Endothelin-1 mRNA Research Articles

The effects of TSH on human vascular endothelial cells and smooth muscle cells

Objective To study the effect of thyroid-stimulating hormone (TSH) on human vascular endothelial cells and smooth muscle cells and to explore the roles of TSH in the development of atherosclerosis.Methods Human vascular endothelial cells and smooth muscle cells were cultured in vitro.MTT method was used to assay the effect of TSH on cell viability.Real-time PCR was used to determine the levels of endothelial nitric oxide synthase (eNOS),prostacyclin(PGI2),endothelin-1 (ET-1),plasminagen activator inhibitor(PAI-1) (mRNA) in endothelial cells and the phenotype transition of smooth muscle cells.The effect of TSH to the cycle of smooth muscle cells was detected by using flow cytometry.Immunocytochemistry and Western blot were used to investigate the expression of the cell cyclin A,D1,and the expression of endothelial cell associated factors eNOS and ET-1.Results Compared with the control group,eNOS and PGI2 mRNA levels decreased while ET-1 and PAI-1 mRNA levels increased when different concentrations of TSH were applied to endothelial cells(P＜0.05).The level of eNOS protein was decreased gradually while the level of ET-1 protein was gradually increased(P＜0.05).Different concentrations of TSH applied to smooth muscle cells could promote the transition of cell cycle phase G2 to phase M and increase the expression of cell cyclin A and D1.Conclusion TSH may damage the function of vascular endothelial cells and promote the proliferation of smooth muscle cells. Key words: Thyroid stimulating hormone; Vascular endothelial cells; Vascular smooth muscle cells; Atherosclerosis

MicroRNA regulation of endothelin-1 mRNA in renal collecting duct cells

AimsRecently, microRNAs (miRNAs) have been implicated in control of Edn1 mRNA in several tissues. Here we examined the role of miRNA action on Edn1 mRNA expression in renal distal collecting duct cells. Main methodsA microarray study was conducted to provide a comprehensive assessment of miRNAs present in a murine inner medullary collecting duct (mIMCD-3) cell line. The experiment was designed as a comparison between mIMCD-3 cells grown in the presence and absence of aldosterone. Argonaute (Ago) immunoprecipitation experiments were used to investigate binding of the RNA induced silencing complex (RISC) to Edn1 mRNA. Key findingsThirty-four miRNAs were detected in very high abundance in mIMCD-3 cells, and a large number of others were present at lower levels. The microarray experiments were validated by quantitative PCR analysis of selected miRNAs. The microarray data, in combination with in silico examination of the Edn1 3′ UTR provided a panel of candidate miRNAs that could act upon the Edn1 expression. Edn1 mRNA was co-immunoprecipitated with an Argonaute protein antibody, and this interaction was blocked by anti-miR-709 oligonucleotides. SignificanceThese results define the miRNA landscape of the mIMCD-3 cell line. Moreover, Edn1 was shown to interact with Argonaute protein suggesting that it is a target of the RNA induced silencing complex (RISC).

Effects of TSH on the function of human umbilical vein endothelial cells

Recent studies have reported that subclinical hypothyroidism (SCH) is associated with atherosclerosis (AS). Thyroid hormone is maintained at normal levels in patients with SCH, whereas TSH is increased. However, the pathogenesis of AS in association with SCH is only partially understood. In addition, endothelial dysfunction plays an important role in the development of AS. The purpose of the present research was to study the direct effect of TSH on human umbilical vein endothelial cells (HUVECs). The expression of some genes associated with endothelial dysfunction after treatment with TSH was evaluated by real-time PCR and western blotting respectively. At first, we showed that the TSH receptor (TSHR) is expressed in HUVECs. We also provide evidence indicating that TSH treatment promotes tumor necrosis factor α-induced endothelial cells interactions by upregulating the expression of the adhesion molecules intercellular adhesion molecule-1. Furthermore, the expression of endothelial nitric oxide synthase (eNOS) and prostacyclin (PGI₂) was significantly attenuated following treatment with TSH in dose- and time-dependent manner. Conversely, the results indicated that TSH upregulated endothelin-1 (ET1) mRNA and protein expression in HUVECs, similar effects were observed for plasminogen activator inhibitor-1 (PAI1) after treatment with various concentrations of TSH. Taken together, these results demonstrate that elevated TSH can promote endothelial dysfunction by altering gene expression in HUVECs.

