ET-1 Protein Levels Research Articles

The effect of varied exercise intensity on antioxidant function, aortic endothelial function, and serum lipids in rats with non-alcoholic fatty liver disease.

This study aimed to compare the effects of diet and exercise of different intensities on antioxidant function, aortic endothelial cell function and serum lipids in NAFLD (nonalcoholic fatty liver disease) rats. Fifty Sprague-Dawley (SD) rats (180-220g) were randomly divided into two experimental groups and fed either a standard rodent chow diet (CON; n=10) or a high-fat diet (HFD; n=40). After 16 weeks, the animals that received the HFD were randomly separated into a high-fat control group (HFC; n=10) or three ex-ercise training groups: HFD and low-intensity exercise (LE; n=10), HFD and moderate-intensity exercise (ME; n=10), and HFD and incremental intensity exercise (IE; n=10). These experimental rats keep sedentary or trained for the next six weeks. A detection kit was used to detect nitric oxide synthase (NOs), nitric oxide (NO), malondialdehyde (MDA) and other markers of aor-tic oxidative stress. The expression levels of endothelial nitric oxide synthase (e-NOS) and endothelin-1 (ET-1) were detected by immunohistochemistry. TC, TG, and other lipid metabolism parameters were detected by an auto-matic analyzer. Exercise with different intensities could improve lipid me-tabolism, enhance antioxidant function, reduce MDA (P<0.01), increase NO (P<0.01), and improve the expression of e-NOS and ET-1 (P<0.01) protein levels in NAFLD rats. Decreased blood lipids were exhibited in all exercise groups. Notably, the moderate-intensity exercise demonstrated more effecton increasing glutathione (GSH) contents (P<0.01) and decreased the ex-pression of ET-1protein levels (P<0.01). The results showed that exercise at different intensities improved lipid metabolism and enhanced anti-oxidation function. Moderate exercise could improve the function of aortic endothelial cells.

Endothelin-converting enzyme-1 expression in acute and chronic liver injury in fibrogenesis

ABSTRACTEndothelin-1 (ET-1) induces contraction, proliferation, and collagen synthesis of activated hepatic stellate cells and is a potent mediator of portal hypertension. Endothelin-converting enzyme-1 (ECE-1) generates ET-1 from the inactive precursor big-endothelin-1. The cellular distribution and activity of ECE-1 in the liver is unknown. Hepatic fibrogenesis was induced in rats by CCl4 administration and secondary biliary cirrhosis after 6 weeks of complete bile duct occlusion (BDO). The tissue ET-1 and ET receptor protein levels were quantified, the ECE-1 isoform mRNAs were measured by RNase protection assay and ECE-1 activity was analyzed. ECE-1a and -b mRNA were upregulated in biliary cirrhosis and in CCl4-injured livers, whereas ECE-1c mRNA remained unchanged. ECE-1 activity was increased after BDO and peaked at 12 h after acute CCl4-intoxication. Tissue levels of ET-1, ETA- and ETB receptors were elevated 7-, 5-, and 4.6-fold in cirrhotic rats, respectively. ECE-1 activity increased following BDO and acute CCl4-intoxication. In conclusion, ECE-1a and -b RNAs are upregulated in fibrogenesis, indicating that these isoforms play a central role in ET-1 generation during fibrogenesis and portal hypertension.

Anti-inflammatory and pro-angiogenic effects of placental mesenchymal stromal cells conditioned media on preeclamptic placental tissue.

