Transient Receptor Potential Vanilloid 4 Antagonist Research Articles

Targeting TRPV4 Channels for Cancer Pain Relief.

Despite the unique and complex nature of cancer pain, the activation of different ion channels can be related to the initiation and maintenance of pain. The transient receptor potential vanilloid 4 (TRPV4) is a cation channel broadly expressed in sensory afferent neurons. This channel is activated by multiple stimuli to mediate pain perception associated with inflammatory and neuropathic pain. Here, we focused on summarizing the role of TRPV4 in cancer etiology and cancer-induced pain mechanisms. Many studies revealed that the administration of a TRPV4 antagonist and TRPV4 knockdown diminishes nociception in chemotherapy-induced peripheral neuropathy (CIPN). Although the evidence on TRPV4 channels' involvement in cancer pain is scarce, the expression of these receptors was reportedly enhanced in cancer-induced bone pain (CIBP), perineural, and orofacial cancer models following the inoculation of tumor cells to the bone marrow cavity, sciatic nerve, and tongue, respectively. Effective pain management is a continuous problem for patients diagnosed with cancer, and current guidelines fail to address a mechanism-based treatment. Therefore, examining new molecules with potential antinociceptive properties targeting TRPV4 modulation would be interesting. Identifying such agents could lead to the development of treatment strategies with improved pain-relieving effects and fewer adverse effects than the currently available analgesics.

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.

TRPV4 Activation during Guinea Pig Airway Smooth Muscle Contraction Promotes Ca2+ and Na+ Influx.

Airway smooth muscle (ASM) contraction is determined by the increase in intracellular Ca2+ concentration ([Ca2+]i) caused by its release from the sarcoplasmic reticulum (SR) or by extracellular Ca2+ influx. Major channels involved in Ca2+ influx in ASM cells are L-type voltage-dependent Ca2+ channels (L-VDCCs) and nonselective cation channels (NSCCs). Transient receptor potential vanilloid 4 (TRPV4) is an NSCC recently studied in ASM. Mechanical stimuli, such as contraction, can activate TRPV4. We investigated the possible activation of TRPV4 by histamine (His)- or carbachol (CCh)-induced contraction in guinea pig ASM. In single myocytes, the TRPV4 agonist (GSK101) evoked an increase in [Ca2+]i, characterized by a slow onset and a plateau phase. The TRPV4 antagonist (GSK219) decreased channel activity by 94%, whereas the Ca2+-free medium abolished the Ca2+ response induced by GSK101. Moreover, GSK101 caused Na+ influx in tracheal myocytes. GSK219 reduced the Ca2+ peak and the Ca2+ plateau triggered by His or CCh. TRPV4 blockade shifted the concentration-response curve relating to His and CCh to the right in tracheal rings and reduced the maximal contraction. Finally, the activation of TRPV4 in single myocytes increased the Ca2+ refilling of the SR. We conclude that contraction of ASM cells after stimulation with His or CCh promotes TRPV4 activation, the subsequent influx of Ca2+ and Na+, and the opening of L-VDCCs. The entry of Ca2+ into ASM cells via TRPV4 and L-VDCCs contributes to optimal smooth muscle contraction.

Modulating TRPV4 Channel Activity in Pro-Inflammatory Macrophages within the 3D Tissue Analog.

