Modulation of the TRPV4 ion channel as a therapeutic target for disease

Megan S. Grace1,2,3, Sara J. Bonvini4, Maria G. Belvisi4, Peter McIntyre2

1. Baker IDI Heart and Diabetes Institute, Melbourne, Australia

2. School of Health and Biomedical Sciences, RMIT University, Bundoora, Melbourne, Australia

3. Department of Physiology, School of Medicine, Nursing and Health Sciences, Monash University, Melbourne, Australia

4. Respiratory Pharmacology, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, UK

Address correspondence to: Dr. Megan Grace, Baker IDI Heart and Diabetes Institute, Level 4 Alfred Centre, 99 Commercial Road, Melbourne, VIC 3004, Australia. E-mail: megan.grace@bakeridi.edu.au; Ph: +61 3 8532 1855

Abstract

Transient Receptor Potential Vanilloid 4 (TRPV4) is a broadly expressed, polymodally gated ion channel that plays an important role in many physiological and pathophysiological processes. TRPV4 knockout mice and several synthetic pharmacological compounds that selectively target TRPV4 are now available, which has allowed detailed investigation in to the therapeutic potential of this ion channel. Results from animal studies suggest that TRPV4 antagonism has therapeutic potential in oedema, pain, gastrointestinal disorders, and lung diseases such as cough, bronchoconstriction, pulmonary hypertension, and acute lung injury. A lack of observed side-effects in vivo has prompted a first-in-human trial for a TRPV4 antagonist in healthy participants and stable heart failure patients. If successful, this would open up an exciting new area of research for a multitude of TRPV4-related pathologies. This review will discuss the known roles of TRPV4 in disease, and highlight the possible implications of targeting this important cation channel for therapy.

Keywords

TRPV4, Inflammation, Pain, Gastrointestinal Disease, Oedema, Respiratory Disease, Vascular Tone

Contents1.Introduction52.Respiratory Disease63.Oedema124.Pain175.Itch206.Gastrointestinal Disease227.Vascular Tone248.TRPV4 Mutations and Human Disease269.Complexes2810.TRPV4 as a Therapeutic Target2911.Future Directions31References33

Abbreviations

4αPDD, 4α-phorbol 12,13-didecanoate; AHR, airway hyperresponsiveness; AKAP, A-kinase anchor protein; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; BKCa, large conductance calcium-activated potassium channel; CFTR, cystic fibrosis transmembrane conductance regulator; COPD, chronic obstructive pulmonary disease; DRG, dorsal root ganglia; EET, epoxyeicosatrienoic acid; FDAB, familial digital arthropathy brachydactily; IBD, inflammatory bowel disease; IBS, irritable bowel syndrome; IKCa, intermediate conductance calcium-activated potassium channel; IPF, idiopathic pulmonary fibrosis; MAPKK, mitogen activated protein kinase kinase; MMP, matrix metalloproteinase; PAR2, protease activated receptor 2; PKC, protein kinase C; RVD, regulatory volume decrease; SKCa, small conductance calcium-activated potassium channel; SNP, single nucleotide polymorphism; TG, trigeminal ganglia; TGFβ, transforming growth factor β; TRPV4, transient receptor potential vanilloid 4; VILI, ventilator induced lung injury

Introduction

Transient Receptor Potential Vanilloid 4 (TRPV4) was isolated from the rat kidney, and originally identified as a vertebrate homologue of the Caenorhabditis elegans gene Osm-9 (Liedtke, et al., 2000). TRPV4 is now known to be a broadly-expressed, polymodally gated, non-selective cation channel (including calcium, potassium, magnesium and sodium) that plays an important role in a multitude of physiological processes. It is an osmosensor (Liedtke, et al., 2000; Strotmann, et al., 2000; Vriens, et al., 2004; Wegierski, et al., 2009); a thermosensor, activated by innocuous warm temperatures in the range of 27-35°C (Liedtke, et al., 2000; Strotmann, et al., 2000; Willette, et al., 2008); can be activated directly by endogenous or exogenous chemical stimuli (Grace, et al., 2014; Moore, et al., 2013; Nilius, et al., 2004; Poole, et al., 2013; Vriens, et al., 2005; Watanabe, et al., 2003); and activated or sensitised indirectly via intracellular signalling pathways ((Alessandri-Haber, et al., 2006; Alessandri-Haber, et al., 2004; Grant, et al., 2007; Zhao, et al., 2014) reviewed in (Darby, et al., 2016)).

Several synthetic pharmacological compounds have been discovered which target TRPV4. Agonists include the phorbol ester 4α-phorbol 12,13-didecanoate (4αPDD), and the more potent GSK1016790A (Klausen, et al., 2009; Thorneloe, et al., 2008). Early studies used the non-selective TRPV4 inhibitors gadolinium and ruthenium red, both of which exhibit a multitude of other effects. Newer, more selective TRPV4 inhibitors include RN-1734, the widely-used HC-067047, and the orally bioavailable GSK2193874 (Everaerts, et al., 2010; Thorneloe, et al., 2012; Vincent, et al., 2009). The most recent generation of pharmacological inhibitors for TRPV4 possess high affinity and specificity. Indeed, TRPV4 inhibitors have been successfully used in vivo without obvious side-effects (Balakrishna, et al., 2014; Thorneloe, et al., 2012), and a novel TRPV4 antagonist recently advanced to Phase I clinical trials (GSK2798745; NCT02119260).

The role of TRPV4 in pathophysiological states is well established, and inflammation contributes to TRPV4’s role in disease processes. Despite this, TRPV4-/- mice are viable and fertile, and display only minor phenotypes. These include altered osmosensation; compromised vascular endothelial function; impaired osteoclast differentiation leading to thicker bones; defective stretch sensation in the bladder wall; and mild hearing, pressure and pain sensory impairments (Cortright & Szallasi, 2009; Earley, et al., 2005; Everaerts, et al., 2010; Gevaert, et al., 2007; Lechner, et al., 2011; Loot, et al., 2008; Marrelli, et al., 2007; Masuyama, et al., 2008; Saliez, et al., 2008; Sonkusare, et al., 2012; Suzuki, et al., 2003a; Tabuchi, et al., 2005; Vriens, et al., 2005). In light of the mild phenotypes observed in TRPV4-/- mice, which would suggest only a minor role of TRPV4 in normal development, it is therefore surprising that mutation of the TRPV4 gene is the direct cause of several disabling and lethal human pathologies, including skeletal dysplasia’s and neuropathies (Lamande, et al., 2011; Loukin, et al., 2010; McNulty, et al., 2015; Nilius & Voets, 2013). The mechanism(s) by which TRPV4 mutation contributes to these diseases is still unclear, but may be due to gain or loss of channel function. This is a focus of ongoing research, aided by the growing number of TRPV4 mutants that are now available for study. The severe pathologies observed with TRPV4 mutations underscore a vital role for normal TRPV4 function in development and the regulation of cellular processes. Alternatively, studies investigating TRPV4-/- suggests that there may be compensatory mechanisms involved when TRPV4 is absent during development that allow generally normal physiological function. Here, we will discuss the known roles of TRPV4 in disease, and highlight the possible implications of targeting this important cation channel for therapy.

Respiratory Disease

Cough

Chronic cough is a major global health problem and is currently the most common reason for patients to visit a doctor in the UK (Schappert & Burt, 2006; Schappert & Rechtsteiner, 2011). Clinically, chronic cough is defined as a cough which lasts for over 8 weeks. It is a common symptom of the inflammatory diseases asthma and chronic obstructive pulmonary disease (COPD) and pulmonary fibrosis, but can also be idiopathic and refractory to treatment. Despite this, there are currently no safe and effective therapies currently in use. Cough occurs as a reflex event initiated following activation of the vagus nerve within the airways. Afferent fibres from within the vagus nerve detect changes in the physical and chemical environment and relay pulmonary information to the CNS (Belvisi, 2002; Brouns, et al., 2012). Once processed by the CNS, a signal is then sent down effector neurons to the diaphragm, respiratory muscles and larynx to cause the cough response.

