Introduction

TRPV4 is a polymodally gated nonselective cation channel [1]. Channel activation leads to permeation of both Ca^2 + and Na⁺, with a permeability ratio of 6.3 [2]. Channel gating can be elicited by mechanical and shear stress, as well as cell swelling [3–6], all of which appear to be secondary to stress-induced activation of phospholipase A₂ (PLA₂) and subsequent synthesis of epoxyeicosatrienoic acids (EETs) [1,2,7,8]. Notably, deformation of cell-detached patches does not activate the channel [2]. Similarly, TRPV4 activation with endocannabinoids appears to be secondary to local hydrolysis of endocannabinoids to arachidonic acid and subsequent EET synthesis [9]. Synthetic phorbol esters such as 4α-phorbol-12,13-didecanoate (4αPDD) [10], small molecule activators GSK1016790A or GSK634775 [11,12], and heat [3] appear to act directly on channel proteins. Thus, there is significant potential for involvement of TRPV4 in lung disease through diverse activation pathways.

TRPV4 Localization in Lungs

Using immunohistochemistry and Western blotting, we and others have documented expression patterns for TRPV4 protein in lung tissue and cells. TRPV4 is expressed in the alveolar septal wall of human, rat, and mouse lung, whereas expression in endothelium of extra-alveolar vessels is sporadic [11–13]. Immunostaining for TRPV4 in human, rat, and mouse lung is shown in Figure 13.1. In vitro, TRPV4 is also expressed in cultured rat lung endothelial cells [13,14]. Pulmonary vascular and airway smooth muscle also expresses TRPV4 in vivo and in vitro [13]. Vascular smooth muscle immunoreactivity for TRPV4 is particularly evident in human and rat lung (see Figure 13.1). TRPV4 is also expressed in airway epithelium and alveolar macrophages [13,15,16]. Though TRPV4 expression has been documented in sensory nerves from skin and colon [17,18], expression in airway sensory nerves has not been directly confirmed. Nonetheless, preliminary functional data supports a role for TRPV4 in Aδ nociceptive afferent fibers from mouse, guinea pig, and human airways [19].

Acute Lung Injury via Direct Channel Activation

Direct activation of TRPV4 with the phorbol ester 4α-phorbol-12,13-didecanoate (4αPDD) or the small molecule agonist GSK1016790A increases the filtration coefficient (K_f) in isolated lungs, associated with disruption of the alveolar septal barrier, alveolar flooding, and lung edema [11–13,20]. The dose-dependent increase in K_f after treatment of isolated rat lung with 4αPDD is shown in Figure 13.1. K_f is a specific and sensitive measure of endothelial permeability, applicable to assessment of lung injury in any isolated mammalian lung [21,22]. The caveat to interpretation of K_f is that it measures the aggregate permeability of the entire perfused surface area in lungs. Interestingly, the TRPV4-mediated acute lung injury is localized to the alveolar septal compartment, with little evidence of injury to endothelium in extra-alveolar vessels. Disruption of the alveolar barrier appears to be due to cell detachment, blebbing, and/or cell swelling rather than interendothelial cell gap formation [13,20]. Similarly, Willette and colleagues have reported that TRPV4 activation leads to detachment of endothelial cells in culture [12]. These perturbations in lungs elicited by TRPV4 activation are dependent on TRPV4-mediated calcium entry, as they are mitigated by perfusion with a low Ca^2 + buffer, pretreatment of lungs with the TRPV4 antagonists, or use of lungs from Trpv4^−/− mice [11,13]. Although the impact of TRPV4 activation on barrier function in lungs is clear, the downstream mechanisms linking TRPV4 activation and endothelial barrier disruption in the lungs needs to be further resolved. In airway epithelium, TRPV4 activation leads to release of the pro-inflammatory matrix metalloproteinase 1 (MMP1) [15]. Our own recent data suggests that TRPV4 activation initiates a signaling cascade leading to increased availability of active MMP2 and MMP9 in mouse lungs [23], which may contribute to matrix degradation and cell detachment.

