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Rationale: Pulmonary arterial hypertension (PAH) is a severe disease characterized by an increase of pulmonary vascular resistance, which is accompanied by functional and structural changes in pulmonary arteries. Microparticles (MPs) have been described as biological vector of endothelial dysfunction in other pathologies. Objectives: The purpose of this work was to characterize circulating MPs during hypoxic PAH and to study their effects on endothelial function. Methods: Male Wistar rats were exposed or not to chronic hypoxia, and normoxic or hypoxic MPs from blood were characterized by flow cytometry. Endothelial cells (ECs) from rat aorta or pulmonary arteries were incubated with MPs, and then expression and phosphorylation of enzymes involved in nitric oxide (NO) and reactive oxygen species productions were analyzed. Hypoxic MPs were injected into rats, and endothelium-dependent relaxation was assessed. Measurements and Main Results: Circulating levels of MPs from hypoxic rats were twofold higher than those present in normoxic rats. In vitro treatment of ECs with hypoxic MPs reduced NO production in aortas and pulmonary arteries by enhancing phosphorylation of endothelial NO synthase at the inhibitory site. Hypoxic MPs increased oxidative stress only in pulmonary ECs via xanthine oxidase and mitochondrial implication. In vivo injection of hypoxic MPs into rat impaired endothelium-dependent relaxation both in aorta and pulmonary arteries. Conclusions: These data provide evidence that hypoxic circulating MPs induce endothelial dysfunction in rat aorta and pulmonary arteries by decreasing NO production. Moreover, MPs display tissue specificity with respect to increased oxidative stress, which occurs only in pulmonary ECs. [ABSTRACT FROM AUTHOR]

Human exposure to manufactured nanoparticles (NPs) is a public health concern. Endothelial cells lining the inner surface of arteries could be one of the primary targets for inhaled nanoparticles. Moreover, it is well known that alteration in calcium signaling is a critical event involved in the physiopathology of cardiovascular diseases. The objective of this study was to assess the role of oxidative stress in carbon black FW2 NPs-induced alteration in calcium signaling and mitochondria in human pulmonary artery endothelial cells. To this end, cells were exposed for 4 or 24 h to FW2 NPs (1-10 μg/cm 2 ) and the following endpoints were studied: (i) production of ROS by fluorimetry and electron paramagnetic resonance, (ii) variation in intracellular calcium concentration by confocal microscopy, and (iii) mitochondrial alteration and apoptosis by confocal microscopy and transmission electronic microscopy. Exposure to FW2 NPs concentration-dependently increases oxidative stress, evidenced by the production of superoxide anion leading to an alteration in calcium content of intracellular organelles, such as endoplasmic reticulum and mitochondria activating, in turn, intrinsic apoptosis. This study provides evidence that FW2 NPs exposure impairs calcium signaling and mitochondria triggered by oxidative stress, and, thus, could act as a cardiovascular disease risk owing to the key role of calcium homeostasis in the control of vascular tone.

The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43 +/- ) and wild-type littermate (Cx43 +/+ ) mice at 12 weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3 weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43 +/- mice were partially protected against CH-PH when compared to Cx43 +/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43 +/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43 +/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy., Competing Interests: Conflict of interest: C. Bouvard has nothing to disclose. Conflict of interest: N. Genet has nothing to disclose. Conflict of interest: C. Phan has nothing to disclose. Conflict of interest: B. Rode has nothing to disclose. Conflict of interest: R. Thuillet has nothing to disclose. Conflict of interest: L. Tu has nothing to disclose. Conflict of interest: P. Robillard has nothing to disclose. Conflict of interest: M. Campagnac has nothing to disclose. Conflict of interest: R. Soleti has nothing to disclose. Conflict of interest: E. Dumas De La Roque has nothing to disclose. Conflict of interest: F. Delcambre has nothing to disclose. Conflict of interest: L. Cronier has nothing to disclose. Conflict of interest: T. Parpaite has nothing to disclose. Conflict of interest: E. Maurat has nothing to disclose. Conflict of interest: P. Berger reports grants from Nycomed, Takeda, Fondation du Souffle and Fonds de Dotation Recherche en Santé Respiratoire, during the conduct of the study; grants and personal fees for lectures, advisory board work and travel to meetings from Novartis, personal fees for lectures and non-financial (travel) support from Chiesi, grants and personal fees for advisory board work and lectures, as well as non-financial (travel) support from Boehringer Ingelheim, personal fees for advisory board work and lectures, as well as non-financial (travel) support from AstraZeneca and Sanofi, personal fees for advisory board work and lectures from Menarini, and personal fees for lectures from TEVA, outside the submitted work. Conflict of interest: J-P. Savineau has nothing to disclose. Conflict of interest: R. Marthan has nothing to disclose. Conflict of interest: C. Guignabert has nothing to disclose. Conflict of interest: V. Freund-Michel has nothing to disclose. Conflict of interest: C. Guibert has nothing to disclose., (Copyright ©ERS 2020.)

Pulmonary arterial adventitial fibroblasts (PAF), the most abundant cellular constituent of adventitia, act as a key regulator of pulmonary vascular wall structure and function from the outside-in. Previous studies indicate that transient receptor potential vanilloid 4 (TRPV4) channel plays an important role in the development of pulmonary hypertension (PH), but no attention has been given so far to its role in adventitial remodeling. In this study, we thus investigated TRPV4 implication in PAF activation occurring in PH. First, we isolated and cultured PAF from rat adventitial intrapulmonary artery. RT-PCR, Western blot, immunostaining, and calcium imaging (fluo-4/AM) showed that PAF express functional TRPV4 channels. In extension of these results, using pharmacological and siRNA approaches, we demonstrated TRPV4 involvement in PAF proliferation (BrdU incorporation) and migration (wound-healing assay). Then, Western blot experiments revealed that TRPV4 activation upregulates the expression of extracellular matrix protein synthesis (collagen type I and fibronectin). Finally, we explored the role of TRPV4 in the adventitial remodeling occurring in PH. By means of Western blot, we determined that TRPV4 protein expression was upregulated in adventitia from chronically hypoxic and monocrotaline rats, two animal models of PH. Furthermore, morphometric analysis indicated that adventitial remodeling is attenuated in PH-induced trpv4 -/- mice. These data support the concept that PAF play an essential role in hypertensive pulmonary vascular remodeling and point out the participation of TRPV4 channel activity in PAF activation leading to excessive adventitial remodeling.

In intrapulmonary arteries (IPA), endothelial cells (EC) respond to mechanical stimuli by releasing vasoactive factors to set the vascular tone. Piezo1, a stretch-activated, calcium-permeable channel, is a sensor of mechanical stress in EC. The present study was undertaken to investigate the implication of Piezo1 in the endothelium-dependent regulation of IPA tone and potential involvement of Piezo1 in pulmonary hypertension, the main disease of this circulation. IPA tone was quantified by means of a myograph in control Piezo1 +/+ mice and in mice lacking endothelial Piezo1 (EC-Piezo1 -/- ). Endothelial intracellular calcium concentration ([Ca 2+ ] i ) and nitric oxide (NO) production were measured, in mouse or human EC, with Fluo-4 or DAF-FM probe, respectively. Immunofluorescent labeling and patch-clamp experiments revealed the presence of Piezo1 channels in EC. Yoda1, a Piezo1 agonist, induced an endothelium-dependent relaxation that was significantly reduced in pulmonary arteries in EC-Piezo1 -/- compared with Piezo1 +/+ mice. Yoda1 as well as mechanical stimulation (by osmotic stress) increased [Ca 2+ ] i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca 2+ ] i increases were reduced in EC from Piezo1 -/- mice or in the presence of Piezo1 inhibitors. Furthermore, deletion of Piezo1 increased α-adrenergic agonist-mediated contraction. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, Piezo1 still mediated arterial relaxation, and deletion of this channel did not impair the development of the disease. The present study thus demonstrates that endothelial Piezo1 contributes to intrapulmonary vascular relaxation by controlling endothelial [Ca 2+ ] i and NO production and that this effect is still present in pulmonary hypertension.

