Transient Receptor Potential Vanilloid 4 Inhibitor Research Articles

Endothelial TRPV4 channel mediates the vasodilation induced by Tanshinone IIA

Tanshinone IIA (TSA), the main lipo-soluble component from the dried rhizome of Salvia miltiorrhiza, has been shown to induce vasodilation. However, the underlying mechanisms remains unclear. This study aimed to investigate the effect of TSA on the vasodilation of small resistant arteries ex vivo. Vascular myography revealed that endothelial denudation reduced significantly the vasodilatory effect of TSA. Blocking transient receptor potential vanilloid 4 (TRPV4) channels prevented TSA-induced vasodilation. Whole-cell patch-clamp analysis revealed that the current passing through TRPV4 channels increased after TSA treatment in endothelial cells (ECs). This was attributed to reduced TRPV4 protein degradation along with its increased expression. The TRPV4 inhibitor HC-067047 lowed nitric oxide (NO) production and TSA-induced expression of endothelial nitric oxide synthase (eNOS). Moreover, it increased the production of cyclic guanosine monophosphate (cGMP) and protein kinase G (PKG). The present results indicate that TSA induces endothelium-dependent vasodilation, which is mediated by the TRPV4-NO-PKG signaling pathway. These findings highlight the potential of TSA, a compound known in traditional Chinese medicine as Danshen (Salvia miltiorrhiza), for future cardiovascular therapeutic strategies.

Effect of TRPV4 Antagonist GSK2798745 on Chlorine Gas-Induced Acute Lung Injury in a Swine Model.

As a regulator of alveolo-capillary barrier integrity, Transient Receptor Potential Vanilloid 4 (TRPV4) antagonism represents a promising strategy for reducing pulmonary edema secondary to chemical inhalation. In an experimental model of acute lung injury induced by exposure of anesthetized swine to chlorine gas by mechanical ventilation, the dose-dependent effects of TRPV4 inhibitor GSK2798745 were evaluated. Pulmonary function and oxygenation were measured hourly; airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, and histopathology were assessed 24 h post-exposure. Exposure to 240 parts per million (ppm) chlorine gas for ≥50 min resulted in acute lung injury characterized by sustained changes in the ratio of partial pressure of oxygen in arterial blood to the fraction of inspiratory oxygen concentration (PaO2/FiO2), oxygenation index, peak inspiratory pressure, dynamic lung compliance, and respiratory system resistance over 24 h. Chlorine exposure also heightened airway response to methacholine and increased wet-to-dry lung weight ratios at 24 h. Following 55-min chlorine gas exposure, GSK2798745 marginally improved PaO2/FiO2, but did not impact lung function, airway responsiveness, wet-to-dry lung weight ratios, airway inflammation, or histopathology. In summary, in this swine model of chlorine gas-induced acute lung injury, GSK2798745 did not demonstrate a clinically relevant improvement of key disease endpoints.

Preliminary study of TRPV4 affects chondrocyte degeneration

To explore and verify that transient receptor potential vanilloid 4(TRPV4) affects chondrocyte degeneration. Neonatal SD rats were selected, primary chondrocytes were extracted, and identified by toluidine blue staining and alcian blue staining;an in vitro chondrocyte inflammation model was constructed by IL-1β, and TRPV4 inhibitor was used to treat chondrocytes under inflammatory conditions, and the chondrocytes were treated by RT-PCR method was used to detect matrix metallopeptidase 13(MMP-13), a disintegrin and metalloproteinase with thrombospondin 5, (ADAMTS-5)、nitric oxide synthase 2(NOS2)、Collagen, type II alpha 1(Col2α1)and aggrecan (Acan) mRNA in chondrocytes; primary chondrocytes were treated with different concentrations of TRPV4 overexpression plasmid, and the optimal overexpression dose was screened. The mRNA expressions of TRPV4, MMP-13, ADAMTS-5, NOS2, Col2α1 and Acan in chondrocytes under the optimal TRPV4 overexpression dose were detected. Toluidine blue staining and Alcian blue staining identified the extracted cells as primary chondrocytes;RT-PCR showed that TRPV4, MMP-13, ADAMTS-5, NOS2 mRNA in chondrocytes treated with TRPV4 inhibitor under inflammatory conditions. The expression of Col2α1 mRNA was significantly decreased (P<0.05), and the expression of Col2α1 mRNA was increased (P<0.05). Although there was no significant difference in the expression of Acan mRNA, the overall trend was also increasing. The expression of Col2α1 and Acan mRNA in chondrocytes was significantly decreased (P<0.05), and the expression of NOS2 mRNA was increased(P<0.05), but there was no significant difference in MMP-13 and ADAMTS-5 (P>0.05). Inhibiting the expression of TRPV4 can down-regulate the expression of genes related to chondrocyte degeneration.

