Endothelin-1 mRNA Expression Research Articles

Effects of endomorphins on human umbilical vein endothelial cells under high glucose

The endomorphin-1 (EM1) and endomorphin-2 (EM2) are endogenous opioid peptides, which modulate extensive bioactivities such as pain, cardiovascular responses, immunological responses and so on. The present study was undertaken to investigate the effects of EM1/EM2 on the primary cultured human umbilical vein endothelial cells (HUVECs) damaged by high glucose. PI AnnexinV-FITC detection was performed to evaluate the apoptosis rate. Levels of nitric oxide (NO) and nitric oxide synthase (NOS) activity were measured by the Griess reaction and the conversion of 3H-arginine to 3H-citrulline, respectively. Endothelin-1 (ET-1) was evaluated by the enzyme-linked immunosorbent assay (ELISA). Cell proliferation was determined by the MTT viability assay. mRNA expression of endothelial nitric oxide synthase (eNOS) and ET-1 were measured by real-time PCR. Our data showed that EM1/EM2 inhibited cell apoptosis. The high glucose induced increase in expression of NO, NOS and ET-1 were significantly attenuated by pretreatment with EM1/EM2 in a dose dependent manner. In addition, EM1/EM2 suppressed the mRNA eNOS and mRNA ET-1 expression in HUVECs under high glucose conditions. Naloxone, the nonselective opioid receptor antagonist, did not influence the mRNA eNOS expression when it was administrated on its own; but it could significantly antagonize the effects induced by EM1/EM2. Furthermore, in all assay systems, EM1 was more potent than EM2. The results suggest that EM1/EM2 have a beneficial effect in protecting against the endothelial dysfunction by high glucose in vitro, and these effects were mediated by the opioid receptors in HUVECs.

Amlodipine Ameliorates Up-Regulation of ET-1 in Left Ventricle of Hypercholesterolemia Rabbits

Objective(s) In addition to antihypertensive effects, amlodipine may exhibit cardiovascular protective effects in heart tissue. The aim of this study was to evaluate the effects of amlodipine and/or high cholesterol diet on blood, heart tissue concentration and mRNA expression of endothelin-1 (ET-1) in male New Zealand white rabbits. Materials and Methods A total of 40 male New Zealand rabbits were divided into four groups: the normal control group, normal group receiving amlodipine, high-cholesterol diet group and high-cholesterol diet with amlodipine group. After 8 weeks, all the animals anesthetized and blood or tissues samples were collected. Results After 8 weeks of a high cholesterol diet, the group with such a diet had a significantly higher ratio of left ventricle (LV) weight to body weight than the control group (P= 0.0001). After treatment with amlodipine for 8 weeks, ET-1 level was reduced considerably in comparison with the control (P= 0.01) and highcholesterol diet groupes (P= 0.01). Amlodipine consumption caused significant reduction (P= 0.01) in the level of ET-1 in heart tissues of high-cholesterol diet group but it had no remarkable effect on the reduction of heart tissue ET-1 in amlodipine group compared with the control group. Conclusion The present study demonstrates that ventricular prepro-ET-1 mRNA quantitatively increases in the highcholesterol diet rabbits which results in development of ventricular hypertrophy. It seems that the treatment with amlodipine retards the progression of LV hypertrophy through attenuation of ET-1 levels independent of lipid changes.

Doxorubicin selectively suppresses mRNA expression and production of endothelin-1 in endothelial cells

Doxorubicin (DXR) is a widely used cytostatic agent, but its administration is limited by its cardiovascular side effects. The endothelium is one of the largest organs in the human body and due to its direct contact with blood; it is exposed to the toxic effects of DXR. The aim of this study was to investigate in endothelial cells the effects of DXR on the expression of genes involved in cardiovascular diseases. We used in vitro cultured human umbilical vein endothelial cells (HUVEC) as a model; gene expression was assessed by SuperArray and qPCR. Out of the 96 representative genes of cardiovascular importance, the expression of only the ET-1 gene changed significantly. ET-1 mRNA expression was 10.9% of the untreated control (p = 0.0049). This result was confirmed by qPCR (2.41% of control, p = 0.0022). DXR also suppressed ET-1 production at protein level (p = 0.0116). Both the early decrease in endothelial ET-1 production in the presence of DXR and the high plasma level of DXR during chemotherapy may influence the toxic effects of the drug.