Recent studies have revealed that particulate matter (PM) exert deleterious effects on vascular function. Pulmonary artery endothelial cells (HPAEC), which are involved in the vasomotricity regulation, can be a direct target of inhaled particles. Modifications in calcium homeostasis and oxidative stress are critical events involved in the physiopathology of vascular diseases. The objectives of this study were to assess the effects of PM 2.5 on oxidative stress and calcium signaling in HPAEC. Different endpoints were studied, (i) intrinsic and intracellular production of reactive oxygen species (ROS) by the H 2 DCF-DA probe, (ii) intrinsic, intracellular and mitochondrial production of superoxide anion (O 2 - ) by electronic paramagnetic resonance spectroscopy and MitoSOX probe, (iii) reactive nitrosative species (RNS) production by Griess reaction, and (vi) calcium signaling by the Fluo-4 probe. In acellular conditions, PM 2.5 leads to an intrinsic free radical production (ROS, O 2 - ) and a 4h-exposure to PM 2.5 (5-15μg/cm 2 ), induced, in HPAEC, an increase of RNS, of global ROS and of cytoplasmic and mitochondrial O 2 - levels. The basal intracellular calcium ion level [Ca 2+ ]i was also increased after 4h-exposure to PM 2.5 and a pre-treatment with superoxide dismutase and catalase significantly reduced this response. This study provides evidence that the alteration of intracellular calcium homeostasis induced by PM 2.5 is closely correlated to an increase of oxidative stress., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Pulmonary arterial hypertension (PAH) is a progressive disease with a poor prognosis. Pulmonary artery smooth muscle cells (PASMCs) play a crucial role in PAH pathophysiology, displaying a hyperproliferative, and apoptotic-resistant phenotype. In the present study, we evaluated the potential therapeutic role of terameprocol (TMP), an inhibitor of cellular proliferation and promoter of apoptosis, in a well-established pre-clinical model of PAH induced by monocrotaline (MCT) and studied the biological pathways modulated by TMP in PASMCs. Wistar rats injected with MCT or saline (SHAM group) were treated with TMP or vehicle. On day 21 after injection, we assessed bi-ventricular hemodynamics and cardiac and pulmonary morphometry. The effects of TMP on PASMCs were studied in a primary culture isolated from SHAM and MCT-treated rats, using an iTRAQ-based proteomic approach to investigate the molecular pathways modulated by this drug. In vivo, TMP significantly reduced pulmonary and cardiac remodeling and improved cardiac function in PAH. In vitro, TMP inhibited proliferation and induced apoptosis of PASMCs. A total of 65 proteins were differentially expressed in PASMCs from MCT rats treated with TMP, some of which involved in the modulation of transforming growth factor beta pathway and DNA transcription. Anti-proliferative effect of TMP seems to be explained, at least in part, by the down-regulation of the transcription factor HMGB1. Our findings support the beneficial role of TMP in PAH and suggest that it may be an effective therapeutic option to be considered in the clinical management of PAH., (© 2016 Wiley Periodicals, Inc.)

In pulmonary arterial endothelial cells, Ca 2+ channels and intracellular Ca 2+ concentration ([Ca 2+ ] i ) control the release of vasorelaxant factors such as nitric oxide and are involved in the regulation of pulmonary arterial blood pressure. The present study was undertaken to investigate the implication of T-type voltage-gated Ca 2+ channels (T-VGCCs, Ca v 3.1 channel) in the endothelium-dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Ca v 3.1 -/- mice. Endothelial [Ca 2+ ] i and NO production were measured, on whole vessels, with the fluo-4 and DAF-fm probes. Acetylcholine (ACh) induced a nitric oxide- and endothelium-dependent relaxation that was significantly reduced in pulmonary arteries from Ca v 3.1 -/- compared to wild type mice as well as in the presence of T-VGCC inhibitors (NNC 55-0396 or mibefradil). ACh also increased endothelial [Ca 2+ ] i and NO production that were both reduced in Ca v 3.1 -/- compared to wild type mice or in the presence of T-VGCC inhibitors. Immunofluorescence labeling revealed the presence of Ca v 3.1 channels in endothelial cells that co-localized with endothelial nitric oxide synthase in arteries from wild type mice. TRPV4-, beta2 adrenergic- and nitric oxide donors (SNP)-mediated relaxation were not altered in Ca v 3.1 -/- compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Ca v 3.1 channels activity. The present study thus demonstrates that T-VGCCs, mainly Ca v 3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca 2+ ] i and ACh-mediated relaxation., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Serotonin (5-HT) is a potent vasoconstrictor agonist and contributes to several vascular diseases including systemic or pulmonary hypertension and atherosclerosis. Although superoxide anion ([Formula: see text]) is commonly associated to cellular damages due to [Formula: see text] overproduction, we previously demonstrated that, in physiological conditions, [Formula: see text] also participates to the 5-HT contraction in intrapulmonary arteries (IPA). Here, we focused on the signaling pathways leading to [Formula: see text] production in response to 5-HT in rat IPA. Using electron paramagnetic resonance on rat IPA, we showed that 5-HT (100 μM)-induced [Formula: see text] production was inhibited by ketanserin (1 μM-an inhibitor of the 5-HT 2 receptor), absence of extracellular calcium, two blockers of voltage-independent calcium permeable channels (RHC80267 50 μM and LOE-908 10 μM) and a blocker of the mitochondrial complex I (rotenone-100 nM). Depletion of calcium from the sarcoplasmic reticulum or nicardipine (1 μM-an inhibitor of the L-type voltage-dependent calcium channel) had no effect on the 5-HT-induced [Formula: see text] production. [Formula: see text] levels were also increased by α-methyl-5-HT (10 μM-a 5-HT 2 receptors agonist) whereas GR127935 (1 μM-an antagonist of the 5-HT 1B/D receptor) and citalopram (1 μM-a 5-HT transporter inhibitor) had no effect on the 5-HT-induced [Formula: see text] production. Peroxynitrites were increased in response to 5-HT (100 μM). In isolated pulmonary arterial smooth muscle cells loaded with rhod-2 or mitosox probes, we respectively showed that 5-HT increased both mitochondrial calcium and [Formula: see text] levels, which were both abrogated in absence of extracellular calcium. Mitochondrial [Formula: see text] levels were also abolished in the presence of rotenone (100 nM). In pulmonary arterial smooth muscle cells loaded with TMRM, we showed that 5-HT transiently depolarized the mitochondrial membrane whereas in the absence of extracellular calcium the mitochondrial membrane depolarisation was delayed and sustained in response to 5-HT. 5-HT decreased the mitochondrial respiratory rate measured with a Clark oxygen electrode. Altogether, in physiological conditions, 5-HT acts on 5-HT 2 receptors and induces an [Formula: see text] production dependent on extracellular calcium and mitochondria.

