Modulation of TRPV4-mediated TNF-α expression in Müller glia and subsequent RGC apoptosis by statins.

In addition to regulating cholesterol synthesis, statins have neuroprotective effects. Apoptosis of retinal ganglion cells (RGCs) causes a gradual loss of visual function in glaucoma. This study aimed to investigate the neuroprotective effect of statins on the RGC apoptosis induced by activated Müller glia. Primary Müller cells and RGCs were cultured from the retina of C57BL6 mice. Müller cells were activated with GSK101, a transient receptor potential vanilloid 4 (TRPV4) agonist, and tumor necrosis factor-alpha (TNF-α) released to the medium was measured using an enzyme-linked immunosorbent assay. Cells were pretreated with simvastatin or lovastatin before GSK101. RGCs were treated with conditioned media from Müller glia cultures, and apoptosis was determined using flow cytometry. TRPV4 activation through GSK101 treatment induced gliosis of Müller cells, and the conditioned media from activated Müller cells was potent to induce RGC apoptosis. Statins suppress both gliosis in Müller cells and subsequent RGC apoptosis. TNF-α release to the media was increased in GSK101-treated Müller cells, and TNF-α in the conditioned media was the critical factor causing RGC apoptosis. The increase in TRPV4-mediated TNF-α expression occurred through the nuclear factor kappa-light chain enhancer of activated B cell pathway activation, which was inhibited by statins. Herein, we showed that statins can modulate gliosis and TNF-α expression in Müller cells, protecting RGCs. These data further support the neuroprotective effect of statins, promoting them as a potential treatment for glaucoma.

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-1ß, 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.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Consideration of Kinase Inhibitors for the Treatment of Hydrocephalus.

Hydrocephalus is a devastating condition characterized by excess cerebrospinal fluid (CSF) in the brain. Currently, the only effective treatment is surgical intervention, usually involving shunt placement, a procedure prone to malfunction, blockage, and infection that requires additional, often repetitive, surgeries. There are no long-term pharmaceutical treatments for hydrocephalus. To initiate an intelligent drug design, it is necessary to understand the biochemical changes underlying the pathology of this chronic condition. One potential commonality in the various forms of hydrocephalus is an imbalance in fluid-electrolyte homeostasis. The choroid plexus, a complex tissue found in the brain ventricles, is one of the most secretory tissues in the body, producing approximately 500 mL of CSF per day in an adult human. In this manuscript, two key transport proteins of the choroid plexus epithelial cells, transient receptor potential vanilloid 4 and sodium, potassium, 2 chloride co-transporter 1, will be considered. Both appear to play key roles in CSF production, and their inhibition or genetic manipulation has been shown to affect CSF volume. As with most transporters, these proteins are regulated by kinases. Therefore, specific kinase inhibitors are also potential targets for the development of pharmaceuticals to treat hydrocephalus.

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.

Calcium influx through TRPV4 channels involve in hyperosmotic stress-induced epithelial-mesenchymal transition in tubular epithelial cells.

The epithelial-mesenchymal transition (EMT) is a biological process that occurs in the pathogenesis of kidney diseases in which injured tubular epithelial cells transform into myofibroblasts. We previously showed that mannitol-mediated hyperosmotic stress induces EMT of tubular epithelial cells. Although Ca2+ signaling is essential for the induction of EMT in tubular epithelial cells, the role of specific calcium channels is unknown. In this study, we assessed the transient receptor potential vanilloid 4 (TRPV4)-mediated Ca2+ influx in the hyperosmolarity-induced EMT. The Fluo-4 assay was used to examine the effect of hyperosmotic stress on the intracellular Ca2+ level of normal rat kidney (NRK)-52E cells. Expression of a mesenchymal marker α-smooth muscle actin (α-SMA) and an epithelial marker E-cadherin was also observed by fluorescence microscopy. The hyperosmotic stress caused a transient increase in intracellular Ca2+ concentration as well as a decrease in E-cadherin and an increase in α-SMA expressions in tubular epithelial cells, indicating the induction of EMT. A TRPV4 channel antagonist inhibited hyperosmotic stress-induced Ca2+ influx and the EMT, whereas, a TRPV4 channel agonist increased Ca2+ influx and EMT induction in tubular epithelial cells without the hyperosmotic stress. These findings suggest that Ca2+ influx through TRPV4 channels contributes to the hyperosmotic stress-induced EMT of tubular epithelial cells.