Inhibition of TRPV4 remodels single cell polarity and suppresses the metastasis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a malignant tumor, frequently causing both intrahepatic and extrahepatic metastases. The overall prognosis of patients with metastatic HCC is poor. Recently, single-cell (sc) polarity is proved to be an innate feature of some tumor cells in liquid phase, and directly involved in the cell adhesion to blood vessel and tumor metastasis. Here, we characterize the maintained sc polarity of HCC cells in a suspension culture, and investigate its roles and regulatory mechanisms during metastasis. We demonstrate that transient receptor potential vanilloid 4 (TRPV4) is a promoting regulator of sc polarity via activating Ca2+-dependent AMPK/MLC/ERM pathway. This attenuates the adhesion of metastatic HCC cells to vascular endothelial cells. The reduction of cancer metastases can result from TRPV4 inhibition, which not only impacts the migration and invasion of tumor cells, but also prevents the adhesion to vascular endothelial cells. Additionally, we discover a brand-new TRPV4 inhibitor called GL-V9 that modifies the degree of sc polarization and significantly decreases the metastatic capacity of HCC cells. Taken together, our data shows that TRPV4 and calcium signal are significant sc polarity regulators in metastatic HCC, and that the pharmacological intervention that results in HCC cells becoming depolarized suggests a promising treatment for cancer metastasis.

TRPV4 inhibitor HC067047 produces antidepressant-like effect in LPS-induced depression mouse model

Inflammation is a crucial component that contributes to the pathogenesis of major depressive disorder. It has been revealed that the nonselective cation channel transient receptor potential vanilloid 4 (TRPV4) profoundly affects a variety of physiological processes, including inflammation. However, its roles and mechanisms in LPS-induced depression are still unclear. Here, for the first time, we found that there was a significant increase in TRPV4 in the hippocampus in a depression mouse model induced by LPS. TRPV4 inhibitor HC067047 or knockdown the hippocampal TRPV4 with TRPV4 shRNA could effectively rescue the aberrant behaviors. Furthermore, TRPV4 inhibitor HC067047 reduced the activation of astrocyte and microglia, decreased expression of CaMKII-NLRP3 inflammasome and increased the expression of neurogenesis marker DCX in the hippocampus. In addition, enhanced neuroinflammation in the serum was also reversed by TRPV4 inhibitor HC067047. Thus, we consider that TRPV4 has an important role in contributing to the depression-like behavior following LPS-induced systemic inflammation.

Amelioration of scopolamine-induced learning and memory impairment by the TRPV4 inhibitor HC067047 in ICR mice

Alzheimer’s disease (AD) is one of the most common causes of neurodegenerative diseases in the elderly. Cholinergic dysfunction is one of the pathological hallmarks of AD and leads to learning and memory impairment. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is involved in learning and memory functions. HC067047, a TRPV4 specific inhibitor, has been reported to protect neurons against cerebral ischemic injury and amyloid-β-(Aβ) 40-induced hippocampal cell death. However, whether HC067047 could improve scopolamine (SCP)-induced cognitive dysfunction in mice is still unknown. The aims of this study were to verify whether HC067047 could ameliorate the SCP-induced learning and memory impairments in mice and to elucidate its underlying mechanisms of action. In this study, we examined the neuroprotective effect of the HC067047 against cognitive dysfunction induced by SCP (5 mg/kg, i.p.), a muscarinic receptor antagonist. The results showed that administration of HC067047 (10 mg/kg, i.p.) significantly ameliorated SCP-induced cognitive dysfunction as assessed by the novel place recognition test (NPRT) and novel object recognition test (NORT). In the Y-maze test, HC067047 significantly enhanced the time spent in the novel arm in SCP mice. To further investigate the molecular mechanisms underlying the neuroprotective effect of HC067047, expression of several proteins involved in apoptosis was examined. The results demonstrated that HC067047 treatment decreased the protein levels of proapoptotic proteins such as Bax and caspase-3 in the hippocampus of SCP mice. In addition, HC067047 enhanced expression of the neurogenesis marker DCX and improved levels of the mature neuronal marker NeuN in SCP mice. These findings suggest the neuroprotective potential of the TRPV4 inhibitor HC067047 for the management of dementia with learning and memory loss.