MiRNA-1 regulates endothelin-1 in diabetes

AimsMicroRNAs (miRNAs) play important roles in several biological processes. In this study, we investigated the role of miR-1, an endothelin-1 (ET-1) targeting miRNA, in endothelial cells (ECs) and tissues of diabetic animals. ET-1 is known to be of pathogenetic significance in several chronic diabetic complications. Main methodsPCR array was used to identify alterations of miRNA expression in ECs exposed to glucose. miR-1 expression was validated by TaqMan real-time PCR assay. Human retinal ECs (HRECs) and human umbilical vein ECs (HUVECs) exposed to various glucose levels with or without miR-1 mimic transfection, and tissues from streptozotocin-induced diabetic animals after two months of follow-up, were examined for miR-1 expression, as well as ET-1 and fibronectin (FN) mRNA and protein levels. Key findingsArray analyses showed glucose-induced alterations of 125 miRNAs (out of 381) in ECs exposed to 25mM glucose compared to 5mM glucose. Fifty-one miRNAs were upregulated and 74 were downregulated. 25mM glucose decreased miR-1 expression and increased ET-1 mRNA and protein levels. miR-1 mimic transfection prevented HG-induced ET-1 upregulation. Furthermore, glucose induced upregulation of FN, which is mediated partly by ET-1, was also prevented by such transfection.Diabetic animals showed decreased miR-1 expression in the retina, heart and kidneys. In parallel, ET-1 mRNA expressions were increased in these tissues of diabetic animals, in association with upregulation of FN. SignificanceThese results indicate a novel glucose-induced mechanism of tissue damage, in which miR-1 regulates ET-1 expressions in diabetes. Identifying such mechanisms may lead to RNA based treatment for diabetic complications.

High Glucose-Induced Increased Expression of Endothelin-1 in Human Endothelial Cells Is Mediated by Activated CCAAT/Enhancer-Binding Proteins

High glucose-induced endothelial dysfunction is partially mediated by the down-stream pathophysiological effects triggered by increased expression of endothelin-1 (ET-1). The molecular control mechanisms of ET-1 synthesis are yet to be discovered. Members of the CCAAT/enhancer-binding proteins (C/EBP) family are important regulators of key metabolic processes, cellular differentiation and proinflammatory genes. In this study, we aimed at elucidating the role of C/EBP in mediating the high glucose effect on ET-1 expression in human endothelial cells (EC). Human umbilical vein cells (EAhy926) and primary cultures of human aortic EC were exposed to high levels of glucose (16.5–25 mM). Real-time PCR, Western blot, enzyme-linked immunosorbent assay, ET-1 promoter-luciferase reporter analysis, and chromatin immunoprecipitation assays were employed to investigate ET-1 regulation. High glucose activated C/EBPα, C/EBPβ, and C/EBPδ in a dose-dependent manner. It also promoted significant increases in ET-1 gene and peptide expression. Chemical inhibition of JNK, p38MAPK and ERK1/2 diminished significantly the high glucose-induced nuclear translocation of C/EBP and ET-1 expression. Silencing of C/EBPα, C/EBPβ or C/EBPδ greatly reduced the high glucose-induced upregulation of ET-1 mRNA, pre-pro-ET-1, and ET-1 secretion. The expression of various C/EBP isoforms was selectively downregulated by siRNA-mediated gene silencing. In silico analysis indicated the existence of typical C/EBP elements within human ET-1 gene promoter. Transient overexpression of C/EBPα, C/EBPβ or C/EBPδ upregulated the luciferase level controlled by the ET-1 gene promoter. The direct interaction of C/EBPα, C/EBPβ or C/EBPδ proteins with the ET-1 promoter in high glucose-exposed EC was confirmed by chromatin immunoprecipitation assay. High glucose-induced ET-1 expression is mediated through multiple mechanisms. We present evidence that members of the C/EBP proinflammatory transcription factors are important regulators of ET-1 in high glucose-exposed human endothelial cells. High glucose-induced activation of C/EBP-related signaling pathways may induce excessive ET-1 synthesis, thus promoting vasoconstriction and dysfunction of the vascular wall cells in diabetes.