Investigating macrophage plasticity emerges as a promising strategy for promoting tissue regeneration and can be exploited by regulating the transient receptor potential vanilloid 4 (TRPV4) channel. The TRPV4 channel responds to various stimuli including mechanical, chemical, and selective pharmacological compounds. It is well documented that treating cells such as epithelial cells and fibroblasts with a TRPV4 agonist enhances the Ca2+ influx to the cells, which leads to secretion of pro-inflammatory cytokines, while a TRPV4 antagonist reduces both Ca2+ influx and pro-inflammatory cytokine secretion. In this work, we investigated the effect of selective TRPV4 modulator compounds on U937-differentiated macrophages encapsulated within three-dimensional (3D) matrices. Despite offering a more physiologically relevant model than 2D cultures, pharmacological treatment of macrophages within 3D collagen matrices is largely overlooked in the literature. In this study, pro-inflammatory macrophages were treated with an agonist, 500 nM of GSK1016790A (TRPV4(+)), and an antagonist, 10 mM of RN-1734 (TRPV4(-)), to elucidate the modulation of the TRPV4 channel at both cellular and extracellular levels. To evaluate macrophage phenotypic alterations within 3D collagen matrices following TRPV4 modulator treatment, we employed structural techniques (SEM, Masson's trichrome, and collagen hybridizing peptide (CHP) staining), quantitative morphological measures for phenotypic assessment, and genotypic methods such as quantitative real-time PCR (qRT-PCR) and immunohistochemistry (IHC). Our data reveal that pharmacological modulation of the macrophage TRPV4 channel alters the cytoskeletal structure of macrophages and influences the 3D structure encapsulating them. Moreover, we proved that treating macrophages with a TRPV4 agonist and antagonist enhances the expression of pro- and anti-inflammatory genes, respectively, leading to the upregulation of surface markers CD80 and CD206. In the TRPV4(-) group, the CD206 gene and CD206 surface marker were significantly upregulated by 9- and 2.5-fold, respectively, compared to the control group. These findings demonstrate that TRPV4 modulation can be utilized to shift macrophage phenotype within the 3D matrix toward a desired state. This is an innovative approach to addressing inflammation in musculoskeletal tissues.

Hypothermia Attenuates Neurotoxic Microglial Activation via TRPV4.

Therapeutic hypothermia (TH) provides neuroprotection. However, the cellular mechanisms underlying the neuroprotective effects of TH are not fully elucidated. Regulation of microglial activation has the potential to treat a variety of nervous system diseases. Transient receptor potential vanilloid 4 (TRPV4), a nonselective cation channel, is activated by temperature stimulus at 27-35°C. Although it is speculated that TRPV4 is associated with the neuroprotective mechanisms of TH, the role of TRPV4 in the neuroprotective effects of TH is not well understood. In the present study, we investigated whether hypothermia attenuates microglial activation via TRPV4 channels. Cultured microglia were incubated under normothermic (37°C) or hypothermic (33.5°C) conditions following lipopolysaccharide (LPS) stimulation. Hypothermic conditions suppressed the expression of pro-inflammatory cytokines, inducible nitric oxide synthase, and the number of phagocytic microglia. AMP-activated protein kinase (AMPK)-NF-κB signaling was inhibited under hypothermic conditions. Furthermore, hypothermia reduced neuronal damage induced by LPS-treated microglial cells. Treatment with TRPV4 antagonist in normothermic culture replicated the suppressive effects of hypothermia on microglial activation and microglia-induced neuronal damage. In contrast, treatment with a TRPV4 agonist in hypothermic culture reversed the suppressive effect of hypothermia. These findings suggest that TH suppresses microglial activation and microglia-induced neuronal damage via the TRPV4-AMPK-NF-κB pathway. Although more validation is needed to consider differences according to age, sex, and specific central nervous system regions, our findings may offer a novel therapeutic approach to complement TH.

TRPV4 blockade alleviates endoplasmic reticulum stress mediated apoptosis in hypoxia–induced cardiomyocyte injury

BackgroundHypoxia-induced myocardial injury remains to be a huge health issue worldwide. Transient receptor potential vanilloid 4 (TRPV4) is a high-flux Ca2+ channel that is involved in numerous cardiovascular diseases. However, the role of TRPV4 in myocardial hypoxic injury remains unclear. Accordingly, this study aimed to investigate the antiapoptotic activity of TRPV4 inhibition and elucidate the underlying mechanisms in myocardial hypoxic injury. MethodsThe ability of TRPV4 to modulate the endoplasmic reticulum stress (ERS) and apoptosis was assessed in vitro through the administration of the TRPV4 antagonist HC-067047 or the agonist GSK1016790A. Additionally, intracellular Ca2+ concentration was measured by Fluo-4 AM. ResultsTRPV4 expression was significantly upregulated in hypoxic H9c2 cells compared with that in normoxic cardiomyocytes, accompanied with increased intracellular Ca2+ levels. Conversely, TRPV4 inhibition alleviated ERS in hypoxic H9c2 cells and prevented apoptosis, whereas TRPV4 agonist exacerbated such events. Furthermore, H9c2 cell apoptosis was attenuated with the administration of 4-PBA, an ERS inhibitor. ConclusionTRPV4 inhibition alleviates hypoxia-induced H9c2 cell apoptosis by mitigating ERS.