Combination Treatment of TRPV4 Agonist with Cisplatin Promotes Vessel Normalization in an Animal Model of Oral Squamous Cell Carcinoma.

Background and Objectives: Oral squamous cell carcinoma (OSCC) is the sixth most common malignancy in the world. Transient receptor potential vanilloid 4 (TRPV4) channel has been shown to be involved in angiogenesis in multiple types of tumors. However, not much is known about TRPV4's involvement in OSCC. Thus, in this study, we investigate the effect of administering a TRPV4 agonist on angiogenesis in OSCC. Materials and Methods: Thirty-six Sprague Dawley (SD) rats were used in this study. 4-nitroquinoline 1-oxide (4NQO) was used to induce OSCC. Cisplatin (an anticancer drug), and GSK1016790A (an agonist for TRPV4) was used in this study. Immunohistochemistry was employed to examine the TRPV4 expression. An RT2 Profiler PCR Array was performed for gene expression analysis of TRPV4, vascular growth factors that correspond directly with angiogenesis, such as angiopoietin (Ang-1 and Ang-2), and tyrosine kinase (Tie-1 and Tie-2) receptors. Tumor vessel maturity was assessed by microvessel density and microvessel-pericyte-coverage index. Results: RT2 profiler PCR array showed significant elevated levels of Ang-1 (2.1-fold change; p < 0.05) and Tie-2 (4.5-fold change; p < 0.05) in OSCC following the administration of a combination of GSK1016790A and cisplatin. Additionally, the combination treatment significantly reduced the microvessel density (p < 0.01) and significantly increased the percentage of microvessels covered with pericytes (p < 0.01) in OSCC. Furthermore, tumor size was significantly reduced (p < 0.05) in rats that received cisplatin alone. The combination treatment also greatly reduced the tumor size; however, the data were not statistically significant. Conclusions: The findings suggest that combining a TRPV4 agonist with cisplatin for treatment of OSCC promote vessels normalization via modulation of Ang-1/Tie-2 pathway.

The nonselective cation channel TRPV4 inhibits angiotensin II receptors.

G-protein-coupled receptors (GPCRs) are a ubiquitously expressed family of receptor proteins that regulate many physiological functions and other proteins. They act through two dissociable signaling pathways: the exchange of GDP to GTP by linked G-proteins and the recruitment of ß-arrestins. GPCRs modulate several members of the transient receptor potential (TRP) channel family of nonselective cation channels. How TRP channels reciprocally regulate GPCR signaling is less well-explored. Here, using an array of biochemical approaches, including immunoprecipitation and fluorescence, calcium imaging, phosphate radiolabeling, and a ß-arrestin-dependent luciferase assay, we characterize a GPCR-TRP channel pair, angiotensin II receptor type 1 (AT1R), and transient receptor potential vanilloid 4 (TRPV4), in primary murine choroid plexus epithelial cells and immortalized cell lines. We found that AT1R and TRPV4 are binding partners and that activation of AT1R by angiotensin II (ANGII) elicits ß-arrestin-dependent inhibition and internalization of TRPV4. Activating TRPV4 with endogenous and synthetic agonists inhibited angiotensin II-mediated G-protein-associated second messenger accumulation, AT1R receptor phosphorylation, and ß-arrestin recruitment. We also noted that TRPV4 inhibits AT1R phosphorylation by activating the calcium-activated phosphatase calcineurin in a Ca2+/calmodulin-dependent manner, preventing ß-arrestin recruitment and receptor internalization. These findings suggest that when TRP channels and GPCRs are co-expressed in the same tissues, many of these channels can inhibit GPCR desensitization.

Lymphangiogenesis and angiogenesis rescue murine ischemic hindlimb via transient receptor potential vanilloid 4.

In this study, we assessed the regulation of transient receptor potential vanilloid 4 (TRPV4) promoting lymphangio/angiogenesis to improve the ischemic hindlimb animal model, and revealed that (1) a TRPV4 agonist improved the blood flow of ischemic hindlimbs by inducing both angiogenesis and lymphangiogenesis; (2) excessive TRPV4 expression was detected on lymphatic endothelial cells (LECs) in the ischemic hindlimb; and (3) hypoxic conditions promoted Ca2+ influx into LECs via TRPV4. It is considered that the upregulation of both lymphatic and blood vessels by activating TRPV4 would be a promising therapeutic strategy for peripheral artery disease.

