Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

Genetically-encoded BRET probes shed light on ligand bias-induced variable ion selectivity in TRPV1 and P2X5/7.

Whether ion channels experience ligand-dependent dynamic ion selectivity remains of critical importance since this could support ion channel functional bias. Tracking selective ion permeability through ion channels, however, remains challenging even with patch-clamp electrophysiology. In this study, we have developed highly sensitive bioluminescence resonance energy transfer (BRET) probes providing dynamic measurements of Ca2+ and K+ concentrations and ionic strength in the nanoenvironment of Transient Receptor Potential Vanilloid-1 Channel (TRPV1) and P2X channel pores in real time and in live cells during drug challenges. Our results indicate that AMG517, BCTC, and AMG21629, three well-known TRPV1 inhibitors, more potently inhibit the capsaicin (CAPS)-induced Ca2+ influx than the CAPS-induced K+ efflux through TRPV1. Even more strikingly, we found that AMG517, when injected alone, is a partial agonist of the K+ efflux through TRPV1 and triggers TRPV1-dependent cell membrane hyperpolarization. In a further effort to exemplify ligand bias in other families of cationic channels, using the same BRET-based strategy, we also detected concentration- and time-dependent ligand biases in P2X7 and P2X5 cationic selectivity when activated by benzoyl-adenosine triphosphate (Bz-ATP). These custom-engineered BRET-based probes now open up avenues for adding value to ion-channel drug discovery platforms by taking ligand bias into account.

Correction: The Naturally Processed CD95L Elicits a c-Yes/Calcium/PI3K-Driven Cell Migration Pathway.

[This corrects the article DOI: 10.1371/journal.pbio.1001090.].

CD95/Fas and metastatic disease: What does not kill you makes you stronger.

CD95 (also known as Fas) is the prototype of death receptors; however, evidence suggests that this receptor mainly implements non-apoptotic signaling pathways such as NF-κB, MAPK, and PI3K that are involved in cell migration, differentiation, survival, and cytokine secretion. At least two different forms of CD95 L exist. The multi-aggregated transmembrane ligand (m-CD95 L) is cleaved by metalloproteases to release a homotrimeric soluble ligand (s-CD95 L). Unlike m-CD95 L, the interaction between s-CD95 L and its receptor CD95 fails to trigger apoptosis, but instead promotes calcium-dependent cell migration, which contributes to the accumulation of inflammatory Th17 cells in damaged organs of lupus patients and favors cancer cell invasiveness. Novel inhibitors targeting the pro-inflammatory roles of CD95/CD95 L may provide attractive therapeutic options for patients with chronic inflammatory disorders or cancer. This review discusses the roles of the CD95/CD95 L pair in cell migration and metastasis.

High-Throughput Screening of Transient Receptor Potential Channel 1 Ligands in the Light of the Bioluminescence Resonance Energy Transfer Technique.

Ion channels are attractive drug targets for many therapeutic applications. However, high-throughput screening (HTS) of drug candidates is difficult and remains very expensive. We thus assessed the suitability of the bioluminescence resonance energy transfer (BRET) technique as a new HTS method for ion-channel studies by taking advantage of our recently characterized intra- and intermolecular BRET probes targeting the transient receptor potential vanilloid type 1 (TRPV1) ion channel. These BRET probes monitor conformational changes during TRPV1 gating and subsequent coupling with calmodulin, two molecular events that are intractable using reference techniques such as automated calcium assay (ACA) and automated patch-clamp (APC). We screened the small-sized Prestwick chemical library, encompassing 1200 compounds with high structural diversity, using either intra- and intermolecular BRET probes or ACA. Secondary screening of the detected hits was done using APC. Multiparametric analysis of our results shed light on the capability of calmodulin inhibitors included in the Prestwick library to inhibit TRPV1 activation by capsaicin. BRET was the lead technique for this identification process. Finally, we present data exemplifying the use of intramolecular BRET probes to study other transient receptor potential (TRP) channels and non-TRPs ion channels. Knowing the ease of use of BRET biosensors and the low cost of the BRET technique, these assays may advantageously be included for extending ion-channel drug screening. SIGNIFICANCE STATEMENT: This study screened a chemical library against TRPV1 ion channel using bioluminescence resonance energy transfer (BRET) molecular probes and compared the results with the ones obtained using reference techniques such as automated calcium assay and automated patch-clamp. Multiparametric analysis of our results shed light on the capability of calmodulin antagonists to inhibit chemical activation of TRPV1 and indicates that BRET probes may advantageously be included in ion channel drug screening campaigns.

