Contribution of Mitochondrial Reactive Oxygen Species to Chronic Hypoxia-Induced Pulmonary Hypertension.

Pulmonary hypertension (PH) resulting from chronic hypoxia (CH) occurs in patients with chronic obstructive pulmonary diseases, sleep apnea, and restrictive lung diseases, as well as in residents at high altitude. Previous studies from our group and others demonstrate a detrimental role of reactive oxygen species (ROS) in the pathogenesis of CH-induced PH, although the subcellular sources of ROS are not fully understood. We hypothesized that mitochondria-derived ROS (mtROS) contribute to enhanced vasoconstrictor reactivity and PH following CH. To test the hypothesis, we exposed rats to 4 weeks of hypobaric hypoxia (PB ≈ 380 mmHg), with control rats housed in ambient air (PB ≈ 630 mmHg). Chronic oral administration of the mitochondria-targeted antioxidant MitoQ attenuated CH-induced decreases in pulmonary artery (PA) acceleration time, increases in right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary arterial remodeling. In addition, endothelium-intact PAs from CH rats exhibited a significantly greater basal tone compared to those from control animals, as was eliminated via MitoQ. CH also augmented the basal tone in endothelium-disrupted PAs, a response associated with increased mtROS production in primary PA smooth muscle cells (PASMCs) from CH rats. However, we further uncovered an effect of NO synthase inhibition with Nω-nitro-L-arginine (L-NNA) to unmask a potent endothelial vasoconstrictor influence that accentuates mtROS-dependent vasoconstriction following CH. This basal tone augmentation in the presence of L-NNA disappeared following combined endothelin A and B receptor blockade with BQ123 and BQ788. The effects of using CH to augment vasoconstriction and PASMC mtROS production in exogenous endothelin 1 (ET-1) were similarly prevented by MitoQ. We conclude that mtROS participate in the development of CH-induced PH. Furthermore, mtROS signaling in PASMCs is centrally involved in enhanced pulmonary arterial constriction following CH, a response potentiated by endogenous ET-1.

Acid-sensing ion channel 1a activates IKCa/SKCa channels and contributes to endothelium-dependent dilation.

Acid-sensing ion channel 1a (ASIC1a) belongs to a novel family of proton-gated cation channels that are permeable to both Na+ and Ca2+. ASIC1a is expressed in vascular smooth muscle and endothelial cells in a variety of vascular beds, yet little is known regarding the potential impact of ASIC1a to regulate local vascular reactivity. Our previous studies in rat mesenteric arteries suggest ASIC1a does not contribute to agonist-induced vasoconstriction but may mediate a vasodilatory response. The objective of the current study is to determine the role of ASIC1a in systemic vasodilatory responses by testing the hypothesis that the activation of endothelial ASIC1a mediates vasodilation of mesenteric resistance arteries through an endothelium-dependent hyperpolarization (EDH)-related pathway. The selective ASIC1a antagonist psalmotoxin 1 (PcTX1) largely attenuated the sustained vasodilatory response to acetylcholine (ACh) in isolated, pressurized mesenteric resistance arteries and ACh-mediated Ca2+ influx in freshly isolated mesenteric endothelial tubes. Similarly, basal tone was enhanced and ACh-induced vasodilation blunted in mesenteric arteries from Asic1a knockout mice. ASIC1a colocalizes with intermediate- and small-conductance Ca2+-activated K+ channels (IKCa and SKCa, respectively), and the IKCa/SKCa-sensitive component of the ACh-mediated vasodilation was blocked by ASIC1a inhibition. To determine the role of ASIC1a to activate IKCa/SKCa channels, we measured whole-cell K+ currents using the perforated-patch clamp technique in freshly isolated mesenteric endothelial cells. Inhibition of ASIC1a prevented ACh-induced activation of IKCa/SKCa channels. The ASIC1 agonist, α/ß-MitTx, activated IKCa/SKCa channels and induced an IKCa/SKCa-dependent vasodilation. Together, the present study demonstrates that ASIC1a couples to IKCa/SKCa channels in mesenteric resistance arteries to mediate endothelium-dependent vasodilation.

Hydrogen sulfide and miR21 are suitable biomarkers of hypoxic exposure.