Volume sensing in the transient receptor potential vanilloid 4 ion channel is cell type-specific and mediated by an N-terminal volume-sensing domain.

Many retinal diseases are associated with pathological cell swelling, but the underlying etiology remains to be established. A key component of the volume-sensitive machinery, the transient receptor potential vanilloid 4 (TRPV4) ion channel, may represent a sensor and transducer of cell swelling, but the molecular link between the swelling and TRPV4 activation is unresolved. Here, our results from experiments using electrophysiology, cell volumetric measurements, and fluorescence imaging conducted in murine retinal cells and Xenopus oocytes indicated that cell swelling in the physiological range activated TRPV4 in Müller glia and Xenopus oocytes, but required phospholipase A2 (PLA2) activity exclusively in Müller cells. Volume-dependent TRPV4 gating was independent of cytoskeletal rearrangements and phosphorylation. Our findings also revealed that TRPV4-mediated transduction of volume changes is dependent by its N terminus, more specifically by its distal-most part. We conclude that the volume sensitivity and function of TRPV4 in situ depend critically on its functional and cell type-specific interactions.

Investigating the effect of TRPV4 inhibition on pulmonary-vascular barrier permeability following segmental endotoxin challenge.

BACKGROUND: Acute Respiratory Distress Syndrome (ARDS) is associated with increased pulmonary-vascular permeability. In the lung, transient receptor potential vanilloid 4 (TRPV4), a Ca2+-permeable cation channel, is a regulator of endothelial permeability and pulmonary edema. We performed a Phase I, placebo-controlled, double-blind, randomized, parallel group, proof-of-mechanism study to investigate the effects of TRPV4 channel blocker, GSK2798745, on pulmonary-vascular barrier permeability using a model of lipopolysaccharide (LPS)-induced lung inflammation. METHODS: Healthy participants were randomized 1:1 to receive 2 single doses of GSK2798745 or placebo, 12 h apart. Two hours after the first dose, participants underwent bronchoscopy and segmental LPS instillation. Total protein concentration and neutrophil counts were measured in bronchoalveolar lavage (BAL) samples collected before and 24 h after LPS challenge, as markers of barrier permeability and inflammation, respectively. The primary endpoint was baseline adjusted total protein concentration in BAL at 24 h after LPS challenge. A Bayesian framework was used to estimate the posterior probability of any percentage reduction (GSK2798745 relative to placebo). Safety endpoints included the incidence of adverse events (AEs), vital signs, 12-lead electrocardiogram, clinical laboratory and haematological evaluations, and spirometry. RESULTS: Forty-seven participants were dosed and 45 completed the study (22 on GSK2798745 and 23 on placebo). Overall, GSK2798745 was well tolerated. Small reductions in mean baseline adjusted BAL total protein (~9%) and neutrophils (~7%) in the LPS-challenged segment were observed in the GSK2798745 group compared with the placebo group; however, the reductions did not meet pre-specified success criteria of at least a 95% posterior probability that the percentage reduction in the mean 24-h post LPS BAL total protein level (GSK2798745 relative to placebo) exceeded zero. Median plasma concentrations of GSK2798745 were predicted to inhibit TRPV4 on lung vascular endothelial cells by ~70-85% during the 24 h after LPS challenge; median urea-corrected BAL concentrations of GSK2798745 were 3.0- to 8.7-fold higher than those in plasma. CONCLUSIONS: GSK2798745 did not affect segmental LPS-induced elevation of BAL total protein or neutrophils, despite blood and lung exposures that were predicted to be efficacious. CLINICALTRIALS. GOV IDENTIFIER: NCT03511105.

When size matters: transient receptor potential vanilloid 4 channel as a volume-sensor rather than an osmo-sensor.

