Endothelin-1 Gene Research Articles

Novel Regulation of Endothelin-1 Promoter Activity by Protein Kinase C

Endothelin-1 (ET-1) is produced in unusually large amounts by the renal collecting duct and acts locally to control renal salt and water excretion and arterial pressure; disorders of collecting duct ET-1 activity can cause marked hypertension. The mechanisms regulating collecting duct ET-1 synthesis are, however, poorly understood. In this study, we investigated the role of protein kinase C (PKC), a known regulator of ET-1 production in endothelial cells, in (1) the control of collecting duct ET-1 production; and (2) the modulation of ET-1 promoter region activity. Cultured rat inner medullary collecting duct (IMCD) cells were studied. Calphostin C, a PKC inhibitor, greatly reduced IMCD ET-1 release. Sustained exposure to phorbol myristate acetate (PMA) also decreased ET-1 secretion. PKC inhibition decreased steady-state ET-1 mRNA content. A brief exposure (15 min) to PMA augmented ET-1 mRNA levels, while prolonged PMA exposure (120 min) reduced ET-1 mRNA content, PKC inhibition did not affect ET-1 mRNA stability. Transfection of ET-1 promoter-luciferase reporter constructs into IMCD cells demonstrated that PKC inhibition reduced activity of only the larger promoter fragments (containing at least 1,725 bp 5' to the ET-1 gene transcription start site). Mutation of a previously identified AP-1 site at -186 in the ET-1 promoter greatly reduced activity of transfected ET-1 promoter-reporter constructs (containing 366 or 1,725 bp 5' to the transcription start site); however, this region appears not to be regulated by PKC in IMCD cells. In summary, PKC stimulates collecting duct ET-1 synthesis via transcriptional activation of the ET-1 promoter. Such transcriptional activation occurs at a heretofore undescribed PKC-regulated region of the ET-1 promoter.

Human Lymphatic Filariasis: Genetic Polymorphism of Endothelin-1 and Tumor Necrosis Factor Receptor II Correlates With Development of Chronic Disease

Hydrocele and elephantiasis are 2 clinically very diverse and often mutually exclusive chronic manifestations of human bancroftian filariasis. Plasma levels of endothelin-1 (ET-1), a major angiogenic factor, and tumor necrosis factor receptors (TNFRs) that regulate host inflammation have been associated with development of chronic filariasis, although their genetic basis are not known. We studied polymorphisms of ET-1 (Ala288Ser) and TNFR-II (Met196Arg) genes by means of the polymerase chain reaction confronting 2 pairs primers method and restriction fragment length polymorphism, respectively. Plasma ET-1 level was measured by enzyme-linked immunosorbent assay. Met196Arg genotype frequency of TNFR-II polymorphism was significantly greater in hydrocele patients, compared with elephantiasis patients (OR, 4.34 [95% CI, 2.04-9.20]). Conversely, a significantly high prevalence of the Ala288Ser mutation of ET-1 was observed in elephantiasis patients, compared with hydrocele cases (OR, 2.15 [95% CI, 1.13-4.10]). Decreased plasma ET-1 levels associated significantly with Ala288Ser mutation in the study population. A combined analysis indicated a 23-fold higher risk for developing elephantiasis in individuals with TNFR-II (Met196Met) and ET-1 mutants (Ala288Ser + Ser288Ser). ET-1 (Ala288Ser) and TNFR-II (Met196Arg) polymorphisms are associated with development of one or the other form of chronic disease in bancroftian filariasis.

