TRPV4 channel mediates adventitial fibroblast activation and adventitial remodeling in pulmonary hypertension.

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.

Connexin-43 is a promising target for pulmonary hypertension due to hypoxaemic lung disease.

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.

Stretch-activated Piezo1 Channel in Endothelial Cells Relaxes Mouse Intrapulmonary Arteries.

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 ([Ca2+]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 [Ca2+]i in mouse or human EC. Consequently, both stimuli increased the production of NO. NO and [Ca2+]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 [Ca2+]i and NO production and that this effect is still present in pulmonary hypertension.

T-type voltage gated calcium channels are involved in endothelium-dependent relaxation of mice pulmonary artery.

In pulmonary arterial endothelial cells, Ca2+ channels and intracellular Ca2+ concentration ([Ca2+]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 Ca2+ channels (T-VGCCs, Cav3.1 channel) in the endothelium-dependent relaxation of intrapulmonary arteries. Relaxation was quantified by means of a myograph in wild type and Cav3.1-/- mice. Endothelial [Ca2+]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 Cav3.1-/- compared to wild type mice as well as in the presence of T-VGCC inhibitors (NNC 55-0396 or mibefradil). ACh also increased endothelial [Ca2+]i and NO production that were both reduced in Cav3.1-/- compared to wild type mice or in the presence of T-VGCC inhibitors. Immunofluorescence labeling revealed the presence of Cav3.1 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 Cav3.1-/- compared to wild type mice. Finally, in chronically hypoxic mice, a model of pulmonary hypertension, ACh relaxation was reduced but still depended on Cav3.1 channels activity. The present study thus demonstrates that T-VGCCs, mainly Cav3.1 channel, contribute to intrapulmonary vascular reactivity in mice by controlling endothelial [Ca2+]i and ACh-mediated relaxation.

Chronic hypoxia aggravates monocrotaline-induced pulmonary arterial hypertension: a rodent relevant model to the human severe form of the disease.

Pulmonary arterial hypertension (PAH) is a severe form of pulmonary hypertension that combines multiple alterations of pulmonary arteries, including, in particular, thrombotic and plexiform lesions. Multiple-pathological-insult animal models, developed to more closely mimic this human severe PAH form, often require complex and/or long experimental procedures while not displaying the entire panel of characteristic lesions observed in the human disease. In this study, we further characterized a rat model of severe PAH generated by combining a single injection of monocrotaline with 4 weeks exposure to chronic hypoxia. This model displays increased pulmonary arterial pressure, right heart altered function and remodeling, pulmonary arterial inflammation, hyperresponsiveness and remodeling. In particular, severe pulmonary arteriopathy was observed, with thrombotic, neointimal and plexiform-like lesions similar to those observed in human severe PAH. This model, based on the combination of two conventional procedures, may therefore be valuable to further understand the pathophysiology of severe PAH and identify new potential therapeutic targets in this disease.

Signaling Pathways Linked to Serotonin-Induced Superoxide Anion Production: A Physiological Role for Mitochondria in Pulmonary Arteries.

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-HT2 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-HT2 receptors agonist) whereas GR127935 (1 µM-an antagonist of the 5-HT1B/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-HT2 receptors and induces an [Formula: see text] production dependent on extracellular calcium and mitochondria.

Calcium signalling induced by in vitro exposure to silicium dioxide nanoparticles in rat pulmonary artery smooth muscle cells.

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 SiO2 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 SiO2 NP, from 1 to 500µg/mL, produced an increase of the [Ca2+]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 (IP3 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.

HMGB1 down-regulation mediates terameprocol vascular anti-proliferative effect in experimental pulmonary hypertension.

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.

Expression and role of connexin-based gap junctions in pulmonary inflammatory diseases.

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.

Caveolae are involved in mechanotransduction during pulmonary hypertension.

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-ß-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.

Involvement of Heme Oxygenase-1 in particulate matter-induced impairment of NO-dependent relaxation in rat intralobar pulmonary arteries.

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.

Effect of hypoxia on TRPV1 and TRPV4 channels in rat pulmonary arterial smooth muscle cells.

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.

Role of Nerve Growth Factor in Development and Persistence of Experimental Pulmonary Hypertension.

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: roles in pulmonary hypertension.

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.

T-type calcium channels are involved in hypoxic pulmonary hypertension.

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.

Stretch-induced Ca2+ signalling in vascular smooth muscle cells depends on Ca2+ store segregation.

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.

Characterization of the components of urban particulate matter mediating impairment of nitric oxide-dependent relaxation in intrapulmonary arteries.

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.

Proteomic remodeling of proteasome in right heart failure.

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.

Biopterin metabolism and eNOS expression during hypoxic pulmonary hypertension in mice.

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.

Beneficial effect of dehydroepiandrosterone on pulmonary hypertension in a rodent model of pulmonary hypertension in infants.

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.