TRPV4: physiological role and therapeutic potential in respiratory diseases.

Naunyn-Schmiedeberg's Arch Pharmacol DOI 10.1007/s00210-014-1058-1

REVIEW

TRPV4: physiological role and therapeutic potential in respiratory diseases Neil M. Goldenberg & Krishnan Ravindran & Wolfgang M. Kuebler

Received: 2 September 2014 / Accepted: 10 October 2014 # Springer-Verlag Berlin Heidelberg 2014

Abstract Members of the family of transient receptor potential (TRP) channels have been implicated in the pathophysiology of a host of lung diseases. The role of these multimodal cation channels in lung homeostasis is thought to stem from their ability to respond to changes in mechanical stimuli (i.e., shear and stretch), as well as to various protein and lipid mediators. The vanilloid subfamily member, TRPV4, which is highly expressed in the majority of lung cell types, is well positioned for critical involvement in several pulmonary conditions, including edema formation, control of pulmonary vascular tone, and the lung response to local or systemic inflammatory insults. In recent years, several pharmacological inhibitors of TRPV4 have been developed, and the current generation of compounds possess high affinity and specificity for TRPV4. As such, we have now entered a time where the therapeutic potential of TRPV4 inhibitors can be systematically examined in a variety of lung diseases. Due to this fact, this review seeks to describe the current state of the art with

N. M. Goldenberg Department of Anesthesia, University of Toronto, Toronto, ON, Canada N. M. Goldenberg : K. Ravindran : W. M. Kuebler (*) The Keenan Research Centre at the Li Ka Shing Knowledge Institute of St. Michael’s Hospital, 30 Bond Street, Bond Wing 2-021, Toronto, ON M5B 1W8, Canada e-mail: [email protected] W. M. Kuebler German Heart Institute, Berlin, Germany W. M. Kuebler Institute of Physiology, Charité-Universitätsmedizin Berlin, Berlin, Germany W. M. Kuebler Departments of Surgery and Physiology, University of Toronto, Toronto, ON, Canada

respect to the role of TRPV4 in pulmonary homeostasis and disease, and to highlight the current and future roles of TRPV4 inhibitors in disease treatment. We will first focus on genera aspects of TRPV4 structure and function, and then will discuss known roles for TRPV4 in pulmonary diseases, including pulmonary edema formation, pulmonary hypertension, and acute lung injury. Finally, both promising aspects and potential pitfalls of the clinical use of TRPV4 inhibitors will be examined. Keywords TRPV4 structure . Transient receptor potential . Acute lung injury

Introduction Transient receptor potential channels Transient receptor potential (TRP) channels form a large superfamily of polymodal cation channels to which a host of cellular functions have been ascribed (Clapham 2003; Flockerzi and Nilius 2014). Originally cloned from Drosophila, six primary subfamilies exist, based on sequence homology: the classical or canonical (TRPC), vanilloid (TRPV), melastatin (TRPM), polycystins (TRPP), mucolipins (TRPML), and ankyrin (TRPA1) (Yin and Kuebler 2010). While overall homology between the channels is low (approximately 20 %), a number of common features exist that are characteristic for TRP family members (Clapham 2003). Each mature channel is made up of four subunits, each of which contains six transmembrane domains, and various subunits can combine to either form a homotetramer or heterotetramer (Schaefer 2005). The quaternary structure of the tetrameric channel, informed by electron cryomicroscopy of TRPV1, is believed to be analogous to that of K+ channels, with the fifth and sixth transmembrane domains of each subunit

Naunyn-Schmiedeberg's Arch Pharmacol

surrounding a central pore, and providing selectivity to the channel (Moiseenkova-Bell et al. 2008; Yellen 2002). Cryoelectron microscopy has also provided key structural insights into the topology of TRPV4 itself, detailing its similarities with the potassium channel, MlotiK1 (Shigematsu et al. 2010). Aside from a restriction to cations, TRP channels are otherwise relatively promiscuous with respect to their transported ion, with precise preferences varying from channel to channel. Both the amino and carboxyl termini are intracellular, and contain important structural and functional domains that regulate channel trafficking and activation state (Clapham 2003) (Hoenderop et al. 2003). Notably, each subunit contains a TRP box, which is a weakly conserved motif between TRP channel subunits, and many subtypes also contain a number of ankyrin repeats at their amino terminus. The carboxyl terminus often contains various domains for protein– protein interaction. TRPV4 The vanilloid subfamily receptor, TRPV4, has been of particular interest in both global physiology and in pulmonary function. At the gene level, TRPV4 is encoded by 15 exons, and has 5 known splice variants in mammalian cells. Consistent with the vast array of reported functions for TRPV4, the channel is almost ubiquitously expressed, and can be found in the brain, lung, heart, vasculature, kidney, bladder, skin, liver, and bone, among other organs. In the lung, TRPV4 has been identified in vascular smooth muscle (Yang et al. 2006), endothelial cells (Earley et al. 2005), and epithelial cells of the alveolae (Alvarez et al. 2006), trachea (Arniges et al. 2004), and bronchi (Fernández-Fernández et al. 2008). Additionally, TRPV4 is highly expressed in several immune cell types that reside in, and play important roles in, lung function, including macrophages (Hamanaka et al. 2010) and neutrophils (Damann et al. 2009). A full-length TRPV4 monomer consists of 871 amino acids, and as with all TRP channels, has 6 transmembrane domains (Fig. 1). While TRPV4 preferentially transports Ca2+ and Mg2+, unlike TRPV5 and TRPV6, TRPV4 is only weakly selective for divalent cations (Voets et al. 2002). While TRPV5 is approximately 100 times as permeable to calcium as it is to sodium, TRPV4, as well as TRPV1 and TRPV2, are less than 10 times more permeable to calcium than sodium. What selectivity that does exist can be abolished by mutating two key aspartate residues (D672 and D682) to alanines. When divalent cations are not present, TRPV4 will transport monovalent cations, and has little preference between these (Nilius et al. 2001). The majority of the TRPV4 protein is found in its long amino terminus, which contains up to six ankyrin repeats, depending on the splice variant in question. This long tail houses several important domains, including a

phosphoinositide binding domain (PBD) and a proline-rich domain, both of which are key features in the regulation of TRPV4 function (to be discussed below). The carboxyl terminus contains a binding site for Ca2+-calmodulin, a PDZ domain, and an oligomerization domain.