Gender Differences In Renal Blood Flow In Response To Endothelin-1 In a Mouse Model Of Sickle Cell Disease

There is increasing evidence that Endothelin-1 (ET-1) plays a significant role in the pathogenesis of complications of sickle cell disease (SCD. SCD patients have elevated levels of endothelin-1 both in plasma and urine and ET-1 has been implicated in SCN, pain modulation, vaso-occlusive episodes, and acute chest syndrome. In the kidney, ET-1 is a potent vasoconstrictor, anti-natriuretic, and hypertrophic factor when signaling through the ETA receptor. ET-1 is released in response to shear stress, hypoxia, thrombin activation, and inflammatory cytokines, all of which are prevalent in SCD. Gender differences exist in other disease models, in which ET-1 plays a contributory pathophysiological role, with males typically exhibiting increased ET-1 mRNA expression, exaggerated ETA-mediated effects, and higher levels of renal injury. Little research exists highlighting the differences between males and females in SCD. It has been observed that the level of Hb F, a major modifier of disease severity in SCD, is higher in females compared to males. In addition, one study examined responses to chronic transfusion therapy, finding males to have increased vascular resistance and resulting decrease in oxygen delivery in response to transfusion when compared to females. We hypothesized that male SCD mice have enhanced ETA mediated responses to ET-1, ultimately leading to renal injury. To test this, we used male and female ‘HbS only' knock in SCD mice to measure renal artery blood flow velocity (BFV) via laser Doppler flowmetry in response to an ET-1 bolus (1nm/kg in 100uL saline). Male mice had an exaggerated decrease in BFV relative to females at 1, 5, and 10 minutes post ET-1 infusion while renal artery diameter remained unchanged throughout the experiment (84.4±5.8 vs. 56±5.7% of BFV baseline at 10 minutes, p<0.05, n=6). A follow up experiment was performed to determine ET-1, ETA receptor, and ETB receptor mRNA expression in the cortex of male and female SCD mice via qPCR. Males and females surprisingly exhibited no significant difference in these parameters although there was a strong trend suggesting females have increased levels of ET-1 mRNA expression compared to males (1.62±0.51 fold increase vs. males, p=0.056, n=5-6). While the exaggerated sensitivity to an ET-1 bolus is partially explained by the relative decrease in ET-1 mRNA expression in males, we nevertheless expected to see differences in ETA/ETB receptor expression. However, receptor-ligand binding could perhaps offer an alternate explanation for this phenomenon. Sex hormones are heavily implicated in other sex differences studies and the potential protective role of estrogen and/or detrimental role for testosterone are both viable explanations for the dichotomy seen in SCD. These data demonstrate a novel sexual dimorphism for SCD, revealing new insights into sex-specific pathophysiology that could have broad effects on treatment strategies in sickle cell nephropathy. Disclosures:No relevant conflicts of interest to declare.

Supplementation of Creatine and Ribose Prevents Apoptosis and Right Ventricle Hypertrophy in Hypoxic Hearts

The simultaneous supplementation of creatine and D-ribose has been shown to reduce apoptosis in vitro in non-irreversibly injured cultured ischemic cardiomyocytes through down-regulation of the signaling mechanisms governing adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (Akt). Here, we test the hypothesis that an analogous mechanism exists in vivo when the challenge is chronic exposure to hypoxia. Five week-old mice were exposed to an atmosphere containing 10% O2 for 10 days. Mice were gavaged daily with vehicle, creatine, D-ribose or creatine + D-ribose. After sacrifice, myocardial and pulmonary tissue were harvested for structural and biochemical analyses. Hypoxia induced right ventricle hypertrophy and left ventricle apoptosis. Both phenotypes were slightly reduced by either creatine or D-ribose, whereas the simultaneous administration of creatine + D-ribose almost completely reversed the effects of hypoxia. Furthermore, creatine + D-ribose diminished the hypoxia-induced increases in the activity of AMPK, Akt and JNK, but not of ERK. Finally, the hypoxia-induced pulmonary overexpression of endothelin-1 mRNA was markedly reduced by creatine + D-ribose. The simultaneous administration of creatine + D-ribose confers additional cardiovascular protection with respect to that observed with either creatine or D-ribose. The mechanism stems from the AMPK and Akt signaling pathways. These findings may form the basis of a paradigm to re-energize non-irreversibly damaged cardiomyocytes, counteracting injury by triggering specific signaling pathways.

