Transient Receptor Potential Vanilloid 4 Activity Research Articles

Increased TRPV4 Channel Expression Enhances and Impairs Blood Vessel Function in Hypertension.

Endothelial cell TRPV4 (transient receptor potential vanilloid 4) channels provide a control point that is pivotal in regulating blood vessel diameter by mediating the Ca2+-dependent release of endothelial-derived vasoactive factors. In hypertension, TRPV4-mediated control of vascular function is disrupted, but the underlying mechanisms and precise physiological consequences remain controversial. Here, using a comprehensive array of methodologies, endothelial TRPV4 channel function was examined in intact mesenteric resistance arteries from normotensive Wistar-Kyoto and spontaneously hypertensive rats. Our results show there is a notable shift in vascular reactivity in hypertension characterized by enhanced endothelium-dependent vasodilation at low levels of TRPV4 channel activation. However, at higher levels of TRPV4 activity, this vasodilatory response is reversed, contributing to the aberrant vascular tone observed in hypertension. The change in response, from dilation to constriction, was accompanied by a shift in intracellular Ca2+ signaling modalities arising from TRPV4 activity. Oscillatory TRPV4-evoked IP3 (inositol triphosphate)-mediated Ca2+ release, which underlies dilation, decreased, while the contraction inducing sustained Ca2+ rise, arising from TRPV4-mediated Ca2+ influx, increased. Our findings also reveal that while the sensitivity of endothelial cell TRPV4 to activation was unchanged, expression of the channel is upregulated and IP3 receptors are downregulated in hypertension. These data highlight the intricate interplay between endothelial TRPV4 channel expression, intracellular Ca2+ signaling dynamics, and vascular reactivity. Moreover, the data support a new unifying hypothesis for the vascular impairment that accompanies hypertension. Specifically, endothelial cell TRPV4 channels play a dual role in modulating blood vessel function in hypertension.

Mechanisms of cerebrospinal fluid and brain interstitial fluid production

Fluid homeostasis is fundamental for brain function with cerebral edema and hydrocephalus both being major neurological conditions. Fluid movement from blood into brain is one crucial element in cerebral fluid homeostasis. Traditionally it has been thought to occur primarily at the choroid plexus (CP) as cerebrospinal fluid (CSF) secretion due to polarized distribution of ion transporters at the CP epithelium. However, there are currently controversies as to the importance of the CP in fluid secretion, just how fluid transport occurs at that epithelium versus other sites, as well as the direction of fluid flow in the cerebral ventricles. The purpose of this review is to evaluate evidence on the movement of fluid from blood to CSF at the CP and the cerebral vasculature and how this differs from other tissues, e.g., how ion transport at the blood-brain barrier as well as the CP may drive fluid flow. It also addresses recent promising data on two potential targets for modulating CP fluid secretion, the Na+/K+/Cl− cotransporter, NKCC1, and the non-selective cation channel, transient receptor potential vanilloid 4 (TRPV4). Finally, it raises the issue that fluid secretion from blood is not constant, changing with disease and during the day. The apparent importance of NKCC1 phosphorylation and TRPV4 activity at the CP in determining fluid movement suggests that such secretion may also vary over short time frames. Such dynamic changes in CP (and potentially blood-brain barrier) function may contribute to some of the controversies over its role in brain fluid secretion.

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<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<0.05) or S1 RBD (10nM, 24 hours, p<0.05) (Fig. C). TRPV4 sensitive current density was significantly increased when incubated with S1 (10nM) or S1 RBD (10nM) for 24 hours (p<0.05 vs. control, respectively, Fig. D-G), whereas co-incubated with HC-067047 (500nM) significantly reversed the S1 (10nM, 24 hours, p<0.05) or S1 RBD (10nM, 24 hours, p<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.

Cell Confluence Modulates TRPV4 Channel Activity in Response to Hypoxia.