Shock Wave-Pretreated Bone Marrow Cells Further Improve Left Ventricular Function After Myocardial Infarction in Rabbits

We tested whether shock wave (SW) offers additional benefits in improving left ventricular (LV) function after acute myocardial infarction (AMI) in rabbits receiving SW-treated autologous bone marrow-derived mononuclear cells (BMDMNCs) transplantation. Saline (750 microL; group 2), BMDMNCs (1.0 x 10(7); group 3), or preimplant SW-treated BMDMNCs (group 4) were implanted into the infarct area of male rabbits 15 minutes after left coronary artery ligation, whereas eight rabbits without AMI served as controls (group 1; n = 8 per group). The results showed that in infarct area of LV, protein expressions of Cx43 and cytochrome C in mitochondria and endothelial nitric oxide synthase mRNA expression were lower in group 2 than in other groups, and decreased in group 3 as compared with groups 1 and 4 (all p values < 0.01). Conversely, mRNA expressions of endothelin-1 and matrix metalloproteinase-9, mitochondrial oxidative stress, and total fibrotic area were higher in group 2 than in other groups (all p values < 0.05). Furthermore, 6-month LV function by 2-D echo/angiogram showed significant impairment in group 2 than in other groups and in group 3 than in groups 1 and 4 (all p values < 0.005). Application of SW-treated autologous BMDMNCs is superior to BMDMNCs alone for preserving LV function after AMI.

Glucose-induced endothelin-1 expression is regulated by ERK5 in the endothelial cells and retina of diabetic ratsThis article is one of a selection of papers published in the two-part special issue entitled 20 Years of Endothelin Research.

Upregulation of endothelin 1 (ET-1) causing blood flow alteration and increased extracellular matrix production are characteristic features of diabetic angiopathy. Several glucose-induced signaling mechanisms cause ET-1 upregulation in diabetic angiopathy. Extracellular signal-regulated kinase 5 (ERK5) is a member of the MAPK family, which plays a key role in cardiovascular development. ERK kinase (MEK) 5 is the specific MEK for ERK5 activation. In this study we examined the role of glucose-induced ERK5 signaling in mediating ET-1 expression in diabetic angiopathy. We investigated retinas from 1-month STZ-induced diabetic rats and human macro- and microvascular endothelial cells to study ERK5-dependent ET-1 alterations. Glucose (25 mmol/L) caused significant upregulation of ET-1 mRNA and downregulation of ERK5 and Kruppel-like factor 2 (KLF2) after 24 h treatment in the endothelial cells. Simultaneously, phospho-ERK5 proteins were reduced. Activation of ERK5 by constitutively active MEK5 (caMEK5) upregulated KLF2 and suppressed ET-1 expression in both cell lines, whereas ERK5 siRNA transfection resulted in decreased ERK5 and KLF2 and increased ET-1 mRNA expression. In addition, caMEK5 prevented glucose-induced upregulation of ET-1. Furthermore, 1 month of diabetes caused a significant increase in retinal ET-1 mRNA and decrease in ERK5 mRNA expression. These data indicate that ERK5 signaling regulates glucose-induced ET-1 expression in diabetes. The ERK5/ET-1 pathway may provide a potential novel target for the treatment of diabetic angiopathy.

Association of Left Atrial Endothelin-1 With Atrial Rhythm, Size, and Fibrosis in Patients With Structural Heart Disease