Vagus nerve fibres express a number of receptors and ion channels which are activated by a wide range of endogenous and exogenous stimuli to cause cough. These include members of the Transient Receptor Potential (TRP) family which detect and respond to a diverse range of stimuli. When these ion channels encounter potentially dangerous stimuli they can act as cellular sensors and induce protective mechanisms such as cough and bronchoconstriction. A number of TRP channels have been shown to play a role in the afferent arm of the cough reflex (reviewed in (Grace, et al., 2013) and (Bonvini, et al., 2015)), including TRPV4 (Bonvini, et al., 2016). TRPV4 activation by specific agonists, including GSK1016790A and hypo-osmolarity, causes depolarisation of airway sensory nerves and cough in conscious guinea pigs, which is blocked by specific antagonists of the channel (Bonvini, et al., 2016). This was shown to be through activation of mechanosensitive Aδ fibres in the vagus, a different mechanism to that caused by activation of TRPV1 and TRPA1 which activate the chemosensitive C fibres to cause airway sensory nerve depolarisation and cough (Bonvini, et al., 2016). Furthermore, a selective P2X3 antagonist AF-353 was shown to inhibit nerve depolarisation and cough induced by TRPV4 in both animals and humans, indicating that TRPV4 can cause the release of ATP (Bonvini, et al., 2016), which in turn acts on P2X3 present on sensory nerves. A further part of the mechanism of activation was also eluded to, as responses to TRPV4 were abolished in vagal tissue from pannexin 1 knockout mice, but responses to TRPV1, TRPA1 agonists and the P2X1/P2X3 agonist αβ-MeATP remained unchanged (Bonvini, et al., 2016). Pannexin 1 is a large conductance ion pore which permits ATP efflux from the cell (Dahl, 2015), therefore suggesting that pannexin is required for TRPV4 mediated ATP release, at least in mouse vagal tissue. It is yet undetermined which cell type is required for TRPV4 induced ATP release, as single cell PCR only found TRPV4 to be expressed in 1/32 nodose neurons (Bonvini, et al., 2016). TRPV4 could instead be activating neighbouring cells which cause the release of ATP and activation of P2X3 on airway sensory nerves. This mechanism is particularly interesting clinically as a P2X3 antagonist was shown to inhibit daytime objective cough frequency in patients with idiopathic chronic cough (Abdulqawi, et al., 2015). This promising clinical data suggests that targeting this axis may provide a novel therapy for treatment of chronic cough (Figure 1). As the antagonist of the ATP receptor P2X3 has shown such efficacy, this suggests ATP plays an important role in chronic cough, and therefore activation of TRPV4, through releasing ATP, is likely to be contributing to the pathology of this condition.

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COPD

COPD is a global health problem and is estimated to become the third leading cause of death worldwide by 2020 (Vestbo, et al., 2013). COPD encompasses a number of disease pathologies including chronic bronchitis, bronchiolitis and emphysema (Bateman, et al., 2008) which are associated with chronic inflammation of the lung, along with progressive, non-reversible airflow limitation (Barnes, 2014). Progressive airflow limitation is caused by small airway narrowing, and remodelling and destruction of the lung parenchyma through chronic inflammation, which increases with the progression of the disease (Hogg, et al., 2004). The lung inflammation associated with COPD is characterised by infiltration of macrophages, neutrophils and CD8+ lymphocytes (Keatings, et al., 1996; Keatings, et al., 1997), and is resistant to anti-inflammatory agents such as glucocorticoids (Barnes, 2013). This obstructive disease of the lung progresses over decades and can lead to death from respiratory failure or by other associated comorbidities (Barnes, 2014).

A key risk factor for the development of COPD is cigarette smoking (Sethi & Rochester, 2000). Baxter et al have recently shown that cigarette smoke-induced release of ATP from human bronchial epithelial cells can be inhibited using a TRPV4 antagonist, and also in TRPV4 and pannexin KO mice (Baxter, et al., 2014). In a similar fashion to what is observed in sensory nerves, the activation of TRPV4 and opening of the pannexin 1 channel result in ATP release. Furthermore, ATP levels are increased in the lungs of COPD patients (Mohsenin & Blackburn, 2006; Mortaz, et al., 2009); and aerosolised ATP induces dyspnea, cough and throat irritation in COPD patients, but not in normal subjects (Basoglu, et al., 2015). This indicates that activation of TRPV4, through release of ATP, contributes to disease pathogenesis. Further to this, Eltom et al. have shown that cigarette smoke induces ATP release which activates the NLRP3 inflammasome via activation of P2X7, leading to the release of IL-1β and IL-18 and inflammation (Eltom, et al., 2011). This is another example where activation of an ATP receptor by ATP contributes to disease pathogenesis, and as TRPV4 leads to ATP release, indicates a potential role of TRPV4.

Baxter et al. also indicated that there was increased TRPV4 gene expression in lung tissue from patients with COPD (Baxter, et al., 2014). Furthermore, a study looking at a population from the International COPD network has found that 7/20 single nucleotide polymorphisms (SNPs) of TRPV4 are associated with the COPD phenotype, indicating an association between TRPV4 protein structure and disease pathogenesis (Zhu, et al., 2009).

Several endogenous agonists of TRPV4 are present in the airways of patients with COPD, including arachidonic acid derivatives (Narumiya, et al., 1999; Watanabe, et al., 2003). In addition to direct activators, indirect activators of TRPV4 are also present in high levels in the COPD airway. The increased neutrophil infiltration into the lungs of patients with COPD leads to higher levels of proteases in the lung, including neutrophil elastase. This is a major protease released during inflammation and has a high circulating concentration and a long half-life of 6-8 hours (Henriksen, 2014). Neutrophil elastase activates the G protein-coupled receptor (GPCR) Protease Activated Receptor 2 (PAR2) through cleavage of the receptor, in a mechanism different to the activation by trypsin (Zhao, et al., 2015). PAR2 also activates TRPV4 through receptor operated gating of the channel, or ‘coupling’ (Grace, et al., 2014; Poole, et al., 2013). Activation of PAR2 leads to a sustained increase of intracellular calcium in HEK293 cells overexpressing TRPV4, compared to non-transfected cells (Poole, et al., 2013). With high circulating levels of neutrophil elastase in the lung in patients with COPD (Henriksen, 2014), this could lead to increased activation of PAR2 and TRPV4, and further contribute to the disease.

Asthma

Asthma is a chronic, inflammatory respiratory disease affecting 300 million people worldwide, with prevalence increasing 50% every decade (Braman, 2006). Asthma is characterised by reversible airway bronchoconstriction, airway hyperresponsiveness (AHR) and symptoms including cough, dyspnoea and wheeze (Lemanske & Busse, 2003). The strongest predictor of asthma is atopy, which is the genetic predisposition for the development of immunoglobulin E (IgE) (Stokes, 2014). The inflammatory phenotype involves Th2 cells, eosinophils, mast cells and CD4+ cells (Barnes, 2008).

A key symptom of asthma is bronchoconstriction. Synthetic TRPV4 agonists cause contraction of airway smooth muscle in vitro in both human and guinea pig tissue (Jia, et al., 2004). A recent study by McAlexander et al. demonstrated that the TRPV4 agonist GSK1016790A induces a long lasting contraction of guinea pig and donor human isolated airways (McAlexander, et al., 2014). This contraction was inhibited following application of cysteinyl leukotriene antagonists and a 5 Lipoxygenase (5LO) inhibitor (McAlexander, et al., 2014), indicating that the TRPV4 agonist may not be causing contraction directly, and instead may be triggering the release of cysteinyl leukotrienes which cause contraction through activation of the cystLt1 receptor on airway smooth muscle.

Similarly to COPD patients, there is increased ATP present in the bronchoalveolar lavage of mice following allergen challenge compared to saline controls (Idzko, et al., 2007), and aerosolised ATP given to asthmatics induces bronchoconstriction, cough and dyspnea (Basoglu, et al., 2005). This indicates that perhaps the TRPV4-ATP axis may also play a role in the asthmatic airway.