Extrapulmonary TRPV4 functions While this review will focus on the role of TRPV4 and TRPV4 inhibition in the lung, many of the properties of TRPV4 in other organs are conserved within pulmonary cell and tissue types. TRPV4 was originally identified as an osmosensor, and the notion that TPV4 was responding to membrane stretch led to its characterization as a mechanosensor (reviewed in Yin and Kuebler 2010). Adding to the model of TRPV4 as a mechanosensor, it has also been shown to be activated by shear stress (Köhler et al. 2006) and viscous loading (reviewed in Garcia-Elias et al. 2014). In addition to its function in mechanosensation, TRPV4, like other TRPV channels, is thermosensitive. The nonnoxious temperature range (24 to 38 °C) activates TRPV4, while other TRPV channels are activated by distinct temperature ranges (Clapham 2003). This allows the array of TRPV channels to function as a rheostat, activating differentially across a broad temperature range. Since the sensitive range of TRPV4 encompasses normal body temperature, it has been suggested that TRPV4 may play a role in normal thermoregulation, governing processes such as peripheral vasodilation and endothelial barrier function (Güler et al. 2002). The precise activity of TRPV4 at various temperatures remains controversial. The ability of TRPV4 to be activated at body temperature positions this channel for potential involvement in normal physiological processes. While some earlier studies did not reveal TRPV4 to be temperature-sensitive (Liedtke et al. 2000; Strotmann et al. 2000), more recent studies using different methods have changed opinion on this issue. Using overexpression models in both mammalian cells and Xenopus oocytes, Güler et al. (2002) were able to show that TRPV4 is activated by physiological temperatures: >27 °C in oocytes and >34 °C in HEK cells. Furthermore, since they found TRPV4 expression in the hypothalamus, they postulated that TRPV4 activity might be involved in mammalian thermoregulation. In comparison, another set of studies indicated that TRPV4 expressed in HEK cells became active at temperatures above 25 °C (Watanabe et al. 2002). Additionally, this same threshold was found for the native channel in aortic endothelial cells. While it now seems clear that TRPV4 is indeed thermosensitive, clearly much work remains in defining both the precise activation temperatures, and the mechanism by which this occurs. The failure to observe temperature-


Ca2+

PBD

TRP

PDZ

ANK Ca2+/CaM

Fig. 1 TRPV4 structure. TRPV4 consists of six transmembrane domains, with both its N- and C-termini in the cytosol. A series of ankyrin repeats (ANK) are found on the N-terminus, the precise number varying depending on the splice variant expression. Additionally, an N-terminal

phosphoinositide binding domain (PBD) is present. The C-terminal contains the TRP box (TRP), as well as a calcium–calmodulin binding region (Ca2+/CaM). Finally, a PDZ domain is also found at the C-terminus

dependent activation of TRPV4 in inside-out membrane patches suggests an endogenous factor is required for this process (Watanabe et al. 2002). Finally, of critical importance is the association of several TRPV4 gene mutations with a variety of skeletal and neurological disease entities. TRPV4-associated skeletal dysplasias include various forms of dwarfism that have been causally linked to the channel mutation (Nilius and Voets 2013). Furthermore, TRPV4 mutations have been found to cause several motor sensory neuropathies related to the CharcotMarie-Tooth syndrome (Nilius and Voets 2013). While such a large number of TRPV4 mutations have been mapped to this wide range of skeletal and neurological diseases, the mechanism by which TRPV4 contributes to skeletal dysgenesis remains unknown, and is a focus of ongoing research.

to oligomerize, which in turn dictates its further trafficking to the plasma membrane. While resident in the ER, TRPV4 has also been shown to interact directly with OS-9 (Wang et al. 2007), a protein associated with the cytosolic face of the ER membrane. While this interaction is present with all TRPV4 splice variants, it is strongest with those that lack full ankyrin domains and are therefore retained in the ER. The interaction between the N-terminus of TRPV4 and OS-9 is thought to protect TRPV4 monomers from ubiquitination and degradation, thereby allowing the formation of mature tetramers to occur. Once allowed to exit the ER, TRPV4 then is packaged for

β-Arr Ub

Intracellular trafficking of TRPV4 PACSIN

As is the case with any integral membrane protein, TRPV4 is synthesized in the endoplasmic reticulum (ER), and subsequently is targeted to the plasma membrane. The precise route taken by TRPV4 from the ER to the plasmalemma is yet to be elucidated, and further work seeking to define this pathway may reveal important regulatory steps in TRPV4 trafficking. However, work from several groups has shed light on the regulation of TRPV4 surface expression (Fig. 2). As stated earlier, splice variants of TRPV4 with different numbers of ankyrin repeats have been demonstrated to have different subcellular localizations; splice variants B, C, and E are retained in the ER, and remain inactive during stimulation of transfected cells with the TRPV4 agonists, arachidonic acid, hypotonicity, or the phorbol ester 4α-phorbol 12,13didecanoate (4α-PDD) (Arniges et al. 2006). These data as well as others suggest that the number of ankyrin repeats present on the TRPV4 subunit dictate the ability of the protein

Golgi

ER

OS-9

Fig. 2 Intracellular trafficking of TRPV4. Like all transmembrane proteins, TRPV4 is synthesized in the endoplasmic reticulum (ER). In the ER, association with the quality control protein, OS-9, delays TRPV4 transit to the Golgi. Expression at the plasma membrane is the net effect of endocytosis and exocytosis. Binding of TRPV4 to PACSIN decreases endocytosis, resulting in increased TRPV4 surface expression. In converse, binding by β-arrestin (β-arr) results in monoubiquitination of TRPV4 and its subsequent endocytosis. Once in the plasma membrane, stimulation of TRPV4 can result in its partitioning into caveolae


transport to the Golgi, and then on for subsequent regulated transport to the plasma membrane. Expression of TRPV4 at the cell surface has been shown to be regulated by the protein PACSIN-3 (Cuajungco et al. 2006), in that overexpression of PACSIN-3 resulted in an increase in plasma membrane-associated TRPV4. Since PACSIN-3 is known to interact with, and inhibit the function of, dynamin, it has been hypothesized that the increased surface expression of TRPV4 in PACSIN-3-transfected cells is due to a decrease in endocytic retrieval of TRPV4 from the plasma membrane (Cuajungco et al. 2006). This is an important observation, which suggests a system of continual cycling of TRPV4 between the cell surface and an intracellular compartment, a process that could potentially be influenced in order to regulate TRPV4 surface activity. Interestingly, when bound by PACSIN-3, TRPV4 is no longer activated by cellular swelling or heat, but continues to be sensitive to the agonist, 4α-PDD (D’hoedt et al. 2008). These data suggest that TRPV4 is activated by different agonists via nonoverlapping mechanisms. Like many surface proteins, activation of TRPV4 can be associated with its downregulation at the cell surface in order to establish a negative feedback loop. In many scenarios, this process is mediated by monoubiquitination of the surface receptor. Indeed, this is the case with TRPV4, which has been shown to be monoubiquitinated by the ubiquitin ligase, ` (Nilius et al. 2001; Wegierski et al. 2006). The activity of AIP4 leads to endocytosis of TRPV4 without degradation, and a subsequent decrease in Ca2+ conductance (Wegierski et al. 2006). In further studies, TRPV4 was found to form a stable complex with the angiotensin II receptor, AT1aR, and that binding of angiotensin II to this receptor resulted in the ubiquitination of TRPV4 and subsequent internalization of both TRPV4 and AT1aR (Arniges et al. 2006; Shukla et al. 2010). The ubiquitination of TRPV4 requires the protein βarrestin, which serves as an adaptor between TRPV4 and AIP4 (Shukla et al. 2010; Wang et al. 2007). This system defines a novel pathway, whereby angiotensin II signaling leads to both downregulation of its own receptor and that of TRPV4. Finally, the precise localization of TRPV4 within the plasma membrane also appears to be tightly regulated, and has important functional consequences. TRPV4 has been shown to associate with caveolin-1 in endothelial cells from systemic vessels (Cuajungco et al. 2006; Saliez et al. 2008). Cavelolin1 is a primary structural component of caveolae, which are plasma membrane microdomains that are required for normal vascular endothelial and smooth muscle cell homeostasis (reviewed in Cuajungco et al. 2006 and Parton and Simons 2007). This association places TRPV4 in close proximity with several proteins of critical importance in vascular biology, most importantly endothelial nitric oxide synthase (eNOS), and this compartmentalization of TRPV4 within caveolae is

required for its function as a systemic vasorelaxant (Saliez et al. 2008). Of particular significance in the lung, caveolae are key sites of nitric oxide production, and other TRP channels, such as TRPC6, have been shown to translocate to caveolae in response to acute hypoxia (Yu et al. 2012). Such translocation is likely critical to the function of TRP channels in the regulation of lung vascular tone, which will be discussed in detail below. Clearly, dynamic regulation of the subcellular localization of TRPV4 is a complex process that remains to be fully unraveled. Further research on this topic will deepen our understanding of TRPV4 biology, and will potentially yield targets for the pharmacological manipulation of TRPV4 activity.

