Published ahead of print on October 25, 2007, doi:10.1165/rcmb.2007-0192OC
© 2008 American Thoracic Society DOI: 10.1165/rcmb.2007-0192OC High Vascular Pressure–Induced Lung Injury Requires P450 Epoxygenase–Dependent Activation of TRPV4Departments of 1 Physiology, 2 Pharmacology, and 3 Pathology, and 4 the Center for Lung Biology, University of South Alabama, Mobile, Alabama; and 5 Departments of Medicine, Neurology, and Neurobiology, Duke University, Durham, North Carolina Correspondence and requests for reprints should be addressed to Mary I. Townsley, Ph.D., Department of Physiology; MSB 3074, University of South Alabama, 307 University Blvd., Mobile, AL 36688. E-mail: mtownsley{at}usouthal.edu
High vascular pressure targets the lung septal network, causing acute lung injury. While calcium entry in septal endothelium has been implicated, the channel involved is not known. This study tested the hypothesis that the vanilloid transient receptor potential channel, TRPV4, is a critical participant in the permeability response to high vascular pressure. Isolated lungs from TRPV4+/+ or TRPV4–/– mice were studied at baseline or during high pressure challenge. Permeability was assessed via the filtration coefficient. Endothelial calcium transients were assessed using epifluorescence microscopy of the lung subpleural network. Light microscopy and point counting were used to determine the alveolar fluid volume fraction, a measure of alveolar flooding. Baseline permeability, calcium intensity, and alveolar flooding were no different in TRPV4+/+ versus TRPV4–/– lungs. In TRPV4+/+ lungs, the high pressure–induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A2 inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure–induced calcium transient in TRPV4+/+ lungs was attenuated with ruthenium red or the epoxygenase inhibitor. High vascular pressure increased the alveolar fluid volume fraction compared with control. In lungs from TRPV4–/– mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic acid–dependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.
Key Words: epoxyeicosatrienoic acid • capillary permeability • respiratory distress syndrome • TRPV cation channels
In lung, high vascular pressure (HiPv) exceeding a threshold of 30 to 50 cm H2O increases endothelial permeability (1–4). While mechanical stress failure of the alveolar septal barrier can occur at higher pressures, leading to overt alveolar flooding (5), the molecular mechanisms underlying the early HiPv-induced increase in endothelial permeability are not well understood. Kuebler and colleagues (6) have reported that moderate HiPv promotes Ca2+ entry into lung endothelium, and acute lung injury is often dependent upon such Ca2+ transients (7–10). Although HiPv-induced lung injury could plausibly be dependent upon Ca2+ entry, this has not been experimentally confirmed nor has a candidate channel been identified.
The Ca2+ permeable channel TRPV4, a member of the vanilloid subfamily of transient receptor potential (TRP) channels, is expressed in the alveolar septal compartment (8). The notion that TRPV4 might subserve HiPv-induced Ca2+ entry in lung endothelium is based on the observation that the channel can be activated by mechanical stress, such as hypotonic cell swelling or shear stress (11–13). In vitro studies have shown that activation of TRPV4 with mechanical stress requires hydrolysis of membrane phospholipids via phospholipase A2 (PLA2) and subsequent arachidonic acid metabolism by cytochrome P450 epoxygenases to form epoxyeicosatrienoic acids or EETs (12, 14, 15). TRPV4 heterologously expressed in HEK-293 cells or the endogenous TRPV4 in aortic endothelium can be activated by EETs, promoting Ca2+ entry (16). Further, we have shown that EETs or direct activation of TRPV4 with 4 Based on these observations, this study was devised to test the hypothesis that HiPv increases lung endothelial permeability by promoting Ca2+ entry via TRPV4 and that this mechanically induced injury process required EET synthesis. To test this hypothesis, lung endothelial permeability, Ca2+ transients in subpleural septal endothelium, and alveolar flooding were assessed in wild-type mice and TRPV4–/– littermates.
