Hypoxic pulmonary vasoconstriction (HPV) optimizes pulmonary ventilation-perfusion matching in regional hypoxia, but promotes pulmonary hypertension in global hypoxia. Ventilation-perfusion mismatch is a major cause of hypoxemia in cystic fibrosis. We hypothesized that cystic fibrosis transmembrane conductance regulator (CFTR) may be critical in HPV, potentially by modulating the response to sphingolipids as mediators of HPV. HPV and ventilation-perfusion mismatch were analyzed in isolated mouse lungs or in vivo. Ca 2+ mobilization and transient receptor potential canonical 6 (TRPC6) translocation were studied in human pulmonary (PASMCs) or coronary (CASMCs) artery smooth muscle cells. CFTR inhibition or deficiency diminished HPV and aggravated ventilation-perfusion mismatch. In PASMCs, hypoxia caused CFTR to interact with TRPC6, whereas CFTR inhibition attenuated hypoxiainduced TRPC6 translocation to caveolae and Ca 2+ mobilization. Ca 2+ mobilization by sphingosine-1-phosphate (S1P) was also attenuated by CFTR inhibition in PASMCs, but amplified in CASMCs. Inhibition of neutral sphingomyelinase (nSMase) blocked HPV, whereas exogenous nSMase caused TRPC6 translocation and vasoconstriction that were blocked by CFTR inhibition. nSMase-and hypoxia-induced vasoconstriction, yet not TRPC6 translocation, were blocked by inhibition or deficiency of sphingosine kinase 1 (SphK1) or antagonism of S1P receptors 2 and 4 (S1P 2/4 ). S1P and nSMase had synergistic effects on pulmonary vasoconstriction that involved TRPC6, phospholipase C, and rho kinase. Our findings demonstrate a central role of CFTR and sphingolipids in HPV. Upon hypoxia, nSMase triggers TRPC6 translocation, which requires its interaction with CFTR. Concomitant SphK1-dependent formation of S1P and activation of S1P 2/4 result in phospholipase C-mediated TRPC6 and rho kinase activation, which conjointly trigger vasoconstriction.

Background:The microRNA-130/301 family regulates pulmonary hypertension (PH), but its breadth of activity remains undefined. Results: Predicted by network analysis, microRNA-130/301 members regulate vasoactive factors such as endothelin-1 for pulmonary vascular cross-talk. Conclusion:The microRNA-130/301 family promotes vasoconstriction in PH. Significance: This microRNA-based mechanism of vascular cross-talk is central to the systems-wide actions of microRNA-130/301 in PH.

The effects of the heptapeptide angiotensin-(1-7) (Ang-(1-7)), via its receptor Mas, oppose many of the effects of the classic angiotensin II signaling pathway, and pharmacological exploitation of this effect is currently actively pursued for a wide range of cardiovascular, neoplastic, or immunological disorders. On the basis of its vasodilatory and antiproliferative properties, Ang-(1-7) has consequentially also been proposed as a novel therapeutic strategy for the treatment of pulmonary arterial hypertension (PAH). In this study, we tested the effectiveness of Ang-(1-7) and its stable, cyclic analog cAng-(1-7) over a range of doses for their therapeutic potential in experimental PAH. In the monocrotaline (MCT) rat model of PAH, Ang-(1-7) or cAng-(1-7) were injected in doses of 30, 100, 300, or 900 μg kg, and effects on pulmonary hemodynamics and vascular remodeling were assessed. Five weeks after MCT injection, right ventricular systolic pressure (RVSP) was significantly reduced for 3 dose groups treated with Ang-(1-7) (100, 300, and 900 μg kg) and for all dose groups treated with cAng-(1-7), as compared to untreated controls, yet the total reduction of RVSP was <50% at best and thus markedly lower than that with a positive treatment control with ambrisentan. Medial-wall thickness in pulmonary arterioles was only slightly reduced, without reaching significance, for any of the tested Ang-(1-7) compounds and doses. The reported moderate attenuation of PAH does not confirm the previously postulated high promise of this strategy, and the therapeutic usefulness of Ang-(1-7) may be limited in PAH relative to that in other cardiovascular diseases.Keywords: pulmonary hypertension, vascular remodeling, renin-angiotensin system. Pulmonary arterial hypertension (PAH) is a rare but ultimately fatal disease defined by an increase in mean pulmonary arterial pressure to >25 mmHg at rest or >30 mmHg during exercise.1 The pathogenesis of PAH is very diverse, in that PAH may be heritable or idiopathic or develop in association with a number of diseases, such as systemic sclerosis or HIV infection. The pathology of PAH is characterized by endothelial dysfunction, which becomes apparent as an imbalance in the release of vasoconstrictive and vasodilatory factors, such as endothelin and nitric oxide (NO), leading to pulmonary vasoconstriction and ultimately promoting structural remodeling of the pulmonary vasculature, resulting in a persistent elevation in pulmonary vascular resistance. 2,3 A series of therapeutic drugs have been approved for the treatment of PAH that target three different pathways: prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase type 5 inhibitors. 4 While these drugs have helped to improve life expectancy and quality of life in PAH patients in the past 2 decades, mortality remains high, as one-third of PAH patients die within 3 years of diagnosis; this emphasizes the need for further research and improved therapeutic options.

