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AIM:To clarify the effect of miR-519d-3p on high glucose-induced human retinal microvascular endothelial cells(HRMEC)dysfunction and angiogenesis, and to elucidate the regulatory mechanism of miR-519d-3p on hypoxia inducible factor 1 subunit alpha(HIF-1α).METHODS: The normal glucose(NG)and high glucose(HG)cell models were established by inducing HRMEC with 5 and 30 mmol/L glucose, respectively. Control group: HG cell model was transfected with negative control mimics; mannitol group: the control group was added with 25 mmol/L mannitol; miR-519d-3p overexpression group: HG cell model was transfected with miR-519d-3p mimics; miR-519d-3p combined with HIF-1α overexpression group: HG cell model was co-transfected with miR-519d-3p mimics and HIF-1α overexpression vector. The expression of miR-519d-3p in each group was tested by real-time fluorescence quantitative PCR. The expression of HIF-1α protein in each group was tested by Western blotting. The binding sites between miR-519d-3p and HIF-1α were detected by luciferase reporter gene assay. The cell proliferation of each group was detected by CCK-8. The cell apoptosis of each group was tested by Hoechst 33342 staining. The protein expression of extracellular fluid inflammatory factors tumor necrosis factor-α(TNF-α), interleukin(IL)-1β and IL-6 in each group was tested by ELISA. The formation of new capillary lumen-like structures was detected by tubule formation assay.RESULTS: Compared with the NG, miR-519d-3p expression was significantly reduced in the HG cell model, while HIF-1α protein expression was significantly increased in the HG(all P<0.01). Compared with the control group, HIF-1α protein expression was significantly reduced in the miR-519d-3p overexpression group(P<0.01). The “CGUGAAA” sequence of miR-519d-3p could specifically bind to the “GCACUUU” sequence of HIF-1α 3'-untranslated region(3'-UTR). Compared with the control group, the miR-519d-3p overexpression group showed a significant increase in 24, 48 and 72h absorbance values, a significant decrease in cell apoptotic rate, a significant decrease in the concentrations of TNF-α, IL-1β and IL-6, and a significant decrease in the number of new capillary lumen-like structures(all P<0.01). Compared with the miR-519d-3p overexpression group, the miR-519d-3p combined with HIF-1α overexpression group showed a significant decrease in 24, 48 and 72h absorbance values, a significant increase in cell apoptotic rate, a significant increase in the concentrations of TNF-α, IL-1β and IL-6, and a significant increase in the number of new capillary lumen-like structures(all P<0.01). There was no difference between the control group and mannitol group in the comparison of the above indicators(all P>0.05).CONCLUSION: miR-519d-3p expression is down-regulated while HIF-1α protein expression is up-regulated in high glucose induced HRMEC model. HIF-1α is a target gene of miR-519d-3p. The miR-519d-3p targets HIF-1α to increase cell proliferation and reduce cell apoptosis and inflammation, thereby alleviating high glucose-induced HRMEC dysfunction and inhibiting angiogenesis.