s / Placenta 35 (2014) A1eA112 A87 increasing doses (0-100mM) on the following outputs 1) heme-oxygenase1 expression (HO-1, key antioxidant enzyme) 2) production of sFlt1 and sEng and 3) assays of endothelial dysfunction 4) endothelial tube formation. Endothelial dysfunction was modeled by adding TNFa or preeclamptic (PE) patient serum (to induce dysfunction) and subsequently adding PPIs. Results: All PPIs induced a significant dose dependent increase in HO-1 mRNA and protein (average 4-5 fold increase). Furthermore, PPIs induced nuclear translocation of Nrf2 (transcription factor). PPIs caused a significant dose dependent reduction in mRNA expression of sFlt1 variants (e15a and i13), and reduced secretion of both sFlt1 and sEng (dose dependently) in all three cell types. PPIs rescued both TNFa and PE serum induced endothelial dysfunction (evidenced by increased VCAM1 and endothelin-1 expression, but this was significantly attenuated by adding PPIs (decreased VCAM and ET-1 mRNA and protein levels). Finally, PPIs reversed TNFainduced disruption of in vitro endothelial cell tube formation. Summary and conclusions: In primary human tissues, PPIs potently 1) induced HO-1, 2) reduced sFlt1 and sEng secretion and 3) quenched endothelial dysfunction 4) rescued endothelial tube formation. PPIs may represent a highly novel candidate therapeutic for severe preeclampsia. Given PPIs are safe in pregnancy, they could be fast-tracked to clinical trials. P2.81-N. ANTI-INFLAMMATORY AND PRO-ANGIOGENIC EFFECTS OF PLACENTAL MESENCHYMAL STROMAL CELLS CONDITIONED MEDIA ON PREE CLAMPTIC PLACENTAL TISSUE. Anna Maria Nuzzo, Domenica Giuffrida, Ettore Piccoli, Cristian Zenerino, Rossella Barrile, Tullia Todros, Alessandro Rolfo Dept. of Surgical Sciences, University of Turin, Turin, Italy Objective: Placenta-derived Mesenchymal Stromal Cells (PDMSCs) are renowned for their unique anti-inflammatory and pro-angiogenic trophic activities. Indeed, we hypothesized that PDMSCs-derived molecules could counteract the production of pro-inflammatory cytokines and antiangiogenic factors typical of preeclamptic (PE) placentae. To verify our hypothesis, herein we investigated the expression of Macrophage-migration Inhibitory Factor (MIF), Tumor Necrosis Factor a (TNF-a) and Interleukin 6 (IL-6) cytokines, and anti-angiogenic soluble Fms-like tyrosine kinase-1 (sFlt1) in PE villous explants treated by PDMSCs conditioned media (CM). Methods: PDMSCs were isolated from control (n1⁄430) and PE (n1⁄430) placentae. At passage 5, control and PE-PDMSC were plated (1x105 cells/ ml) in DMEM without FBS. Conditioned media (CM) were collected after 48h. PE (n1⁄448) villous explants were treated for 72h using control PDMSCs CM. MIF, TNF-a, IL-6 and sFlt1 mRNA expression were evaluated by Real Time PCR. Results:Normal PDMSCs CM treatment significantly decreasedMIF, TNF-a, IL-6 and sFlt-1 mRNA levels (p<0.05, 3 Fold decrease) in PE villous explants relative to untreated controls. Conclusion: Our results suggest that PDMSCs CM could be used to neutralize the exacerbated inflammation and impaired angiogenesis typical of PE placentae. These data open to the development of innovative PDMSCs-based therapeutic tools for the treatment of preeclampsia. P2.82. A COMBINATION OF SINGLE NUCLEOTIDE POLYMORPHISMS IN THE 3’UNTRANSLATED REGION OF HLA-G IS ASSOCIATED WITH PREECLAMPSIA Kevin Quach , Stephanie Grover , Shlomit Kenigsberg , Clifford Librach a,b,c CReATe Fertility Centre, Toronto, Ontario, Canada; Women’s College Hospital, Toronto, Ontario, Canada; University of Toronto, Toronto,

Endothelin Axis Is Upregulated in Human and Rat Right Ventricular Hypertrophy

Right ventricular (RV) function is the most important determinant of morbidity and mortality in pulmonary arterial hypertension (PAH). Endothelin (ET)-1 receptor antagonists (ERAs) are approved therapies for PAH. It is not known whether ERAs have effects on the RV, in addition to their vasodilating/antiproliferative effects in pulmonary arteries. We hypothesized that the ET axis is upregulated in RV hypertrophy (RVH) and that ERAs have direct effects on the RV myocardium. RV myocardial samples from 34 patients with RVH were compared with 16 nonhypertrophied RV samples, and from rats with normal RV versus RVH attributable to PAH. Confocal immunohistochemistry showed that RVH myocardial ET type A (but not type B) receptor and ET-1 protein levels were increased compared with the nonhypertrophied RVs and positively correlated with the degree of RVH (RV thickness/body surface area; r(2)=0.838 and r(2)=0.818, respectively; P<0.01). These results were recapitulated in the rat model. In modified Langendorff perfusions, ERAs (BQ-123 and bosentan 10(-7,-6,-5) mol/L) decreased contractility in the hypertrophied, but not normal RV, in a dose-dependent manner (P<0.01). Patients and rats with PAH have an upregulation of the myocardial ET axis in RVH. This might be a compensatory mechanism to preserve RV contractility, as the afterload increases. ERAs use might potentially worsen RV function, and this could explain some of the peripheral edema noted clinically with these agents. Further studies are required to evaluate the effects of ERAs on the RV in patients with RVH and PAH.

