The role of endothelin-1 and the endothelin B receptor in the pathogenesis of hepatopulmonary syndrome in the rat

Endothelin-1 (ET-1) stimulation of endothelial nitric oxide synthase (eNOS) via pulmonary endothelial endothelin B (ET(B)) receptors and pulmonary intravascular macrophage accumulation with expression of inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1) are implicated in experimental hepatopulmonary syndrome (HPS) after common bile duct ligation (CBDL). Our aim was to evaluate the role of ET-1 in the development of experimental HPS. The time course of molecular and physiological changes of HPS and the effects of selective endothelin receptor antagonists in vivo were assessed after CBDL. Effects of ET-1 on intralobar pulmonary vascular segment reactivity and on eNOS expression and activity in rat pulmonary microvascular endothelial cells (RPMVECs) were also evaluated. Hepatic and plasma ET-1 levels increased 1 week after CBDL in association with a subsequent increase in pulmonary microvascular eNOS and ET(B) receptor levels and the onset of HPS. Selective ET(B) receptor inhibition in vivo significantly decreased pulmonary eNOS and ET(B) receptor levels and ameliorated HPS. CBDL pulmonary artery segments had markedly increased ET(B) receptor mediated, nitric oxide dependent vasodilatory responses to ET-1 compared with controls and ET-1 triggered an ET(B) receptor dependent stimulation of eNOS in RPMVECs. Pulmonary intravascular macrophages also accumulated after CBDL and expressed HO-1 and iNOS at 3 weeks. Selective ET(B) receptor blockade also decreased macrophage accumulation and iNOS production. In conclusion, ET-1 plays a central role in modulating pulmonary micovascular tone in experimental HPS.     

Nitric oxide impacts endothelin-1 gene expression in intrapulmonary arteries of chronically hypoxic rats.

This study aimed to investigate whether nitric oxide (NO) could inhibit the elevated endothelin-1 (ET-1) gene expression by pulmonary artery endothelial cells or smooth muscle cells in chronically hypoxic rats by use of in situ hybridization. Male Wistar rats (n = 40) were randomly divided into 1-week hypoxia group, 1-week hypoxia with L-arginine (L-arg) group, 1-week hypoxia with N(omega)-nitro-L-arginine methyl ester (L-NAME) group, 2-week hypoxia group, 2-week hypoxia with L-arg group, and 2-week hypoxia with L-NAME group. All rats were put into a normobaric hypoxic chamber with an oxygen concentration of 10 +/- 0.5% for hypoxic challenge. The results showed that most pulmonary arteries had 1-50% of the endothelial cells showing positive signals for ET-1 expression in hypoxic rats, which was significantly suppressed by L-arg. L-NAME, however, significantly augmented ET-1 gene expression in pulmonary artery endothelial cells and smooth muscle cells. The results suggest that endogenous NO markedly inhibits ET-1 mRNA expression in both pulmonary artery endothelial cells and smooth muscle cells in chronically hypoxic rats, which may be one of the mechanisms by which NO modulates hypoxic pulmonary circulation.     

Sensitivity to endothelin-1 is decreased in isolated livers of endothelial constitutive nitric oxide synthase knockout mice

Background  

Hepatic sinusoidal resistance is regulated by vasoactive factors including endothelin-1 (ET-1) and nitric oxide (NO). In the absence of NO, vasoconstrictor response to endothelin is expected to predominate. Therefore, we hypothesized sensitivity to endothelin to be increased in mice lacking the endothelial cell NO synthase gene. Response of vascular resistance to endothelin was assessed in the in situ perfused liver of endothelial constitutive nitric oxide synthase (ecNOS) knockout and wild type mice. Livers were also harvested for RNA and protein isolation for quantitative PCR and Western blotting, respectively. The expression of endothelin receptors, isoenzymes of NO synthase, heme-oxygenase and adrenomedullin was quantified.     