Polymorphisms in the Endothelin-1 and Endothelin A Receptor Genes and Survival in Patients with Locoregionally Advanced Nasopharyngeal Carcinoma

We aimed to investigate the prognostic role of endothelin-1 (EDN1) and endothelin A receptor (EDNRA) gene polymorphisms in patients with locoregionally advanced nasopharyngeal carcinoma (NPC). Two hundred three consecutive patients with locoregionally advanced NPC were enrolled. Seven potentially functional polymorphisms in the EDN1 and EDNRA genes were determined by ligase detection reaction-PCR method from prospectively collected blood samples. The influence of the genetic polymorphisms on patient overall survival (OS) was analyzed using Cox proportional hazards model, Kaplan-Meier method, and the log-rank test. The 5-year OS in patients with EDNRA/H323H TT, TC, and CC genotypes were 81.3%, 62.1%, and 75.0%, respectively (P = 0.004). Patients carrying the heterozygous (TC) or homozygous variant (CC) genotype in EDNRA/H323H were combined for analysis, which revealed that the 5-year OS in patients with TC/CC genotypes was significantly lower than those with the wild-type TT genotype (63.2% vs. 81.3%; P = 0.002). Multivariate analysis showed that EDNRA/H323H polymorphism (HR: 1.95; 95% CI: 1.18-3.23; P = 0.009) and N classification (HR: 1.35; 95% CI: 1.03-1.79; P = 0.03) were independent significant prognostic factors for OS in patients with locoregionally advanced NPC. In contrast, the EDN1 polymorphisms revealed no prognostic value. The EDNRA/H323H polymorphism was a novel and independent prognostic marker for patients with locoregionally advanced NPC. The analysis of EDNRA/H323H polymorphism may help identify patient subgroups at high risk for poor disease outcome.

Loss of visceral pain following colorectal distension in an endothelin‐3 deficient mouse model of Hirschsprung's disease

Endothelin peptides and their endogenous receptors play a major role in nociception in a variety of different organs. They also play an essential role in the development of the enteric nervous system. Mice with deletions of the endothelin-3 gene (lethal spotted mice, ls/ls) develop congenital aganglionosis. However, little is known about how nociception might be affected in the aganglionic rectum of mice deficient in endothelin-3. In this study we investigated changes in spinal afferent innervation and visceral pain transmission from the aganglionic rectum in ls/ls mice. Electromyogram recordings from anaesthetized ls/ls mice revealed a deficit in visceromotor responses arising from the aganglionic colorectum in response to noxious colorectal distension. Loss of visceromotor responses (VMRs) in ls/ls mice was selective, as no reduction in VMRs was detected after stimulation of the bladder or somatic organs. Calcitonin gene related peptide (CGRP) immunoreactivity, retrograde neuronal tracing and extracellular afferent recordings from the aganglionic rectum revealed decreased colorectal spinal innervation, combined with a reduction in mechanosensitivity of rectal afferents. The sensory defect in ls/ls mice is primarily associated with changes in low threshold wide dynamic range rectal afferents. In conclusion, disruption of endothelin 3 gene expression not only affects development and function of the enteric nervous system, but also specific classes of spinal rectal mechanoreceptors, which are required for visceral nociception from the colorectum.

The impact of endothelin-1 genetic analysis and job strain on ambulatory blood pressure

ObjectiveAn interaction between the endothelin-1 gene (EDN1), blood pressure (BP) and social determinants has been previously found. Using a well-characterized cohort of participants, the impact of associations between genetic factors and job strain on BP was evaluated. MethodsA cross-sectional analysis of five polymorphisms covering the EDN1, of which 2 were previously reported to be associated with BP, was performed. Study subjects had previously completed a baseline evaluation including 24-h ambulatory BP monitoring and an assessment of job strain. This report presents the findings for 184 subjects who gave DNA samples for genetic analysis. One-way analysis of variance (ANOVA) was performed between each genetic marker and 24-h systolic blood pressure (SBP) and diastolic blood pressure (DBP), as well as two-way ANOVAs to test the interaction effect with job strain. ResultsTrends for relationships were observed between SBP and two polymorphisms: rs10478694 and rs5369. An interaction between job strain and those heterozygous for two polymorphisms showed higher SBP (P=.029 and .008) and a tendency for higher DBP. All findings were more significant when analyses were confined to the 139 Caucasian subjects. ConclusionThis is the first study to report an interaction between the EDN1 gene, job strain and BP, supporting previous evidence of a role of this gene in the interaction between environmental stress and ambulatory BP. Given the limited sample size, the results should be considered preliminary, and further studies are required.

