EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia.

Ephrin family receptor tyrosine kinases are mediators of angiogenesis that may also regulate endothelial barrier function in the lung. Previous work has demonstrated that stimulation of EphA ephrin receptors causes increased vascular leak in the intact lung and increased permeability in cultured endothelial cells. Whether EphA receptors are involved in the permeability changes associated with lung injury is unknown. We studied this question in young rats exposed to viral respiratory infection combined with exposure to moderate hypoxia, a previously described lung injury model. We found that the EphA2 receptor is expressed in normal lung and that EphA2 expression is markedly upregulated in the lungs of hypoxic infected (HV) rats compared with normal control animals. Immunohistochemistry showed increased EphA2 expression principally in areas of edematous alveolar septae. In HV rats, EphA2 antagonism with either the soluble decoy receptor EphA2/Fc or with monoclonal anti-EphA2 antibody reduced albumin extravasation and histological evidence of edema formation (P<0.01). Vascular leak in HV rats is mediated in large part by increased lung endothelin (ET) levels. In HV rats, ET receptor antagonism with bosentan resulted in reduced EphA2 mRNA and protein expression (P<0.01). Experiments with cultured rat lung microvascular endothelial cells demonstrated that ET increases endothelial EphA2 expression. These results suggest that EphA2 expression is increased in lung injury, contributes to vascular leak in the injured lung, and is regulated in endothelial cells by ET. EphA2 may be a previously unrecognized contributor to the pathophysiology of lung injury.

Endothelin B receptor deficiency predisposes to pulmonary edema formation via increased lung vascular endothelial cell growth factor expression.

Endothelin (ET) may contribute to pulmonary edema formation, particularly under hypoxic conditions, and decreases in ET-B receptor expression can lead to reduced ET clearance. ET increases vascular endothelial cell growth factor (VEGF) production in vitro, and VEGF overexpression in the lung causes pulmonary edema in vivo. We hypothesized that pulmonary vascular ET-B receptor deficiency leads to increased lung ET, that excess ET increases lung VEGF levels, promoting pulmonary edema formation, and that hypoxia exaggerates these effects. We studied these hypotheses in ET-B receptor-deficient rats. In normoxia, homozygous ET-B-deficient animals had significantly more lung vascular leak than heterozygous or control animals. Hypoxia increased vascular leak regardless of genotype, and hypoxic ET-B-deficient animals leaked more than hypoxic control animals. ET-B-deficient animals had higher lung ET levels in both normoxia and hypoxia. Lung HIF-1alpha and VEGF content was greater in the ET-B-deficient animals in both normoxia and hypoxia, and both HIF-1alpha and VEGF levels were reduced by ET-A receptor antagonism. Both ET-A receptor blockade and VEGF antagonism reduced vascular leak in hypoxic ET-B-deficient animals. We conclude that ET-B receptor-deficient animals display an exaggerated lung vascular protein leak in normoxia, that hypoxia exacerbates that leak, and that this effect is in part attributable to an ET-mediated increase in lung VEGF content.

Endothelial EphA receptor stimulation increases lung vascular permeability.

Mediators of angiogenesis such as VEGFs and angiopoietins may regulate pulmonary vascular permeability under normal and pathological conditions. Ephrin family receptor tyrosine kinases are expressed in the vasculature and also regulate angiogenesis under some circumstances, but whether they also modulate lung vascular permeability is unknown. We hypothesized that stimulation of lung endothelial EphA receptors with ephrin-a1 ligand would alter pulmonary vascular permeability and tested this idea in vivo and in vitro. We found that ephrin-a1 ligand and EphA2 receptors are expressed in distal normal lung vasculature and that their expression is increased in injured lung, suggesting a link to mechanisms of increased permeability. Intravenous injection of ephrin-a1 caused a large increase in the leakage of labeled albumin into the lungs of rats within 30 min (293 +/- 27 vs. 150 +/- 6 ng/mg dry lung, P < 0.01), along with histological evidence of the formation of endothelial disruptions. In cultured lung vascular endothelial cells, stimulation with ephrin-a1 increased monolayer permeability by 44% (P < 0.01), a permeability change similar to that seen with VEGF stimulation of the same cells. Ephrin-a1 stimulation in vivo and in vitro was associated with histological evidence for disruptions of tight and adherens junctions. These observations describe a novel role for ephrin-a1 and EphA receptors in the regulation of vascular permeability in the lung.

Hypoxia upregulates lung microvascular neurokinin-1 receptor expression.

Subacute exposure to moderate hypoxia can promote pulmonary edema formation. The tachykinins, a family of proinflammatory neuropeptides, have been implicated in the pathogenesis of pulmonary edema in some settings, including the pulmonary vascular leak associated with exposure to hypoxia. The effects of hypoxia on tachykinin receptor and peptide expression in the lung, however, remain poorly understood. We hypothesized that subacute exposure to moderate hypoxia increases lung neurokinin-1 (NK-1) receptor expression as well as lung substance P levels. We tested this hypothesis by exposing weanling Sprague-Dawley rats to hypobaric hypoxia (barometric pressure 0.5 atm) for 0, 24, 48, or 72 h. Hypoxia led to time-dependent increases in lung NK-1 receptor mRNA expression and lung NK-1 receptor protein levels at 48 and 72 h of exposure (P < 0.05). Immunohistochemistry and in situ NK-1 receptor labeling with substance P-conjugated fluorescent nanocrystals demonstrated that hypoxia increased NK-1 expression primarily in the pulmonary microvasculature and in alveolar macrophages. Hypoxia also led to increases in lung substance P levels by 48 and 72 h (P < 0.05) but led to a decrease in preprotachykinin mRNA levels (P < 0.05). We conclude that subacute exposure to moderate hypoxia upregulates lung NK-1 receptor expression and lung substance P peptide levels primarily in the lung microvasculature. We speculate that this effect may contribute to the formation of pulmonary edema in the setting of regional or environmental hypoxia.