Animals Male or female wild-type (TRPV4+/+) and null (TRPV4–/–) littermate mice were used at 8 to 10 weeks of age (17). Animal protocols were approved by the Institutional Animal Care and Use Committee of the University of South Alabama, adhering to the NIH guidelines for the care and use of laboratory animals.
Isolated Lung Preparation
HiPv-Induced Ca2+ Response in Subpleural Microvessels
Morphometric Analysis of Alveolar Flooding by Light Microscopy
Statistics Additional details regarding materials and methods are available in the online supplement.
Pulmonary hemodynamics, including total pulmonary vascular resistance, were similar in lungs from TRPV4+/+ and TRPV4–/– mice (Table 1). Further, these measures were not altered by the choice of perfusate [Ca2+]. There were no differences with respect to baseline Kf between TRPV4+/+ or TRPV4–/– mice. Lowering perfusate temperature to 26°C affected neither the baseline endothelial permeability nor hemodynamics.
We first needed to document the pressure threshold for the HiPv-induced increase in endothelial permeability in mouse lung. Results are shown in Figure 1. At a perfusate temperature of 26°C, Kf remained near baseline until lungs were challenged with Pv exceeding 30 cm H2O. Above this threshold, Kf increased in a pressure-dependent fashion. Although there were no significant differences in baseline Kf as a function of perfusate temperature, increasing the temperature to 33°C resulted in a left-shift in the pressure–response curve. Since at 33°C, Pv ranging from 28 to 34 cm H2O reliably increased Kf, we used Pv in this range as the test HiPv and a perfusate temperature of 33°C for the remaining experiments.
Figure 2 summarizes the results for paired baseline and HiPv Kf measurements in wild-type lungs. Kf increased 5.3-fold with HiPv in this group (P < 0.05). To determine a role for extracellular Ca2+ in this response, we used a low Ca2+/Ca2+ addback paradigm (7, 8). There was no difference in the baseline Kf when lungs were perfused with normal Ca2+ or low Ca2+ buffer. However, the HiPv-induced increase in Kf was prevented in low Ca2+ (0.02 mM) perfusate. The addition of Ca2+ to achieve a physiologic concentration of 2.2 mM was required to restore the HiPv-induced increase in Kf (P < 0.01 versus baseline and low Ca2+). In separate lungs from wild-type mice, blockade of TRPV channels with ruthenium red significantly attenuated the permeability response to HiPv (P < 0.001). Further, specific knockout of TRPV4 also blunted the permeability response to HiPv (P < 0.001). These results support the notion that Ca2+ entry via TRPV4 plays an important role in HiPv-induced increase in lung endothelial permeability.
To evaluate the potential involvement of the P450-mediated arachidonic acid metabolism in the HiPv-induced permeability response, we used pharmacologic inhibitors to target several steps in this cascade. As shown in Figure 3, the HiPv-induced increase in Kf in wild-type lungs (repeated from Figure 2 for comparison) was significantly attenuated by pretreatment with PLA2 inhibitor MAFP (P < 0.001) or the P450 epoxygenase inhibitor PPOH (P < 0.001). These results provide support for the notion that P450 epoxygenase metabolites are involved in the permeability response to HiPv.
To verify that HiPv produced significant Ca2+ entry in lung subpleural microvascular endothelium, we turned to fluorescence microscopy. A representative image of a subpleural microvascular network from a wild-type lung and an area chosen for assessment (boxed) are shown in Figure 4A. Serial images of an assessment area illustrating the impact of HiPv in lung from wild-type and knockout mice are shown in Figure 4B. The Ca2+ response to HiPv, as determined by Fluo-4 fluorescence intensity, was reproducible in wild-type lung (Figure 4C). As shown in Figure 4D, there was no significant difference in fluorescence intensity at baseline among the five experimental groups studied. HiPv elicited a Ca2+ response in the lungs from wild-type mouse, an effect which was significantly attenuated by pretreatment with the P450 epoxygenase inhibitor PPOH or the TRPV channel blocker ruthenium red. Similarly, the response was attenuated in TRPV4–/– mice. Though there was a tendency for Ca2+ intensity to increase in these latter groups, HiPv-induced recruitment of Ca2+ entry pathways other than TRPV4 was considered unlikely, since the response to HiPv remained the same in knockout lungs pretreated with the nonspecific Ca2+ entry blocker GdCl3.