Transient Receptor Potential Vanilloid 4 channel participates in mouse ventricular electrical activity

Transient Receptor Potential Vanilloid 4 (TRPV4) channel is a calcium permeable channel (P Ca /P Na ∼ 10). Its expression was 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 Ca v 1.2 was confirmed using western-blots biotinylation, immunoprecipitation and immunostaining experiments. Then, electrocardiograms (ECGs) and patch-clamp recordings showed shortened QTc and action potential (AP) duration in trpv4 −/− compared to trpv4 +/+ mice. Thus, TRPV4 activator GSK1016790A produced a transient and dose-dependent increase in AP duration at 90% of repolarization (APD 90 ) in trpv4 +/+ , but not in trpv4 −/− myocytes or when combined with TRPV4 inhibitor GSK2193874 (100 nM). Hence, GSK1016790A increased CaT amplitude in trpv4 +/+ but not in trpv4 −/− myocytes, suggesting that TRPV4 carries an inward Ca 2+ current in myocytes. Conversely, TRPV4 inhibitor GSK2193874 (100 nM) alone reduced APD 90 in trpv4 +/+ but not in trpv4 −/− myocytes, suggesting that TRPV4 prolongs AP duration (APD) 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 accordance with what 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.

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 activates the Cdc42/N-wasp pathway to promote glioblastoma invasion by altering cellular protrusions

The invasion ability of glioblastoma (GBM) causes tumor cells to infiltrate the surrounding brain parenchyma and leads to poor outcomes. Transient receptor potential vanilloid 4 (TRPV4) exhibits a remarkable role in cancer cell motility, but the contribution of TRPV4 to glioblastoma metastasis is not fully understood. Here, we reported that TRPV4 expression was significantly elevated in malignant glioma compared to normal brain and low-grade glioma, and TRPV4 expression was negatively correlated with the prognosis of glioma patients. Functionally, stimulation of TRPV4 promoted glioblastoma cell migration and invasion, and repression of TRPV4 hindered the migration and invasion of glioblastoma cells in vitro. Molecularly, TRPV4 strongly colocalized and interacted with skeletal protein-F-actin at cellular protrusions, and TRPV4 regulated the formation of invadopodia and filopodia in glioblastoma cells. Furthermore, the Cdc42/N-wasp axis mediated the effect of TRPV4-regulated cellular protrusions and invasion. Foremost, TRPV4 inhibitor treatment or downregulation of TRPV4 significantly reduced the invasion-growth of subcutaneously and intracranially transplanted glioblastoma in mice. In conclusion, the TRPV4/Cdc42/wasp signaling axis regulates cellular protrusion formation in glioblastoma cells and influences the invasion-growth phenotype of glioblastoma in vivo. TRPV4 may serve as a prognostic factor and specific therapeutic target for GBM patients.

Urgent reconsideration of lung edema as a preventable outcome in COVID-19: inhibition of TRPV4 represents a promising and feasible approach.

Lethality of coronavirus disease (COVID-19) during the 2020 pandemic, currently still in the exponentially accelerating phase in most countries, is critically driven by disruption of the alveolo-capillary barrier of the lung, leading to lung edema as a direct consequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We argue for inhibition of the transient receptor potential vanilloid 4 (TRPV4) calcium-permeable ion channel as a strategy to address this issue, based on the rationale that TRPV4 inhibition is protective in various preclinical models of lung edema and that TRPV4 hyperactivation potently damages the alveolo-capillary barrier, with lethal outcome. We believe that TRPV4 inhibition has a powerful prospect at protecting this vital barrier in COVID-19 patients, even to rescue a damaged barrier. A clinical trial using a selective TRPV4 inhibitor demonstrated a benign safety profile in healthy volunteers and in patients suffering from cardiogenic lung edema. We argue for expeditious clinical testing of this inhibitor in COVID-19 patients with respiratory malfunction and at risk for lung edema. Perplexingly, among the currently pursued therapeutic strategies against COVID-19, none is designed to directly protect the alveolo-capillary barrier. Successful protection of the alveolo-capillary barrier will not only reduce COVID-19 lethality but will also preempt a distressing healthcare scenario with insufficient capacity to provide ventilator-assisted respiration.