Interaction with other channel proteins TRPV4 has been shown to have important interactions— physically and functionally—with other membrane channels. Several such complexes have been defined; one involves TRPV4, ryanodine receptors (RyRs), and large-conductance Ca2+-activated K+ channels (BKCa) (Earley et al. 2005). Ca2+ currents in cerebral artery smooth muscle cells elicited by treatment with epoxyeicosatrienoic acids (EETs) resulted in Ca2+ release from the sarcoplasmic reticulum via RyR (Earley et al. 2005). The subsequent spike in Ca2+ results in K+ release via BKCa channels. This sequence of events was inhibited following TRPV4 antisense treatment, suggesting that intracellular Ca2+ release via RyR and subsequent activation of BKCa channels was initially triggered by the influx of extracellular Ca2+ via TRPV4 in the form of a Ca2+-dependent Ca2+ release. Activation of Ca2+-sensitive K+ channels following localized Ca2+ influx (so called “sparklets”) via as few as three functional TRPV4 channels per cell has also been shown for intermediate (IK)- and small (SK)-conductance K+ channels, and can trigger endothelial-dependent vasodilation of resistance arteries (Sonkusare et al. 2012). Notably, the hyperpolarization that results from K+ efflux via Ca2+-sensitive K+ channels will increase the electrochemical driving force for Ca2+ entry via TRPV4, thereby establishing a positive potentiating and, potentially, detrimental feedback loop between Ca2+ entry and K+ exit. As stated previously, TRP channels can form heterodimers with each other. One such heterodimer has been described involving TRPV4 and TRPC1 (Ma et al. 2010). In these experiments, a heteromeric channel containing TRPV4 and TRPC1 was found to translocate to the plasma membrane in response to depletion of intracellular Ca2+ stores (Ma et al. 2010, 2011). This heteromer translocated with greater efficiency than either channel subtype alone, and was found to comprise a functional store-operated Ca2+ channel (SOCC). This result is particularly intriguing, since the interpretation of


such data is that by associating with TRPC1, TRPV4 is able to take on the role of a TRPC channel, and function as a SOCC. Conversely, TRPC1, which has debatable channel function on its own, becomes a bona fide Ca2+ channel via its interaction with TRPV4. Such an arrangement is a key example of the multitude of potential functions of TRP channels; different subcellular and tissue localizations and different protein–protein interactions are able to confer a huge array of functions upon this already large family of channel proteins. Another example for a heteromeric TRP channel assembly is the recent identification by atomic force microscopy of an interaction of TRPP2 with both, TRPV4 and TRPC1 (Stewart et al. 2010). Assembly of these three subunits from notably three different TRP families can form a heteromeric flow-sensitive cation channel; yet, flow-induced cation currents are abolished by pore-dead mutants for each of these three TRP isoforms, suggesting that all three TRPs contribute to the ion permeation pore of the channels (Du et al. 2014). Cooperation, at least at the functional level, has also been demonstrated between TRPV4 and TRPC6 in mechanical hyperalgesia and nociceptor sensitization, in that antisense to TRPC6, but not to TRPC1, reversed the mechanical hyperalgesia induced by a thermal injury or the TRPV4-selective agonist 4α-PDD (Alessandri-Haber et al. 2009). However, whether TRPV4 really forms heteromeric channel complexes with TRPC6, or whether this cooperativity is merely based on functional interdependence, remains to be elucidated. Given the large size of the intracellular domains of TRPV4, it is not surprising that a variety of intracellular signaling proteins exert regulatory effects on TRPV4 activity (Fig. 3). One important nonprotein modulator of TRPV4 function is the membrane phospholipid, phosphatidylinositol 4,5bisphosphate (PIP2) (Garcia-Elias et al. 2013). PIP2 is almost exclusively localized at the inner leaflet of the plasma membrane, and is a powerful signaling molecule, responsible for a huge array of cellular processes (Di Paolo and De Camilli 2006). Importantly, it is also a source of the fundamental second messengers, diacylglycerol (DAG) and inositol triphosphate (IP3), both of which are key regulators of Ca2+ signaling and activation of the protein kinase c (PKC) family. PIP2 is a negatively charged membrane phospholipid, and as such, interacts with a variety of positively charged protein domains (Yeung et al. 2006). Consistent with these systems, TRPV4 has also been shown to interact with plasma membrane PIP2 in heterologous expression systems in epithelial and neural cells types (Fernandes et al. 2008; Garcia-Elias et al. 2013). The N-terminal PBD of TRPV4 interacts with PIP2, and is thought to stabilize the intracellular tail of TRPV4 in an open conformation along the inner surface of the plasmalemma (Garcia-Elias et al. 2013) (Fig. 3a). Functionally, depletion of PIP2—which could occur physiologically as a result of G protein or PKC activation—makes the channel unresponsive to heat or osmotic stimuli, but maintains

activation by EETs or 4α-PDD (Garcia-Elias et al. 2013). Overexpression of PACSIN also blocks PIP2–TRPV4 association and modifies channel activation (Fig. 3b). These data demonstrate the importance of the lipid environment as a key modulator of TRPV4 agonist specificity. Calcium, which is transported preferentially by TRPV4, also regulates TRPV4 activity. The response of TRPV4 to intracellular Ca2+ is under debate. In a heterologous expression system, it has been shown that increasing intracellular Ca2+ inhibits channel function (Watanabe et al. 2003a). However, also in an overexpression model, Strotmann et al. (2003) have demonstrated that increasing intracellular Ca2+ activates TRPV4 through the direct binding of Ca2+–calmodulin to TRPV4. This second observation would be an intriguing one whereby the potential for positive feedback exists between Ca2+ and TRPV4. This hypothesis requires further experiments to be resolved, however, since other groups have found that Ca2+–calmodulin binding to TRPV4 is, in fact, inhibitory at least in some cell systems (Phelps et al. 2010). In addition to the mechanisms discussed above, several other intracellular signaling processes are capable of modulating TRPV4 channel activity. Both protein kinase A (PKA) and PKC have been found to phosphorylate TRPV4, which has been linked to enhanced TRPV4 channel activation (Fan et al. 2009). Importantly, A-kinase anchor proteins (AKAPs), which bind to both PKA and PKC and distribute these kinases to discrete locations within the cell, seem to play an important role in the signaling between TRPV4 and PKA/PKC. Specifically, AKAP150 has been shown to interact with TRPV4, and to mediate TRPV4 regulation via PKC in a dynamic fashion. In cerebral artery myocytes, some plasma membrane-bound TRPV4 is basally bound to AKAP. When the cells are stimulated with angiotensin II, increased signaling through G proteincoupled receptors leads to increased PKC activity. This, via interaction with TRPV4/AKAP, results in phosphorylation of TRPV4, which increases its open probability, and allows for the formation of local Ca2+ sparklets (Mercado et al. 2014). The interplay between TRPV4, PKC, and AKAP is seen again in the communication between endothelial and smooth muscle cells as myoendothelial projections (MEPs) which link the two cell type. Here, stimulation of muscarinic receptors with acetylcholine results in TRPV4-mediated Ca2+ influx, specifically at the MEPs (Sonkusare et al. 2014). MEP-based TRPV4 is bound by AKAP, which is required for the spatial specificity of this response, as in the example above using angiotensin II (Mercado et al. 2014). At MEPs, Ca2+ influx is further stimulated by cooperative opening of TRPV4 channels in a PKC- and AKAP-dependent fashion (Sonkusare et al. 2014). Clearly, complex mechanisms exist in order to localize the effect of agonists upon TRPV4 activity, in order to generate local Ca2+ flux, while preventing the deleterious effects of a global increase in [Ca2+]i. It appears that signaling through PKC and AKAP are key aspects of this functional and spatial segregation.

