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Objective To investigate the expression of endothelin-1 (ET-1), ET-2, ET-3, endothelin receptor A (ETRA), ETRB and endothelin coverting enzyme (ECE) in the retinas of 8-week-old diabetic SD rats. Methods The retinal gene expression profiles of healthy and 8-week-old diabetic rats were constructed with restriction fragment differential display polymerase chained reaction (RFDD-PCR), and the differential expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE was verified using semi-quantitative RT-PCR and Western blotting analysis. Results The results of RFDD-PCR showed that the expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE was up-regulated in diabetic retina. The results of semi-quantitative RT-PCR and Western blotting analysis showed that the expression levels of the six genes and proteins (relative D ratio) in diabetic group were significantly higher than those in the normal retinas (P<0. 05 and P<0. 01, respectively). Conclusion The expression of ET-1, ET-2, ET-3, ETRA, ETRB and ECE is up-regulated in diabetic retina, suggesting that the six genes may be involved in the pathgenesis of diabetic retina.
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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.
Subject(s)
Animals , Male , Rats , Aspartic Acid Endopeptidases/antagonists & inhibitors , Body Weight , Heart Ventricles/physiopathology , Hypertension, Pulmonary/chemically induced , Lentivirus/genetics , Lung/anatomy & histology , Metalloendopeptidases/antagonists & inhibitors , Monocrotaline/toxicity , Pulmonary Artery/drug effects , RNA, Small Interfering/metabolism , Rats, Sprague-Dawley , Receptor, Endothelin A/genetics , Survival Rate , Tumor Necrosis Factor-alpha/metabolism , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Aim To investigate the effects of iptakalim(IPT),a novel K_(ATP) opener,on the functions of endothelin system in human pulmonary artery endothelial cells.Methods Primary cultured human pulmonary artery endothelial cells were incubated with different concentrations iptakalim for 24 h.The levels of ET-1 in medium were observed by radioimmunoassay.Reverse transcription polymerase chain reaction(RT-PCR)was performed to analyze the expression of ET-1 and ECE.Results When endothelial cells were incubated with IPT at concentrations above 10 μmol·L~(-1),the levels of ET-1 release in medium and the levels of ET-1 mRNA were significantly inhibited.When endothelial cells were incubated with IPT at concentrations above 1 μmol·L~(-1),the levels of ECE mRNA were significantly inhibited.Conclusions IPT can inhibit the expression of ET-1 and ECE mRNA from human pulmonary artery endothelial cells, thus it inhibits the secretion of ET-1 from endothelial cells. Iptakalim may serve as a promising candidate drug to treat pulmonary hypertension.
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Aim: To prepare chitosan nanoparticlescarrying gene and study its characteristics in vitro and in vivo.Methods:12-ACSs was dissolved in 0.05mol/L sodium acetate buffer to form a solution of 1mg/ml and a DNA(plasmid-encoding antisense ECE ) solution of 0.1mg/ml was dissolved in 25mmol/L Na2SO4.The charge ratio of components is selected as 2/1 for 12-ACSs /DNA complex.The complex was prepared by mixing 12-ACSs solution with DNA solution prior to observation by using transmission electron microscopy.Using Electrophoretic Retardation of DNA Migration detection,DNA precipitation,Binding Equilibration and DNase Resistivity Assay,the formation of 12-ACSs /DNA complex was determined and its stability was simultaneously evaluated.MTT Cell Proliferation Assays was performed on investigation of the cytotoxicity of 12-ACSs /DNA nanoparticles in bronchial epithelial cells (16HBE).The transfection efficiency of 12-ACSs /DNA nanoparticles in vitro and in vivo was investigated in 16HBE cells and Balb/c mice.Results: 12-ACSs /DNA nanoparticles (100~150nm) were observed by transmission electron microscopy.12-ACSs can protect the plasmid encoding antisence-ECE from degradation by DNase I.12-ACSs can transfer plasmid-encoding antisense ECE into 16HBE cells and into the airway epithelium in mice.As shown by fluorescent microscopy,green fluorescent protein reporter can be observed in the transfected cells as well as in the airway epithelium of the treated mice.And it showed a lower cell cytotocixity in cultured 16HBE cells and in mice treated with12-ACSs /DNA nanoparticles.Conclusion: In summery,the chitosan can be used as an effective protectant for DNA as well as an enhancer for in vitro gene transfection.
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Aim: To investigate the effects of iptakalim hydrochloride (Ipt), a new antihypertensive drug, on the functions of endothelin system. Methods: Radioimmunoassay was used to test the plasma endothelin (ET) concentration of rats and endothelin released from cultured vasocular endothelial cells. The expressions of ET and endothelin converting enzyme (ECE) were determined by RT-PCR. The effects of endothelin on vascular tone were studied in isolated rat aorta. The pulmonary artery pressure was measured in anaesthetized rats. Results: Circled digit one In stroke-prone spontaneously hypertensive rats (SHRsp), the plasma levels of endothelin were increased, which could be reversed by the treatment with Ipt at the doses of 0.25, 1.0 and 4.0 mg·kg -1 po, once a day, for 3 months. Circled digit two In cultured neonatal bovine aortic endothelial cells (BAEC), Ipt at the concentrations of 1-1000 μmol ·L-1, inhibited the secretion of ET in a concentration-dependent manner. Circled digit three Under the same experimental conditions, Ipt also inhibited the expressions of ET and ECE in BAEC. Circled digit four In isolated preparations derived from rat aorta, the vascular contraction evoked by ET-1 was antagonized by Ipt at the concentrations of 0.5-100 μmol·L-1 in a concentration-dependent manner. The vasorelaxative effects of Ipt were attenuated significantly in buffer without Ca2+. Circled digit five In anaesthetized SD rats, intrapulmonary artery administration of ET-1 induced vascular contraction in vivo, resulting in intrapulmonary artery hypertension, which was prevented by iptakalim hydrochloride at the doses of 0.5-1.0 mg·kg-1. But it had no effects on normal pulmonary artery pressure. Conclusion: Ipt could antagonize the functions of endothelin system. Its characteristics include inhibiting the expression of ET-1 and ECE, inhibiting the releasing of ET-1, reversing the increased plasma levels of ET-1 under pathological condition, and preventing intrapulmonary artery hypertension induced by ET-1.
