SPARC, a Novel Regulator of Vascular Cell Function in Pulmonary Hypertension.

BACKGROUND: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMCs) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVECs). On reexposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, the authors aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. METHODS: After microarray analysis, the authors assessed the role of SPARC (secreted protein acidic and rich in cysteine) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients, as well as from chronically hypoxic mice. In vitro studies were conducted in primary human PASMCs and PMVECs. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an adeno-associated virus-mediated Sparc knockdown approach. RESULTS: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent reexposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes downregulated at all reoxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. TGF-ß1 (transforming growth factor ß1) or HIF2A (hypoxia-inducible factor 2A) signaling pathways induced SPARC expression in human PASMCs. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMCs, but not PMVECs, proliferation. Coculture and conditioned medium experiments revealed that PMVECs-secreted SPARC acts as a paracrine factor triggering PASMCs proliferation. Contrary to the authors' expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably because of counter-regulatory proproliferative signaling. However, adeno-associated virus-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. CONCLUSIONS: This study provides evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most likely by affecting vascular cell function.

Shear force sensing of epithelial Na+ channel (ENaC) relies on N-glycosylated asparagines in the palm and knuckle domains of αENaC.

Mechanosensitive ion channels are crucial for normal cell function and facilitate physiological function, such as blood pressure regulation. So far little is known about the molecular mechanisms of how channels sense mechanical force. Canonical vertebrate epithelial Na+ channel (ENaC) formed by α-, ß-, and γ-subunits is a shear force (SF) sensor and a member of the ENaC/degenerin protein family. ENaC activity in epithelial cells contributes to electrolyte/fluid-homeostasis and blood pressure regulation. Furthermore, ENaC in endothelial cells mediates vascular responsiveness to regulate blood pressure. Here, we provide evidence that ENaC's ability to mediate SF responsiveness relies on the "force-from-filament" principle involving extracellular tethers and the extracellular matrix (ECM). Two glycosylated asparagines, respectively their N-glycans localized in the palm and knuckle domains of αENaC, were identified as potential tethers. Decreased SF-induced ENaC currents were observed following removal of the ECM/glycocalyx, replacement of these glycosylated asparagines, or removal of N-glycans. Endothelial-specific overexpression of αENaC in mice induced hypertension. In contrast, expression of αENaC lacking these glycosylated asparagines blunted this effect. In summary, glycosylated asparagines in the palm and knuckle domains of αENaC are important for SF sensing. In accordance with the force-from-filament principle, they may provide a connection to the ECM that facilitates vascular responsiveness contributing to blood pressure regulation.

Enhanced Shear Force Responsiveness of Epithelial Na+ Channel's (ENaC) δ Subunit Following the Insertion of N-Glycosylation Motifs Relies on the Extracellular Matrix.

Members of the Degenerin/epithelial Na+ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC-which is less responsive to shear force (SF)-increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with ß and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC's SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction.

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.

Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions.

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O2-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H2O2. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

Gadolinium released by the linear gadolinium-based contrast-agent Gd-DTPA decreases the activity of human epithelial Na+ channels (ENaCs).