Study on the antiulcer effects of Veronicastrum axillare on gastric ulcer in rats induced by ethanol based on tumor necrosis factor-α (TNF-α) and endothelin-1 (ET-1)

ObjectiveTo assess whether Veronicastrum axillare (V. axillare) can ameliorate ethanol-induced gastric mucosal lesions in rats, reduce the production of pro-inflammatory cytokines, suppress apoptosis and improve local microcirculation disturbances. MethodsTotally 48 male Sprague-Dawley rats were randomly divided into six groups, eight rats in each group. Rats in the normal group and the model group were administered with 0.9% normal saline respectively. Rats in the positive group and ranitidine group were administered with 0.18% ranitidine suspension by intragastric administration respectively. Those in the high dose V. axillare group, the medium dose V. axillare group and the low dose V. axillare group were administrated with V. axillare at the daily dose of 2.8 g/kg, 1.4 g/kg and 0.7 g/kg by intragastric administration. Gastric mucosal lesions were produced by intragastric administration of absolute ethanol. Water extract of V. axillare was successively injected for 14 d and last day was injected 1 h before ethanol administration. Gastric mucosal ulcer index and ulcer inhibitory rate were counted by improved Guth methods. The tissue sections were made for pathological histology analysis. Also, we measured the concentrations of tumor necrosis factor-α (TNF-α) and endothelin-1 (ET-1) in gastric mucosal, as an index of the pro-inflammatory cytokines, apoptosis and local microcirculation. Besides, the mRNA contents of TNF-α and ET-1 were measured to verify effects on gene expression by real-time fluorescent quantitative PCR. ResultsWater extract of V. axillare significantly ameliorated the gastric mucosal lesions induced by ethanol administration (P<0.01). Pro-inflammatory cytokines, TNF-α and ET-1 were increased after ethanol administration and significantly reduced by water extract of V. axillare. The expressions of TNF-α and ET-1 mRNA were also be inhibited by water extract of V. axillare. ConclusionCurrent evidences show water extract of V. axillare is effective for defending against ethanol-induced gastric mucosal lesions, significantly inhibiting the production of pro-inflammatory cytokines and the expressions of TNF-α and ET-1 mRNA, which may be useful for inhibiting apoptosis and improving local microcirculation.

Chemotherapy-mediated p53-dependent DNA damage response in clear cell renal cell carcinoma: role of the mTORC1/2 and hypoxia-inducible factor pathways

The DNA-damaging agent camptothecin (CPT) and its analogs demonstrate clinical utility for the treatment of advanced solid tumors, and CPT-based nanopharmaceuticals are currently in clinical trials for advanced kidney cancer; however, little is known regarding the effects of CPT on hypoxia-inducible factor-2α (HIF-2α) accumulation and activity in clear cell renal cell carcinoma (ccRCC). Here we assessed the effects of CPT on the HIF/p53 pathway. CPT demonstrated striking inhibition of both HIF-1α and HIF-2α accumulation in von Hippel–Lindau (VHL)-defective ccRCC cells, but surprisingly failed to inhibit protein levels of HIF-2α-dependent target genes (VEGF, PAI-1, ET-1, cyclin D1). Instead, CPT induced DNA damage-dependent apoptosis that was augmented in the presence of pVHL. Further analysis revealed CPT regulated endothelin-1 (ET-1) in a p53-dependent manner: CPT increased ET-1 mRNA abundance in VHL-defective ccRCC cell lines that was significantly augmented in their VHL-expressing counterparts that displayed increased phosphorylation and accumulation of p53; p53 siRNA suppressed CPT-induced increase in ET-1 mRNA, as did an inhibitor of ataxia telangiectasia mutated (ATM) signaling, suggesting a role for ATM-dependent phosphorylation of p53 in the induction of ET-1. Finally, we demonstrate that p53 phosphorylation and accumulation is partially dependent on mTOR activity in ccRCC. Consistent with this result, pharmacological inhibition of mTORC1/2 kinase inhibited CPT-mediated ET-1 upregulation, and p53-dependent responses in ccRCC. Collectively, these data provide mechanistic insight into the action of CPT in ccRCC, identify ET-1 as a p53-regulated gene and demonstrate a requirement of mTOR for p53-mediated responses in this tumor type.

