1906

EDN1

ET1|HDLCQ7|PPET1;endothelin 1;EDN1;endothelin 1

endothelin-1|preproendothelin-1

6p24.1

protein-coding

Count: Edn1; 24323

AIM: To study the effects of endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) on the mechanism of hypoxic pulmonary hypertension (HPH). METHODS: We studied 4 groups of age-controlled male rats, i.e., normal control for 2 weeks group (N2), normal control for 3 weeks group (N3), exposed to hypoxia for 2 weeks group (H2) and for 3 weeks group (H3). Chronic HPH rat models were established by chronic hypobaric hypoxia [(10.0% +/- 0.5% O2] for 2 and 3 weeks, respectively. The rats were anesthetized and fixed, and the levels of mean pulmonary artery pressure (mPAP) and carotid arterial pressure (CAP) were measured using catheters by a microcomputer via transducers. The weight ratio of right ventricle (RV) and left ventricle and septum (LV + S) [RV/ (LV+S)] were determined. The contents of ET-1 in plasma of pulmonary artery and carotid artery and in homogenates of lung and systemic arteries were determined by radioimmunoassay, and the contents of VEGF in serum of pulmonary artery and carotid artery were determined by ABC-ELISA. RESULTS: HPH rat models were established successfully. Compared with control groups, the values of ET-1 were both enhanced in carotid artery and pulmonary artery plasma in model groups (P < 0.01). In the HPH groups, the level of pulmonary artery plasma ET-1 was significantly lower than that of carotid artery plasma, but just the reverse was ET-1 in control rats. The levels of ET-1 in homogenates of lungs from HPH models were significantly higher than those in homogenates of lungs from control groups (P < 0.01), and markedly higher than those in homogenates of systemic arteries from HPH rats (P < 0.01) SThe values of VEGF in serum of pulmonary artery from H3 group were significantly higher than those from control groups and H2 group (P < 0.01). In serum of carotid artery, the values of VEGF from the HPH models were higher than those from the control groups (P < 0.01). CONCLUSION: ET-1 and VEGF play important roles in the pathogenesis of HPH. The result that ET-1 concentration around pulmonary arteries was significantly higher than that around systemic arteries may be one of the mechanisms accounting for the different reaction of them to hypoxia.

AIM: To investigate the role of vascular endothelial growth factor (VEGF) and endothelin-1 (ET-1) on pulmonary vascular structural remodeling in rats and pika. METHODS: The Wistar rats which reside at 2 260 m were carried to 3 417 m. After they were fed 24 hours,2 weeks and 3 weeks respectively, the level of VEGF and ET-1 were measured using a kit by ELISA method. Pulmonary tissue was taken out to stain with elastica-Van Gieson. The amount of pulmonary arteries (< 100 microm) and the component ratio of MA, PMA,and NMA were calculated by using a light microscope. The ratio of right ventricle weight to left ventricle plus septum weight (RV/LV + S) were measured. RESULTS: The ET-1 was significantly different in pika as compared with 24 h, 2 weeks, 3 weeks hypoxic rats (P < 0.01) respectively. The levels of VEGF in 2 weeks, 3 weeks rats were much higher than that of pika but no difference was found between pika and 24 h hypoxic rats. The ratio of MA, PMA obviously increased, and NMA decreased significantly, right ventricular hypertrophy was developed in differ groups of hypoxic rats. CONCLUSION: The VEGF and ET-1 participate the muscularization of pulmonary vessels during hypoxia and play an important role in the process of hypoxic pulmonary hypertension in rats, however the VEGF and ET-1 may be maintainable only normal organic function in pika.

pulmonary hypertension associated with human immunodeficiency virus (HIV) infection also involves injury to the lung endothelium. However, the pathogenesis of HIV-induced pulmonary hypertension is not known; we hypothesized that HIV or secreted viral proteins could play a role in vascular injury and the increased frequency of pulmonary hypertension observed in HIV-infected patients. Here, we report that exposure of HIV-1 gp120 proteins to primary human lung microvascular endothelial cells causes apoptosis, as assessed by TUNEL assay, Annexin-V staining, and DNA laddering. Using ribonuclease protection assay and Western blotting we find that gp120-induced apoptosis of lung endothelial cells involves a down-regulation in Bcl-xl mRNA and proteins. In addition, gp120 significantly increases secretion of the potent vasoconstrictor endothelin-1 by human lung endothelial cells. These data suggest that secreted HIV gp120 proteins induce lung endothelial cell injury and could contribute to the development of HIV-associated pulmonary hypertension.