Ventilator-Induced Lung Injury (VILI)

Although positive pressure mechanical ventilation is a lifesaving intervention in the setting of adult respiratory distress syndrome (ARDS) and acute lung injury (ALI), mechanical ventilation with excessive tidal volumes actually contributes to ventilator-induced lung injury (VILI) and increases mortality. Specifically, a large-scale clinical trial showed that decreasing the tidal volume from 12 mL/kg to 6 mL/kg reduced mortality by 22% [24]. The severity of VILI has been generally recognized to be both time and pressure (or volume) dependent in experimental studies as well as in clinical settings [25–28]. The syndrome includes a rapid increase in vascular permeability followed by cytokine release and inflammatory cell infiltrates [25,26,28]. The rapid onset of the increase in vascular permeability, compared to the slower increase in cytokine levels and mobilization of inflammatory cell infiltrates some hours later, suggests a rapid intracellular signaling cascade of events followed by recruitment of inflammatory cells, which may then amplify the lung injury [29–31].

An increase in endothelial cell intracellular Ca^2 + is a necessary component for increased vascular permeability induced by most mediators, and the permeability response to lung overdistention appears to be no exception [32–34]. Ca^2 + entry occurs within seconds after mechanical perturbation and thus is one of the most rapid responses to mechanical strain in both isolated lungs and cultured cell preparations [35–37]. Early studies by Parker and colleagues on VILI in isolated rat lungs indicated that high peak inflation pressure (PIP) increases lung vascular permeability for the total lung, as well as individual arterial, venular, and capillary vascular segments, and these increases were attenuated by gadolinium, an inhibitor of stretch-activated cation channels [33,38].

The identity of these stretch-activated cation channels involved in initiating VILI was established by Hamanaka et al. [39], who measured K_f in isolated lungs of wild-type (WT) and Trpv4^−/− mice ventilated with high and low PIP. High PIP ventilation increased K_f in lungs of WT mice at 35 °C, an effect significantly augmented by increasing the perfusate temperature to 40 °C. The increase in K_f induced by high PIP in WT lungs was abolished by the TRPV antagonist ruthenium red and absent in lungs from Trpv4^−/− mice at both 35 °C and 40 °C (Figure 13.2). In parallel studies, Hamanaka and colleagues showed that intracellular Ca^2 + transients elicited in intact lungs during high PIP were abolished in the TRPV4 knockout (KO) lungs and ruthenium red treated lungs. Further, a morphometric evaluation of edema distribution indicated significant alveolar flooding in WT lungs compared to lungs from Trpv4^−/− mice.

The mechanism linking mechanical strain in VILI to activation of TRPV4 involves synthesis of arachidonic acid epoxygenase metabolites. In one of the first publications on TRPV4 in endothelial cells, Watanabe et al. reported that EETs derived from metabolism of arachidonic acid by P450 epoxygenases elicited TRPV4-mediated Ca^2 + entry [9]. Alvarez et al. subsequently reported that EETs increased K_f in rat lungs [40,41]. As hypotonicity-induced cell swelling, a form of stretch, activates TRPV4 in a mechanism involving EET synthesis [7,8], we considered such a link in VILI (and in high vascular pressure-induced lung injury). Hamanaka et al. found that methanandamide (a competitive inhibitor of anandamide-derived arachidonic acid synthesis) and miconazole (a P450 inhibitor) abolished the increase in K_f induced by high PIP ventilation [39]. These findings were consistent with earlier work from the Parker laboratory that implicated PLA₂ activation and subsequent synthesis of arachidonic acid metabolites in initiating the acute pulmonary vascular permeability increase in response to high PIP ventilation. Specifically, Yoshikawa et al. [42] showed that mice deficient in Clara cell secretory protein (CCSP), an inhibitor of cytosolic PLA₂ activity, had an increased susceptibility to acute VILI, whereas inhibition of PLA₂ with arachidonyl trifluoromethyl ketone attenuated the lung vascular permeability increases and edema in both CCSP^−/− and WT mice after 2 and 4 h of high PIP ventilation. Similarly, Miyahara et al. [43] observed that either a cytosolic PLA₂ inhibitor or a combination of cyclooxygenase, lipoxygenase, and P450 epoxygenase inhibitors prevented VILI-induced increase in K_f in isolated mouse lungs.