Transient receptor potential vanilloid 4 channel participates in mouse ventricular electrical activity.

The TRPV4 channel is a calcium-permeable channel (PCa/PNa ∼ 10). Its expression has been reported in ventricular myocytes, where it is involved in several cardiac pathological mechanisms. In this study, we investigated the implication of TRPV4 in ventricular electrical activity. Left ventricular myocytes were isolated from trpv4+/+ and trpv4-/- mice. TRPV4 membrane expression and its colocalization with L-type calcium channels (Cav1.2) was confirmed using Western blot biotinylation, immunoprecipitation, and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4-/- compared with trpv4+/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD90) in trpv4+/+ but not in trpv4-/- myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased calcium transient (CaT) amplitude in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 carries an inward Ca2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD90 in trpv4+/+ but not in trpv4-/- myocytes, suggesting that TRPV4 prolongs AP duration in basal condition. Finally, introducing TRPV4 parameters in a mathematical model predicted the development of an inward TRPV4 current during repolarization that increases AP duration and CaT amplitude, in accord with what was found experimentally. This study shows for the first time that TRPV4 modulates AP and QTc durations. It would be interesting to evaluate whether TRPV4 could be involved in long QT-mediated ventricular arrhythmias.NEW & NOTEWORTHY Transient receptor potential vanilloid 4 (TRPV4) is expressed at the membrane of mouse ventricular myocytes and colocalizes with non-T-tubular L-type calcium channels. Deletion of trpv4 gene in mice results in shortened QT interval on electrocardiogram and reduced action potential duration of ventricular myocytes. Pharmacological activation of TRPV4 channel leads to increased action potential duration and increased calcium transient amplitude in trpv4-/- but not in trpv4-/- ventricular myocytes. To the contrary, TRPV4 channel pharmacological inhibition reduces action potential duration in trpv4+/+ but not in trpv4-/- myocytes. Integration of TRPV4 channel in a computational model of mouse action potential shows that the channel carries an inward current contributing to slowing down action potential repolarization and to increase calcium transient amplitude, similarly to what is observed experimentally. This study highlights for the first time the involvement of TRPV4 channel in ventricular electrical activity.

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.

Disrupting the CD95-PLCγ1 interaction prevents Th17-driven inflammation.

CD95L is a transmembrane ligand (m-CD95L) that is cleaved by metalloproteases to release a soluble ligand (s-CD95L). Unlike m-CD95L, interaction between s-CD95L and CD95 fails to recruit caspase-8 and FADD to trigger apoptosis and instead induces a Ca2+ response via docking of PLCγ1 to the calcium-inducing domain (CID) within CD95. This signaling pathway induces accumulation of inflammatory Th17 cells in damaged organs of lupus patients, thereby aggravating disease pathology. A large-scale screen revealed that the HIV protease inhibitor ritonavir is a potent disruptor of the CD95-PLCγ1 interaction. A structure-activity relationship approach highlighted that ritonavir is a peptidomimetic that shares structural characteristics with CID with respect to docking to PLCγ1. Thus, we synthesized CID peptidomimetics abrogating both the CD95-driven Ca2+ response and transmigration of Th17 cells. Injection of ritonavir and the CID peptidomimetic into lupus mice alleviated clinical symptoms, opening a new avenue for the generation of drugs for lupus patients.

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.

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.

Localized Store-Operated Calcium Influx Represses CD95-Dependent Apoptotic Effects of Rituximab in Non-Hodgkin B Lymphomas.