Hypoxia is the reduction of alveolar partial pressure of oxygen ([Formula: see text]). Military members and people who practice recreational activities from moderate to high altitudes are at risk for hypoxic exposure. Hypoxemia's signs and symptoms vary from asymptomatic to severe responses, such as excessive hypoxic ventilatory responses and residual neurobehavioral impairment. Therefore, it is essential to identify hypoxia-induced biomarkers to indicate people with exposure to hypoxia. Advances have been made in understanding physiological responses to hypoxia, including elevations in circulating levels of endothelin 1 (ET-1) and microRNA 21 (miR-21) and reduction in circulating levels of hydrogen sulfide (H2S). Although the levels of these factors change upon exposure to hypoxia, it is unclear if these changes are sustained on return to normoxia. We hypothesize that hypoxia-induced ET-1 and miR-21 remain elevated, whereas hypoxia-reduction in H2S sustains after returning to normoxic conditions. To test this hypothesis, we exposed male rats to 6 h of 12% O2 and measured circulating levels of ET-1 and miR-21, pre, during, and posthypoxia. We found that ET-1 plasma levels increased in response to hypoxia but returned to normal levels within 30 min after the restoration of normoxia. miR-21 plasma levels and transdermal H2S emissions decreased in response to hypoxia, remaining decreased on return to normoxia, thus following the biomarker criteria. Therefore, this study supports a unique role for plasma miR21 and transdermal H2S as hypoxia biomarkers that could be used to identify individuals after exposure to hypoxia.

Smooth muscle Acid-sensing ion channel 1a as a therapeutic target to reverse hypoxic pulmonary hypertension.

Acid-sensing ion channel 1a (ASIC1a) is a voltage-independent, non-selective cation channel that conducts both Na+ and Ca2+. Activation of ASIC1a elicits plasma membrane depolarization and stimulates intracellular Ca2+-dependent signaling pathways in multiple cell types, including vascular smooth muscle (SM) and endothelial cells (ECs). Previous studies have shown that increases in pulmonary vascular resistance accompanying chronic hypoxia (CH)-induced pulmonary hypertension requires ASIC1a to elicit enhanced pulmonary vasoconstriction and vascular remodeling. Both SM and EC dysfunction drive these processes; however, the involvement of ASIC1a within these different cell types is unknown. Using the Cre-LoxP system to generate cell-type-specific Asic1a knockout mice, we tested the hypothesis that SM-Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling, whereas EC-Asic1a opposes the development of CH-induced pulmonary hypertension. The severity of pulmonary hypertension was not altered in mice with specific deletion of EC-Asic1a (TekCre-Asic1a fl/fl). However, similar to global Asic1a knockout (Asic1a -/-) mice, mice with specific deletion of SM-Asic1a (MHCCreER-Asic1a fl/fl) were protected from the development of CH-induced pulmonary hypertension and right heart hypertrophy. Furthermore, pulmonary hypertension was reversed when deletion of SM-Asic1a was initiated in conditional MHCCreER-Asic1a fl/fl mice with established pulmonary hypertension. CH-induced vascular remodeling was also significantly attenuated in pulmonary arteries from MHCCreER-Asic1a fl/fl mice. These findings were additionally supported by decreased CH-induced proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) from Asic1a -/- mice. Together these data demonstrate that SM-, but not EC-Asic1a contributes to CH-induced pulmonary hypertension and vascular remodeling. Furthermore, these studies provide evidence for the therapeutic potential of ASIC1a inhibition to reverse pulmonary hypertension.

Acid-sensing ion channel 1 contributes to pulmonary arterial smooth muscle cell depolarization following hypoxic pulmonary hypertension.

Pulmonary hypertension is characterized by sustained vasoconstriction and remodelling of the small pulmonary arteries, which is associated with persistent depolarization of the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs). It is well-known that the underlying mechanism of this depolarization includes inhibition of K+ channels; however, whether other ion channels contribute to this depolarization is unknown. We previously reported that acid-sensing ion channel 1 (ASIC1), a non-selective cation channel (NSCC) that conducts both Na+ and Ca2+ , is present in PASMCs and contributes to the development of chronic hypoxia (CH)-induced pulmonary hypertension. Therefore, we tested the hypothesis that ASIC1-mediated Na+ influx contributes to PASMC Em regulation following CH-induced pulmonary hypertension. Using sharp electrode intracellular recordings in isolated, pressurized small pulmonary arteries from rats and mice, we show that exposure to CH leads to PASMC membrane depolarization compared with control animals, and this is independent of intraluminal pressure-induced depolarization. In addition to a decrease in PASMC whole-cell K+ currents following CH, we demonstrate that whole-cell NSCC currents are increased and essential to the persistent CH-induced Em depolarization in PASMCs. Both the specific inhibitor of ASIC1, psalmotoxin 1, and global knockout of ASIC1 (Asic1-/- ) prevents CH-induced Em depolarization and largely inhibits whole-cell NSCC currents, without affecting whole-cell K+ currents. Our results show a combination of factors, including inhibition of K+ efflux and augmented Na+ influx, mediate CH-induced PASMC depolarization. Furthermore, this study demonstrates a novel role for ASIC1 in the regulation of Em in PASMCs during CH-induced pulmonary hypertension. KEY POINTS: In pulmonary hypertensive patients and animal models of pulmonary hypertension, the resting membrane potential (Em ) of pulmonary arterial smooth muscle cells (PASMCs) is persistently depolarized. In addition to the well-established reduction of K+ conductance, we show that non-selective cation channel currents are increased and essential to the persistent Em depolarization in PASMCs following chronic hypoxia (CH)-induced pulmonary hypertension. The current study provides novel evidence that acid-sensing ion channel 1 (ASIC1)-mediated Na+ influx induces membrane depolarization and regulates Em in PASMCs following CH exposure. Although fairly quiescent under control conditions, our findings demonstrate a pathological function of ASIC1 in the development of chronic hypoxia-induced pulmonary hypertension.