BACKGROUND: In non-thromboembolic pulmonary hypertension, endothelin (ET)-1 levels are increased and correlate with the hemodynamic severity of the disease. Whether such correlations exist in chronic thromboembolic pulmonary hypertension (CTEPH) is unknown, nor whether ET-1 levels correlate with hemodynamic outcome after pulmonary endarterectomy (PEA). METHODS AND RESULTS: ET-1 levels were determined by ELISA. ET-levels were increased in 35 CTEPH patients (1.62+/-0.21 pg/ml) compared with healthy controls (n=11: 0.75+/-0.06 pg/ml, p<0.02). ET-1 levels correlated (all p<0.0001) with mean pulmonary artery pressure (mPAP) (r=0.70), cardiac index (r=-0.76), total pulmonary resistance (r=0.72), mixed venous oxygen saturation (r=-0.87), and the distance walked in the 6-min walk test (r=-0.59; p<0.005; n=23). Three months after PEA, ET-1 levels had decreased (p<0.002), and were similar between patients with and without residual pulmonary hypertension (p=0.4). Preoperative ET-1 levels, however, were higher in patients with bad postoperative outcome; that is, patients who either died because of persistent pulmonary hypertension or had residual pulmonary hypertension after PEA (2.68+/-0.48 pg/ml, and 1.13+/-0.15 pg/ml, respectively; p<0.002). The levels also correlated with hemodynamic outcome after PEA (mPAP: r=0.67, p<0.0001). By receiver-operator characteristic curve analysis, ET-1>1.77 pg/ml detected a bad postoperative outcome with a sensitivity and specificity of 79% and 85%, respectively, and a likelihood ratio of 5.2. CONCLUSION: ET-1 levels in CTEPH closely correlated with the hemodynamic and clinical severity of disease in a large cohort of patients. Preoperative ET-1 levels may be useful for better identification of patients at risk for persistent pulmonary hypertension after PEA.

PURPOSE: According to current knowledge, endothelin (ET)-1 plays an important role in the pathogenesis of systemic sclerosis (SSc). We assessed ET plasma levels in SSc patients according to the clinical presentation and the presence of complications such as pulmonary arterial hypertension (PAH). METHODS: Sixty-three consecutive patients with SSc were included. The control group included 17 healthy patients. ET plasma level was determined for all patients. Pulmonary function test and pulmonary high resolution computed tomography were performed in 44 patients and echocardiography in 51 patients, to screen for PAH, always confirmed by a right heart catheterization. RESULTS: ET plasmatic levels were higher in SSc patients than in healthy group subjects but the difference was not significant (3.72+/-1.13 vs 3.40+/-0.71 pmol/l, p=0.27). ET plasmatic levels were significantly higher in patients with PAH than in patients without PAH (4.28+/-0.65 vs 3.62+/-1.07 pmol/l, p=0.04) and in patients with anticentromere antibodies (3.96+/-1.11 vs 3.19+/-1.12 pmol/l, p=0.03). There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure (r=0.34, p=0.013). The best cut-off value for ET plasmatic level to discriminate patients affected by PAH was determined by ROC curve method: 4.1 pmol/l (sensibility 85.7%, specificity 66%). CONCLUSION: ET plasmatic levels were higher in SSc patients affected by PAH and patients with anticentromere antibodies. There was a positive linear correlation between ET plasmatic levels and systolic pulmonary arterial pressure. Assessment of ET plasmatic levels for detection and monitoring of pulmonary hypertension during SSc is warranted in larger prospective studies.

BACKGROUND: Endothelin-1 (ET-1) and Nitric Oxide (NO) are crucial mediators for establishing pulmonary artery hypertension (PAH). We tested the hypothesis that their imbalance might also occur in COPD patients with PAH. METHODS: The aims of the study were to measure exhaled breath condensate (EBC) and circulating levels of ET-1, as well as exhaled NO (FENO) levels by, respectively, a specific enzyme immunoassay kit, and by chemiluminescence analysis in 3 groups of subjects: COPD with PAH (12), COPD only (36), and healthy individuals (15). In order to evaluate pulmonary-artery systolic pressure (PaPs), all COPD patients underwent Echo-Doppler assessment. RESULTS: Significantly increased exhaled and circulating levels of ET-1 were found in COPD with PAH compared to both COPD (p < 0.0001) only, and healthy controls (p < 0.0001). In COPD with PAH, linear regression analysis showed good correlation between ET-1 in EBC and PaPs (r = 0.621; p = 0.031), and between arterial levels of ET-1 and PaPs (r = 0.648; p = 0.022), while arterial levels of ET-1 inversely correlated with FEV1%, (r = -0.59, p = 0.043), and PaPs negatively correlated to PaO2 (r = -0.618; p = 0.032). Significantly reduced levels of FENO were found in COPD associated with PAH, compared to COPD only (22.92 +/- 11.38 vs.35.07 +/- 17.53 ppb; p = 0.03). Thus, we observed an imbalanced output in the breath between ET-1 and NO, as expression of pulmonary endothelium and epithelium impairment, in COPD with PAH compared to COPD only. Whether this imbalance is an early cause or result of PAH due to COPD is still unknown and deserves further investigations.