Darbepoetin alfa suppresses tumor necrosis factor-α-induced endothelin-1 production through antioxidant action in human aortic endothelial cells: Role of sialic acid residues

Recombinant human erythropoietin (r-HuEPO) is widely used to correct anemia in end-stage renal disease patients, who commonly suffer from atherosclerosis. Endothelin-1 (ET-1) has been implicated in the pathogenesis of atherosclerosis. Here, we tested whether darbepoetin alfa, a hypersialylated analogue of r-HuEPO, regulates tumor necrosis factor-α (TNF-α)-induced ET-1 production in human aortic endothelial cells, and sought to identify the signal pathways involved. Darbepoetin alfa attenuated TNF-α-induced ET-1 production. It also diminished TNF-α-induced reactive oxygen species (ROS) accumulation and subsequent activation of c-Jun NH2-terminal kinase (JNK), which regulates the DNA-binding activities of both AP-1 and NF-κB required for ET-1 gene transcription. Like a JNK inhibitor, darbepoetin alfa did not affect IκBα degradation or p65 nuclear translocation, but did inhibit mitogen- and stress-activated protein kinase 1 (MSK1) activation and attenuated p65 phosphorylation (serine 276), effects that may account for the reduction in NF-κB DNA-binding activity. Desialylation completely abolished darbepoetin alfa's inhibitory effects on TNF-α-induced ROS accumulation, MSK1 activation, and ET-1 gene expression, without affecting its stimulation of STAT5 activity. These data demonstrate that darbepoetin alfa suppresses TNF-α-induced ET-1 production through its antioxidant action and suggest that the sialic acid residues of darbepoetin alfa are essential for its antioxidant effect, possibly by scavenging ROS.

Clinical significance of the presence of endothelin-1 gene rs1800541 and rs5370 SNPs in patients with acute ischemic stroke

Clinical significance of the presence of endothelin-1 gene rs1800541 and rs5370 SNPs in patients with acute ischemic stroke

SNP-by-fitness and SNP-by-BMI interactions from seven candidate genes and incident hypertension after 20 years of follow-up: the CARDIA Fitness Study

The association of SNPs from seven candidate genes, including genotype-by-baseline fitness and genotype-by-baseline body mass index (BMI) interactions, with incident hypertension over 20 years was investigated in 2663 participants (1301 blacks, 1362 whites) of the Coronary Artery Risk Development in Young Adults Study (CARDIA) Study. Baseline cardiorespiratory fitness was determined from duration of a modified Balke treadmill test. A total of 98 SNPs in blacks and 89 SNPs in whites from seven candidate genes were genotyped. Participants that became hypertensive (295 blacks and 146 whites) had significantly higher blood pressure and BMI (both races), and lower fitness (blacks only) at baseline than those who remained normotensive. Markers at the PPARGC1A and BDKRB2 genes were nominally associated with greater risk of hypertension, while one marker each at the BDKRB2 and NOS3 genes were nominally associated with lower risk. The association of baseline fitness with risk of hypertension was nominally modified by genotype at markers within the ACE, AGT, BDKRB2, and NOS3 genes in blacks and the BDKRB2, EDN1, and PPARGC1A genes in whites. BDKRB2 rs4900318 showed nominal interactions with baseline fitness on the risk of hypertension in both races. The association of baseline BMI with risk of hypertension was nominally modified by GNB3 rs2301339 genotype in whites. None of the above associations were statistically significant after correcting for multiple testing. We found that SNPs in these candidate genes did not modify the association between baseline fitness or BMI and risk of hypertension in CARDIA participants.

