Todd C. Carpenter

Unique, Highly Proliferative Growth Phenotype Expressed by Embryonic and Neointimal Smooth Muscle Cells Is Driven by Constitutive Akt, mTOR, and p70S6K Signaling and Is Actively Repressed by PTEN

Background—At distinct times during embryonic development and after vascular injury, smooth muscle cells (SMCs) exhibit a highly proliferative, serum-independent growth phenotype. The aim of the present study was to evaluate the functional role of S6 ribosomal protein (S6RP) and upstream positive and negative regulators in the control of SMC serum-independent growth. Methods and Results—We previously reported increased expression of S6RP mRNA was associated with this unique growth phenotype. Using immunohistochemistry and Western blot analysis, we report high levels of total and phospho-S6RP and increased levels of Akt and p70S6K phosphorylation, upstream positive regulators of S6RP, in rat embryonic aortas and adult balloon-injured carotid arteries compared with quiescent adult aortas and uninjured carotid arteries. Western blot analysis demonstrated that cultured embryonic and neointimal SMCs that exhibited serum-independent growth capabilities expressed high levels of S6RP and constitutively active Akt, mTOR, and p70S6K. Pharmacological and molecular inhibition of phosphatidylinositol 3-kinase (PI3K) signaling pathways, using PI3K inhibitors, rapamycin, or dominant-negative Akt adenovirus, suppressed embryonic and neointimal SMC serum-independent growth. Finally, decreased activity of PTEN, an endogenous negative regulator of PI3K signaling, was associated with high in vivo SMC growth rates, and morpholino-mediated loss of endogenous PTEN induced a serum-independent growth phenotype in cultured serum-dependent SMCs. Conclusions—The possibility exists that cells that exhibit a distinct embryonic-like growth phenotype different from traditional SMCs are major contributors to intimal thickening. Growth of SMCs that exhibit this phenotype is dependent on constitutive Akt and mTOR/p70S6K signaling and is actively inhibited through the timed acquisition of the endogenously produced growth suppressor PTEN.

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.

Endothelin B Receptor Deficiency Predisposes to Pulmonary Edema Formation via Increased Lung Vascular Endothelial Cell Growth Factor Expression

Abstract— 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-1&agr; and VEGF content was greater in the ET-B–deficient animals in both normoxia and hypoxia, and both HIF-1&agr; 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.

Predisposition of infants with chronic lung disease to respiratory syncytial virus-induced respiratory failure: a vascular hypothesis.

Background. Respiratory syncytial virus (RSV) causes the highest rate of severe respiratory infections and mortality in infants and children worldwide. Preterm infants with underlying chronic lung disease (CLD), including bronchopulmonary dysplasia (BPD), are among those at high risk for severe morbidity, long term sequelae and mortality postinfection. The definition of CLD/BPD has evolved and is currently described as a disease of restricted lung development (i.e. impaired alveolar and pulmonary vascular development). This article describes potential mechanisms by which RSV infection causes respiratory failure in the infant with BPD. Methods and results. The opinions expressed in this article are based on a review of recent investigations into the mechanisms through which RSV infections could cause excessive pulmonary edema formation and subsequent respiratory failure in the infant with CLD. Although alveolar overinflation and atelectasis are well-described patterns of RSV-induced respiratory illness in this infant population, the finding of pulmonary edema is a complex, multifactorial process that is less well understood. Experimental evidence suggests that RSV infection in infants with CLD/BPD not only causes increases in pulmonary vascular reactivity but also precipitates pulmonary edema formation via multiple mechanisms (e.g. nonuniform elevations in pulmonary artery pressure, endothelial injury, alveolar epithelial damage and impairments of native alveolar liquid clearance mechanisms). Conclusions. Novel therapies for managing RSV-induced respiratory failure in the infant with CLD/BPD must consider factors responsible for the substantial pulmonary vascular component of this illness.

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.

Eph-A2 Promotes Permeability and Inflammatory Responses to Bleomycin-Induced Lung Injury

Stimulation by the ephrin-A1 ligand of the EphA2 receptor increases endothelial permeability. Lung injury increases the expression of EphA2, but the role of EphA2 in such injury is not well understood. To determine whether EphA2 contributes to changes in permeability and inflammation in the injured lung, we studied wild-type (WT) and EphA2 knockout (KO) mice, using isolated, perfused lung (IPL) preparations and a model of bleomycin-induced lung injury. We also studied the response of endothelial cells to ephrin-A1. In the IPL preparations, ephrin-A1 increased the filtration coefficient in WT mice, but not in EphA2 KO mice, demonstrating that EphA2 regulates vascular permeability. In early bleomycin injury in WT mice, the expression of both EphA2 and ephrin-A1 increased. EphA2 KO animals were protected from lung injury, showing less water and alveolar protein in the lungs than WT mice, consistent with reduced permeability. Bleomycin caused less accumulation of lung leukocytes in EphA2 KO animals than in WT animals, suggesting that EphA2 regulates inflammation. To determine whether EphA2 deficiency alters the production of chemokines, CXCL1 and CCL2 in the lungs were measured. After bleomycin injury, EphA2 KO animals produced less CXCL1 and CCL2 than WT animals. Because NF-κβ mediates the production of chemokines, the effect of the ephrin-A1 ligand on the activation of NF-κβ and the expression of chemokines was measured in endothelial cells. Ephrin-a1 significantly increased NF-κβ nuclear translocation and the expression of chemokine mRNA. This study demonstrates that the expression of EphA2 increases in the injured lung, and not only contributes to changes in permeability, but also plays a previously unrecognized role in promoting inflammatory responses.

Hypoxia reduces airway epithelial sodium transport in rats

Ascent to high altitude leads to pulmonary edema formation in some individuals. Recent laboratory evidence supports the hypothesis that hypoxia may impair the function of the alveolar epithelium and thus augment edema accumulation via reduced clearance of lung liquid. We investigated the effect of hypobaric hypoxia on epithelial sodium transport in adult Sprague-Dawley rats by measuring the nasal transepithelial potential difference (PD) as an index of airway sodium transport. Baseline PDs were similar to those previously reported in other species. Administration of amiloride resulted in a significant fall in nasal PD, as did ouabain administration for 24 h (-27.8 vs. -18.8 mV; P = 0.001; n = 5 rats). Exposure to hypobaric hypoxia (0.5 atm) for 24 h caused a significant fall in nasal PD (-23.7 vs. -18.8 mV; P = 0.002; n = 15 rats), which was not additive to the changes in nasal PD produced by amiloride or ouabain. We conclude that subacute exposure to moderate hypobaric hypoxia can inhibit sodium transport by the airway epithelium in rats.Ascent to high altitude leads to pulmonary edema formation in some individuals. Recent laboratory evidence supports the hypothesis that hypoxia may impair the function of the alveolar epithelium and thus augment edema accumulation via reduced clearance of lung liquid. We investigated the effect of hypobaric hypoxia on epithelial sodium transport in adult Sprague-Dawley rats by measuring the nasal transepithelial potential difference (PD) as an index of airway sodium transport. Baseline PDs were similar to those previously reported in other species. Administration of amiloride resulted in a significant fall in nasal PD, as did ouabain administration for 24 h (-27.8 vs. -18.8 mV; P = 0.001; n = 5 rats). Exposure to hypobaric hypoxia (0.5 atm) for 24 h caused a significant fall in nasal PD (-23.7 vs. -18.8 mV; P = 0.002; n = 15 rats), which was not additive to the changes in nasal PD produced by amiloride or ouabain. We conclude that subacute exposure to moderate hypobaric hypoxia can inhibit sodium transport by the airway epithelium in rats.