Acetaminophen‐induced liver injury is mediated by the ion channel TRPV4

Overdosing of the analgesic acetaminophen (APAP) is one of the most common causes for acute liver failure in modern countries. Although the exact molecular mechanisms mediating hepatocellular necrosis are still elusive, it is preceded by oxidative stress triggered by excessive levels of the metabolite N-acetyl-para-benzoquinone imine (NAPQI). Here, we describe the role of the redox-sensitive transient receptor potential (TRP) ion channel TRP vanilloid 4 (TRPV4) for APAP-induced hepatoxicity. Both pharmacological inhibition and genetic deletion of TRPV4 ameliorate APAP-induced necrosis in mouse and human hepatocytes in vitro. Liver injury caused by a systemic overdose of APAP is reduced in TRPV4-deficient mice and in wild-type mice treated with a TRPV4 inhibitor. The reduction of hepatotoxicity accomplished by systemic TRPV4 inhibition is comparable to the protective effects of the antioxidant N-acetyl-cysteine. Although TRPV4 does not modulate intrahepatic levels of glutathione, both its inhibition and genetic deletion attenuate APAP-induced oxidative and nitrosative stress as well as mitochondrial membrane depolarization. NAPQI evokes a calcium influx by activating heterologously expressed TRPV4 channels and endogenous TRPV4 channels in hepatoma cells but not in primary mouse hepatocytes. Taken together, our data suggest that TRPV4 mediates APAP-induced hepatotoxicity and thus may be a suitable target for treatment of this critical side effect.-Echtermeyer, F., Eberhardt, M., Risser, L., Herzog, C., Gueler, F., Khalil, M., Engel, M., Vondran, F., Leffler, A. Acetaminophen-induced liver injury is mediated by the ion channel TRPV4.

TRPV4 Mediates the Macrophage Phagocytic and Cytokine Response to Leptin

Abstract Obesity is characterized by chronic activation of the innate immune system. Leptin is an adipokine upregulated in obesity that has been shown to stimulate macrophage function through an unidentified signal. Our lab recently showed that the mechanosensitive calcium channel, transient receptor potential vanilloid 4 (TRPV4) mediates macrophage phagocytosis in response to infection. This study seeks to determine if TRPV4 also mediates the macrophage response to leptin. Bone marrow derived macrophages were isolated from 7–10 week old WT and TRPV4 KO mice and incubated in the presence of pathophysiological range leptin concentrations (0.1–1μg/mL; 24 hr) ± LPS (E coli 0111:B4 as a positive control) ± TRPV4 inhibitor. Macrophage phagocytosis was measured as uptake of fluorescently-labeled E coli particles. Intracellular calcium was measured using the FLIPR Calcium 5 Assay in the presence of a selective TRPV4 agonist (GSK). MIP-2 and IL-8 secretion was measured by ELISA. Leptin increases phagocytosis of E coli particles by 17% (59% of the LPS response) and TRPV4 activity by 24% (51% of the LPS response). The phagocytic response and calcium influx are abrogated by either pharmacologic inhibition or deletion of TRPV4. Secretion of MIP-2 and IL-8 in response to leptin increases by 8-fold after pharmacologic inhibition of TRPV4. Our data show that TRPV4 mediates macrophage phagocytosis of E coli particles, and suppresses proinflammatory cytokine release, in response to leptin. In addition, we show that TRPV4 function is increased by leptin. In vitro results predict that TRPV4 enhances host defense and suppresses inflammation in the setting of obesity-induced hyperleptinemia, thus potentially modulating pneumonia severity in obese patients.

Discovery of Pyrrolidine Sulfonamides as Selective and Orally Bioavailable Antagonists of Transient Receptor Potential Vanilloid-4 (TRPV4).

A novel series of pyrrolidine sulfonamide transient receptor potential vanilloid-4 (TRPV4) antagonists was developed by modification of a previously reported TRPV4 inhibitor (1). Several core-structure modifications were identified that improved TRPV4 activity by increasing structural rigidity and reducing the entropic energy penalty upon binding to the target protein. The new template was initially discovered as a minor regio-isomeric side product formed during routine structure-activity relationship (SAR) studies, and further optimization resulted in highly potent compounds with a novel pyrrolidine diol core. Further improvements in potency and pharmacokinetic properties were achieved through SAR studies on the sulfonamide substituent to give an optimized lead compound GSK3395879 (52) that demonstrated the ability to inhibit TRPV4-mediated pulmonary edema in an in vivo rat model. GSK3395879 is a tool for studying the biology of TRPV4 and an advanced lead for identifying new heart failure medicines.