Endothelin‐1 gene regulation

Over two decades of research have demonstrated that the peptide hormone endothelin-1 (ET-1) plays multiple, complex roles in cardiovascular, neural, pulmonary, reproductive, and renal physiology. Differential and tissue-specific production of ET-1 must be tightly regulated in order to preserve these biologically diverse actions. The primary mechanism thought to control ET-1 bioavailability is the rate of transcription from the ET-1 gene (edn1). Studies conducted on a variety of cell types have identified key transcription factors that govern edn1 expression. With few exceptions, the cis-acting elements bound by these factors have been mapped in the edn1 regulatory region. Recent evidence has revealed new roles for some factors originally believed to regulate edn1 in a tissue or hormone-specific manner. In addition, other mechanisms involved in epigenetic regulation and mRNA stability have emerged as important processes for regulated edn1 expression. The goal of this review is to provide a comprehensive overview of the specific factors and signaling systems that govern edn1 activity at the molecular level.

Teratogenic Effects of Blue Cohosh (Caulophyllum thalictroides) in Japanese Medaka (Oryzias latipes) Are Probably Mediated through GATA2/EDN1 Signaling Pathway

Blue cohosh (Caulophyllum thalictroides) (BC) has been used widely to induce labor and to treat other uterine conditions. However, the safety and effectiveness of this herbal product has not yet been evaluated by the US Food and Drug Administration (FDA). Several conflicting reports indicated that the root extract of BC is a teratogen and, by some unknown mechanisms, is able to induce cardiovascular malfunctions in new-born babies. To understand the mechanism, we have used Japanese medaka (Oryzias latipes) embryo-larval development as the experimental model and the methanolic extract of BC root as the teratogen. The embryo mortality, hatching efficiency, and morphological abnormalities in craniofacial and cardiovascular systems are considered for the evaluation of BC toxicity. Our results indicate that BC is able to disrupt cardiovascular and craniofacial cartilage development in medaka embryo in a dose and developmental stage-specific manner. Moreover, embryos in precirculation are to some extent more resistant to BC than ones with circulation. By using subtractive hybridization, we have observed that gata2 mRNA was differentially expressed in the circulating embryos after BC treatment. As GATA-binding sequences are required for the expression of the endothelin1 (edn1) gene and edn1 expressed in blood vessels and craniofacial cartilages, we have extended our investigations to edn1 gene expression regulation by BC. We found that edn1, furin1, and endothelin receptor A (ednrA) genes are developmentally regulated; endothelin converting enzyme mRNA (ece1) maintained a steady-state level throughout development. Circulating medaka embryos (3 days post fertilization, dpf) exposed to BC (10 microg/mL) for 48 h have increased levels of gata2, ece1, and preproenodthelin (preproedn1) mRNA contents; however, other mRNAs (furin and ednrA) remained unaltered. Therefore, the enhanced expression of gata2 mRNA followed by ece1 and preproedn1 mRNA by BC might be able to induce vasoconstriction and cardiovascular defects and disrupt craniofacial cartilages in medaka embryos. We conclude that cardiovascular and craniofacial defects in medaka embryogenesis by BC are probably mediated through a GATA2-EDN1 signaling pathway.

Endothelial Cell–Derived Endothelin-1 Promotes Cardiac Fibrosis in Diabetic Hearts Through Stimulation of Endothelial-to-Mesenchymal Transition

Persistently high plasma endothelin-1 (ET-1) levels in diabetic patients have been associated with the development of cardiac fibrosis, which results from the deposition of extracellular matrix and fibroblast recruitment from an as-yet unknown source. The underlying mechanism, however, remains elusive. Here, we hypothesize that ET-1 might contribute to the accumulation of cardiac fibroblasts through an endothelial-to-mesenchymal transition in diabetic hearts. We induced diabetes mellitus in vascular endothelial cell-specific ET-1 knockout [ET-1(f/f);Tie2-Cre (+)] mice and their wild-type littermates using the toxin streptozotocin. Gene expression and histological and functional parameters were examined at 8, 24, and 36 weeks after the induction of diabetes mellitus. Diabetes mellitus increased cardiac ET-1 expression in wild-type mice, leading to mitochondrial disruption and myofibril disarray through the generation of superoxide. Diabetic mice also showed impairment of cardiac microvascularization and a decrease in cardiac vascular endothelial growth factor expression. ET-1 further promotes cardiac fibrosis and heart failure through the accumulation of fibroblasts via endothelial-to-mesenchymal transition. All of these features were abolished in ET-1(f/f);Tie2-Cre (+) hearts. Targeted ET-1 gene silencing by small interfering RNA in cultured human endothelial cells ameliorated high glucose-induced phenotypic transition and acquisition of a fibroblast marker through the inhibition of transforming growth factor-beta signaling activation and preservation of the endothelial cell-to-cell contact regulator VE-cadherin. These results provide new insights suggesting that diabetes mellitus-induced cardiac fibrosis is associated with the emergence of fibroblasts from endothelial cells and that this endothelial-to-mesenchymal transition process is stimulated by ET-1. Targeting endothelial cell-derived ET-1 might be beneficial in the prevention of diabetic cardiomyopathy.