BACKGROUND: Gadolinium-based-contrast-agents (GBCAs) are used for magnetic-resonance-imaging and associated with renal and cardiovascular adverse reactions caused by released Gd3+ ions. Gd3+ is also a modulator of mechano-gated ion channels, including the epithelial Na+ channel (ENaC) that is expressed in kidney epithelium and the vasculature. ENaC is important for salt-/water homeostasis and blood pressure regulation and a likely target of released Gd3+ from GBCAs causing the above-mentioned adverse reactions. Therefore this study examined the effect of Gd3+ and GBCAs on ENaC's activity. METHODS: Human αßγENaC was expressed in Xenopus laevis oocytes and exposed to Gd3+, linear (Gd-DTPA, Magnevist) or cyclic (Dotarem) GBCAs. Transmembrane ion-currents (IM) were recorded by the two-electrode-voltage-clamp technique and Gd3+-release by Gd-DTPA was confirmed by inductively coupled plasma-mass spectrometry. RESULTS: Gd3+ exerts biphasic effects on ENaC's activity: ≤0.3mmol/l decreased IM which was preventable by DEPC (modifies histidines). Strikingly Gd3+≥0.4mmol/l increased IM and this effect was prevented by cysteine-modifying MTSEA. Linear Gd-DTPA and Magnevist mimicked the effect of ≤0.3mmol/l Gd3+, whereas the chelator DTPA showed no effect. Gd3+ and Gd-DTPA increased the IC50 for amiloride, but did not affect ENaC's self-inhibition. Interestingly, cyclic Gd-DOTA (Dotarem) increased IM to a similar extent as its chelator DOTA, suggesting that the chelator rather than released Gd3+ is responsible for this effect. CONCLUSION: These results confirm Gd3+-release from linear Gd-DTPA and indicate that the released Gd3+ amount is sufficient to interfere with ENaC's activity to provide putative explanations for GBCA-related adverse effects.

Use of FRET-Sensor 'Mermaid' to Detect Subtle Changes in Membrane Potential of Primary Mouse PASMCs.

Subtle changes in the membrane potential of pulmonary arterial smooth muscle cells (PASMCs) are pivotal for controlling pulmonary vascular tone, e.g., for initiating Hypoxic Pulmonary Vasoconstriction, a vital mechanism of the pulmonary circulation. In our study, we evaluated the ability of the fluorescence resonance energy transfer (FRET)-based voltage-sensor Mermaid to detect such subtle changes in membrane potential. Mouse PASMCs were isolated and transduced with Mermaid-encoding lentiviral vectors before the acceptor/donor emission ratio was assessed via live cell FRET-imaging. Mermaid's sensitivity was tested by applying specific potassium chloride (KCl) concentrations. These KCl concentrations were previously validated by patch clamp recordings to induce depolarization with predefined amplitudes that physiologically occur in PASMCs. Mermaid's emission ratio dose-dependently increased upon depolarization with KCl. However, Mermaid formed unspecific intracellular aggregates, which limited the usefulness of this voltage sensor. When analyzing the membrane rim only to circumvent these unspecific signals, Mermaid was not suitable to resolve subtle changes in the membrane potential of ≤10 mV. In summary, we found Mermaid to be a suitable alternative for reliably detecting qualitative membrane voltage changes of more than 10 mV in primary mouse PASMCs. However, one should be aware of the limitations associated with this voltage sensor.

Experimental Setup for Investigation of Acute Mitochondrial Oxygen Sensing in Primary Cells.

The ability to sense and respond to acute changes in oxygen is essential for the viability of cells and organisms. To study molecular mechanisms of acute oxygen sensing, we established a setup for the adjustment of acute hypoxic conditions in cultured cells, exemplified here for the use of primary pulmonary arterial smooth muscle cells (PASMCs). The mitochondrial electron transport chain (ETC) is the main consumer of oxygen but recently also emerged as essential oxygen sensor suggesting that the ETC itself adapts its electron flux to oxygen availability. To test this assumption and to experimentally manipulate electron flux through the ETC, we used alternative oxidase (AOX), which bypasses the cytochrome pathway of the ETC when blocked. The described combination of our experimental setup and AOX allowed us in previous publications unprecedented insights into the role of the ETC in cellular oxygen sensing and cellular response mechanisms in living cells. Against this background, we here describe and discuss this method in detail, which will allow transfer to other cell types and research questions.

Mitochondrial oxygen sensing of acute hypoxia in specialized cells - Is there a unifying mechanism?

