Disruption of the alveolar septal barrier leads to acute lung injury, patchy alveolar flooding, and hypoxemia. Although calcium entry into endothelial cells is critical for loss of barrier integrity, the cation channels involved in this process have not been identified. We hypothesized that activation of the vanilloid transient receptor potential channel TRPV4 disrupts the alveolar septal barrier. Expression of TRPV4 was confirmed via immunohistochemistry in the alveolar septal wall in human, rat, and mouse lung. In isolated rat lung, the TRPV4 activators 4alpha-phorbol-12,13-didecanoate and 5,6- or 14,15-epoxyeicosatrienoic acid, as well as thapsigargin, a known activator of calcium entry via store-operated channels, all increased lung endothelial permeability as assessed by measurement of the filtration coefficient, in a dose- and calcium-entry dependent manner. The TRPV antagonist ruthenium red blocked the permeability response to the TRPV4 agonists, but not to thapsigargin. Light and electron microscopy of rat and mouse lung revealed that TRPV4 agonists preferentially produced blebs or breaks in the endothelial and epithelial layers of the alveolar septal wall, whereas thapsigargin disrupted interendothelial junctions in extraalveolar vessels. The permeability response to 4alpha-phorbol-12,13-didecanoate was absent in TRPV4(-/-) mice, whereas the response to thapsigargin remained unchanged. Collectively, these findings implicate TRPV4 in disruption of the alveolar septal barrier and suggest its participation in the pathogenesis of acute lung injury.

. 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...

Endothelial progenitor cells (EPCs) have been isolated postnatally from bone marrow, blood, and both the intima and adventitia of conduit vessels. However, it is unknown whether EPCs can be isolated from the lung microcirculation. Thus we sought to determine whether the microvasculature possesses EPCs capable of de novo vasculogenesis. Rat pulmonary artery (PAEC) and microvascular (PMVEC) endothelial cells were isolated and selected by using a single-cell clonogenic assay. Whereas the majority of PAECs (approximately 60%) were fully differentiated, the majority of PMVECs (approximately 75%) divided, with approximately 50% of the single cells giving rise to large colonies (>2,000 cells/colony). These highly proliferative cells exhibited the capacity to reconstitute the entire proliferative hierarchy of PMVECs, unveiling the existence of resident microvascular endothelial progenitor cells (RMEPCs). RMEPCs expressed endothelial cell markers (CD31, CD144, endothelial nitric oxide synthase, and von Willenbrand factor) and progenitor cell antigens (CD34 and CD309) but did not express the leukocyte marker CD45. Consistent with their origin, RMEPCs interacted with Griffonia simplicifolia and displayed restrictive barrier properties. In vitro and in vivo Matrigel assays revealed that RMEPCs possess vasculogenic capacity, forming ultrastructurally normal de novo vessels. Thus the pulmonary microcirculation is enriched with EPCs that display vasculogenic competence while maintaining functional endothelial microvascular specificity.

Pulmonary microvascular endothelial cells possess both highly proliferative and angiogenic capacities, yet it is unclear how these cells sustain the metabolic requirements essential for such growth. Rapidly proliferating cells rely on aerobic glycolysis to sustain growth, which is characterized by glucose consumption, glucose fermentation to lactate, and lactic acidosis, all in the presence of sufficient oxygen concentrations. Lactate dehydrogenase A converts pyruvate to lactate necessary to sustain rapid flux through glycolysis. We therefore tested the hypothesis that pulmonary microvascular endothelial cells express lactate dehydrogenase A necessary to utilize aerobic glycolysis and support their growth. Pulmonary microvascular endothelial cell (PMVEC) growth curves were conducted over a 7-day period. PMVECs consumed glucose, converted glucose into lactate, and acidified the media. Restricting extracellular glucose abolished the lactic acidosis and reduced PMVEC growth, as did replacing glucose with galactose. In contrast, slow-growing pulmonary artery endothelial cells (PAECs) minimally consumed glucose and did not develop a lactic acidosis throughout the growth curve. Oxygen consumption was twofold higher in PAECs than in PMVECs, yet total cellular ATP concentrations were twofold higher in PMVECs. Glucose transporter 1, hexokinase-2, and lactate dehydrogenase A were all upregulated in PMVECs compared with their macrovascular counterparts. Inhibiting lactate dehydrogenase A activity and expression prevented lactic acidosis and reduced PMVEC growth. Thus PMVECs utilize aerobic glycolysis to sustain their rapid growth rates, which is dependent on lactate dehydrogenase A.