Effects of sFlt-1 and alpha 2-macroglobulin on vascular endothelial growth factor-induced endothelin-1 upregulation in human microvascular endothelial cells

IntroductionSoluble fms-like tyrosine kinase-1 (sFlt-1) is a vascular endothelial growth factor (VEGF) binding protein and potent antagonist of VEGF. Alpha 2 macroglobulin (α2M) is another major binding protein for circulating VEGF, which is present in human plasma at higher concentration (2–4 mg/mL) than sFlt-1. This study investigated the effects of sFlt-1 and α2M on VEGF-induced endothelin-1 (ET-1) upregulation in human microvascular endothelial cell-1 (HMEC-1). MethodsHMEC-1 was cultured and incubated with varying concentrations of sFlt-1 and α2M in combination with VEGF. ET-1 mRNA expression in the cells was measured by real time RT-PCR and ET-1 protein by western blot analysis. ResultsET-1 expression in HMEC-1 incubated with VEGF significantly increased in time- and dose-dependent manners. Next, HMEC-1 was treated with the sFlt-1 (10–1000 ng/mL) or α2M (10–10000 ng/mL) in the presence of VEGF (10 ng/mL). We found that sFlt-1 induced a significant decrease of ET-1 expression upregulated by VEGF, while α2M did not affect the VEGF-induced ET-1 expression. ConclusionssFLT-1 suppressed the VEGF-induced the ET-1 expression of HMEC-1. However, α2M did not show a significant effect on the ET-1 expression that was induced by VEGF. The results suggest that a certain proportion of the bound form α2M-VEGF have a biological action involved in the pathophysiology of preeclampsia.

Changes of hypoxia-inducible factor-1α, endothelin-1 and adrenomedullin in newborn rats with hypoxia-induced pulmonary hypertension

Objective To provide the foundation of theory on prevention of hypoxia-induced pulmonary hypertension(HPH) by investigating the concentrations in the serum and expression levels in the lung tissue, and by studying the roles of hypoxia-inducible factor-1α(HIF-1α), endothelin-1(ET-1) and adrenomedullin (ADM) in newborn rats with HPH. Methods Seventy-two newborn rats were randomly divided into hypoxic group(hypoxia exposure) and normoxic group.The mean pulmonary arteria pressure(mPAP), concentrations in the serum and expression levels in the lung tissue of the above-mentioned factors were measured in these neonatal rats after 3, 5, 7, 10, 14 and 21 days, including hypoxic group and normoxic group, respectively.The morphology of pulmonary arterioles was observed and the vascular remodeling indexes were measured. Results 1.Compared with the normoxic group, the mPAP increased with the prolonged hypoxic time in the hypoxic group(P<0.05).2.Concentrations in the serum and expression levels in the lung tissue of HIF-1α were higher than those of the normoxic group on 3, 5, 7, 10, 14 days(all P<0.05). The serum concentration of ET-1 elevated significantly in the hypoxic group at each time point, and ET-1 mRNA increased only on 3 and 5 days of the hypoxia group.The serum concentration of ADM level in the hypoxic group was higher than that of the normoxic group on 3 and 5 days, but lower than the normoxic group after 14 and 21 days of hypoxia, and the expression of ADM mRNA in the lung tissue was higher on 3 and 5 days in the hypoxic group.3.Morphology and ultrastructure of the pulmonary arteries change were found after 7 days of hypoxia exposure. Conclusions HIF-1α may regulate ET-1 and ADM in the pathogenesis of HPH.ET-1 plays a powerful role by pulmonary vasoconstriction and vascular remodeling, whereas ADM displays the protective effects on blood vessels in the early stages of HPH.Pulmonary vascular remodeling occurs in the hypoxic group after 7 days of hypoxia. Key words: Hypoxia-inducible factor-1α; Endothelin-1; Adrenomedullin; Hypertension; Rat

Endothelin-1 Single Nucleotide Polymorphisms and Risk of Pulmonary Metastatic Osteosarcoma