Naunyn-Schmiedeberg's Arch Pharmacol Fig. 3 Regulation of TRPV4 activation. a When PIP2 is present in the plasma membrane, TRPV4 is held in an open conformation by its PI binding domain. In this state, the channel can be activated by heat and osmotic stretch. In contrast, in b, when PIP2 is absent, and/or TRPV4 is bound by PACSIN, the channel adopts a closed conformation, rendering it insensitive to stretch and heat, but still sensitive to the chemical agonists, 4-αPDD or EETs. c Binding of TRPV4 by Ca2+/ calmodulin yields different results depending on the system used (see text for details), but may be an activator or inhibitor of channel function. d Phosphorylation at the indicated residues by PKA (green residues) or PKC (blue residues) increases channel activation

A

Heat Osmolarity

B

4-αPDD EET

PIP2

PACSIN

C

D Acvaon/Inhibion

Acvaon

S189 S824

S162 T175

Ca/CaM

The previous identification of an inhibitory role of cGMP on TRPV4 by our group furthermore suggests negative regulation of TRPV4 via PKG (Yin et al. 2008). In HEK cells expressing TRPV4-TRPP2 heteromeric channels, flowinduced Ca2+ entry was shown to be negatively regulated by cGMP; however, abolishment of this effect by point mutations at two putative PKG phosphorylation sites on TRPP2 suggests TRPP2 rather than TRPV4 as a phosphorylation target for PKG in this system (Du et al. 2012). Conversely, direct evidence for TRPV4 phosphorylation by either cGKI or cGKII is yet lacking. Overall, the effects of such posttranslational modifications are still controversial, and have been reviewed in detail elsewhere (Garcia-Elias et al. 2014).

Pharmacological modulators of TRPV4 Activators TRPV4 is activated by a variety of physiological and pharmacological stimuli. One of the earliest physiological activators of TRPV4 to be described was non-noxious heat. In

general, TRPV channels are activated by specific, largely non-overlapping temperature ranges, and as such, this subfamily of channels can serve as a rheostat for temperature sensation (reviewed in Clapham 2003). For instance, TRPV4 is activated at temperatures between 24 and 38 °C, while TRPV1 is activated at temperatures greater than 43 °C, and TRPV2 if temperature is greater than 52 °C (Clapham 2003; Watanabe et al. 2002). Hypo-osmolarity and the ensuing mechanical stretch are also key physiological activators of TRPV4. The mechanism by which cellular swelling activates TRPV4 seems to involve activation of phospholipase A2 (PLAs), which produces arachidonic acid (AA) (Vriens et al. 2004). AA is subsequently metabolized to form EETs, which then directly activate the channel. Functionally, mechanosensation by TRPV4 is required for normal osmoregulation (Liedtke and Friedman 2003), and for bladder function (Everaerts et al. 2010). This phenomenon is also seen in flow-induced vasodilation (Mendoza et al. 2010), and for endothelial monolayer reorientation in response to cyclic strain (Thodeti et al. 2009). Several chemical mediators are also potent activators of TRPV4. The phorbol ester, 4α-PDD, triggers Ca2+ influx


through TRPV4. 4α-PDD binds TRPV4 between transmembrane domains 3 and 4, and its activity on the channel is not mediated by activation of PKC enzymes (Klausen et al. 2009). The arachidonic acid metabolites, EETs, are also TRPV4 activators, and are thought to be the downstream effectors of other stimuli for TRPV4, including the endocannabinoid anandamide (Watanabe et al. 2003b) and—vide supra—cellular swelling (Vriens et al. 2004). Intriguingly, EETs are also produced in the lung during hypoxia (Keserü et al. 2008), and are thus a potential mechanism for the known role of TRPV4 in the pulmonary vascular response to both acute (Goldenberg et al. 2014) and chronic (Yang et al. 2012) hypoxia (vide infra). Finally, a small molecule created by GlaxoSmithKline, named GSK1016790A, is also a useful TRPV4 agonist (Thorneloe et al. 2008). This molecule is approximately 300 times more potent than 4α-PDD when tested in a heterologous expression system, and causes bladder contraction in mice (Thorneloe et al. 2008). Treatment of mice, rats, or dogs with this compound resulted in dose-dependent circulatory collapse (Willette et al. 2008), an effect that was largely mediated by endothelial barrier dysfunction and widespread microvascular leak. In vitro, GSK1016790A causes contraction and gap formation in cultured endothelial cells. While a useful agonist for laboratory studies, the extreme potency of this compound, and the above systemic adverse effects, make its clinical usefulness questionable. Inhibitors A number of pharmacological inhibitors of TRPV4 have been used in research and clinical applications over the years (Fig. 4). As time has progressed, these molecules have developed increasing specificity for TRPV4, which was an issue with earlier compounds. One of the earliest inhibitors of TRPV4, to use this term in the broadest sense, was gadolinium. Gadolinium, which is most commonly used as a contrast agent for magnetic resonance imaging, was identified in the 1980s as an inhibitor of stretch-activated ion channels (Yang and Sachs 1989). Even earlier data indicated that gadolinium could block Ca2+ entry into chromaffin cells in response to acetylcholine (Bourne and Trifaró 1982). Indeed, gadolinium is capable of blocking TRPV4-mediated Ca2+ currents (Ryskamp et al. 2011). Clearly, this molecule has a much wider array of targets than simply TRPV4 including, e.g., voltage-gated, L-type Ca2+ channels (Lacampagne et al. 1994) and store-operated Ca2+ channels (Bourne and Trifaró 1982), and is commonly viewed these days as a nonselective inhibitor of extracellular Ca2+ entry. The promiscuity of gadolinium has limited its use as an agent for determining TRPV4 function experimentally, or for blocking the channel clinically. A similarly nonspecific, yet widely used, compound for studying TRPV4 is ruthenium red (RuR). RuR is a polyvalent