pulmonary arterial hypertension (PAH) is the major complication of systemic sclerosis (SSc) and the main cause of morbi-mortality. It is important to find predictors for this vascular problem. The objective of this study was to determine the serum levels of different biomarkers in patients with SSc and secondary PAH and to compare them with those of healthy control subjects to define their potential role as predictors of PAH. Cross-section study in which 20 patients with SSc were included. PAH was diagnosed by echocardiogram. The optical densities of endoglin (Eng), endothelin-1 (ET-1), platelet-derived growth factor (PDGF), tumoral necrosis factor alpha (TNF-alpha), Transforming growth factor beta 2 (TGF-beta2) and Interleukin 8 (IL-8) were measured in 20 patients with SSc and 20 healthy controls matched by sex. The differences found between the group of patients with PAH and the control group were (mean or median and range): ET-1 (0.20; 0.10-0.35 vs. 0.16; 0.10-0.24; P = 0.0276), IL-8 (195.7; 45.5-504 vs. 118.9; 23-299.5; P = 0.0364), TNF-alpha (0.70; 0.50-0.96 vs. 0.48; 0.38-0.65; P = 1 x 10(-8)) and Eng (0.95; 0.57-1.72 vs. 0.75; 0.57-0.89; P = 0.0028). A correlation was found between the progression of the disease and the development of Raynaud's phenomenon (Rho: 0.67 and P = 0.0011), ET-1 and Eng (Rho: 0.53 and P = 0.0196), and between IL-8 and Eng (Rho: 0.68 and P = 0.0019). In conclusions, the elevation of the serum levels of Eng and ET-1 could represent a useful tool as PAH biomarkers. Nevertheless, the diagnostic value of these markers needs to be determined by prospective studies.

Endothelin (ET)-1 causes long-lasting vasoconstriction and vascular remodeling by interacting with specific G-protein-coupled receptors in pulmonary artery smooth muscle cells (PASMCs), and thus plays an important role in the pathophysiology of pulmonary arterial hypertension. The two-pore domain K(+) channel, TASK-1, controls the resting membrane potential in human PASMCs (hPASMCs), and renders these cells sensitive to a variety of vasoactive factors, as previously shown. ET-1 may exert its vasoconstrictive effects in part by targeting TASK-1. To clarify this, we analyzed the ET-1 signaling pathway related to TASK-1 in primary hPASMCs. We employed the whole-cell patch-clamp technique combined with TASK-1 small interfering RNA (siRNA) in hPASMC and the isolated, perfused, and ventilated mouse lung model. We found that ET-1 depolarized primary hPASMCs by phosphorylating TASK-1 at clinically relevant concentrations. The ET sensitivity of TASK-1 required ET(A) receptors, phospholipase C, phosphatidylinositol 4,5-biphosphate, diacylglycerol, and protein kinase C in primary hPASMCs. The ET-1 effect on membrane potential and TASK-1 was abrogated using TASK-1 siRNA. This is the first time that the background K(+) channel, TASK-1, has been identified in the ET-1-mediated depolarization in native hPASMC, and might represent a novel pathologic mechanism related to pulmonary arterial hypertension.

Macrophage derived-endothelin-1 (ET-1) has been suggested to contribute to a number of chronic lung diseases. Whether the ET-1 cascade from non-vascular sources (inflammatory cells) also contributes to pulmonary artery hypertension (PAH) and in particular to heritable PAH (HPAH) with known bone morphogenetic protein type 2 receptor (BMPR2) mutations is not known. We tested this notion using bone marrow-derived macrophages (BMDM; precursors of tissue macrophages) isolated from ROSA26rtTAXTetO(7)-tet-BMPR2(R899X) mice (model of PAH with universal expression of a mutated BMPR2 gene) with and without activation by LPS and in human lung tissue from HPAH with BMPR2 mutations and idiopathic PAH (IPAH). At baseline ET(A) and ET(B) receptors and endothelin converting enzyme (ECE) gene expression was reduced in BMPR2 mutant BMDM compared with controls. In control BMDM, LPS resulted in increased ppET-1 gene expression and ET-1 in culture media, whereas ET(A) and ET(B) receptor and ECE gene expression was decreased. These findings were more severe in BMPR2 mutant BMDM. Antagonism of the ET(B) receptor resulted in increased ET-1 in the media, suggesting that decreased ET-1 uptake by the ET(B) receptor contributes to the elevation. While ET-1 expression was demonstrated in lung macrophages from controls and IPAH and HPAH patients, ET(A) and ET(B) expression was decreased in the HPAH, but not IPAH, patients compared with controls. We conclude that reduced expression of macrophage ET-1 receptors in HPAH increases lung ET-1 and may contribute to the pathogenesis and maintenance of HPAH. This is the first description of protein expression that distinguishes HPAH from IPAH in patients.

Abnormalities of signalling for the transforming growth factor beta (TGFbeta) family of peptides, including bone morphogenic proteins (BMP), have been described in heritable pulmonary arterial hypertension (PAH). TGFbeta can modulate synthesis of the vasoconstrictor and mitogen, endothelin-1 (ET-1), a mediator that contributes to the pathogenesis of PAH. BMP-9 is a circulating peptide recently recognized to affect endothelial function. The stimuli for increased microvascular endothelial production of ET-1 in PAH are unknown. We therefore studied the effects of BMP-9 on ET-1 production by human lung blood microvascular endothelial cells (HMVEC-LBl) in vitro. In vitro, BMP-9 increased ET-1 production by HMVEC-LBl. The effect was identical to TGFbeta-1, but BMP-9 and TGFbeta-1 combined further increased ET-1 levels by 29%. As compared to TGFbeta-1, BMP-9 induced more potent and rapid phosphorylation of Smad 1/5, the downstream signalling molecules of the activin-like kinase 1 (ALK-1) receptor. Moreover, as has been previously shown for endothelial cells of other origin, BMP-9 also induced Smad 2 phosphorylation in HMVEC-LBl. In conclusion, BMP-9 stimulates ET-1 production by HMVEC-LBl in vitro. BMP-9 signals via several Smad pathways. These studies provide novel mechanisms for the potentiation of PAH.CI - Copyright (c) 2010 Elsevier Inc. All rights reserved.