Transient receptor potential vanilloid 4 (TRPV4) is a polymodal Ca2+-permeable channel involved in various hypoxia-sensitive pathophysiological phenomena. Different tools are available to study channel activity, requiring cells to be cultured at specific optimal densities. In the present study, we examined if cell density may influence the effect of hypoxia on TRPV4 activity. Transiently TRPV4-transfected HEK293T cells were seeded at low or high densities corresponding to non-confluent or confluent cells, respectively, on the day of experiments, and cultured under in vitro normoxia or hypoxia. TRPV4-mediated cytosolic Ca2+ responses, single-channel currents, and Ca2+ influx through the channel were measured using Ca2+ imaging/microspectrofluorimetric assay, patch-clamp, and Bioluminescence Resonance Energy Transfer (BRET), respectively. TRPV4 plasma membrane translocation was studied using confocal microscopy, biotinylation of cell surface proteins, and BRET. Our results show that hypoxia exposure has a differential effect on TRPV4 activation depending on cell confluence. At low confluence levels, TRPV4 response is increased in hypoxia, whereas at high confluence levels, TRPV4 response is strongly inhibited, due to channel internalization. Thus, cell density appears to be a crucial parameter for TRPV4 channel activity.

Emergent Temporal Signaling in Human Trabecular Meshwork Cells: Role of TRPV4-TRPM4 Interactions.

Trabecular meshwork (TM) cells are phagocytic cells that employ mechanotransduction to actively regulate intraocular pressure. Similar to macrophages, they express scavenger receptors and participate in antigen presentation within the immunosuppressive milieu of the anterior eye. Changes in pressure deform and compress the TM, altering their control of aqueous humor outflow but it is not known whether transducer activation shapes temporal signaling. The present study combines electrophysiology, histochemistry and functional imaging with gene silencing and heterologous expression to gain insight into Ca2+ signaling downstream from TRPV4 (Transient Receptor Potential Vanilloid 4), a stretch-activated polymodal cation channel. Human TM cells respond to the TRPV4 agonist GSK1016790A with fluctuations in intracellular Ca2+ concentration ([Ca2+]i) and an increase in [Na+]i. [Ca2+]i oscillations coincided with monovalent cation current that was suppressed by BAPTA, Ruthenium Red and the TRPM4 (Transient Receptor Potential Melastatin 4) channel inhibitor 9-phenanthrol. TM cells expressed TRPM4 mRNA, protein at the expected 130-150 kDa and showed punctate TRPM4 immunoreactivity at the membrane surface. Genetic silencing of TRPM4 antagonized TRPV4-evoked oscillatory signaling whereas TRPV4 and TRPM4 co-expression in HEK-293 cells reconstituted the oscillations. Membrane potential recordings suggested that TRPM4-dependent oscillations require release of Ca2+ from internal stores. 9-phenanthrol did not affect the outflow facility in mouse eyes and eyes from animals lacking TRPM4 had normal intraocular pressure. Collectively, our results show that TRPV4 activity initiates dynamic calcium signaling in TM cells by stimulating TRPM4 channels and intracellular Ca2+ release. It is possible that TRPV4-TRPM4 interactions downstream from the tensile and compressive impact of intraocular pressure contribute to homeostatic regulation and pathological remodeling within the conventional outflow pathway.

TRPV4 Stimulation Level Regulates Ca2+-Dependent Control of Human Corneal Endothelial Cell Viability and Survival.

The functional contribution of transient receptor potential vanilloid 4 (TRPV4) expression in maintaining human corneal endothelial cells (HCEC) homeostasis is unclear. Accordingly, we determined the effects of TRPV4 gene and protein overexpression on responses modulating the viability and survival of HCEC. Q-PCR, Western blot, FACS analyses and fluorescence single-cell calcium imaging confirmed TRPV4 gene and protein overexpression in lentivirally transduced 12V4 cells derived from their parent HCEC-12 line. Although TRPV4 overexpression did not alter the baseline transendothelial electrical resistance (TEER), its cellular capacitance (Ccl) was larger than that in its parent. Scanning electron microscopy revealed that only the 12V4 cells developed densely packed villus-like protrusions. Stimulation of TRPV4 activity with GSK1016790A (GSK101, 10 µmol/L) induced larger Ca2+ transients in the 12V4 cells than those in the parental HCEC-12. One to ten nmol/L GSK101 decreased 12V4 viability, increased cell death rates and reduced the TEER, whereas 1 µmol/L GSK101 was required to induce similar effects in the HCEC-12. However, the TRPV4 channel blocker RN1734 (1 to 30 µmol/L) failed to alter HCEC-12 and 12V4 morphology, cell viability and metabolic activity. Taken together, TRPV4 overexpression altered both the HCEC morphology and markedly lowered the GSK101 dosages required to stimulate its channel activity.

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.

Cardiac TRPV4 channels.