These data support the notion that the HiPv-induced permeability response is related to TRPV4. However, an increase in Kf does not elucidate the compartment of the lung targeted by the injury (8). Light microscopy (Figure 5) showed that HiPv caused alveolar flooding in lungs from wild-type mice, compared with that in wild-type or TRPV4–/– controls or in the TRPV4–/– HiPv group. To quantify the degree of alveolar edema in each of these groups, we determined the alveolar fluid volume fraction (Vaf/Vas). In wild-type control lungs fixed after one 15-minute baseline Kf at low venous pressure (15 cm H2O), Vaf/Vas was 0.01 ± 0.01, no different than that in previous reports (8). When the Vaf/Vas was reassessed in a separate group of wild-type control lungs after both baseline and final Kf measures at low venous pressure, Vaf/Vas was not significantly increased (0.07 ± 0.02). HiPv (30 cm H2O) did increase Vaf/Vas significantly to 0.73 ± 0.08 in wild-type lung. In contrast, in lungs from TRPV4–/– mice, Vaf/Vas after HiPv (0.17 ± 0.08) was no different compared with that in low pressure controls (0.14 ± 0.07). Neither result in TRPV4–/– mice was significantly different from that in wild-type controls. These data confirmed that HiPv leads to TRPV4-dependent alveolar flooding.
In the present study, we provide the first evidence implicating TRPV4 in HiPv-induced lung injury. HiPv increased Kf, with a threshold of approximately 30 cm H2O. This response in lung from wild-type mice was dependent upon temperature, PLA2 activation, and P450 epoxygenase-mediated arachidonic acid metabolism. Further, the HiPv-induced increased in Kf was attenuated in low extracellular Ca2+ and by pharmacologic blockade or knockout of the TRPV4 channel. Fluorescence and light microscopy directly confirmed the impact of HiPv on Ca2+ entry and on alveolar flooding, as well as the dependence of these responses to HiPv on TRPV4. Much of the earlier investigation into the impact of HiPv in lung presumed that this mechanical stress led to structural alterations in the endothelial barrier. This was thought to result in a stretched pore phenomenon, whereby HiPv elicited leak of fluid and protein into the lung interstitium and/or alveolar space (1, 2, 19). HiPv does indeed increase endothelial permeability when Pv exceeds approximately 30 to 50 cm H2O in mammalian lung (2–4, 20–22). Using vascular corrosion casting with low viscosity prepolymerized methyl methacrylate, we have shown that the alveolar septal barrier is the site of leak in the pulmonary circulation when HiPv increases permeability (23). Higher pressures can evoke frank stress failure, with disruption of the endothelial and/or epithelial layers of the alveolar septal barrier. West and colleagues (5) have suggested that the stress failure threshold is directly related to the thickness of the septal barrier. A thicker septal barrier, such as that in horse, apparently confers some mechanical advantage and the alveolar septal wall is able to withstand higher pressures—that is, the threshold is increased (5, 24). The relation between barrier thickness and the threshold for the permeability response to HiPv mimics the relation for stress failure. Thresholds for HiPv-induced increase in Kf do indeed increase (28, 31, and 37 cm H2O; current study and Refs. 4 and 22) as harmonic mean thickness of the blood gas barrier increases in mouse, rat, and dog (0.44, 0.39, and 0.66 µm) (24, 25), when a similar approach to measurement of Kf and similar duration of HiPv challenge are used. However, this relationship does not necessarily rule out participation from HiPv-induced activation of signaling, which initiates barrier dysfunction. Mechanical stress, such as that induced by increased shear forces at the endothelial lumenal surface, is well known to promote Ca2+ entry into the endothelium (26). However, since the early literature provided clear evidence for mechanical disruption of the septal barrier with HiPv, the notion that mechanical stress in lung may act in part by initiating intracellular signaling pathways which impact barrier function has only recently been investigated. Parker and Ivey (22) found that isoproterenol could protect against HiPv-induced acute lung injury, suggesting active regulation of barrier integrity