Mediators of asthma: nitric oxide

Endogenous nitric oxide is an ubiquitous gaseous molecule that regulates many aspects of human airway biology including the modulation of airway and vascular smooth muscle tone. It is generated from the three different enzymes nitric oxide synthases (NOS) -1, -2 and -3 which are all expressed in pulmonary cells. NOS-1 is localised primarily to neuronal structures, where NO is a mediator of the inhibitory Non-Adrenergic Non-Cholinergic System and NOS-3 is present in endothelial cells. While these enzymes are constitutively expressed, NOS-2 is an inducible enzyme independent of calcium and highly induced in inflammatory diseases such as allergic asthma, where NO may act beneficial or deleterious depending on the site of and amount of generation. The use of NO-donor compounds or classical unselective NOS inhibitors did not lead to significant therapeutical effects in asthmatic patients. Insights on the precise role of NO in asthma can only be achieved by targeting NO generation selectively. More potent and selective NOS-2 inhibitors have to clarify a role of NOS-modification based therapy in clinical routine. NO can also be detected in the exhaled air. Increased levels of exhaled NO in asthmatic patients may be useful for a non-invasive determination of airway inflammation.     

冠状动脉搭桥材料的内皮细胞分泌的一氧化氮和内皮素水平研究

目的检测冠状动脉搭桥患者桡动脉和大隐静脉内皮细胞所分泌的一氧化氮(NO)和内皮素(ET)水平,并与脐静脉内皮细胞相比较,了解内皮细胞的功能。方法利用剩余的冠状动脉搭桥移植材料,采用Ⅱ型胶原酶消化的方法获取内皮细胞并培养,收集培养细胞的上清液,利用试剂盒检测内皮细胞分泌的NO和ET水平。结果与人正常脐静脉内皮细胞相比,冠状动脉搭桥材料桡动脉和大隐静脉内皮细胞分泌的NO水平显著降低(P<0.05),ET水平显著增高(P<0.05)。结论冠状动脉搭桥患者桡动脉和大隐静脉的内皮细胞与脐静脉内皮细胞相比,所分泌的NO和ET差异有统计学意义,冠状动脉搭桥患者搭桥材料的内皮细胞分泌功能可能存在功能障碍。     

Dehydroepiandrosterone upregulates soluble guanylate cyclase and inhibits hypoxic pulmonary hypertension

OBJECTIVE: It has been reported that dehydroepiandrosterone is a pulmonary vasodilator and inhibits chronic hypoxia-induced pulmonary hypertension. Additionally, dehydroepiandrosterone has been shown to improve systemic vascular endothelial function. Thus, we hypothesized that chronic treatment with dehydroepiandrosterone would attenuate hypoxic pulmonary hypertension by enhancing pulmonary artery endothelial function. METHODS AND RESULTS: Rats were randomly assigned to five groups. Three groups received food containing 0, 0.3, or 1% dehydroepiandrosterone during a 3-wk-exposure to simulated high altitude (HA). The other 2 groups were kept at Denver's low altitude (LA) and received food containing 0 or 1% dehydroepiandrosterone. Dehydroepiandrosterone dose-dependently inhibited hypoxic pulmonary hypertension (mean pulmonary artery pressures after treatment with 0, 0.3, and 1% dehydroepiandrosterone=45+/-5, 33+/-2*, and 25+/-1*# mmHg, respectively. *P<0.05 vs. 0% and # vs. 0.3%). Dehydroepiandrosterone (1%, 3 wks) treatment started after rats had been exposed to 3-wk hypoxia also effectively reversed established hypoxic pulmonary hypertension. Pulmonary artery rings isolated from both LA and HA rats treated with 1% dehydroepiandrosterone showed enhanced relaxations to acetylcholine and sodium nitroprusside, but not to 8-bromo-cGMP. In the pulmonary artery tissue from dehydroepiandrosterone-treated LA and HA rats, soluble guanylate cyclase, but not endothelial nitric oxide synthase, protein levels were increased. CONCLUSION: These results indicate that the protective effect of dehydroepiandrosterone against hypoxic pulmonary hypertension may involve upregulation of pulmonary artery soluble guanylate cyclase protein expression and augmented pulmonary artery vasodilator responsiveness to nitric oxide.     