Expression and clinical relevance of uPA and ET-1 in non-small cell lung cancer

背景与目的肺癌是世界第一大恶性肿瘤，其发病率及死亡率居高不下，本研究旨在探讨uPA和ET-1蛋白在非小细胞肺癌中的表达状况及其在临床诊断和预后判断方面的应用价值。方法采用组织微阵列联合免疫组织化学染色技术，研究155例非小细胞肺癌中uPA和ET-1蛋白的表达情况，分析其与临床病理参数的相关性。结果uPA阴性/弱、中度和高表达在鳞癌中的比例分别为12.3%、64.4%、23.3%，在腺癌中分别为12.2%、53.7%、34.1%，在全部病例中分别为12.3%、58.7%、29.0%。ET-1在鳞癌中阴性/弱、中度和高表达分别为2.7%、42.5%、54.8%，在腺癌中分别为11.0%、30.5%、58.5%，在全部病例中分别为7.1%、36.1%、56.8%。uPA和ET-1同时高表达多见于无淋巴结转移的腺癌中（P=0.017）。uPA高表达或与ET-1同时高表达的腺癌患者具有较长的术后生存时间（P=0.007, P=0.016）。结论检测uPA和ET-1蛋白表达水平变化可能有助于非小细胞肺癌的预后评估。

Modulating the hypoxia-inducible factor signaling pathway as a therapeutic modality to regulate retinal angiogenesis

Hypoxia-inducible factor (HIF) signaling cascade plays a critical role in angiogenesis by activating the transcription of genes encoding angiogenic growth factors. This study evaluated the effects of YC-1, a HIF-1 inhibitor, on the morphological, biochemical and molecular changes in human retinal microvascular endothelial cells. We found that YC-1 suppressed vascular endothelial cell proliferation, migration and tube formation, while it significantly increased the proteasome activity. Moreover, YC-1 induced a G0/G1 cell-cycle arrest, whereas it exerted only an insignificant proapoptotic effects. Under normoxia or hypoxia, YC-1 did not alter the morphology or the cell viability. Additionally, under hypoxic conditions, YC-1 downregulated HIF-2α, VEGF, EPO, ET-1, and MMP-9 mRNA and protein levels, this was accompanied by a significant decrease in the MMP-9 activity. YC-1 decreased the basal expression of HIF-1α protein under normoxia, whereas it inhibited HIF-1α protein synthesis, stability, and nuclear translocation mechanisms under hypoxia. Furthermore, in a 3D collagen matrix model using mouse retinal explants cultured under normoxic and hypoxic conditions, YC-1; (1) inhibited outgrowth of new vessel sprouts; (2) reduced VEGF expression; (3) dramatically decreased the vessels immunoreactivities for CD31 and von Willebrand Factor (vWF); and (4) was highly effective in reducing the vascular density within the retina, compared to controls. These findings indicate that YC-1 possesses several antiangiogenic properties, both in vitro and ex vivo, which could be exploited as valuable therapeutic potentials to inhibit formation and the growth of new retinal vessels in the hypoxic retina.

Chronic high-sodium diet increases aortic wall endothelin-1 expression in a blood pressure-independent fashion in rats.

Vascular endothelin (ET)-1 is upregulated in several forms of salt-induced hypertension. It is unclear to what extent these effects are primary or secondary to endothelial damage. We hypothesized that a high-sodium diet (HNa) increases vascular ET-1 production independent of arterial blood pressure changes. We investigated the effect of chronic HNa with and without ET(A) blockade on circulating and aortic ET-1 protein levels as well as aortic expression of ET-1 and ET(A) messenger RNA (mRNA) in inbred Wistar-Kyoto (WKY) and congenic ET(B)-deficient rats. Comparing WKY rats fed a low-sodium diet (LNa) with those fed HNa for 3 weeks, aortic wall ET-1 protein is significantly increased in response to HNa (331 +/- 43 pg/g tissue for LNa vs. 557 +/- 34 pg/gm tissue for HNa). HNa also increased aortic wall ET-1 mRNA levels by 40%, as determined by quantitative reverse transcriptase polymerase chain reaction. We then compared rats chronically treated with the ET(A)-selective antagonist, ABT-627, while receiving either LNa or HNa. There were no differences in arterial blood pressure (mean arterial pressure 89 +/- 1 mm Hg for WKY on LNa; 90 +/- 3 for WKY on HNa; 91 +/- 2 for ET(B)-deficient/ABT-627-treated on HNa) or heart rate. However, aortic wall ET-1 protein levels were 4-fold higher in the HNa group. Further, HNa increased aortic wall ET-1 mRNA (approximately 1.5- to 3-fold) and ET(A) mRNA (approximately 2- to 7-fold), independent of activation of ET(B). Therefore, the expression of ET-1 mRNA by the aortic wall is increased in response to chronic high dietary sodium in WKY rats in the absence of changes in arterial blood pressure.