could offset stresses induced by mechanical perturbation. Subsequently, they reported that another type of mechanically induced lung injury, that secondary to over-ventilation, increased Kf in a Ca2+ entry-dependent fashion (10). More recently, confocal microscopy was used to clearly document that HiPv promotes Ca2+ entry into subpleural septal endothelium via a gadolinium-sensitive channel in rat lung (6). An increase in left atrial pressure from 5 to 20 cm H2O rapidly increased endothelial cell Ca2+ in a reversible fashion. Our data showed that increasing PV from 5 to 30 cm H2O produced an immediate increase in endothelial Ca2+ fluorescence, which recovered after vascular pressure was returned to baseline (data not shown). This data suggests the possibility for Ca2+ entry as a contributor to signaling regulating endothelial barrier function. The attenuation of the HiPv permeability response in wild-type lungs when a low Ca2+ perfusate was used, along with the reappearance of the permeability response upon Ca2+ addback, supports this conclusion. We considered a role for TRPV4 as the mechanosensitive cation channel activated by HiPv. This hypothesis developed from the observation that endothelial TRPV4 is activated by mechanical stresses, including hypotonic cell swelling and shear stress (11, 12, 18). Similarly, knockdown of TRPV4 expression in renal tubular epithelium with siRNA abolished hypotonicity-induced Ca2+ influx (13). We had previously reported that activation of TRPV4 led to Ca2+ entry–dependent injury and disruption of the alveolar septal barrier in both rat and mouse lung (8), the same compartment as that targeted by HiPv. The current study has provided clear-cut evidence that TRPV4 is a key element of signaling leading to HiPv-induced Ca2+ entry–dependent lung injury. Both the Ca2+ influx in subpleural endothelium and the permeability response in isolated lung observed in TRPV4+/+ lung challenged with HiPv were lacking after TRPV blockade or in TRPV4–/– littermates. Since gadolinium did not cause further attenuation of the HiPv-induced Ca2+ response in TRPV4–/– lung, we conclude that TRPV4 is the sole cation channel involved. Although TRPV4 was originally described as a channel activated by hypotonicity, it also appears to participate in the Ca2+ entry–dependent regulation of lung endothelial permeability induced by the mechanical stress deriving from HiPv. There are several potential mechanisms by which HiPv could regulate TRPV4, including direct mechanical gating or gating secondary to generation of some mechanically elicited second messenger. Force applied to the endothelium eliciting membrane stretch could directly activate the channel (13). The amino-terminal domain of TRPV4 includes three ankyrin repeats that appear to anchor the channel to the cytoskeleton (27). Deletion of these ankyrin repeats did abolish heat-induced activation of heterologously expressed TRPV4 (28). However, Liedtke and coworkers showed that this deletion only attenuated, but did not prevent, the activation by hypotonic stimuli (29). Their results suggested that direct perturbation of the membrane cytoskeleton was not absolutely essential for hypotonicity-induced gating of TRPV4. Alternatively, we considered whether HiPv activated TRPV4 indirectly, via a signaling pathway involving arachidonic acid metabolism. Arachidonic acid metabolites generated via P450 epoxygenases (such as 5,6-EET) activate TRPV4 in HEK-293 cells and in aortic endothelial cells, promoting Ca2+ entry (30). Both hypotonic cell swelling and shear stress increase cytosolic PLA2 activity in endothelial cells and activate TRPV4, a process that requires EET synthesis (11, 12). Our previous work has shown that the increase in lung endothelial permeability elicited by EETs requires extracellular Ca2+ and that TRPV4 mediates the EET-dependent permeability response (7, 8). In the current study we show that inhibition of PLA2 or P450 epoxygenases limits the HiPv-induced Ca2+ transient in subpleural endothelium as well as the permeability response in isolated lung. Our results support the notion that HiPv activates TRPV4 by a signaling cascade that sequentially leads to activation of PLA2, PLA2-dependent release of arachidonic acid from membrane phospholipids, and