Cystic fibrosis

Cystic fibrosis is an autosomal recessive disease caused by mutations in the gene for cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan, et al., 1989; Welsh, et al., 2001). Patients present with chronic bacterial infection, neutrophilic inflammation, and mucus in the airways along with progressive bronchiectasis, which is the cause of the majority of morbidity and death of sufferers (Welsh, et al., 2001). Mutations in the CFTR lead to altered electrophysiological properties across the airway epithelia, where CFTR dependent chloride and bicarbonate secretion along with epithelial sodium channel mediated sodium absorption are altered (Welsh, et al., 2001), leading to mucus accumulation and impaired clearance.

Arniges et al found that activation of TRPV4 by hypotonic solution does not trigger the normal regulatory mechanism in an epithelial cell line lacking CFTR, indicating that TRPV4 is linked to the CFTR protein (Arniges, et al., 2004). TRPV4 is activated by osmotic stimuli, and hypotonic cell swelling is known to trigger a regulatory mechanism known as Regulatory Volume Decrease (RVD) to return cells to their normal size (Fernandez-Fernandez, et al., 2002). This involves the loss of osmolytes and water via activation of Cl- channels (Giraldez, et al., 1988; Valverde, et al., 2000) and K+ channels (Fernandez-Fernandez, et al., 2002; Lock & Valverde, 2000), including Ca2+ dependent K+ ion channels. These ion channels are particularly important as epithelial RVD is normally triggered by changes in the intracellular Ca2+ concentration (Pasantes-Morales & Morales Mulia, 2000). In normal cells, a RVD response is induced following Ca2+ entry through the TRPV4 channel (Arniges, et al., 2004). However in a human cystic fibrosis epithelial cell line, cells were shown to express TRPV4, but no hypotonicity induced Ca2+ entry and no RVD in response to hypotonic stress (Arniges, et al., 2004). This indicates that hypotonic activation of TRPV4 channels could therefore be CFTR dependent (Arniges, et al., 2004). TRPV4 activation by hypotonic stimuli is a key mechanism in the control of cell volume in human airway epithelial cells and is impaired in cystic fibrosis epithelia, indicating that TRPV4 could play a role in the pathophysiology of cystic fibrosis and contribute to the observed increase in sodium absorption. TRPV4 is a Ca2+ permeable cation channel, and Na+ absorption is decreased following cystosolic Ca2+ increases (Mall, et al., 2000). A faulty TRPV4 channel may lead to a reduced Ca2+ response to mechanical stimulation and therefore increased sodium absorption (Arniges, et al., 2004).

Pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, irreversible and usually lethal scarring disease of the lung with unknown cause (King, et al., 2011). Patients present with shortness of breath and a dry cough (King, et al., 2011). Until recently, only lung transplant had been shown to extend the life of sufferers (King, et al., 2011). However, in 2014 the drugs perfinidone and nifetedanib were approved by the FDA as treatment for IPF. Both drugs show efficacy in patients with mild to moderate functional impairment by inhibiting functional decline and disease progression (reviewed in (Spagnolo, et al., 2015)). Incidence and prevalence of the disease increases with age and following diagnosis there is a median survival rate of 2.5-3.5 years (King, et al., 2011). At the cellular level, IPF is driven by aberrant wound healing mechanisms, which are thought to be driven by myofibroblasts. Myofibroblasts localise to fibrotic lesions and aid to remodel the extracellular matrix and drive pathological fibrosis through the release of mediators (Chapman, 2004; Hinz, et al., 2012; King, et al., 2011). Myofibroblasts can be characterised by their expression of α-smooth muscle actin, and their differentiation is regulated by TGFβ (Hinz, et al., 2012; King, et al., 2011; Marinkovic, et al., 2012; Muro, et al., 2008; Spagnolo, et al., 2015).

Rahaman et al. recently outlined a major role for TRPV4 in IPF (Rahaman, et al., 2014). In a classical model of lung fibrosis, TRPV4-/- mice treated with bleomycin were unaffected, with 75% less collagen accumulation and less myofibroblast accumulation as measured by levels of α smooth muscle actin compared to the wild-type controls (Rahaman, et al., 2014). Furthermore, TRPV4 was functionally expressed in primary human and murine lung fibroblasts, and Ca2+ influx induced by TRPV4 was two times greater in cells from patients with IPF, indicating that TRPV4 activity is upregulated in IPF (Rahaman, et al., 2014). This TRPV4 mediated Ca2+ influx was shown to promote myofibroblast differentiation in areas of pathological stiffness (Rahaman, et al., 2014). The TRPV4 channel was also required for TGFβ1 induced lung myofibroblast differentiation, as indicated through knockdown of the gene using siRNA, and a selective antagonist, which both inhibited differentiation. Therefore, this data suggests that targeting TRPV4 may be a beneficial therapeutic target for the treatment of IPF.

However, TRPV4 also has a protective effect in IPF under certain conditions. A further study has shown that under pathological matrix stiffness conditions, TRPV4 is essential for LPS mediated macrophage phagocytosis (Scheraga, et al., 2016). Macrophage TRPV4 was sensitised by stiff matrix conditions in the lung, which are found in conditions such as IPF and an infected lung. When exposed to LPS, there is a phenotypic change in the macrophage resulting in increased phagocytosis and a change in cytokine expression leading to an increased ability for bacterial clearance and a faster resolution of respiratory infection (Scheraga, et al., 2016). Under these conditions of respiratory infection with an underlying lung injury, targeting TRPV4 may instead show a protective effect in the lung.

Oedema

Epithelial and endothelial barriers are characterised by intercellular tight junctions and adherens junctions that control the exchange of fluid and proteins from the vasculature to the surrounding tissues. Damage, for example during pathological states or mechanical injury, disrupts barrier integrity and leads to the uncontrolled movement of fluid and plasma proteins through intercellular gaps, leading to oedema formation (Figures 2 and 3). Oedema characterises life-threatening conditions (Goldenberg, et al., 2011; Matthay & Zemans, 2011; Thorneloe, et al., 2012). For example, pulmonary oedema causes difficulty breathing and restricts oxygen absorption in the lungs, and can lead to respiratory failure, cardiac arrest, hypoxia, and death (Aman, et al., 2012; Egermayer, et al., 1999; Huh, et al., 2012; Thorneloe, et al., 2012). Moreover, brain oedema is an important pathological process in many central nervous system diseases, which can disturb physiological neuronal function and amplify tissue damage (Iuso & Krizaj, 2016; Jie, et al., 2015).

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Current therapies for oedema involve treatment of the underlying cause, ventilation, diuretics, and surgery. However, TRPV4 was recently identified as a critical regulator of endothelial barrier integrity, and is a promising pharmacological target (Balakrishna, et al., 2014; Hamanaka, et al., 2010; Hamanaka, et al., 2007; Jian, et al., 2008; Jie, et al., 2015; Thorneloe, et al., 2012). TRPV4 is known to be prominently expressed on endothelial and epithelial cells (Alvarez, et al., 2006; Arniges, et al., 2004; Hamanaka, et al., 2007; Reiter, et al., 2006; Thorneloe, et al., 2012), and a wide body of research has confirmed an important role for TRPV4 in oedema formation. An early study established that TRPV4 activation results in epithelial permeability in vitro via two distinct pathways. A rapid increase in transcellular conductance was mediated by activation of large conductance calcium-activated potassium (BKCa) channels; whereas, a delayed downregulation of tight junction claudin proteins led to an increase in paracellular permeability and altered tight junction structure (Reiter, et al., 2006). An important role for TRPV4 was substantiated in vivo where a TRPV4 agonist caused lung endothelial permeability, which could be inhibited by the non-selective TRP blocker ruthenium red, and was absent in TRPV4-/- mice (Alvarez, et al., 2006). Moreover, high-doses of a TRPV4 agonist were found to lead to circulatory collapse in mice via a nitric oxide-independent mechanism (Willette, et al., 2008). These studies provided strong evidence that TRPV4 activation could be involved in disrupting the integrity of epithelial and endothelial barriers, indicating a possible role in oedema formation in states of inflammation or tissue injury.

