Objective: To investigate the clinical significance of endothelin A receptor (ETAR) expression in high-grade serous ovarian carcinoma (HGSOC). To design ETAR carboxyl terminal (ETAR-C) amino acids derived polypeptide and to study the inhibitory effect on ovarian epithelial carcinoma cells in vitro. Methods: (1) A total of 126 patients who received surgical treatment and were diagnosed with HGSOC by postoperative pathological examination in Central Hospital of Xuzhou from January 1, 2007 to December 31, 2017 were selected. All patients had completed clinicopathological data and follow-up data. Cancer tissue samples were collected and ETAR mRNA expression in HGSOC tissues was detected by reverse transcript-PCR. The clinical significance was analyzed. (2) ETAR-C fusion polypeptide was designed based on the sequence of carboxyl terminal amino acids of ETAR, expressed and purified in vitro. The effects of ETAR-C fusion polypeptide on migration and invasion ability of ovarian cancer SKOV3 and CAOV3 cells were detected by scratch test and invasion test, respectively. The effect of ETAR-C fusion polypeptide on chemosensitivity of cisplatin-resistant ovarian cancer SKOV3/cDDP and CAOV3/cDDP cells was determined by methyl thiazolyl tetrazolium (MTT) colorimetric assay. The effect of ETAR-C fusion polypeptide on β-arrestin-1 expression in ovarian cancer SKOV3 and CAOV3 cells was detected by western blot. Results: (1) The relative expression level of ETAR mRNA in HGSOC tissues was 18.6±5.1. Patients with HGSOC were divided into high ETAR mRNA expression (n=76) and low ETAR mRNA expression (n=50) with 61.7% as cut-off value analyzed by X-Tile software. High expression of ETAR mRNA was significantly correlated with abdominal water volume, platinum drug resistance, and cancer antigen 125 (CA₁₂₅) value in HGSOC patients (all P<0.05), but was not related to the age of patients with HGSOC and the size of postoperative residual lesions (all P>0.05). The 5-year progression free survival rates were 18.4% and 28.0%, and the 5-year overall survival rates were 38.2% and 52.0% in HGSOC patients with high and low ETAR mRNA expression respectively, there were statistically significant differences (P=0.046, P=0.034). (2) The results of scratch test and invasion test showed that the scratch healing rate and cell invasion rate of SKOV3 or CAOV3 cells treated with endothelin-1 (ET-1) and ET-1+ETAR-C were respectively compared, and the differences were statistically significant (all P<0.05). MTT assay showed that the inhibition rates of ETAR-C fusion polypeptide treated in SKOV3/cDDP and CAOV3/cDDP cells were significantly higher than those of control cells after the addition of 4, 6, 8, 10, 12, and 24 μg/ml cisplatin (all P<0.05). Western blot analysis showed that the relative expression levels of β-arrestin-1 in SKOV3 or CAOV3 cells treated with ET-1 and ET-1+ETAR-C were 1.85±0.09 and 1.13±0.09 (SKOV3 cells), 2.14±0.15 and 1.66±0.12 (CAOV3 cells), respectively. The differences were statistically significant (all P<0.05). Conclusions: The prognosis of HGSOC patients with high expression of ETAR mRNA is significantly worse than those with low expression of ETAR mRNA. ETAR might be a new target for HGSOC treatment. The ETAR-C fusion polypeptide that interferes with the interaction of ETAR and β-arrestin-1 has good inhibitory effect on ovarian cancer cells in vitro, and might have clinical application potential.
Female ; Humans ; Amino Acids/therapeutic use* ; beta-Arrestins/therapeutic use* ; Cell Line, Tumor ; Cisplatin/pharmacology* ; Clinical Relevance ; Ovarian Neoplasms/pathology* ; Receptor, Endothelin A/therapeutic use* ; RNA, Messenger/metabolism*

