Pulmonary edema resulting from high pulmonary venous pressure (PVP) is a major cause of morbidity and mortality in heart failure (HF) patients, but current treatment options demonstrate substantial limitations. Recent evidence from rodent lungs suggests that PVP-induced edema is driven by activation of pulmonary capillary endothelial transient receptor potential vanilloid 4 (TRPV4) channels. To examine the therapeutic potential of this mechanism, we evaluated TRPV4 expression in human congestive HF lungs and developed small-molecule TRPV4 channel blockers for testing in animal models of HF. TRPV4 immunolabeling of human lung sections demonstrated expression of TRPV4 in the pulmonary vasculature that was enhanced in sections from HF patients compared to controls. GSK2193874 was identified as a selective, orally active TRPV4 blocker that inhibits Ca(2+) influx through recombinant TRPV4 channels and native endothelial TRPV4 currents. In isolated rodent and canine lungs, TRPV4 blockade prevented the increased vascular permeability and resultant pulmonary edema associated with elevated PVP. Furthermore, in both acute and chronic HF models, GSK2193874 pretreatment inhibited the formation of pulmonary edema and enhanced arterial oxygenation. Finally, GSK2193874 treatment resolved pulmonary edema already established by myocardial infarction in mice. These findings identify a crucial role for TRPV4 in the formation of HF-induced pulmonary edema and suggest that TRPV4 blockade is a potential therapeutic strategy for HF patients.
. TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs. Am J Physiol Lung Cell Mol Physiol 293: L923-L932, 2007. First published July 27, 2007; doi:10.1152/ajplung.00221.2007.-We have previously implicated calcium entry through stretch-activated cation channels in initiating the acute pulmonary vascular permeability increase in response to high peak inflation pressure (PIP) ventilation. However, the molecular identity of the channel is not known. We hypothesized that the transient receptor potential vanilloid-4 (TRPV4) channel may initiate this acute permeability increase because endothelial calcium entry through TRPV4 channels occurs in response to hypotonic mechanical stress, heat, and P-450 epoxygenase metabolites of arachidonic acid. Therefore, permeability was assessed by measuring the filtration coefficient (K f) in isolated perfused lungs of C57BL/6 mice after 30-min ventilation periods of 9, 25, and 35 cmH2O PIP at both 35°C and 40°C. Ventilation with 35 cmH2O PIP increased Kf by 2.2-fold at 35°C and 3.3-fold at 40°C compared with baseline, but Kf increased significantly with time at 40°C with 9 cmH2O PIP. Pretreatment with inhibitors of TRPV4 (ruthenium red), arachidonic acid production (methanandamide), or P-450 epoxygenases (miconazole) prevented the increases in Kf. In TRPV4 Ϫ/Ϫ knockout mice, the high PIP ventilation protocol did not increase Kf at either temperature. We have also found that lung distention caused Ca 2ϩ entry in isolated mouse lungs, as measured by ratiometric fluorescence microscopy, which was absent in TRPV4 Ϫ/Ϫ and ruthenium red-treated lungs. Alveolar and perivascular edema was significantly reduced in TRPV4 Ϫ/Ϫ lungs. We conclude that rapid calcium entry through TRPV4 channels is a major determinant of the acute vascular permeability increase in lungs following high PIP ventilation. pulmonary edema; P-450 epoxygenases; stretch-activated cation channel; vascular permeability; Ca 2ϩ channels; epoxyeicosatrienoic acids; temperature ACUTE LUNG INJURY (ALI) and the acute respiratory distress syndrome (ARDS) are life-threatening conditions caused by a variety of pathologic processes and affect over 200,000 patients in the United States each year (41). Although positive pressure mechanical ventilation is a life-saving intervention in the setting of ARDS and ALI, clinical trials have demonstrated that mechanical ventilation with excessive tidal volumes actually contributes to lung injury and increases mortality (6). Although clinicians and researchers have been interested in ventilator-induced lung injury (VILI) for decades, the molecular mechanisms driving this process remain incompletely understood (7,8,27,48).Many previous investigators have reported that high airway pressures and lung volumes can increase pulmonary endothelial and epithelial permeability (7,8,27). An altered ion channel activity occurs within seconds in response to mechanical stress, and an increase in intracellular Ca 2ϩ concentration ([C...
