ETA Receptor mRNA Research Articles

Study of the clinical significance of ETAR mRNA expression in high-grade serous ovarian cancer and the inhibitory effect of ETAR derived fusion polypeptide on cancer progression

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.

Effects of Coriandrum Sativum Extract and Simvastatin in Isopreterenol Induced Heart Failure in Rats

Abstract Heart failure is a syndrome, caused due to structural and functional cardiac abnormalities, characterized by changes in the hemodynamic and neurohumoral mechanisms. It is becoming a major health burden worldwide. More effective therapies are desperately needed. Coriandrum sativum (C. sativum), a traditional spice crop has been known to possess many biological and medicinal properties. The present study was designed to investigate the cardioprotective efficacy of C. sativum in rat model of isoproterenol induced heart failure. Heart failure was produced by injecting isoproterenol subcutaneously (85 mg/kg twice at an interval of 24 h). Oral efficacy of seed extract was assessed on hemodynamic profile, antioxidant enzyme activities, lipid peroxidation, lipid profile, atherogenic indices, mRNA and protein expression of endothelin receptors (ETA and ETB) and histopathology. Treatment of heart failure rats with C. sativum orally (1g/kg b.wt) improved the altered hemodynamics, restored the cardiac antioxidant enzymes armory, attenuated oxidative stress, improved lipid profile, lowered atherogenic indices, decreased the levels of ETA and ETB receptor mRNA and protein, and restored the cardiac morphology. In conclusion, our results suggest C. sativum to be a cardioprotective agent in heart failure, possibly by the virtue of its ability to alleviate oxidative stress, improve lipid profile and endothelial dysfunction.

Nephron prorenin receptor deficiency alters renal medullary endothelin-1 and endothelin receptor expression.

The endothelin (ET) and prorenin/renin/prorenin receptor (PRR) systems have opposing physiological effects on collecting duct (CD) salt and water reabsorption. It is unknown if the CD ET and renin/PRR systems interact, hence we examined the effects of deleting CD renin or nephron PRR on CD ET system components. PRR knockout (KO) mice were polyuric and had markedly increased urinary ET-1 and inner medullary CD (IMCD) ET-1 mRNA. PRR KO mice had greatly increased IMCD ETA receptor mRNA and protein, while ETB mRNA and protein were decreased. Water loaded wild-type mice with similar polyuria as PRR KO mice had modestly increased urinary ET-1 excretion and inner medullary ET-1 mRNA, while inner medullary ETA and ETB mRNA or protein expression were unaffected. In contrast to PRR KO, CD prorenin/renin KO did not alter ET system components. Taken together, these results suggest that the nephron PRR is involved in regulating CD ET system expression, but this effect may be independent of CD-derived renin.

Up-regulation of contractile endothelin receptors by airborne fine particulate matter in rat mesenteric arteries via activation of MAPK pathway.

Fine particle matters (PM2.5) is a well-known risk factor for cardiovascular diseases. However, the underlying molecular mechanisms are largely unknown. Vascular hyper-reactivity plays an important roles in the pathogenesis of cardiovascular diseases. The present study was designed to investigate a hypothesis that PM2.5 up-regulated endothelin receptors in mesenteric artery and the potential underlying mechanisms. Rat mesenteric arteries were cultured with PM2.5. The artery contractile responses were recorded by a sensitive myograph. ETB and ETA receptor expressions of mRNA and protein were assessed by quantitative real-time PCR, Western blotting, and immunohistochemistry, respectively. Results showed that ETB receptor agonist, sarafotoxin 6c induced a negligible contraction in fresh artery segments, while ETA receptor agonist, ET-1 induced an obvious contraction. After organ culture, the contraction curve mediated by ETB and ETA receptors were shifted toward the left. PM2.5 1.0μg/ml cultured for 16h further enhanced ETB and ETA receptor-mediated contractile responses with a markedly increased maximal contraction. The organ culture enhanced ETB and ETA receptor mRNA and protein levels from fresh arteries, which were further increased by PM2.5. The U0126 (MEK/ERK1/2 inhibitor) and SB203580 (p38 inhibitor) significantly attenuated both organ cultured-induced and PM2.5-induced up-regulation of ETB receptor. U0126 also suppressed organ culture-increased and PM2.5-increased expressions of ETA receptor. SB203580 only suppressed PM2.5-induced enhanced expressions of ETA receptor In conclusion, airborne PM2.5 up-regulates ETB and ETA receptors of mesenteric artery via p38 MAPK and MEK/ERK1/2 MAPK pathways.