Membrane depolarization is required for pressure-dependent pulmonary arterial tone but not enhanced vasoconstriction to endothelin-1 following chronic hypoxia.

Enhanced vasoconstriction is increasingly identified as an important contributor to the development of pulmonary hypertension. Chronic hypoxia results in enhanced Rho kinase mediated Ca2+ sensitization contributing to pressure-dependent pulmonary arterial tone as well as augmented vasoconstriction to endothelin-1 and depolarizing stimuli. We sought to investigate the interaction between these vasoconstrictor stimuli in isolated, pressurized, pulmonary arteries. We used the K+ ionophore, valinomycin, to clamp membrane potential (Vm) to investigate the role of membrane depolarization in endothelin-1 and pressure-dependent constriction, and endothelin-1 receptor inhibitors to determine whether membrane depolarization or stretch signal through endothelin-1 receptors. Clamping Vm prevented pressure-dependent tone, but not enhanced vasoconstriction to endothelin-1 following chronic hypoxia. Furthermore, endothelin-1 receptor inhibition had no effect on either pressure-dependent tone or vasoconstriction to KCl. As Src kinases contribute to both pressure-dependent tone and enhanced endothelin-1 vasoconstriction following chronic hypoxia, we further investigated their role in depolarization-induced vasoconstriction. Inhibition of Src kinases attenuated enhanced vasoconstriction to KCl. We conclude that membrane depolarization contributes to pressure-dependent tone but not enhanced vasoconstriction to ET-1, and that Src kinases serve as upstream mediators facilitating enhanced Rho kinase-dependent vasoconstriction following chronic hypoxia.

Vasoconstrictor Mechanisms in Chronic Hypoxia-Induced Pulmonary Hypertension: Role of Oxidant Signaling.

Elevated resistance of pulmonary circulation after chronic hypoxia exposure leads to pulmonary hypertension. Contributing to this pathological process is enhanced pulmonary vasoconstriction through both calcium-dependent and calcium sensitization mechanisms. Reactive oxygen species (ROS), as a result of increased enzymatic production and/or decreased scavenging, participate in augmentation of pulmonary arterial constriction by potentiating calcium influx as well as activation of myofilament sensitization, therefore mediating the development of pulmonary hypertension. Here, we review the effects of chronic hypoxia on sources of ROS within the pulmonary vasculature including NADPH oxidases, mitochondria, uncoupled endothelial nitric oxide synthase, xanthine oxidase, monoamine oxidases and dysfunctional superoxide dismutases. We also summarize the ROS-induced functional alterations of various Ca2+ and K+ channels involved in regulating Ca2+ influx, and of Rho kinase that is responsible for myofilament Ca2+ sensitivity. A variety of antioxidants have been shown to have beneficial therapeutic effects in animal models of pulmonary hypertension, supporting the role of ROS in the development of pulmonary hypertension. A better understanding of the mechanisms by which ROS enhance vasoconstriction will be useful in evaluating the efficacy of antioxidants for the treatment of pulmonary hypertension.

NFATc3 regulation of collagen V expression contributes to cellular immunity to collagen type V and hypoxic pulmonary hypertension.

Chronic hypoxia (CH)-induced pulmonary hypertension (PH) results, in part, from T helper-17 (TH17) cell-mediated perivascular inflammation. However, the antigen(s) involved is unknown. Cellular immunity to collagen type V (col V) develops after ischemia-reperfusion injury during lung transplant and is mediated by naturally occurring (n)TH17 cells. Col5a1 gene codifies for the α1-helix of col V, which is normally hidden from the immune system within type I collagen in the extracellular matrix. COL5A1 promoter analysis revealed nuclear factor of activated T cells, cytoplasmic 3 (NFATc3) binding sites. Therefore, we hypothesized that smooth muscle NFATc3 upregulates col V expression, leading to nTH17 cell-mediated autoimmunity to col V in response to CH, representing an upstream mechanism in PH development. To test our hypothesis, we measured indexes of PH in inducible smooth muscle cell (SMC)-specific NFATc3 knockout (KO) mice exposed to either CH (380 mmHg) or normoxia and compared them with wild-type (WT) mice. KO mice did not develop PH. In addition, COL5A1 was one of the 1,792 genes differentially affected by both CH and SMC NFATc3 in isolated intrapulmonary arteries, which was confirmed by RT-PCR and immunostaining. Cellular immunity to col V was determined using a trans vivo delayed-type hypersensitivity assay (Tv-DTH). Tv-DTH response was evident only when splenocytes were used from control mice exposed to CH but not from KO mice, and mediated by nTH17 cells. Our results suggest that SMC NFATc3 is important for CH-induced PH in adult mice, in part, by regulating the expression of the lung self-antigen COL5A1 protein contributing to col V-reactive nTH17-mediated inflammation and hypertension.