Abstract 2363: Positive inter-regulation between β-catenin and endothelin signaling in ovarian cancer cells: Epigenetic regulation of gene transcription

Abstract Although endothelin-1 (ET-1) and the endothelin A receptor (ETAR) regulate different steps of ovarian tumor progression, the molecular mechanism controlling the expression of ET-1 in this tumor is unknown. We previously demonstrated that ET-1 activates β-catenin/TCF4 transcriptional activity to promote cell invasion and metastasis. Here, we show that ET-1 gene is directly regulated by β-catenin in ovarian cancer cells. Thus, inhibition of β-catenin signalling results in lowered ET-1 promoter activity and expression, while enhanced β-catenin signalling leads to further activation of this gene. Chromatin immunoprecipitation (ChIP) demonstrated that, after ET-1 stimulus, β-catenin and its cognate DNA binding partner, TCF4, are recruited on the specific DNA element within the ET-1 promoter. Unravelling the role of the scaffold protein β-arrestin-1 as nucleus chaperone controlling β-catenin transcription activity, we demonstrated that β-arrestin-1 is required for the activation of ET-1 promoter, and for the recruitment of β-catenin/TCF4 on this promoter. In agreement with these findings, immunoprecipitation assay demonstrated that ET-1 promotes the interaction between endogenous β-catenin and β-arrestin-1 both in the cytoplasm and in the nucleus. Moreover, β-arrestin-1 siRNA leads to the loss of ET-1 mRNA expression and ET-1 secretion, as well as a significant inhibition of ET-1 promoter activity, thus suggesting the critical role of β-arrestin-1 in the β-catenin-dependent ET-1 gene expression. Further experiments showed that ET-1 promotes the nuclear association between histone acetyltransferase p300 and β-arrestin and the recruitment of p300 on the ET-1 promoter, resulting in H3 and H4 histone acetylation and enhanced ET-1 transcription. Besides ET-1, β-arrestin-1 represents a platform for achieving signal specificity that converges on β-catenin-mediated transcription of defined genes, such as cyclin D1 and matrix metalloprotease (MMP)-2, but not MMP-9. Moreover, ETAR blockade with the specific ETAR antagonist, zibontentan (ZD4054), abrogates both the engagement of β-arrestin and the interplay between ET-1 and the β-catenin in controlling gene transcription. In an intraperitoneal model of ovarian cancer metastasis, HEY cells expressing the mutant β-arrestin-1, or treatment with zibontentan, significantly reduced peritoneal tumor-cell dissemination and β-catenin expression, highlighting the importance of β-arrestin-mediated signaling in metastasis formation. Altogether these results reveal a positive inter-regulation between β-catenin and ET-1 that amplify the ET-1/ETAR autocrine loop in ovarian cancer cells, in which β-arrestin-1 acts as a nuclear messenger mediating epigenetic mechanism in β-catenin-mediated ET-1 transcription. Supported by AIRC, AstraZeneca Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2363.