Effects of CASZI on bronchopulmonary development of neonatal rats.

The effect of CASZI on bronchopulmonary development in neonatal rats was investigated. Forty SD rats were randomly divided into the control group (n=20) and the experimental group (n=20) using a random number table. The experimental group was treated with hyperoxia to establish the bronchopulmonary dysplasia (BPD) model, while the control group received no treatment. At 14 days of experiment, rats in each group were treated, and lung tissues were taken for hematoxylin and eosin (H&E) staining. Radial alveolar count (RAC) and morphological changes in lung tissues were observed under a microscope, and the thickness of respiratory membrane was detected via IPP6 image. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting were used to detect the expression levels of CASZI, Rho kinase, RhoA and ET-A receptor in lung tissues. There were no obviously pathological changes in lung tissues of neonatal rats in the control group, while BPD could be seen in lung tissue structures of neonatal rats in the experimental group. RAC in experimental group was significantly lower than that in the control group (P<0.05), and the thickness of respiratory membrane was significantly higher than that in the control group (P<0.05). CASZI mRNA expression level in the experimental group was significantly lower than that in control group (P<0.05), and Rho kinase, RhoA and ET-A receptor mRNA expression levels were significantly higher than those in the control group (P<0.05). CASZI protein expression level in the experimental group was significantly lower than that in the control group (P<0.05), and the Rho kinase, RhoA and ET-A receptor protein expression levels were significantly higher than those in the control group (P<0.05). In conclusion, CASZI expression was downregulated during the development of BPD. CASZI downregulation may have an effect on the pulmonary microvascular regeneration through RhoA/ROCK signaling pathway, thus participating in the pathogenesis of BPD.

Effect of electroacupuncture stimulation on expression of angiotensinogen, angiotensin II type 1 receptor, endothelin-1, and endothelin a receptor mRNA in spontaneously hypertensive rat aorta

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

The effects of MEK1/2 inhibition on cigarette smoke exposure-induced ET receptor upregulation in rat cerebral arteries

Cigarette smoking, a major stroke risk factor, upregulates endothelin receptors in cerebral arteries. The present study examined the effects of MEK1/2 pathway inhibition on cigarette smoke exposure-induced ET receptor upregulation. Rats were exposed to the secondhand smoke (SHS) for 8weeks followed by intraperitoneal injection of MEK1/2 inhibitor, U0126 for another 4weeks. The urine cotinine levels were assessed with high-performance liquid chromatography. Contractile responses of isolated cerebral arteries were recorded by a sensitive wire myograph. The mRNA and protein expression levels of receptor and MEK/ERK1/2 pathway molecules were examined by real-time PCR and Western blotting, respectively. Cerebral artery receptor localization was determined with immunohistochemistry. The results showed the urine cotinine levels from SHS exposure group were significantly higher than those from the fresh group. In addition, the MEK1/2 inhibitor, U0126 significantly reduced SHS exposure-increased ETA receptor mRNA and protein levels as well as contractile responses mediated by ETA receptors. The immunoreactivity of increased ETA receptor expression was primarily cytoplasmic in smooth muscle cells. In contrast, ETB receptor was noted in endothelial cells. However, the SHS-induced decrease in endothelium-dependent relaxation was unchanged after U0126 treatment. Furthermore, SHS increased the phosphorylation of MEK1/2 and ERK1/2 protein in cerebral arteries. By using U0126 could inhibit the phosphorylated ERK1/2 protein but not MEK1/2. Taken together, our data show that treatment with MEK1/2 pathway inhibitor offsets SHS exposure-induced ETA receptor upregulation in rat cerebral arteries.