Coupling of store-operated calcium entry to vasoconstriction is acid-sensing ion channel 1a dependent in pulmonary but not mesenteric arteries.

Although voltage-gated Ca2+ channels (VGCC) are a major Ca2+ entry pathway in vascular smooth muscle cells (VSMCs), several other Ca2+-influx mechanisms exist and play important roles in vasoreactivity. One of these is store-operated Ca2+ entry (SOCE), mediated by an interaction between STIM1 and Orai1. Although SOCE is an important mechanism of Ca2+ influx in non-excitable cells (cells that lack VGCC); there is debate regarding the contribution of SOCE to regulate VSMC contractility and the molecular components involved. Our previous data suggest acid-sensing ion channel 1a (ASIC1a) is a necessary component of SOCE and vasoconstriction in small pulmonary arteries. However, it is unclear if ASIC1a similarly contributes to SOCE and vascular reactivity in systemic arteries. Considering the established role of Orai1 in mediating SOCE in the systemic circulation, we hypothesize the involvement of ASIC1a in SOCE and resultant vasoconstriction is unique to the pulmonary circulation. To test this hypothesis, we examined the roles of Orai1 and ASIC1a in SOCE- and endothelin-1 (ET-1)-induced vasoconstriction in small pulmonary and mesenteric arteries. We found SOCE is coupled to vasoconstriction in pulmonary arteries but not mesenteric arteries. In pulmonary arteries, inhibition of ASIC1a but not Orai1 attenuated SOCE- and ET-1-induced vasoconstriction. However, neither inhibition of ASIC1a nor Orai1 altered ET-1-induced vasoconstriction in mesenteric arteries. We conclude that SOCE plays an important role in pulmonary, but not mesenteric, vascular reactivity. Furthermore, in contrast to the established role of Orai1 in SOCE in non-excitable cells, the SOCE response in pulmonary VSMCs is largely mediated by ASIC1a.

Intermittent Hypoxia Augments Pulmonary Vasoconstrictor Reactivity through PKCß/Mitochondrial Oxidant Signaling.

Pulmonary vasoconstriction resulting from intermittent hypoxia (IH) contributes to pulmonary hypertension (pHTN) in patients with sleep apnea (SA), although the mechanisms involved remain poorly understood. Based on prior studies in patients with SA and animal models of SA, the objective of this study was to evaluate the role of PKCß and mitochondrial reactive oxygen species (mitoROS) in mediating enhanced pulmonary vasoconstrictor reactivity after IH. We hypothesized that PKCß mediates vasoconstriction through interaction with the scaffolding protein PICK1 (protein interacting with C kinase 1), activation of mitochondrial ATP-sensitive potassium channels (mitoKATP), and stimulated production of mitoROS. We further hypothesized that this signaling axis mediates enhanced vasoconstriction and pHTN after IH. Rats were exposed to IH or sham conditions (7 h/d, 4 wk). Chronic oral administration of the antioxidant Tempol or the PKCß inhibitor LY-333531 abolished IH-induced increases in right ventricular systolic pressure and right ventricular hypertrophy. Furthermore, scavengers of O2- or mitoROS prevented enhanced PKCß-dependent vasoconstrictor reactivity to endothelin-1 in pulmonary arteries from IH rats. In addition, this PKCß/mitoROS signaling pathway could be stimulated by the PKC activator PMA in pulmonary arteries from control rats, and in both rat and human pulmonary arterial smooth muscle cells. These responses to PMA were attenuated by inhibition of mitoKATP or PICK1. Subcellular fractionation and proximity ligation assays further demonstrated that PKCß acutely translocates to mitochondria upon stimulation and associates with PICK1. We conclude that a PKCß/mitoROS signaling axis contributes to enhanced vasoconstriction and pHTN after IH. Furthermore, PKCß mediates pulmonary vasoconstriction through interaction with PICK1, activation of mitoKATP, and subsequent mitoROS generation.

Altered Lipid Domains Facilitate Enhanced Pulmonary Vasoconstriction after Chronic Hypoxia.