BACKGROUND: It is unclear why some patients, who undergo complete repair or palliative surgery for congenital heart disease (CHD), still develop irreversible pulmonary artery hypertension (PAH). There is no consensus to preoperationally assess the reversible and irreversible pulmonary vasculopathy seen in PAH. METHODS AND RESULTS: The peri-operative pulmonary hemodynamic data of 16 CHD patients (reversible PAH, n = 6; irreversible PAH, n = 10) were analyzed. The lung biopsies were also performed during surgery for defining histopathological characteristics as well as immunohistochemical expression of endothelin-1 (ET-1), endothelin-1 receptors (ETR), and its downstream signaling markers in the small pulmonary arteries and arterioles. Neointimal formation and neoangiogenesis was characterized by increased intimal layer immunoreactivity for alpha-SMA, Factor VIII, CD34, and VEGF. Neointimal formation was found in 90% of patients and neoangiogenesis was found in 80% of patients with irreversible PAH. Neither was present in the reversible PAH group and the control group. expression of ET-1 and ETR in the neointimal layer of the pulmonary arterioles was upregulated in irreversible PAH, and immunoreactivity of phospho-Akt, phospho-ERK1/2, and phospho-mTOR was also increased in irreversible PAH. CONCLUSIONS: Increased expression of ET-1, ETR, and activation of signaling pathways were observed in the pulmonary arteries and arterioles of irreversible PAH patients associated with CHD. Activation of these pathways might in turn lead to neointimal formation and neoangiogenesis and thus might contribute to irreversible pulmonary vascular abnormalities.

This study analyses the frequency and the potential role of two polymorphisms, the +134del/insA, located in the gene encoding for Endothelin-1 (EDN1), and the His323His in the gene encoding for Endothelin receptor type A (EDNRA) in a cohort of 98 consecutive patients with pulmonary arterial hypertension from two different Cardiology Units (Mid-South of Italy), and in 100 healthy Caucasian subjects randomly recruited from the same area. Cardiac anatomy and function were analysed by non invasive diagnostic imaging techniques (Echocardiography standard m-mode, 2D, colour-Doppler) and by invasive studies (cardiac catheterization). Molecular screening of the region of interest was performed by automated sequencing. At univariate analysis, patients with the His323His TT genotype show a lower cardiac index (2 +/- 0.6 vs. 2.3 +/- 0.6; p = 0.05) and a higher indexed pulmonary vascular resistance (18.8 +/- 9.6 vs. 14.2 +/- 6.9; p = 0.01) at cardiac catheterization. A logistic multivariate model shows idiopathic disease (p = 0.01; OR = 3.8; CI = 1.3-11) and indexed pulmonary vascular resistances (p = 0.01; OR = 1.1; CI = 1-1.2) as independent predictors of TT genotype. Our findings may suggest a potential link between specific genotypes in the EDNRA gene and susceptibility for PAH.

BACKGROUND: Bone morphogenetic proteins (BMPs) and their receptors, such as bone morphogenetic protein receptor (BMPR) II, have been implicated in a wide variety of disorders including pulmonary arterial hypertension (PAH). Similarly, endothelin-1 (ET-1), a mitogen and vasoconstrictor, is upregulated in PAH and endothelin receptor antagonists are used in its treatment. We sought to determine whether there is crosstalk between BMP signalling and the ET-1 axis in human pulmonary artery endothelial cells (HPAECs), possible mechanisms involved in such crosstalk and functional consequences thereof. METHODOLOGY/PRINCIPAL FINDING: Using western blot, real time RT-PCR, ELISA and small RNA interference methods we provide evidence that in HPAECs BMP-9, but not BMP-2, -4 and -6 significantly stimulated ET-1 release under physiological concentrations. This release is mediated by both Smad1 and p38 MAPK and is independent of the canonical Smad4 pathway. Moreover, knocking down the ALK1 receptor or BMPR II attenuates BMP-9 stimulated ET-1 release, whilst causing a significant increase in prepro ET-1 mRNA transcription and mature peptide release. Finally, BMP-9 induced ET-1 release is involved in both inhibition of endothelial cell migration and promotion of tubule formation. CONCLUSIONS/SIGNIFICANCE: Although our data does not support an important role for BMP-9 as a source of increased endothelial ET-1 production seen in human PAH, BMP-9 stimulated ET-1 production is likely to be important in angiogenesis and vascular stability. However, increased ET-1 production by endothelial cells as a consequence of BMPR II dysfunction may be clinically relevant in the pathogenesis of PAH.