Abstract 408: Endothelin-1 and Vitamin D 3 Potentiated Calcification and Outward Vascular Remodeling in Leptin-deficient ob/ob Mice

Vascular remodeling, which is an adaptive response of the vessel to specific stimuli has a pivotal role in cardiovascular disease, such as aneurysm formation. In that scope, serum endothelin-1 (ET-1) which is increased in diabetic patients, may contribute to atherosclerosis and aneurysm development. We hypothesized that leptin-deficient ob/ob mice may demonstrate an exaggerated vascular remodeling associated with vascular calcification after Vitamin D 3 in vivo . We further investigated the role of ET-1 in vascular smooth muscle cells (SMC) osteochondrogenic differentiation and calcification in vitro . We used leptin-deficient (obob, n=12) and C57BL/6 mice (C57, n=12) injected with saline (Cont) or Vitamin D 3 8x10 4 IU (VitD) i.p. daily for 14 days. Results are shown as Mean±SEM and considered statistically significant if p≤.05 by ANOVA. Aorta from ob/obVitD increased both external and internal elastic lamina circumferential length (3478±269.7μm and 3186±262.6μm) versus (vs.) ob/obCont (2841±74.73μm and 2657±71.37μm) vs. C57VitD (2611±39.62μm and 2333±41.79μm) and vs. C57Cont (2569±164.6μm and 2351±167.7μm), thus characterizing outward vascular remodeling. We found that vascular calcification area strongly correlated with increased total vessel area (r 2 =0.8, p=0.003). We also incubated primary SMC isolated either from obob or from C57 aorta without (Cont) or with ET-1 50nM (ET) for 0-72h. ObobET SMC increased calcification vs. obobCont (1,69±0.025 vs. 1.03±0.03) after 14 days p&lt;.05, n=3 and C57ET did not calcify (0.97±0.02 vs. C57Cont 1±0.05), p=NS. Concomitantly, obobET SMC increased osteochondrogenic differentiation, by modulating RUNX2 (1.23±0.03 vs. obobCont 1±0.01, p&lt;.05 n=3) after 48h, which did not occur in C57. Furthermore, ERk1/2 dephosphorylation peaked after 30min in C57ET in comparison to 60min in obobET SMC. Concurrently, VitD increased both ETA and ETB receptor (4.00±1.04 and 4.25±1.25) in obob aorta vs. paired control in vivo . Nevertheless, C57VitD increased ETA receptor mRNA expression (5.44±1.08) only. In conclusion, we uncovered signaling pathways that may connect Vitamin D and ET-1 calcifying effect to induce excessive outward remodeling in type 2 diabetes.