Atrial fibrillation (AF) promotes atrial remodeling and can develop secondary to heart failure or mitral valve disease. Cardiac endothelin-1 (ET-1) expression responds to wall stress and can promote myocyte hypertrophy and interstitial fibrosis. We tested the hypothesis that atrial ET-1 is elevated in AF and is associated with AF persistence. Left atrial appendage tissue was studied from coronary artery bypass graft, valve repair, and/or Maze procedure in patients in sinus rhythm with no history of AF (SR, n=21), with history of AF but in SR at surgery (AF/SR, n=23), and in AF at surgery (AF/AF, n=32). The correlation of LA size with atrial protein and mRNA expression of ET-1 and ET-1 receptors (ETAR and ETBR) was evaluated. LA appendage ET-1 content was higher in AF/AF than in SR, but receptor levels were similar. Immunostaining revealed that ET-1 and its receptors were present both in atrial myocytes and in fibroblasts. ET-1 content was positively correlated with LA size, heart failure, AF persistence, and severity of mitral regurgitation. Multivariate analysis confirmed associations of ET-1 with AF, hypertension, and LA size. LA size was associated with ET-1 and MR severity. ET-1 mRNA levels were correlated with genes involved in cardiac dilatation, hypertrophy, and fibrosis. Elevated atrial ET-1 content is associated with increased LA size, AF rhythm, hypertension, and heart failure. ET-1 is associated with atrial dilatation, fibrosis, and hypertrophy and probably contributes to AF persistence. Interventions that reduce atrial ET-1 expression and/or block its receptors may slow AF progression.

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.

Effects of resveratrol on NO secretion stimulated by insulin and its dependence on SIRT1 in high glucose cultured endothelial cells

To investigate the effects of resveratrol on the secretion of NO induced by insulin in high glucose cultured primary human umbilical vein endothelial cells (HUVEC). HUVEC were treated with 1 μmol/l resveratrol for 24 h before cultured in high glucose medium for 48 h, then all cells were stimulated by 100 nmol/l insulin for 30 min. Method based on nitric acid reductase was used to analyze the NO contents in the supernatant. Cells were collected to analyze the expression of eNOS, endothelin-1, E-selectin, and SIRT1. In order to investigate the dependence of resveratrol on SIRT1, the effects of resveratrol on cells treated by SIRT1 siRNA were also examined. Compared with control cells, high glucose decreased the secretion of NO induced by insulin. Resveratrol treatment increased the expression of SIRT1 and the secretion of NO. After interfering the expression of SIRT1 using SIRT1 siRNA, the effects of resveratrol on the NO secretion induced by insulin was impaired. Resveratrol also counteracted other pro-atherosclerotic effects of high glucose, including the up-regulating roles of high glucose on the expression of endothelin-1 mRNA and E-selectin mRNA, and the down-regulating roles of high glucose on the expression of eNOS mRNA and the basal NO secretion without the stimulating of insulin. Resveratrol can improve the NO stimulating function of insulin in high glucose cultured HUVEC in SIRT1-dependent manner. Thus, our results imply that resveratrol may have the preventive roles of atherosclerosis in diabetic patients.

Concomitant Endothelin‐1 Overexpression in Lung Transplant Donors and Recipients Predicts Primary Graft Dysfunction

Primary graft dysfunction (PGD) causes significant morbidity following lung transplantation (LTX). Mortality is high in PGD and therapeutic strategies are limited. To investigate whether endothelin-1 (ET-1) that mediates increased vascular permeability and edema formation in lung grafts can predict PGD, ET-1 mRNA expression was examined in lung tissue biopsies of 105 donors and recipients obtained shortly before LTX. Serum ET-1 concentration was assessed by ELISA. PGD grade was diagnosed and scored by oxygenation and radiological characteristics according to ISHLT guidelines. PGD grade 3 developed in 11% of patients. ET-1 mRNA expression was significantly increased in both donor (p < 0.0001) and recipient (p = 0.01) developing PGD as compared to no PGD group. Pretransplant ET-1 serum concentrations were elevated in recipients with PGD as compared to no PGD group (p < 0.0001), although serum ET-1 was not different between donors whose grafts developed PGD grades 0-3. In regression analysis, concomitant elevated donor tissue ET-1 and recipient serum ET-1 predicted PGD grade 3. This study indicates that pretransplant ET-1 mRNA overexpression in donors associated with elevated pretransplant serum ET-1 in recipients contribute to PGD development and that their assessment might be beneficial to predict PGD and to identify recipients who could benefit from a targeted ET-1 blockade.

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.