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Mechanical Injury

Positive pressure mechanical ventilation is a life-saving intervention that mechanically assists or replaces spontaneous breathing. However, this technique can cause injury to the airway tissues (termed ventilator induced lung injury, or VILI), potentially leading to increased permeability and oedema, bleeding of the alveoli, decreased lung compliance, and complete alveolar collapse. VILI is a complex syndrome that is induced by the cellular response to mechanical stress. It had previously been shown that vascular permeability associated with VILI involves an increase in intracellular Ca2+ concentration which is prevented by the non-selective stretch-activated cation channel blocker gadolinium (Naruse & Sokabe, 1993; Parker, et al., 1998). Hamanaka and colleagues (Hamanaka, et al., 2007) subsequently identified TRPV4 as the candidate mechanically-sensitive Ca2+-permeable channel that leads to endothelial permeability associated with VILI. The authors demonstrated that inhibition of signalling pathways that lead to TRPV4 activation (arachidonic acid metabolism by cytochrome p450 epoxygenases), direct antagonism of TRPV4 (with the non-selective inhibitor ruthenium red), or knockdown of the TRPV4 gene, all abrogate acute VILI in mice (Hamanaka, et al., 2007). In a murine model of overventilation (tidal volume 20 mL/kg body weight), the more selective TRPV4 inhibitor HC067047 or genetic knockdown was also shown to protect mice from oedema formation, cytokine release and histological signs of lung injury (Michalick, et al., 2013). Moreover, TRPV4 expressed on infiltrating macrophages are important in the airway response to VILI, whereby perfusion of TRPV4-/- mouse lungs with macrophages isolated from wild-type mice can restore VILI, which is usually blunted in knockout animals (Hamanaka, et al., 2010).

Vascular Pressure Induced Injury

Similar to VILI, promotion of acute lung injury and tissue permeability by high vascular pressure is mediated by TRPV4 activation and Ca2+ influx into lung endothelium in a murine isolated perfused lung model (Jian, et al., 2008). Under these conditions, TRPV4 was also purported to be indirectly activated via phospholipase A2 (PLA2) and cytochrome p450 epoxygenases (Jian, et al., 2008). High pulmonary venous pressure is a major cause of morbidity and mortality in heart failure patients, and TRPV4 has since been shown to be important in pulmonary oedema associated with heart failure in a disease-relevant animal model (Thorneloe, et al., 2012). Importantly, a TRPV4 inhibitor both prevented the development of oedema when given prior to myocardial infarction; and stimulated resolution of oedema when given following myocardial infarction (Thorneloe, et al., 2012). This study provides strong evidence for the potential use of TRPV4 blockade as a therapeutic strategy for people who are at risk of or suffering from lung injury and oedema, particularly in heart failure patients.

Chemical Injury

Acute lung injury can also be caused by the inhalation of toxic gases, smoke or acid, and aspiration of gastric acid. In murine models of chemically induced lung injury (intra-tracheal instillation of hydrochloric acid or inhalation of chlorine gas), Balakrishna and colleagues observed increases in the level of N-acyl amides in the bronchoalveolar lavage fluid and lung tissue (Balakrishna, et al., 2014). N-acyl amides are fatty acid-derived products that are related to anandamide and similar to endocannabinoids, and are known activators of several TRP channels, including TRPV4. Moreover, their results support an important role for TRPV4 expressed on infiltrating immune cells, where a TRPV4 inhibitor or genetic knockdown resulted in significantly lower levels of infiltrating macrophages and neutrophils, and associated chemokines and cytokines, following hydrochloric acid or chlorine gas treatment (Balakrishna, et al., 2014). Drug toxicity-induced pulmonary oedema associated with cancer patients taking interleukin-2 therapy has also been linked to TRPV4 activation (Huh, et al., 2012). Using a lung-on-a-chip device, the authors demonstrated that a TRPV4 inhibitor abolished the increase in vascular permeability induced by interleukin-2 treatment under physiological conditions that mimic breathing movements (involving 10% cyclic mechanical strain) (Huh, et al., 2012). Importantly, TRPV4 activation in the lung activates matrix metalloproteinases (MMPs) 2 and 9. MMP2 and MMP9 likely contribute to lung injury by degrading components of the alveolar basement membrane, as well as non-matrix components such as integrins and intercellular targets (e.g. E-cadherin) (Villalta, et al., 2014).

Sepsis

Sepsis is a fatal disease characterised by systemic inflammation and oedema formation (Dalsgaard, et al., 2016). There are conflicting results on the potential role of TRPV4 in sepsis pathogenesis. It was recently shown that gene deletion or pharmacological antagonism of TRPV4 did not protect mice in an LPS-induced model of sepsis (Sand, et al., 2015). Surprisingly, the pathology of sepsis even appeared to be slightly exaggerated with loss of TRPV4 signalling, with TRPV4/- mice exhibiting a higher sepsis severity score. It is important to note that endotoxaemic wild-type mice did not exhibit overt oedema, which is unusual, as the inflammation associated with sepsis is known to cause vascular leak (Ivady, et al., 2011). By contrast, TRPV4-/- mice showed significantly increased oedema in the kidneys, and slightly elevated oedema in the liver and spleen (Sand, et al., 2015). In contrast, Dalsgaard and colleagues (Dalsgaard, et al., 2016) demonstrated a role for TRPV4 channels in both lipopolysaccharide and cecal-ligation-and-puncture models of sepsis in mice. In this study, inhibition of TRPV4 prevented sepsis-induced inflammation, preserved endothelial cell function and integrity, and reduced mortality (Dalsgaard, et al., 2016).

Brain Oedema

A role for TRPV4 in brain oedema has also been identified. Brain oedema disturbs physiological neural function, amplifies tissue damage, and is important in the pathophysiology of many central nervous system diseases, such as cerebral ischemia, traumatic brain injury, and epilepsy (Jie, et al., 2015). A recent study demonstrated an important role for TRPV4 in brain oedema associated with stroke (Jie, et al., 2015). It was shown in mice that a TRPV4 antagonist reduced brain water content and Evans Blue extravasation 48 hours after middle cerebral artery occlusion relative to control (Jie, et al., 2015). The TRPV4 antagonist also abrogated the observed increase in MMP9 activity and loss of zonula occludens-1 (a tight junction protein) in the hippocampus following artery occlusion, thereby helping to preserve the integrity of the blood brain barrier and limit brain oedema (Jie, et al., 2015).

The cellular mechanisms leading to brain oedema and neuronal damage are linked to impaired astrocytic ion and water transport via Aquaporin (AQP) channels (Iuso & Krizaj, 2016). AQP channels function as low-resistance channels for water to cross the cell membrane, and are the primary determinant of membrane osmotic water permeability (Sidhaye, et al., 2006). When cells are exposed to a hypotonic extracellular environment, they display a RVD response, which serves as a mechanism to prevent detrimental swelling in response to hypo-osmotic stress (Benfenati, et al., 2011; Sidhaye, et al., 2006). Disruptions in cell volume regulation can have deleterious consequences for cell signalling, barrier dysfunction, cell viability, and enzyme function; and can result in irreversible metabolic injury which occurs in a wide range of brain pathologies (Iuso & Krizaj, 2016; Sidhaye, et al., 2006). AQP4 and TRPV4 ion channels co-localise in astrocytes, forming a complex which is important for the RVD response in these cells (Benfenati, et al., 2011). Interestingly, astrocytes from AQP4-/- mice, and astrocytes treated with TRPV4 siRNA fail to respond to hypotonic stress. It has been suggested that AQP4-mediated water fluxes promote the activation of a swelling sensor, whereas calcium entry through TRPV4 channels reciprocally modulate volume regulation, swelling and AQP4 expression (Jo, et al., 2015). This reciprocal interaction between TRPV4 and AQP4 is thought to play a key role in astroglial swelling, volume regulation and reorganisation of downstream signalling pathways (Benfenati, et al., 2011; Jo, et al., 2015). This research indicates that astrocytic swelling and brain oedema could be controlled by therapeutically targeting either AQP4 or TRPV4 channels (Benfenati, et al., 2011; Iuso & Krizaj, 2016).