TRPV4 activates cytosolic phospholipase A2 via Ca2+ -dependent PKC/ERK1/2 signalling in controlling hypertensive contraction.

Activation of TRPV4 (transient receptor potential vanilloid 4) has been reported to result in endothelium-dependent contraction in the aortae of hypertensive mice. This contraction involved increased cPLA2 (cytosolic phospholipase A2 ) activity. The mechanism by which TRPV4 regulates cPLA2 activity to induce contraction in hypertension, however, is unknown. Through measurements of arterial tension and protein level, we showed that high-salt diet induced hypertension increases activity of PKC (protein kinase C) and ERK1/2 (extracellular signal-regulated kinase 1/2). GSK1016790A, a TRPV4 agonist and ACh (acetylcholine) induced contractions were suppressed by Go6983, a PKC inhibitor and PD98059, an ERK1/2 inhibitor. TRPV4 activation increased activity of PKC and ERK1/2 in endothelial cells from hypertensive mice and this response was suppressed by HC067047, a TRPV4 inhibitor and BAPTA/AM, a Ca2+ chelator. PLA2 assay and western blotting showed that blocking of PKC or ERK1/2 inhibited TRPV4 or ACh-induced cPLA2 activity. Enzyme immunoassay showed that GSK1016790A or ACh triggered the release of PGF2α (prostaglandin F2α ) was reduced by inhibition of PKC or ERK1/2. These data further suggest Ca2+ /PKC/ERK1/2 axis as a novel mechanism for TRPV4 in the activation of cPLA2 in hypertension.

GSK2193874 treatment at heatstroke onset reduced cell apoptosis in heatstroke mice

Heatstroke is still a potentially fatal threat during summer heat waves, despite improved prevention and treatment. It is reported that the transient receptor potential vanilloid 4 (TRPV4) inhibitor may protect septicemia mice. Many aspects of heatstroke have been defined, from the sepsis-mimic inflammatory response to hyperthermia. Hence, TRPV4 may be a therapeutic target for heatstroke. The results in murine models of heatstroke verified that GSK2193874, as a selected TRPV4 inhibitor, was injected at heatstroke onset, and then reduced the reduction of core temperature, the death rate, wet/dry ratio of the lung, levels of tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, coagulation indicators, the degree of organ injury, and caspase-3/7 activity (P&lt;0.05). But GSK2193874 treatment before heat stress did not improve the symptoms of heatstroke mice. Therefore, TRPV4 should be involved in heatstroke-induced injury. Timely GSK2193874 administration may be useful to reduce heatstroke-induced injury. TRPV4 may be a potential new therapeutic target in fatal heatstroke.

Role of mitochondrial reactive oxygen species (ROS) and TRPV4 activation in microvascular endothelial cell dysfunction in PAH.