Cytotoxic activity of TRPV4 antagonist RN-1734 in G-361 human melanoma cancer cell line

Purpose: Intracellular calcium (Ca2+) signaling plays a role in many cellular events, such as cell proliferation and differentiation, gene transcription, oxidative stress, the antioxidant system, and apoptosis. Transient receptor potential vanilloid 4 (TRPV4) channels are non-selective cation (Ca2+) channels. The present study aims to investigate the cytotoxic activity of RN-1734, a transient receptor potential vanilloid 4 (TRPV4) antagonist, in the G361 human melanoma cancer cell line. 
 Materials and Methods: The effects of RN-1734 on G361 cell viability at concentrations of 1, 5, 25, 50, and 100 μM were measured using the 3-(4,5-dimethylthiazol-2-il)-2,5-diphenyltetrazolium bromide (MTT) method. Total antioxidant status (TAS) and total oxidant status (TOS) levels were determined using a ready-made commercial kit, after which oxidative stress index (OSI) values were calculated. To determine the apoptotic effects of RN-1734, Bcl-2, Bax, and p53 expression levels, caspase-3 and -8 activities were examined via quantitative real-time PCR analysis. 
 Results: G361 cell viability significantly decreased to 82.72, 72.81, 56.36, 39.16 and 18.96% in RN-1734 groups (1, 5, 25, 50 and 100 μM) compared to the control group (100.00%). At IC50 concentration (39.48 μM), RN-1734 application (3.35 mmol/g prot.-TAS, 45.87 μmol/g prot.-TOS, and 1501.97 AU-OSI) increased the TAS level (2.17 mmol/g prot.) and decreased the TOS level (55.41 μmol/g prot.) and OSI value (3142.76 AU) compared to the control group. 
 Conclusion: Our findings show that RN-1734 may be a novel therapeutic approach to treating melanoma by decreasing the cell viability of G361 human melanoma cancer cells.

TRPV4 Activation and its Intracellular Modulation Mediated by Kinin Receptors Contribute to Painful Symptoms Induced by Anastrozole.

Anastrozole, an aromatase inhibitor, induces painful musculoskeletal symptoms, which affect patients' quality of life and lead to therapy discontinuation. Efforts have been made to understand the mechanisms involved in these painful symptoms to manage them better. In this context, we explored the role of the Transient Receptor Potential Vanilloid 4 (TRPV4), a potential transducer of several nociceptive mechanisms, in anastrozole-induced musculoskeletal pain in mice. Besides, we evaluated the possible sensibilization of TRPV4 by signalling pathways downstream, PLC, PKC and PKCε from kinin B2 (B2R) and B1 (B1R) receptors activation in anastrozole-induced pain. Anastrozole caused mechanical allodynia and muscle strength loss in mice. HC067047, TRPV4 antagonist, reduced the anastrozole-induced mechanical allodynia and muscle strength loss. In animals previously treated with anastrozole, the local administration of sub-nociceptive doses of the TRPV4 (4α-PDD or hypotonic solution), B2R (Bradykinin) or B1R (DABk) agonists enhanced the anastrozole-induced pain behaviours. The sensitizing effects induced by local injection of the TRPV4, B2R and B1R agonists in animals previously treated with anastrozole were reduced by pre-treatment with TRPV4 antagonist. Furthermore, inhibition of PLC, PKC or PKCε attenuated the mechanical allodynia and muscle strength loss induced by TRPV4, B2R and B1R agonists. The generation of painful conditions caused by anastrozole depends on direct TRPV4 activation or indirect, e.g., PLC, PKC and PKCε pathways downstream from B2R and B1R activation. Thus, the TRPV4 channels act as sensors of extracellular and intracellular changes, making them potential therapeutic targets for alleviating pain related to aromatase inhibitors use, such as anastrozole.