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Objective To explore the impact of Hedgehog protein on human retinal microvascular endothelial cell(HRMEC) and its signaling pathway. Methods The cultured HRMECs were divided into normal control group,0. 5μmol/L agonist group and 1. 0μmol/L agonist group,and were cultured in medium with final concentration of 0,0. 5 and 1. 0μmol/L Hedgehog agonist,respectively;HRMECs cultured in high glucose medium were divided into high glucose control group,1. 5μmol/L inhibitor group and 2. 5μmol/L inhibitor group. Erismodegib,the Smoothed inhibitor with final concentration of 0,1. 5 and 2. 5 μmol/L was added into corresponding group,respectively. MTS method and Transwell cell migration method were used to detect the proliferation( A490 value) and relative mobility of HRMEC. The phosphorylation of PLCγ1, Akt and Erk proteins were detected by Western blot. Results The relative expression of Hedgehog protein in the high glucose control group was 6. 24±0. 11,which was significantly higher than 1. 00±0. 00 in the normal control group(t=667. 573,P<0. 001). The A490 value was 1. 349±0. 050 and 1. 422±0. 053,and the relative mobility rate was 2. 34±0. 14 and 3. 59±0. 32 in the 0. 5μmol/L agonist group and the 1. 0μmol/L agonist group, respectively, which were significantly higher than 1. 203 ± 0. 101 and 1. 00 ± 0. 00 in the normal control group(all at P<0. 01). The A490 value was 0. 849±0. 010 and 0. 737±0. 030,and the relative mobility rate was 0. 43 ± 0. 02 and 0. 27 ± 0. 01 in the 1. 5 μmol/L inhibitor group and the 2. 5 μmol/L inhibitor group, respectively,which were significantly lower than 1. 000±0. 040 and 1. 00±0. 00 in the high glucose control group(all at P<0. 01). The phosphorylation ratios of PLCγ1,Akt and Erk in the 0. 5μmol/L agonist group and the 1. 0μmol/L agonist group were significantly higher than those in the normal control group ( all at P<0. 01 ) . The phosphorylation ratios of PLCγ1,Akt and Erk in the 1. 5μmol/L inhibitor group and the 2. 5μmol/L inhibitor group were significantly lower than those in the high glucose control group ( all at P<0. 01 ) . Conclusions High glucose induces the expression of Hedgehog protein in HRMEC. Hedgehog protein may regulate the function of HRMEC by regulating the phosphorylation of PLCγ1,Akt and Erk in G Protein-coupled receptors pathway.
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Objective@#To explore the impact of Hedgehog protein on human retinal microvascular endothelial cell(HRMEC)and its signaling pathway.@*Methods@#The cultured HRMECs were divided into normal control group, 0.5 μmol/L agonist group and 1.0 μmol/L agonist group, and were cultured in medium with final concentration of 0, 0.5 and 1.0 μmol/L Hedgehog agonist, respectively; HRMECs cultured in high glucose medium were divided into high glucose control group, 1.5 μmol/L inhibitor group and 2.5 μmol/L inhibitor group.Erismodegib, the Smoothed inhibitor with final concentration of 0, 1.5 and 2.5 μmol/L was added into corresponding group, respectively.MTS method and Transwell cell migration method were used to detect the proliferation(A490 value)and relative mobility of HRMEC.The phosphorylation of PLCγ1, Akt and Erk proteins were detected by Western blot.@*Results@#The relative expression of Hedgehog protein in the high glucose control group was 6.24±0.11, which was significantly higher than 1.00±0.00 in the normal control group(t=667.573, P<0.001). The A490 value was 1.349±0.050 and 1.422±0.053, and the relative mobility rate was 2.34±0.14 and 3.59±0.32 in the0.5 μmol/L agonist group and the 1.0 μmol/L agonist group, respectively, which were significantly higher than 1.203±0.101 and 1.00±0.00 in the normal control group(all at P<0.01). The A490 value was 0.849±0.010 and 0.737±0.030, and the relative mobility rate was 0.43±0.02 and 0.27 ±0.01 in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group, respectively, which were significantly lower than 1.000±0.040 and 1.00±0.00 in the high glucose control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 0.5 μmol/L agonist group and the 1.0 μmol/L agonist group were significantly higher than those in the normal control group(all at P<0.01). The phosphorylation ratios of PLCγ1, Akt and Erk in the 1.5 μmol/L inhibitor group and the 2.5 μmol/L inhibitor group were significantly lower than those in the high glucose control group(all at P<0.01).@*Conclusions@#High glucose induces the expression of Hedgehog protein in HRMEC.Hedgehog protein may regulate the function of HRMEC by regulating the phosphorylation of PLCγ1, Akt and Erk in G Protein-coupled receptors pathway.