Pain

The ability to perceive pain is controlled by a group of primary somatosensory neurons, termed nociceptors, which originate in the dorsal root ganglia (DRG) and trigeminal ganglia (TG). Nociceptive neurons detect noxious mechanical, chemical and thermal stimuli, and transmit messages to the central nervous system. The capacity to detect pain is generally advantageous, providing the stimulus to withdraw from noxious stimuli and guard injured tissue. However, the majority of chronic pain conditions are associated with altered activity and excitability of peripheral nociceptors, leading to allodynia (pain caused by a stimulus that does not normally evoke pain) and hyperalgesia (heightened pain from a normally painful stimulus) (Sousa-Valente, et al., 2014). One of the main drivers of this alteration in nociceptor activity is the sensitization or over-activation of membrane-bound ion channels, which are responsible for the conversion of noxious stimuli into neuronal excitation (Sousa-Valente, et al., 2014).

A number of TRP channels, including TRPV4, are functionally expressed in cells of the DRG and TG, and constitute the major class of detectors and transducers in nociceptive neurons (Mickle, et al., 2015). A large body of pharmacological and genetic research has confirmed that these channels are important in the generation and transduction of pain. Thus, like other TRP channels, TRPV4 is a potential target for the development of novel analgesics. However, controversy exists around the expression of TRPV4 in sensory neurons. Alexander and colleagues reported that TRPV4 is important for sensing noxious pressure in mice, and that TRPV4 is expressed at high levels in DRG and TG tissue measured by western blotting (Alexander, et al., 2013). In contrast, using immunohistochemistry, no TRPV4 was detected in neurons after 18-24 hours in culture. In functional assays, only 17 out of 261 wild-type mouse neurons responded to the potent, selective TRPV4 agonist GSK1016790A, compared to 1 out of 252 neurons from TRPV4-/- mice. Furthermore, no difference was observed in fura2-calcium responses of cultured neurons from wild-type or TRPV4-/- mice treated with hypo-osmotic solution or with 4αPDD (Alexander, et al., 2013). The authors concluded that 4αPDD is not a selective agonist because it activates neurons in TRPV4-/- DRG cultures, and that most TRPV4 staining in western blots is likely due to expression in non-neuronal cells (Alexander, et al., 2013). Indeed, TRPV4 is expressed in 73% of satellite glial cells from murine dorsal root ganglia, which form a sheath around neurons of sensory ganglia, and can regulate neuronal excitability in pain and inflammatory states (Rajasekhar, et al., 2015). TRPV4 is also expressed in other non-neuronal cells which are associated with pain conditions. For example, its expression in skin keratinocytes is important for the initiation of nociception, itch and inflammation (Basbaum, et al., 2009; Chung, et al., 2004; Julius, 2013; Nilius, 2007). ATP release from keratinocytes may trigger the activation of nociceptors, further complicating mechanisms of pain generation involving TRPV4 (Mihara, et al., 2011).

Inflammatory pain

The development of tissue hypersensitivity associated with inflammation is mediated by changes in the sensitivity of nociceptive neurons. TRPV4 plays a major role in mechanical pain and hyperalgesia in the colon, which is substantially attenuated in TRPV4-/- mice (Brierley, et al., 2008; Sipe, et al., 2008). There is also evidence of a role for both TRPV4 in pain associated with chronic pancreatitis and colitis (Ceppa, et al., 2010; D'Aldebert, et al., 2011; Zhang, et al., 2015). Moreover, UVB radiation was recently shown to activate TRPV4 in epidermal cells to cause the release of endothelin 1, which may underlie a component of sunburn-induced allodynia and hyperalgesia (Moore, et al., 2013). A role for TRPV4 in inflammatory thermal hyperalgesia is unclear, with some studies showing no effects on normal acute responses to noxious heat, but impaired inflammatory thermal hyperalgesia; whereas, others demonstrate deficits in acute heat responses, but no effect in inflammatory pain models (Alexander, et al., 2013; Huang, et al., 2011; Lee, et al., 2005; Liedtke & Friedman, 2003; Suzuki, et al., 2003a; Todaka, et al., 2004).

Roles for TRPV4 in inflammatory hyperalgesia and hypotonicity-induced pain have been demonstrated using TRPV4-/- mice. Inflammatory hyperalgesia is associated with chronic inflammatory conditions, and high local levels of inflammatory mediators and activated proteases (Alessandri-Haber, et al., 2006). Activation of TRPV4 is thought to induce transcriptional regulation and release of cytokines and tachykinins (substance P and calcitonin gene-related peptide) (Brierley, et al., 2008; Vergnolle, et al., 2010) which contribute to chronic pain. TRPV4 is sensitized or opened by the activation of Protease Activated Receptor 2 (PAR2) by digestion with trypsin, which reveals the N-terminal activating peptide SLIGKV. This activation is mimicked by the synthetic peptides SLIGKV and the equivalent mouse sequence, SLIGRL, which activates TRPV4 by a PLA2- and src-kinase dependent mechanism (Poole, et al., 2013) . It was subsequently found that activation of PAR2 by the proteases cathepsin S (Zhao, et al., 2014) and neutrophil elastase (Zhao, et al., 2015) at more distal sites on the GPCR N-terminus, also revealed activating peptides that signal to open TRPV4 by a PKC- and PKA-dependent mechanism. In a mouse model of PAR2-dependent mechanical hyperalgesia, the pain behaviour was inhibited by the c-Abl and src tyrosine kinase inhibitor bafetinib, but not by the related compound dasatinib (Grace, et al., 2014). This indicates specificity of bafetinib for the activation pathway, and may help to narrow the search for the tyrosine kinase(s) which are responsible for TRPV4 channel opening downstream of PAR2 activation.

Presence of the inflammatory mediator prostaglandin E2 has also been shown to contribute to TRPV4-mediated nocifensive behaviours to small changes in osmolarity (Alessandri-Haber, et al., 2005; Alessandri-Haber, et al., 2003), which may be important in pathological pain states associated with diseases such as diabetes and aquadinia (Levine & Alessandri-Haber, 2007). Moreover, the mechanism by which mechanical stimuli, such as membrane stretch and shear stress, activate TRPV4 is not well understood, but is thought to involve indirect signalling. TRPV4 is activated by hypotonic solutions (causing membrane stretch due to osmotic entry of water into the cell) both in transfected cells (Vriens, et al., 2004) and in vivo (Alessandri-Haber, et al., 2003; Vergnolle, et al., 2010). When expressed in HeLa and HEK293 cells, TRPV4 is opened by high levels of shear stress (Baratchi, et al., 2013; Soffe, et al., 2015; Wegierski, et al., 2009); and TRPV4 interacts with integrins and src kinases in sensory neurons of the DRG (Alessandri-Haber, et al., 2008). These mechanisms may underlie transduction of mechanical stimuli that open TRPV4 via tyrosine-kinase-dependent pathway(s) (Wegierski, et al., 2009).

Neuropathic Pain

Neuropathic pain is a complex chronic pain state that is associated with damage, dysfunction or injury of the nervous system. Studies using TRPV4-/- mice have demonstrated a role for TRPV4 in neuropathic mechanical hyperalgesia caused by Taxol toxicity (Alessandri-Haber, et al., 2004), and by chronic DRG compression, acting via a p38 MAPK pathway (Qu, et al., 2016). Although it is not yet clear which cell types are involved in pain generation, these data suggest a role for neuronal TRPV4. TRPV4 is expressed in many cell types, including sensory neurons and motor neurons (Alessandri-Haber, et al., 2003; Suzuki, et al., 2003a; Zimon, et al., 2010), glia, epithelia and endothelia (Alexander, et al., 2013; Hartmannsgruber, et al., 2007; Suzuki, et al., 2003b) and these cells could all contribute to pain conditions.

Itch

The sensation of itch plays a protective role in daily life, eliciting the scratch reflex to remove the associated irritant. Itch was classically thought to involve weak activation of nociceptors, and was therefore described as a “subthreshold pain”. However, pain and itch pathways have since been differentiated, and are now known to involve distinct as well as overlapping neuronal mechanisms (Han, et al., 2013; Toth & Biro, 2013). The circuitry controlling itch is complex, involving numerous pruritogenic substances that are released from neurons and cutaneous cells, including keratinocytes, mast cells, endothelial cells and immune cells (Toth & Biro, 2013). TRPV1, TRPV3 and TRPA1 have been implicated in itch processes either causing the release of pruritogens, or activating neurons to facilitate the sensation of itch (Ikoma, et al., 2006). TRPV1 is associated with histaminergic itch; TRPA1 with chloroquine and SLIGRL; and TRPV3 mutations with pruritic dermatitis and Olmsted syndrome (Imamachi, et al., 2009; Lin, et al., 2012; Liu, et al., 2011; Wilson, et al., 2011; Yoshioka, et al., 2009). These recent findings help to explain why antihistamine treatments are often ineffective, as many causes of itch are not histamine-related (Mollanazar, et al., 2015). Despite the recent developments in this field, chronic itch is a common problem, and remains a clinical challenge.

