Protective Effect And Mechanism Of Osteoprotegerin On Human Umbilical Vein Endothelial Cells Cultured With High Glucose

BackgroundDiabetic angiopathy is a major cause of morbidity and mortality in diabetes mellitus, the mechanisms of diabetic angiopathy caused by high glucose are still unclear. Vascular endothelial cells attaching to blood vessel wall can directly affect the stability and function of the blood vessel wall. Recent studies show that high glucose in vitro can induce apoptosis in vascular endothelial cells. Increased cell apoptosis may contribute to the lossing of endothelial integrity and endothelial dysfunction. Endothelial cell apoptosis is a primary cause leading to the origin and development in diabetic angiopathy. All these suggest that apoptosis of endothelial cells is closely associated with diabetic vascular complications.The TSC/mTORCl pathyway can modulate apoptosis and may play an important role in diabetic vascular complications.Osteoprotegerin (OPG) is a member of the tumor-necrosis factor receptor superfamily which can restrain osteoclastogenesis and increase bone mineral density. Clinical studies suggest that the level of serum OPG obviously rising in diabetic patients is connected to diabetic vascular complications and endothelial dysfunction. In one view, the result of’the more serious of the disease, the higher level of the OPG’which is based on these clinical observations can be relatively intuitive to speculate that OPG may be a risk factor of diabetic angiopathy. While another widely accepted view is that elevated level of OPG is a compensated defensive mechanism which can avoid blood vessel injury, so some experts call OPG’vasculoprotegerin’. However, the effect and mechanism of OPG on vascular endothelial cells cultured with high glucose is still unknown.ObjectiveThe purpose of study is to investigate the protective effect and mechanism of OPG on the injury of human umbilical vein endothelial cells (HUVECs) induced by high glucose in vitro, and to provide the basis for the effects of OPG on treatment and prevention of diabetic angiopathy in the future.MethodsⅠ. The damage model of HUVECs induced by high glucose in vitro.The cultured HUVECs were randomly divided into normal glucose group (5.5mmol/L glucose) and high glucose groups (11mmol/L,22mmol/L,33mmol/L,44mmol/L glucose). At different time point (24h,48h,72h,96h), cell viability was measured by MTT, cell apoptosis and cell cycle were assessed by flow cytometry.Ⅱ. Protective effect and mechanism of OPG on HUVECs cultured with high glucose.Part-1The cultured HUVECs were treated with normal glucose (5.5mmol/L), high glucose (33mmol/L), high glucose+OPG (33mmol/L glucose+0.5μg/mL,1μg/mL,2μg/mL OPG) as well as mannitol (5.5mmol/L glucose+27.5mmol/L mannitol) for48h, respectively. Cell apoptosis was assessed by flow cytometry. Apoptotic nuclei were detected using Hoechst33258staining and analyzed by fluorescence microscopy at350-nm excitation and460-nm emission. Migration ability was deteced using scratch test and analyzed by inverted phase contrast microscope.Part-2The cultured HUVECs were treated with normal glucose (5.5mmol/L), high glucose (33mmol/L), high glucose+OPG (33mmol/L glucose+2μg/mL OPG) as well as high glucose+rapamycin (33mmol/L glucose+10ng/mL rapamycin) for48h, respectively. The expression levels of Tuberin, P-Tuberin, S6K, P-S6K, Bcl-2and Bax protein were measured by western blot analysis using specific antibodies. Ⅲ. Statistical analysisData were presented as mean±standard deviation (SD) and analysed with SPSS13.0software. Results were determined using ANOVA followed by Bonferroni test using one trial analysis. Statistical significance was set at P<0.05.ResultsⅠ. The damage model of HUVECs induced by high glucose in vitro.1) Morphological change of HUVECs cultured with nomal glucose (inverted phase contrast microscope).About4h after inoculation, HUVECs partly began to adhere to the flask and stretch out pseudopodia. About24h after inoculation, all the cells adhered to the culture bottle, presented a monolayer arrangement and a variety of shapes, such as rounded, short spindle or flat polygonal shape. During3～4days after inoculation, HUVECs coalesced together and aligned like cobble-stone shape. During5～6days after inoculation, HUVECs presented a dense monolayer and then grew slowly.2) Cell viability was demonstrated by trypan blue dye exclusion test before each experiment.Living cells were excluded to trypan blue because of cell membrane integrity. Cells with incomplete membrane were inclined to be stained by trypan blue. The results of trypan blue dye exclusion test revealed that cell viability of HUVEC before each experiment were over95%.3) Growth curve of HUVECs cultured with normal glucose (detected by MTT).Growth curve of HUVECs cultured with normal glucose was detected by MTT from day1to7. HUVECs were almost invariable and still in the period of retention on day1after inoculation. On the2nd day, the amount of the cells increased significantly. During3～5days, cells grew rapidly and in the logarithmic growth phase. On the6th day, cells grew stably and in the period of plateau.4) Effect of high glucose on proliferation in HUVECs (detected by MTT).The cell vitality in normal glucose group was gradually increased from24h to72h and decreased at96h. The cell vitality in11mmol/L,22mmol/L glucose group was gradually increased from24h through48h to96h, compared with normal glucose group, the viability of HUVECs in both