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@#AIM: To investigate the effects of insulin on syndecan-1 expression, cellular permeability and proliferation in human retinal microvascular endothelial cells. <p>METHODS: Cells were treated with 100nmol/L and 1 000nmol/L insulin for 48h respectively. Expression of protein and mRNA were detected by western blot and quantitative real-time polymerase chain reaction. Cellular proliferation and permeability were examined by methods of methylthiazolyl tetrazolium and horseradish peroxidase. <p>RESULTS: With treatment of insulin, protein and mRNA of syndecan-1 both increased obviously, and the effect of high level insulin was more significant. After treated with insulin, cellular proliferation and permeability both enhanced, and the effects of high level insulin were stronger. <p>CONCLUSION: Insulin can up-regulate syndecan-1 protein and mRNA in cultured human retinal microvascular endothelial cells, and increase cellular permeability and proliferation.
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Background Retninal neovascular diseases caused by hypoxia has become a major blinding disease,which is lack of effective chemical treatment currently,it's important to study the molecuar mechanism of the disease,so as to guide the clinical medication.Objective This study was to explore the effect of 15 (S)-hydroxyeicosate traenoic acid (15-HETE) on the proliferation of hypoxic retinal microvascular endothelial cells (RMVECs) and its probable mechanism.Methods RMVECs were isolated from C57BL/6J mice and incubated and then identified with anti-Ⅷ factor antibody by immunochemistry and immunofluorescence.The cells were divided into the normoxia group and the hypoxia group.The hypoxia cell models were established by treated with 125 μmol/L CoCl2.The cells were cultured with serum-free DMEM containing endothelial cell growth supplement (ECGS)and high glucose for 48 hours,and then different concentrations of 15-HETE (0.0,0.1,1.0,5.0 μmol/L) were added in the medium for 48 hours respectively to subgroup the groups.The proliferation of the cells (absorbance,A) was detected using MTT.The relative expression levels of protein and mRNA of hypoxia inducible factor-1α (HIF-1α),bcl-2 and caspase-3 were assayed by reverse transcription PCR (RT-RCR)and Western blot.Results The cells showed the positive response for anti-Ⅷ factor antibody with the positive rate of (94.38 ±4.25)％.No significant difference was found in the cell proliferation of various groups under the normoxia condition (F =0.283,P =0.837),but under the hypoxia condition,the proliferation values were significantly different among various groups (F =702.582,P＜0.001).The cell proliferation value in the 1.0 μmol/L 15-HETE group and 5.0 μmol/L 15-HETE group was lower than that of the simple hypoxia group respectively(both at P＜0.05).The inhibitory rates in the 0.1,1.0,5.0 μ mol/L 15-HETE groups were (1.09±0.31) ％,(21.09± 3.53) ％ and (49.86 ±4.15) ％,showing a dosedependent manner.No significant difference was seen in the expression levels of bcl-2,caspase-3 and HIF-1α mRNA in various groups under the normoxia conditions.However,compared with the simple normoxia group,the relative expressions of bcl-2 mRNA and HIF-1α mRNA in the cells were increased by 1.53 folds and 1.7 folds in the simple hypoxia group respectively,and caspasse-3 mRNA expression decreased by 70％ (all at P ＜ 0.05).Under the normoxia condition,the expression of bcl-2 and pro-caspase-3 protein in the cells were not significantly different among the various groups (P＞0.05),however,the expressions of bcl-2 and pro-caspase-3 proteins were elevated by 1.6 folds and 1.9 folds in the hypoxia group in compared with the normoxia group (P＜0.05).Compared with the simple hypoxia group,the expressions of bcl-2 and pro-caspase-3 were lowed by 40.4％ and 42.5％ in the 5.0 μmol/L 15-HETE group (P＜0.05).Conclusions 15-HETE inhibits the proliferation of RMVECs and therefore suppresses neovascularization by down-regulating the expressions of HIF-1α and bcl-2 and the activation of caspase-3 in a dose-dependent manner.