TRPV4 is implicated in scratching behaviour associated with serotonin-induced itch in mice (Akiyama, et al., 2016). Serotonin, histamine, SLIGRL (a PAR2/MrgprA3 agonist), or chloroquine were injected intradermally. Serotonin-induced scratching was significantly abrogated by a selective TRPV4 inhibitor and TRPV4 knockdown, compared to controls (Akiyama, et al., 2016). In this study, the response to other pruritogens was not affected (Akiyama, et al., 2016).. Another study suggests that TRPV4 contributes to non-neuronal histamine-induced but not chloroquine-induced itch in keratinocyte-specific TRPV4-/- mice, and calcium influx in isolated mouse keratinocytes (Chen et al., 2016). Downstream of TRPV4, the mechanism mediating non-neuronal histamine-induced itch may involve signalling via ERK phosphorylation (Chen et al., 2016). A further study has demonstrated that TRPV4 partially mediates histamine and chloroquine-induced itch, and that this requires TRPV1-mediated facilitation (Kim et al., 2016). Histamine is one of the best-characterised pruritogens in humans, and plays a role in a wide variety of itch pathologies, including ocular and nasal allergic reactions; chloroquine is a medication used to prevent and treat malaria, and is known to cause itch as a side-effect; and upregulation of serotonin is associated with atopic dermatitis, psoriasis and contact dermatitis (Akiyama, et al., 2016; Thurmond, et al., 2015). Though research in the field of TRP-related itch is still relatively young, and the current literature is somewhat contradictory, these studies provide encouraging evidence suggesting that TRPV4 could be an attractive pharmacological target to treat various itch pathologies.

Gastrointestinal Disease

Inflammatory bowel diseases (IBD) encompass a group of inflammatory conditions of the colon and small intestine, of which Crohn's disease and ulcerative colitis are the principal types. Irritable bowel syndrome (IBS) also manifests in chronic low-grade inflammation, and is characterised by chronic abdominal pain, discomfort, bloating, and diarrhoea or constipation. TRPV4 is expressed widely throughout the gastrointestinal tract, including the ileal and colonic tissues, DRG neurons, and fine nerve fibres associated with blood vessels in the submucosa and serosa (Brierley, et al., 2008; Cenac, et al., 2008; Holzer, 2011). It has been suggested that TRPV4 activation may affect innate immunity, directing intestinal epithelial cells toward a pro-inflammatory phenotype, thus causing or enhancing bowel dysfunction, and maintaining an inflammatory state (D'Aldebert, et al., 2011; Fichna, et al., 2012). Indeed, colitis is associated with an increase in TRPV4 expression in human and mouse colonic tissues and intestinal epithelial cells compared to healthy controls; and serosal blood vessels in colitis patients are more densely innervated by TRPV4-positive sensory fibres compared to patients with an uninflamed colon (Brierley, et al., 2008; D'Aldebert, et al., 2011; Fichna, et al., 2012) (Figure 4). In addition, inhibition of TRPV4 alleviates colitis and pain associated with intestinal inflammation in an animal model (Fichna, et al., 2012).

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The pro-inflammatory mediators histamine and serotonin are released by tissues of IBS patients, and are implicated in the development of visceral hypersensitivity (Cenac, et al., 2010). Moreover, proteolytic activity and PAR2-activating enzymes are increased in patients with IBD and IBS (Cenac, et al., 2007; Hyun, et al., 2008), and activation of the PAR2 receptor is thought to play a major pro-inflammatory role in animal models (Cenac, et al., 2002; Hyun, et al., 2008). Histamine, serotonin and proteases have been reported to potentiate the cellular response to TRPV4 activation via diverse intracellular signalling pathways involving PLA2, PKA, C and D, mitogen activated protein kinase kinase (MAPKK), and tyrosine kinases (Cenac, et al., 2008; Cenac, et al., 2010; Grace, et al., 2014; Grant, et al., 2007; Poole, et al., 2013; Sipe, et al., 2008).

Levels of fatty acid metabolites that directly activate the TRPV4 ion channel (epoxyeicosatrienoic acids, EETs) are also increased in the colon of patients with IBS (Cenac, et al., 2015). Direct TRPV4 activation causes tissue damage and visceral pain, leading to signs of colitis, including oedema, hyperaemia and mucus secretion, release of pro-inflammatory cytokines and invading inflammatory cells. These symptoms developed 3-6 hours following a single administration of TRPV4 agonist (4αPDD), and resolved within 24 hours (Cenac, et al., 2008; Cenac, et al., 2010; D'Aldebert, et al., 2011). Furthermore, repeated colonic administration of 4αPDD for 7 days caused chronic inflammation, again mimicking the parameters of colitis (D'Aldebert, et al., 2011; Vergnolle, 2014). In contrast to TRPV4-induced pain states, including visceral pain (Brierley, et al., 2008; Cenac, et al., 2008; Cenac, et al., 2015; Sipe, et al., 2008), TRPV4-induced colitis was not mediated by a neurogenic mechanism (D'Aldebert, et al., 2011).

The above evidence strongly suggests that TRPV4 plays an important role in gastrointestinal disorders, where the inflammatory environment acts to both enhance the level of TRPV4 expression and increase channel activation (Cenac, et al., 2015; D'Aldebert, et al., 2011). Therefore, within the gastrointestinal tract TRPV4 inhibitors hold potential both as anti-inflammatory drugs, and as potential therapeutics for patients with abdominal pain (Holzer, 2011).

Vascular Tone

The endothelial cell layer is crucial in controlling vascular smooth muscle tone. In response to vasoactive factors and shear stress elicited by blood flow, the vascular endothelium secretes substances which control vessel tone by inducing vasodilation (nitric oxide, NO; prostacyclin, PGI2; and endothelium-derived hyperpolarising factor, EDHF) (Kohler & Hoyer, 2007). This is an important mechanism by which the endothelium protects the vessels against damage, and endothelial dysfunction contributes to several cardiovascular pathologies, such as atherosclerosis and hypertension (Kohler & Hoyer, 2007).

TRPV4 is expressed in vascular endothelial cells in rodents (Watanabe, et al., 2002; Zhang, et al., 2009) and humans (Sullivan, et al., 2012), and has also been identified in vascular smooth muscle cells (Senadheera, et al., 2013) and astrocytic endfeet processes which wrap around blood vessels in the CNS (Benfenati, et al., 2007). There is mounting evidence of subtle vascular phenotypes in TRPV4-/- mice suggesting that TRPV4 plays a role in regulation of vascular tone and reactivity. Pressure myography experiments with TRPV4-/- mice have demonstrated that shear stress induced vasodilation in mouse carotid artery (Hartmannsgruber, et al., 2007) and mesenteric arteries (Mendoza, et al., 2010) is dependent on TRPV4 expression. Acetylcholine-induced dilation of mesenteric arteries was also shown to be TRPV4 dependent (Zhang, et al., 2009), and TRPV4-induced dilation of peripheral resistance arteries influenced arterial pressure (Earley, et al., 2009). Moreover, endothelial TRPV4 channels are necessary for dilation of cerebral arteries, and have been implicated in cerebrovascular pathologies related to Alzheimer’s disease, since recovery is impaired in TRPV4 null mice or after treatment with the antagonist HC067047 (Zhang, et al., 2013). In human tissues, flow induced dilation mediated by endothelial TRPV4 channels in coronary arterioles occurs via signaling through Ca2+ entry and mitochondrial ROS signaling (Bubolz, et al., 2012; Zheng, et al., 2013).