Chronic hypoxia (CH) augments depolarization-induced pulmonary vasoconstriction through superoxide-dependent, Rho kinase-mediated Ca2+ sensitization. Nicotinamide adenine dinucleotide phosphate oxidase and EGFR (epidermal growth factor receptor) signaling contributes to this response. Caveolin-1 regulates the activity of a variety of proteins, including EGFR and nicotinamide adenine dinucleotide phosphate oxidase, and membrane cholesterol is an important regulator of caveolin-1 protein interactions. We hypothesized that derangement of these membrane lipid domain components augments depolarization-induced Ca2+ sensitization and resultant vasoconstriction after CH. Although exposure of rats to CH (4 wk, ∼380 mm Hg) did not alter caveolin-1 expression in intrapulmonary arteries or the incidence of caveolae in arterial smooth muscle, CH markedly reduced smooth muscle membrane cholesterol content as assessed by filipin fluorescence. Effects of CH on vasoreactivity and superoxide generation were examined using pressurized, Ca2+-permeabilized, endothelium-disrupted pulmonary arteries (∼150 µm inner diameter) from CH and control rats. Depolarizing concentrations of KCl evoked greater constriction in arteries from CH rats than in those obtained from control rats, and increased superoxide production as assessed by dihydroethidium fluorescence only in arteries from CH rats. Both cholesterol supplementation and the caveolin-1 scaffolding domain peptide antennapedia-Cav prevented these effects of CH, with each treatment restoring membrane cholesterol in CH arteries to control levels. Enhanced EGF-dependent vasoconstriction after CH similarly required reduced membrane cholesterol. However, these responses to CH were not associated with changes in EGFR expression or activity, suggesting that cholesterol regulates this signaling pathway downstream of EGFR. We conclude that alterations in membrane lipid domain signaling resulting from reduced cholesterol content facilitate enhanced depolarization- and EGF-induced pulmonary vasoconstriction after CH.

PKCß and reactive oxygen species mediate enhanced pulmonary vasoconstrictor reactivity following chronic hypoxia in neonatal rats.

Reactive oxygen species (ROS), mitochondrial dysfunction, and excessive vasoconstriction are important contributors to chronic hypoxia (CH)-induced neonatal pulmonary hypertension. On the basis of evidence that PKCß and mitochondrial oxidative stress are involved in several cardiovascular and metabolic disorders, we hypothesized that PKCß and mitochondrial ROS (mitoROS) signaling contribute to enhanced pulmonary vasoconstriction in neonatal rats exposed to CH. To test this hypothesis, we examined effects of the PKCß inhibitor LY-333,531, the ROS scavenger 1-oxyl-2,2,6,6-tetramethyl-4-hydroxypiperidine (TEMPOL), and the mitochondrial antioxidants mitoquinone mesylate (MitoQ) and (2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl)triphenylphosphonium chloride (MitoTEMPO) on vasoconstrictor responses in saline-perfused lungs (in situ) or pressurized pulmonary arteries from 2-wk-old control and CH (12-day exposure, 0.5 atm) rats. Lungs from CH rats exhibited greater basal tone and vasoconstrictor sensitivity to 9,11-dideoxy-9α,11α-methanoepoxy prostaglandin F2α (U-46619). LY-333,531 and TEMPOL attenuated these effects of CH, while having no effect in lungs from control animals. Basal tone was similarly elevated in isolated pulmonary arteries from neonatal CH rats compared with control rats, which was inhibited by both LY-333,531 and mitochondria-targeted antioxidants. Additional experiments assessing mitoROS generation with the mitochondria-targeted ROS indicator MitoSOX revealed that a PKCß-mitochondrial oxidant signaling pathway can be pharmacologically stimulated by the PKC activator phorbol 12-myristate 13-acetate in primary cultures of pulmonary artery smooth muscle cells (PASMCs) from control neonates. Finally, we found that neonatal CH increased mitochondrially localized PKCß in pulmonary arteries as assessed by Western blotting of subcellular fractions. We conclude that PKCß activation leads to mitoROS production in PASMCs from neonatal rats. Furthermore, this signaling axis may account for enhanced pulmonary vasoconstrictor sensitivity following CH exposure.NEW & NOTEWORTHY This research demonstrates a novel contribution of PKCß and mitochondrial reactive oxygen species signaling to increased pulmonary vasoconstrictor reactivity in chronically hypoxic neonates. The results provide a potential mechanism by which chronic hypoxia increases both basal and agonist-induced pulmonary arterial smooth muscle tone, which may contribute to neonatal pulmonary hypertension.

Augmented Pulmonary Vasoconstrictor Reactivity after Chronic Hypoxia Requires Src Kinase and Epidermal Growth Factor Receptor Signaling.