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Background To optimize the culture method of human retinal microvascular endothelial cells is very important for the study of retinal angiogenesis disease.Human retinal microvascular endothelial cells have been successfully cultured in previous studies,but further improvement of the culture method to harvest higher yields and purity cells is still needed.Objective This study was to design a modified method to isolate and purify human retinal microvascular endothelial cells much easily and quickly,and to compare the expression of specific markers of vascular endothelial cells,factor Ⅷ and CD31/CD34 in the cells.Methods The use of human donor eyeballs was approved by the Ethic Commission of Zhongshan Ophthalmic Center of Sun Yat-sen University.The retina tissue from healthy donor was isolated and digested by the two-step digestion method with 2％ trypsin and 0.133％ collagenase Ⅳ.Human retinal microvascular endothelial cells were collected and plated in 60 mm dishes coated by 0.1％ fibronectin and cultured in endothelial cell-specialized medium supplemented with 10％ fetal bovine serum,0.3 mg/L β-endothelial cell growth factor (ECGF) and 100 ng/L sodium heparin.During the culturing,the growth situation of the cells was monitored by morphological observation,and immunohistochemical staining was performed to probe vascular endothelial cell-specific membrane protein CD31,CD34 and factor Ⅷ for identification of the cell purity.Results Human retinal microvascular endothelial cells were isolated successfully from the retina by the twostep digestion method.The primary cultured cells adhered to well 72 hours later and achieved confluence with the typical cobblestone appearance 9 to 10 days after cultured.The cells exhibited the blue nuclei and reddish cytoplasm by regular haematoxylin and eosin stain and showed a strong positive response for CD31,CD34 and factor Ⅷ by immunohistochemistry.The positive dye of CD31 and CD34 was lower than Ⅷ factor in both endothelial cells.Conclusions Modified culture method of human retinal microvascular endothelial cells can improve cell culture result and purify target cells.
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Background Researches have demonstrated that ocular hypertension induces the ischemia-reperfusion of retina and further leads to the degeneration of retinal ganglion cells,but its mechanism is beyond understanding.Objective The present study aims to observe the effects of static pressure on the morphology,proliferation activity and viability of cultured retinal microvascular endothelial cells (RMECs) and evaluate the expression of ET-1 and NO in these cells under variant static pressure.Methods RMECs were isolated from 30 healthy Wistar rats and cultured using explant culture method by Ⅷ factor antibody and PECAM-1 antibody.The static pressure of 1.33kPa,2.67kPa,5.33kPa and 10.67kPa was used in culture bottle respectively.The RMECs without static pressure were used as normal control group.The morphology of RMECs under the different static pressure was observed by inverted phase contrast microscopy,and the number of RMECs was counted using the counting plate.Cellular viability was studied by trypan blue staining.The changes of ET-1 and NO_2~-/NO_3~-,two metabolic products of NO,in the medium were detected by radioimmunoassay and Griess's nitrate reductase method.The expression of ET-1,eNOS and iNOS mRNA in RMECs was analyzed by semi-quantitative RT-PCR 24 hours after treatment of variant static pressure.Results Cultured RMECs sticked well at 24 hours and reached to confluence at 48 hours and showed the red fluorescence for Ⅷ factor antibody and PECAM-1 antibody.Enlargement of nuclei,extenders of cell bodies and suspension of RMECs in medium were observed.The number of RMECs was gradually increased.The cell viability was reduced with the raise of static pressure among these four groups(F=12.205,P＜0.01;F=11.180,P＜0.01).The static pressure increased the content of ET-1 released by RMECs in 2.67kPa,5.33kPa and 10.67kPa of static pressure groups,and concentrations of NO_2~-/NO_3~- in the medium showed a significant increase in 5.33kPa and 10.67kPa of static pressure groups compared with normal and 1.33kPa of static pressure groups(P＜0.01).The expressions of ET-1 mRNA,eNOS mRNA and iNOS mRNA were considerably enhanced in 5.33kPa and 10.67kPa of static pressure groups compared with normal control group(P＜0.01).Conclusion Raised static pressure causes the alteration of RMGCs structure and morphology.Static pressure could upregulate the expressions of ET-1,eNOS and iNOS mRNA in RMECs and increase the release of ET-1 and NO.This pathway might be one of pathologic mechanisms of retinal injury induced by high intraocular pressure.