BACKGROUND AND OBJECTIVES: Elevated endothelin (ET)-1 level is strongly correlated with the pathogenesis of pulmonary arterial hypertension (PAH). Expression level of nicotinamide adenine dinucleotide phosphate oxidase (NOX) 4 is increased in the PAH patients. Ambrisentan, a selective endothelin receptor A (ERA) antagonist, is widely used in PAH therapy. The current study was undertaken to evaluate the effects of ambrisentan treatment in the monocrotaline (MCT)-induced PAH rat model. METHODS: Rats were categorized into control group (C), monocrotaline group (M) and ambrisentan group (Am). The M and Am were subcutaneously injected 60 mg/kg MCT at day 0, and in Am, ambrisentan was orally administered the day after MCT injection for 4 weeks. The right ventricle (RV) pressure was measured and pathological changes of the lung tissues were observed by Victoria blue staining. Protein expressions of ET-1, ERA, endothelial nitric oxide synthase (eNOS) and NOX4 were confirmed by western blot analysis. RESULTS: Ambrisentan treatment resulted in a recovery of the body weight and RV/left ventricle+septum at week 4. The RV pressure was lowered at weeks 2 and 4 after ambrisentan administration. Medial wall thickening of pulmonary arterioles and the number of intra-acinar arteries were also attenuated by ambrisentan at week 4. Protein expression levels of ET-1 and eNOS were recovered at weeks 2 and 4, and ERA levels recovered at week 4. CONCLUSIONS: Ambrisentan administration resulted in the recovery of ET-1, ERA and eNOS protein expression levels in the PAH model. However, the expression level of NOX4 remained unaffected after ambrisentan treatment.
Animals ; Arteries ; Arterioles ; Blotting, Western ; Body Weight ; Endothelin Receptor Antagonists ; Endothelins ; Gene Expression ; Heart Ventricles ; Humans ; Hypertension ; Hypertension, Pulmonary ; Lung ; Models, Animal ; Monocrotaline ; NADP ; NADPH Oxidase ; Nitric Oxide Synthase Type III ; Oxidoreductases ; Rats ; Receptors, Endothelin ; Victoria

BACKGROUND: Extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling by remodeling the 6-O-sulfation of heparan sulfate proteoglycans on the cell surface. The present study examined the effects of Sulfs on angiotensin II (Ang II)-induced hypertensive mediator expression and vascular smooth muscle cells (VSMCs) proliferation in spontaneously hypertensive rats (SHR). METHODS: Ang II receptors, 12-lipoxygenase (12-LO), and endothelin-1 (ET-1) messenger RNA (mRNA) expressions in SHR VSMCs were analyzed by real-time polymerase chain reaction and Western blotting. VSMCs proliferation was determined by [³ H]-thymidine incorporation. RESULTS: Basal Sulfs mRNAs expression and enzyme activity were elevated in SHR VSMCs. However, Sulfs had no effect on the basal or Ang II-induced 12-LO and ET-1 mRNA expression in SHR VSMCs. The inhibition of Ang II-induced 12-LO and ET-1 expression by blockade of the Ang II type 2 receptor (AT₂ R) pathway was not observed in Sulf1 siRNA-transfected SHR VSMCs. However, Sulf2 did not affect the action of AT₂ R inhibitor on Ang II-induced 12-LO and ET-1 expression in SHR VSMCs. The down-regulation of Sulf1 induced a reduction of AT₂ R mRNA expression in SHR VSMCs. In addition, the inhibition of Ang II-induced VSMCs proliferation by blockade of the AT₂ R pathway was mediated by Sulf1 in SHR VSMCs. CONCLUSION: These findings suggest that extracellular sulfatase Sulf1 plays a modulatory role in the AT₂ R pathway that leads to an Ang II-induced hypertensive effects in SHR VSMCs.
Angiotensin II* ; Angiotensins* ; Arachidonate 12-Lipoxygenase ; Blotting, Western ; Down-Regulation ; Endothelin-1 ; Heparan Sulfate Proteoglycans ; Hypertension ; Muscle, Smooth, Vascular* ; Rats, Inbred SHR* ; Real-Time Polymerase Chain Reaction ; Receptor, Angiotensin, Type 2* ; RNA, Messenger ; Sulfatases