Pulmonary Expression of iNOS and HO-1 Protein Is Upregulated in a Rat Model of Prehepatic Portal Hypertension

Portal hypertension is associated with a wide range of pulmonary pathophysiologies, ranging from portopulmonary hypertension to hepatopulmonary syndrome. Although the clinical and pathological features of pulmonary dysfunction in this setting have been extensively characterized, the underlying biology is not well understood. Specifically, the role of mediators that regulate mesenteric vascular hemodynamics in portal hypertension, such as nitric oxide and endothelin, have not been studied in the lung. Using a rat model of prehepatic portal hypertension with preserved hepatic function, we examined pulmonary elaboration of endothelial nitric oxide synthase (NOS), inducible NOS, heme oxygenase- 1 (HO-1), heme oxygenase-2 (HO-2), endothelin-1 mRNA, and protein. In comparison to sham controls, portal hypertensive animals exhibited significantly increased pulmonary iNOS and HO-1 mRNA and protein. Cyclic GMP was significantly increased in portal hypertensive lung tissue, suggesting activation of guanylyl cyclase by the endproducts of iNOS and/or HO-1 activity. Using immunohistochemical analysis, iNOS expression was localized to the vascular endothelium, while HO-1 localized to bronchiolar epithelium and macrophages. These results suggest that production of nitric oxide and carbon monoxide may contribute to the pulmonary pathology associated with portal hypertension.     

Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension

OBJECTIVE: The regulation of pulmonary prostacyclin synthesis is not completely understood. We tested the hypothesis that prostacyclin production is predominantly stimulated by hemodynamic factors, such as increased shear-stress, and is thus increased in rats with chronic hypoxic pulmonary hypertension. METHODS: To this end, we determined pulmonary prostacyclin synthase (PGIS) gene expression, circulating levels of the stable prostacyclin metabolite 6-keto prostaglandin F(1alpha) (6-keto-PGF(1alpha)), pulmonary endothelin (ET)-1 gene expression, and ET-1 plasma levels in rats exposed to 4 weeks of hypoxia (10% O(2)) in the presence or absence of either the nitric oxide (NO) donor molsidomine (MD, 15 mg/kg/day) or the ET-A receptor antagonist LU135252 (LU, 50 mg/kg/day). RESULTS: Right ventricular systolic pressure (RVSP), the cross-sectional medial vascular wall area of pulmonary arteries, and ET-1 production increased significantly during hypoxia. PGIS mRNA levels increased 1.7-fold, and 6-keto-PGF(1alpha) plasma levels rose from 8.2+/-0.8 to 12.2+/-2.2 ng/ml during hypoxia (each P<0.05 vs. normoxic controls). MD and LU reduced RVSP and pulmonary vascular remodeling similarly (each P<0.05 vs. hypoxia), but only MD inhibited pulmonary ET-1 formation (P<0.05 vs. hypoxia). Nevertheless, both drugs attenuated the increase in PGIS gene expression and plasma 6-keto-PGF(1alpha) levels (each P<0.05 vs. hypoxia). CONCLUSION: Our data suggest that prostacyclin production in hypertensive rat lungs is predominantly increased by hemodynamic factors while hypoxia, NO and ET-1 per are less important stimuli, and that this increase may serve as a compensatory mechanism to partially negate the hypoxia-induced elevation in pulmonary vascular tone.     

Expression of tumour necrosis factor alpha and accumulation of fibronectin in coronary artery restenotic lesions retrieved by atherectomy.

BACKGROUND--The formation of coronary artery neointima experimentally induced in piglets after cardiac transplantation is related to an immune-inflammatory reaction associated with increased expression of T cells and inflammatory mediators (tumour necrosis factor alpha and interleukin 1 beta) and upregulation of fibronectin. In vivo blockade of tumour necrosis factor alpha in rabbits after cardiac transplantation results in reduced neointimal formation. The objective of this study was to investigate the hypothesis that coronary restenosis after atherectomy or percutaneous balloon angioplasty is associated with a similar inflammatory cascade initiated by mechanical injury. METHODS--Specimens taken at coronary atherectomy were analysed from 16 patients. Nine had had the procedure performed twice, firstly, to remove a primary lesion, and secondly, to remove a restenotic lesion. Seven had percutaneous balloon angioplasty after removal of restenotic tissue. Coronary atherectomy specimens were analysed by immunohistochemistry for the presence of T cells, macrophages, major histocompatibility complex II, interleukin 1 beta, tumour necrosis factor alpha, fibronectin, and the receptor for hyaluronan mediated motility. RESULTS--The groups were clinically and angiographically similar with equivalent lumens before and after atherectomy. Restenotic lesions had increased expression of tumour necrosis factor alpha and fibronectin compared with the primary lesions (P < 0.05 for both). There was also a trend towards a greater number of T cells and increased expression of interleukin 1 beta. CONCLUSIONS--Restenosis is associated with increased expression of tumour necrosis factor alpha and fibronectin, suggesting that an immune-inflammatory reaction probably contributes to neointimal formation and may represent a form of wound healing and repair secondary to mechanical injury.     