The role of TRPV4 in the regulation of retinal ganglion cells apoptosis in rat and mouse

Retinal ganglion cell (RGC) damages are common in glaucoma, causing atrophy of the optic papilla, visual field damage, and visual loss. Transient receptor potential vanilloid 4 (TRPV4) is significantly expressed in the eyeball and is sensitive to mechanical and osmotic pressure. However, the specific role and mechanism of TRPV4 in glaucoma and RGC progression remain unclear. TRPV4 expression was detected in RGCs under different pressure culture conditions. We also explored the pressure effect on TRPV4 expression and the role and mechanism behind the functional regulation of RGCs. Immunofluorescence staining, western blotting, and TUNEL were utilized in this study. Our results established that TRPV4 was expressed in RGCs. TRPV4 expression was decreased at 40 mmHg and 60 mmHg, and the expression of BAX at 40 mmHg, 60 mmHg. Additionally, the expression of caspase 9 protein increased at 40 mmHg with the pressure increase compared with the conventional culture group. TUNEL staining revealed that the apoptosis rate of RGCs was elevated at 40 mmHg and 60 mmHg, compared with the traditional culture group. Therefore, the expression of BAX and caspase 9 increased, along with the apoptosis rate of RGCs compared with the control group. However, after TRPV4 antagonist treatment, the expression of BAX and caspase 9 decreased, and the apoptosis rate of RGCs decreased. Thus, TRPV4 may affect the mitochondrial apoptosis pathway, such as BAX and caspase 9, leading to the apoptosis of RGCs. The antagonists of TRPV4 could provide a new idea for clinically treating acute glaucoma.

Activation of Transient Receptor Potential Vanilloid 4 (TRPV4) channels opens Connexin Hemichannels in Lens Epithelial Cells

ATP release from the lens via connexin hemichannels has been hypothesized as a response to TRPV4 activation when the lens is subjected to osmotic swelling. To explore the apparent linkage between TRPV4 activation and hemichannel opening we performed patch-clamp recordings on cultured mouse lens epithelial cells exposed to the TRPV4 agonist GSK1016790A (GSK) in the presence or absence of the TRPV4 antagonist HC067047 (HC). GSK was found to cause a fast, variable and generally large non-selective increase of whole cell membrane conductance evident as a larger membrane current (Im) over a wide voltage range. This response was prevented by HC. The GSK-induced Im increase was proportionally larger at negative voltages and coincided with fast depolarization and the simultaneous disappearance of an outward current, likely a K + current. The initial presence of this outward current in control conditions appeared to be a reliable predictor of a cell’s response to GSK treatment. In some studies, recordings from single cells combining cell-attached and whole-cell patch clamp configurations, were obtained. This novel approach revealed events with a channel conductance 180-270 pS following GSK application through the patch pipette on the cell-attached side. The findings are consistent with TRPV4-dependent opening of connexin hemichannels. Ongoing studies are aimed at confirming the hemichannels identity. NIH: 5R01EY029171-04 and 2R01EY009532-28A1. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P=0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047significantly decreased afferent discharge to mechanical stimulation (P=0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P=0.019, ΔMAP: P=0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Pharmacological activation of transient receptor potential vanilloid 4 promotes triggering of the swallowing reflex in rats.

The swallowing reflex is an essential physiological reflex that allows food or liquid to pass into the esophagus from the oral cavity. Delayed triggering of this reflex is a significant health problem in patients with oropharyngeal dysphagia for which no pharmacological treatments exist. Transient receptor potential channels have recently been discovered as potential targets to facilitate triggering of the swallowing reflex. However, the ability of transient receptor potential vanilloid 4 (TRPV4) to trigger the swallowing reflex has not been studied. Here, we demonstrate the involvement of TRPV4 in triggering the swallowing reflex in rats. TRPV4 immunoreactive nerve fibers were observed in the superior laryngeal nerve (SLN)-innervated swallowing-related regions. Retrograde tracing with fluorogold revealed localization of TRPV4 on approximately 25% of SLN-afferent neurons in the nodose-petrosal-jugular ganglionic complex. Among them, approximately 49% were large, 35% medium, and 15% small-sized SLN-afferent neurons. Topical application of a TRPV4 agonist (GSK1016790A) to the SLN-innervated regions dose-dependently facilitated triggering of the swallowing reflex, with the highest number of reflexes triggered at a concentration of 250 μM. The number of agonist-induced swallowing reflexes was significantly reduced by prior topical application of a TRPV4 antagonist. These findings indicate that TRPV4 is expressed on sensory nerves innervating the swallowing-related regions, and that its activation by an agonist can facilitate swallowing. TRPV4 is a potential pharmacological target for the management of oropharyngeal dysphagia.