Pulmonary metastases are the major cause of death of osteosarcoma (OS) patients. Endothelin-1 (ET-1) reportedly plays an important role in OS metastasis. In the present study, we for the first time explored the association of ET-1 SNPs with the risk of pulmonary metastatic OS. We genotyped three SNPs (rs1800541, rs2070699 and rs5370) in the ET-1 gene in a case-control study, using 260 pairs of age-, sex-, residence area- and tumor location-matched subjects. Patients with pulmonary metastatic OS and patients with localized high-grade (stage IIB) OS were enrolled as cases and controls, respectively. The G allele at rs1800541 was found associated with reduced risk of pulmonary metastatic OS after adjustment for body mass index, systolic blood pressure, diastolic blood pressure and the plasma ET-1 level (P=10-4; adjusted OR, 0.55; 95% CI, 0.42-0.70), while the G allele at rs2070699 was not significantly associated with the risk of pulmonary metastatic OS (P=0.15; adjusted OR, 1.15; 95% CI, 0.87-1.50). The mRNA and the secreted protein levels of ET-1 in primary OS cell cultures (POCCs) established from surgically resected primary OS in the rs1800541 TT homozygotes were higher than those from the TG heterozygotes (P<0.05), who in turn showed higher ET-1 mRNA and secreted ET-1 levels than the GG homozygotes (P<0.05). In the control subjects, the rs1800541 TT homozygotes showed an 18.4% relapse rate, significantly higher than that of the GG homozygotes (0%) (P<0.01). On the other hand, the GG homozygotes showed a 71.4% complete recovery rate, significantly higher than that of the TG heterozygotes (7.3%) and the TT homozygotes (0%) (P<0.01). This study provides the first evidence of an association between the ET-1 gene SNPs and the risk of pulmonary metastatic OS.

Alterations in circulating angiogenic and anti‐angiogenic factors in type 2 diabetic patients with neuropathy

Diabetic peripheral neuropathy (DPN) is one of the most common diabetic chronic complications. There is an increased attention directed towards the role of angiogenic factors including vascular endothelial growth factor (VEGF) and anti-angiogenic factors including soluble endoglin (sEng) as contributors to diabetic microvascular complications including neuropathy. The purposes of this study were to determine the role of these angiogenesis regulators in the prognosis of DPN. The study group included 60 patients with type 2 diabetes mellitus (T2DM) and 20 clinically healthy individuals. The patients were divided into two groups. Group I included 20 T2DM patients without peripheral neuropathy, and Group II consisted of 40 T2DM patients with DPN. In all groups, plasma VEGF, sEng and endothelin-1 (ET-1), nitric oxide and ET-1 mRNA were estimated. Plasma levels of VEGF, sEng, ET-1 and nitric oxide were significantly elevated in diabetic patients (Groups I and II) compared with healthy control subjects, with a higher increase in their levels in patients with DPN compared with diabetic patients without peripheral neuropathy. Measurement of plasma levels of angiogenesis-related biomarkers in high-risk diabetic patients might identify who later develop DPN, thus providing opportunities for early detection and targets for novel treatments.

Gestational Hypoxia Induces Preeclampsia-Like Symptoms via Heightened Endothelin-1 Signaling in Pregnant Rats

Preeclampsia is a life-threatening pregnancy disorder. However, its pathogenesis remains unclear. We tested the hypothesis that gestational hypoxia induces preeclampsia-like symptoms via heightened endothelin-1 (ET-1) signaling. Time-dated pregnant and nonpregnant rats were divided into normoxic and hypoxic (10.5% O2 from the gestational day 6-21) groups. Chronic hypoxia had no significant effect on blood pressure or proteinuria in nonpregnant rats but significantly increased blood pressure on day 12 (systolic blood pressure, 111.7 ± 6.1 versus 138.5 ± 3.5 mm Hg; P=0.004) and day 20 (systolic blood pressure, 103.4 ± 4.6 versus 125.1 ± 6.1 mm Hg; P=0.02) in pregnant rats and urine protein (μg/μL)/creatinine (nmol/μL) ratio on day 20 (0.10 ± 0.01 versus 0.20 ± 0.04; P=0.04), as compared with the normoxic control group. This was accompanied with asymmetrical fetal growth restriction. Hypoxia resulted in impaired trophoblast invasion and uteroplacental vascular remodeling. In addition, plasma ET-1 levels, as well as the abundance of prepro-ET-1 mRNA, ET-1 type A receptor and angiotensin II type 1 receptor protein in the kidney and placenta were significantly increased in the chronic hypoxic group, as compared with the control animals. Treatment with the ET-1 type A receptor antagonist, BQ123, during the course of hypoxia exposure significantly attenuated the hypoxia-induced hypertension and other preeclampsia-like features. The results demonstrate that chronic hypoxia during gestation induces preeclamptic symptoms in pregnant rats via heightened ET-1 and ET-1 type A receptor-mediated signaling, providing a molecular mechanism linking gestational hypoxia and increased risk of preeclampsia.