Waardenburg syndrome (WS) is a rare genetic disorder, including clinical features of pigmentary abnormalities of irides, skin, hair and sensorineural hearing loss and facial dysmorphism. Among the four types, WS type IV (Waardenburg-Shah syndrome) additionally represents Hirschsprung's disease. Mutations in the SOX10, END3, or EDNRB genes are known to cause WS type IV. Here, we report a 6 year-old girl who was diagnosed as WS type IV by typical clinical manifestations, including skin hypopigmentation, heterochromia of both irides, unilateral sensorineural hearing loss, mild developmental delay and Hirschsprung's disease. The diagnosis was confirmed by molecular genetic analysis of EDNRB. Two novel EDNRB mutations were identified, and each mutation was segregated from each of her parents. During the follow-up period, the patient underwent a surgery for spleen torsion and was medically managed due to recurrent enterocolitis. Also, she suffered from impaired immunity including Hirschsprung's associated enterocolitis.
Diagnosis ; Endothelins* ; Enterocolitis ; Female ; Follow-Up Studies ; Hair ; Hearing Loss, Sensorineural ; Hirschsprung Disease ; Humans ; Hypopigmentation ; Molecular Biology ; Parents ; Receptor, Endothelin B ; Receptors, Endothelin* ; Skin ; Spleen ; Waardenburg Syndrome

BACKGROUND: Extracellular sulfatases (Sulfs), sulfatase 1 (Sulf1) and sulfatase 2 (Sulf2), play a pivotal role in cell signaling by remodeling the 6-O-sulfation of heparan sulfate proteoglycans on the cell surface. The present study examined the effects of Sulfs on angiotensin II (Ang II)-induced hypertensive mediator expression and vascular smooth muscle cells (VSMCs) proliferation in spontaneously hypertensive rats (SHR).METHODS: Ang II receptors, 12-lipoxygenase (12-LO), and endothelin-1 (ET-1) messenger RNA (mRNA) expressions in SHR VSMCs were analyzed by real-time polymerase chain reaction and Western blotting. VSMCs proliferation was determined by [³ H]-thymidine incorporation.RESULTS: Basal Sulfs mRNAs expression and enzyme activity were elevated in SHR VSMCs. However, Sulfs had no effect on the basal or Ang II-induced 12-LO and ET-1 mRNA expression in SHR VSMCs. The inhibition of Ang II-induced 12-LO and ET-1 expression by blockade of the Ang II type 2 receptor (AT₂ R) pathway was not observed in Sulf1 siRNA-transfected SHR VSMCs. However, Sulf2 did not affect the action of AT₂ R inhibitor on Ang II-induced 12-LO and ET-1 expression in SHR VSMCs. The down-regulation of Sulf1 induced a reduction of AT₂ R mRNA expression in SHR VSMCs. In addition, the inhibition of Ang II-induced VSMCs proliferation by blockade of the AT₂ R pathway was mediated by Sulf1 in SHR VSMCs.CONCLUSION: These findings suggest that extracellular sulfatase Sulf1 plays a modulatory role in the AT₂ R pathway that leads to an Ang II-induced hypertensive effects in SHR VSMCs.
Angiotensin II ; Angiotensins ; Arachidonate 12-Lipoxygenase ; Blotting, Western ; Down-Regulation ; Endothelin-1 ; Heparan Sulfate Proteoglycans ; Hypertension ; Muscle, Smooth, Vascular ; Rats, Inbred SHR ; Real-Time Polymerase Chain Reaction ; Receptor, Angiotensin, Type 2 ; RNA, Messenger ; Sulfatases

OBJECTIVETo observe the effect of electroacupuncture (EA) stimulation on the expressions of angiotensinogen (AGT), angiotensin II type 1 receptor (AT1R), endothelin-1 (ET1), and endothelin A receptor (ETAR) mRNA in spontaneously hypertensive rat (SHR) aorta.

METHODSEighteen male SHRs were randomly divided into three groups, an SHR group, an SHR Baihui (DU 20) and Zusanli (ST 36) acupoint (SHR-AP) group, and an SHR non-acupoint (SHR-NAP) group, with 6 rats in each group. Six Wistar rats were used as a control. Rats in the SHR-AP group were stimulated by DU 20 and ST 36 acupoints, both of which were connected with EA. EA was handled one time every Monday, Wednesday and Friday, for total 24 times (8 weeks). SHRNAP rats were acupointed at a 15°angle flat into 0.5 cm to two points, which were 1 and 2 cm from rail tip separately. EA parameters were the same as the SHR-AP rats. SHR control rats and Wistar rats were fixed without EA. Real-time quantitative polymerase chain reaction (PCR) was used to measure AGT, AT1R, ET1, and ETAR mRNA expression in rat aorta.