Pulmonary artery hypertension (PAH) causes right ventricular failure and possibly even death by a progressive increase in pulmonary vascular resistance. Bone marrow-derived mesenchymal stem cell therapy has provided an alternative treatment for ailments of various organs by promoting cell regeneration at the site of pathology. The purpose of this study was to investigate changes of pulmonary haemodynamics, pathology and expressions of various genes, including ET (endothelin)-1, ET receptor A (ERA), endothelial nitric oxide synthase (NOS) 3, matrix metalloproteinase (MMP) 2, tissue inhibitor of matrix metalloproteinase (TIMP), interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in monocrotaline (MCT)-induced PAH rat models after bone marrow cell (BMC) transfusion. The rats were grouped as the control (C) group, monocrotaline (M) group, and BMC transfusion (B) group. M and B groups received subcutaneous (sc) injection of MCT (60 mg/kg). BMCs were transfused by intravenous injection at the tail 1 week after MCT injection in B group. Results showed that the average RV pressure significantly decreased in the B group compared with the M group. RV weight and the ratio of RH/LH+septum significantly decreased in the B group compared to the M group. Gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha significantly decreased in week 4 in the B group compared with the M group. In conclusion, BMC transfusion appears to improve survival rate, RVH, and mean RV pressure, and decreases gene expressions of ET-1, ERA, NOS 3, MMP 2, TIMP, IL-6, and TNF-alpha.

BACKGROUND: Idiopathic pulmonary arterial hypertension (IPAH) is a poorly understood complex disorder, which results in progressive remodeling of the pulmonary artery that ultimately leads to right ventricular failure. A two-hit hypothesis has been implicated in pathogenesis of IPAH, according to which the vascular abnormalities characteristic of PAH are triggered by the accumulation of genetic and/or environmental insults in an already existing genetic background. The multifactor dimensionality reduction (MDR) analysis is a statistical method used to identify gene-gene interaction or epistasis and gene-environment interactions that are associated with a particular disease. The MDR method collapses high-dimensional genetic data into a single dimension, thus permitting interactions to be detected in relatively small sample sizes. AIM: To identify and characterize polymorphisms/genes that increases the susceptibility to IPAH using MDR analysis. MATERIALS AND METHODS: A total of 77 IPAH patients and 100 controls were genotyped for eight polymorphisms of five genes (5HTT, EDN1, NOS3, ALK-1, and PPAR-gamma2). MDR method was adopted to determine gene-gene interactions that increase the risk of IPAH. RESULTS: With MDR method, the single-locus model of 5HTT (L/S) polymorphism and the combination of 5HTT(L/S), EDN1(K198N), and NOS3(G894T) polymorphisms in the three-locus model were attributed to be the best models for predicting susceptibility to IPAH, with a P value of 0.05. CONCLUSION: MDR method can be useful in understanding the role of epistatic and gene-environmental interactions in pathogenesis of IPAH.

The ovarian hormone 17beta-estradiol (E2beta) attenuates chronic hypoxia-induced pulmonary hypertension. We hypothesized that E2beta attenuates this response to hypoxia by decreasing pulmonary expression of the vasoactive and mitogenic peptide endothelin-1 (ET-1). To test this hypothesis, we measured preproET-1 mRNA and ET-1 peptide levels in the lungs of adult female normoxic and hypoxic (24 h or 4 wk at barometric pressure = 380 mmHg) rats with intact ovaries and in hypoxic ovariectomized (OVX) rats administered E2beta or vehicle via subcutaneous osmotic pumps. Hypoxic exposure increased lung preproET-1 mRNA levels in OVX vehicle-treated rats, but not in rats with intact ovaries. In addition, E2beta replacement prevented hypoxia-mediated increases in preproET-1 mRNA and ET-1 peptide expression. Considering that hypoxic induction of ET-1 gene expression is mediated by a hypoxia-inducible transcription factor(s) (HIF), we further hypothesized that E2beta-induced attenuation of pulmonary ET-1 expression during hypoxia results from decreased HIF activity. We found that E2beta abolished HIF-dependent increases in reporter gene activity. Further experiments demonstrated that overexpression of the transcriptional coactivator cAMP response element binding protein (CREB) binding protein (CBP)/p300, a factor common to both the estrogen receptor and HIF pathways, eliminated E2beta-mediated attenuation of hypoxia-induced ET-1 promoter activity. We conclude that E2beta inhibits hypoxic induction of ET-1 gene expression by interfering with HIF activity, possibly through competition for limiting quantities of CBP/p300.

BACKGROUND/PURPOSE: Although high levels of endothelin-1 (ET-1) in plasma may be relevant in certain pathophysiologic states, such as pulmonary hypertension accompanying congenital diaphragmatic hernia (CDH), experimental evidence favors a local, paracrine, or autocrine role for ET-1 in most tissues. Evidence of ET-1 production has been documented in fetal heart tissue where it exerts growth-enhancing and mitogenic effects. ET-1 also has a potent positive inotrope action on cardiac muscle. ET-1 -/- homozygous mice display a wide variety of cardiac anomalies, which also are features of the human and of the experimental CDH. Autopsy reports have shown that total heart weight is reduced significantly in the presence of CDH, and animal models have documented the presence of cardiac hypoplasia associated with CDH. Experimental and clinical studies have shown that prenatal exposure to corticosteroids improves cardiovascular function in the immediate newborn period. The aim of this study was to determine cardiac gene expression of ET-1 and of its receptor ET(A) and the cardiac ET-1 content in the heart of nitrofen-induced CDH in rats and to evaluate the effect of antenatal Dexamethasone (Dex) treatment. METHODS: A CDH model was induced in pregnant rats after administration of 100 mg of nitrofen on day 9.5 of gestation (term, 22 days). Dex (0.25 mg/kg) was given by intraperitoneal injection on days 18.5 and 19.5 of gestation. Cesarean section was performed on day 21 of gestation. The fetuses were divided into 3 groups: group I, control (n = 8); group II, nitrofen-induced CDH (n = 8); group III, nitrofen-induced CDH with antenatal Dex treatment (n = 8). ET-1 protein was measured using ELISA. RT-PCR was performed to evaluate the relative amount of ET-1 and ET(A) mRNA expression. RESULTS: There was a reduction in ET-1 mRNA (P <.05) and in ET(A) mRNA (P <.01) in the heart of CDH group compared with controls. ET-1 protein level also was reduced in heart of CDH compared with controls. Antenatal Dex treatment increased significantly both ET-1 mRNA and protein levels in the heart of CDH animals (P <.05 and P <.01, respectively). CONCLUSIONS: The reduced cardiac ET-1 gene expression and ET-1 synthesis may be responsible for the heart hypoplasia associated with CDH. Prenatal corticosteroids increase the cardiac production of ET-1, and this may enhance heart growth and cardiac inotropism at birth.CI - Copyright 2002, Elsevier Science (USA). All rights reserved.