KEY POINTS: Mammalian cells are frequently exposed to stressors causing volume changes. The transient receptor potential vanilloid 4 (TRPV4) channel translates osmotic stress into ion flux. The molecular mechanism coupling osmolarity to TRPV4 activation remains elusive. TRPV4 responds to isosmolar cell swelling and osmolarity translated via different aquaporins. TRPV4 functions as a volume-sensing ion channel irrespective of the origin of the cell swelling. ABSTRACT: Transient receptor potential channel 4 of the vanilloid subfamily (TRPV4) is activated by a diverse range of molecular cues, such as heat, lipid metabolites and synthetic agonists, in addition to hyposmotic challenges. As a non-selective cation channel permeable to Ca2+ , it transduces physical stress in the form of osmotic cell swelling into intracellular Ca2+ -dependent signalling events. Its contribution to cell volume regulation might include interactions with aquaporin (AQP) water channel isoforms, although the proposed requirement for a TRPV4-AQP4 macromolecular complex remains to be resolved. To characterize the elusive mechanics of TRPV4 volume-sensing, we expressed the channel in Xenopus laevis oocytes together with AQP4. Co-expression with AQP4 facilitated the cell swelling induced by osmotic challenges and thereby activated TRPV4-mediated transmembrane currents. Similar TRPV4 activation was induced by co-expression of a cognate channel, AQP1. The level of osmotically-induced TRPV4 activation, although proportional to the degree of cell swelling, was dependent on the rate of volume changes. Importantly, isosmotic cell swelling obtained by parallel activation of the co-expressed water-translocating Na+ /K+ /2Cl- cotransporter promoted TRPV4 activation despite the absence of the substantial osmotic gradients frequently employed for activation. Upon simultaneous application of an osmotic gradient and the selective TRPV4 agonist GSK1016790A, enhanced TRPV4 activation was observed only with subsaturating stimuli, indicating that the agonist promotes channel opening similar to that of volume-dependent activation. We propose that, contrary to the established paradigm, TRPV4 is activated by increased cell volume irrespective of the molecular mechanism underlying cell swelling. Thus, the channel functions as a volume-sensor, rather than as an osmo-sensor.

Aortic smooth muscle TRPV4 channels regulate vasoconstriction in high salt-induced hypertension.

Recent studies have focused on the contribution of vascular endothelial transient receptor potential vanilloid 4 (TRPV4) channels to hypertension. However, in hypertension, TRPV4 channels in vascular smooth muscle remain unexplored. In the present study, we performed wire myograph experiments in isolated aortas from endothelial cell specific TRPV4 channel knockout (TRPV4EC-/-) mice to demonstrate that GSK1016790A (a specific TRPV4 channel agonist) triggered aortic smooth muscle-dependent contractions from mice on a normal-salt diet, and the contractions were enhanced in high-salt diet (HSD) mice. Intracellular Ca2+ concentration ([Ca2+]i) and Ca2+ imaging assays showed that TRPV4-induced [Ca2+]i was significantly higher in aortic smooth muscle cells (ASMCs) from HSD-induced hypertensive mice, and application of an inositol trisphosphate receptor (IP3R) inhibitor markedly attenuated TRPV4-induced [Ca2+]i. IP3R2 expression was enhanced in ASMCs from HSD-induced hypertensive mice and the contractile response induced by TRPV4 was inhibited by the IP3R inhibitor. Whole-transcriptome analysis by RNA-seq and western blot assays revealed the involvement of interferon regulatory factor 7 (IRF7) in TRPV4-IRF7-IP3R2 signaling in HSD-induced hypertension. These results suggested that TRPV4 channels regulate smooth muscle-dependent contractions in high salt-induced hypertension, and this contraction involves increased [Ca2+]i, IP3R2, and IRF7 activity. Our study revealed a considerable effect of TRPV4 channels in smooth muscle-dependent contraction in mice during high-salt induced hypertension.

Activation of TRPV4 induces intraneuronal tau hyperphosphorylation via cholesterol accumulation.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel, whose aberrant function in neurons has been reported to participate in the progression of brain disorders, including Alzheimer's disease (AD). However, the influence of TRPV4 activation on tau hyperphosphorylation in AD has not yet been elucidated. Since disturbed brain cholesterol homeostasis is considered to be related to excessive tau phosphorylation, this study aimed to explore whether dysregulation of TRPV4 affects tau phosphorylation and whether it involves cholesterol unbalance. Our data indicated that TRPV4 activation increased tau phosphorylation in the cortex and hippocampus of P301S tauopathy mouse model and aggravated its cognitive decline. In addition, we detected that TRPV4 activation upregulated cholesterol levels in primary neurons, and the elevation of cholesterol promoted hyperphosphorylation of tau. TRPV4 knockdown improved tau hyperphosphorylation by reducing intracellular cholesterol accumulation. Our results suggest that activation of TRPV4 may take part in the pathological mechanism of AD by promoting intraneuronal tau hyperphosphorylation in a cholesterol-dependent manner.