The development and use of nanomaterials, especially engineered nanoparticles (NP), is expected to provide many benefits. But at the same time the development of such materials is also feared because of their potential human health risks. Indeed, NP display some characteristics similar to ultrafine environmental particles which are known to exert deleterious cardiovascular effects including pro-hypertensive ones. In this context, the effect of NP on calcium signalling, whose deregulation is often involved in hypertensive diseases, remain poorly described. We thus assessed the effect of SiO 2 NP on calcium signalling by fluorescence imaging and on the proliferation response in rat pulmonary artery smooth muscle cells (PASMC). In PASMC, acute exposure to SiO 2 NP, from 1 to 500μg/mL, produced an increase of the [Ca 2+ ] i . In addition, when PASMC were exposed to NP at 200μg/mL, a proliferative response was observed. This calcium increase was even greater in PASMC isolated from rats suffering from pulmonary hypertension. The absence of extracellular calcium, addition of diltiazem or nicardipine (L-type voltage-operated calcium channel inhibitors both used at 10μM), and addition of capsazepine or HC067047 (TRPV1 and TRPV4 inhibitors used at 10μM and 5μM, respectively) significantly reduced this response. Moreover, this response was also inhibited by thapsigargin (SERCA inhibitor, 1μM), ryanodine (100μM) and dantrolene (ryanodine receptor antagonists, 10μM) but not by xestospongin C (IP 3 receptor antagonist, 10μM). Thus, NP induce an intracellular calcium rise in rat PASMC originating from both extracellular and intracellular calcium sources. This study also provides evidence for the implication of TRPV channels in NP induced calcium rise that may highlight the role of these channels in the deleterious cardiovascular effects of NP., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Connexins are transmembrane proteins that can generate intercellular communication channels known as gap junctions. They contribute to the direct movement of ions and larger cytoplasmic solutes between various cell types. In the lung, connexins participate in a variety of physiological functions, such as tissue homeostasis and host defence. In addition, emerging evidence supports a role for connexins in various pulmonary inflammatory diseases, such as asthma, pulmonary hypertension, acute lung injury, lung fibrosis or cystic fibrosis. In these diseases, the altered expression of connexins leads to disruption of normal intercellular communication pathways, thus contributing to various pathophysiological aspects, such as inflammation or tissue altered reactivity and remodeling. The present review describes connexin structure and organization in gap junctions. It focuses on connexins in the lung, including pulmonary bronchial and arterial beds, by looking at their expression, regulation and physiological functions. This work also addresses the issue of connexin expression alteration in various pulmonary inflammatory diseases and describes how targeting connexin-based gap junctions with pharmacological tools, synthetic blocking peptides or genetic approaches, may open new therapeutic perspectives in the treatment of these diseases., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Caveolae are stiff plasma membrane microdomains implicated in various cell response mechanisms like Ca(2+) signaling and mechanotransduction. Pulmonary arterial smooth muscle cells (PASMC) transduce mechanical stimuli into Ca(2+) increase via plasma membrane stretch-activated channels (SAC). This mechanotransduction process is modified in pulmonary hypertension (PH) during which stretch forces are increased by the increase in arterial blood pressure. We propose to investigate how caveolae are involved in the pathophysiology of PH and particularly in mechanotransduction. PASMC were freshly isolated from control rats (Ctrl rats) and rats suffering from PH induced by 3 wk of chronic hypoxia (CH rats). Using a caveolae disrupter (methyl-β-cyclodextrin), we showed that SAC activity measured by patch-clamp, stretch-induced Ca(2+) increase measured with indo-1 probe and pulmonary arterial ring contraction to osmotic shock are enhanced in Ctrl rats when caveolae are disrupted. In CH rats, SAC activity, Ca(2+), and contraction responses to stretch are all higher compared with Ctrl rats. However, in contrast to Ctrl rats, caveolae disruption in CH-PASMC, reduces SAC activity, Ca(2+) responses to stretch and arterial contractions. Furthermore, by means of immunostainings and transmission electron microscopy, we observed that caveolae and caveolin-1 are expressed in PASMC from both Ctrl and CH rats and localize close to subplasmalemmal sarcoplasmic reticulum (ryanodine receptors) and mitochondria, thus facilitating Ca(2+) exchanges, particularly in CH. In conclusion, caveolae are implicated in mechanotransduction in Ctrl PASMC by buffering mechanical forces. In PH-PASMC, caveolae form a distinct Ca(2+) store facilitating Ca(2+) coupling between SAC and sarcoplasmic reticulum., (Copyright © 2016 the American Physiological Society.)

Particulate air pollution exerts deleterious effects on cardiovascular system. We previously described that exposure to urban particulate matter (SRM1648) impairs nitric oxide (NO, a major vasculoprotective factor) responsiveness in intrapulmonary arteries. As Heme Oxygenase-1 (HO-1) is induced by urban particles in some cell types and is known to alter NO-dependent signaling pathway, the objective was to characterize HO-1 involvement in SRM1648-induced impairment of NO-dependent relaxation in intrapulmonary arteries. Rat intrapulmonary artery rings were exposed or not to Co (III) Protoporphyrin IX Chloride (HO-1 inducer) or SRM1648 in the absence or presence of Cr (III) Mesoporphyrin IX Chloride (HO-1 activity inhibitor). NO-dependent relaxation was assessed with DEA-NOnoate (DEA-NO) on pre-contracted arteries. HO-1 and soluble guanylyl-cyclase (sGC) mRNA and protein expressions were assessed by qRT-PCR and Western blotting, respectively. SRM1648 or Co (III) Protoporphyrin IX Chloride exposure (24) impaired DEA-NO-dependent relaxation. The SRM-induced alteration of DEA-NO responsiveness was partially prevented by Cr (III) Mesoporphyrin IX Chloride. Co (III) Protoporphyrin IX Chloride induced HO-1 mRNA and protein expressions, whereas SRM1648 only induced HO-1 protein expression without affecting its mRNA level. Exposure to either SRM1648 or to Co (III) Protoporphyrin IX Chloride did not affect the expression levels of sGC. In conclusion, this study provides some evidence that impairment of NO signaling pathway in intrapulmonary arteries involves HO-1. Therefore it highlights the role of HO-1 in particulate matter-induced detrimental effects in pulmonary circulation., (Copyright © 2016. Published by Elsevier Ltd.)

Transient receptor potential (TRP) channels of the vanilloid subfamily, mainly TRPV1 and TRPV4, are expressed in pulmonary artery smooth muscle cells (PASMC) and implicated in the remodeling of pulmonary artery, a landmark of pulmonary hypertension (PH). Among a variety of PH subtypes, PH of group 3 are mostly related to a prolonged hypoxia exposure occurring in a variety of chronic lung diseases. In the present study, we thus investigated the role of hypoxia on TRPV1 and TRPV4 channels independently of the increased pulmonary arterial pressure that occurs during PH. We isolated PASMC from normoxic rat and cultured these cells under in vitro hypoxia. Using microspectrofluorimetry and the patch-clamp technique, we showed that hypoxia (1 % O2 for 48 h) significantly increased stretch- and TRPV4-induced calcium responses. qRT-PCR, Western blotting, and immunostaining experiments revealed that the expression of TRPV1 and TRPV4 was not enhanced under hypoxic conditions, but we observed a membrane translocation of TRPV1. Furthermore, hypoxia induced a reorganization of the F-actin cytoskeleton, the tubulin, and intermediate filament networks (immunostaining experiments), associated with an enhanced TRPV1- and TRPV4-induced migratory response (wound-healing assay). Finally, as assessed by immunostaining, exposure to in vitro hypoxia elicited a significant increase in NFATc4 nuclear localization. Cyclosporin A and BAPTA-AM inhibited NFATc4 translocation, indicating the activation of the Ca(2+)/calcineurin/NFAT pathway. In conclusion, these data point out the effect of hypoxia on TRPV1 and TRPV4 channels in rat PASMC, suggesting that these channels can act as direct signal transducers in the pathophysiology of PH.