The Association Between Polymorphism of Lys198Asn of Endothelin-1 Gene and Arterial Hypertension Risk in Kazakh People

Aim: We researched frequency distribution of genotypes and alleles Lys198Asn of polymorphism of endothelin -1 gene associated with arterial hypertension development in Kazakh people Method: The research included 120 patients suffering arterial hypertension of 2-3 degree, average, high and very high degree of risk, and control group included 80 actually healthy person of Kazakh nationality. Result: The research discovered that development of arterial hypertension in the present ethnic group associated with polymorphism of endothelin-1 gene. Conclusion: At that, allele 198Asn and AsnAsn genotype of endothelin-1 gene act as marker of increased risk of arterial hypertension development.

Simvastatin stimulates production of the antiapoptotic protein Bcl-2 via endothelin-1 and NFATc3 in SH-SY5Y cells.

The use of statins for the prevention or treatment of different neurodegenerative diseases has generated considerable interest albeit with some controversy. Mechanisms of statin-induced neuroprotection are not well understood. Recently, we reported that simvastatin stimulated neuronal gene expression and protein levels of the major antiapoptotic protein Bcl-2 in vivo and in vitro; suppression of Bcl-2 in SH-SY5Y cells reduced simvastatin neuroprotection; effects were independent of cholesterol and other products of the 3-hydroxy-3-methylglutaryl-CoA reductase pathway. Endothelin-1 (ET-1) can increase Bcl-2 abundance via the transcription factor nuclear factor of activated thymocytes (NFATc), and simvastatin was reported to increase ET-1 gene expression. We tested the hypothesis that simvastatin stimulation of Bcl-2 involves up-regulation of ET-1 and binding of NFATc to Bcl-2 promoter sites in SH-SY5Y human neuroblastoma cells. Simvastatin increased both intracellular and secreted ET-1 protein levels. Exogenous ET-1 increased Bcl-2 protein abundance, which was inhibited by ET-1 receptor antagonists. Simvastatin increased translocation of NFATc3 to the nucleus while reducing nuclear NFATc1 and having no effect on NFATc4. Endothelin-1 also increased NFATc3 levels in the nucleus, and this increase was inhibited by ET-1 receptor antagonists. Treatment of cells with simvastatin stimulated binding of NFATc3 to the Bcl-2 promoter. We report novel findings showing that up-regulation of Bcl-2 by simvastatin involves ET-1 and the transcription factor NFATc3. Discovering how statins can selectively alter a specific NFATc isoform that leads to an increase in an antiapoptotic protein will provide a new approach to understanding statin-induced neuroprotection and conditions outside the brain in which apoptosis contributes to pathophysiology.

Effects of transgenic endothelin‐2 overexpression on diabetic cardiomyopathy in rats

Transgenic overexpression of human endothelin-2 in rats was used to characterize the contribution of endothelin to diabetic cardiomyopathy. Diabetes mellitus was induced by streptozotocin in transgenic rats and transgene-negative controls. Nondiabetic animals were included as well to form a 4-group study design. Heart morphological and molecular alterations were analysed following 6 months of hyperglycaemia. Plasma endothelin concentrations were significantly higher in both transgenic groups than in wild-type groups (nondiabetic: 3.5 +/- 0.4 vs. 2.1 +/- 0.2, P < 0.05; diabetic: 4.5 +/- 0.4 vs. 2.5 +/- 0.4 fmol mL(-1), P < 0.01). Diabetes induced cardiac hypertrophy in both wild-type and transgenic rats and showed the highest myocardial interstitial tissue volume density in diabetic transgenic rats (1.5 +/- 0.07%) as compared with nondiabetic transgenic (1.1 +/- 0.03%), nondiabetic wild-type (0.8 +/- 0.01%) and diabetic wild-type rats (1.1 +/- 0.03%; P < 0.01 for all comparisons). A similar pattern with the most severe changes in the enothelin-2 transgenic, diabetic animals was observed for hypertrophy of the large coronary arteries and the small intramyocardial arterioles respectively. Cardiac mRNA expression of endothelin-1, endothelin receptors type A and B were altered in some degree by diabetes or transgenic overexpression of endothelin-2, but not in a uniform manner. Blood pressure did not differ between any of the four groups. Overexpression of the human endothelin-2 gene in rats aggravates diabetic cardiomyopathy by more severe coronary and intramyocardial vessel hypertrophy and myocardial interstitial fibrosis. This transgenic intervention provides further and independent support for a detrimental, blood pressure-independent role of endothelins in diabetic cardiac changes.