HC-067047

GSK2193874

Fig. 4 Chemical structures of selected TRPV4 inhibitors

cationic dye, with a long history of clinical uses including immunosuppressive and antimalarial applications (Allardyce and Dyson 2001). Ruthenium compounds have also been used on a limited scale as chemotherapeutic and photosensitizing agents (Schmitt et al. 2008). Like gadolinium, RuR is unfortunately highly nonspecific and blocks signaling through most TRPV channels, members of the TRPM and TRPA family, as well as some unrelated channels and transporters including the mitochondrial Ca2+ uniporter or the ryanodine receptor (Güler et al. 2002). In spite of its nonspecificity, it is highly avid, being a useful channel inhibitor at nanomolar concentrations. While the low cost and clinical safety of RuR are attractive, its usefulness as a TRPV4 inhibitor remains limited. Newer compounds have appeared with a far more favorable profile in terms of TRPV4 specificity and affinity. One such compound is HC-067047, which was found after a highthroughput screen of compounds capable of blocking 4αPDD-induced Ca2+ currents in human cells overexpressing TRPV4 (Everaerts et al. 2010). At doses that blocked TRPV4 function, mice displayed no adverse behaviors or gross signs of sickness. Importantly, at submicromolar concentrations, other than TRPV4, only TRPM8 and hERG were inhibited partially by HC-067047. Other tested channels in the TRPV, TRPC, TRPA, and TRPM subfamilies, as well as voltage-gated Na+ and Ca2+ channels, were not affected by HC-067047 administration (Everaerts et al. 2010). This favorable profile, with an IC50 ranging from 17 to 133 nM, depending on species, makes HC-067047 a far more powerful tool for studying TRPV4 than previous inhibitors. However, its clinical safety profile remains untested. Possibly the largest body of work regarding inhibition of TRPV4 has been performed by Thorneloe et al. (2012) at GlaxoSmithKline, who have screened and characterized a variety of compounds. The most promising, and best


characterized, of these is the compound GSK2193874 (′874). This small molecule was identified using high-throughput screening, and displays a remarkable specificity for both rodent and human TRPV4, as demonstrated by a screen against approximately 200 other channel proteins, including other TRPV subfamily members (Thorneloe et al. 2012). The ′874 compound was able to block TRPV4 Ca2+ responses due to hypotonicity, the activator GSK634775, and blocked TRPV4mediated edema formation in animal models of congestive heart failure (Thorneloe et al. 2012). In the same publication, another compound with an even greater inhibitor potency than ′874, and excellent specificity, called GSK2263095, was also tested in dogs. This compound was capable of inhibiting pressure-induced pulmonary edema formation in isolate perfused canine lungs (Thorneloe et al. 2012). However, a key advantage of ′874 lies in its oral bioavailability, meaning it can potentially be dosed repeatedly for chronic use (Thorneloe et al. 2012). Regarding potential adverse effects, an exhaustive screen has not been completed, but heart rate, systemic blood pressure, and osmoregulation were unaffected by ′874 in rats, and endothelial barrier function was maintained in mice. These results represent the most important data to date regarding a TRPV4 inhibitor with the specificity, affinity, and apparent safety for potential use in human trials. While safety of this inhibitor in humans is yet to be shown, ′874 may represent an important therapy for a variety of pulmonary and extrapulmonary disease states. A final strategy for the clinical inhibition of TRPV4 activity that bears mention involves blocking a downstream effector of TRPV4, namely phosphodiesterase 5 (PDE5). Previous work carried out in our laboratory demonstrated that cGMP could inhibit TRPV4-mediated Ca2+ signaling in endothelial cells (Yin et al. 2008). Importantly, the PDE5 inhibitor, sildenafil, attenuated TRPV4-mediated endothelial Ca2+ entry and pulmonary edema formation in ex vivo and in vivo models of congestive heart failure (Yin et al. 2008). These data establish a potential role for PDE5 inhibition as an indirect route for preventing the adverse physiology associated with TRPV4 activation. While such an intervention would clearly be nonspecific, the advantage of such a therapy is that PDE5 inhibitors have a long history of clinical safety (Yin and Kuebler 2010).

TRPV4 in pulmonary disease Hydrostatic pulmonary edema Hydrostatic lung edema is the result of either increased hydrostatic pressure or decreased oncotic pressure in the pulmonary capillaries, leading to fluid leak across the microvasculature and its subsequent accumulation in the alveoli and

interstitium. This type of edema is commonly caused by left heart failure, which leads to higher end diastolic left ventricular pressure that is then transmitted backward to the pulmonary microcirculation, resulting in fluid extravasation. While the resulting fluid filtration across the pulmonary capillaries was initially believed to be a mere result of an imbalance in transcapillary Starling forces, more recent studies identified that increases in endothelial permeability in response to elevated vascular pressure contribute importantly to the pathology of hydrostatic lung edema (Parker and Ivey 1997). Such increases in endothelial permeability can be triggered by endothelial Ca2+ influx, resulting in activation of signaling pathways that cause cytoskeletal reorganization and loss of interendothelial junctional proteins (Tiruppathi et al. 2006). Broadly speaking, increased cytosolic Ca2+ results in phosphorylation of the myosin light chain via myosin light chain kinase (MLCK), and disruption of VE–cadherin-containing adherens junctions (Sandoval et al. 2001). Parker et al. (1998) first suggested that stretch-gated ion channels may mediate increases in endothelial Ca2+ that may be responsible for the increase in lung vascular permeability after high-pressure overinflation. Stretch-activated cation channels had been previously identified in endothelial cells by patch-clamp technique and Ca2+ imaging in cultured cells, but their identity remained elusive at that time (Lansman et al. 1987; Naruse and Sokabe 1993; Thorneloe et al. 2008). Using real-time fluorescence imaging in the intact lung, our group was able to directly visualize for the first time that increases in lung hydrostatic pressure caused a gadolinium-inhibitable increase in lung endothelial Ca2+ (11943655) that in turn triggered the exocytosis of Weibel–Palade bodies and the expression of Pselectin (Kuebler et al. 1999; Thorneloe et al. 2008) as well as stimulated nitric oxide synthesis from endothelial NO synthase (Kaestle et al. 2007; Kuebler et al. 2003; Willette et al. 2008). The identity of these channels remained, however, unknown for some time, until the identification of TRPV4 as key mediator of this stretch-induced lung endothelial Ca2+ entry and the resulting acute increase in vascular permeability (Hamanaka et al. 2007; Yang and Sachs 1989) (Bourne and Trifaró 1982; Jian et al. 2008; Yin et al. 2008). TRPV4 was originally identified as a stretch-gated channel, activated by cell swelling as a result of hypotonic osmotic stress (Lacampagne et al. 1994; Nilius et al. 2001). As such, TRPV4 was classified as a functional osmoreceptor in the central nervous system, controlling vasopressin release from the magnocellular neurosecretory cells of the hypothalamus (reviewed in Allardyce and Dyson 2001 and O’Neil and Heller 2005). In the endothelium, the application of fluid shear stress to the cell surface, in combination with direct membrane stretch, results in Ca2+ influx through TRPV4 (Güler et al. 2002; Köhler et al. 2006). This response to shear stress is highly temperature-sensitive, with no flow-induced TRPV4 activation observed between 25 and 35 °C (Everaerts et al.