Rationale: Pulmonary hypertension (PH) is characterized by a progressive elevation in mean pulmonary arterial pressure, often leading to right ventricular failure and death. Growth factors play significant roles in the pathogenesis of PH, and their targeting may therefore offer novel therapeutic strategies in this disease., Objectives: To evaluate the nerve growth factor (NGF) as a potential new target in PH., Methods: Expression and/or activation of NGF and its receptors were evaluated in rat experimental PH induced by chronic hypoxia or monocrotaline and in human PH (idiopathic or associated with chronic obstructive pulmonary disease). Effects of exogenous NGF were evaluated ex vivo on pulmonary arterial inflammation and contraction, and in vitro on pulmonary vascular cell proliferation, migration, and cytokine secretion. Effects of NGF inhibition were evaluated in vivo with anti-NGF blocking antibodies administered both in rat chronic hypoxia- and monocrotaline-induced PH., Measurements and Main Results: Our results show increased expression of NGF and/or increased expression/activation of its receptors in experimental and human PH. Ex vivo/in vitro, we found out that NGF promotes pulmonary vascular cell proliferation and migration, pulmonary arterial hyperreactivity, and secretion of proinflammatory cytokines. In vivo, we demonstrated that anti-NGF blocking antibodies prevent and reverse PH in rats through significant reduction of pulmonary arterial inflammation, hyperreactivity, and remodeling., Conclusions: This study highlights the critical role of NGF in PH. Because of the recent development of anti-NGF blocking antibodies as a possible new pain treatment, such a therapeutic strategy of NGF inhibition may be of interest in PH.

Mitochondria are essential cell organelles responsible for ATP production in the presence of oxygen. In the pulmonary vasculature, mitochondria contribute to physiological intracellular signalling pathways through production of reactive oxygen species and play the role of oxygen sensors that coordinate hypoxic pulmonary vasoconstriction. Mitochondria also play a pathophysiological role in pulmonary hypertension (PH). This disease is characterized by increased pulmonary arterial pressure and remodelling of pulmonary arteries, leading to increased pulmonary vascular resistance, hypertrophy of the right ventricle, right heart failure and ultimately death. Mitochondrial alterations have been evidenced in PH in pulmonary arteries and in the right ventricle, in particular a chronic shift in energy production from mitochondrial oxidative phosphorylation to glycolysis. This shift, initially described in cancer cells, may play a central role in PH pathogenesis. Further studies of these metabolic mitochondrial alterations in PH may therefore open new therapeutic perspectives in this disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Aims: Pulmonary hypertension (PH) is the main disease of pulmonary circulation. Alteration in calcium homeostasis in pulmonary artery smooth muscle cells (PASMCs) is recognized as a key feature in PH. The present study was undertaken to investigate the involvement of T-type voltage-gated calcium channels (T-VGCCs) in the control of the pulmonary vascular tone and thereby in the development of PH., Methods and Results: Experiments were conducted in animals (rats and mice) kept 3-4 weeks in either normal (normoxic) or hypoxic environment (hypobaric chamber) to induce chronic hypoxia (CH) PH. In vivo, chronic treatment of CH rats with the T-VGCC blocker, TTA-A2, prevented PH and the associated vascular hyperreactivity, pulmonary arterial remodelling, and right cardiac hypertrophy. Deletion of the Cav3.1 gene (a T-VGCC isoform) protected mice from CH-PH. In vitro, patch-clamp and PCR experiments revealed the presence of T-VGCCs (mainly Cav3.1 and Cav3.2) in PASMCs. Mibefradil, NNC550396, and TTA-A2 inhibited, in a concentration-dependent manner, T-VGCC current, KCl-induced contraction, and PASMC proliferation., Conclusion: The present study demonstrates that T-VGCCs contribute to intrapulmonary vascular reactivity and is implicated in the development of hypoxic PH. Specific blockers of T-VGCCs may thus prove useful for the therapeutic management of PH., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

Aim: Calcium is a key second messenger that can be mobilized from both the extracellular medium and intracellular calcium stores. Pulmonary arterial smooth muscle cells (PASMCs) respond to stretch by a calcium increase, a mechanism enhanced during pulmonary hypertension (PH). We investigated the role of the spatial organization between plasma membrane stretch-activated channels (SACs) and intracellular calcium stores [sarcoplasmic reticulum (SR), mitochondria, and lysosomes) in response to stretch., Methods and Results: Studies were performed in freshly isolated PASMCs from both control and two different rat models of PH (chronically hypoxic and monocrotaline-treated rats). Co-immunolabellings revealed that the subcellular segregation between each subtype of SR ryanodine receptors (RyR1, RyR2, and RyR3), SERCA2 pumps (SERCA2a and SERCA2b), mitochondria, or lysosomes in freshly isolated PASMCs differs from control and PH PASMCs. In control PASMCs, stretching the membrane activates a Ca(2+) influx through SACs. This influx is amplified by cell hyperpolarization, a calcium release by subplasmalemmal RyR1 and is then buffered by mitochondria. In two different PH rat models, the calcium response to stretch is enhanced due to hyper-reactivity of SACs and a greater calcium amplification by all RyR subtypes., Conclusion: The spatial organization of RyR and calcium stores in PASMCs is important for cell signalling and plays a causal role in PH., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

We have previously shown that exposure to urban particulate matter (UPM) impairs endothelial nitric oxide (NO) bioactivity in intrapulmonary arteries. As UPM is composed of heterogeneous constituents, the aim of this study was to clarify the class of pollutants responsible for such effect. Extracts (aqueous, acidic or organic) were prepared from SRM1648, an UPM sample collected in St. Louis (MO, USA). The metal composition of extracts as well as endotoxin content was determined. The effects of each extract, metal mixture and endotoxin were evaluated on endothelium-dependent relaxation to acetylcholine (reflecting endothelial NO production) in rat isolated intrapulmonary arteries. Aqueous or organic SRM1648 pretreatment altered acetylcholine-induced relaxation, similar to that induced by native SRM1648. Organic extract induced similar attenuation of acetylcholine relaxation than organic-treated SRM1648, whereas aqueous extract had no effect. Acidic pretreatment, which impoverished metal and endotoxin content of SRM1648, prevented the impairment of acetylcholine-induced relaxation. However, neither the acidic extract enriched in metals, nor a metal mixture representative of SRM1648 content, modified acetylcholine relaxation, while endotoxin impaired it. Polymyxin B, which chelates endotoxin, prevented SRM1648-induced decrease in relaxation to acetylcholine. It is concluded that SRM1648-induced impairment of endothelial NO-dependent relaxation in intrapulmonary arteries unlikely involved a soluble factor released by vascular cells during UPM exposure, but rather an organic extractible and acidic-sensitive constituents of UPM. Endotoxin, but not metals, may be responsible for UPM-induced impairment of endothelial NO-dependent relaxation., (Copyright © 2013 John Wiley & Sons, Ltd.)