The anti-CD20 mAb, rituximab, is routinely used to treat B cell malignancies. However, a majority of patients relapse. An improvement in the complete response was obtained by combining rituximab with chemotherapy, at the cost of increased toxicity. We reported that rituximab induced the colocalization of both the Orai1 Ca(2+) release-activated Ca(2+) channel (CRAC) and the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule 1 with CD20 and CD95 into a cluster, eliciting a polarized store-operated Ca(2+) entry (SOCE). We observed that blocking this Ca(2+) entry with downregulation of Orai1, pharmacological inhibitors, or reducing calcemia with hypocalcemic drugs sensitized human B lymphoma cell lines and primary human lymphoma cells to rituximab-induced apoptosis in vitro, and improved the antitumoral effect of rituximab in xenografted mice. This revealed that Ca(2+) entry exerted a negative feedback loop on rituximab-induced apoptosis, suggesting that associating CRAC channel inhibitors or hypocalcemic agents with rituximab may improve the treatment of patients with B cell malignancies. The calcium-dependent proteins involved in this process appear to vary according to the B lymphoma cell type, suggesting that CRAC-channel targeting is likely to be more efficient than calcium-dependent protein targeting.

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.

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.

The naturally processed CD95L elicits a c-yes/calcium/PI3K-driven cell migration pathway.

Patients affected by chronic inflammatory disorders display high amounts of soluble CD95L. This homotrimeric ligand arises from the cleavage by metalloproteases of its membrane-bound counterpart, a strong apoptotic inducer. In contrast, the naturally processed CD95L is viewed as an apoptotic antagonist competing with its membrane counterpart for binding to CD95. Recent reports pinpointed that activation of CD95 may attract myeloid and tumoral cells, which display resistance to the CD95-mediated apoptotic signal. However, all these studies were performed using chimeric CD95Ls (oligomerized forms), which behave as the membrane-bound ligand and not as the naturally processed CD95L. Herein, we examine the biological effects of the metalloprotease-cleaved CD95L on CD95-sensitive activated T-lymphocytes. We demonstrate that cleaved CD95L (cl-CD95L), found increased in sera of systemic lupus erythematosus (SLE) patients as compared to that of healthy individuals, promotes the formation of migrating pseudopods at the leading edge of which the death receptor CD95 is capped (confocal microscopy). Using different migration assays (wound healing/Boyden Chamber/endothelial transmigration), we uncover that cl-CD95L promotes cell migration through a c-yes/Ca²âº/PI3K-driven signaling pathway, which relies on the formation of a CD95-containing complex designated the MISC for Motility-Inducing Signaling Complex. These findings revisit the role of the metalloprotease-cleaved CD95L and emphasize that the increase in cl-CD95L observed in patients affected by chronic inflammatory disorders may fuel the local or systemic tissue damage by promoting tissue-filtration of immune cells.

CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) ß2, and prevents death-inducing signaling complex formation.

The death receptor CD95 plays a pivotal role in immune surveillance and immune tolerance. Binding of CD95L to CD95 leads to recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates caspase-8 and caspase-10. Efficient formation of the CD95/FADD/caspase complex, known as the death-inducing signaling complex (DISC), culminates in the induction of apoptosis. We show that cells exposed to CD95L undergo a reorganization of the plasma membrane in which the Ca(2+) release-activated Ca(2+) channel Orai1 and the endoplasmic reticulum-resident activator stromal interaction molecule 1 colocalize with CD95 into a micrometer-sized cluster in which the channel elicits a polarized entry of calcium. Orai1 knockdown and expression of a dominant negative construct (Orai1E106A) reveal that on CD95 engagement, the Orai1-driven localized Ca(2+) influx is fundamental to recruiting the Ca(2+)-dependent protein kinase C (PKC) ß2 to the DISC. PKCß2 in turn transiently holds the complex in an inactive status, preventing caspase activation and transmission of the apoptotic signal. This study identifies a biological role of Ca(2+) and the Orai1 channel that drives a transient negative feedback loop, introducing a lag phase in the early steps of the CD95 signal. We suggest that these localized events provide a time of decision to prevent accidental cell death.

NGF increases Connexin-43 expression and function in pulmonary arterial smooth muscle cells to induce pulmonary artery hyperreactivity.

