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. 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.

Lung injury is a broad descriptor that can be applied to conditions ranging from mild interstitial edema without cellular injury to massive and fatal destruction of the lung. This review addresses those methods that can be readily applied to rats and mice whose small size limits the techniques that can be practically used to assess injury. The methodologies employed range from nonspecific measurement of edema formation to techniques for calculating values of specific permeability coefficient for the microvascular membrane in lung. Accumulation of pulmonary edema can be easily and quantitatively measured using gravimetric methods and indicates an imbalance in filtration forces or restrictive properties of the microvascular barrier. Lung compliance can be continuously measured, and light and electron microscopy can be used regardless of lung size to detect edema and structural damage. Increases in fluid and/or protein flux due to increased permeability must also be separated from those due to increased filtration pressure for mechanistic interpretation. Although an increase in the initial lung albumin clearance compared with controls matched for size and filtration pressure is a reliable indicator of endothelial dysfunction, calculated alterations in capillary filtration coefficient K(f,c), reflection coefficient sigma, and permeability-surface area product PS are the most accurate indicators of increased permeability. Generally, PS and K(f,c) will increase and sigma will decrease with vascular injury, but derecruitment of microvascular surface area may attenuate the affect on PS and K(f,c) without altering measurements of sigma.

High peak inspiratory pressures (PIP) during mechanical ventilation can induce lung injury. In the present study we compare the respective roles of high tidal volume with high PIP in intact immature rabbits to determine whether the increase in capillary permeability is the result of overdistension of the lung or direct pressure effects. New Zealand White rabbits were assigned to one of three protocols, which produced different degrees of inspiratory volume limitation: intact closed-chest animals (CC), closed-chest animals with a full-body plaster cast (C), and isolated excised lungs (IL). The intact animals were ventilated at 15, 30, or 45 cmH2O PIP for 1 h, and the lungs of the CC and C groups were placed in an isolated lung perfusion system. Microvascular permeability was evaluated using the capillary filtration coefficient (Kfc). Base-line Kfc for isolated lungs before ventilation was 0.33 +/- 0.31 ml.min-1.cmH2O-1.100g-1 and was not different from the Kfc in the CC group ventilated with 15 cmH2O PIP. Kfc increased by 850% after ventilation with only 15 cmH2O PIP in the unrestricted IL group, and in the CC group Kfc increased by 31% after 30 cmH2O PIP and 430% after 45 cmH2O PIP. Inspiratory volume limitation by the plaster cast in the C group prevented any significant increase in Kfc at the PIP values used. These data indicate that volume distension of the lung rather than high PIP per se produces microvascular damage in the immature rabbit lung.