Loss of TRPV4 Cation Channel Inhibition of Macrophage Infiltration and Neovascularization in a Mouse Cornea

Corneal injury–associated inflammation could induce inward-growing neovascularization from the periphery of the tissue. Such neovascularization could cause stromal opacification and curvature disturbance, and both potentially impair visual function. In this study, we determined the effects of the loss of transient receptor potential vanilloid 4 (TRPV4) expression on the development of neovascularization in the corneal stroma in mice by producing a cauterization injury in the central area of the cornea. New vessels were immunohistochemically labeled with anti-TRPV4 antibodies. TRPV4 gene knockout suppressed the growth of such CD31-labeled neovascularization in association with the suppression of infiltration of macrophages and tissue messenger RNA expression of the vascular endothelial cell growth factor A level. Treatment of cultured vascular endothelial cells with supplementation of HC-067047 (0.1 μM, 1 μM, or 10 μM), a TRPV4 antagonist, attenuated the formation of a tube-like structure with sulforaphane (15 μM, for positive control) that modeled the new vessel formation. Therefore, the TRPV4 signal is involved in injury-induced macrophagic inflammation and neovascularization activity by vascular endothelial cells in a mouse corneal stroma. TRPV4 could be a therapeutic target to prevent unfavorable postinjury neovascularization in the cornea.

Potential Role of TRPV4 in Stretch-Induced Ghrelin Secretion and Obesity.

Obesity is an important health problem, which can be prevented through appetite control. Ghrelin is an appetite-stimulating hormone considered to promote obesity. Thus, we examined whether gastric stretching affects ghrelin secretion. We investigated the role of transient receptor potential vanilloid 4 (TRPV4) in gastric glands in the regulation of ghrelin secretion. TRPV4 immunostaining was performed in tissue samples from 57 patients who underwent gastrectomy. TRPV4 expression was compared between patients with (body mass index (BMI) ≥ 30) and without (BMI <30) obesity. For in vitro experiments, we used MGN3-1 cells, a ghrelin-producing cell line derived from mice. To investigate the bioactivity of TRPV4, MGN3-1 cells were treated with TRPV4 agonists and antagonists, and changes in intracellular Ca2+ concentration were confirmed. The concentration of ghrelin in the cell supernatant was measured using the ELISA with and without 120% stretch stimulation. TRPV4 expression was significantly higher in patients with obesity than in those without at all sites, except the fornix. Immunostaining confirmed the expression of TRPV4 in MGN3-1 cells. TRPV4 agonist administration increased intracellular Ca2+ concentration and ghrelin secretion in MGN3-1 cells, whereas the administration of the agonist combined with the antagonist decreased intracellular Ca2+ concentration and ghrelin secretion. Ghrelin secretion significantly increased in response to a 120% stretch in MGN3-1 cells. However, secretion was not increased by stretch when cells were treated with a TRPV4 antagonist. TRPV4 regulates ghrelin secretion in response to stretch in the stomach, which may affect body weight.