Chronic hypoxia augments pressure- and agonist-induced pulmonary vasoconstriction through myofilament calcium sensitization. NADPH oxidases contribute to the development of pulmonary hypertension, and both epidermal growth factor receptor and Src kinases can regulate NADPH oxidase. We tested the hypothesis that Src-epidermal growth factor receptor (EGFR) signaling mediates enhanced vasoconstrictor sensitivity after chronic hypoxia through NADPH oxidase-derived superoxide generation. Protocols employed pharmacological inhibitors in isolated, pressurized rat pulmonary arteries to examine the contribution of a variety of signaling moieties to enhanced vascular tone after chronic hypoxia. Superoxide generation in pulmonary arterial smooth muscle cells was assessed using the fluorescent indicator dihydroethidium. Indices of pulmonary hypertension were measured in rats treated with the EGFR inhibitor gefitinib. Inhibition of NADPH oxidase, Rac1 (Ras-related C3 botulinum toxin substrate 1), and EGFR abolished pressure-induced pulmonary arterial tone and endothelin-1 (ET-1)-dependent calcium sensitization and vasoconstriction after chronic hypoxia. Consistently, chronic hypoxia augmented ET-1-induced superoxide production through EGFR signaling, and rats treated chronically with gefitinib displayed reduced right ventricular pressure and diminished arterial remodeling. Src kinases were also activated by ET-1 after chronic hypoxia and contributed to enhanced basal arterial tone and vasoconstriction in response to ET-1. A role for matrix metalloproteinase 2 to mediate Src-dependent EGFR activation is further supported by our findings. Our studies support a novel role for an Src kinase-EGFR-NADPH oxidase signaling axis to mediate enhanced pulmonary vascular smooth muscle Ca2+ sensitization, vasoconstriction, and pulmonary hypertension after chronic hypoxia.

Loss of acid-sensing ion channel 2 enhances pulmonary vascular resistance and hypoxic pulmonary hypertension.

Acid-sensing ion channels (ASICs) are voltage-insensitive cation channels that contribute to cellular excitability. We previously reported that ASIC1 in pulmonary artery smooth muscle cells (PASMC) contribute to pulmonary vasoreactivity and vascular remodeling during the development of chronic hypoxia (CH)-induced pulmonary hypertension. However, the roles of ASIC2 and ASIC3 in regulation of pulmonary vasoreactivity and the development of CH-induced pulmonary hypertension are unknown. We tested the hypothesis that ASIC2 and ASIC3 contribute to increased pulmonary vasoreactivity and development of CH-induced pulmonary hypertension using ASIC2- and ASIC3-knockout (-/-) mice. In contrast to this hypothesis, we found that ASIC2-/- mice exhibit enhanced CH-induced pulmonary hypertension compared with WT and ASIC3-/- mice. This response was not associated with a change in ventilatory sensitivity or systemic cardiovascular function but was instead associated with direct changes in pulmonary vascular reactivity and pulmonary arterial morphology in ASIC2-/- mice. This increase in reactivity correlated with enhanced pulmonary arterial basal tone, elevated basal PASMC [Ca2+] and store-operated calcium entry (SOCE) in PASMC from ASIC2-/- mice. This increase in PASMC [Ca2+] and vasoreactivity was dependent on ASIC1-mediated Ca2+ influx but was not contingent upon an increase in ASIC1 mRNA or protein expression in PASMC from ASIC2-/- mice. Together, the results from this study demonstrate an important role for ASIC2 to regulate pulmonary vascular reactivity and for ASIC2 to modulate the development of CH-induced pulmonary hypertension. These data further suggest that loss of ASIC2 enhances the contribution of ASIC1 to overall pulmonary vascular reactivity.NEW & NOTEWORTHY This study demonstrates that loss of ASIC2 leads to increased baseline pulmonary vascular resistance, enhanced responses to a variety of vasoconstrictor stimuli, and greater development of hypoxic pulmonary hypertension. Furthermore, these results suggest that loss of ASIC2 enhances the contribution of ASIC1 to pulmonary vascular reactivity.

Redox Regulation of Ion Channels and Receptors in Pulmonary Hypertension.

Significance: Pulmonary hypertension (PH) is characterized by elevated vascular resistance due to vasoconstriction and remodeling of the normally low-pressure pulmonary vasculature. Redox stress contributes to the pathophysiology of this disease by altering the regulation and activity of membrane receptors, K+ channels, and intracellular Ca2+ homeostasis. Recent Advances: Antioxidant therapies have had limited success in treating PH, leading to a growing appreciation that reductive stress, in addition to oxidative stress, plays a role in metabolic and cell signaling dysfunction in pulmonary vascular cells. Reactive oxygen species generation from mitochondria and NADPH oxidases has substantial effects on K+ conductance and membrane potential, and both receptor-operated and store-operated Ca2+ entry. Critical Issues: Some specific redox changes resulting from oxidation, S-nitrosylation, and S-glutathionylation are known to modulate membrane receptor and ion channel activity in PH. However, many sites of regulation that have been elucidated in nonpulmonary cell types have not been tested in the pulmonary vasculature, and context-specific molecular mechanisms are lacking. Future Directions: Here, we review what is known about redox regulation of membrane receptors and ion channels in PH. Further investigation of the mechanisms involved is needed to better understand the etiology of PH and develop better targeted treatment strategies.

