characterised largely by fibroblast, smooth muscle and endothelial cell proliferation, which results in lumen obliteration. Chronic hypoxia elicits expression of mitogens, growth factors and cytokines by fibroblasts and endothelial cells, and also the suppression of endothelial nitric oxide synthase. Although hypoxic pulmonary vascular remodelling is associated with medial hypertrophy, many in vitro studies have found that hypoxia does not lead to a direct increase in smooth muscle cell proliferation. This paradox is not well understood and this review aims to examine the various reasons why this might be so. The present authors reviewed data from in vitro studies and also considered whether hypoxia could act on adjacent cells such as fibroblasts and endothelial cells to trigger smooth muscle cell proliferation. It is possible that hypoxia is sensed by fibroblasts, endothelial cells, or both, and relayed to adjacent pulmonary artery smooth muscle cells by intercellular signalling, causing proliferation. The present article reviews the data from in vitro studies of hypoxia on the three cellular components of the pulmonary vascular wall, namely endothelial cells, smooth muscle cells and fibroblasts.

Activation of TRPC6 Channels Is Essential for Lung Ischaemia–Reperfusion Induced Oedema in Mice

Lung ischaemia–reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/−) or the classical transient receptor potential channel 6 (TRPC6−/−) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2y/− cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE.

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates K(v) channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.

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.

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

Hypoxia-Dependent Reactive Oxygen Species Signaling in the Pulmonary Circulation: Focus on Ion Channels

SIGNIFICANCE An acute lack of oxygen in the lung causes hypoxic pulmonary vasoconstriction, which optimizes gas exchange. In contrast, chronic hypoxia triggers a pathological vascular remodeling causing pulmonary hypertension, and ischemia can cause vascular damage culminating in lung edema. RECENT ADVANCES Regulation of ion channel expression and gating by cellular redox state is a widely accepted mechanism; however, it remains a matter of debate whether an increase or a decrease in reactive oxygen species (ROS) occurs under hypoxic conditions. Ion channel redox regulation has been described in detail for some ion channels, such as Kv channels or TRPC6. However, in general, information on ion channel redox regulation remains scant. CRITICAL ISSUES AND FUTURE DIRECTIONS In addition to the debate of increased versus decreased ROS production during hypoxia, we aim here at describing and deciphering why different oxidants, under different conditions, can cause both activation and inhibition of channel activity. While the upstream pathways affecting channel gating are often well described, we need a better understanding of redox protein modifications to be able to determine the complexity of ion channel redox regulation. Against this background, we summarize the current knowledge on hypoxia-induced ROS-mediated ion channel signaling in the pulmonary circulation.

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.

Mitochondrial Complex IV Subunit 4 Isoform 2 Is Essential for Acute Pulmonary Oxygen Sensing

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 a critical step in the signaling pathway underlying HPV. However, the identity of the primary oxygen sensor and the mechanism of superoxide release in acute hypoxia, as well as its relevance for chronic pulmonary oxygen sensing, remain unresolved. Objectives: To investigate the role of the pulmonary-specific isoform 2 of subunit 4 of the 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 PASMCs was absent in Cox4i2−/− PASMCs and was dependent on cysteine residues of Cox4i2. HPV could be inhibited by mitochondrial superoxide inhibitors proving the functional relevance of superoxide release for HPV. Mitochondrial hyperpolarization, which can promote mitochondrial superoxide release, was detected during acute hypoxia in wild-type but not Cox4i2−/− PASMCs. Downstream signaling determined by patch-clamp measurements showed decreased hypoxia-induced cellular membrane depolarization in Cox4i2−/− PASMCs compared with wild-type PASMCs, which could be normalized by the 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. Conclusions: 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.

Impact of Short- and Medium-Chain Fatty Acids on Mitochondrial Function in Severe Inflammation

BACKGROUND Sepsis is a severe inflammatory disorder with a high mortality in intensive care units mostly due to multiorgan failure. Mitochondrial dysfunction is regarded as a key factor involved in the pathogenesis of septic disorders, leading to a decline in energy supply. The aim of the present study was to evaluate whether application of short-chain fatty acids (SCFAs) and medium-chain fatty acids (MCFAs) could improve mitochondrial function and thus might serve as a potential energy source under inflammatory conditions. MATERIALS AND METHODS As an experimental approach, starved human endothelial cells and monocytes were incubated with hexanoic acid, heptanoic acid, octanoic acid, or glucose and subsequently subjected to high-resolution respirometry to assess mitochondrial function under baseline conditions. In a second set of experiments, cells were pretreated with tumor necrosis factor-α to mimic inflammation and sepsis. RESULTS We demonstrated that addition of SCFAs and MCFAs increases mitochondrial respiratory capacity at baseline and inflammatory conditions in both cell types. None of the fatty acids induced changes in mitochondrial DNA content or the generation of proinflammatory cytokines, indicating a beneficial safety profile. CONCLUSION We deduce that SCFAs and MCFAs are suitable and safe sources of energy under inflammatory conditions with the capability to partly restore mitochondrial respiration.

Rhodiola: an ordinary plant or a promising future therapy for pulmonary hypertension? a brief review

Pulmonary hypertension (PH) is a chronic, complex, and progressive disease that eventuates in fatality. Research efforts over the past decades have resulted in therapeutic options that improve quality of life and prolong survival of patients, but they do not offer a cure. We propose a philosophical model that a disturbed balance of yin and yang results in pulmonary vascular remodeling, the hallmark of PH pathology. The model may be useful in exploring the wisdom of traditional Chinese medicine and incorporating it into mainstream PH research. In this context, the medicinal plant Rhodiola can be of profound interest owing to its variety of health-friendly attributes. Rhodiola has been shown to be beneficial in high-altitude-related symptoms and acute exacerbation of PH; moreover, improvement of PH has been demonstrated experimentally in chronically hypoxic rats. The beneficial effects of Rhodiola in PH may be attributable to its potential targeting of the signaling pathways, such as endothelin-1, nitric oxide, vascular endothelial growth factor, angiotensin-converting enzyme, nuclear factor κ-B, tumor necrosis factor α, and interleukin-6. Alterations in these mediators are implicated in PH pathogenesis, the characteristics of which include chronic pulmonary vasoconstriction, vasoproliferation, and vascular inflammation. Salidroside, one of the compounds extracted from Rhodiola, has been found to provide therapeutic benefits in experimental PH. As the data are limited and the field is in its infancy, further studies including in-depth analysis of the therapeutic effects on various animal models of PH are desirable. We believe that future PH research should place an adequate and special emphasis on exploring and promoting the potential of traditional Chinese medicine, and to this end, the medicinal plant Rhodiola offers a promising field on which to embark.