Effects of Hyperinsulinemia on Blood Pressure in High-Fat Diet Fed Rats

Objective. To determine the mechanisms of hyperinsulinemia-induced elevation in blood pressure in rats. Methods. Male Sprague-Dawley rats were divided into normal diet (normal control) and high-fat diet group. After 36 weeks of feeding, high-fat diet group was further randomized into high-fat diet control group and streptozocin treatment group. Plasma insulin, endothelin-1(ET-1), norepinephrine (NE), aldosterone, and angiotensin II levels were measured. AT1 receptor, ET-1, and ETA receptor mRNA expression in the aorta was evaluated by real-time PCR. Results. After 9 months, systolic blood pressure (SBP) in high-fat diet group was higher than in the normal control group (155.4±1.6 vs.132.1±5.3 mmHg, P<0.01). The levels of plasma insulin in high-fat diet group were higher than in normal control group (116.63±12.31μIU/mL versus 29.10±4.92μIU/mL, P<0.01). High-fat diet group also exhibited higher plasma levels of ET-1, NE, aldosterone, and angiotensin II,and a lower 24 h urinary sodium excretion than the normal control group (P<0.05). The expression of AT1 receptor, ET-1 and ETA receptors in the aorta in the high-fat diet group was greater than in the normal control group (P<0.05). Streptozocin treatment reduced SBP by an average of 20.9±3.3 mmHg (P<0.05), and reduced insulin level from 110.63±14.86μIU/mL to 39.45±6.59μIU/mL (P<0.01). The streptozocin group also showed a higher level of urine sodium excretion, and a lower level of plasma ET-1 and NE than in the high-fat diet control group (P<0.05). Conclusions. Hyperinsulinemia following high-fat diet is associated with an elevation in blood pressure. Sodium retention, increased plasma endothelin-1 and noradrenaline, as well as activation of renin-angiotensin system may all contribute to the blood pressure elevation.

Endothelin-1 contributes to the progression of renal injury in sickle cell disease via reactive oxygen species.

Endothelin-1 (ET-1) is increased in patients with sickle cell disease and may contribute to the development of sickle cell nephropathy. The current study was designed to determine whether ET-1 acting via the ETA receptor contributes to renal injury in a mouse model of sickle cell disease. Adult, humanized HbSS (homozygous for sickle Hb) mice had increased ET-1 mRNA expression in both the cortex and the glomeruli compared with mice heterozygous for sickle and Hb A (HbAS controls). In the renal cortex, ETA receptor mRNA expression was also elevated in HbSS (sickle) mice although ETB receptor mRNA expression was unchanged. Ligand binding assays confirmed that sickle mice had increased ETA receptors in the renal vascular tissue when compared with control mice. In response to PKC stimulation, reactive oxygen species production by isolated glomeruli from HbSS sickle mice was increased compared with that from HbSA controls, an effect that was prevented by 1 week in vivo treatment with the selective ETA antagonist, ABT-627. Protein and nephrin excretion were both elevated in sickle mice, effects that were also significantly attenuated by ABT-627. Finally, ETA receptor antagonism caused a significant reduction in mRNA expression of NADPH oxidase subunits, which may contribute to nephropathy in sickle cell disease. These data support a novel role for ET-1 in the progression of sickle nephropathy, specifically via the ETA receptor, and suggest a potential role for ETA receptor antagonism in a treatment strategy.

MiR-30a inhibits endothelin A receptor and chemoresistance in ovarian carcinoma.

Drug resistance remains the major clinical barrier to successful treatment in epithelial ovarian carcinoma (EOC) patients, and the evidence of microRNA involvement in drug resistance has been recently emerging. Endothelin-1 (ET-1)/ETA receptor (ETAR) axis is aberrantly activated in chemoresistant EOC cells and elicits pleiotropic effects promoting epithelial-to-mesenchymal transition (EMT) and the acquisition of chemoresistance. However, the relationship between ETAR and miRNA is still unknown. Hence, in this study we evaluated whether dysregulation of miRNA might enhance ETAR expression in sensitive and resistant EOC cells. Based on bioinformatic tools, we selected putative miRNA able to recognize the 3′UTR of ETAR. An inverse correlation was observed between the expression levels of miR-30a and ETAR in both EOC cell lines and tumor samples. miR-30a was found to specifically bind to the 3′UTR of ETAR mRNA, indicating that ETAR is a direct target of miR-30a. Overexpression of miR-30a decreased Akt and mitogen activated protein kinase signaling pathway activation, cell proliferation, invasion, plasticity, EMT marker levels, and vascular endothelial growth factor release. Interestingly, ectopic expression of miR-30a re-sensitized platinum-resistant EOC cells to cisplatinum-induced apoptosis. Consistently, resistant EOC xenografts overexpressing miR-30a resulted in significantly less tumor growth than controls. Together our study provides a new perspective on the regulatory mechanism of ETAR gene. Interestingly, our findings highlight that blockade of ETAR regulatory axis is the mechanism underlying the tumor suppressor function of miR-30a in chemoresistant EOC cells.