RETRACTED: Epigenetic control of endothelin-1 axis affects invasiveness of breast carcinoma cells with bone tropism

Here, we report a complex regulation of endothelin-1 (ET-1) axis driven by epigenetic reactions in 1833-bone metastatic cells, emphasizing the importance in skeletal metastasis from breast carcinoma. Inhibitors of histone deacetylases, trichostatin A (TSA), and of DNA methylases, 5'-Azacytidine (Aza), caused, respectively, reduction and increase in 1833 cell invasiveness, without affecting the basal migration of parental MDA-MB231 cells. Of note, in the two cell lines exposed to Aza the blockade of the ET-1 receptor ETAR with BQ-123 oppositely changed invasive properties. Even if in MDA-MB231 cells the ET-1 axis was scarcely influenced by epigenetic reactions, ETAR remarkably decreased after Aza. In contrast, in 1833 cells Aza exposure enhanced ET-1 coupled to ETAR wild type, being also ETAR truncated form increased, and invasiveness was stimulated. Under demethylation, the increase in ET-1 steady state protein level in 1833 clone seemed regulated at transcriptional level principally via Ets1 transcription factor. In fact, actinomycin D almost completely prevented ET-1 mRNA induction due to Aza. Only in 1833 cells, TSA exposure inactivated ET-1 axis, with reduction of the expression of ET-1 and ETAR mutated form, in agreement with Matrigel invasion decrease. This treatment favoured the ET-1 repressional control, taking place at the level of mRNA stability due to the 3'-untranslated region in the ET-1 gene, and also decreased transcription via NF-kB. Environmental conditions that alter the balance between epigenetic reactions might, therefore, affect metastasis migratory mode influencing ET-1 axis.

Angiotensin II Induces Endothelin-1 Expression in Human Hepatic Stellate Cells

Both angiotensin (Ang)-II and endothelin-1 (ET-1) are involved in the pathogenesis of liver fibrosis. Activated hepatic stellate cells (HSCs) are considered a key effector of liver fibrosis. To explore the effect of Ang-II on ET-1 expression in cultured human HSCs and the underlying mechanisms. Human HSCs were treated with Ang-II in different concentrations (0.1, 0.5, 1, 5, or 10nM) for different lengths of time (0.5, 1, 2, 4, or 6h) with or without transcription inhibitor actinomycin D, Ang-II type 1 (AT1) receptor blocker losartan, AT2 receptor blocker PD123177, or different kinase inhibitors. Ang-II increased the ET-1 mRNA level in a statistically significant dose- and time-dependent manner within 4h, which led to dose-dependent up-regulation of the ET-1 protein level. Actinomycin D (1mg/ml), losartan (50μM), and phosphatidylinositol-3 kinase inhibitor LY294002 (50μM) abolished the promoting effect of Ang-II on ET-1 expression. Ang-II (10nM) significantly increased the expression of α-smooth muscle actin and type I collagen in HSCs, which was abolished by losartan, LY294002, ET A receptor blocker BQ123, and ET-1 siRNA, but not PD123177 and ET B receptor blocker BQ788. Ang-II induces ET-1 expression in human HSCs via the AT1 receptor by the PI3K/Akt signaling pathway. The ET-1/ET A receptor axis could mediate the promoting effects of Ang-II on HSCs' transdifferentiation into myofibroblast-like cells. This is the first evidence of crosstalk between the Ang-II/AT1 axis and the ET-1 system in regard to the pathogenesis of liver fibrosis.

Association of Endothelin-1 Expression and Cartilaginous Endplate Degeneration in Humans

BackgroundInflammatory cytokines are involved in intervertebral disc (IVD) degeneration. Endothelin-1 (ET-1), a 21-amino-acid cytokine implicated with cartilage degradation, is secreted by vascular endothelial cells and also by many other cell types. The expression of ET-1 in human IVD cartilage endplate (CEP) and its role in disc degeneration have not been explored.Methods and FindingsThe expression of ET-1 in degenerated CEP was analyzed by immunohistochemical staining and Western blotting; ET-1 was demonstrated in cartilaginous endplate cells (CECs) by immunofluorescent staining. The ET-1 mRNA expression and protein production by CECs stimulated by tumor necrosis factor alpha (TNF-α), a pro-inflammatory cytokine, were determined by real-time PCR analysis and Western blotting, respectively. The matrix metalloprotease-1 (MMP-1), MMP-13 and tissue inhibitor of metalloproteases-1 (TIMP-1) levels in the supernatant of cultured CECs treated with ET-1 were determined using enzyme-linked immunosorbent assays. Nitric oxide (NO) release and nitric oxide synthase (NOS) activity were measured using a spectrophotometric assay. The apoptosis of CECs by ET-1 was measured by an Annexin V-FITC detection assay. The production of ET-1 in degenerated cartilage endplate was significantly higher than normal CEP. The results showed that ET-1 was expressed by CECs and modulated by TNF-α in a dose-dependent manner. ET-1 increased production of MMP-1 and MMP-13, decreased TIMP-1 production, and induced NO and NOS release by cultured CECs. The direct stimulation of CECs by ET-1 did not promote cell apoptosis.ConclusionThe study results suggest that ET-1 played a pivotal role in human CEP degeneration, and may be a new target for development of therapies for this condition.