The mechanism of opening of TRPV4 in response to shear stress is not known. However, shear stress can increase cell surface expression of TRPV4 and can sensitize TRPV4 to a selective agonist in human umbilical vein endothelial cells (Baratchi, et al., 2015). In addition, TRPV4 surface expression in mesenteric resistance arteries is concentrated in myoendothelial projections facing the vascular smooth muscle (Sonkusare, et al., 2014) and this may facilitate signaling between the smooth muscle and endothelium through small- (SKCa) and intermediate-conductance (IKCa) calcium-activated potassium channels (Sonkusare, et al., 2012). TRPV4 Ca2+ entry and surface expression is attenuated by inhibition of myosin light chain kinase in rat pulmonary microvascular endothelial cells (Parker, et al., 2013) and by inhibitors of exocytotosis (Baratchi, et al., 2015).

The arachidonic acid derived metabolites EETs cause vascular smooth muscle cell hyperpolarisation and vascular relaxation independently of NO and PGI2 (Campbell & Fleming, 2010). EETs are TRPV4 agonists, and their effect on vessel tone is thought to involve TRPV4 (Campbell & Fleming, 2010; Earley, et al., 2005; Earley, et al., 2009; Loot, et al., 2008; Plant & Strotmann, 2007; Vriens, et al., 2005; Vriens, et al., 2004). TRPV4-/- experiments suggest that the channel is also involved in NO and EDHF dependent vascular relaxation, as these mechanisms are impaired in small mesenteric arteries of TRPV4-/- mice (Zhang, et al., 2009). Moreover, the plant derivative eugenol, known to have antihypertensive properties, was recently shown to cause vasorelaxation via TRPV4 activation in rat mesenteric arteries (Peixoto-Neves, et al., 2015); and GPCR agonists such as acetylcholine (Adapala, et al., 2011), angiotensin II and proteases (Saifeddine, et al., 2015) can modulate TRPV4 in blood vessels. The NO mediated signaling pathway downstream of TRPV4 activation is suggested to involve activation of IKca and SKCa ion channels, and activation of nitric oxide synthase (NOS) (Peixoto-Neves, et al., 2015). Thus a model is emerging in which physiological activation of TRPV4, via direct activation by exogenous and endogenous agonists, GPCR signaling and blood-flow induced shear stress, act through endothelium-dependent hyperpolarization factors to modulate vascular tone. This suggests that TRPV4 activation could be a therapeutic target for the treatment of cardiovascular pathologies such as hypertension. However, the clinical usefulness of such therapies could be limited by the adverse effects that are mediated by TRPV4 activation.

TRPV4 Mutations and Human Disease

The clinical manifestations of a large number of autosomal dominant TRPV4 mutations have been described, and the number of identified mutations continues to grow, as does the clinical spectrum of disease. It remains to be determined how mutations within the same region of the TRPV4 protein can lead to very different disease outcomes, of which there are three gross phenotypes: skeletal dysplasia’s, arthropathies and neuropathies, discussed below. Also of note is the apparent lack of, or mild effect of TRPV4 mutations on other tissues in which TRPV4 is highly expressed, such as the lung (Nilius & Voets, 2013). These questions highlight our current lack of understanding about how TRPV4 functions in vivo, and suggests that there are further mechanism(s) regulating TRPV4 function that are not yet understood. The development of a targeted and effective pharmacological means to help treat TRPV4 channelopathies may depend on a more in-depth understanding of the aetiology of these diseases, and the differential regulation of tissue-specific TRPV4 function.

Skeletal Dysplasia’s, Arthropathies and Neuropathy

Mutations of TRPV4 that are associated with the development of bone, cartilage and joints (i.e. skeletal dysplasia’s and arthropathies) have recently been reviewed (Nishimura, et al., 2012). The more than 40 mutations that occur in TRPV4 do not map to a single region, but are distributed throughout the coding regions of TRPV4, with many clustering between transmembrane domains 5 and 6.

Skeletal dysplasia’s encompass a diverse group of more than 200 diseases that affect bone and cartilage growth. The severity of disease ranges from mild, resulting in scoliosis and shortened stature; to severe, which can cause joint contracture, hip dislocation, fetal akinesia, and death (McNulty, et al., 2015; Nilius & Voets, 2013). For example, more than 50 mutation sites spread throughout the trpv4 gene are reported to cause brachyolmias, which are a rare form of skeletal dysplasia’s that result in platyspondyly, scoliosis, a short trunk, and mild short stature (Nilius & Voets, 2013). Skeletal dysplasia’s are reported to be caused by gain of function of the TRPV4 channel, leading to higher basal and stimulated whole-cell currents or Ca2+ signals in heterologous expression systems (Loukin, et al., 2010; Nilius & Voets, 2013). Thus, it has been suggested that skeletal dysplasia’s may result from mutations that cause an increase in calcium flux into chondrocytes through mutant TRPV4 channels, leading to upregulation of follistatin, inhibition of bone morphogenetic protein growth factors and reduced sensitivity of cells to bone morphogenetic protein signalling. This would lead to dysfunction of the normal chondrocyte hypertrophy cascade and altered endochondral ossification, resulting in improper bone formation (McNulty, et al., 2015; Saitta, et al., 2014; Weinstein, et al., 2014).

A cluster of 3 independent mutations affecting amino acid residues 270-273 in the third finger loop of the cytoplasmic N-terminal ankyrin repeat region of TRPV4, cause a familial digital arthropathy brachydactily (FDAB) (Lamande, et al., 2011). FDAB appears to be a developmental disease of chondrocyte function, which affects the fingers and toe joints, leading to aggressive osteoarthropathy and consequent shortening of the phalanges. FDAB patients generally appear to be clinically normal at birth, developing irregularities and painful osteoarthritis in the joints of their fingers and toes over the first decade of life (Lamande, et al., 2011). The FDAB mutations show a distinct phenotype to skeletal dysplasia’s, exhibiting reduced activity in heterologous systems which suggest loss of channel function; and are interesting because they are immediately adjacent to the site of mutations to residue 269, which causes Charcot–Marie-Tooth disease type 2C, an autosomal dominant neuropathy (Lamande, et al., 2011; Landoure, et al., 2010). This class of TRPV4 mutations are also associated with congenital distal spinal muscular atrophy and scapuloperoneal spinal muscular atrophy (Landoure, et al., 2010); reviewed in (Zimon, et al., 2010).

Hyponatremia

A single TRPV4 allele resulting from a polymorphism which results in a proline to serine substitution at residue 19 in the proximal N-terminus, results in a high propensity for carriers to develop hyponatremia (Tian, et al., 2009). The allele results in a less robust response to mild hypotonic stress compared to wild-type human TRPV4, when expressed in HEK293 cells, and is consistent with TRPV4 playing a role in water and ion balance in the kidney.

Complexes

Heteromultimerisation, complex formation, protein-protein interactions, and different subcellular and tissue localisations of TRP channels confer the potential for an immense array of functions throughout the body. TRPV4 protein is co-immunoprecipitated with many intracellular signalling molecules, such as the IP3 receptor through a calmodulin binding domain in the C-terminus (Garcia-Elias, et al., 2008); PACSIN3 through a proline rich domain in the cytoplasmic N-terminus (Garcia-Elias, et al., 2013); beta arrestin-1 (Shukla, et al., 2010); and AKAP proteins (Fan, et al., 2009; Mercado, et al., 2014). TRPV4 association with AKAP proteins is thought to enhance PKC-dependent phosphorylation of TRPV4, and facilitate communication between endothelial and smooth muscle cells via myoendothelial projections (Fan, et al., 2009; Mercado, et al., 2014; Sonkusare, et al., 2014). TRPV4 is also known to have important physical and functional interactions with other membrane channels, for example it is able to form functional heteromeric complexes with TRPP2 (Kottgen, et al., 2008) and TRPC1 (Ma, et al., 2011). TRPV4 association with TRPC1 is particularly interesting, as it appears to allow TRPV4 to function as a store-operated Ca2+ channel, and allows TRPC1 (which may not function as a channel on its own) to act as a legitimate Ca2+ channel (Goldenberg, et al., 2015). In DRG neurons, TRPV4 has been co-immunoprecipitated from rat dorsal root ganglion with integrins and Src kinase, which may be involved in sensing mechanical hyperalgesia (Alessandri-Haber, et al., 2008). Physical complexes between TRPV4 and aquaporins may regulate cell volume in astrocytes (Benfenati, et al., 2011). In vascular endothelial cells, TRPV4 is associated with the RyR2 ryanodine receptor and the BKCa ion channel (Earley, et al., 2005) and is functionally coupled to KCa2.3 (SK3) in endothelial cells in normal and streptozotocin-induced diabetic rats (Ma, et al., 2013). Functional coupling through activation of Ca2+-sensitive K+ channels following localised Ca2+ influx through TRPV4 in resistance arteries has also been shown for IK and SK channels, and can trigger endothelial-dependent vasodilation (Sonkusare, et al., 2012). Although it is currently difficult to envisage how such physical and functional interactions contribute to TRPV4 function, it will be important to understand the potential for such interactions to affect the responses of TRPV4 to activating stimuli in different cellular environments in order to identify tissue specific mechanisms of modulation of the ion channel (Darby, et al., 2016).