groups was lower than that in normal glucose group during24h-72h. The cell vitality in33mmol/L,44mmol/L glucose group was gradually decreased from24h through48h to96h, compared with normal glucose group, the viability of HUVECs in both groups was lower than that in normal glucose group and in a time-dose dependent manner (P<0.05)5) Cell apoptosis was assessed by flow cytometry (Annexin v-FITC/PI double staining).Cell apoptosis was increased in both normal glucose group and high glucose group in time-dependent manner. Compared with normal glucose group, early and late apoptosis ratio of HUVECs in all high glucose groups were both notably elevated (P<0.05) and in a time-dose dependent manner.6) Cell cycle was analyzed by flow cytometry (PI staining).Cell cycle had no noticeable change at24h in both normal glucose group and high glucose group. At the same time point (48h,72h,96h), compared with normal glucose group, the proportion of G0/G1phase in high glucose group was obviously higher than that in normal glucose group, while the proportion of S phase and G2/M phase was significantly lower than that in normal glucose group in a dose-dependent manner.II. Protective effect and mechanism of OPG on HUVECs cultured with high glucose.1) Effect of OPG on apoptosis in HUVECs cultured with high glucose (Annexin v-FITC/PI double staining).Compared with normal glucose group, the apoptosis of HUVECs was dramatically increased in high glucose group (P<0.05). Compared with normal glucose group or high glucose group, the apoptosis of HUVECs in high glucose+OPG group was significantly lower than that in high glucose group, which was still higher than that in normal glucose group (P<0.05). There was no statistically difference between hyperosmolar control group and normal glucose group.2) Effect of OPG on morphological change in HUVECs cultured with high glucose (Hoechst33258staining).Apoptotic nuclei were detected using Hoechst33258staining and analyzed by fluorescence microscopy. Compared with normal glucose group, the apoptosis of HUVECs was dramatically increased in high glucose group. Compared with normal glucose group or high glucose group, the apoptosis of HUVECs in high glucose+OPG group was significantly lower than that in high glucose group, which was still higher than that in normal glucose group. No significant difference was found between hyperosmolar control group and normal glucose group.3) Effect of OPG on migration ability in HUVECs cultured with high glucose (detected using scratch test)HUVECs were treated with normal glucose, high glucose as well as high glucose+OPG for24h, respectively, and were analyzed by inverted phase contrast microscope. Compared with normal glucose group, the migration ability of HUVECs was dramatically decreased in high glucose group. Compared with normal glucose group or high glucose group, the migration ability of HUVECs in high glucose+OPG group was higher than that in high glucose group, which was still lower than that in normal glucose group.4) The expression levels of Tuberin, P-Tuberin, S6K, P-S6K, Bcl-2and Bax protein were measured by western blot.To investigate the protective mechanism of OPG on HUVEC induced by high glucose, the upstream (Tuberin, P-Tuberin) and downstream (S6K, P-S6K) of mTORC1pathway was determined by western blot. To clarify whether Tuberin/S6K pathway was associated with apoptotic signals, the expression levels of Bcl-2and Bax were determined by western blot. To determine whether inhibition of mTORC1pathway would restrain the activation of apoptosis pathway, HUVECs were pretreated with the mTORCl inhibitor rapamycin for24h, then treated the cells with high glucose (33mmol/L) for48h, the expression levels of Tuberin, P-Tuberin, S6K, P-S6K, Bcl-2and Bax protein were measured by western blot. Compared with normal glucose group, the expression levels of Tuberin, P-Tuberin, S6K, P-S6K and Bax were increased markedly (P<0.05), and the expression of Bcl-2was decreased significantly in high glucose group (P<0.05). Compared with normal glucose group or high glucose group, the expression levels of P-Tuberin, P-Tuberin/Tuberin, S6K, P-S6K/S6K, Bax in high glucose+OPG group were significantly lower than that in high glucose group, which was still higher than that in normal glucose group (P< 0.05),and the expression of Bcl-2in high glucose+OPG group was significantly higher than that in high glucose group, which was still lower than that in normal glucose group (P<0.05). No significant difference was found between high glucose+OPG group and high glucose+rapamycin group.ConclusionOur data suggest that1. High glucose can inhibit HUVECs proliferation and increase the cell apoptosis as well as block the cell cycle in G1/S phase in a time and dose dependent manner. HUVECs treated with33mmol/L glucose for48h are considered to the damage model of HUVECs induced by high glucose in vitro.2. The mechanism of HUVECs apoptosis induced by high glucose may be related to TSC/mTORCl pathway. High glucose enhances tuberin phosphorylation and mTORCl activation (measured by S6K phosphorylation) to activate apoptosis signal pathyways (upregulation of Bax and downregulation of Bcl-2) in HUVECs.3. Protective effect and mechanism of OPG on HUVECs cultured with high glucose may be association with TSC/mTORC1pathway. OPG decrease tuberin phosphorylation and mTORC1activation (measured by S6K phosphorylation) to inactivate apoptosis signal pathyways (downregulation of Bax and upregulation of Bcl-2) in HUVECs.