Transient Receptor Potential Vanilloid 4 (TRPV4) is expressed in numerous cell types within the heart, yet the expression levels, subcellular localization, and functional relevance of TRPV4 in cardiac myocytes is under-appreciated. Recent data indicate a critical role of TRPV4 in both atrial and ventricular myocyte biology, with expression levels and channel function increasing following pathological scenarios including ischemia, myocardial infarction, mechanical stress, and inflammation. Excessive activation of TRPV4 at the cellular level contributes to enhanced Ca2+ entry which predisposes the cardiac myocyte to pro-arrhythmic Ca2+ overload and electrophysiological abnormalities. At the organ level, excessive TRPV4 activity associates with cardiac hypercontractility, cardiac damage, ventricular arrhythmia, and atrial fibrillation. This manuscript chapter describes the emerging literature on TRPV4 in cardiac myocytes in physiology and disease.

The transient receptor potential vanilloid 4 (TRPV4) ion channel mediates protease activated receptor 1 (PAR1)-induced vascular hyperpermeability

AbstractEndothelial barrier disruption is a hallmark of tissue injury, edema, and inflammation. Vascular endothelial cells express the G protein-coupled receptor (GPCR) protease acctivated receptor 1 (PAR1) and the ion channel transient receptor potential vanilloid 4 (TRPV4), and these signaling proteins are known to respond to inflammatory conditions and promote edema through remodeling of cell–cell junctions and modulation of endothelial barriers. It has previously been established that signaling initiated by the related protease activated receptor 2 (PAR2) is enhanced by TRPV4 in sensory neurons and that this functional interaction plays a critical role in the development of neurogenic inflammation and nociception. Here, we investigated the PAR1–TRPV4 axis, to determine if TRPV4 plays a similar role in the control of edema mediated by thrombin-induced signaling. Using Evans Blue permeation and retention as an indication of increased vascular permeability in vivo, we showed that TRPV4 contributes to PAR1-induced vascular hyperpermeability in the airways and upper gastrointestinal tract of mice. TRPV4 contributes to sustained PAR1-induced Ca2+ signaling in recombinant cell systems and to PAR1-dependent endothelial junction remodeling in vitro. This study supports the role of GPCR–TRP channel functional interactions in inflammatory-associated changes to vascular function and indicates that TRPV4 is a signaling effector for multiple PAR family members.The thrombin receptor PAR1 and mechanosensitve ion channel TRPV4 are known to modulate endothelial barriers and contribute to edema, but functional interactions between these targets are not well understood. Utilizing cell signalling studies and in vivo models, the authors have demonstrated that PAR1 activation leads to functional upregulation of endothelial TRPV4 activity, to promote vascular leakage in airways and the gastrointestinal system.

TRPV4 disrupts mitochondrial transport and causes axonal degeneration via a CaMKII-dependent elevation of intracellular Ca2+

The cation channel transient receptor potential vanilloid 4 (TRPV4) is one of the few identified ion channels that can directly cause inherited neurodegeneration syndromes, but the molecular mechanisms are unknown. Here, we show that in vivo expression of a neuropathy-causing TRPV4 mutant (TRPV4R269C) causes dose-dependent neuronal dysfunction and axonal degeneration, which are rescued by genetic or pharmacological blockade of TRPV4 channel activity. TRPV4R269C triggers increased intracellular Ca2+ through a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism, and CaMKII inhibition prevents both increased intracellular Ca2+ and neurotoxicity in Drosophila and cultured primary mouse neurons. Importantly, TRPV4 activity impairs axonal mitochondrial transport, and TRPV4-mediated neurotoxicity is modulated by the Ca2+-binding mitochondrial GTPase Miro. Our data highlight an integral role for CaMKII in neuronal TRPV4-associated Ca2+ responses, the importance of tightly regulated Ca2+ dynamics for mitochondrial axonal transport, and the therapeutic promise of TRPV4 antagonists for patients with TRPV4-related neurodegenerative diseases.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Cytokine and inflammatory mediator effects on TRPV4 function in choroid plexus epithelial cells.