2010; O’Neil and Heller 2005). In the lung, the pulmonary microvascular bed is not only exposed to the combination of stretch and shear stress, but also to longitudinal strain associated with the respiratory cycle, thus providing a unique role for mechanotransduction by TRPV4. The dynamic equilibrium between recruitment and derecruitment of various capillary segments furthermore results in oscillatory shear stress events acting on the pulmonary capillary endothelium (Everaerts et al. 2010; Yin and Kuebler 2010). Biochemically, it is has been postulated that the ankyrin repeat domains at the intracellular amino terminus play a role in mechanosensation, potentially by anchoring TRPV4 to the cytoskeleton (Nilius et al. 2004; Thorneloe et al. 2012). Interestingly, TRPV4 has been shown to bring filamentous actin in a regulated fashion (Shin et al. 2012; Thorneloe et al. 2012). Phosphorylation of TRPV4 by serum glucocorticoidinduced kinase 1 (SGK1) leads to enhanced binding of TRPV4 to actin (Shin et al. 2012; Thorneloe et al. 2012). This sets up an intriguing possibility whereby the mechanosensitivity of TRPV4 may be modulated by protein kinases. Through its mechanosensory role at the endothelial plasma membrane, TRPV4 has been shown to govern the endothelial Ca2+ and permeability response to hydrostatic pressure. Pharmacological activation of TRPV4 by 4α-PDD resulted in increased endothelial permeability in isolated rat lungs, as assessed by gravimetric filtration coefficient (Kf) measurements, and this effect was reversed by ruthenium red administration (Alvarez et al. 2006; Thorneloe et al. 2012). Similarly, endothelial permeability can be increased by the TRPV4 activator GSK1016790 (Thorneloe et al. 2012). Conversely, TRPV4 knockout mice displayed minimal edema (measured by lung wet/dry ratios), and diminished intracellular endothelial Ca2+ elevation in response to pressure (Yin et al. 2008), a phenotype which is examined elsewhere in this review. Chronic pressure stress The endothelial response to chronically elevated hydrostatic pressure, as is seen for instance in congestive heart failure (CHF), is a distinct form that is seen during acutely elevated pressure. The lung is thought to adapt to chronic pressure stress, becoming less permeable as a result of this physiological change (Ivey et al. 1998; Kerem et al. 2010; Yin et al. 2008). This occurs via changes in the structure of the alveolocapillary barrier (Huang et al. 2001; Yin and Kuebler 2010), improved alveolar fluid absorption (Kaestle et al. 2007; Parker and Ivey 1997), among others. Data regarding the role of TRPV4 expression and function in CHF, and in the adaptations detailed above, are contradictory. Kerem et al. reported decreased TRPV4 expression in CHF rats, along with a decrease in mean intracellular Ca2+ concentrations (Kerem et al.

2010; Tiruppathi et al. 2006). In CHF rats, TRP channel activation failed to produce an endothelial Ca2+ response, which was attributed to TRPV4 downregulation (Kerem et al. 2010; Sandoval et al. 2001). However, using immunolabeling of human lungs, Thornloe et al. demonstrated enhanced TRPV4 expression in CHF patients as compared to controls (Parker et al. 1998; Thorneloe et al. 2012). In both acute and chronic CHF animal models, GSK2198374 was protective against edema formation, although TRPV4 expression was not examined (Thorneloe et al. 2012). Whether these observed differences with respect to TRPV4 expression and function in lungs of subjects with CHF is species-specific, or due to another phenomenon, remains unclear at this time. Pulmonary vascular contractility and pulmonary hypertension Pulmonary vascular tone is maintained via the relaxation and contraction of the pulmonary artery smooth muscle cell (PASMC). Increased tone in the pulmonary vasculature— which, at baseline, is in most species maximally dilated (Hampl and Herget 2000)—leads to elevated pulmonary arterial pressure, and—if persistent over time—to pulmonary hypertension, which is defined as a mean pulmonary arterial pressure >25 mmHg at rest (Simonneau et al. 2013). The local interaction between the endothelium and the vascular smooth muscle is a major determinant of myogenic tone. Vasoactive molecules produced by the endothelium, including nitric oxide, endothelin-1, and prostaglandins, act upon the PASMC to modulate pulmonary vascular resistance (Barnes and Liu 1995). Regulation of pulmonary vascular tone is largely Ca 2+ -dependent; Ca 2+ influx into the sarcolemma of PASMC results in myosin light chain kinase activation, phosphorylation of the myosin light chain, and subsequent smooth muscle contraction (Sylvester et al. 2012). Thus, there is a potential role for TRPV4, as a Ca2+ permeable channel located both at the endothelial and smooth muscle membranes in the contractile response of pulmonary vessels. Indeed, Ca2+ influx through TRPV4 on the PASMC membrane has been shown to be a critical component of the response of the pulmonary vasculature to agonist-induced vasoconstriction. Inhibition of TRPV4 using HC-067047 resulted in a decrease in the maximal response to serotonin in murine pulmonary arteries, and a rightward shift in the concentration-response curve, that was similarly observed in PAs from TRPV4 knockout mice (Xia et al. 2013). Furthermore, HC-067047 reduced the peak Ca2+ response to serotonin in PASMCs from wild-type mice, an effect that was mirrored in PASMC isolated from TRPV4 knockout animals (Xia et al. 2013). TRPV4 has additionally been implicated in the phenomenon of hypoxic pulmonary vasoconstriction, whereby pulmonary arterioles constrict in response to acute hypoxia in order to divert pulmonary blood flow toward better-ventilated lung regions, therefore optimizing ventilation–perfusion matching.


Pharmacologically inhibiting TRPV4 using HC-067047 attenuated the hypoxic response both in isolated mouse lungs and in vivo; furthermore, TRPV4 knockout mice also showed a reduced HPV response (Goldenberg et al. 2014). TRPV4 inhibition also resulted in a marked decrease in the peak intracellular Ca2+ concentration in response to hypoxia in cultured human PASMC (Goldenberg et al. 2014). While a precise mechanism for this effect has yet to be demonstrated, several possibilities exist. One such model for the role of TRPV4 in acute hypoxia would be that EETs—which have been shown to be increased during acute hypoxia (Keserü et al. 2008; Wang et al. 2012)—activate TRPV4. Additionally, there is a possibility for the functional cooperation of TRPV4 and TRPC6 in HPV. TRPC6 is also required for HPV (Tang et al. 2010; Urban et al. 2012), and both TRPV4 and TRPC6 have been shown to localize to caveolae in the plasma membrane. Additionally, TRPV4 and TRPC6 display synergy during pathological nociception (AlessandriHaber et al. 2009), providing evidence that such a functional coupling can exist in another system. Such interactions between TRPV4 and other TRP channels during hypoxia form the basis for ongoing research in this field. While HPV represents an acute response to hypoxia, chronic exposure to hypoxic conditions can lead to the development of pulmonary hypertension. Chronic hypoxia results in the upregulation of TRPV4 in rat pulmonary arteries, and TRPV4 knockout mice develop less severe hypoxia-induced pulmonary hypertension than controls (Xia et al. 2013). TRPV4 was also shown to be upregulated in rat PAs chronically exposed to hypoxia, and was additionally involved in the associated increase in myogenic tone (Yang et al. 2012). Hypoxic PASMCs had elevated levels of basal intracellular Ca2+, which was reduced by pretreatment with ruthenium red (Yang et al. 2012). Moreover, TRPV4 knockout mice were resistant to the development of chronic hypoxia-induced pulmonary hypertension (Goldenberg et al. 2014; Xia et al. 2013). Therefore, TRPV4 is intimately involved in the response of the pulmonary vasculature to both acute and chronic hypoxia, and use of TRPV4 inhibitors may allow clinicians to modify this response in the face of various disease states. However, the role of TRPV4 in vascular beds outside of the lung cannot be ignored when discussing the potential therapeutic use of TRPV4 inhibitors in human subjects. As is the case with many vasoactive mediators (such as serotonin and hypoxia), TRPV4 exerts opposite effects upon the pulmonary and systemic vasculature. In both renal and mesenteric vessels, stimulation of TRPV4 results in vasodilation (Chen et al. 2014). In such systemic vessels, TRPV4 stimulates Ca2+dependent activation of eNOS, resulting in vasodilation (Adapala et al. 2011; Willette et al. 2008). In contrast, in the lung, TRPV4 appears to acts primarily at the level of the smooth muscle cell. While the precise mechanism for the differential effect of TRPV4 in the lung and systemic