Expression of endothelin-1 system in a pig model of endotoxic shock

Endothelin (ET)-1 is a potent vasoconstrictive peptide and it is involved in the pathogenesis of septic shock. Blockade of ET-1 receptors abolishes the LPS-induced pulmonary hypertension and worsens the LPS-dependent systemic hypotension, but the role of ET-1 in sepsis remains uncertain. To determine the role of ET-1 in cardiovascular and respiratory derangement in a porcine model of endotoxemic shock we evaluated ET-1 plasma levels and ET-1 mRNA and protein levels in lung, liver, and heart as well as Endothelin Converting Enzyme-1, ET A and ET B receptors mRNA in the same tissues. Twelve piglets were randomised to sham operated or to LPS-treated (40 μg/kg/h for 4 h) groups. During the experiment, respiratory and circulatory parameters have been recorded and blood samples collected. At the end of the experiment the animals were sacrificed and tissue samples collected for real-time quantitative PCR and ELISA test. LPS infusion evokes a large increase in ET-1 plasma concentration, and in tissues mRNA levels, associated with an increase in pulmonary arterial pressure, as well as in pulmonary and systemic vascular resistances, and a decrease in stroke volume. LPS infusion caused also a derangement of respiratory mechanics, evidenced by an increase in resistance and a decrease in compliance of the respiratory system. ET A and ET B receptor mRNA levels were markedly decreased in liver and lung and slightly increased in heart, evidencing that ET receptor subtypes were differentially regulated in the major organs of endotoxin treated pigs. In conclusion our data show the presence of a continuative and differentially regulated stimulating mechanism of ET-1 expression during pig endotoxaemia as well as a fundamental role of ET-1 system in the cardiovascular and respiratory derangement.

Coxsackievirus B3 infection compromises endothelial-dependent vasodilation of coronary resistance arteries.

The mechanisms of coronary artery dysfunction in coxsackievirus B3 (CVB3)-mediated viral myocarditis are poorly understood. We used pressure myography of mouse septal coronary arteries to determine the early and late effects of CVB3 infection on vascular function. Male CD-1 mice (age 6-7 weeks) were infected with CVB3 (1.75 x 10(10) pfu, i.p.). Control mice were injected with PBS. Mice were killed at 3, 7, and 42 days post infection, and the ventricular septal artery was dissected and mounted on a pressure myograph. Pressure-induced myogenic tone was similar in CVB3-infected and sham-infected mice at 3 and 7 days post infection. However, at 42 days post infection constriction of septal arteries to pressures equal to or less than 60 mm Hg was enhanced in CVB3-infected mice compared with sham controls. Agonist-induced vasodilation, as assessed by response to acetylcholine (1 nM-3 microM), was unaltered at early time points (days 3 and 7) in CVB3-infected mice. At later time points (day 42), there was a significant decrease in ACh-induced vasodilation in CVB3-infected mice. Bosentan, an ET-1 (ETA and ETB) receptor antagonist, did not completely ameliorate the reduced ACh-induced vasodilation in 42-day infected mice, indicating that ET-1 does not contribute to vascular dysfunction. Smooth muscle function, as measured by constriction to KCl or dilation to sodium nitroprusside, was unchanged in infected mice at early and late time points. Immunohistochemistry and ET-1 immunoassay were then performed to assess ET-1 levels in CVB3- and sham-infected hearts. There were no differences in ET-1 protein localization or levels at 42 days post infection in sham- and CVB3-infected animals. Finally, in situ hybridization and TUNEL staining were performed to assess viral localization and cell death in CVB3-infected hearts. There was no detectable CVB3 or TUNEL positivity in the endothelium of coronary arteries. Therefore, late impairment of endothelial-dependent vasorelaxation of coronary resistance vessels in CVB3-induced myocarditis does not appear to involve altered ET-1 expression but may be secondary to decreased stimulated NO secretion by the endothelium.

Endothelin 1 type a receptor antagonism prevents vascular dysfunction and hypertension induced by 11beta-hydroxysteroid dehydrogenase inhibition: role of nitric oxide.