cytochrome P450 epoxygenase-dependent synthesis of EETs from arachidonic acid, followed by activation of TRPV4. Collectively, our data suggest that TRPV4 is the cation channel that mediates the HiPv-induced increase in lung endothelial permeability. However, recent work by us and others has highlighted the potential for discrete targeting of extra-alveolar or septal microvascular endothelium in acute lung injury, an effect that can be masked when permeability is assessed by measurement of whole lung Kf (8, 9, 23). We previously reported that TRPV4 is expressed in the septal compartment of lungs from humans, rats, and mice, and that activation of TRPV4 via EETs results in disruption of the septal endothelial barrier and alveolar flooding in rat and mouse lung (8). In contrast, activation of store-operated cation channels in extra-alveolar endothelium had no impact on alveolar septal barrier integrity or the alveolar fluid volume fraction (8). In the current study, we found that the increase in Vaf/Vas observed in wild-type lung after HiPv was absent in TRPV4–/– lung. These data confirm previous reports that HiPv targets the alveolar septal compartment, evoking disruption of alveolar septal capillaries and alveolar flooding (5, 22–24, 31), and suggest that this is due to HiPv-induced activation of TRPV4. There are several potential limitations or caveats which should be considered. First, one might argue that our findings in mouse lung may not predict responses to HiPv in lungs from larger mammals, particularly since edema accumulation was evident even in the control Pv experiments in the TRPV4–/– mice. However, pulmonary edema in and of itself does not impact the measure of Kf (32). As a case in point, our measures of baseline hemodynamics and Kf in the mouse lung (Table 1) are no different than those we have reported in rat, dog, or pig lung, when normalized for lung mass (4, 8, 33), and in our hands, these measures remain stable in control mouse lungs that are perfused for up to 3 hours (data not shown). Second, Kf tended to increase after HiPv, despite perfusion with low Ca2+ perfusate, pharmacologic blockade of TRPV4, or key steps in the signaling cascade regulating TRPV4, or deletion of TRPV4. We considered whether the apparent residual Ca2+ response to HiPv may be due to release of Ca2+ from intracellular stores. However, such release is not likely sufficient to explain the residual increase in Kf when TRPV4-mediated Ca2+ entry is blocked. Our conclusion is based on the observation that store depletion and the resultant store-operated Ca2+ entry does not produce inter-endothelial cell gap formation in pulmonary microvascular endothelium nor evoke alveolar flooding (8, 9). Further, since the Ca2+ response in TRPV4–/– lungs was not altered in the presence of gadolinium chloride, a nonspecific cation channel blocker, we suggest that the residual increase in Kf not attributed to TRPV4 is likely due to frank mechanically induced disruption of the alveolar septal barrier, that is, to stress failure (5). We conclude that TRPV4 plays a critical role in eliciting a permeability response to HiPv in the mouse lung. Gating of TRPV4 during HiPv requires activation of PLA2, synthesis of EETs from arachidonic acid, and EET-dependent activation of TRPV4. This scenario may represent a conserved signaling pathway in vascular endothelium linking mechanical stress to Ca2+ entry. In lung alveolar septal endothelium, TRPV4 activation evokes barrier disruption and alveolar flooding. These findings have clear relevance for the clinical setting, where acute lung injury is well recognized to be localized to this compartment (34).
The authors appreciate the technical expertise and assistance provided by Sue Barnes, Mita Patel, and Frieda McDonald.
This work was supported by P01 HL066299. This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org Originally Published in Press as DOI: 10.1165/rcmb.2007-0192OC on October 25, 2007 Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript. Received in original form May 31, 2007 Accepted in final form October 4, 2007
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-phorbol didecanoate (4
ali A, Hudspeth AJ, Friedman JM, Heller S. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 2000;103:525–535.