Diverse Roles of TRPV4 in Macrophages: A Need for Unbiased Profiling.

Transient receptor potential vanilloid 4 (TRPV4) is a non-selective mechanosensitive ion channel expressed by various macrophage populations. Recent reports have characterized the role of TRPV4 in shaping the activity and phenotype of macrophages to influence the innate immune response to pathogen exposure and inflammation. TRPV4 has been studied extensively in the context of inflammation and inflammatory pain. Although TRPV4 activity has been generally described as pro-inflammatory, emerging evidence suggests a more complex role where this channel may also contribute to anti-inflammatory activities. However, detailed understanding of how TRPV4 may influence the initiation, maintenance, and resolution of inflammatory disease remains limited. This review highlights recent insights into the cellular processes through which TRPV4 contributes to pathological conditions and immune processes, with a focus on macrophage biology. The potential use of high-throughput and omics methods as an unbiased approach for studying the functional outcomes of TRPV4 activation is also discussed.

[Exploration on effect mechanism of Miao medicinal acupuncture therapy in treatment of knee osteoarthritis based on TRPV ion channel].

OBJECTIVE: To observe the effect of Miao medicinal acupuncture therapy on transient receptor potential vanilloid (TRPV) channel in knee joint synovial tissue of the rabbits with knee osteoarthritis (KOA) model and to explore the mechanism of Miao medicinal acupuncture therapy in treatment of KOA. METHODS: Of 34 New Zealand male rabbits, 6 rabbits were selected randomly as the normal group. KOA model was established in the rest rabbits by injecting a mixture of papain and L-cysteine in right knee joints. The 24 successfully modeled rabbits were randomized into a model group, a Miao medicinal acupuncture therapy group, a dermal needle group and a smearing group, 6 rabbits in each one. In the Miao medicinal acupuncture therapy group, Miao medicinal acupuncture therapy was adopted, in which, the roller type of dermal needle was used on the surface of right knee joint [a rectangle shape formed by "Xuehai" (SP 10), "Liangqiu" (ST 34), "Yanglingquan" (GB 34) and "Yinlingquan" (SP 9)], rolling in a "" shape, on which, Miao medicinal solution was smeared in advance. In the dermal needle group, the rolling stimulation was exerted on the right the right knee joint surface with the roller type of dermal needle. In the smearing group, Miao medicinal solution was smeared on the right knee joint surface. The intervention was given once every two days, 3 times weekly and the intervention was exerted consecutively for 4 weeks. Successively, on day 1, 21, 28, 35, 42 and 49 of experiment, paw withdrawal threshold (von Frey threshold) after mechanical stimulation was detected in the rabbits. HE staining was adopted to observe the histomorphological changes of the right knee joint cartilage in the rabbits. ELISA was used to determine the contents of interleukin-1 (IL-1ß) and tumor necrosis factor-α (TNF-α) in the right knee synovial fluid. Western blot method and real-time PCR were used to determine the relative expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue of the rabbits. RESULTS: Compared with the normal group, on day 49 of experiment, von Frey threshold was reduced significantly in the rabbits of the model group (P<0.01), the integrety of cartilage surface of knee joint was seriously damaged, the structural layers were disordered, the chondrocytes were clustered, the tide lines were distorted and the matrix staining disappeared. The contents of IL-1ß and TNF-α of the right knee synovial fluid were increased significantly (P<0.01), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were increased significantly (P<0.01). Compared with the model group, on day 49 of experiment, von Frey threshold was increased significantly in each of the Miao medicinal acupuncture therapy group, the dermal needle group and the smearing group (P<0.01). The right knee joint cartilage was complete in morphology, the structure clear was in layer, the cells were arranged in order and the matrix staining was uniform. The contents of IL-1ß and TNF-α of the right knee synovial fluid were reduced significantly (P<0.01, P<0.05), and the relative expressions of protein and mRNA of TRPV1 and TRPV4 in the synovial tissue of the right knee were reduced significantly (P<0.01). Compared with either the dermal needle group or the smearing group, on day 49 of experiment, von Frey threshold was increased significantly in the Miao medicinal acupuncture therapy group (P<0.01). The right knee joint cartilage surface was even, the structure layers were clear. The contents of IL-1ß and TNF-α of the right knee synovial fluid were reduced significantly (P<0.05), and the relative expressions of protein of TRPV1 and TRPV4 in the synovial tissue were reduced (P<0.01, P<0.05). Compared with the smearing group, the relative expression of TRPV1 mRNA in the synovial tissue was reduced significantly in the Miao medicinal acupuncture therapy group (P<0.05). CONCLUSION: Miao medicinal acupuncture therapy plays a role in treatment of KOA probably through inhibiting the expressions of IL-1ß and TNF-α of knee synovial fluid and down-regulating the expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue.