The choroid plexus (CP), composed of capillaries surrounded by a barrier epithelium, is the main producer of cerebrospinal fluid (CSF). The CP epithelium regulates the transport of ions and water between the blood and the ventricles, contributing to CSF production and composition. Several studies suggest a connection between the cation channel transient receptor potential vanilloid-4 (TRPV4) and transepithelial ion movement. TRPV4 is a nonselective, calcium-permeable cation channel present in CP epithelia reported to be activated by cytokines and inflammatory mediators. Utilizing the PCP-R (porcine choroid plexus-Riems) cell line, we investigated the effects of various cytokines and inflammatory mediators on TRPV4-mediated activity. Select proinflammatory cytokines (TNF-α, IL-1β, TGF-β1) had inhibitory effects on TRPV4-stimulated transepithelial ion flux and permeability changes, whereas anti-inflammatory cytokines (IL-10, IL-4, and IL-6) had none. Quantitative mRNA analysis showed that these cytokines had no effect on TRPV4 transcription levels. Inhibition of the transcription factor NF-κB, involved in the production and regulation of several inflammatory cytokines, inhibited TRPV4-mediated activity, suggesting a link between TRPV4 and cytokine production. Contrary to published studies, the proinflammatory mediator arachidonic acid (AA) had inhibitory rather than stimulatory effects on TRPV4-mediated responses. However, inhibition of AA metabolism also caused inhibitory effects on TRPV4, suggesting a complex interaction of AA and its metabolites in the regulation of TRPV4 activity. Together these data imply that TRPV4 activity is involved in the inflammatory response; it is negatively affected by proinflammatory mediators. Furthermore, arachidonic acid metabolites, but not arachidonic acid itself, are positive regulators of 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.

Role of TRPV4‐TAZ signaling axis in epithelial‐mesenchymal transition

Epithelial to mesenchymal transition (EMT) is a critical event in development, fibrogenesis, and oncogenesis. Emerging data support a role for both a mechanical signal, and a biochemical signal, e.g., transforming growth factor β1 (TGFβ1) in EMT. Here, we report evidence that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel sensitive to both matrix stiffness and TGFβ1 stimuli, is a likely mediator of EMT. We found that antagonism of TRPV4 blocked both matrix stiffness‐ and TGFβ1‐induced EMT in normal human primary epidermal keratinocytes (NHEKs), as determined by changes in morphology and alterations in expression of EMT markers including E‐cadherin, N‐cadherin, and α‐smooth muscle actin (α‐SMA). In a murine skin fibrosis model, TRPV4 deletion resulted in decreased expression of the mesenchymal marker, α‐SMA, and increased expression of epithelial marker, E‐cadherin. Mechanistically, we found that: i) TRPV4 activity was essential for the nuclear translocation of TAZ (transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1; ii) Antagonism of TRPV4 activity inhibited both matrix stiffness‐induced and TGFβ1‐induced expression of TAZ proteins; and iii) TRPV4 antagonism suppressed both matrix stiffness‐induced and TGFβ1‐induced activation of Smad2/3, but not of AKT. Altogether, these data identify a novel role for TRPV4 in regulating EMT in response to both matrix stiffness and TGFβ1.Support or Funding InformationAHA (13SDG17310007), Startup grant from University of Maryland, NIH (1R01EB024556‐01), and NSF (CMMI‐1662776) grants to Shaik RahamanThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

TRPV4-mediated calcium signaling in mesenchymal stem cells regulates aligned collagen matrix formation and vinculin tension

Microarchitectural cues drive aligned fibrillar collagen deposition in vivo and in biomaterial scaffolds, but the cell-signaling events that underlie this process are not well understood. Utilizing a multicellular patterning model system that allows for observation of intracellular signaling events during collagen matrix assembly, we investigated the role of calcium (Ca2+) signaling in human mesenchymal stem cells (MSCs) during this process. We observed spontaneous Ca2+ oscillations in MSCs during fibrillar collagen assembly, and hypothesized that the transient receptor potential vanilloid 4 (TRPV4) ion channel, a mechanosensitive Ca2+-permeable channel, may regulate this signaling. Inhibition of TRPV4 nearly abolished Ca2+ signaling at initial stages of collagen matrix assembly, while at later times had reduced but significant effects. Importantly, blocking TRPV4 activity dramatically reduced aligned collagen fibril assembly; conversely, activating TRPV4 accelerated aligned collagen formation. TRPV4-dependent Ca2+ oscillations were found to be independent of pattern shape or subpattern cell location, suggesting this signaling mechanism is necessary for aligned collagen formation but not sufficient in the absence of physical (microarchitectural) cues that force multicellular alignment. As cell-generated mechanical forces are known to be critical to the matrix assembly process, we examined the role of TRPV4-mediated Ca2+ signaling in force generated across the load-bearing focal adhesion protein vinculin within MSCs using an FRET-based tension sensor. Inhibiting TRPV4 decreased tensile force across vinculin, whereas TRPV4 activation caused a dynamic unloading and reloading of vinculin. Together, these findings suggest TRPV4 activity regulates forces at cell-matrix adhesions and is critical to aligned collagen matrix assembly by MSCs.