Bidens pilosa is an Asteraceae widely used in traditional medicine for the treatment of various ailments including pain and inflammation. The present work was undertaken to assess the analgesic and antiinflammatory properties of the ethyl acetate fraction of methylene chloride/methanol (1:1) extract of leaves of Bidens pilosa at the gradual doses of 50, 100 and 200 mg/kg in mice and rats, respectively. The analgesic properties of Bidens pilosa were investigated using the acetic acid writhing, hot plate, capsaicin and formalin-induced pain models. This was followed by a study of the antiinflammatory properties using carrageenan, dextran, histamine and serotonin to induce acute inflammation in rat hind paw. The extract provided a significant (p < 0.01) reduction in pain induced by all four models of nociception. It also presented significant (p < 0.05) antiinflammatory activity in all four models of acute inflammation. These results show that the ethyl acetate fraction of methylene chloride/methanol (1:1) of Bidens pilosa has both analgesic and antiinflammatory properties. The qualitative analysis of the fraction by the high-performance liquid chromatography (HPLC) fingerprint revealed the presence of two flavonoids, namely quercetin and iso-okanin, known to have antiinflammatory and antinociceptive properties, which could be responsible for the analgesic and antiinflammatory effects observed.

The development of right heart failure (RHF) is characterized by alterations of right ventricle (RV) structure and function, but the mechanisms of RHF remain still unknown. Thus, understanding the RHF is essential for improved therapies. Therefore, identification by quantitative proteomics of targets specific to RHF may have therapeutic benefits to identify novel potential therapeutic targets. The objective of this study was to analyze the molecular mechanisms changing RV function in the diseased RHF and thus, to identify novel potential therapeutic targets. For this, we have performed differential proteomic analysis of whole RV proteins using two experimental rat models of RHF. Differential protein expression was observed for hundred twenty six RV proteins including proteins involved in structural constituent of cytoskeleton, motor activity, structural molecule activity, cytoskeleton protein binding and microtubule binding. Interestingly, further analysis of down-regulated proteins, reveals that both protein and gene expressions of proteasome subunits were drastically decreased in RHF, which was accompanied by an increase of ubiquitinated proteins. Interestingly, the proteasomal activities chymotrypsin and caspase-like were decreased whereas trypsin-like activity was maintained. In conclusion, this study revealed the involvement of ubiquitin-proteasome system (UPS) in RHF. Three deregulated mechanisms were discovered: (1) decreased gene and protein expressions of proteasome subunits, (2) decreased specific activity of proteasome; and (3) a specific accumulation of ubiquitinated proteins. This modulation of UPS of RV may provide a novel therapeutic avenue for restoration of cardiac function in the diseased RHF., (© 2013.)

Tetrahydrobiopterin (BH4), which fosters the formation of and stabilizes endothelial NO synthase (eNOS) as an active dimer, tightly regulates eNOS coupling / uncoupling. Moreover, studies conducted in genetically-modified models demonstrate that BH4 pulmonary deficiency is a key determinant in the pathogenesis of pulmonary hypertension. The present study thus investigates biopterin metabolism and eNOS expression, as well as the effect of sepiapterin (a precursor of BH4) and eNOS gene deletion, in a mice model of hypoxic pulmonary hypertension. In lungs, chronic hypoxia increased BH4 levels and eNOS expression, without modifying dihydrobiopterin (BH2, the oxidation product of BH4) levels, GTP cyclohydrolase-1 or dihydrofolate reductase expression (two key enzymes regulating BH4 availability). In intrapulmonary arteries, chronic hypoxia also increased expression of eNOS, but did not induce destabilisation of eNOS dimers into monomers. In hypoxic mice, sepiapterin prevented increase in right ventricular systolic pressure and right ventricular hypertrophy, whereas it modified neither remodelling nor alteration in vasomotor responses (hyper-responsiveness to phenylephrine, decrease in endothelium-dependent relaxation to acetylcholine) in intrapulmonary arteries. Finally, deletion of eNOS gene partially prevented hypoxia-induced increase in right ventricular systolic pressure, right ventricular hypertrophy and remodelling of intrapulmonary arteries. Collectively, these data demonstrate the absence of BH4/BH2 changes and eNOS dimer destabilisation, which may induce eNOS uncoupling during hypoxia-induced pulmonary hypertension. Thus, even though eNOS gene deletion and sepiapterin treatment exert protective effects on hypoxia-induced pulmonary vascular remodelling, increase on right ventricular pressure and / or right ventricular hypertrophy, these effects appear unrelated to biopterin-dependent eNOS uncoupling within pulmonary vasculature of hypoxic wild-type mice.

Background: Pulmonary hypertension (PH) is a disease that affects the adult or infant population. Dehydroepiandrosterone (DHEA), a steroid hormone, has been previously shown to prevent and to reverse PH in an adult rat model. We thus investigated its effect in a rat-pup model of chronic hypoxic PH., Methods: Animals were maintained for 3 wk in a hypobaric chamber to induce PH, with or without concomitant treatment with DHEA (30 mg/kg every alternate day)., Results: DHEA significantly reduced mean pulmonary artery pressure (measured by right cardiac catheterization), pulmonary artery remodeling (evaluated by histology), and right-ventricular hypertrophy (measured by echography and by the Fulton index). At the level of the pulmonary artery smooth muscle cell (PASMC), DHEA increased activity and expression of the large-conductance Ca2+-activated potassium channel (BKCa) (assessed by means of the patch clamp technique). DHEA also inhibited both serotonin- and KCl-induced contraction and smooth muscle cell proliferation., Conclusion: Collectively, these results indicate that DHEA prevents PH in infant rats and may therefore be clinically relevant for the management of PH in human infants.

Pulmonary hypertension (PH) is characterized by a progressive elevation of pulmonary arterial pressure due to alterations of both pulmonary vascular structure and function. This disease is rare but life-threatening, leading to the development of right heart failure. Current PH treatments, designed to target altered pulmonary vascular reactivity, include vasodilating prostanoids, phosphodiesterase-5 inhibitors and endothelin-1 receptor antagonists. Although managing to slow the progression of the disease, these molecules still do not cure PH. More effective treatments need to be developed, and novel therapeutic strategies, targeting in particular vascular remodelling, are currently under investigation. Reactive oxygen species (ROS) are important physiological messengers in vascular cells. In addition to atherosclerosis and other systemic vascular diseases, emerging evidence also support a role of ROS in PH pathogenesis. ROS production is increased in animal models of PH, associated with NADPH oxidases increased expression, in particular of several Nox enzymes thought to be the major source of ROS in the pulmonary vasculature. These increases have also been observed in vitro and in vivo in humans. Moreover, several studies have shown either the deleterious effect of agents promoting ROS generation on pulmonary vasculature or, conversely, the beneficial effect of antioxidant agents in animal models of PH. In these studies, ROS production has been directly linked to pulmonary vascular remodelling, endothelial dysfunction, altered vasoconstrictive responses, inflammation and modifications of the extracellular matrix, all important features of PH pathophysiology. Altogether, these findings indicate that ROS are interesting therapeutic targets in PH. Blockade of ROS-dependent signalling pathways, or disruption of sources of ROS in the pulmonary vasculature, targeting in particular Nox enzymes, represent promising new therapeutic strategies in this disease.