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Objective To study the effet of ET-1 and ECE on endothelial proliferation of autografted vein.Methods An animal model of the autogenous vein graft was established in 80 Wistar rats.The expression of ECE and EF-1 gene was determined by RT-PCR and immunohistological method to test the mRNA and protein expresion level respectively.Results PCNA positive smooth muscle cells appeared 6 hours after transplantation,increased with time,and reached a peak at 1 to 2 weeks.After 2 weeks,PCNA positive SMC in the tunica media began to decline and recovered to the 6 hour level at 8 weeks after the operation.mRNA of ECE increased with the time after the operation,reached a peak after 1-2 weeks,declined afterwards,and became stable at around 8 weeks.Expression change of ET1 was similar to the change of ECE,reached the peak after 1-2 weeks and was stable after 8 weeks(r=0.975).Conclusions The time and pattern of change of the pathologic process of intimal hyperplasia are in agreement with the expression of ET-1 and ECE,and suggests that ECE is involved in the process of intimal hyperplasia of vein graft.
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Objective To investigate changes of endothelin-1(ET-1) and endothelin-converting enzyme (ECE) in different time intervals after autograft vein implantation. Methods A model of autogenous vein graft was established by interposition of the jugular vein into abdominal aorta in 80 Wistar rats. RT-PCR and immunohistochemistry were employed to test mRNA and protein level of ECE, ET-1 and proliferating cell nuclear antigen (PCNA). Results Positive PCNA appeared at 6 hours after transplantation, with time reaching a peak at 1 to 2 week. ECE mRNA increased with time reaching a peak after 1-2 weeks and stabilizing around 8 weeks. ET-1 expression underwent similar tendence with ECE, reaching a peak after 1-2 weeks and stabilizing at 8 weeks at the protein level. Expression of ET-1 and ECE were closely related by the time pattern after vein autograft (r=0.975). Conclusions The process of intimal hyperplasia in its occurrence and pattern of change are related with dynamics of ET-1 and ECE. ECE may lead to intimal hyperplasia of the autografted vein through a passway of ECE to ET-1 to SMC.
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AIM: To investigate the effect of hypoxia on glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ?-actin and endothelin-converting enzyme (ECE)-2 mRNA levels in cultured mouse brain astrocytes (AC). METHODS: AC from neonatal mouse brain was incubated for 24 h in serum-free medium under hypoxic or normoxic conditions. The amount of transferred RNA was estimated using ethidium bromide stained 28S rRNA and 18S rRNA. The levels of tested mRNA were evaluated by Northern blot RNA hybridization. RESULTS: The GAPDH mRNA was up-regulated to 503.0% of the normoxic controls in hypoxic AC (n=10. P
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Objective To investigate the expression of endothelin converting enzyme (ECE) mRNA in patients with acute cerebral infarction (ACI) and its clinical significance.Methods Blood samples from 40 patients with ACI (patient group) within 72 hours after the onset of ACI and 28 gender and age-matched healthy subjects (control group) were collected on admission. Plasma concentrations of endothelin-1 (ET-1) as well as the serum levels of cholesterol, triglyceride, low density lipoprotein,high density lipoprotein, apoA1, apoB, lipoprotein a and fasting plasma glucose in each sample were measured and analyzed. Additionally, semi-quantitative RT-PCR was performed to check the ECE mRNA level in the blood cells. European stroke scale (ESS) was used to evaluate ACI patients' neurological deficit on admission.Results (1) ECE mRNA could be detected in every blood sample from either patient group or control group. However, the ECE mRNA level increased significantly in the patient group compared with that in the control group (0.31?0.092 versus 0.25?0.10, t=2.46, P=0.016). (2) The plasma ET-1 concentration in patient group was also significantly higher than that of control subjects (183.27?56.63pg/ml versus 156.47?34.24 pg/ml, t=2.23, P=0.029). (3) Plasma ET-1 concentration was negatively correlated with ECE mRNA level in the control group (r=-0.452, P=0.021). However, the result in the patient group was not the same as the control group. (4) The ET-1 concentration and ECE mRNA level in the patients had histories of hypertension, diabetes mellitus and stroke were not significantly different from those in the patients without these histories. (5) No significant correlation existed between plasma ET-1 concentration and ECE mRNA level and age of the patient, ESS score, fasting plasma glucose and serum lipid. Conclusions ECE mRNA level is significantly increased in the early stage of ACI, which may be associated with the acute-phase reaction of cerebral infarction and may have deleterious effects on the development of neuronal injury. Our results suggest the protective reflection in the endothelin system of normal human body may be disturbed by the onset of ACI. The relationship between ECE mRNA level and neurological deficit degree, stroke risk factors is worthy for further study.