Research progress of TRPV4 in ocular pathophysiological function / 国际眼科杂志(Guoji Yanke Zazhi)

Transient receptor potential vanilloid receptor 4(TRPV4)is a non-selective cation channel responsible for sensing changes in cell swelling, temperature, mechanical stretch, shear stress and osmotic pressure by regulating transmembrane calcium signaling and thereby influencing gene expression, cell morphology, and cytoskeletal construction. TRPV4 is widely expressed throughout the body. Intraocularly, TRPV4 is functionally expressed in the cornea, lens, ciliary body, trabecular meshwork and retina. In this article, the expression and physiopathological functions of TRPV4 in various tissues of the eye were described. With the in-depth study of TRPV4 in ocular pathophysiological functions, TRPV4 may become a potential drug target in corneal injury repair, glaucoma and retinal angiogenesis, but further in-depth study is still needed.

Osteoporotic Pain is Associated with Increased Transient Receptor Vanilloid 4 Expression in the Dorsal Root Ganglia of Ovariectomized Osteoporotic Rats: A Pilot Basic Study.

INTRODUCTION: Osteoporosis can produce a persistent state of pain known as osteoporotic pain. One proposed mechanism of this pathology is increased calcitonin gene-related peptide (CGRP; a marker related to inflammatory pain) expression in the dorsal root ganglia (DRG) innervating osteoporotic vertebrae. Alternatively, a previous study revealed that axial loading caused osteoporotic pain in a rodent model of coccygeal vertebrae compression. Because this compression model is associated with trauma, additional mechanistic studies of osteoporotic pain in the absence of trauma are required. The current study aimedto evaluate the expression and relative distribution of transient receptor potential vanilloid 4 (TRPV4), a pain-related mechanoreceptor, in ovariectomized (OVX) osteoporotic rats. METHODS: CGRP-immunoreactive (-ir) and TRPV4-ir DRG neurons innervating the L3 vertebrae of Sprague-Dawley rats were labeled with a neurotracer, FluoroGold. Intravertebral pH was also measured during the neurotracer procedure. TRPV4-ir/CGRP-ir FluoroGold-positive DRG neurons were quantified in sham control and OVX rats (n = 10, ea). The threshold for statistical significance was set at P < 0.05. RESULTS: There was no statistical difference in the number of FluoroGold-positive DRG neurons between groups; however, there were significantly more CGRP-ir/TRPV4-ir FluoroGold-positive DRG neurons in the OVX group compared with the sham control group (P < 0.05) as well as the significantly increased molecular production of each peptide. Intravertebral pH was also lower in the OVX group compared with the sham control group (P < 0.05). CONCLUSION: Sensory neurons innervating osteoporotic vertebrae exhibited increased expression of co-localized CGRP and TRPV4 in OVX osteoporotic rats. Additionally, intravertebral pH was low in the vicinity osteoporotic vertebrae. Considering that TRPV4 is a mechanosensitive nociceptor that is activated in acidic environments, its upregulation may be associated with the pathology of osteoporotic pain derived from microinflammation involved in osteoporosis.

Intravesical Activation of the Cation Channel TRPV4 Improves Bladder Function in a Rat Model for Detrusor Underactivity.