Prostaglandin F2.ALPHA. Differentially Affects mRNA Expression Relating to Angiogenesis, Vasoactivation and Prostaglandins in the Early and Mid Corpus Luteum in the Cow

Administration of prostaglandin (PG) F(2alpha) in cattle during the mid-luteal phase (Days 8-12 of the estrous cycle) drastically reduces the plasma progesterone concentrations and the volume of the corpus luteum (CL). However, PGF(2alpha) does not induce luteolysis during the early luteal phase (up to Day 5 of the estrous cycle). To characterize the possible distinct difference in acute response to a luteolytic dose of PGF(2alpha) administration, we determined various mRNA expressions in the early and mid CL relating to angiogenesis, vasoactivation and PG-related factors at 30 min after PGF(2alpha) injection in cyclic cows. The experiments were conducted on Day 4 (early CL) and Days 10-12 (mid CL). Cows were either injected with 500 microg PGF(2alpha) analogue or saline as the control (early CL control, n=5; early CL PGF(2alpha) treated, n=5; mid CL control, n=5; mid CL PGF(2alpha) treated, n=7). Thirty min after injection of PGF(2alpha) or saline, the cows were ovariectomized transvaginally, and the CL tissues were collected from regions designated as the periphery and center of the CL. Administration of PGF(2alpha) up-regulated the mRNA expressions of angiogenic-related factors such as vascular endothelial growth factors, vasohibin, fibroblast growth factor 2 and insulin-like growth factor-II in the early CL, whereas PGF(2alpha) down-regulated these mRNA expressions in the mid CL. In the vasoactive factors, PGF(2alpha) stimulated the mRNA expressions of endothelin-1, angiotensin converting enzyme, endothelial nitric oxide synthase (NOS) and inducible NOS in the periphery area of the mid CL, but not in the early CL. However, PGF(2alpha) drastically down-regulated PGF(2alpha) receptor mRNA expression in both regions of the early and mid CL. The results indicated a clear difference in the acute action of PGF(2alpha) depending not only on the luteal phase (immature vs. mature) but also the region (periphery vs. center) within the CL at 30 min after PGF(2alpha) injection in the cow.

Physiological concentrations of dietary polyphenols regulate vascular endothelial cell expression of genes important in cardiovascular health

Previous cell culture-based studies have shown potential health beneficial effects on gene expression of dietary polyphenols, including those found in red wine and green tea. However, these studies have tended to use higher concentrations (2-100 microm) than those observed in blood (0.1-1 microm) after consuming polyphenol-rich foods or beverages. The present study investigated effects of physiological concentrations of different classes of dietary polyphenol on the expression of genes important in cardiovascular health (endothelial NO synthase (eNOS), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF)) by cultured vascular endothelial cells (human umbilical vein endothelial cells) in the absence or presence of H2O2. Resveratrol and quercetin (0.1-1 microm) increased eNOS and VEGF mRNA expression particularly in the absence of H2O2 (50 microm) and decreased H2O2-induced ET-1 mRNA expression (P < 0.001 for polyphenol x H2O2 interactions). Similarly, resveratrol and quercetin decreased endothelin secretion into the media, blocking the stimulatory effect of 50 microm-H2O2 (P < 0.001 for polyphenol x H2O2 interaction). Of the nine other polyphenols tested, only epigallocatechin gallate had similar effects on both the eNOS and ET-1 mRNA expression, but to a lesser extent than resveratrol at an equimolar concentration (0.1 microm). The observed effects on gene expression would be expected to result in vasodilation and thereby reduced blood pressure. Since only three of the eleven polyphenols tested had biological activity, it is unclear whether particular structures are important or whether the effects might relate to the relatively high antioxidant capacities of the three active polyphenols.

Transcriptional coactivator p300 regulates glucose-induced gene expression in endothelial cells