We have previously implicated transient receptor potential vanilloid 4 (TRPV4) channels and alveolar macrophages in initiating the permeability increase in response to high peak inflation pressure (PIP) ventilation. Alveolar macrophages were harvested from TRPV4(-/-) and TRPV4(+/+) mice and instilled in the lungs of mice of the opposite genotype. Filtration coefficients (K(f)) measured in isolated perfused lungs after ventilation with successive 30-min periods of 9, 25, and 35 cmH(2)O PIP did not significantly increase in lungs from TRPV4(-/-) mice but increased >2.2-fold in TRPV4(+/+) lungs, TRPV4(+/+) lungs instilled with TRPV4(-/-) macrophages, and TRPV4(-/-) lungs instilled with TRPV4(+/+) macrophages after ventilation with 35 cmH(2)O PIP. Activation of TRPV4 with 4-alpha-phorbol didecanoate (4alphaPDD) significantly increased intracellular calcium, superoxide, and nitric oxide production in TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. Cross-sectional areas increased nearly 3-fold in TRPV4(+/+) macrophages compared with TRPV4(-/-) macrophages after 4alphaPDD. Immunohistochemistry staining of lung tissue for nitrotyrosine revealed increased amounts in high PIP ventilated TRPV4(+/+) lungs compared with low PIP ventilated TRPV4(+/+) or high PIP ventilated TRPV4(-/-) lungs. Thus TRPV4(+/+) macrophages restored susceptibility of TRPV4(-/-) lungs to mechanical injury. A TRPV4 agonist increased intracellular calcium and reactive oxygen and nitrogen species in harvested TRPV4(+/+) macrophages but not TRPV4(-/-) macrophages. K(f) increases correlated with tissue nitrotyrosine, a marker of peroxynitrite production.
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 1/1 or TRPV4 2/2 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 1/1 versus TRPV4 2/2 lungs. In TRPV4 1/1 lungs, the high pressure-induced permeability response was significantly attenuated by low calcium perfusate, the TRPV antagonist ruthenium red, the phospholipase A 2 inhibitor methyl arachidonyl fluorophosphonate, or the P450 epoxygenase inhibitor propargyloxyphenyl hexanoic acid. Similarly, the high pressure-induced calcium transient in TRPV4 1/1 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 2/2 mice, permeability, calcium intensity, and alveolar fluid volume fraction were not increased. These data support a role for P450-derived epoxyeicosatrienoic aciddependent regulation of calcium entry via TRPV4 in the permeability response to high vascular pressure.Keywords: epoxyeicosatrienoic acid; capillary permeability; respiratory distress syndrome; TRPV cation channels In lung, high vascular pressure (HiPv) exceeding a threshold of 30 to 50 cm H 2 O 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 Ca 21 entry into lung endothelium, and acute lung injury is often dependent upon such Ca 21 transients (7-10). Although HiPv-induced lung injury could plausibly be dependent upon Ca 21 entry, this has not been experimentally confirmed nor has a candidate channel been identified.The Ca 21 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 Ca 21 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 phospholipas...
influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the ␣1G T-type Ca 2ϩ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca 2ϩ , immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K ϩ perfusate induces Ca 2ϩ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca 2ϩ entry in septal endothelium evoked by the selective TRPV4 activator 4␣-phorbol-12,13-didecanoate (4␣-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of ␣1G abolishes depolarization-induced Ca 2ϩ entry and surface expression of P-selectin but does not prevent 4␣-PDD-activated Ca 2ϩ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4␣-PDD-activated Ca 2ϩ entry and the increase in permeability, while not impacting depolarization-induced Ca 2ϩ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca 2ϩ entry through ␣1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.
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