Abstract 11093: Spike Proteins Increase Endothelial Calcium via TRPV4

Introduction: Endothelial dysfunction plays a central role in the pathogenesis of acute respiratory distress syndrome (ARDS) with COVID-19. Transient receptor potential vanilloid 4 (TRPV4), a cation channel ubiquitously expressed, can regulate inflammatory cytokines that play key roles in in acute lung injury/ARDS. However, it is unknown whether spike proteins can affect TRPV4 activity and related Ca 2+ signaling in pulmonary microvascular endothelial cells. Hypothesis: We hypothesized that spike protein causes activation of TRPV4 channels, resulting in increases in intracellular Ca 2+ , may lead to pulmonary endothelial dysfunction. Methods: Intracellular Ca 2+ concentrations in human lung microvascular endothelial cells (HLMECs) were measured by calcium imaging in the presence of SARS CoV-2 Spike protein S1, receptor-binding domain (RBD) of S1, or protein S2 with or without co-incubation of the selective TRPV4 antagonist (HC-067047). Results: The intracellular Ca 2+ concentration of HLMECs was significantly increased when incubated with S1 (1, 10nM) or S1 RBD (1, 10nM) for 12, 24, 48 hours, relative to control or S2 (p&lt;0.05, respectively, Fig. A, B). Co-incubation of HC-067047 (500nM) significantly attenuated Ca 2+ intracellular influx upon treatment with S1 (10nM, 24 hours, p&lt;0.05) or S1 RBD (10nM, 24 hours, p&lt;0.05) (Fig. C). TRPV4 sensitive current density was significantly increased when incubated with S1 (10nM) or S1 RBD (10nM) for 24 hours (p&lt;0.05 vs. control, respectively, Fig. D-G), whereas co-incubated with HC-067047 (500nM) significantly reversed the S1 (10nM, 24 hours, p&lt;0.05) or S1 RBD (10nM, 24 hours, p&lt;0.05) induced increases of TRPV4 sensitive current density (Fig. D-G). Conclusions: The of SARS CoV-2 Spike protein S1 and S1 RBD caused the activation of TRPV4 channels, resulting in increased intracellular Ca 2+ , may lead to pulmonary endothelial dysfunction.

Blockage of TRPV4 Downregulates the Nuclear Factor-Kappa B Signaling Pathway to Inhibit Inflammatory Responses and Neuronal Death in Mice with Pilocarpine-Induced Status Epilepticus.

The blockage of transient receptor potential vanilloid 4 (TRPV4) inhibits inflammation and reduces hippocampal neuronal injury in a pilocarpine-induced mouse model of temporal lobe epilepsy. However, the underlying mechanisms remain largely unclear. NF-κB signaling pathway is responsible for the inflammation and neuronal injury during epilepsy. Here, we explored whether TRPV4 blockage could affect the NF-κB pathway in mice with pilocarpine-induced status epilepticus (PISE). Application of a TRPV4 antagonist markedly attenuated the PISE-induced increase in hippocampal HMGB1, TLR4, phospho (p)-IκK (p-IκK), and p-IκBα protein levels, as well as those of cytoplasmic p-NF-κB p65 (p-p65) and nuclear NF-κB p65 and p50; in contrast, the application of GSK1016790A, a TRPV4 agonist, showed similar changes to PISE mice. Administration of the TLR4 antagonist TAK-242 or the NF-κB pathway inhibitor BAY 11-7082 led to a noticeable reduction in the hippocampal protein levels of cleaved IL-1β, IL-6 and TNF, as well as those of cytoplasmic p-p65 and nuclear p65 and p50 in GSK1016790A-injected mice. Finally, administration of either TAK-242 or BAY 11-7082 greatly increased neuronal survival in hippocampal CA1 and CA2/3 regions in GSK1016790A-injected mice. Therefore, TRPV4 activation increases HMGB1 and TLR4 expression, leading to IκK and IκBα phosphorylation and, consequently, NF-κB activation and nuclear translocation. The resulting increase in pro-inflammatory cytokine production is responsible for TRPV4 activation-induced neuronal injury. We conclude that blocking TRPV4 can downregulate HMGB1/TLR4/IκK/κBα/NF-κB signaling following PISE onset, an effect that may underlie the anti-inflammatory response and neuroprotective ability of TRPV4 blockage in mice with PISE.

Role of transient receptor potential vanilloid 4 in therapeutic antifibrotic effects of pirfenidone.

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, fatal lung disorder characterized by aberrant extracellular matrix deposition in the interstitium. Pirfenidone is an antifibrotic agent used to treat patients with IPF. Pirfenidone shows a pleiotropic mode of action, but its underlying antifibrotic mechanism is unclear. Transient receptor potential vanilloid 4 (TRPV4), which is a mechanosensitive calcium channel, was recently shown to be related to pulmonary fibrosis. To clarify the antifibrotic mechanisms of pirfenidone, we investigated whether TRPV4 blockade has a pharmacological effect in a murine model of pulmonary fibrosis and whether pirfenidone contributes to suppression of TRPV4. Our synthetic TRPV4 antagonist and pirfenidone treatment attenuated lung injury in the bleomycin mouse model. TRPV4-mediated increases in intracellular calcium were inhibited by pirfenidone. In addition, TRPV4-stimulated interleukin-8 release from cells was reduced and a delay in cell migration was abolished by pirfenidone. Furthermore, pirfenidone decreased TRPV4 endogenous ligands in bleomycin-administered mouse lungs and their production by microsomes of human lungs. We found TRPV4 expression in the bronchiolar and alveolar epithelium and activated fibroblasts of the lungs in patients with IPF. Finally, we showed that changes in forced vital capacity of patients with IPF treated with pirfenidone were significantly correlated with metabolite levels of TRPV4 endogenous ligands in bronchoalveolar lavage fluid. These results suggest that the antifibrotic action of pirfenidone is partly mediated by TRPV4 and that TRPV4 endogenous ligands in bronchoalveolar lavage fluid may be biomarkers for distinguishing responders to pirfenidone.