Sustained hyperglycemia in diabetes causes alteration of a large number of transcription factors and mRNA transcripts, leading to tissue damage. We investigated whether p300, a transcriptional coactivator with histone acetyl transferase activity, regulates glucose-induced activation of transcription factors and subsequent upregulation of vasoactive factors and extracellular matrix (ECM) proteins in human umbilical vein endothelial cells (HUVECs). HUVECs were incubated in varied glucose concentrations and were studied after p300 small interfering RNA (siRNA) transfection, p300 overexpression, or incubation with the p300 inhibitor curcumin. Histone H2AX phosphorylation and lysine acetylation were examined for oxidative DNA damage and p300 activation. Screening for transcription factors was performed with the Luminex system. Alterations of selected transcription factors were validated. mRNA expression of p300, endothelin-1 (ET-1), vascular endothelial growth factor (VEGF), and fibronectin (FN) and its splice variant EDB(+)FN and FN protein production were analyzed. HUVECs in 25 mmol/l glucose showed increased p300 production accompanied by increased binding of p300 to ET-1 and FN promoters, augmented histone acetylation, H2AX phosphorylation, activation of multiple transcription factors, and increased mRNA expression of vasoactive factors and ECM proteins. p300 overexpression showed a glucose-like effect on the mRNA expression of ET-1, VEGF, and FN. Furthermore, siRNA-mediated p300 blockade or chemical inhibitor of p300 prevented such glucose-induced changes. Similar mRNA upregulation was also seen in the organ culture of vascular tissues, which was prevented by p300 siRNA transfection. Data from these studies suggest that glucose-induced p300 upregulation is an important upstream epigenetic mechanism regulating gene expression of vasoactive factors and ECM proteins in endothelial cells and is a potential therapeutic target for diabetic complications.

Green Tea Polyphenol Epigallocatechin Gallate Reduces Endothelin-1 Expression and Secretion in Vascular Endothelial Cells: Roles for AMP-Activated Protein Kinase, Akt, and FOXO1

Epigallocatechin gallate (EGCG), a green tea polyphenol, promotes vasodilation by phosphatidylinositol 3-kinase-dependent activation of Akt and endothelial nitric oxide synthase to stimulate production of nitric oxide. Reduction in endothelin-1 (ET-1) synthesis may also increase bioavailability of nitric oxide. We hypothesized that the phosphatidylinositol 3-kinase-dependent transcription factor FOXO1 may mediate effects of EGCG to regulate expression of ET-1 in endothelial cells. EGCG treatment (10 microm, 8 h) of human aortic endothelial cells reduced expression of ET-1 mRNA, protein, and ET-1 secretion. We identified a putative FOXO binding domain in the human ET-1 promoter 51 bp upstream from the transcription start site. Trans-activation of a human ET-1 (hET-1) promoter luciferase reporter was enhanced by coexpression of a constitutively nuclear FOXO1 mutant, whereas expression of a mutant FOXO1 with disrupted DNA binding domain did not trans-activate the hET-1 promoter. Disrupting the hET-1 putative FOXO binding domain by site-directed mutagenesis ablated promoter activity in response to overexpression of wild-type FOXO1. EGCG stimulated time-dependent phosphorylation of Akt (S(473)), FOXO1 (at Akt phosphorylation site T(24)), and AMP-activated protein kinase alpha (AMPK alpha) (T(172)). EGCG-induced nuclear exclusion of FOXO1, FOXO1 binding to the hET-1 promoter, and reduction of ET-1 expression was partially inhibited by the AMPK inhibitor Compound C. Basal ET-1 protein expression was enhanced by short interfering RNA knock-down of Akt and reduced by short interfering RNA knock-down of FOXO1 or adenovirus-mediated expression of dominant-negative Foxo1. We conclude that EGCG decreases ET-1 expression and secretion from endothelial cells, in part, via Akt- and AMPK-stimulated FOXO1 regulation of the ET-1 promoter. These findings may be relevant to beneficial cardiovascular actions of green tea.

Effects of behind legs vibrations on erectile function in male rabbits

To study the effects of behind legs vibrations on erectile function in male rabbits through the concentration of plasma reproductive hormone and the expression of nitric oxide synthase (eNOS), endothelin-1 (ET-1) mRNA in vibrated male rabbits. 30 male adult rabbits were assigned randomly to A group (vibration power: 3.02 m/s(2)), B group (vibration power: 6.13 m/s(2)), C group (vibration power: 12.26 m/s(2)) and control group. The concentration of expression of eNOS, ET-1 mRNA were measured with RT-PCR after rated for 30 days. (1) Compared with 0 days vibration, after exposure to vibration for 10, 20, 30 days, the A, B, C group concentration of plasma T, LH are much lower (P < 0.05), the concentration of plasma E2 is much higher. (2) Compared with control group after exposed for 30 days, the expression of ET-1 mRNA [B group:(17.39 +/- 4.59) x 104; C group: (36.21 +/- 13.13) x 104 ] were much higher and expression of eNOS mRNA [A group: (19.11 +/- 6.83) x 104; B group: (11.86 +/- 3.15) x 104; C group: (4.68 +/- 3.26) x 104] was much lower, there were significant differences (P < 0.01). The vibration of behind legs in rabbits resulted the concentration of plasma T, LH are much lower, the concentration of plasma E2 is much higher, increased the expression of eNOS mRNA, decreased the expression of eNOS mRNA, then vary the erectile function.