Abstract 3580: Downregulated miR-30a promotes acquisition of chemoresistance by targeting endothelin A receptor in ovarian carcinoma

Abstract Chemotherapy is the preferred therapeutic approach for the therapy of epithelial ovarian carcinoma (EOC), but a successful long-term treatment is prevented by the development of drug resistance. Endothelin-1 (ET-1)/ETA receptor (ETAR) axis is upregulated in chemoresistant EOC and associated with acquisition of chemoresistance and epithelial-mesenchymal transition (EMT) phenotype and poor prognosis. Therefore, interference with ET-1/ETAR signaling pathway has emerged as a promising strategy for cancer therapy. Because dysregulation of miRNAs has been associated with cancer development and drug-resistance, in this study we evaluated whether altered expression of miRNAs might regulate ETAR expression in 2008 and A2780 sensitive and resistant EOC cell lines. Based on bioinformatics tools, we selected putative miRNAs able to recognize the 3′UTR of ETAR. Among these, only miR-30a resulted downregulated in chemoresistant EOC cells compared with parental cells. Luciferase reporter assays using wild-type and mutant ETAR 3′ -UTR constructs, and transfection of miR-30a mimic showed that miR-30a targets the 3′ -UTR of ETAR mRNA. Indeed ectopic expression of miR-30a inhibited ETAR-driven signaling pathways. Interestingly, miR-30a re-introduction in chemoresistant EOC cells impaired their capacity to promote the invasive process and reverted EMT phenotype. Moreover, miR-30a overexpression was able to restore drug sensitivity by enhancing the susceptibility of resistant EOC cells to cisplatinum-induced apoptosis, as supported by the reduced viability and increased cleavage of caspase 7 and PARP. Similarly, we demonstrated that the treatment with macitentan, a potent ETAR antagonist with significant affinity for ETBR, counteracted the effects elicited by ETAR pathway in chemoresistant EOC cells restoring drug-sensitivity and regulating cell plasticity. In preclinical model of resistant EOC, macitentan treatment reduced tumor growth, vascularization, intravasation and metastatic progression. The combination of macitentan and cisplatinum inhibited tumor growth of resistant EOC xenografts more effective then chemotherapy alone. In addition, lower expression of miR-30a and higher expression of ETAR in platinum-resistant specimens from EOC patients were significantly associated with poor clinical outcome, representing potential predictive markers of chemoresistance. This study for the first time demonstrated miR-30a-dependent regulation of ETAR for chemoresistance of EOC, further supporting that the regulation of ETAR represents a valid therapeutic target for circumventing drug resistance. Citation Format: Rosanna Sestito, Roberta Cianfrocca, Laura Rosanò, Elisa Semprucci, Piera Tocci, Valeriana Di Castro, Gabriella Ferrandina, Anna Bagnato. Downregulated miR-30a promotes acquisition of chemoresistance by targeting endothelin A receptor in ovarian carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3580. doi:10.1158/1538-7445.AM2015-3580

Expression and hypoxic regulation of the endothelin system in endocrine cells of human and rat pancreatic islets.