Maturational changes of endothelial vasoactive factors and pulmonary vascular tone at birth.

The aim of this study was to determine which endothelial factors were involved in the decrease of pulmonary vascular resistance at birth, and how they changed with maturation. Response of intrapulmonary artery rings precontracted with prostaglandin F2alpha were studied from piglets aged <2 h, 2-3 day, 10 day and adult pigs for pharmacological responses to acetylcholine (ACh) and cromakalim (CMK) in the presence and the absence of the nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NA), the adenosine triphosphate sensitive potassium (K(ATP)) channel blocker, glibenclamide and the endothelin (ET)-A receptor antagonist, BQ123. In situ hybridization and immunochemistry studies were performed in lung tissues of the same animals in order to determine the expression of NOS and ET. There was a small contractile effect of ACh in the newborn. Relaxation to ACh, which was blocked by L-NA and reduced by glibenclamide, only appeared from the age of 3 days. The significantly greater relaxation to CMK in rings without endothelium (p<0.05) was abolished by BQ123 in the newborn, and then disappeared by 2 days of age. Glibenclamide had a greater inhibitory effect on relaxation induced by CMK at 10 days than in the newborn and 2 days old piglets. NOS expression was low in pulmonary arteries of the newborn and increased by 2 days of age whereas the converse was seen with ET expression. It is concluded that: 1) relaxant response to acetylcholine was absent at birth and appeared at 2 days; 2) the reduced relaxant response to cromakalin in rings with endothelium at birth could be blocked by BQ123; and 3) the expression of endothelin decreased whereas the expression of nitric oxide synthase increased from birth to 2 days of age.     

Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers

Cigarette smoking has been associated with alterations in the structure and endothelial function of pulmonary arteries. Nitric oxide (NO) and endothelin-1 are endothelium-derived mediators with opposite effects on vascular tone and cell growth. To investigate whether cigarette smoking could induce changes in the synthesis of these mediators in pulmonary arteries, we compared the expression of both endothelial NO synthase (eNOS) and endothelin-1 in the lungs of smokers with that in nonsmokers. Lung tissue samples of 23 smokers and nine nonsmokers were studied. Expression of eNOS and endothelin-1 in pulmonary artery endothelium was evaluated by immunohistochemistry. In protein extracts of lung tissue, the content of eNOS protein was assessed by Western blot analysis and that of endothelin-1 by radioimmunoassay. The immunohistochemical expression of eNOS in arterial endothelium and the eNOS protein content in lung tissue were lower in the smokers than in the nonsmokers. No differences were shown in cell expression and protein content of endothelin-1 between both groups. We conclude that cigarette smoking is associated with reduced expression of eNOS in pulmonary arteries. The diminished synthesis of nitric oxide may contribute to the alterations in the structure and endothelial function of pulmonary vessels in cigarette-smoke-induced respiratory disease.     

Attenuation of NF-kappaB signalling in rat cardiomyocytes at birth restricts the induction of inflammatory genes

OBJECTIVES AND BACKGROUND: The expression of inflammatory genes in the heart plays an important role in organ dysfunction. However, the contribution of cardiomyocytes to this process, and in particular to the synthesis of high concentrations of nitric oxide and prostaglandins, has not been analyzed in detail. For this reason, cultured isolated cardiomyocytes were used to evaluate the response to pro-inflammatory stimuli. METHODS AND RESULTS: Isolated cultured foetal, neonatal, and adult rat cardiomyocytes were stimulated with lipopolysaccharide and cytokines, and the expression of nitric oxide synthase-2 (NOS-2) and cyclooxygenase-2 (COX-2) in these cells was investigated. Only cultured foetal cardiomyocytes expressed NOS-2 and COX-2 under these conditions, whereas the neonatal counterparts required various days in culture to gain this response. Analysis of the NF-kappaB signalling pathway showed an impaired activation of IkappaB kinase in response to lipopolysaccharide and cytokines in cells maintained in culture for 1 day. These data were confirmed by DNA microarray analysis. However, other early signalling pathways, such as the p38 and Erk MAPKs, were not affected by the time in culture. CONCLUSIONS: Neonatal and adult cardiomyocytes are resistant to the expression of pro-inflammatory genes due to impairment in the activation of IkappaB kinase, a process that might contribute to preventing rapid organ dysfunction in the course of various inflammatory pathologies, such as septic shock and myocarditis.     