Effects of an Endothelin Receptor Antagonist on a Model of Hypertensive Retinopathy

Hypertensive retinopathy manifests itself as progressive retinal microvascular pathology in response to aberrant blood flow. The current study sought to evaluate whether dysfunction of the vasoactive endothelin-1 (ET-1) system is involved in the pathogenesis of hypertension-induced retinopathy in an animal model of systemic hypertension. The endothelin receptor antagonist, bosentan, was administered to spontaneously hypertensive rats (SHRs) and comparisons were made with untreated SHRs and normotensive Wistar Kyoto (WKY) rats. The retinal mRNA expression of ET-1, ET-converting enzyme-1, ET_A and ET_B receptors and the basement membrane proteins, laminin β1, collagen IV and fibronectin was quantified using real-time RT-PCR. In addition, retinal arteriole and/or capillary bed damage was assessed by qualitative and quantitative microscopy. mRNA for the ET_A receptor was increased in SHRs, when compared to WKY control animals (p < 0.001). Treatment with bosentan in SHRs significantly reduced the expression of ET-1 (p < 0.05), and both the ET_A (p < 0.0001) and ET_B (p < 0.05) receptor subtypes. The laminin β1, collagen IV and fibronectin mRNA expression was significantly higher in SHRs when compared to WKY control animals (p < 0.001). Treatment with bosentan abolished these responses and also the appearance of various microvascular lesions. ET-mediated vasoregulation abnormalities in the retinal microvasculature could play an associative role in lesion formation during hypertensive retinopathy.

Ethanol-induced expression of ET-1 and ET-BR in liver sinusoidal endothelial cells and human endothelial cells involves hypoxia-inducible factor-1alpha and microrNA-199.

Chronic alcohol consumption leads to inflammation and cirrhosis of the liver. In this study, we observed that liver sinusoidal endothelial cells (LSEC) derived from ethanol-fed rats showed several fold increases in the mRNA expression of endothelin-1 (ET-1), hypoxia-inducible factor-1alpha (HIF-1alpha), and inflammatory cytochemokines compared with control rat LSEC. We also observed the same results in acute ethanol-treated LSEC from control rats and human dermal microvascular endothelial cells. Ethanol-mediated ET-1 expression involved NADPH oxidase and HIF-1alpha activation. Furthermore, ethanol increased the expression of the ET-1 cognate receptor ET-BR in Kupffer cells and THP-1 monocytic cells, which also involved HIF-1alpha activation. Promoter analysis and chromatin immunoprecipitation showed that hypoxia response element sites in the proximal promoter of ET-1 and ET-BR were required for the binding of HIF-1alpha to up-regulate their expression. We showed that microRNAs, miR-199 among several microRNAs, attenuated HIF-1alpha and ET-1 expression, while anti-miR-199 reversed the effects, suggesting that ethanol-induced miR-199 down-regulation may contribute to augmented HIF-1alpha and ET-1 expression. Our studies, for the first time to our knowledge, show that ethanol-mediated ET-1 and ET-BR expression involve HIF-1alpha, independent of hypoxia. Additionally, ethanol-induced ET-1 expression in rat LSEC is regulated by miR-199, while in human endothelial cells, ET-1 expression is regulated by miR-199 and miR-155, indicating that these microRNAs may function as novel negative regulators to control ET-1 transcription and, thus, homeostatic levels of ET-1 to maintain microcirculatory tone.