The success of pancreatic islet transplantation depends largely on the capacity of the islet graft to survive the initial phase immediately after transplantation until revascularization is completed. Endothelin-1 (ET-1) is a strong vasoconstrictor which has been involved in solid organ graft failure but is also known to be a potent mitogenic/anti-apoptotic factor which could also potentially enhance the survival of the transplanted islets. Characterization of the endothelin system with regard to a potential endothelin agonist/antagonist treatment. Regulated expression of the endothelin system in human and rat pancreatic islets and beta-cell lines was assessed by means of immunohistochemistry, competition binding studies, western blot, RT-PCR, real-time PCR and transplant studies. ET-1, ETA- and ETB-receptor immunoreactivity was identified in the endocrine cells of human and rat pancreatic islets. The corresponding mRNA was detectable in rat beta-cell lines and isolated rat and human pancreatic islets. Competition binding studies on rat islets revealed binding sites for both receptor types. ET-1 stimulated the phosphorylation of mitogen-activated protein kinase, which was prevented by ETA- and ETB-receptor antagonists. After exposure to hypoxia equal to post-transplant environment oxygen tension, mRNA levels of ET-1 and ETB-receptor of human islets were robustly induced whereas ETA-receptor mRNA did not show significant changes. Immunostaining signals for ET-1 and ETA-receptor of transplanted rat islets were markedly decreased when compared to native pancreatic sections. In pancreatic islets, ET-1 and its receptors are differentially expressed by hypoxia and after transplantation. Our results provide the biological basis for the study of the potential use of endothelin agonists/antagonists to improve islet transplantation outcome.

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.

Altered Endothelin Receptor Expression and Affinity in Spontaneously Hypertensive Rat Cerebral and Coronary Arteries

BackgroundHypertension is associated with arterial hyperreactivity, and endothelin (ET) receptors are involved in vascular pathogenesis. The present study was performed to examine the hypothesis that ET receptors were altered in cerebral and coronary arteries of spontaneously hypertensive rats (SHR).Methodology/Principal FindingsCerebral and coronary arteries were removed from SHR. Vascular contraction was recorded using a sensitive myograph system. Real-time PCR and Western blotting were used to quantify mRNA and protein expression of receptors and essential MAPK pathway molecules. The results demonstrated that both ETA and ETB receptor-mediated contractile responses in SHR cerebral arteries were shifted to the left in a nonparallel manner with increased maximum contraction compared with Wistar-Kyoto (WKY) rats. In SHR coronary arteries, the ETA receptor-mediated contraction curve was shifted to the left in parallel with an increased pEC50 compared with the arteries in WKY rats. There was no significant increase in ETB receptor-mediated contraction in SHR coronary arteries. ETA receptor mRNA and protein expression was increased in SHR cerebral arteries compared with the arteries in WKY rats. However, ETA receptor mRNA and protein levels in coronary arteries and ETB receptor protein levels in cerebral and coronary arteries remained unchanged in SHR compared with WKY rats. Meanwhile, phosphorylated ERK1/2 protein was significantly increased in SHR brain and heart vessels.Conclusions/SignificanceIn SHR cerebral arteries, ETA receptor expression was upregulated. ETA receptor affinity was increased in coronary arteries, and ETB receptor affinity was increased in cerebral arteries. The ERK1/2 activation may be involved in the receptor alterations.

Altered expression and signal transduction of endothelin‐1 receptors in heritable and idiopathic pulmonary arterial hypertension

Human pulmonary arterial smooth muscle cells (PASMC) were isolated from elastic pulmonary arteries dissected from lungs of individuals with and without pulmonary arterial hypertension (PAH). Reflecting increased smooth muscle constriction in cells from PAH subject, Ca(2+) influx in response to endothelin-1 (ET-1) increased in all the PAH PASMC populations relative to the normal donor control cells. The ETA receptor mRNA levels remained unchanged, whereas the ETB receptor mRNA levels decreased in both heritable and idiopathic PAH-derived PASMC. All the PASMC populations expressed considerably higher ETA compared to ETB receptor number. Both ETA and ETB receptor numbers were reduced in bone morphogenetic protein receptor type II (BMPR2) mutation PAH. ETB receptors showed a particular reduction in number. Phospho-antibody array analysis of normal and BMPR2 deletion PASMC illustrated ERK and Akt activation to be the most prominent and to be taking place principally through ETB receptors in normal PASMC, but primarily through ETA receptors in PASMC from BMPR2 PAH subjects. Additionally in the PAH cells the total relative ET-1 signal response was markedly reduced. Western analysis from the BMPR2 PASMC duplicated the array results, whereas PASMC from iPAH subjects showed variability with most samples continuing to signal through ETB. In sum, these results indicate that generally both receptors are reduced in PAH particularly ETB, and that ETB signaling through protein kinases becomes markedly reduced in BMPR2 PASMC, while it continues in IPAH. Importantly, the data suggest that caution must be taken when applying ET-1 receptor antagonist therapy to PAH patients.