RationaleIn pulmonary arterial hypertension (PAH), endothelial cells (ECs) exhibit increased migration/proliferation leading to formation of vaso‐occlusive lesions. The mechanisms underlying this pro‐migratory/proliferative state remain unclear. Increased ROS and intracellular Ca2+ concentration ([Ca2+]i) are known to promote migration and proliferation in other vascular beds, and there is evidence for increased oxidant stress and basal [Ca2+]i in ECs from humans with PAH. Although the exact source of increased EC oxidant stress in PAH ECs is not fully known, mitochondria are a significant source of ROS in ECs, and mitochondrial dysfunction is present in human PAH ECs. However, the links between ROS production, elevated [Ca2+]i and enhanced migration and proliferation in PAH ECs are incompletely understood.We previously showed that exogenous ROS increased [Ca2+]i in lung microvascular endothelial cells (MVECs) via the transient receptor potential vanilloid‐4 (TRPV4) calcium channel. To study endogenous ROS production and the possible role of ROS‐induced Ca2+ influx in promoting MVEC migration and proliferation in PAH, we isolated MVECs from the lungs of rats subjected to normoxia (N‐MVEC) or SU5416/hypoxia (SuHx‐MVEC), a robust model of PAH, and examined ROS production, [Ca2+]i, mitochondrial morphology/function and cellular migration and proliferation.MethodsCa2+ was measured using Fura‐2AM‐loaded MLMVEC in a flow chamber perfused with Krebs buffer. Mitochondrial morphology measurements were made using validated algorithms on confocal images of mitochondria in N‐ and SuHx‐MVEC transfected with MitoRFP. ROS measurements were made using the ratiometric ROS sensor roGFP. Migration and proliferation were measured using transwell assay and BrDU incorporation, respectively.ResultsROS levels were increased in SuHx‐MVEC and attenuated following treatment with MitoQ (MQ) a mitochondrion‐specific antioxidant. In SuHx‐MVECs, mitochondrial number, network fragmentation and activation of the fission‐inducing protein dynamin‐related protein 1 were increased, while oxidative phosphorylation and mitochondrial membrane potential were both decreased. Basal [Ca2+]i, migration and proliferation were all increased in SuHx‐MVEC, but similarly attenuated following treatment with: 1) MQ; 2) a global ROS scavenger (TEMPOL); or 3) HC‐067047, a specific TRPV4 inhibitor. While total TRPV4 expression was unchanged between N‐ and SuHx‐MVEC, activation of membrane bound TRPV4 (using the specific agonist GSK1016790A ‐ GSK) produced greater [Ca2+]i influx in SuHx‐MVEC. Biotinylation of TRPV4 revealed greater membrane‐bound expression of TRPV4 in SuHx‐MVEC. Interestingly, quenching mitochondrial ROS attenuated GSK‐induced [Ca2+]i influx in SuHx‐MVEC.ConclusionThese data suggest that, in SuHx‐MVEC, mitochondrial ROS promote membrane translocation of TRPV4, leading to increased basal [Ca2+]i and enhanced migration and proliferation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPV4 Enhances Cardiomyocyte Calcium Transients and Cardiac Contractility Following Hypoosmotic Stress and Ischemia-Reperfusion

The Transient Receptor Potential (TRP) ion channel family plays an important role in cardiomyocyte calcium homeostasis, particularly in diseased states. Here, we test the hypothesis that the osmotically-activated TRP Vanilloid 4 (TRPV4) channel enhances cardiomyocyte calcium transients in the aged heart following hypoosmotic stress. Expression of TRPV4 was low in cardiomyocytes of Young (3-6 month) mice, but significantly increased in cardiomyocytes of Aged (24-26 month) mice. In cardiomyocytes of Aged, hypoosmotic stress (250 mOsm) induced an increase in calcium transient amplitude (F/F0:3.6±0.1 hypoosmotic versus 2.9±0.2 isosmotic, P<0.05). This effect was prevented by the TRPV4 inhibitor HC067047 (1 μM, F/F0:2.9±0.1 hypoosmotic) and was absent in cardiomyocytes of Young mice (F/F0:3.2±0.1 hypoosmotic versus 2.9±0.2 isosmotic). However, cardiomyocytes of Young mice with cardiac-specific TRPV4 Overexpression exhibited enhanced calcium transient amplitude following hypoosmotic stress (F/F0:3.5±0.2 hypoosmotic versus 2.4±0.1 isosmotic, P<0.05) that was prevented by HC067047 (F/F0:2.6±0.1 hypoosmotic). Ischemia-reperfusion (I-R) injury is a pathological scenario associated with pronounced osmotic stress on cardiomyocytes. We therefore monitored left-ventricular pressure development in Langendorff-perfused hearts of Young, Aged, Young TRPV4 Overexpressor, and Aged TRPV4 knock-out mice subjected to 45 minute global ischemia followed by reperfusion. Prior to ischemia, all hearts exhibited similar contractility (dP/dtMax range: 1934±206 to 2226±62 mmHg/s). Following I-R, hearts of Aged mice exhibited enhanced contractile performance (dP/dtMax: 2770±180 mmHg/s) versus hearts of Aged TRPV4 knock-out mice (dP/dtMax: 1400±300 mmHg/s, P<0.05). Similarly, hearts of Young TRPV4 Overexpressor mice exhibited enhanced contractile performance (dP/dtMax: 2750±180 mmHg/s) versus hearts of Young mice (dP/dtMax: 1650±260 mmHg/s, P<0.05). In conclusion, TRPV4 enhances calcium transients following hypoosmotic stress and contributes to hypercontractility following I-R. Our findings have potential clinical implications in the treatment of elderly populations at increased risk of myocardial infarction and reperfusion injury.