Acclimation to acute hypoxia through cardiorespiratory responses is mediated by specialized cells in the carotid body and pulmonary vasculature to optimize systemic arterial oxygenation and thus oxygen supply to the tissues. Acute oxygen sensing by these cells triggers hyperventilation and hypoxic pulmonary vasoconstriction which limits pulmonary blood flow through areas of low alveolar oxygen content. Oxygen sensing of acute hypoxia by specialized cells thus is a fundamental pre-requisite for aerobic life and maintains systemic oxygen supply. However, the primary oxygen sensing mechanism and the question of a common mechanism in different specialized oxygen sensing cells remains unresolved. Recent studies unraveled basic oxygen sensing mechanisms involving the mitochondrial cytochrome c oxidase subunit 4 isoform 2 that is essential for the hypoxia-induced release of mitochondrial reactive oxygen species and subsequent acute hypoxic responses in both, the carotid body and pulmonary vasculature. This review compares basic mitochondrial oxygen sensing mechanisms in the pulmonary vasculature and the carotid body.

Deletion of classical transient receptor potential 1, 3 and 6 alters pulmonary vasoconstriction in chronic hypoxia-induced pulmonary hypertension in mice.

Chronic hypoxia-induced pulmonary hypertension (CHPH) is a severe disease that is characterized by increased proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs) leading to pulmonary vascular remodeling. The resulting increase in pulmonary vascular resistance (PVR) causes right ventricular hypertrophy and ultimately right heart failure. In addition, increased PVR can also be a consequence of hypoxic pulmonary vasoconstriction (HPV) under generalized hypoxia. Increased proliferation and migration of PASMCs are often associated with high intracellular Ca2+ concentration. Recent publications suggest that Ca2+-permeable nonselective classical transient receptor potential (TRPC) proteins-especially TRPC1 and 6-are crucially involved in acute and sustained hypoxic responses and the pathogenesis of CHPH. The aim of our study was to investigate whether the simultaneous deletion of TRPC proteins 1, 3 and 6 protects against CHPH-development and affects HPV in mice. We used a mouse model of chronic hypoxia as well as isolated, ventilated and perfused mouse lungs and PASMC cell cultures. Although right ventricular systolic pressure as well as echocardiographically assessed PVR and right ventricular wall thickness (RVWT) were lower in TRPC1, 3, 6-deficient mice, these changes were not related to a decreased degree of pulmonary vascular muscularization and a reduced proliferation of PASMCs. However, both acute and sustained HPV were almost absent in the TRPC1, 3, 6-deficient mice and their vasoconstrictor response upon KCl application was reduced. This was further validated by myographical experiments. Our data revealed that 1) TRPC1, 3, 6-deficient mice are partially protected against development of CHPH, 2) these changes may be caused by diminished HPV and not an altered pulmonary vascular remodeling.

A Microfluidic System for Simultaneous Raman Spectroscopy, Patch-Clamp Electrophysiology, and Live-Cell Imaging to Study Key Cellular Events of Single Living Cells in Response to Acute Hypoxia.

The ability to sense changes in oxygen availability is fundamentally important for the survival of all aerobic organisms. However, cellular oxygen sensing mechanisms and pathologies remain incompletely understood and studies of acute oxygen sensing, in particular, have produced inconsistent results. Current methods cannot simultaneously measure the key cellular events in acute hypoxia (i.e., changes in redox state, electrophysiological properties, and mechanical responses) at controlled partial pressures of oxygen (pO2 ). The lack of such a comprehensive method essentially contributes to the discrepancies in the field. A sealed microfluidic system that combines i) Raman spectroscopy, ii) patch-clamp electrophysiology, and iii) live-cell imaging under precisely controlled pO2 have therefore been developed. Merging these modalities allows label-free and simultaneous observation of oxygen-dependent alterations in multiple cellular redox couples, membrane potential, and cellular contraction. This technique is adaptable to any cell type and allows in-depth insight into acute oxygen sensing processes underlying various physiologic and pathologic conditions.

Amelioration of elastase-induced lung emphysema and reversal of pulmonary hypertension by pharmacological iNOS inhibition in mice.

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 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. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.1/issuetoc.

Cytochrome P450 epoxygenase-derived 5,6-epoxyeicosatrienoic acid relaxes pulmonary arteries in normoxia but promotes sustained pulmonary vasoconstriction in hypoxia.