Leptin and endothelin‐1 mediated increased extracellular matrix protein production and cardiomyocyte hypertrophy in diabetic heart disease

We investigated the role of leptin and its interaction with endothelin 1 (ET-1) in fibronectin (FN) synthesis and cardiomyocyte hypertrophy, two characteristic features of diabetic cardiomyopathy. Endothelial cells [human umbilical vein endothelial cells (HUVECs)] were examined for FN production and neonatal rat cardiomyocytes for hypertrophy, following incubation with glucose, ET-1, leptin and specific blockers. FN, ET-1, leptin and leptin receptors mRNA expression and FN protein were measured. Myocytes were also morphometrically examined. Furthermore, hearts from streptozotocin-diabetic rats were analysed. Glucose caused increased FN mRNA and protein expression in HUVECs and cardiomyocytes hypertrophy along with upregulation of ET-1 mRNA, leptin mRNA and protein. Glucosemimetic effects were seen with leptin and ET-1. Leptin receptor antagonist (leptin quadruple mutant) and dual endothelin A endothelin B (ETA/ETB) receptor blocker bosentan normalized such abnormalities. Hearts from the diabetic animals showed hypertrophy and similar mRNA changes. These data indicate that in diabetes increased FN production and cardiomyocyte hypertrophy may be mediated through leptin with its interaction with ET-1.

-Lipoic acid prevents cisplatin-induced acute kidney injury in rats

Cisplatin-induced nephropathy has been related to increased lipid peroxide formation and decreased activity of antioxidant enzymes in the kidney. The present study aimed to examine whether treatment with alpha-lipoic acid (alpha-LA) prevents the cisplatin-induced nephrotoxicity. Two groups of rats were treated with cisplatin, one of which being cotreated with alpha-LA. The control group was treated with vehicle only. Four days later, the expression of aquaporins and sodium transporters was determined in the kidney by immunoblotting and immunohistochemistry. The arginine vasopressin-stimulated generation of cAMP was measured by radioimmunoassay. The expression of nitric oxide synthases (NOS) was determined by immunoblotting. The mRNA expression of endothelin-1 (ET-1) and tumour necrosis factor (TNF)-alpha was measured by real-time PCR. Apoptosis was examined by TUNEL staining. Following the treatment with cisplatin, urinary volume and fractional excretion of sodium increased. Accordingly, the expression of aquaporins 1-3, Na,K-ATPase, NHE3 and NKCC2 was decreased. The expression of adenylyl cyclase VI and vasopressin-stimulated cAMP generation was decreased. The expression of inducible NOS was increased, while that of endothelial NOS decreased. The ET-1 expression was increased. TUNEL-positive cells were increased, in association with an increased expression of TNF-alpha. alpha-LA treatment prevented dysregulation of these parameters and resumed the renal function. alpha-LA may prevent the cisplatin-induced nephrotoxicity, possibly through preserving the activities of NO and ET systems and inhibiting the development of apoptosis.

Teratogenic Effects of Blue Cohosh is Mediated through GATA2 Signaling Pathway

Blue cohosh (Caulophyllum thalictroides) (BC) is a perennial herb used by Native American Indian women to induce labor and for the treatment of other uterine complications. Several studies indicated that BC was not absolutely safe for the fetus and able to induce perinatal stroke and ischemia in newborn babies [1]. A recent chemical analysis identified 15 alkaloid-triterpene compounds present in BC [2] and some of them are potential teratogens. We used Japanese medaka (Oryzias latipes) embryo-larval development as our experimental model to verify the teratogenic potency of BC during embryogenesis. We observed that BC was able to induce cardiovascular defects in medaka embryo during development; however, total protein, RNA and several transcription factor mRNAs (emx2, en2, iro3, otx2, shh1, wnt1 and zic5) which were expressed in central nervous system (CNS) of medaka embryo during embryogenesis remained unaltered. Further, we have used subtractive hybridization technique to identify BC-sensitive genes in medaka embryogenesis. We have observed that transcription factor GATA2 was over expressed by BC and in situ hybridization analysis indicated that GATA2 over expression was occurred in CNS. Analysis by semi-quantitative reverse transcriptase polymerase Chain reaction (rt-PCR) indicated that GATA2 mRNA expression was very rapid (significantly increased within 15 min of BC exposure). We predict that teratogenic effects of BC are due to over expression of GATA2 gene that can induce the expression of endothelin-1 mRNA in the cerebral microvessels and peripheral vessels, and thus cause dysfunction of cerebrovascular and cardiovascular system of Japanese Medaka during development.