Dehydroepiandrosterone (DHEA) is a steroid hormone derived from cholesterol synthesized by the adrenal glands. DHEA and its 3β-sulphate ester (DHEA-S) are the most abundant circulating steroid hormones. In human, there is a clear age-related decline in serum DHEA and DHEA-S and this has suggested that a relative deficiency in these steroids may be causally related to the development of a series of diseases associated with aging including cardiovascular diseases (CVD). This commentary aims to highlight the action of DHEA in CVD and its beneficial effect in therapy. We thus discuss the possible impact of serum DHEA decline and DHEA supplementation in diseases such as hypertension, coronary artery disease and atherosclerosis. More specifically, we provide evidence for a beneficial action of DHEA in the main disease of the pulmonary circulation: pulmonary hypertension. We also examine the potential cellular mechanism of action of DHEA in terms of receptors (membrane/nuclear) and associated signaling pathways (ion channels, calcium signaling, PI3K/AKT/eNos pathway, cGMP, RhoA/RhoK pathway). We show that DHEA acts as an anti-remodeling and vasorelaxant drug. Since it is a well-tolerated and inexpensive drug, DHEA may prove to be a valuable molecule in CVD but it deserves further studies both at the molecular level and in large clinical trials., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Dehydroepiandrosterone (DHEA) prevents chronic hypoxia-induced pulmonary hypertension and associated right ventricle dysfunction in rats. In this animal model, reoxygenation following hypoxia reverses pulmonary hypertension but not right ventricle dysfunction. We thus studied the effect of DHEA on the right ventricle after reoxygenation, i.e. after a normoxic recovery phase secondary to chronic hypoxia in rats. Right ventricle function was assessed in vivo by Doppler echocardiography and in vitro by the isolated perfused heart technique in three groups of animals: control, recovery (21 days of hypoxia followed by 21 days of normoxia) and recovery DHEA (30 mg · kg(-1) every 2 days during the recovery phase). Right ventricle tissue was assessed by optical and electron microscopy. DHEA abolished right ventricle diastolic dysfunction, as the echographic E wave remained close to that of controls (mean ± SD 76.5 ± 2.4 and 79.7 ± 1.7 cm · s(-1), respectively), whereas it was diminished to 40.3 ± 3.7 in the recovery group. DHEA also abolished right ventricle systolic dysfunction, as shown by the inhibition of the increase in the slope of the pressure-volume curve in isolated heart. The DHEA effect was related to cardiac myocytes proliferation. In conclusion, DHEA prevents right ventricle dysfunction in this animal model by preventing cardiomyocyte alteration.

There is a growing body of evidence indicating that transient receptor potential (TRP) channels are implicated in calcium signaling and various cellular functions in the pulmonary vasculature. The aim of this study was to investigate the expression, functional role, and coupling to reticulum calcium channels of the type 4 vanilloid TRP subfamily (TRPV4) in the pulmonary artery from both normoxic (Nx) and chronically hypoxic (CH) rats. Activation of TRPV4 with the specific agonist 4α-phorbol-12,13-didecanoate (4α-PDD, 5 μM) increased the intracellular calcium concentration ([Ca(2+)](i)). This effect was significantly reduced by a high concentration of ryanodine (100 μM) or chronic caffeine (5 mM) that blocked ryanodine receptor (RyR) but was insensitive to xestospongin C (10 μM), an inositol trisphosphate receptor antagonist. Inhibition of RyR1 and RyR3 only with 10 μM of dantrolene did not attenuate the 4α-PDD-induced [Ca(2+)](i) increase. Western blotting experiments revealed the expression of TRPV4 and RyR2 with an increase in both receptors in pulmonary arteries from CH rats vs. Nx rats. Accordingly, the 4α-PDD-activated current, measured with patch-clamp technique, was increased in pulmonary artery smooth muscle cells (PASMC) from CH rats vs. Nx rats. 4α-PDD increased isometric tension in artery rings, and this response was also potentiated under chronic hypoxia conditions. 4α-PDD-induced calcium response, current, and contraction were all inhibited by the selective TRPV4 blocker HC-067047. Collectively, our findings provide evidence of the interplay between TRPV4 and RyR2 in the Ca(2+) release mechanism and contraction in PASMC. This study provides new insights onto the complex calcium signaling in PASMC and point out the importance of the TRPV4-RyR2 signaling pathway under hypoxic conditions that may lead to pulmonary hypertension.

Pulmonary hypertension, the main disease of the pulmonary circulation, is characterized by an increase in pulmonary vascular resistance, involving proliferation and migration of pulmonary arterial smooth muscle cells (PASMC). However, cellular and molecular mechanisms underlying these phenomena remain to be identified. In the present study, we thus investigated in rat intrapulmonary arteries (1) the expression and the functional activity of TRPV1 and TRPV4, (2) the PASMC migration triggered by these TRPV channels, and (3) the associated reorganization of the cytoskeleton. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis demonstrated expression of TRPV1 and TRPV4 mRNA in rat intrapulmonary arteries. These results were confirmed at the protein level by western blot. Using microspectrofluorimetry (indo-1), we show that capsaicin and 4α-phorbol-12,13-didecanoate (4α-PDD), selective agonists of TRPV1 and TRPV4, respectively, increased the intracellular calcium concentration of PASMC. Furthermore, stimulation of TRPV1 and TRPV4 induced PASMC migratory responses, as assessed by two different methods (a modified Boyden chamber assay and a wound-healing migration assay). This response cannot seem to be attributed to a proliferative effect as assessed by BrdU and Wst-1 colorimetric methods. Capsaicin- and 4α-PDD-induced calcium and migratory responses were inhibited by the selective TRPV1 and TRPV4 blockers, capsazepine and HC067047, respectively. Finally, as assessed by immunostaining, these TRPV-induced migratory responses were associated with reorganization of the F-actin cytoskeleton and the tubulin and intermediate filament networks. In conclusion, these data point out, for the first time, the implication of TRPV1 and TRPV4 in rat PASMC migration, suggesting the implication of these TRPV channels in the physiopathology of pulmonary hypertension.