Prevention of Ventilator-Induced Lung Edema by Inhalation of Nanoparticles Releasing Ruthenium Red

The acute respiratory distress syndrome (ARDS), a devastating lung disease that has no cure, is exacerbated by life-supportive mechanical ventilation that worsens lung edema and inflammation through the syndrome of ventilator-induced lung injury. Recently, the membrane ion channel transient receptor potential vanilloid 4 (TRPV4) on alveolar macrophages was shown to mediate murine lung vascular permeability induced by high-pressure mechanical ventilation. The objective of this study was to determine whether inhalation of nanoparticles (NPs) containing the TRPV4 inhibitor ruthenium red (RR) prevents ventilator-induced lung edema in mice. Poly-lactic-co-glycolic acid NPs containing RR were evaluated in vitro for their ability to block TRPV4-mediated calcium signaling in alveolar macrophages and capillary endothelial cells. Lungs from adult C57BL6 mice treated with nebulized NPs were then used in ex vivo ventilation perfusion experiments to assess the ability of the NPs to prevent high-pressure mechanical ventilation-induced lung edema. Poly-lactic-co-glycolic acid NPs (300 nm) released RR for 150 hours in vitro, and blocked TRPV4-mediated calcium signaling in cells up to 7 days after phagocytosis. Inhaled NPs deposited in alveoli of spontaneously breathing mice were rapidly phagocytosed by alveolar macrophages, and blocked increased vascular permeability from high-pressure mechanical ventilation for 72 hours in ex vivo ventilation perfusion experiments. These data offer proof of principle that inhalation of NPs containing a TRPV4 inhibitor prevents ventilator damage for several days, and imply that this novel drug delivery strategy could be used to target alveolar macrophages in patients at risk of ventilator-induced lung injury before initiating mechanical ventilation.

TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury

The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function.

Abstract 133: Inhibition of TRPV4 is Protective of the Brain and Cerebral Circulation During Ischemic Stroke

Background: Transient receptor potential vanilloid 4 (TRPV4) channels are highly calcium permeable channels expressed in brain and cerebral endothelium and are regulated by a diverse array of stimuli including shear stress, cell swelling and second messengers. We hypothesized that inhibition of TRPV4 during postischemic reperfusion would be protective of the brain and cerebral endothelium. Methods: Transient MCAO was used for all experiments. Infarction (by TTC) was measured in TRPV4 knockout mice (n=3) or C57B mice treated upon reperfusion with the TRPV4 inhibitor HC067047 (1mg/kg; n=7) or vehicle (n=8) after 90 minutes of ischemia with 24 hours of reperfusion. To determine the effect of TRPV4 inhibition on BBB permeability, hyperglycemic male Wistar rats (3 days by STZ) were treated with HC067047 (1mg/kg; n=6) or vehicle (n=6) upon reperfusion after 2 hours of ischemia and edema measured at 2 hours by wet:dry weights. Lastly, basal tone of MCAs isolated from male Wistar rats after 2 hours of ischemia with 2 hours of reperfusion (MCAO, n=7) or sham controls (Sham, n=6) was measured before and after addition of HC067047 (1μM) at 75 mmHg. Results: Infarct volume was significantly reduced in TRPV4 knockout mice and with HC067047 treatment vs. vehicle (17.8 ± 2.8% for KO, 22.9 ± 2.7% for HC067047 vs. 39.8 ± 3.3% for vehicle; p&lt;0.05). Hyperglycemic stroke caused considerable edema formation the ipsilateral vs. contralateral brain in vehicle-treated rats (81.38 ± 0.21% vs. 78.64 ± 0.12%; p&lt;0.01) that was prevented by treatment with HC067047 (78.58 ± 0.22% vs. 78.24 ± 0.11%;p&gt;0.05). MCAO diminished basal tone of MCAs vs. sham (16.3 ± 3.3 vs. 26.4 ± 2.8%; p&lt;0.05) that was restored by HC067047 (25.9 ± 3.0%; p&lt;0.05). The constriction to HC067047 did not occur in MCA that were denuded of endothelium or from sham animals. Conclusion: These results suggest that postischemic reperfusion activates TRPV4 in cerebral endothelium to cause dilation and BBB permeability. The decreased infarct size after TRPV4 inhibition suggests that activation of TRPV4 during ischemia contributes to neuronal cell death. Thus, inhibition of TRPV4 may be an effective treatment for acute ischemic stroke.