High-altitude hypoxia causes pulmonary hypertension in humans and animals. Endothelin-1 (ET-1) is a novel and long-lasting vasoconstrictor. However, no study has dealt with the effects of a hypobaric hypoxic environment (HHE) on ET-1 activity in the brain. We examined 134 male rats permanently exposed to the equivalent of 5500 m altitude for 1 to 8 weeks. In these HHE rats, the mean pulmonary arterial pressure was significantly raised. The level of ET-1 protein, measured by enzyme immunoassay, increased rapidly in the lungs on exposure to HHE, but decreased in the brain. The level of ET-1 mRNA, measured by semiquantitative RT-PCR, was raised at 1, 4, and 6 weeks' exposure in the lungs and at 4 or more weeks' exposure in 3 of 8 brain regions. By in situ hybridization and immunohistochemistry of brain sections, ET-1 mRNA and protein were detected in the endothelial cells, neurons, and astrocyte-like cells in control rats. In HHE rats, the immunoreactive intensity for ET-1 protein decreased rapidly with time in these cells within the brain, although a few weakly ET-1 protein-positive cells were detected until 8 weeks' exposure to HHE. Only a few weakly ET-1 mRNA-positive endothelial cells were detected in any HHE rats. Although the reactivity for ET-1 mRNA had decreased significantly in neurons and astrocyte-like cells at 1 and 2 weeks' exposure to HHE, it was again strong in both types of cells at 4 weeks' exposure to HHE. These results raise the possibility that during exposure to HHE, ET-1 production in the lung may play a role in the development of pulmonary hypertension, while a decrease in ET-1 production within the brain may help to protect neurons by preventing or limiting the constriction of cerebral microvessels during the hypoxia induced by HHE.

The high mortality in patients with congenital diaphragmatic hernia (CDH) has been attributed to pulmonary hypoplasia and persistent pulmonary hypertension (PPH). Endothelin-1 (ET-1), nitric oxide (NO), and calcitonin gene-related peptide (CGRP) have been reported to be important vasoactive mediators in the perinatal pulmonary circulation. The exact mechanism by which these vasoactive mediators interact to regulate the perinatal pulmonary vascular tone in CDH with PPH is not fully understood. We hypothesized that the altered pulmonary vascular reactivity in CDH is due to imbalance in vasoactive mediators. This study was designed to investigate mRNA expression of ET-1, eNOS, and CGRP in CDH lung in the perinatal period. A CDH model was induced in pregnant rats following administration of nitrofen. In control animals, the same dose of olive oil was given without nitrofen. Cesarean section was performed on day 21 of gestation. The newborn rats were intubated and ventilated, and ventilation was continued for 1-6 h. Left lungs were collected from both groups at 0, 1, and 6 h after ventilation (n=8 in each group). Reverse transcriptase-polymerase chain reaction on lung tissue was performed to evaluate the relative level of ET-1, eNOS, and CGRP mRNA expression. The results showed a significant increase in ET-1 mRNA in CDH lung at 1 and 6 h after ventilation compared with controls. In CDH lung, eNOS mRNA and CGRP mRNA levels were significantly increased at 1 h but were similar to control values at 6 h after ventilation. The increased expression of vasoconstrictor ET-1 mRNA and vasodilators eNOS mRNA and CGRP mRNA in the CDH lung at 1 h after ventilation suggests that pulmonary vascular tone is rapidly changing after birth. An imbalance in the production of vasoconstrictors and vasodilators by the CDH lung may contribute to high pulmonary vascular resistance.