Cholesterol Regulation of Pulmonary Endothelial Calcium Homeostasis.

Cholesterol is a key structural component and regulator of lipid raft signaling platforms critical for cell function. Such regulation may involve changes in the biophysical properties of lipid microdomains or direct protein-sterol interactions that alter the function of ion channels, receptors, enzymes, and membrane structural proteins. Recent studies have implicated abnormal membrane cholesterol levels in mediating endothelial dysfunction that is characteristic of pulmonary hypertensive disorders, including that resulting from long-term exposure to hypoxia. Endothelial dysfunction in this setting is characterized by impaired pulmonary endothelial calcium entry and an associated imbalance that favors production vasoconstrictor and mitogenic factors that contribute to pulmonary hypertension. Here we review current knowledge of cholesterol regulation of pulmonary endothelial Ca2+ homeostasis, focusing on the role of membrane cholesterol in mediating agonist-induced Ca2+ entry and its components in the normal and hypertensive pulmonary circulation.

Interleukin-6 trans-signaling contributes to chronic hypoxia-induced pulmonary hypertension.

Interleukin-6 (IL-6) is a pleotropic cytokine that signals through the membrane-bound IL-6 receptor (mIL-6R) to induce anti-inflammatory ("classic-signaling") responses. This cytokine also binds to the soluble IL-6R (sIL-6R) to promote inflammation ("trans-signaling"). mIL-6R expression is restricted to hepatocytes and immune cells. Activated T cells release sIL-6R into adjacent tissues to induce trans-signaling. These cellular actions require the ubiquitously expressed membrane receptor gp130. Reports show that IL-6 is produced by pulmonary arterial smooth muscle cells (PASMCs) exposed to hypoxia in culture as well as the medial layer of the pulmonary arteries in mice exposed to chronic hypoxia (CH), and IL-6 knockout mice are protected from CH-induced pulmonary hypertension (PH). IL-6 has the potential to contribute to a broad array of downstream effects, such as cell growth and migration. CH-induced PH is associated with increased proliferation and migration of PASMCs to previously non-muscularized vessels of the lung. We tested the hypothesis that IL-6 trans-signaling contributes to CH-induced PH and arterial remodeling. Plasma levels of sgp130 were significantly decreased in mice exposed to CH (380 mmHg) for five days compared to normoxic control mice (630 mmHg), while sIL-6R levels were unchanged. Consistent with our hypothesis, mice that received the IL-6 trans-signaling-specific inhibitor sgp130Fc, a fusion protein of the soluble extracellular portion of gp130 with the constant portion of the mouse IgG1 antibody, showed attenuation of CH-induced increases in right ventricular systolic pressure, right ventricular and pulmonary arterial remodeling as compared to vehicle (saline)-treated control mice. In addition, PASMCs cultured in the presence of IL-6 and sIL-6R showed enhanced migration but not proliferation compared to those treated with IL-6 or sIL-6R alone or in the presence of sgp130Fc. These results indicate that IL-6 trans-signaling contributes to pulmonary arterial cell migration and CH-induced PH.

Role of acid-sensing ion channels in hypoxia- and hypercapnia-induced ventilatory responses.

Previous reports indicate roles for acid-sensing ion channels (ASICs) in both peripheral and central chemoreception, but the contributions of ASICs to ventilatory drive in conscious, unrestrained animals remain largely unknown. We tested the hypotheses that ASICs contribute to hypoxic- and hypercapnic-ventilatory responses. Blood samples taken from conscious, unrestrained mice chronically instrumented with femoral artery catheters were used to assess arterial O2, CO2, and pH levels during exposure to inspired gas mixtures designed to cause isocapnic hypoxemia or hypercapnia. Whole-body plethysmography was used to monitor ventilatory parameters in conscious, unrestrained ASIC1, ASIC2, or ASIC3 knockout (-/-) and wild-type (WT) mice at baseline, during isocapnic hypoxemia and during hypercapnia. Hypercapnia increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice, but there were no differences between ASIC1-/-, ASIC2-/-, or ASIC3-/- and WT. Isocapnic hypoxemia also increased respiratory frequency, tidal volume, and minute ventilation in all groups of mice. Minute ventilation in ASIC2-/- mice during isocapnic hypoxemia was significantly lower compared to WT, but there were no differences in the responses to isocapnic hypoxemia between ASIC1-/- or ASIC3-/- compared to WT. Surprisingly, these findings show that loss of individual ASIC subunits does not substantially alter hypercapnic or hypoxic ventilatory responses.