BACKGROUND: Improvement of bladder emptying by modulating afferent nerve activity is an attractive therapeutic strategy for detrusor underactivity. Transient receptor potential vanilloid 4 (TRPV4) is a sensory ion channel in urothelial cells that contribute to the detection of bladder filling. OBJECTIVE: To investigate the potential benefit of intravesical TRPV4 agonists in a pelvic nerve injury rat model for detrusor underactivity. DESIGN, SETTING, AND PARTICIPANTS: Female wild-type and Trpv4 knockout rats underwent sham surgery or bilateral pelvic nerve injury (bPNI). Four weeks later, rats underwent cystometry with infusion of the TRPV4 agonist GSK1016790A. Bladders were harvested for in vitro pharmacological studies, quantitative reverse polymerase chain reaction and immunohistochemistry. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Data are expressed as median ± interquartile range. Statistical comparisons were made using the Mann-Witney U test and Wilcoxon signed rank test as appropriate. RESULTS AND LIMITATIONS: Rats with bPNI showed a phenotype characteristic of detrusor underactivity with lower-amplitude voiding contractions, decreased voiding frequency, and increased postvoid residual. Intravesical application of GSK1016790A increased voiding frequency and reduced postvoid residual in wild-type, but not Trpv4-/-, rats. In isolated bladder strips, GSK1016790A did not induce relevant contractions, indicating that the observed improvements in bladder function are the result of increased afferent signalling through TRPV4 activation, rather than a local effect on the detrusor. The altered urinary phenotype of Trpv4-/- mice was not apparent in the Trpv4-/- rat model, suggesting species-related functional variations. Our results are limited to the preclinical setting in rodents. CONCLUSIONS: Intravesical activation of TRPV4 improves bladder dysfunction after bPNI by increasing afferent signalling. PATIENT SUMMARY: We demonstrate that the sensory protein transient receptor potential vanilloid 4 (TRPV4) can be targeted to improve bladder function in animals that have iatrogenic injury to the nerves innervating the bladder. Further research is required to determine whether these results can be translated to patients with an underactive bladder.

Exploration on effect mechanism of Miao medicinal acupuncture therapy in treatment of knee osteoarthritis based on TRPV ion channel / 中国针灸

OBJECTIVE@#To observe the effect of Miao medicinal acupuncture therapy on transient receptor potential vanilloid (TRPV) channel in knee joint synovial tissue of the rabbits with knee osteoarthritis (KOA) model and to explore the mechanism of Miao medicinal acupuncture therapy in treatment of KOA.@*METHODS@#Of 34 New Zealand male rabbits, 6 rabbits were selected randomly as the normal group. KOA model was established in the rest rabbits by injecting a mixture of papain and L-cysteine in right knee joints. The 24 successfully modeled rabbits were randomized into a model group, a Miao medicinal acupuncture therapy group, a dermal needle group and a smearing group, 6 rabbits in each one. In the Miao medicinal acupuncture therapy group, Miao medicinal acupuncture therapy was adopted, in which, the roller type of dermal needle was used on the surface of right knee joint [a rectangle shape formed by "Xuehai" (SP 10), "Liangqiu" (ST 34), "Yanglingquan" (GB 34) and "Yinlingquan" (SP 9)], rolling in a " shape, on which, Miao medicinal solution was smeared in advance. In the dermal needle group, the rolling stimulation was exerted on the right the right knee joint surface with the roller type of dermal needle. In the smearing group, Miao medicinal solution was smeared on the right knee joint surface. The intervention was given once every two days, 3 times weekly and the intervention was exerted consecutively for 4 weeks. Successively, on day 1, 21, 28, 35, 42 and 49 of experiment, paw withdrawal threshold (von Frey threshold) after mechanical stimulation was detected in the rabbits. HE staining was adopted to observe the histomorphological changes of the right knee joint cartilage in the rabbits. ELISA was used to determine the contents of interleukin-1 (IL-1β) and tumor necrosis factor-α (TNF-α) in the right knee synovial fluid. Western blot method and real-time PCR were used to determine the relative expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue of the rabbits.@*RESULTS@#Compared with the normal group, on day 49 of experiment, von Frey threshold was reduced significantly in the rabbits of the model group (@*CONCLUSION@#Miao medicinal acupuncture therapy plays a role in treatment of KOA probably through inhibiting the expressions of IL-1β and TNF-α of knee synovial fluid and down-regulating the expressions of protein and mRNA of TRPV1 and TRPV4 in knee synovial tissue.