BACKGROUND: Endothelin-1 (ET-1) has potent vasoconstrictor and hypertrophic actions. Pharmacological antagonists of endothelin receptors attenuate cardiac hypertrophy, have been approved for treatment of pulmonary hypertension, and are under investigation for treatment of heart failure. To investigate the role of ET-1 in the heart, we created mice with cardiomyocyte deletion of ET-1. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of ET-1 are phenotypically normal when young. Remarkably, as the mice age or when young animals are subjected to aortic banding, they develop an unexpected phenotype of progressive systolic dysfunction and cardiac dilation. Echocardiography, necropsy, histology, and molecular phenotype confirm a dilated cardiomyopathy. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling analysis reveals greater abundance of apoptotic nuclei in the ET-1-deficient hearts. Transcriptional and Western analyses suggest enhanced tumor necrosis factor (TNF)-mediated apoptosis with increases in caspase-8 activity. These ET-1-deficient hearts also have diminished nuclear factor (NF)-kappaB activity, resulting in diminution of downstream inhibitors of TNF signaling. CONCLUSIONS: Local ET-1 gene expression is necessary to maintain normal cardiac function and cardiomyocyte survival in mice with both age and hemodynamic stress. This cardiac-protective effect is mediated by paracrine ET-1 modulation of TNF-related apoptosis, in part through upregulation of NF-kappaB signaling.

The aryl hydrocarbon receptor (AHR) is a basic helix-loop-helix Per-Arnt-Sim transcription factor that mediates induction of metabolic enzymes and toxicity of certain environmental pollutants. Although AHR knockout (KO) mice develop cardiac hypertrophy, conflicting reports associate this pathology with hypotension or endothelin (ET)-1-dependent hypertension. Because hypertension occurred at modest altitude, we tested the hypothesis that loss of AHR increases the sensitivity to hypoxia-induced ET-1, contributing to systemic hypertension. We found that AHR KO mice were hypertensive at modest altitude (1632 m) but hypotensive at low altitude (225 m). When AHR KO mice residing at 1632 m were exposed to the partial pressure of inspired oxygen (PIO(2)) at sea level for 11 days, blood pressure declined to levels measured at 225 m. Although plasma ET-1 in AHR KO mice was significantly elevated at 1632 m and decreased at 225 m and sea level PIO(2), pulmonary prepro-ET-1 mRNA was significantly reduced at 1632 m and decreased further at 225 m and sea level PIO(2). Blood gas analysis revealed that AHR KO mice were hypoxemic, hypercapnic, and acidotic at 1632 m, values that were attenuated and normalized after 24 hours and 11 days under sea level PIO(2), respectively. Lastly, AHR inactivation in endothelial cells by small interfering RNA significantly reduced basal prepro-ET-1 mRNA but did not alter hypoxia-induced expression. Our studies establish the AHR KO mouse as a model in which modest decreases in PIO(2) lead to hypoxemia, increased plasma ET-1, and systemic hypertension without increased pulmonary prepro-ET-1 mRNA expression.

Macrophage derived-endothelin-1 (ET-1) has been suggested to contribute to a number of chronic lung diseases. Whether the ET-1 cascade from non-vascular sources (inflammatory cells) also contributes to pulmonary artery hypertension (PAH) and in particular to heritable PAH (HPAH) with known bone morphogenetic protein type 2 receptor (BMPR2) mutations is not known. We tested this notion using bone marrow-derived macrophages (BMDM; precursors of tissue macrophages) isolated from ROSA26rtTAXTetO(7)-tet-BMPR2(R899X) mice (model of PAH with universal expression of a mutated BMPR2 gene) with and without activation by LPS and in human lung tissue from HPAH with BMPR2 mutations and idiopathic PAH (IPAH). At baseline ET(A) and ET(B) receptors and endothelin converting enzyme (ECE) gene expression was reduced in BMPR2 mutant BMDM compared with controls. In control BMDM, LPS resulted in increased ppET-1 gene expression and ET-1 in culture media, whereas ET(A) and ET(B) receptor and ECE gene expression was decreased. These findings were more severe in BMPR2 mutant BMDM. Antagonism of the ET(B) receptor resulted in increased ET-1 in the media, suggesting that decreased ET-1 uptake by the ET(B) receptor contributes to the elevation. While ET-1 expression was demonstrated in lung macrophages from controls and IPAH and HPAH patients, ET(A) and ET(B) expression was decreased in the HPAH, but not IPAH, patients compared with controls. We conclude that reduced expression of macrophage ET-1 receptors in HPAH increases lung ET-1 and may contribute to the pathogenesis and maintenance of HPAH. This is the first description of protein expression that distinguishes HPAH from IPAH in patients.

Macrophage derived-endothelin-1 (ET-1) has been suggested to contribute to a number of chronic lung diseases. Whether the ET-1 cascade from non-vascular sources (inflammatory cells) also contributes to pulmonary artery hypertension (PAH) and in particular to heritable PAH (HPAH) with known bone morphogenetic protein type 2 receptor (BMPR2) mutations is not known. We tested this notion using bone marrow-derived macrophages (BMDM; precursors of tissue macrophages) isolated from ROSA26rtTAXTetO(7)-tet-BMPR2(R899X) mice (model of PAH with universal expression of a mutated BMPR2 gene) with and without activation by LPS and in human lung tissue from HPAH with BMPR2 mutations and idiopathic PAH (IPAH). At baseline ET(A) and ET(B) receptors and endothelin converting enzyme (ECE) gene expression was reduced in BMPR2 mutant BMDM compared with controls. In control BMDM, LPS resulted in increased ppET-1 gene expression and ET-1 in culture media, whereas ET(A) and ET(B) receptor and ECE gene expression was decreased. These findings were more severe in BMPR2 mutant BMDM. Antagonism of the ET(B) receptor resulted in increased ET-1 in the media, suggesting that decreased ET-1 uptake by the ET(B) receptor contributes to the elevation. While ET-1 expression was demonstrated in lung macrophages from controls and IPAH and HPAH patients, ET(A) and ET(B) expression was decreased in the HPAH, but not IPAH, patients compared with controls. We conclude that reduced expression of macrophage ET-1 receptors in HPAH increases lung ET-1 and may contribute to the pathogenesis and maintenance of HPAH. This is the first description of protein expression that distinguishes HPAH from IPAH in patients.