A major cause of death after influenza virus infection is lung injury due to a bacterial superinfection, yet the mechanism is unknown. Death has been attributed to virus-induced immunosuppression and bacterial overgrowth, but this hypothesis is based on data from the preantibiotic era and animal models that omit antimicrobial therapy. Because of diagnostic uncertainty, most patients with influenza receive antibiotics, making bacterial overgrowth unlikely. Respiratory failure after superinfection presents as acute respiratory distress syndrome, a disorder characterized by lung microvascular leak and edema. The objective of this study was to determine whether the influenza virus sensitizes the lung endothelium to leak upon exposure to circulating bacterial-derived molecular patterns from Staphylococcus aureus. In vitro as well as in vivo models of influenza followed by S. aureus superinfection were used. Molecular mechanisms were explored using molecular biology, knockout mice, and human autopsy specimens. Influenza virus infection sensitized human lung endothelium to leak when challenged with S. aureus, even at low doses of influenza and even when the pathogens were given days apart. Influenza virus increased endothelial expression of TNFR1 both in vitro and in intact lungs, a finding corroborated by human autopsy specimens of patients with influenza. Leak was recapitulated with protein A, a TNFR1 ligand, and sequential infection caused protein A-dependent loss of IκB, cleavage of caspases 8 and 3, and lung endothelial apoptosis. Mice infected sequentially with influenza virus and S. aureus developed significantly increased lung edema that was protein A and TNFR1 dependent. Influenza virus primes the lung endothelium to leak, predisposing patients to acute respiratory distress syndrome upon exposure to S. aureus.

BackgroundDevelopment of right ventricular (RV) hypertension eventually contributes to RV and left ventricular (LV) myocardial fibrosis and dysfunction. The molecular mechanisms are not fully elucidated.Methods and ResultsPulmonary artery banding was used to induce RV hypertension in rats in vivo. Then, we evaluated cardiac function and regional remodeling 6 weeks after pulmonary artery banding. To further elucidate mechanisms responsible for regional cardiac remodeling, we also mimicked RV hypertensive stress by cyclic mechanical stretching applied to confluent cultures of cardiac fibroblasts, isolated from the RV free wall, septal hinge points, and LV free wall. Echocardiography and catheter evaluation demonstrated that rats in the pulmonary artery banding group developed RV hypertension with leftward septal displacement, LV compression, and increased LV end‐diastolic pressures. Picrosirius red staining indicated that pulmonary artery banding induced marked RV fibrosis and dysfunction, with prominent fibrosis and elastin deposition at the septal hinge points but less LV fibrosis. These changes were associated with proportionally increased expressions of integrin‐β1 and profibrotic signaling proteins, including phosphorylated Smad2/3 and transforming growth factor‐β1. Moreover, mechanically stretched fibroblasts also expressed significantly increased levels of α‐smooth muscle actin, integrin‐β1, transforming growth factor‐β1, collagen I deposition, and wrinkle formation on gel assays, consistent with myofibroblast transformation. These changes were not observed in parallel cultures of mechanically stretched fibroblasts, preincubated with the integrin inhibitor (BTT‐3033).ConclusionsExperimentally induced RV hypertension triggers regional RV, hinge‐point, and LV integrin β1‐dependent mechanotransduction signaling pathways that eventually trigger myocardial fibrosis via transforming growth factor‐β1 signaling. Reduced LV fibrosis and preserved global function, despite geometrical and pressure aberrations, suggest a possible elastin‐mediated protective mechanism at the septal hinge points.