Background and Purpose: Dehydroepiandrosterone (DHEA) and its sulfated form, DHEAS, are the most abundant steroid hormones in the mammalian blood flow. DHEA may have beneficial effects in various pathophysiological conditions such as cardiovascular diseases or deterioration of the sense of well-being. However to date, the cellular mechanism underlying DHEA action remains elusive and may involve ion channel modulation. In this study, we have characterized the effect of DHEA on T-type voltage-activated calcium channels (T-channels), which are involved in several cardiovascular and neuronal diseases., Key Results: Using the whole-cell patch-clamp technique, we demonstrate that DHEA inhibits the three recombinant T-channels (Ca(V)3.1, Ca(V)3.2 and Ca(V)3.3) expressed in NG108-15 cell line, as well as native T-channels in pulmonary artery smooth muscle cells. This effect of DHEA is both concentration (IC(50) between 2 and 7μM) and voltage-dependent and results in a significant shift of the steady-state inactivation curves toward hyperpolarized potentials. Consequently, DHEA reduces window T-current and inhibits membrane potential oscillations induced by Ca(V)3 channels. DHEA inhibition is not dependent on the activation of nuclear androgen or estrogen receptors and implicates a PTX-sensitive Gi protein pathway. Functionally, DHEA and the T-type inhibitor NNC 55-0396 inhibited KCl-induced contraction of pulmonary artery rings and their effect was not cumulative., Conclusions: Altogether, the present data demonstrate that DHEA inhibits T-channels by a Gi protein dependent pathway. DHEA-induced alteration in T-channel activity could thus account for its therapeutic action and/or physiological effects., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objectives: It was previously shown that dehydroepiandrosterone (DHEA) reverses chronic hypoxia-induced pulmonary hypertension (PH) in rats, but whether DHEA can improve the clinical and hemodynamic status of patients with PH associated to chronic obstructive pulmonary disease (PH-COPD) has not been studied whereas it is a very severe poorly treated disease., Patients and Methods: Eight patients with PH-COPD were treated with DHEA (200mg daily orally) for 3 months. The primary end-point was the change in the 6-minute walk test (6-MWT) distance. Secondary end-points included pulmonary hemodynamics, lung function tests and tolerance of treatment., Results: The 6-MWT increased in all cases, from 333m (median [IQR]) (257; 378) to 390m (362; 440) (P<0.05). Mean pulmonary artery pressure decreased from 26mmHg (25; 27) to 21.5mmHg (20; 25) (P<0.05) and pulmonary vascular resistance from 4.2UI (3.5; 4.4) to 2.6UI (2.5; 3.8) (P<0.05). The carbon monoxide diffusing capacity of the lung (DLCO % predicted) increased significantly from 27.4% (20.1; 29.3) to 36.4% (14.6; 39.6) (P<0.05). DHEA treatment did not change respiratory parameters of gas exchange and the 200mg per day of DHEA used was perfectly tolerated with no side effect reported., Conclusion: DHEA treatment significantly improves 6-MWT distance, pulmonary hemodynamics and DLCO of patients with PH-COPD, without worsening gas exchange, as do other pharmacological treatments of PH (trial registration NCT00581087)., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Background: Pulmonary hypertension (PH) is characterized by arterial vascular remodelling and alteration in vascular reactivity. Since gap junctions are formed with proteins named connexins (Cx) and contribute to vasoreactivity, we investigated both expression and role of Cx in the pulmonary arterial vasoreactivity in two rat models of PH., Methods: Intrapulmonary arteries (IPA) were isolated from normoxic rats (N), rats exposed to chronic hypoxia (CH) or treated with monocrotaline (MCT). RT-PCR, Western Blot and immunofluorescent labelling were used to study the Cx expression. The role of Cx in arterial reactivity was assessed by using isometric contraction and specific gap junction blockers. Contractile responses were induced by agonists already known to be involved in PH, namely serotonin, endothelin-1 and phenylephrine., Results: Cx 37, 40 and 43 were expressed in all rat models and Cx43 was increased in CH rats. In IPA from N rats only, the contraction to serotonin was decreased after treatment with 37-43Gap27, a specific Cx-mimetic peptide blocker of Cx 37 and 43. The contraction to endothelin-1 was unchanged after incubation with 40Gap27 (a specific blocker of Cx 40) or 37-43Gap27 in N, CH and MCT rats. In contrast, the contraction to phenylephrine was decreased by 40Gap27 or 37-43Gap27 in CH and MCT rats. Moreover, the contractile sensitivity to high potassium solutions was increased in CH rats and this hypersensitivity was reversed following 37-43Gap27 incubation., Conclusion: Altogether, Cx 37, 40 and 43 are differently expressed and involved in the vasoreactivity to various stimuli in IPA from different rat models. These data may help to understand alterations of pulmonary arterial reactivity observed in PH and to improve the development of innovative therapies according to PH aetiology.

Chronic hypoxia alters mitochondrial energy metabolism. In the heart, oxidative capacity of both ventricles is decreased after 3 weeks of chronic hypoxia. The aim of this study was to evaluate the reversal of these metabolic changes upon normoxia recovery. Rats were exposed to a hypobaric environment for 3 weeks and then subjected to a normoxic environment for 3 weeks (normoxia-recovery group) and compared with rats maintained in a normoxic environment (control group). Mitochondrial energy metabolism was differentially examined in both left and right ventricles. Oxidative capacity (oxygen consumption and ATP synthesis) was measured in saponin-skinned fibers. Activities of mitochondrial respiratory chain complexes and antioxidant enzymes were measured on ventricle homogenates. Morphometric analysis of mitochondria was performed on electron micrographs. In normoxia-recovery rats, oxidative capacities of right ventricles were decreased in the presence of glutamate or palmitoyl carnitine as substrates. In contrast, oxidation of palmitoyl carnitine was maintained in the left ventricle. Enzyme activities of complexes III and IV were significantly decreased in both ventricles. These functional alterations were associated with a decrease in numerical density and an increase in size of mitochondria. Finally, in the normoxia-recovery group, the antioxidant enzyme activities (catalase and glutathione peroxidase) increased. In conclusion, alterations of mitochondrial energy metabolism induced by chronic hypoxia are not totally reversible. Reactive oxygen species could be involved and should be investigated under such conditions, since they may represent a therapeutic target.

Smooth muscles are widely distributed in mammal body through various systems such as circulatory, respiratory, gastro-intestinal and urogenital systems. The smooth muscle cell (SMC) is not only a contractile cell but is able to perform other important functions such as migration, proliferation, production of cytokines, chemokines, extracellular matrix proteins, growth factors and cell surface adhesion molecules. Thus, SMC appears today as a fascinating cell with remarkable plasticity that contributes to its roles in physiology and disease. Most of the SMC functions are dependent on a key event: the increase in intracellular calcium concentration ([Ca(2+)](i)). Calcium entry from the extracellular space is a major step in the elevation of [Ca(2+)](i) in SMC and involves a variety of plasmalemmal calcium channels, among them is the superfamily of transient receptor potential (TRP) proteins. TRPC (canonical), TRPM (melastatin), TRPV (vanilloid) and TRPP (polycystin), are widely expressed in both visceral (airways, gastrointestinal tract, uterus) and vascular (systemic and pulmonary circulation) smooth muscles. Mainly, TRPC, TRPV and TRPM are implicated in a variety of physiological and pathophysiological processes such as: SMC contraction, relaxation, growth, migration and proliferation; control of blood pressure, arterial myogenic tone, pulmonary hypertension, intestinal motility, gastric acidity, uterine activity during parturition and labor. Thus it is becoming evident that TRP are major element of SMC calcium homeostasis and, thus, appear as novel drug targets for a better management of diseases originating from SMC dysfunction.

Stretch-activated channels (SACs) act as membrane mechanotransducers since they convert physical forces into biological signals and hence into a cell response. Pulmonary arterial smooth muscle cells (PASMCs) are continuously exposed to mechanical stimulations e.g., compression and stretch, that are enhanced under conditions of pulmonary arterial hypertension (PAH). Using the patch-clamp technique (cell-attached configuration) in PASMCs, we showed that applying graded negative pressures (from 0 to -60 mmHg) to the back end of the patch pipette increases occurrence and activity of SACs. The current-voltage relationship (from -80 to +40 mV) was almost linear with a reversal potential of 1 mV and a slope conductance of 34 pS. SACs were inhibited in the presence of GsMTx-4, a specific SACs blocker. Using microspectrofluorimetry (indo-1), we found that hypotonic-induced cell swelling increases intracellular Ca(2+) concentration ([Ca(2+)](i)). This [Ca(2+)](i) increase was markedly inhibited in the absence of external Ca(2+) or in the presence of the following blockers of SACs: gadolinium, streptomycin, and GsMTx-4. Interestingly, in chronically hypoxic rats, an animal model of PAH, SACs were more active and hypotonic-induced calcium response in PASMCs was significantly higher (nearly a two-fold increase). Moreover, unlike in normoxic rats, intrapulmonary artery rings from hypoxic rats mounted in a Mulvany myograph, exhibited a myogenic tone sensitive to SAC blockers. In conclusion, this work demonstrates that SACs in rat PASMCs can be activated by membrane stretch as well as hypotonic stimulation and are responsible for [Ca(2+)](i) increase. The link between SACs activation-induced calcium response and myogenic tone in chronically hypoxic rats suggests that SACs are an important element for the increased pulmonary vascular tone in PAH and that they may represent a molecular target for PAH treatment., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This review describes the cellular and molecular mechanism heterogeneity of dehydroepiandrosterone (DHEA) and its putative therapeutic role in vascular remodeling diseases such as pulmonary artery hypertension (PAH). PAH is characterized by enhanced pulmonary artery smooth muscle cell (PASMC) proliferation, constriction and resistance to apoptosis, all of which contribute to increase the pulmonary artery wall thickness, resistance and therefore pressure. The etiology of PAH remains elusive. Nonetheless, the implications of endothelial dysfunction (decreased nitric oxide generation and increased endothelin production etc), PASMC K(+) channel/mitochondrial axis disruption (voltage-gated K(+) channel (Kv1.5) downregulation and mitochondrial membrane potential hyperpolarization) and the activation of survival pathways such as PI3K/Akt are now accepted. Therefore, a drug able to target all of these abnormalities would be of a great therapeutic interest for the treatment of PAH. We and others have demonstrated that DHEA, a clinically available drug with a low adverse effect profile, is able to achieve these effects. In several animal models of vascular remodeling diseases such as PAH, DHEA has been demonstrated to be a good anti-proliferative and pro-apoptotic drug, decreasing vascular remodeling, and a potent vasodilator. A better understanding of the DHEA mechanisms of action may allow the development of new and better therapies to treat vascular remodeling diseases such as pulmonary hypertension., (Copyright 2010. Published by Elsevier Inc.)