TRPV4 as a Therapeutic Target

TRPV4 antagonism has therapeutic potential in a diverse number of diseases, including oedema, pain, gastrointestinal disorders, and lung diseases such as cough, bronchoconstriction, pulmonary hypertension, ALI and ARDs. Indeed, the role of TRPV4 in humans and its suitability as a therapeutic target is currently being tested in a Phase I, first in human trial of the GlaxoSmithKline inhibitor GSK2798745 (clinicaltrials.gov NCT02119260). This trial aims to investigate the safety and tolerability of the compound in healthy subjects and stable heart failure patients for single and repeat oral administration. The results of the trial are eagerly awaited, due to the positive outcomes and lack of side-effects reported with the GSK2193874 compound in rodent models of heart failure (Thorneloe, et al., 2012). Two further clinical studies have also been registered, a Phase I study looking to validate a method for evaluation of pulmonary oedema, with the eventual aim of testing TRPV4 inhibitors in this context (NCT02135861); and a Phase II study to evaluate the effect of TRPV4 inhibition on pulmonary gas transfer and respiration in congestive heart failure patients (NCT02497937).

Due to the widespread expression and multitude of effects of TRPV4 throughout the body, there are some safety concerns around global TRPV4 agonism or antagonism. These concerns largely revolve around the fact that TRPV4 can have both beneficial and detrimental effects within the same tissue. For example, TRPV4 is a critical regulator of endothelial barrier integrity, and TRPV4 inhibitors could be used to prevent pulmonary oedema associated with several disorders (including sepsis, ALI, ARDS and heart failure). But their use in this circumstance may be contraindicated by the fundamentally important role that TRPV4 plays in the complex signalling cascade that mediates hypoxic pulmonary vasoconstriction (Goldenberg, et al., 2015; Yang, et al., 2012). This mechanism helps to redistribute blood flow from poorly ventilated to more well aerated lung areas, and inhibition could be detrimental to patients with lung disease (Goldenberg, et al., 2015; Morty & Kuebler, 2014; Yang, et al., 2012). Similarly, TRPV4 inhibition could play opposing roles in the development and treatment of conditions such as ARDs. In one instance, antagonism of the pro-inflammatory effects of TRPV4 could be detrimental, for example in the early stages of sepsis TRPV4 blockade could impair the immune response by inhibiting macrophage migration and ROS production, and reducing neutrophil infiltration. By contrast, in the treatment of ARDs, the barrier-stabilising and anti-inflammatory effects of TRPV4 inhibition could be beneficial (Goldenberg, et al., 2015). A great deal of evidence also exists showing that TRPV4 plays a vital role in the maintenance of systemic vascular tone, and that modifying TRPV4 activity can have profound effects on the regulation of vascular reactivity and blood pressure. It is encouraging to note that global inhibition of TRPV4 did not affect mean arterial pressure or heart rate in rodents (Thorneloe, et al., 2012). However, it remains to be seen whether TRPV4 inhibition affects vascular control in human beings under varying conditions including at rest, during physical exertion, and in disease states (Goldenberg, et al., 2015).

In circumstances under which global TRPV4 antagonism may be contraindicated, an alternative approach would be to target the signalling pathways upstream of TRPV4 activation. This review highlights the diverse signalling mechanisms that are associated with TRPV4-related pathologies. This opens up the possibility of targeting select signalling pathway(s) that are associated with a particular disorder. For example, targeting of the kinase(s) involved in specific pain or gastrointestinal pathologies (e.g. tyrosine kinases, src, PKA, PKC, MAPKK) could provide therapeutic potential. It may also be possible to block the formation of complexes or interactions that allow TRPV4 to function in select tissues. Furthermore, it has been suggested that targeted deletions and mutations of the folding recognition domain in the C-terminus of TRPV4 could be used to selectively down-regulate TRPV4 activity to treat overactive TRPV4-mediated pathologies, such as skeletal dysplasia’s (Lei, et al., 2013).

Future Directions

Results from the ongoing and upcoming clinical trials will provide a basis for future human research in to TRPV4-related pathologies. Further trials will reveal the true potential of direct TRPV4 antagonists to be used as therapeutic agents for a wide range of disease states. In conjunction, more should be done to investigate alternative options to direct inhibition under conditions where global TRPV4 inhibition is contraindicated, namely targeting of the upstream signalling pathways that ultimately lead to abnormal TRPV4 function.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figure Legends

Figure 1. Proposed hypothesis for the TRPV4/Pannexin/ATP axis. The TRPV4 ion channel is activated, leading to Ca2+ influx, which subsequently leads to ATP release through the Pannexin 1 ion channel. ATP can then act in an autocrine or paracrine fashion to activate P2X receptors on neighbouring cells. In sensory neurons, ATP release can activate the P2X3 ion channel to cause sensory nerve activation and cough. In epithelial cells, cigarette smoke induced ATP release is inhibited in Pannexin 1 and TRPV4 knockout animals.

Figure 2. Mechanisms of TRPV4 contributing to pulmonary oedema. Mechanical injury of the lungs, high vascular pressures, chemical injury, infection, and inflammation cause failure of the alveolar-capillary barrier, leading to degradation of tight junctions and adherens junctions between the epithelial and endothelial barriers. This allows the influx of white blood cells including neutrophils and macrophages, and leakage of fluid into the alveoli, causing pulmonary oedema. TRPV4 is expressed on the alveolar epithelial and vascular endothelial cells, and white blood cells, and is critically important to the oedematous process. Loss of TRPV4 in any of these cells results in a milder phenotype. Activation of nerves and neurogenic inflammation also causes oedema. TRPV4 is also expressed on nerve endings, but a role for neuronal TRPV4 in pulmonary oedema has not yet been investigated.

Figure 3. Cellular mechanisms of TRPV4 induced pulmonary oedema. Mechanical stress, chemical stimulation and intracellular signalling (including PLA2 and CYP450) can lead to TRPV4 activation and calcium influx in to the cell, which has a multitude of effects. In mammary epithelial cells, calcium entry can 1) rapidly trigger calcium-activated large conductance potassium channels (BKCa) and an increase in transcellular conductance; and 2) cause a delayed downregulation of tight junction claudin proteins, an increase in paracellular permeability and altered tight junction structure, via an unidentified signalling pathway. TRPV4 activation in the lung also activates MMP2 and MMP9, which may disrupt adherens and tight junctions, and degrade the alveolar basement membrane. TRPV4 expressed on infiltrating macrophages may contribute to lung injury and oedema by increasing reactive oxygen species (ROS) and nitrous oxide (NO) production.

Figure 4. TRPV4 and gastrointestinal disease. Inflammatory bowel disease (IBD) and irritable bowel syndrome (IBS) lead to enhanced TRPV4 expression within the colonic tissues, and innervating afferent neurons. The density of TRPV4-expressing nerve fibres innervating the serosal blood vessels is also increased. High levels of pro-inflammatory mediators such as histamine, serotonin, PAR2 activating peptides, and epoxyeicosatrienoic acids (EETs) in patients with IBD and IBS either directly or indirectly lead to TRPV4 sensitisation or activation. Augmented TRPV4 activity can result in enhanced inflammation, mucous secretion, oedema and visceral pain.

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