Activation of transient receptor potential vanilloid 4 is involved in pressure overload-induced cardiac hypertrophy.

Previous studies, including our own, have demonstrated that transient receptor potential vanilloid 4 (TRPV4) is expressed in hearts and implicated in cardiac remodeling and dysfunction. However, the effects of TRPV4 on pressure overload-induced cardiac hypertrophy remain unclear. In this study, we found that TRPV4 expression was significantly increased in mouse hypertrophic hearts, human failing hearts, and neurohormone-induced hypertrophic cardiomyocytes. Deletion of TRPV4 attenuated transverse aortic constriction (TAC)-induced cardiac hypertrophy, cardiac dysfunction, fibrosis, inflammation, and the activation of NFκB - NOD - like receptor pyrin domain-containing protein 3 (NLRP3) in mice. Furthermore, the TRPV4 antagonist GSK2193874 (GSK3874) inhibited cardiac remodeling and dysfunction induced by TAC. In vitro, pretreatment with GSK3874 reduced the neurohormone-induced cardiomyocyte hypertrophy and intracellular Ca2+ concentration elevation. The specific TRPV4 agonist GSK1016790A (GSK790A) triggered Ca2+ influx and evoked the phosphorylation of Ca2+/calmodulin-dependent protein kinase II (CaMKII). But these effects were abolished by removing extracellular Ca2+ or GSK3874. More importantly, TAC or neurohormone stimulation-induced CaMKII phosphorylation was significantly blocked by TRPV4 inhibition. Finally, we show that CaMKII inhibition significantly prevented the phosphorylation of NFκB induced by GSK790A. Our results suggest that TRPV4 activation contributes to pressure overload-induced cardiac hypertrophy and dysfunction. This effect is associated with upregulated Ca2+/CaMKII mediated activation of NFκB-NLRP3. Thus, TRPV4 may represent a potential therapeutic drug target for cardiac hypertrophy and dysfunction after pressure overload.

Changes in TRPV4, AQP1, and AQP4 in a Genetic Model of Hydrocephalus Treated with a TRPV4 Antagonist

Hydrocephalus occurs as a result of cerebrospinal fluid (CSF) accumulating in the brain’s ventricles causing ventriculomegaly. Hydrocephalus can affect various cell types in the brain, and this study focuses on choroid plexus epithelial cells (CPe) and astrocytes. The choroid plexus is a tight epithelial structure within the ventricular system, responsible for producing CSF. Astrocytes are supportive cells that serve in blood‐brain barrier maintenance and brain fluid/electrolyte regulation. The CP and astrocytes contain channels and transporters that are important in regulating both CSF and brain interstitial fluid. These include transient receptor potential vanilloid 4 (TRPV4), aquaporin 1 (AQP1), and aquaporin 4 (AQP4). TRPV4 is a mechanosensitive channel and has been implicated in osmotic regulation. AQPs are a family of membrane proteins that facilitate water transport in various tissues. The CPe expresses AQP1 and TRPV4 which may be important in CSF production. Astrocytes express TRPV4 and AQP4 on their endfeet to regulate cell volume and vascular permeability. Previous studies done by the Blazer‐Yost laboratory have shown increases in AQP1, but not TRPV4 mRNA of CPe from hydrocephalic animals compared to wildtype. This study further examined changes in localization and expression of TRPV4, AQP1, and AQP4 in a rodent model of hydrocephalus.A single missense point mutation in the Transmembrane 67 (TMEM67) protein, that causes a ciliopathy resulting in hydrocephalus and polycystic kidney disease (PKD) in our rodent model, is orthologous to the human Meckel Gruber syndrome type 3 (MKS3). Treatment with the TRPV4 antagonist, RN1734, ameliorates hydrocephalus in our genetic model of hydrocephalus. Using immunohistochemical techniques, TRPV4, AQP1, and AQP4 antibodies were used to examine localization in untreated and RN1734 treated hydrocephalic animals at P15. Changes of AQP4 and TRPV4 were observed in the subventricular zone of homozygous animals. RTqPCR was used to quantitate mRNA from the CPe and subventricular region of the cortex. Preliminary RTqPCR showed increased AQP1, decreased TRPV4 and no change in AQP4 mRNA in the cortex and CPe of P15 untreated and RN1734 treated hydrocephalic animals compared to wild‐type. These data suggests that the changes occurring may be posttranslational. To observe this, Western blots were utilized to look at changes in protein. The protein expression of AQP1 and AQP4 may be increased in untreated cortical tissue from hydrocephalic animals, while total TRPV4 remaining unchanged. AQP1 and AQP4 may be still increased, while total TRPV4 decreased in animals treated with RN1734. These results provide further characterization of the role of several channels and transporters in the pathophysiology of hydrocephalus. Future studies will explore post‐translational changes in protein expression and membrane localization to compliment these studies. Examining channels and transporters can elucidate how brain fluid regulation may be altered in hydrocephalus and produce targets for pharmacological treatment in the future.