Effect of NADPH oxidase inhibitor apocynin on the expression of hypoxia-induced factor-1α and endothelin-1 in rat carotid body exposed to chronic intermittent hypoxia

The effects of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin on the enhanced hypoxia induced factor-1α (HIF-1α) and endothelin-1 (ET-1) expression, elevated systolic blood pressure under chronic intermittent hypoxia (CIH) condition and its action mechanism were investigated. Thirty healthy 8-week old Sprague-Dawley (SD) male rats were randomly divided into three groups (n=10 each): sham group, CIH group, and apocynin-treated CIH group. Tail artery systolic blood pressure was measured by tail-cuff method. Real-time fluorescence quantitative polymerase chain reaction (PCR) was used to detect the mRNA expression of HIF-1α and ET-1 in the carotid body, and the HIF-1α protein expression was examined by using Western blotting. The levels of malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by using colorimetric method. In addition, the plasma ET-1 and HIF-1α levels were measured by using enzyme-linked immunosorbent assay. It was found that CIH exposure was associated with increased MDA levels, and apocynin-treated CIH animals showed reduction in MDA levels. Apocynin treatment prevented CIH-induced hypertension as well as CIH-induced decrease in SOD. The increases of HIF-1α and ET-1 mRNA along with HIF-1α protein expression in the carotid body, and elevated circulating HIF-1α and ET-1 levels were observed in CIH-exposed animals. Treatment with apocynin significantly decreased the ET-1 mRNA, HIF-1α protein expression and circulating HIF-1α level in CIH-exposed animals, and there was no statistically significant difference in the HIF-1α mRNA expression between CIH group and apocynin-treated group. These results indicated that apocynin alleviated CIH-induced hypertension by inhibiting NADPH oxidase, further leading to the reduced vasoconstrictor ET-1 level and oxidative stress. HIF-1α/ET-1 system signal pathway may interact with CIH-induced NADPH oxidase-dependent oxidative stress. Inhibition of NADPH oxidase activity may hopefully serve as a useful strategy for prevention and treatment of obstructive sleep apnea hypopnea syndrome-induced hypertension.

The Effect of Aldosterone on the miRNA Content of a Murine Inner Medullary Collecting Duct Cell

MicroRNAs (miRNAs) are a family of small noncoding RNAs that have been shown to regulate gene expression by inhibiting translation or targeting specific mRNAs for degradation. miRNAs play important roles in the regulation of metabolism in the healthy cell and in the pathogenesis of many diseases including diabetic nephropathy and hypertension‐related renal disease.Aldosterone is the major known hormone responsible for sodium retention in the kidney collecting duct. Our working hypothesis is that aldosterone action alters the miRNA content in the inner medullary collecting duct resulting in increased translation and stabilization of mRNAs involved in sodium reabsorption.Here we report the miRNA landscape in mIMCD‐3 cells using Toray 3D‐GeneTM miRNA microarray analysis to evaluate 1080 miRNAs. Microarray analysis identified 35 highly expressed miRNAs, as well as several miRNAs whose levels appear to be affected by the presence of aldosterone. Two aldosterone responsive miRNAs affected levels of serum/glucocorticoid regulated kinase 1and sodium/potassium‐transporting ATPase subunit beta‐1 mRNA. Argonaute pull‐down experiments also demonstrated that endothelin‐1 mRNA is a target of the RNA‐induced silencing complex indicating miRNA action on this transcript.This work was supported by NIH NIDDK 1RO1DK082680 to B.D.C and C.S.W. and a 5T32DK076541–04 pre‐doctoral fellowship to M.E.J. Additional funding support and miRNA microarrays were provided by Toray Industries, Inc.

HLA-B35 and dsRNA Induce Endothelin-1 via Activation of ATF4 in Human Microvascular Endothelial Cells

Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.