Design and Optimization of Sulfone Pyrrolidine Sulfonamide Antagonists of Transient Receptor Potential Vanilloid-4 with in Vivo Activity in a Pulmonary Edema Model.

Pulmonary edema is a common ailment of heart failure patients and has remained an unmet medical need due to dose-limiting side effects associated with current treatments. Preclinical studies in rodents have suggested that inhibition of transient receptor potential vanilloid-4 (TRPV4) cation channels may offer an alternative-and potentially superior-therapy. Efforts directed toward small-molecule antagonists of the TRPV4 receptor have led to the discovery of a novel sulfone pyrrolidine sulfonamide chemotype exemplified by lead compound 6. Design elements toward the optimization of TRPV4 activity, selectivity, and pharmacokinetic properties are described. Activity of leading exemplars 19 and 27 in an in vivo model suggestive of therapeutic potential is highlighted herein.

TRPV4 regulates matrix stiffness and TGFβ1-induced epithelial-mesenchymal transition.

Substrate stiffness (or rigidity) of the extracellular matrix has important functions in numerous pathophysiological processes including fibrosis. Emerging data support a role for both a mechanical signal, for example, matrix stiffness, and a biochemical signal, for example, transforming growth factor β1 (TGFβ1), in epithelial‐mesenchymal transition (EMT), a process critically involved in fibrosis. Here, we report evidence showing that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive channel, is the likely mediator of EMT in response to both TGFβ1 and matrix stiffness. Specifically, we found that: (a) genetic ablation or pharmacological inhibition of TRPV4 blocked matrix stiffness and TGFβ1‐induced EMT in normal mouse primary epidermal keratinocytes (NMEKs) as determined by changes in morphology, adhesion, migration and alterations of expression of EMT markers including E‐cadherin, N‐cadherin (NCAD) and α‐smooth muscle actin (α‐SMA), and (b) TRPV4 deficiency prevented matrix stiffness‐induced EMT in NMEKs over a pathophysiological range. Intriguingly, TRPV4 deletion in mice suppressed expression of mesenchymal markers, NCAD and α‐SMA, in a bleomycin‐induced murine skin fibrosis model. Mechanistically, we found that: (a) TRPV4 was essential for the nuclear translocation of YAP/TAZ (yes‐associated protein/transcriptional coactivator with PDZ‐binding motif) in response to matrix stiffness and TGFβ1, (b) TRPV4 deletion inhibited both matrix stiffness‐ and TGFβ1‐induced expression of YAP/TAZ proteins and (c) TRPV4 deletion abrogated both matrix stiffness‐ and TGFβ1‐induced activation of AKT, but not Smad2/3, suggesting a mechanism by which TRPV4 activity regulates EMT in NMEKs. Altogether, these data identify a novel role for TRPV4 in regulating EMT.

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.

PIP2 depletion promotes TRPV4 channel activity in mouse brain capillary endothelial cells.

We recently reported that the inward-rectifier Kir2.1 channel in brain capillary endothelial cells (cECs) plays a major role in neurovascular coupling (NVC) by mediating a neuronal activity-dependent, propagating vasodilatory (hyperpolarizing) signal. We further demonstrated that Kir2.1 activity is suppressed by depletion of plasma membrane phosphatidylinositol 4,5-bisphosphate (PIP2). Whether cECs express depolarizing channels that intersect with Kir2.1-mediated signaling remains unknown. Here, we report that Ca2+/Na+-permeable TRPV4 (transient receptor potential vanilloid 4) channels are expressed in cECs and are tonically inhibited by PIP2. We further demonstrate that depletion of PIP2 by agonists, including putative NVC mediators, that promote PIP2 hydrolysis by signaling through Gq-protein-coupled receptors (GqPCRs) caused simultaneous disinhibition of TRPV4 channels and suppression of Kir2.1 channels. These findings collectively support the concept that GqPCR activation functions as a molecular switch to favor capillary TRPV4 activity over Kir2.1 signaling, an observation with potentially profound significance for the control of cerebral blood flow.

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.