RESULTSEA stimulation significantly reduced rat aorta vascular AGT, ET1, ETAR and AT1R mRNA expressions in the SHR-AP and SHR-NAP groups (P <0.01). Among these four genes, AT1R mRNA expression was significantly lower in the SHR-AP than in the SHR-NAP group (P <0.01).

CONCLUSIONEA could reduce the AT1R mRNA expression in SHR-AP rat aorta, indicating a potential mechanism for the hypotensive effects of EA.

Angiotensinogen ; genetics ; metabolism ; Animals ; Aorta ; metabolism ; physiopathology ; Blood Pressure ; Electroacupuncture ; Endothelin-1 ; genetics ; metabolism ; Gene Expression Regulation ; Male ; RNA, Messenger ; genetics ; metabolism ; Rats, Inbred SHR ; Receptor, Angiotensin, Type 1 ; genetics ; metabolism ; Receptor, Endothelin A ; genetics ; metabolism

Pulmonary arterial hypertension (PAH) causes right ventricular failure due to a gradual increase in pulmonary vascular resistance. The purposes of this study were to confirm the engraftment of human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) placed in the correct place in the lung and research on changes of hemodynamics, pulmonary pathology, immunomodulation and several gene expressions in monocrotaline (MCT)-induced PAH rat models after hUCB-MSCs transfusion. The rats were grouped as follows: the control (C) group; the M group (MCT 60 mg/kg); the U group (hUCB-MSCs transfusion). They received transfusions via the external jugular vein a week after MCT injection. The mean right ventricular pressure (RVP) was significantly reduced in the U group after the 2 week. The indicators of RV hypertrophy were significantly reduced in the U group at week 4. Reduced medial wall thickness in the pulmonary arteriole was noted in the U group at week 4. Reduced number of intra-acinar muscular pulmonary arteries was observed in the U group after 2 week. Protein expressions such as endothelin (ET)-1, endothelin receptor A (ERA), endothelial nitric oxide synthase (eNOS) and matrix metalloproteinase (MMP)-2 significantly decreased at week 4. The decreased levels of ERA, eNOS and MMP-2 immunoreactivity were noted by immnohistochemical staining. After hUCB-MSCs were administered, there were the improvement of RVH and mean RVP. Reductions in several protein expressions and immunomodulation were also detected. It is suggested that hUCB-MSCs may be a promising therapeutic option for PAH.
Animals ; Cytokines/metabolism ; Disease Models, Animal ; Endothelin-1/metabolism ; Fetal Blood/*cytology ; Gene Expression Regulation/drug effects ; Hemodynamics ; Humans ; Hypertension, Pulmonary/chemically induced/*therapy ; Hypertrophy, Right Ventricular/physiopathology ; Immunohistochemistry ; Lung/metabolism/pathology ; Male ; Matrix Metalloproteinase 2/metabolism ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology/metabolism ; Monocrotaline/toxicity ; Nitric Oxide Synthase Type III/metabolism ; Pulmonary Artery/pathology ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/metabolism

OBJECTIVETo investigate the relationship between the 3 polymorphic loci of endothelin receptor type A (EDNRA) gene and intracranial aneurysms.

METHODSThree EDNRA gene polymorphisms (rs5335, rs6842241, and rs6841581) were genotyped using polymerase chain reaction-restriction fragment length polymorphism analysis. The genotype and allele frequencies were calculated in the patients and the control group to analyze the association between the gene and the size of aneurysms.

RESULTSNo significant difference was found in the distribution of the EDNRA gene genotypes or allele frequencies between the patients and the control subjects. Only GG genotype of rs6841581 was found to significantly correlate with the size of aneurysms.

CONCLUSIONEDNRA gene rs6841581 has significant associations with the size of intracranial aneurysms, indicating a possible role of EDNRA in the genetic mechanisms of intracranial aneurysms and subarachnoid hemorrhage.