Nitric Oxide Inhibits TRPV4‐mediated Hemichannel Opening in Porcine Nonpigmented Ciliary Epithelium

Previous studies showed the presence of connexin 43 (Cx43), connexin 50 (Cx50), and pannexin 1 (Panx1) in the nonpigmented ciliary epithelium (NPE) of the eye, as well as evidence of hemichannel (HC) activity. In the lens, mechanosensitive Transient Receptor Potential Vanilloid 4 (TRPV4)‐mediated calcium entry stimulates hemichannel activity. The aim here is to test for TRPV4 expression in the NPE, determine whether TRPV4 activation causes HC opening, and study the influence of nitric oxide.Immunohistochemistry was used to probe for TRPV4 in porcine ciliary body. Propidium iodide (PI) uptake and ATP release by primary cultured NPE was used to assess HC opening. PI and ATP are large molecules (MW 668 and 507, respectively) that pass through open hemichannels but not the plasma membrane. ATP in the bathing medium was measured by luciferase luminometry and PI uptake by fluorimetry.Abundant expression of TRPV4 was detected in the NPE cell layer of the ciliary body. Cultured NPE cells displayed an increase in PI uptake when exposed to a TRPV4 agonist GSK 1016790A (10 nM) or to hypoosmotic solution (200 mOsm) (Control 4.65 ± 0.24 vs GSK 10.26 ± 0.32 and Hypo 8.26 ± 0.42 fluorescence units per mg protein; n=6, p&lt;0.001). PI uptake also was increased in NPE cells cultured on a flexible membrane and subjected to cyclic stretch for 1‐10 min (10%, 0.5 Hz), the peak response was at at 2 min (Control 1.5 ± 0.03 vs stretch 2.5 ± 0.10; n=6, p&lt;0.001). The increase in PI uptake caused by hypoosmotic solution or by cyclic stretch was abolished by a selective TRPV4 antagonist HC 067047 (10 μM). Hyposmotic solution increased ATP release from 1.12 ± 0.24 to 2.81± 0.27 (n=12, p&lt;0.001) while cyclic stretch increased ATP release from 0.20 ± 0.03 to 0.40 ± 0.03 pmoles/mg protein (n=6, p&lt;0.001). ATP release responses caused by hypoosmotic solution and cyclic stretch were inhibited by HC 067047. Hypoosmotic solution‐induced ATP release was also inhibited by connexin inhibitory peptide, Gap‐27 (200 μM) and nitric oxide (NO) donors, sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine (SNAP).TRPV4 channels are expressed by the NPE. The ability of HC 067047 and Gap‐27 to prevent ATP release and PI uptake points to TRPV4‐dependent opening of connexin HCs in NPE cells. Suppression of ATP release by SNP and SNAP indicates the hemichannel opening mechanism is inhibited by NO.