Sera From Diabetic Foot Ulcer Patients Induced Inflammatory And Senescence Of Vascular Endothelial Via H3K27me3 Demethylation

Objective:To analyze the effect of sera from diabetic foot ulcer patients(The following is referred to as diabetic foot ulcer sera)on the activation and injury of vascular endothelial cells,and the underlying epigenetic mechanisms.Methods:1?The primary isolation and culture of human umbilical vein endothelial cells(HUVECs):Cells were obtained by incubating human umbilical vein with equal volume mixed 0.1%collagenase type II and 0.25%trypsin at 37°C for 20~30 min.Then the cells were cultured with ECM complete medium,and then the morphological of the cells were observed.2?The identification of HUVECs:The expression of CD31 factor-related antigen specific to HUVECs was detected by immunofluorescence.3?HUVECs culture and sample treatment:HUVECs were maintained in ECM complete medium to logarithmic phase,randomly divided into healthy control group and diabetic foot ulcer group,HUVECs were cultured with the conditioned medium containing 5%different human serum,and were sampled at different time points accordingly for the corresponding assays.4?Effect of sera from diabetic foot ulcer on the cell viability detected by Cell Counting Kit-8.5?Effect of sera from diabetic foot ulcer on the cell senescence measured by?-galactosidase in situ staining assay.6?Effect of sera from diabetic foot ulcer on the cell cycle analysis with flow cytometry.7?Effect of sera from diabetic foot ulcer on the cell migration and repairing ability scratch test.8?Gene expression of related factors,such as Endothelial NOS(eNOS),receptor for advanced glycation end-products(RAGE),pro-inflammatory factors(IL-1?,IL-6,IL-12,TNF-?),adhesion molecules(ICAM1,VCAM1)as well as growth factors(TGF-?,VEGF192),determined by real-time fluorescence quantitative RT-PCR.9?The expression of JMJD3 and methylation status of histone H3K27 in HUVECs monitored by immunofluorescence staining.Results:1?The morphology of HUVECs:The adherent cells were single or clustering,spindle or spherical in the early period.With the cells growth,they were round or ellipse,single-layer wall,looked like paving stone.2?The identification of HUVECs:The immunofluorescence staining showed that the cultured cells had the presence of CD31 factor antigen specific to HUVECs,which indicated that the cultured cells were HUVECs.3?Effect of sera from diabetic foot ulcer on HUVECs viability:CCK-8 test found that the sera did not significantly inhibit the viability of umbilical vein endothelial cells in patients with diabetic foot ulcer.4?Effect of sera from diabetic foot ulcer on HUVECs senescence:The cell senescence?-galactosidase in situ staining assay showed that the percentage of?-galactosidase staining positive cells in diabetic foot ulcer group was increased significantly,compared with that of the healthy control group P<0.0001;The histone H3K27me2/3 specific demethylase inhibitor GSK-J4 treated diabetic foot ulcer group could significantly reduce the positive cell percentage,compared with the diabetic foot ulcer group P<0.0001.It indicated that sera from diabetic foot ulcer are capable of promoting HUVECs cellular senescence,and the inhibition of H3K27me2/3demethylation could effectively reverse the senescence of HUVECs induced by sera of diabetic foot ulcer.5?Effect of sera from diabetic foot ulcer on the HUVECs cell cycle:The results of flow cytometry showed that the sera of diabetic foot ulcer could cause endothelial cell proliferation disorder.We treated HUVECs with diabetic foot ulcer sera for 48h,the G2-M phase arrest of HUVECs were induced,and the percentage of cells arrested in G2 phase were 3.11±0.51%,which was significantly higher than the healthy control group 1.50±0.17%,P<0.01,the difference was statistically significant.The percentage of cells in G1 phase was only 70.50±2.00%,compared with that of the healthy control group 75.30±1.30%,P<0.05,there was also statistical difference;However,the inhibitor GSK-J4 can effectively inhibit the proliferation disorder of endothelial cells caused by diabetic foot ulcer sera.The percentage of G2 cells in the GSK-J4 inhibitor-containing diabetic foot ulcer group is only 1.91±0.26%,which is significantly lower than that of diabetic foot ulcer group(P<0.05).6?Effect of sera from diabetic foot ulcer on the HUVECs cell migration and repairing ability:The results of the scratch test showed that there were no significant difference in the scratch repair area between the two groups at each time point observed,but there were a significant difference in the cell density per unit area of the repaired part.Within the scratch repairing area,the cell density of the diabetic foot ulcer group is 252.68±14.19 per mm~2,decreased significantly when compared with those of the healthy control group,368.26±14.65 per mm~2,P<0.0001.7?Effect of sera from diabetic foot ulcer on the HUVECs gene expression of factors related to the abovementioned biological processes:RT-PCR analysis showed that the mRNA levels of eNOS,RAGE122 and pro-inflammatory factors(IL-1?,IL-6,IL-12,TNF-?)were all upregulated significantly in diabetic foot ulcer group when compared with those of healthy control group,the diabetic foot ulcer sera treatment down-regulated the expressions of ICAM-1,VCAM-1,VEGF192 and TGF-?.However,this effect was reversed by adding GSK-J4 treatment.8?The expression of JMJD3 and methylation status of histone H3K27 in endothelial cells:By immunofluorescence staining,we found that diabetic foot ulcer sera treatment significantly up-regulated JMJD3 expression.Meanwhile,the expression of H3K27me3 was significantly down-regulated in the diabetic foot ulcer group,with significant increase of f H3K27me2 and H3K27me.However,we proved that GSK-J4 can inhibit diabetic foot ulcer induced down-regulation of H3K27me3,and restore H3K27me3 into a normal level along with the decrease of H3K27me2/1back to control levels.Discussion:Our findings indicate that the sera of diabetic foot ulcers induce human endothelium senescence,cell cycle and functional disorder by activating JMJD3signaling which down-regulating the global methylation level of H3K27 and causing the expression disorder of endothelial function,cell adhesion and inflammation related genes.JMJD3 signaling pathway is involved in the process of endothelial cell senescence,cell cycle and dysfunction induced by the sera of diabetic foot ulcers.