Genetic polymorphisms in endothelin-receptor-subtype-a-gene as susceptibility factor for obstructive sleep apnea syndrome

Introduction Traits of obstructive sleep apnea syndrome (OSAS) such as impaired ventilatory control, craniofacial abnormalities, and concomitant cardiovascular diseases are associated with modified endothelin-1 gene (EDN-1) or endothelin-receptor-subtype-a (EDNRA) gene. The endothelin system regulates the cardiovascular homeostasis. EDN-1 interacts mainly with EDNRA for signal transduction. In our study we investigate associations of EDNRA-polymorphisms (four frequent polymorphisms with an allele frequency >5%) and OSAS severity. Methods Three hundred ninety-three patients older than 18 years, of Caucasian origin and with OSAS (AHI > 5/h and daytime sleepiness) were investigated by cardiorespiratory polysomnography. In addition 58 control subjects with healthy sleep were recruited from nearly 300 volunteers. We analysed the EDNRA-polymorphisms E335E, H323H, G-231A and G+70C by polymerase-chain-reaction, restriction-fragment-length-polymorphism and real-time-PCR. Results Carrier of the mutant G-231A allele had a significantly lower AHI ( p = 0.03, OR 0.53, 95% CI 0.3–0.94) when comparing patients and controls. When comparing OSAS severity groups without controls we could not detect significant correlations for the four investigated EDNRA-polymorphisms. Our data confirm that BMI ( p < 0.001) and male gender ( p = 0.02) are significantly associated with AHI. The allele frequencies were similar. Discussion The genetic investigation of OSA remains important. Our control group was relatively small and we investigated 4 reasonable candidates out of more than 100 EDNRA-polymorphisms. The detected protective effect of the mutant G-231A allele needs further confirmation. Gene based research in OSAS should use genome wide scan and should still consider the endothelin system.

Gene Expression of the Endothelin-1 in Vasospastic Flap Pedicle – an Experimental Study on a Porcine Model

The aim of this study was to evaluate the amount of Endothelin-1 (ET-1) gene expression in the vasospastic vessel of the flap pedicle to prove or disprove the role of ET-1 gene expression in pathogenesis of mechanically induced vasospasm. The vasospasm was induced by the tension on the pedicle of the pedicled caudal superficial epigastric flap on 8 pigs. Laser Doppler was used for peripheral blood flow measurement. Specimens from the vasospastic vessel (group of specimens B) and from the flap border with no vasospasm (control group A) were taken 2 h after the stimulus initiation. Detection of ET-1 mRNA by Quantitative Real-Time RT-PCR was performed. β-actin was selected as an acceptable reference gene. Relative gene expression data were given as the n-fold change in transcription of target genes normalized to the endogenous control. Relative gene expressions and time indicators of vasospasm were compared in both groups. No significant difference of the ET-1 gene expressions was found between groups A and B (p = 0.505). No correlation between the duration of vasospasm and ET-1 gene expression was found as well (p = 0.299). In conclusion, the expression of the ET-1 gene in the mechanically induced vasospastic vessel of the pedicled flap was not significantly increased. In this study, the causality of the vasospasm pathogenesis and gene expression of ET-1 was not proven.

Propofol Depresses Angiotensin II–induced Cell Proliferation in Rat Cardiac Fibroblasts