Chronic pulmonary hypertension in infancy and childhood is characterized by a fixed and progressive increase in pulmonary arterial pressure and resistance, pulmonary arterial remodeling, and right ventricular hypertrophy and systolic dysfunction. These abnormalities are replicated in neonatal rats chronically exposed to hypoxia from birth in which increased activity of Rho-kinase (ROCK) is critical to injury, as evidenced by preventive effects of ROCK inhibitors. Our objective in the present study was to examine the reversing effects of a late or rescue approach to treatment with a ROCK inhibitor on the pulmonary and cardiac manifestations of established chronic hypoxic pulmonary hypertension. Rat pups were exposed to air or hypoxia (13% O(2)) from postnatal day 1 and were treated with Y-27632 (15 mg/kg) or saline vehicle by twice daily subcutaneous injection commencing on day 14, for up to 7 days. Treatment with Y-27632 significantly attenuated right ventricular hypertrophy, reversed arterial wall remodeling, and completely normalized right ventricular systolic function in hypoxia-exposed animals. Reversal of arterial wall remodeling was accompanied by increased apoptosis and attenuated content of endothelin (ET)-1 and ET(A) receptors. Treatment of primary cultured juvenile rat pulmonary artery smooth muscle cells with Y-27632 attenuated serum-stimulated ROCK activity and proliferation and increased apoptosis. Smooth muscle apoptosis was also induced by short interfering RNA-mediated knockdown of ROCK-II, but not of ROCK-I. We conclude that sustained rescue treatment with a ROCK inhibitor reversed both the hemodynamic and structural abnormalities of chronic hypoxic pulmonary hypertension in juvenile rats and normalized right ventricular systolic function. Attenuated expression and activity of ET-1 and its A-type receptor on pulmonary arterial smooth muscle was a likely contributor to the stimulatory effects of ROCK inhibition on apoptosis. In addition, our data suggest that ROCK-II may be dominant in enhancing survival of pulmonary arterial smooth muscle.

PURPOSE: Chronic pulmonary hypertension (PH) therapy is poorly investigated in intensive care. Our aim was to evaluate haemodynamic and neuroendocrine effects of the dual endothelin-1 (ET-1) blocker tezosentan in monocrotaline (MCT)-induced PH. METHODS: Male Wistar rats (180-200 g, n = 194) randomly received 60 mg kg(-1) MCT or vehicle, subcutaneously, and 2 days later, a subgroup of MCT-injected rats was gavaged with 300 mg kg(-1) day(-1) bosentan (MCT BOS, n = 46), while another (MCT, n = 125) and control rats (Ctrl, n = 23) received vehicle. At 25-30 days, 48 h after interrupting bosentan, rats randomly underwent either a dose-response evaluation (0.5-20 mg kg(-1), n = 7 each group) or a 4 h perfusion of tezosentan (20 mg kg(-1) in 10 min + 10 mg g(-1) h(-1)) or vehicle (n = 8 per group, each). Haemodynamics, including blood gas analysis, were evaluated after thoracotomy under anaesthesia. After plasma, right ventricle (RV) and lung collection, plasma ET-1, cytokines, nitrate and 6-keto-PGF1alpha, and lung and right ventricular gene expression and cyclooxygenase (COX) and nitric oxide synthase (NOS) activities were quantified. RESULTS: Monocrotaline resulted in PH, RV dilation and decreased cardiac output (CO) that were attenuated in MCT BOS. pulmonary hypertension was attenuated by tezosentan without systemic hypotension. Tezosentan increased CO without changing ventilation-perfusion matching. Both bosentan and tezosentan reduced ET-1 and cytokine plasma levels and tissue expression, and inducible NOS and COX-2 RV activities. Bosentan increased nitrate plasma levels and non inducible NOS activities whereas tezosentan decreased circulating 6-keto-PGF1alpha but increased lung COX-1 activity. CONCLUSIONS: Tezosentan may be useful for haemodynamic handling and bosentan replacement in critically ill PH patients exerting important beneficial neuroendocrine and anti-inflammatory actions.

The assessment of the indicators of functional state of endothelium (endothelin-1 level and von Willebrand factor activity) was implemented in healthy children and patients with bronchopulmonary pathology with normal and high pressure in pulmonary artery It is established that pulmonary hypertension in children with chronic bronchopulmonary pathology is associated with the endothelium dysfunction (increase of endothelin-1 concentration and activity of von Willebrand factor). The direct dependence of evidence of the pulmonary hypertension from the level of endothelin-1 and activity of von Willebrand factor is proved. The increase of the level of endothelin-1 and the activity of von Willebrand factor is a risk factor of the development of pulmonary hypertension in children with chronic bronchopulmonary pathology.FAU - Agapitov, L I