Although these findings might be interpreted as a straightforward impact of stretch on lung endothelium, leading to EET synthesis and TRPV4 activation, other work suggests that the mechanism of VILI is more complex. Macrophages have been proposed as the major initiators of lung injury during mechanical ventilation because these cells produce copious amounts of pro-inflammatory cytokines during in vitro cyclical stretch compared to other lung cell types [44,45]. Further, TRPV4 agonists elicit Ca^2 + entry and activate both human and murine macrophages [46,47]. More recently, macrophage depletion studies indicated protection against the lung permeability increase elicited by injurious ventilation [48,49]. The pattern of injury progression in VILI appears similar to that for other insults such as ischemia-reperfusion injury or sepsis, in that the early phase of injury is coordinated by lung macrophages, whereas the late phase of tissue injury is neutrophil dependent [50–52]. Both VILI and LPS-induced lung injury are attenuated by depletion of alveolar macrophages [39,48,49]. Alveolar macrophages activated by stretch secrete reactive oxygen species and platelet activating factor, which then activates endothelial NADPH oxidases [49,52,53]. Hamanaka et al. showed that VILI was blocked (Figure 13.3) by genetic deletion of TRPV4 and restored by instilling TRPV4-competent alveolar macrophages [46]. They also observed that 4αPDD-induced TRPV4 activation in mouse alveolar macrophages increased intracellular calcium, mitochondrial superoxide, and nitric oxide production and cell spreading on a glass surface (Figure 13.4), responses that were absent in macrophages lacking TRPV4. Further, the addition of WT macrophages restored lung nitrotyrosine staining in Trpv4^−/− mice to the high level observed in WT lungs following high-pressure ventilation [46]. Collectively, these results are indicative of peroxynitrite formation with VILI due to production of excess superoxide and nitric oxide production [54]. The link between stress transmission and TRPV4 channel gating in macrophages may be direct. Alveolar macrophages are rapidly activated by high-volume and pressure ventilation and become firmly adherent within minutes after initiation of ventilation [48,49]. Although stretch-activated calcium transients in endothelial cells appear to be mediated by β₁ integrins [35,55], macrophage adhesion and migration are dependent on β₂ and β₃ integrins [56,57]. Thus, direct cell membrane distortion due to surface tension-dependent compression of macrophages likely leads to their activation and adherence to the alveolar surface at high lung distending pressures.

Aside from clinical trials that definitively identified that low-volume ventilation reduced mortality, there has been little progress toward development of therapeutic interventions targeting TRPV4 in VILI. However, Jurek et al. recently proposed an approach with promise for clinical applicability, utilizing an intratracheal aerosol of ruthenium red-coated nanoparticles to target the critical macrophage TRPV4 in VILI [58]. Alveolar macrophages rapidly phagocytosed the nanoparticles, and a significant lung ruthenium red content was attained within minutes. Importantly, inhaled ruthenium red nanoparticles blocked the increase in lung microvascular permeability caused by high airway pressure mechanical ventilation. In the intact lung, treatment with inhaled ruthenium red nanoparticles was effective in blocking the K_f response to high-pressure mechanical ventilation for up to 3 days (Figure 13.5). Ruthenium red nanoparticles blocked calcium transients induced by 4αPDD in both alveolar macrophages and capillary endothelial cells, but they did not affect endothelial calcium transients due to ATP-dependent store depletion [58]. Development of such therapeutic strategies is important due not only to the impact of VILI itself, but also potentially due to exacerbation of underlying injury by mechanical ventilation and resultant multiple organ failure [59]. Huh and colleagues demonstrated that mechanical stretch dramatically amplifies interleukin-2 mediated injury to alveolar epithelium and endothelium in a lung-on-a-chip pulmonary edema model, effects completely abrogated by coadministration of a TRPV4 inhibitor [60].