Prevention of renal ischemia-reperfusion injury by short hairpin RNA of endothelin A receptor in a rat model

Endothelin A receptor (ETaR) is a key molecule involved in a variety of biological events such as vessel contraction and inflammatory response in ischemia-reperfusion (I/R) injury. RNA interference using short hairpin RNA (shRNA) is a powerful tool to silence gene expression. Here, the effect of ETaR shRNA on I/R injury in rats was studied. A more effective shRNA sequence out of two constructed into plasmid vectors was selected using the A-10 cell line, and was then applied to a rat model. Twenty-eight male Sprague-Dawley rats were randomized into four groups: Sham, shRNA, vector and phosphate-buffered saline (PBS). Renal I/R injury was induced by clamping the left renal pedicle for one hour followed by reperfusion for 24 h. ETaR shRNA (100 μg) plasmid was administered by renal vein injection 48 h before clamping. The expression of both ETaR mRNA and protein was lowered by ETaR shRNA treatment compared with that in the vector and PBS groups; serum creatinine and blood urea nitrogen were significantly decreased; the semi-quantitative score of renal structural damage was improved; the mRNA level of endothelin 1 (ET-1), tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), macrophage inflammatory protein 2 (MIP-2) and monocyte chemoattractant protein 1 (MCP-1) was reduced, but nitric oxide (NO) production in kidney tissues was increased (P < 0.05). In conclusion, ETaR shRNA partially silenced ETaR expression in I/R injury kidneys, reduced the mRNA level of ET-1, inflammatory mediators including TNF-α, IL-6, MIP-2 and MCP-1, increased NO production, and ultimately improved renal function and structure.

CYP2J3 Gene Delivery Reduces Insulin Resistance via Upregulation of eNOS in Fructose-treated Rats

Accumulating evidence suggests that cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs) which play important roles in various pathophysiological processes. Interestingly, CYP-derived eicosanoids are vasodilatory, at least in part through their ability to activate eNOS and subsequent NO release. This study investigated the roles of eNOS in CYP2J3 gene delivery reducing blood pressure and improving insulin resistance in fructose-treated rats. CYP2J3 overexpression in vivo increased EET generation, reduced blood pressure and reversed insulin resistance as determined by insulin resistance index (HOMA-IR). Furthermore, administration of eNOS inhibitor L-NMMA significantly and partially abolished the beneficial effects of CYP2J3 gene delivery on hypertension and insulin resistance induced by fructose intake, and possible mechanism is associated with increased ET-1, ETA-receptor mRNA expression and reduced sensitivity of insulin to peripheral tissues and organs characterized by reduced activity of IRS-1/PI3K/AKT and AMPK signalling pathways. These data provide direct evidence that CYP2J3-derived EETs may alleviate insulin resistance at least in part through upregulated eNOS expression.