Acute coronary artery injury in dogs following administration of CI-1034, an endothelin A receptor antagonist

The objective of this study was to characterize acute coronary artery injury evoked by the endothelin A receptor (ET_AR) antagonist, CI-1034. Male dogs (n=5) were intravenously administered CI-1034 at 120 mg/kg for 4 d. Control animals (n=3) received vehicle. Macroscopically, drug-related hemorrhage was observed in the right coronary groove and atrium. Histologically, drug-related coronary changes were characterized as medial hemorrhage and necrosis, with mixed inflammatory-cell infiltrates in the adventitia and media. Immunohistochemistry staining indicated increased expression of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and S100A8/A9 (within in monocytes and neutrophils) proteins in coronary arteries of CI-1034-treated animals. However, there were similar expression levels of endothelial nitric oxide synthase (eNOS) among control and CI-1034-treated animals. Significant drug-related nitric oxide (NO) accumulation occurred on days 1 through 4 in serum. Increased interleukin (IL)-6 and fibrinogen in plasma and serum amyloid A (SAA) occurred on days 2 through 5 in CI-1034-treated animals. Increased levels of NO accumulation in serum; increased IL-6 and fibrinogen levels in plasma; increased SAA levels; and increased expressions of iNOS, cleaved caspase-3, and S100A8/A9 complex appear to be characteristic of CI-1034-induced acute vascular injury in dogs.     

Quantitative analysis of endothelin and nitric oxide synthase in rat model of postobstructive pulmonary vasculopathy

There is increasing evidence that endothelin (ET) and endothelial nitric oxide synthase (eNOS) may contribute various kinds of pulmonary vascular remodeling, including postobstructive pulmonary vasculopathy (POPV), which resulted from chronic ligation of unilateral pulmonary artery. The aim of this study was to investigate the expression of ET-1, ET-A receptor, ET-B receptor, and eNOS quantitatively in POPV rats. One month after a left thoracotomy with either left main pulmonary artery ligation (ligated group) or no ligation (control group), rat pulmonary arteries and lungs were used for Western blot analysis using specific antibodies against ET-1, ET-A receptor, ET-B receptor, and eNOS. ET-A receptor was more highly expressed in the pulmonary arteries of ligated rats compared to the control. The expression of ET-1, ET-B receptor,and eNOS was not different between ligated and control rats. These findings suggest that ET-A receptor overexpression would play a main role for pulmonary arterial remodeling in POPV rats, whereas eNOS may serve as a compensatory mediator.     

Elevated vascular endothelial growth factor is correlated with elevated erythropoietin in stable, young cystic fibrosis patients

Angiogenesis is an important mechanism of airway remodeling in lung disease. We previously demonstrated that serum vascular endothelial growth factor (VEGF) is elevated in cystic fibrosis (CF) patients and declines with therapy for pulmonary exacerbation. We hypothesized that VEGF is elevated early in the course of CF and is associated with markers of tissue hypoxia. A prospective, single-visit evaluation of thirty stable infants and children with CF was performed. Serum was analyzed for VEGF and for other markers of tissue hypoxia (erythropoietin (EPO), insulin-like growth factor binding protein-1 (IGFBP-1)) and for inflammatory mediators (IL-1 beta, IL-6, IL-8, and tumor necrosis factor alpha (TNFα)) using Meso Scale multi-spot serum immunoassays. Measurements were correlated between assay groups; and with age in months and pulmonary function (FEV0.5 or FEV1). VEGF, EPO, TNFα and IL-8 were elevated compared to published normative values. VEGF levels were not significantly correlated with any inflammatory mediators. However, VEGF correlated with EPO (r=0.505; P<0.05). There was no correlation between lung function and markers of inflammation or tissue hypoxia. VEGF is elevated in young, stable infants and children suggesting angiogenesis as a contributing mechanism for early lung disease in CF. VEGF elevation does not show significant correlation with inflammatory mediators known to be increased in CF, but is significantly correlated with EPO levels. We propose that VEGF elevation and angiogenesis contribute to early lung disease and may result from a direct tissue hypoxia pathway in CF.