Serotonin (5-hydroxytryptamine; 5-HT) is a potent pulmonary vasoconstrictor and mitogenic agent whose plasma level is increased in pulmonary hypertensive patients. Thus, we explored the signalling pathways involved in the contractile response to 5-HT in human pulmonary arteries (HPAs). Intact and beta-escin permeabilised rings from HPAs mounted in an organ bath system were used to assess both tension and myofilament Ca(2+)-sensitisation. Microspectrofluorimetry was used for intracellular Ca(2+) recordings in cultured HPA smooth muscle cells. Voltage-operated Ca(2+) channel blockers (nitrendipine and nifedipine) partially reduced the contraction to 5-HT. Thapsigargin or cyclopiazonic acid (CPA), known to deplete sarcoplasmic reticulum Ca(2+) stores, also partially inhibited the contraction, whereas removal of extracellular Ca(2+) under these conditions further inhibited the contraction. Changing from Ca(2+)-free to Ca(2+) containing solution, in the presence of nitrendipine and CPA, a protocol known to stimulate store-operated Ca(2+) channels, induced HPA contractions that were blocked by nickel. Nickel or gadolinium also reduced the contraction to 5-HT. Finally, 5-HT increased intracellular Ca(2+) responses in cultured HPA smooth muscle cells and myofilament Ca(2+)-sensitisation in HPA rings. Collectively, these results indicate that voltage-operated and voltage-independent Ca(2+) channels, as well as Ca(2+) release and myofilament Ca(2+)-sensitisation, participate in 5-HT-induced contraction in HPAs.

Background: Endothelial control of vascular smooth muscle plays a major role in the resulting vasoreactivity implicated in physiological or pathological circulatory processes. However, a comprehensive understanding of endothelial (EC)/smooth muscle cells (SMC) crosstalk is far from complete. Here, we have examined the role of gap junctions and reactive oxygen species (ROS) in this crosstalk and we demonstrate an active contribution of SMC to endothelial control of vasomotor tone., Methodology/principal Findings: In small intrapulmonary arteries, quantitative RT-PCR, Western Blot analyses and immunofluorescent labeling evidenced connexin (Cx) 37, 40 and 43 in EC and/or SMC. Functional experiments showed that the Cx-mimetic peptide targeted against Cx 37 and Cx 43 ((37,43)Gap27) (1) reduced contractile and calcium responses to serotonin (5-HT) simultaneously recorded in pulmonary arteries and (2) abolished the diffusion in SMC of carboxyfluorescein-AM loaded in EC. Similarly, contractile and calcium responses to 5-HT were decreased by superoxide dismutase and catalase which, catabolise superoxide anion and H(2)O(2), respectively. Both Cx- and ROS-mediated effects on the responses to 5-HT were reversed by L-NAME, a NO synthase inhibitor or endothelium removal. Electronic paramagnetic resonance directly demonstrated that 5-HT-induced superoxide anion production originated from the SMC. Finally, whereas 5-HT increased NO production, it also decreased cyclic GMP content in isolated intact arteries., Conclusions/significance: These data demonstrate that agonist-induced ROS production in SMC targeting EC via myoendothelial gap junctions reduces endothelial NO-dependent control of pulmonary vasoreactivity. Such SMC modulation of endothelial control may represent a signaling pathway controlling vasoreactivity under not only physiological but also pathological conditions that often implicate excessive ROS production.

In smooth muscle cells, agonists such as neurotransmitters or hormones can induce an increase in [Ca(2+)](i) via a release of intracellular stored calcium or/and an influx of extracellular calcium. The calcium entry pathway operates through a variety of plasmalemmal calcium channels which involve voltage-dependent and voltage-independent calcium channels. Voltage-independent calcium channels include (1) receptor-operated channels (ROCs) activated by agonist-receptor interaction and, in the majority of cases, the downstream signal transduction proteins, (2) store-operated channels (SOCs) activated by the emptying of intracellular Ca(2+) store (mainly the sarcoplasmic reticulum), (3) mechanosensitive or stretch-activated channels (SACs) activated by membrane stretch. Generally, voltage-independent calcium channels are calcium permeable non-selective cation channels with electrophysiological differences, complex regulatory mechanisms and pharmacology. Although the molecular identity of voltage-independent calcium channels is not yet fully elucidated, there are growing evidences that these channels correspond to a new family of membrane proteins encoded by mammalian homologues of specific transient receptor potential (TRP) genes. Several types of TRP proteins are ubiquitously expressed in smooth muscle cells and variations in the expression depend on tissue and species. More recently, other proteins such as Orai1 and STIM1 proteins have been also proposed as participating in the molecular identity of voltage-independent calcium channels. These channels control phenomena such as smooth muscle cells proliferation and/or contraction.

In the present study, we investigated the implication of transient receptor potential vanilloid (TRPV)-related channels in the 5-hydroxytryptamine (5-HT)-induced both intracellular calcium response and mitogenic effect in rat pulmonary arterial smooth muscle cells (PASMC). Using microspectrofluorimetry (indo-1 as Ca(2+) fluorescent probe) and the patch-clamp technique (in whole-cell configuration), we found that 5-HT (10 microM) induced a transient intracellular calcium mobilization followed by a sustained calcium entry. This latter was partly blocked by an inhibitor of cytochrome P450 epoxygenase (17-ODYA) and insensitive to cyclo-oxygenase and lipoxygenase inhibitors (indomethacin and CDC), suggesting the involvement of arachidonic acid metabolization by cytochrome P450 epoxygenase. This calcium influx was also sensitive to Ni(2+) and to ruthenium red, a TRPV channel blocker, and mimicked by 4alpha-phorbol-12,13-didecanoate (4alpha-PDD), a TRPV4 channel agonist. In patched PASMC, 5-HT and 4alpha-PDD-activated TRPV4-like ruthenium red sensitive currents with typical characteristics. Furthermore, 5-HT induced a ruthenium red sensitive increase in BrdU incorporation levels in PASMC. The present study provides evidence that 5-HT activates a TRPV4-like current, potentially involved in PASMC proliferation. The signalling pathway between proliferation and ion channel activation remains to be determined and may represent a molecular target for the treatment of vascular diseases such as pulmonary hypertension.