AIMS: The aim of the study was to investigate the role of cytochrome P450 (CYP) epoxygenase-derived epoxyeicosatrienoic acids (EETs) in sustained hypoxic pulmonary vasoconstriction (HPV). METHODS: Vasomotor responses of isolated mouse intrapulmonary arteries (IPAs) were assessed using wire myography. Key findings were verified by haemodynamic measurements in isolated perfused and ventilated mouse lungs. RESULTS: Pharmacological inhibition of EET synthesis with MS-PPOH, application of the EET antagonist 14,15-EEZE or deficiency of CYP2J isoforms suppressed sustained HPV. In contrast, knockdown of EET-degrading soluble epoxide hydrolase or its inhibition with TPPU augmented sustained HPV almost twofold. All EET regioisomers elicited relaxation in IPAs pre-contracted with thromboxane mimetic U46619. However, in the presence of KCl-induced depolarization, 5,6-EET caused biphasic contraction in IPAs and elevation of pulmonary vascular tone in isolated lungs, whereas other regioisomers had no effect. In patch-clamp experiments, hypoxia elicited depolarization in pulmonary artery smooth muscle cells (PASMCs), and 5,6-EET evoked inward whole cell currents in PASMCs depolarized to the hypoxic level, but not at their resting membrane potential. CONCLUSIONS: The EET pathway substantially contributes to sustained HPV in mouse pulmonary arteries. 5,6-EET specifically appears to be involved in HPV, as it is the only EET regioisomer able to elicit not only relaxation, but also sustained contraction in these vessels. 5,6-EET-induced pulmonary vasoconstriction is enabled by PASMC depolarization, which occurs in hypoxia. The discovery of the dual role of 5,6-EET in the regulation of pulmonary vascular tone may provide a basis for the development of novel therapeutic strategies for treatment of HPV-related diseases.

Bypassing mitochondrial complex III using alternative oxidase inhibits acute pulmonary oxygen sensing.

Mitochondria play an important role in sensing both acute and chronic hypoxia in the pulmonary vasculature, but their primary oxygen-sensing mechanism and contribution to stabilization of the hypoxia-inducible factor (HIF) remains elusive. Alteration of the mitochondrial electron flux and increased superoxide release from complex III has been proposed as an essential trigger for hypoxic pulmonary vasoconstriction (HPV). We used mice expressing a tunicate alternative oxidase, AOX, which maintains electron flux when respiratory complexes III and/or IV are inhibited. Respiratory restoration by AOX prevented acute HPV and hypoxic responses of pulmonary arterial smooth muscle cells (PASMC), acute hypoxia-induced redox changes of NADH and cytochrome c, and superoxide production. In contrast, AOX did not affect the development of chronic hypoxia-induced pulmonary hypertension and HIF-1α stabilization. These results indicate that distal inhibition of the mitochondrial electron transport chain in PASMC is an essential initial step for acute but not chronic oxygen sensing.

NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and death worldwide. Peroxynitrite, formed from nitric oxide, which is derived from inducible nitric oxide synthase, and superoxide, has been implicated in the development of emphysema, but the source of the superoxide was hitherto not characterized. Here, we identify the non-phagocytic NADPH oxidase organizer 1 (NOXO1) as the superoxide source and an essential driver of smoke-induced emphysema and pulmonary hypertension development in mice. NOXO1 is consistently upregulated in two models of lung emphysema, Cybb (also known as NADPH oxidase 2, Nox2)-knockout mice and wild-type mice with tobacco-smoke-induced emphysema, and in human COPD. Noxo1-knockout mice are protected against tobacco-smoke-induced pulmonary hypertension and emphysema. Quantification of superoxide, nitrotyrosine and multiple NOXO1-dependent signalling pathways confirm that peroxynitrite formation from nitric oxide and superoxide is a driver of lung emphysema. Our results suggest that NOXO1 may have potential as a therapeutic target in emphysema.

Author Correction: NADPH oxidase subunit NOXO1 is a target for emphysema treatment in COPD.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.