Actin polymerization contributes to enhanced pulmonary vasoconstrictor reactivity after chronic hypoxia.

Chronic hypoxia (CH) augments basal and endothelin-1 (ET-1)-induced pulmonary vasoconstrictor reactivity through reactive oxygen species (ROS) generation and RhoA/Rho kinase (ROCK)-dependent myofilament Ca2+ sensitization. Because ROCK promotes actin polymerization and the actin cytoskeleton regulates smooth muscle tension, we hypothesized that actin polymerization is required for enhanced basal and ET-1-dependent vasoconstriction after CH. To test this hypothesis, both end points were monitored in pressurized, endothelium-disrupted pulmonary arteries (fourth-fifth order) from control and CH (4 wk at 0.5 atm) rats. The actin polymerization inhibitors cytochalasin and latrunculin attenuated both basal and ET-1-induced vasoconstriction only in CH vessels. To test whether CH directly alters the arterial actin profile, we measured filamentous actin (F-actin)-to-globular actin (G-actin) ratios by fluorescent labeling of F-actin and G-actin in fixed pulmonary arteries and actin sedimentation assays using homogenized pulmonary artery lysates. We observed no difference in actin polymerization between groups under baseline conditions, but ET-1 enhanced actin polymerization in pulmonary arteries from CH rats. This response was blunted by the ROS scavenger tiron, the ROCK inhibitor fasudil, and the mDia (RhoA effector) inhibitor small-molecule inhibitor of formin homology domain 2. Immunoblot analysis revealed an effect of CH to increase both phosphorylated (inactive) and total levels of the actin disassembly factor cofilin but not phosphorylated cofilin-to-total cofilin ratios. We conclude that actin polymerization contributes to increased basal pulmonary arterial constriction and ET-1-induced vasoconstrictor reactivity after CH in a ROS- and ROCK-dependent manner. Our results further suggest that enhanced ET-1-mediated actin polymerization after CH is dependent on mDia but independent of changes in the phosphorylated cofilin-to-total cofilin ratio. NEW & NOTEWORTHY This research is the first to demonstrate a role for actin polymerization in chronic hypoxia-induced basal pulmonary arterial constriction and enhanced agonist-induced vasoconstrictor activity. These results suggest that a reactive oxygen species-Rho kinase-actin polymerization signaling pathway mediates this response and may provide a mechanistic basis for the vasoconstrictor component of pulmonary hypertension.

RhoA increases ASIC1a plasma membrane localization and calcium influx in pulmonary arterial smooth muscle cells following chronic hypoxia.

Increases in pulmonary arterial smooth muscle cell (PASMC) intracellular Ca2+ levels and enhanced RhoA/Rho kinase-dependent Ca2+ sensitization are key determinants of PASMC contraction, migration, and proliferation accompanying the development of hypoxic pulmonary hypertension. We previously showed that acid-sensing ion channel 1a (ASIC1a)-mediated Ca2+ entry in PASMC is an important constituent of the active vasoconstriction, vascular remodeling, and right ventricular hypertrophy associated with hypoxic pulmonary hypertension. However, the enhanced ASIC1a-mediated store-operated Ca2+ entry in PASMC from pulmonary hypertensive animals is not dependent on an increase in ASIC1a protein expression, suggesting that chronic hypoxia (CH) stimulates ASIC1a function through other regulatory mechanism(s). RhoA is involved in ion channel trafficking, and levels of activated RhoA are increased following CH. Therefore, we hypothesize that activation of RhoA following CH increases ASIC1a-mediated Ca2+ entry by promoting ASIC1a plasma membrane localization. Consistent with our hypothesis, we found greater plasma membrane localization of ASIC1a following CH. Inhibition of RhoA decreased ASIC1a plasma membrane expression and largely diminished ASIC1a-mediated Ca2+ influx, whereas activation of RhoA had the opposite effect. A proximity ligation assay revealed that ASIC1a and RhoA colocalize in PASMC and that the activation state of RhoA modulates this interaction. Together, our findings show a novel interaction between RhoA and ASIC1a, such that activation of RhoA in PASMC, both pharmacologically and via CH, promotes ASIC1a plasma membrane localization and Ca2+ entry. In addition to enhanced RhoA-mediated Ca2+ sensitization following CH, RhoA can also activate a Ca2+ signal by facilitating ASIC1a plasma membrane localization and Ca2+ influx in pulmonary hypertension.