Low Blood Pressure in Endothelial Cell–Specific Endothelin 1 Knockout Mice

Endothelin (ET) 1 is a potent vasoconstrictor peptide produced by vascular endothelial cells and implicated in various pathophysiologic states involving abnormal vascular tone. Homozygous ET-1 null mice have craniofacial and cardiac malformations that lead to neonatal death. To study the role of ET-1 in adult vascular physiology, we generated a mouse strain (ET-1(flox/flox);Tie2-Cre mice) in which ET-1 is disrupted specifically in endothelial cells. ET-1 peptide levels in plasma, heart, lung, kidney, and brain homogenates were reduced by 65% to 80% in these mice. mRNA levels for ET receptors were unaltered except that the ET(A) receptor mRNA was upregulated in the heart. ET-1(flox/flox);Tie2-Cre mice had mean blood pressures 10 to 12 mm Hg lower than genetic controls. In contrast, the blood pressure of mice systemically heterozygous for the ET-1 null allele (ET-1(dlox/+) mice) was unchanged compared with wild-type littermates. Despite the lower basal blood pressure, acute pharmacological responses to angiotensin II, captopril, phenylephrine, bradykinin, N(G)-nitro-L-arginine methyl ester, and exogenous ET-1 were normal in ET-1(flox/flox);Tie2-Cre mice. These results support an essential role of endothelial-derived ET-1 in the maintenance of basal vascular tone and blood pressure. Normal pharmacological responses of ET-1(flox/flox);Tie2-Cre mice suggest that the renin-angiotensin system, the adrenergic system, and NO are not significantly altered by endothelial ET-1. Taken in conjunction with other lines of genetically altered mice, our results provide evidence for a paracrine vasoregulatory pathway mediated by endothelial cell-derived ET-1 acting on the vascular smooth muscle ET(A) receptor.

Abstract 2551: Acquisition of epithelial-mesenchymal transition phenotype of chemoresistant ovarian cancer cells is linked with activation of the endothelin signaling pathway

Abstract Chemoresistance is a major challenge in ovarian cancer (OC). High-throughput analysis has shown that the endothelin A receptor (ETAR) is overexpressed in post-chemotherapy OCs, suggesting a role in the chemoresistance onset. To identify ETAR-driven molecular determinants involved in chemoresistance to cisplatin and paclitaxel, we used the A2780 WT, cisplatin-resistant A2780 CIS, and taxol-resistant A2780 TAX human OC cells. ET-1 and ETAR mRNA and protein expression as well as ET-1 secretion were higher in the resistant OC cells, associated with increased ET-1-induced MAPK and AKT activation and cell proliferation. These effects were reverted by treatment with zibotentan (ZD4054), a specific ETAR antagonist. In sensitive OC cells, zibotentan enhanced the number of early apoptotic cells, similar to cisplatinum. Furthermore, zibotentan sensitized resistant A2780 CIS cells to cisplatinum-induced apoptosis, confirming the role of ET-1 as survival factor. Given the documented role of the epithelial to mesenchymal transition (EMT) in the chemoresistance development and the ability of the ET-1/ETAR axis to regulate EMT effectors, such as Snail and β-catenin, we analyzed whether ETAR-driven EMT molecular changes were linked with the chemoresistance in EOC cells. Decreased expression of the epithelial adhesion molecule E-cadherin and an increased expression of mesenchymal markers, such as Snail and Slug, the principal transcriptional repressor of E-cadherin, as well as Snail promoter activity were observed in sensitive OC cells after ET-1 treatment. Moreover, these effects were upregulated in resistant OC cells and were blocked in the presence of zibotentan, providing molecular evidence that the ETAR-mediated EMT signaling in EOC can represent a “salvage pathway” occurring during chemoresistance development. In sensitive A2780, as well as in resistant A2780 CIS and TAX xenografts, zibotentan induced a significant tumor growth inhibition, associated with reduction of Snail and upregulation of E-cadherin, suggesting a specific therapeutic window in which combination therapy of zibotentan with cytotoxic drugs is effective also in resistant OC. Finally immunohistochemical analysis of human OC, with different responses to chemotherapy, showed that ET-1 and ETAR are overexpressed in the resistant tumors, associated with upregulation of E-cadherin and with downregulation of N-cadherin and β-catenin. In conclusion, our study suggest that ETAR blockade with the specific antagonist zibotentan can revert EMT and increase the sensitivity to chemotherapeutic agents, representing a potential strategy to overcome chemoresistance in OC. Supported by AIRC, Ministero della Salute, 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 2551.