AIMS: Pulmonary hypertension (PH) is characterised by an increase in pulmonary arterial pressure, ultimately leading to right ventricular failure and death. We have previously shown that nerve growth factor (NGF) plays a critical role in PH. Our objectives here were to determine whether NGF controls Connexin-43 (Cx43) expression and function in the pulmonary arterial smooth muscle, and whether this mechanism contributes to NGF-induced pulmonary artery hyperreactivity. METHODS AND RESULTS: NGF activates its TrkA receptor to increase Cx43 expression, phosphorylation, and localization at the plasma membrane in human pulmonary arterial smooth muscle cells, thus leading to enhanced activity of Cx43-dependent GAP junctions as shown by Lucifer Yellow dye assay transfer and fluorescence recovery after photobleaching -FRAP- experiments. Using both in vitro pharmacological and in vivo SiRNA approaches, we demonstrate that NGF-dependent increase in Cx43 expression and activity in the rat pulmonary circulation causes pulmonary artery hyperreactivity. We also show that, in a rat model of PH induced by chronic hypoxia, in vivo blockade of NGF or of its TrkA receptor significantly reduces Cx43 increased pulmonary arterial expression induced by chronic hypoxia and displays preventive effects on pulmonary arterial pressure increase and right heart hypertrophy. CONCLUSIONS: Modulation of Cx43 by NGF in pulmonary arterial smooth muscle cells contributes to NGF-induced alterations of pulmonary artery reactivity. Since NGF and its TrkA receptor play a role in vivo in Cx43 increased expression in PH induced by chronic hypoxia, these NGF/Cx43-dependent mechanisms may therefore play a significant role in human PH pathophysiology.

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.

Pathophysiological Roles of the TRPV4 Channel in the Heart.

The transient receptor potential vanilloid 4 (TRPV4) channel is a non-selective cation channel that is mostly permeable to calcium (Ca2+), which participates in intracellular Ca2+ handling in cardiac cells. It is widely expressed through the body and is activated by a large spectrum of physicochemical stimuli, conferring it a role in a variety of sensorial and physiological functions. Within the cardiovascular system, TRPV4 expression is reported in cardiomyocytes, endothelial cells (ECs) and smooth muscle cells (SMCs), where it modulates mitochondrial activity, Ca2+ homeostasis, cardiomyocytes electrical activity and contractility, cardiac embryonic development and fibroblast proliferation, as well as vascular permeability, dilatation and constriction. On the other hand, TRPV4 channels participate in several cardiac pathological processes such as the development of cardiac fibrosis, hypertrophy, ischemia-reperfusion injuries, heart failure, myocardial infarction and arrhythmia. In this manuscript, we provide an overview of TRPV4 channel implications in cardiac physiology and discuss the potential of the TRPV4 channel as a therapeutic target against cardiovascular diseases.

OP2113, a new drug for chronic hypoxia-induced pulmonary hypertension treatment in rat.

BACKGROUND AND PURPOSE: Pulmonary hypertension (PH) is a cardiovascular disease characterised by an increase in pulmonary arterial (PA) resistance leading to right ventricular (RV) failure. Reactive oxygen species (ROS) play a major role in PH. OP2113 is a drug with beneficial effects on cardiac injuries that targets mitochondrial ROS. The aim of the study was to address the in vivo therapeutic effect of OP2113 in PH. EXPERIMENTAL APPROACH: PH was induced by 3 weeks of chronic hypoxia (CH-PH) in rats treated with OP2113 or its vehicle via subcutaneous osmotic mini-pumps. Haemodynamic parameters and both PA and heart remodelling were assessed. Reactivity was quantified in PA rings and in RV or left ventricular (LV) cardiomyocytes. Oxidative stress was detected by electron paramagnetic resonance and western blotting. Mitochondrial mass and respiration were measured by western blotting and oxygraphy, respectively. KEY RESULTS: In CH-PH rats, OP2113 reduced the mean PA pressure, PA remodelling, PA hyperreactivity in response to 5-HT, the contraction slowdown in RV and LV and increased the mitochondrial mass in RV. Interestingly, OP2113 had no effect on haemodynamic parameters, both PA and RV wall thickness and PA reactivity, in control rats. Whereas oxidative stress was evidenced by an increase in protein carbonylation in CH-PH, this was not affected by OP2113. CONCLUSION AND IMPLICATIONS: Our study provides evidence for a selective protective effect of OP2113 in vivo on alterations in both PA and RV from CH-PH rats without side effects in control rats.