Effect Of Adrenomedullin Antagonist ADM_22-52 On High Glucose-induced Human Retinal Vascular Endothelial Cells

Background and ObjectiveDiabetic retinopathy (DR) is one of the most common and severe micro-vascularcomplication of diabetics which might be lead to impairment of vision and eventuallyblindness. An early clinical signs of DR include micro aneurysms and hemorrhages, whilethe later signs are dilated, tortuous, irregular and narrow retinal blood vessels which calledretinal ischemia that ultimately results in neovascularization. An important sign ofproliferative diabetic retinopathy (PDR) is the neovascularization. The incidence of DR isincreasing year by year and turns into the main cause of blindness. Adrenomedullin (ADM),a kind of variety biological active peptide, is widely participated in the activities of thebody. Although several research suggests that it have effect on the process of the formationof new blood vessels and tumor formation, the role of ADM on retinal vascular endothelialcells has remained obscure. This study was to explore the effect of ADM22-52on highglucose-induced human retinal vascular endothelial cells and its mechanism. In this study,we used CCK-8assay, scratch wound assay and tube formation assay to observe the effectof ADM22-52on proliferation, migration and capillary formation of high glucose-inducedhuman retinal microvascular endothelial cells (HRECs) in order to provide new therapeutictargets for DR.Materials and Methods1. HRECs were divided into three groups, cultured with different concentrations ofglucose (5mM,15mM,30mM). After culture for24h, the RNA of cell was isolated, andthen theADM expression was quantified.2. HRECs were cultured with different concentrations of glucose (5mM,30mM,30mM+100ng/ml ADM22-52,30mM+1000ng/ml ADM22-52), and were grown toover-confluence. A wound of appropriate width was made by a200μl tip, followed by removing the floating cells and incubated with serum-free medium containing differentconcentrations of glucose with or without ADM22-52and then cultured the6-well plate in at37°C in a5%CO2incubator. The migration monolayer was photographed at0h,12h, and24h. The migration distance was measured by Photoshop CS2.3. About4×103of HRECs were seeded into per well of96–well plate in mediumcontain different concentration of glucose (5mM,30mM,30mM+10ng/ml ADM22-52,30mM+100ng/ml ADM22-52,30mM+1000ng/ml ADM22-52) and allowed to adhere for24h.After cells adherent into the bottom of the plate, the cells were cultured with serum-freemedium to starvation for24h. Then, the cells were treated with different concentration ofglucose and ADM22-52for24h, the proliferative activity was determined by CCK-8assayaccording to the manufacture’s instruction. In brief,10μl CCK-8was added to each wellfollowed by incubation for an additional2-4h. When the colour medium changed from redto yellow, the absorbance value at a wave-length of450nm was detected.4. HRECs had been cultured under different medium with or without ADM22-52,which were seeded on the solidify Matrigel immediately at a density of1.5×104cells perwell. Then, the plates were placed in a humidified atmosphere of5%CO2and95%air at37°C for8h to allow the formation of capillary-like structure.5. After culture under different medium with or without ADM22-52for8h, the RNAof cell was isolated and then the expression of VEGF、TSP-1、ADAMTS-1was quantifiedusing RT-PCR.6. After culture under different medium with or without ADM22-52for24h, theprotein of cells was extracted and VEGF and PI3K expression was detected by Westernblot technique.Results1. RT-PCR results show that compared with5mM glucose,30mM glucose couldpromote the expression ofADM and VEGF (P<0.05).2. Compared with the5mM glucose,30mM glucose could obviously promote cellmigration and tube formation, while this ability could be inhibited byADM22-52(P<0.05).3. CCK-8assay result showed1000ng/ml ADM22-52could inhibit cell proliferation(P<0.05), while glucose have no effect on the proliferation of cells (P>0.05). 4. The result of RT-PCR showed that5μg/ml ADM22-52could inhibit the expressionof VEGF and promote the expression of ADAMTS-1which was induced by high glucose(P<0.05).5. The result of Western blot showed that5μg/ml ADM22-52could inhibit theexpression of VEGF and PI3K protein (P<0.05).Conclusion1. High glucose could promote the migration, tube formation and the expression ofADM and VEGF.2. ADM22-52inhibited HRECs proliferation, migration, tube formation which wasinduced by high glucose.3. ADM22-52could inhibit the expression of VEGF and PI3K; promote the expressionofADAMTS-1.