Propofol may have beneficial effects on the prevention of angiotensin II (Ang II)-induced cardiac fibroblast proliferation via its antioxidative properties. The authors hypothesized that propofol may alter Ang II-induced cell proliferation and aimed to identify the putative underlying signaling pathways in rat cardiac fibroblasts. Cultured rat cardiac fibroblasts were pretreated with propofol then stimulated with Ang II; cell proliferation and endothelin-1 gene expression were examined. The effect of propofol on Ang II-induced nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity were also examined. The effect of propofol on nitric oxide production and protein kinase B and endothelial nitric oxide synthase phosphorylations were also tested to elucidate the intracellular mechanism of propofol in proliferation. Ang II (100 nm) increased cell proliferation and endothelin-1 expression, which were partially inhibited by propofol (10 or 30 microm). Propofol also inhibited Ang II-increased nicotinamide adenine dinucleotide phosphate-oxidase activity, reactive oxygen species formation, extracellular signal-regulated kinase phosphorylation, and activator protein 1-mediated reporter activity. Propofol was also found to increase nitric oxide generation and protein kinase B and nitric oxide synthase phosphorylations. Nitric oxide synthase inhibitor (N-nitro-L-arginine methylester) and the short interfering RNA transfection for protein kinase B or endothelial nitric oxide synthase markedly attenuated the inhibitory effect of propofol on Ang II-induced cell proliferation. The authors' results suggest that propofol prevents cardiac fibroblast proliferation by interfering with the generation of reactive oxygen species and involves the activation of the protein kinase B-endothelial nitric oxide synthase-nitric oxide pathway.

EDNRA variants associate with smooth muscle mRNA levels, cell proliferation rates, and cystic fibrosis pulmonary disease severity

Airway inflammation and pulmonary disease are heterogeneous phenotypes in cystic fibrosis (CF) patients, even among patients with the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Endothelin, a proinflammatory peptide and smooth muscle agonist, is increased in CF airways, potentially contributing to the pulmonary phenotype. Four cohorts of CF patients were screened for variants in endothelin pathway genes to determine whether any of these variants associated with pulmonary function. An initial cohort of 808 CF patients homozygous for the common CF mutation, DeltaF508, showed significant association for polymorphisms in the endothelin receptor A gene, EDNRA (P = 0.04), but not in the related endothelin genes (EDN1, EDN2, EDN3, or EDNRB) or NOS1, NOS2A, or NOS3. Variants within EDNRA were examined in three additional cohorts of CF patients, 238 patients from Seattle, WA, 303 from Ireland and the U.K., and 228 from Cleveland, OH, for a total of 1,577 CF patients. The three additional groups each demonstrated a significant association between EDNRA 3'-untranslated region (UTR) variant rs5335 and pulmonary function (P = 0.002). At the molecular level, single nucleotide primer extension assays suggest that the effect of the variants is quantitative. EDNRA mRNA levels from cultured primary tracheal smooth muscle cells are greater for the allele that appears to be deleterious to lung function than for the protective allele, suggesting a mechanism by which increased receptor function is harmful to the CF airway. Finally, cell proliferation studies using human airway smooth muscle cells demonstrated that cells homozygous for the deleterious allele proliferate at a faster rate than those homozygous for the protective allele.

Renin, endothelial no synthase and endothelin gene expression in the 2Kidney-1clip goldblatt model of long-term renovascular hypertension

ObjectiveNumerous reports have shown the influence of renin, nitric oxide (NO) and the endothelin (ET) systems for regulation of blood pressure and renal function. Furthermore, interactions between these peptides have been reported. Aim of our study was to investigate the relative contribution of these compounds in long-term renovascular hypertension/renal ischemia.MethodsHypertension/left-sided renal ischemia was induced using the 2K1C-Goldblatt rat model. Renal renin, ET-1, ET-3 and endothelial NO synthase (eNOS) gene expression was measured by means of RNAse protection assay at different timepoints up to 10 weeks after induction of renal artery stenosis.ResultsPlasma renin activity and renal renin gene expression in the left kidney were increased in the clipped animals while eNOS expression was unchanged. Furthermore, an increase in ET-1 expression and a decrease of ET-3 expression was detected in early stenosis.ConclusionsWhile renin is obviously involved in regulation of blood pressure and renal function in unilateral renal artery stenosis, ET-1, ET-3 and endothelium derived NO do not appear to play an important role in renal adaptation processes in long-term renal artery stenosis, although ET-1 and ET-3 might be involved in short-term adaptation processes.