Endothelin-mediated increases in lung VEGF content promote vascular leak in young rats exposed to viral infection and hypoxia.

Viral respiratory infections increase the susceptibility of young animals to hypoxia-induced pulmonary edema formation. Previous work has shown that increased lung levels of endothelin (ET) contribute to this effect, though the mechanisms by which ET promotes vascular leak remain uncertain. Both in vitro and in vivo evidence suggests that ET can upregulate the production of VEGF, which is known to increase vascular permeability. We hypothesized that increases in lung ET promote increases in lung VEGF, which in turn increases vascular leak in the lung. Weanling rats were exposed to moderate hypoxia for 24 h while recovering from a mild viral respiratory infection, to hypoxia alone, or to viral infection alone. Lung VEGF mRNA and protein content were measured by RT-PCR and Western blotting, respectively. Animals exposed to hypoxia + virus demonstrated significant increases in lung VEGF mRNA and protein content. Immunohistochemical studies showed increased VEGF expression in alveolar septa and small pulmonary vessels in those animals. ET receptor blockade with bosentan prevented this increase in lung VEGF content, suggesting that ET promotes VEGF accumulation in the lung in this setting. Animals exposed to hypoxia + virus also demonstrated substantial increases in lung albumin extravasation, and those increases were blocked by both ET receptor blockade and VEGF antagonism. These findings suggest that ET-driven increases in lung VEGF content can contribute to the formation of pulmonary edema.

Hypoxia reversibly inhibits epithelial sodium transport but does not inhibit lung ENaC or Na-K-ATPase expression.

Hypoxia reduces alveolar liquid clearance and the nasal potential difference, a marker of airway epithelial sodium transport. The mechanisms underlying this impaired epithelial sodium transport in vivo remain uncertain. We hypothesized that epithelial sodium transport impaired by hypoxia would recover quickly with reoxygenation and that hypoxia decreases the expression of lung epithelial sodium channels and Na,K-ATPases. We studied adult rats exposed to normoxia, hypoxia (Fi(O(2)) = 0.1) for 24 h, or hypoxia followed by recovery in normoxia. Nasal potential differences decreased by 40% with hypoxia (P < 0.001), returning to baseline levels with reoxygenation. Lung Na,K-ATPase activity decreased by 40% with hypoxia (P = 0.003), recovering to baseline levels with reoxygenation. Lung expression of mRNA encoding for epithelial sodium channel (ENaC)-alpha, -beta, and -gamma or for Na,K-ATPase-alpha(1) did not change significantly with hypoxia or recovery nor did lung expression of ENaC-alpha, ENaC-beta, Na,K-ATPase-alpha(1), or Na,K-ATPase-beta(1) protein. We conclude that subacute exposure to moderate hypoxia reversibly impairs airway epithelial sodium transport and lung Na,K-ATPase activity but that those changes are not due to changes in the lung expression of sodium-transporting proteins.

Cross talk between the GABA(A) receptor and the Na-K-Cl cotransporter is mediated by intracellular Cl-.

It has been suggested that the GABA(A) receptor-mediated depolarization in immature neurons depends on a high intracellular Cl(-) concentration maintained by Na-K-Cl cotransporter isoform1 (NKCC1). We previously found that activation of the GABA(A) receptor led to stimulation of NKCC1. This stimulation could be a result of GABA(A) receptor-mediated Cl(-) efflux. However, a loss of intracellular Cl(-) is associated with cell shrinkage, membrane depolarization, as well as a rise of intracellular Ca(2+) concentration ([Ca(2+)](i)). To determine which cellular mechanism is underlying NKCC1 stimulation, we investigated changes of intracellular Cl(-) content, [Ca(2+)](i), cell volume, and NKCC1 activity following GABA(A) receptor activation. The basal levels of intracellular (36)Cl were 0.70 +/- 0.04 micromol/mg protein. The intracellular (36)Cl content decreased to 0.53 +/- 0.03 micromol/mg protein in response to 30 microM muscimol (P < 0.05). The loss of intracellular (36)Cl was blocked by 10 microM bicuculline. Muscimol triggered a rise in [Ca(2+)](i), but did not cause cell shrinkage. In contrast, 10-50 mM [Cl(-)](o) or hypertonic HEPES-MEM resulted in reversible cell shrinkage (P < 0.05). Moreover, the GABA-mediated stimulation of NKCC1 activity was not abolished either by removal of extracellular Ca(2+) or BAPTA-AM. An increase in phosphorylation of NKCC1 was detected under both 10 mM [Cl(-)](o) and muscimol conditions. These results suggest that a GABA-mediated loss of intracellular Cl(-), but not a subsequent rise in [Ca(2+)](i) or shrinkage, leads to stimulation of NKCC1. This stimulation may be an important positive feedback mechanism to maintain intracellular Cl(-) level and GABA function in immature neurons.