Adult ; Aged ; Case-Control Studies ; Female ; Gene Frequency ; Genotype ; Humans ; Intracranial Aneurysm ; genetics ; pathology ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Receptor, Endothelin A ; genetics

The study is to investigate the effect of angiotensin II (Ang II) and its receptor blockers on migration and endothelin-1 (ET-1) expression of rat vascular adventitial fibroblast subpopulations. Vascular adventitial fibroblasts were individually expanded by using cloning rings, and the effects of Ang II on the migration of adventitial fibroblast subpopulations were evaluated by Transwell. Fluorescence quantitative-PCR detected the expression of preproET-1 mRNA induced by Ang II, and its receptor antagonists losartan and PD-123319. The concentration of ET-1 was determined by ELISA. It showed that spindle shaped and epithelioid shaped cells were isolated by using cloning rings, named as spindle cells and round cells. RT-PCR showed that fibroblast subpopulations did not have leukocytes, endothelial cells and smooth muscle cells, namely pure cell lines. Compared with respective control cells, two subpopulations had transferring ability. Ang II significantly improved round cells migration in a concentration-dependent manner, and had no obvious influence on spindle cells migration. Ang II (1 x 10(-8) - 1 x 10(-6) mol x L(-1)) significantly increased the expression of preproET-1 mRNA in round cells (P < 0.01), and had no significant effect on the expression of preproET-1 mRNA in spindle cells. Losartan blocked the expression of preproET-1 mRNA induced by Ang II in round cells, and had no significant effect on the expression of preproET-1 mRNA in spindle cells. The effects of Ang II and ET-1 receptor inhibitors on the release of ET-1 were similar to the expression of preproET-1 mRNA. The results indicate that there are two cell subpopulations: round cells and spindle cells in rat vascular adventitial fibroblasts. Ang II significantly improved cells migration, and increased the expression of ET-1 in round cell subpopulation. It suggested that there may be different migratory mechanisms in two cell subpopulations, and the two subpopulations may play a different role in vascular remodeling and reparative process.
Angiotensin II ; pharmacology ; Angiotensin Receptor Antagonists ; pharmacology ; Animals ; Cell Movement ; drug effects ; Cells, Cultured ; Endothelin-1 ; genetics ; metabolism ; Fibroblasts ; cytology ; metabolism ; Imidazoles ; pharmacology ; Losartan ; pharmacology ; Male ; Pyridines ; pharmacology ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vasoconstrictor Agents ; pharmacology

The purpose of this study was to investigate the therapeutic effects of small hairpin RNA (shRNA) targeting endothelin-converting enzyme (ECE)-1 in monocrotaline (MCT)-induced pulmonary hypertensive rats. Ninty-four Sprague-Dawley rats were divided into three groups: control (n = 24), MCT (n = 35) and shRNA (n = 35). Four-week survival rate in the shRNA group was significantly increased compared to that in the MCT group. The shRNA group showed a significant improvement of right ventricular (RV) pressure compared with the MCT group. The MCT and shRNA groups also showed an increase in RV/(left ventricle + septum) ratio and lung/body weight. Plasma endothelin (ET)-1 concentrations in the shRNA group were lower than those in the MCT group. Medial wall thickness of pulmonary arterioles were increased after MCT injection and was significantly decreased in the shRNA group. The number of intra-acinar muscular pulmonary arteries was decreased in the shRNA group. The mRNA expressions of ET-1 and ET receptor A (ETA) were significantly decreased in the shRNA group in week 4. The protein levels of ETA were decreased in the shRNA group in week 2. The protein levels of tumor necrosis factor-alpha and vascular endothelial growth factor were decreased in the shRNA group in week 4. In conclusion, the gene silencing with lentiviral vector targeting ECE-1 could be effective against hemodynamic, histopathological and gene expression changes in pulmonary hypertension.
Animals ; Aspartic Acid Endopeptidases/*antagonists & inhibitors/blood/genetics ; Body Weight ; Heart Ventricles/physiopathology ; Hypertension, Pulmonary/chemically induced/*enzymology/mortality ; Lentivirus/genetics ; Lung/anatomy & histology/metabolism/pathology ; Male ; Metalloendopeptidases/*antagonists & inhibitors/blood/genetics ; Monocrotaline/toxicity ; Pulmonary Artery/drug effects/physiopathology ; RNA, Small Interfering/*metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/genetics/metabolism ; Survival Rate ; Tumor Necrosis Factor-alpha/metabolism ; Vascular Endothelial Growth Factor A/metabolism