circulation remains unclear—and could involve varying TRPV4 expression levels, eNOS activity, or other factors— this holistic view of TRPV4 function must be taken when preparing to provide TRPV4 inhibition to human patients. Acute lung injury Acute respiratory distress syndrome (ARDS) is defined as acute hypoxic respiratory failure, bilateral pulmonary infiltrates on chest radiography, and pulmonary edema that is not due to fluid overload or a cardiac cause (ARDS Definition Task Force et al. 2012). ARDS is a severe manifestation of acute lung injury (ALI); while this term is outdated in humans, it remains useful in describing a pathological entity in animal models. ARDS can arise as a result of a host of insults, including those originating in the lung (i.e., pneumonia), or systemic inflammation (i.e., sepsis or severe trauma). Additionally, ARDS can be the direct result of acid inhalation following aspiration of gastric contents, or of positive pressure ventilation with high peak airway pressures. Ventilatorinduced lung injury (VILI), as with the other noted causes of ALI/ARDS, remains an important entity during the care of critically ill patients, and is a large contributor to their mortality (ARDSNet 2000). The hallmark of ARDS is disruption of both the epithelial and endothelial barriers, leading to proteinrich alveolar edema fluid, as well as a massive immune reaction characterized by pulmonary cytokine secretion, and granulocyte infiltration (Ware and Matthay 2000). As such, mediators of this process reside in both the lung and in immune cells. As a known sensor of mechanical stimuli, TRPV4 was hypothesized to play a role in the pathogenesis of ARDS and VILI. Indeed, TRPV4, acting in both the lung and in the cells of the innate immune system, has been shown to be a key mediator of ALI/ARDS (Fig. 5). Activated in response to both highway ventilator pressures and elevated temperature, TRPV4 is involved in the formation of pulmonary edema during overventilation both in vitro (Hamanaka et al. 2007) and in vivo (Michalick et al. 2013). Inhibition of TRPV4 with ruthenium red, or genetic deletion of TRPV4, protected isolated mouse lungs from edema formation in response to high ventilation pressures, as seen by Kf measurements and analysis of lung wet-to-dry ratios (Hamanaka et al. 2007). Additionally, inhibition of mediators known to activate TRPV4—including EETs and anandamide—also protected lungs from elevations in Kf in response to high airway pressure. Overventilation also stimulated an increase in cytosolic Ca2+ in the alveolar capillary endothelium, which was not seen in lungs isolated from TRPV4 knockout mice, or in lungs treated with ruthenium red (Hamanaka et al. 2007). Similarly, TRPV4 inhibition by HC-067047 or genetic TRPV4 deficiency protected mice from edema formation, protein extravasation, cytokine release, and histological signs of lung injury in a murine model of 2-h overventilation

Naunyn-Schmiedeberg's Arch Pharmacol Fig. 5 TRPV4 in acute lung injury. TRPV4 is expressed in alveolar epithelial cells (green), pulmonary microvascular endothelial cells (pink), and neutrophils and macrophages. During acute lung injury, TRPV4mediated calcium influx into endothelial cell results in cell contraction and barrier dysfunction. Concurrently, transmigration of neutrophils and macrophages into the airspaces occurs, as does the formation of pulmonary edema. In the inflammatory cells, TRPV4 activation results in enhanced migration and production of reactive oxygen species (ROS). Loss of TRPV4 from any of these cell types results in a milder ALI phenotype. See text for details

with tidal volumes of 20 mL/kg bw (Michalick et al. 2013). Furthermore, activation of TRPV4 with 4α-PDD resulted in structural disruption of the alveolar septal barrier (Alvarez et al. 2006). Taken together, these results demonstrate a pivotal role for TRPV4 in the pathogenesis of a devastating clinical entity, and set the stage for the potential of TRPV4 inhibition in the treatment or prevention of ARDS in response to positive pressure ventilation. Further evidence for the importance of pulmonary TRPV4 in ALI was recently provided by a series of studies of acid inhalation and chlorine gas-induced ALI (Balakrishna et al. 2014). Importantly, these insults are both chemical stimuli for ALI, as opposed to the physical stimuli detailed above. The TRPV4 inhibitor tested in these studies, GSK2220691, was able to block both the increase in pulmonary edema and the decrease in systemic blood pressure seen in mice pretreated with the TRPV4 agonist, GSK1016790 (Balakrishna et al. 2014). These results indicate that this novel inhibitor is capable of blocking TRPV4 activity in both the pulmonary and systemic circulations. Following acid inhalation, an intraperitoneal injection of GSK2220691 decreased lung inflammation, as indicated by lower levels of neutrophils and macrophages in bronchoalveolar lavage fluid (BALF). A similar effect was observed after acid inhalation in TRPV4 knockout mice (Balakrishna et al. 2014). Both TRPV4 knockout and treatment with the inhibitor resulted in improvements in histological lung injury scores as well. TRPV4 inhibition or knockdown also rescued the deleterious effects of acid exposure or chlorine inhalation on lung

mechanics, resulting in improved elastance and oxygenation as compared to controls. Importantly, these effects were all seen when the TRPV4 inhibitor was administered after induction of lung injury, suggesting that TRPV4 inhibition could be a viable therapeutic target for the treatment of chemical lung injury (Balakrishna et al. 2014). The relevant source of TRPV4 in ALI remains unclear, but may involve contributions from both pulmonary and extrapulmonary (i.e., inflammatory and immune) sources. ARDS is often the pulmonary manifestation of a syndrome of systemic inflammation. As such, it is clear that inflammatory cells have a central role in this entity. Since TRPV4 is expressed in several immune lineages (Damann et al. 2009; Spinsanti et al. 2008), researchers have also probed the role of TRPV4 in the cells of the innate immune system during ARDS. In a series of studies in isolated perfused mouse lungs, TRPV4 knockout resulted in decreased sensitivity to VILI (Hamanaka et al. 2010). Surprisingly, perfusion of knockout lungs with macrophages isolated from wild-type mice resensitized TRPV4 knockout lungs to VILI. These data established a role for macrophage TRPV4 in VILI for the first time. Phenotypically, macrophages isolated from wild-type mice exhibited an increase in intracellular Ca2+ in response to 4α-PDD that was not seen in knockout cells (Hamanaka et al. 2010). Additionally, wild-type macrophages produced reactive oxygen species when stimulated by 4α-PDD, and this effect was not seen in wild-type cells. Taken together, these data show that extrapulmonary TRPV4 plays an important role in VILI; specifically, TRPV4 is required in the