Aims Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia that diverts blood flow from poorly ventilated to better aerated lung areas to optimize ventilation-perfusion matching. Yet, the exact sensory and signaling mechanisms by which hypoxia triggers pulmonary vasoconstriction remain incompletely understood. Recently, ATP release via pannexin 1 (Panx1) and subsequent signaling via purinergic P2Y receptors has been identified as regulator of vasoconstriction in systemic arterioles. Here, we probed for the role of Panx1-mediated ATP release in HPV and chronic hypoxic pulmonary hypertension (PH). Methods and Results Pharmacological inhibition of Panx1 by probenecid, spironolactone, the Panx1 specific inhibitory peptide (10Panx1) and genetic deletion of Panx1 specifically in smooth muscle attenuated HPV in isolated perfused mouse lungs. In pulmonary artery smooth muscle cells (PASMC), both spironolactone and 10Panx1 attenuated the increase in intracellular Ca2+ concentration ([Ca2+]i) in response to hypoxia. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Unexpectedly, ATP release in response to hypoxia was not detectable in PASMC, and inhibition of purinergic receptors or ATP degradation by ATPase failed to attenuate HPV. Rather, transient receptor potential vanilloid 4 (TRPV4) antagonism and Panx1 inhibition inhibited the hypoxia-induced [Ca2+]i increase in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP-P2Y-TRPV4 pathway. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. Conclusion In the present study we identify Panx1 as novel regulator of HPV. Yet, the role of Panx1 in HPV was not attributable to ATP release and downstream signaling via P2Y receptors or TRPV4 activation, but relates to a role of Panx1 as direct or indirect modulator of the PASMC Ca2+ response to hypoxia. Panx1 did not affect the development of chronic hypoxic PH. Translational perspective Hypoxic pulmonary vasoconstriction (HPV) optimizes lung ventilation-perfusion matching, but also contributes to pulmonary pathologies including high altitude pulmonary edema (HAPE) or chronic hypoxic pulmonary hypertension. Here, we demonstrate that pharmaceutical inhibition as well as genetic deletion of the hemichannel pannexin-1 (Panx1) in pulmonary artery smooth muscle cells attenuates the physiological HPV response. Panx1 deficiency did, however, not prevent the development of chronic hypoxic pulmonary hypertension in mice. Panx1 inhibitors such as the mineralocorticoid receptor antagonist spironolactone may thus present a putative strategy for the prevention or treatment of HAPE, yet not for chronic hypoxic lung disease.

Our data indicate a novel interplay between ROCK and [Ca2+]i signalling in HPV via PTEN, in that ROCK mediates interaction of PTEN and TRPC6 which then conjointly translocate to caveolae allowing for Ca2+ influx into and subsequent contraction of PASMC.

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to alveolar hypoxia by which pulmonary blood flow is redirected from poorly ventilated areas to better aerated lung regions. Sustained HPV plays a critical role in the development of pulmonary hypertension (PH) but the exact signaling pathways underlying HPV remain incompletely defined. Recently, ATP release via Pannexin 1 (Panx1) and subsequent purinergic signaling have been linked to transient receptor potential vanlloid 4 (TRPV4) channel mediated Ca2+ signaling in systemic vasoconstriction. Here, we probed for a similar role of Panx1 in HPV and the development of PH.To test the role of Panx1, we measured HPV as increase in pulmonary artery pressure (PAP) in response to hypoxia in lungs of endothelial cell (Cdh5‐CreERT2/Panx1fl/fl) and smooth muscle cell specific (SMMHC‐CreERT2/Panx1fl/fl) Panx1‐KO mice. Isolated perfused lungs (IPL) of C57Bl6/J and Panx1‐deficient mice were ventilated with normoxic (20% O2; 5% CO2, 75% N2) or hypoxic gas (1% O2; 5% CO2, 94% N2) and PAP was recorded via a pulmonary artery catheter. To elucidate the role of Panx1 in the development of PH, C57Bl6/J as well as Panx1‐KO mice were kept under hypoxic conditions (10% O2) for 5 weeks followed by measurement of right ventricular systolic pressure, echocardiography and histology.Panx1 protein was expressed in human pulmonary artery endothelial cells as well as in human pulmonary artery smooth muscle cells (hPASMCs). Genetic deletion of Panx1 in smooth muscle cells, yet not in endothelial cells reduced the HPV response in isolated mouse lungs. In lungs of C57Bl6/J mice, Panx1‐inhibition by the non‐specific Panx1 inhibitors spironolactone or probenecid and the Panx1 specific inhibitory peptide (10Panx1) effectively attenuated HPV. Yet, genetic deletion of Panx1 in either endothelial or smooth muscle cells did not prevent the development of PH in mice. Interestingly, ATP release in response to hypoxia was not detectable in PASMC, and ATP degradation as well as inhibition of purinergic receptors failed to affect HPV. Rather, TRPV4 antagonism and Panx1 inhibition inhibited the hypoxia‐induced increase of intracellular Ca2+ concentration ([Ca2+]i) in PASMC in an additive manner, suggesting that Panx1 regulates [Ca2+]i independently of the ATP‐P2Y‐TRPV4 signaling axis. In line with this notion, Panx1 overexpression increased the [Ca2+]i response to hypoxia in HeLa cells. Here we identify Panx1 as novel regulator of HPV. The role of Panx1 in HPV was, however, not attributable to ATP release and downstream purinergic receptor signaling or TRPV4 activation, but may involve a direct role of Panx1 as Ca2+ channel. In contrast to its role in HPV, Panx1 does not seem to contribute to chronic hypoxic PH.