The enzyme 11beta-hydroxysteroid dehydrogenase (11beta-HSD) prevents inappropriate activation of the nonselective mineralocorticoid receptors by glucocorticoids. Renal activity of 11beta-HSD is decreased in patients with apparent mineralocorticoid excess (SAME), licorice-induced hypertension, and essential hypertension. Although expressed in vascular cells, the role of 11beta-HSD in the regulation of vascular tone remains to be determined. lycyrrhizic acid (GA; 50 mg/kg IP, twice daily for 7 days) caused a significant inhibition of 11beta-HSD activity and induced hypertension in Wistar-Kyoto rats (157 versus 127 mm Hg in controls; P<0.01). After 11beta-HSD inhibition, aortic endothelial nitric oxide (NO) synthase (eNOS) protein content, nitrate tissue levels, and acetylcholine-induced release of NO were blunted (all P<0.05 versus controls). In contrast, vascular prepro-endothelin (ET)-1 gene expression, ET-1 protein levels, and vascular reactivity to ET-1 were enhanced by GA treatment (P<0.05 versus controls). Chronic ET(A) receptor blockade with LU135252 (50 mg. kg(-1). d(-1)) normalized blood pressure, ET-1 tissue content, vascular reactivity to ET-1, vascular eNOS protein content, and nitrate tissue levels and improved NO-mediated endothelial function in GA-treated rats (P<0.05 to 0.01 versus untreated and verapamil-treated controls). In human endothelial cells, GA increased production of ET-1 in the presence of corticosterone, which indicates that activation of the vascular ET-1 system by 11beta-HSD inhibition can occur independently of changes in blood pressure but is dependent on the presence of glucocorticoids. Chronic ET(A) receptor blockade normalizes blood pressure, prevents upregulation of vascular ET-1, and improves endothelial dysfunction in 11beta-HSD inhibitor-induced hypertension and may emerge as a novel therapeutic approach in cardiovascular disease associated with reduced 11beta-HSD activity.

Endothelin-1 production during the acute chest syndrome in sickle cell disease.

To investigate the role of the endothelial-derived vasoactive mediator endothelin (ET-1) in the acute chest syndrome (ACS), we incubated bovine pulmonary artery endothelial cells (BPAEC) with red blood cells (equivalent to a hematocrit of 20%) and/or autologous plasma (1:10 dilution) from two patients during ACS and during routine clinic visits. Cellular RNA was analyzed for ET-1 transcripts by Northern analysis and ET-1 protein levels in BPAEC supernatants and in plasma measured by radioimmunoassay. ET-1 mRNA expression and protein levels increased in BPAEC exposed to plasma obtained during ACS; in contrast, exposure to plasma obtained during routine clinic visits did not alter BPAEC ET-1 mRNA expression or protein levels. Plasma ET-1 level was elevated during ACS, decreased during resolution, and remained slightly elevated during routine clinic visits. Plasma obtained from one patient 4 d prior to hospitalization for vasoocclusive crisis contained the highest ET-1 level and markedly increased BPAEC ET-1 mRNA expression and protein levels. In both patients, BPAEC ET-1 mRNA and protein expression in vitro and plasma ET-1 levels in vivo correlated with stage of disease and occurred in the absence of direct erythrocyte contact in vitro. These observations suggest that ET-1 production contributes to development of ACS.

Vascular cell interactions modulate the expression of endothelin-1 and platelet-derived growth factor BB.

Endothelin (ET)-1 and platelet-derived growth factor (PDGF) BB are important regulators of vascular cell growth and contractile function. We tested the hypothesis that interactions between endothelial cells (ECs) and vascular smooth muscle cells (VSMCs) regulate the expression of these two factors. Bovine and rat cocultures of EC-VSMC that allowed cell contact and exchange of soluble mediators were studied. In bovine cocultures, ET-1 and PDGF B transcription decreased. Minimum levels of transcription were 39 +/- 9% of control for ET-1 and 31 +/- 6% of control for PDGF B. In contrast, steady-state mRNA levels transiently increased in coculture; maximum mRNA levels were 579 +/- 248 and 267 +/- 41% of control for ET-1 and PDGF B, respectively. ET-1 and PDGF BB protein levels were decreased in bovine cocultures. The amount of ET-1 in cocultures was 51% less and the amount of PDGF BB was 25% less than that in homotypic cultures. In rat cocultures, ET-1 protein levels were transiently increased compared with homotypic cultures; the maximum difference was 99 +/- 26%. These results indicate that EC-VSMC interactions regulate ET-1 and PDGF BB expression in EC-VSMC cocultures via multiple mechanisms. Furthermore, the interactive regulation appears to be species specific. These data suggest a novel mechanism for the regulation of these two vasoactive factors that may play an important role in cardiovascular physiology and/or pathology.