macrophage for normal calcium signaling and cell spreading and morphology, and TRPV4 function in the macrophage is a necessary component of VILI. Furthermore, TRPV4 is expressed in human macrophages, suggesting this concept may be translatable to human subjects (Thorneloe et al. 2012). To add further support for the role of extrapulmonary TRPV4 in lung injury, recent work from our group has demonstrated that neutrophil TRPV4 is also a key component of the pathway to ARDS (Tang et al. 2013). Following induction of ALI with either acid instillation or administration of bacterial lipopolysaccharide (LPS), TRPV4 knockout mice had increased survival as compared to wild-type, and developed less severe lung injury as assessed by edema formation, protein leakage, cytokine production, impaired gas exchange and respiratory mechanics, and histological signs of tissue injury. Additionally, wild-type lungs were more sensitive to edema formation in response to platelet activating factor (PAF) than control lungs. Strikingly, perfusion of knockout lungs with wild-type blood restored the effects of PAF on pulmonary edema (Tang et al. 2013), indicating that a key role of TRPV4 in ALI relates to its expression on circulating immune cells rather than in the lung parenchyma. As was found with macrophages, neutrophils lacking TRPV4 had significant phenotypic differences from controls. Neutrophils isolated from knockout mice had a dysfunctional Ca2+ response to both PAF and EETs, and displayed less ROS generation in response to stimulation as well. Furthermore, neutrophils lacking TRPV4 had deficiencies in transmigration and in Rac activation assays. Clearly, TRPV4 has an important role in immune cells, and this effect on innate immunity is required for the normal development of ALI in response to chemical, physical, and infectious stimuli. The notion of a more generalized role of TRPV4 in inflammatory disease processes is further substantiated by the recent reports that TRPV4 blockade can protect against experimental colitis in mice (Fichna et al. 2012) while conversely, TRPV4 agonists trigger joint inflammation in rats (Denadai-Souza et al. 2012). These data present crucial understanding of a complex set of disease entities, and may also provide insight into potential offtarget effects from the clinical delivery of TRPV4 inhibitors.

Considerations for TRPV4 inhibition in clinical use The clinical use of TRPV4 inhibitors may provide exciting new treatments for a host of devastating diseases, including hypoxic lung diseases, ALI/ARDS, CHF, and pulmonary hypertension. Additionally, TRPV4 has been shown to be involved in airway smooth muscle cell proliferation (Zhao et al. 2014). Importantly, TRPV4 activation produced smooth muscle contraction, which could be blocked by 5lipoxygenase inhibition (McAlexander et al. 2014).

Intriguingly, activation of TRPV4 did not directly induce airway smooth muscle cell contraction; this phenomenon is indirect, and mediated through leukotrienes. These data suggest a potential role for TRPV4 blockade in the treatment of asthma and other reactive airway diseases. Furthermore, a genetic screen of families susceptible to the development of COPD has also identified single nucleotide polymorphisms in the TRPV4 locus that are associated with the severity of this devastating condition (Zhu et al. 2009). Clearly, TRPV4 manipulation may hold real promise as a treatment for a variety of disease states. Indeed, a safety trial for the inhibitor, GSK2798745, in healthy controls and patients with stable heart failure is currently underway (NCT02119260). A further study that will begin recruitment soon seeks to use MRI to evaluate vascular permeability and lung water in healthy controls and patients with heart failure (NCT02135861). These trials represent the forefront of clinical inquiry into the role of TRPV4 in humans, and its suitability as a therapeutic target in respiratory disease. While this work is rightfully exciting, several important safety concerns remain untested, especially given the multiplicity of TRPV4 effects and its widespread expression throughout the body. Additionally, many of these effects potentially represent double-edged swords in clinical practice. For instance, the edemapreventing effect of TRPV4 inhibition makes TRPV4 inhibition a perspective treatment for cardiogenic pulmonary edema. However, TRPV4 is also required for the appropriate lung vascular response to acute (Goldenberg et al. 2014) and chronic (Yang et al. 2012) hypoxia. The acute response, namely hypoxic pulmonary vasoconstriction, is fundamentally important for ventilation–perfusion matching in the face of regional alveolar hypoxia, as would be expected during cardiogenic pulmonary edema. Furthermore, a great deal of experimental evidence exists showing that TRPV4 has an important role in the maintenance of systemic vascular tone, and that modifying TRPV4 activity can have profound effects on systemic blood pressure. While it is encouraging that TRPV4 blockade in mice did not cause hypotension (Thorneloe et al. 2012), potentially due to activation of compensatory mechanisms of vascular tonus regulation, it remains to be seen whether TRPV4 inhibition may interfere with systematic hemodynamics in human subjects at rest, during exercise, and in cardiovascular disease, respectively. At the very least, this consideration could limit the usefulness of TRPV4 blockade to patients with stable, compensated heart failure, who would not be expected to suffer from oxygenation deficits. TRPV4 is also highly expressed in the kidney, with immunoreactivity demonstrated along most of the renal vasculature, as well as in the collecting system (Chen et al. 2014; Pochynyuk et al. 2013). In the kidney, TRPV4 plays a critical role in osmosensation and in the cellular response to fluid flow and shear stress (Pochynyuk et al. 2013). Developmentally,


TRPV4 is involved in the pathogenesis of polycystic kidney disease (Zaika et al. 2013). Due to these important renal functions of TRPV4, vigilance for off-target effects of TRPV4 inhibition in the kidney during any early clinical study would be prudent. While it is difficult to predict the net renal effect of TRPV4 inhibition in a human subject, the possibility does exist for the development of electrolyte disturbances or hemodynamic abnormalities when TRPV4 inhibitors are administered systemically. However, in a more positive light, the expression and functional importance of TRPV4 in the kidney could lead to other exciting therapeutic avenues for TRPV4 inhibition. Such possibilities are, however, beyond the scope of this review. Similarly, the utility of TRPV4 antagonists in the treatment of ARDS will require a balancing act between the pulmonary and extrapulmonary effects of TRPV4. In frank ARDS, the barrier-stabilizing effect of TRPV4 blockade, coupled with its anti-inflammatory properties, may represent a much-needed “two-pronged attack” against this complicated disease state. By targeting both the lung and the leukocyte, perhaps TRPV4 inhibition will yield better results than previous experimental agents. Furthermore, by preventing the development of ARDS, TRPV4 blockade can also be predicted to decrease subsequent rates of lung fibrosis, patient morbidity, and hospital length of stay. However, in specific conditions leading to ARDS, the potential effects of TRPV4 antagonism on innate immunity may be undesirable. For instance, in the early stages of sepsis, a therapy that blocks macrophage migration and ROS production, and also inhibits acute inflammation by neutrophils, may be detrimental. However, in a case of hyperactive immunity, this effect may be a benefit. Again, only further trial will reveal the true results, and careful stratification of patients may be required to differentiate those who may profit from TRPV4 inhibition from those where a similar intervention may potentially have detrimental effect. Acknowledgments NMG is supported by The PSI Foundation and The Canadian Anesthesiologists’ Society. WMK is supported by operating grants from the Deutsche Forschungsgemeinschaft (DFG), the Canadian Institutes of Health Research (CIHR), and the Heart & Stroke Foundation Canada.

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