Mechanism And Effects Of Glucose On Endostatin Expression In Vascular Endothelial Cells

Endostatin is an anti-angiogenic 20 kDa carboxyl-terminal fragment derived from collagenⅩⅧthat specifically inhibits endothelial proliferation and migration in vitro and potently inhibits angiogenesis which is balanced by such angiogenic factors as VEGF. Recent studies reveal that both atherosclerosis (AS) and diabetic retinopathy (DR), which are major manifestations of vascular complication of diabetes mellitus (DM), are subject to angiogenesis. Therefore, endostatin is administered to inhibit growth of blood vessel and is considered a new therapy strategy in dealing with vascular complication of DM. However, up to now there is few report on this study at home and abroad. This study is begun with measuring ES level in patient blood with DM, then focused on ES gene expression and ES protein release in vascular endothelial cells cultured with high glucose. We aim to make clear the relationship between ES and vascular complication of DM, to observe ES expression and release with in vitro experiment and accordingly to investigate the mechanism of ES release. The study in this field may provide important new pharmacological information on both ES and other potential drugs to retard angiogenesisMethods1. The determination of endostatin ,VEGF levels in 100 T2DM patients including 34 cases without macroangiopathy (group N),40 cases with only one kind of macroangiopathy (group A),26 cases with much more macroangiopathy (group B) and 30 healthy control subjects (group C) was made by ELISA assay. 2. Human umbilical vein endothelial cells (HUVEC) were cultured in DEME media with or without high glucose and were divided into 3 groups randomly: control group(with 5.6mmol/L Glu), high glucose groups (with 11.2 , 22.4mmol/L Glu respectively). After24, 48, 72, 96 hour(s), total RNA and protein were extracted. Semi-quantative RT-PCR and Western blot were used to detect the expression of ES.3. HUVEC were cultured in DEME media for 72 hours with 5.6,11.2,22.4mmol/L glucose respectively. The expression of ES protein was determined by Western-blot and the level of nitric oxide (NO) in the supernatant of cell culture was measured by nitrate -reductase method. Those HUVEC incubated with 11.2mmol/L glucose for 72 hours (also as control group) were stimulated by NO synthase inhibitor L-NAME, NO synthase-independent NO donor sodium nitroprusside (SNP) and soluble guanylate cyclase inhibitor ODQ respectively. Western-blot was employed to determine ES protein expression.Results1. The levels of endostatin and VEGF in T2DM patients complicated with macroangiopathy (group A, group B) were significantly higher than those without macroangiopathy (group N, group C), [endostatin: (32.44±15.6) ng/ml, (35.1±20.2) ng/ml vs (11.2±8.6) ng/ml, (9.94±6.7) ng/ml; VEGF: (133.54±36.8) pg/ml, (302.1±52.4) pg/ml vs (90.2±42.4) pg/ml, (81.34±33.5) pg/ml, P＜0.01]. There was no significant difference between endostatin, VEGF levels in group C and those in group N. It was not endostatin levels but VEGF levels that were significantly elevated between group A and group B in turn[(133.5±36.8) pg/ml vs (302.1±52.4) pg/ml, P＜0.01]. There was a significantly positive correlation between endostatin and VEGF levels in Group A (r=0.540, P<0.01).2. The level of ES expression in HUVEC treated with 11.2 mmol/L Glu was increased with prolonging exposure time at both mRNA and protein levels, especially when exposuring for 72 and 96 hours, there was significant increase compared with control group(P_72h＞0.05, P_96h＜0.01); When treated HUVEC with 22.4mmol/L Glu for 24, 48 and 72 hour(s), both gene expression and protein release of endostatin were increased with time-dependent manner. There was significant increase in ES expression when they were incubated for 72 hours, but a significant decrease for 96 hours compared with control group (P＜0.05); For control group, there was significant decrease in protein release of ES incubated for 96 hours compared with those for 24 hours(P＜0.05), but there was no significant change in expression of ES mRNA during the observed period.3. The level of ES protein expression and NO concentration in HUVEC were increased with significant glucose dose-dependent manner during short period(P＜0.05, compared with 5.6mmol/LGlu); the reduction of ES protein expression was observed after L-NAME and ODQ with 0.372-fold , 0.406-fold intensivity compared with control group respectively (P＜0.01). The level of ES protein expression was induced by SNP with 1.32-fold increase (P＜0.01)Conclusion1. The study suggests that the variation of serum VEGF levels is closely associated with the initiation and progression of T2DM macroangiopathy and meanwhile VEGF together with endostatin possibly plays an important role on the pathological course of AS based on homeostasis regulation.2. For certain limited period, the expression of endostatin was induced with time-dependent manner in HUVEC cultured with high concentration of glucose, but it was suppressed with prolonging exposure time. And the reduction in ES expression at both mRNA and protein levels maybe part of the reason for those vascular disease complicated with DM chronically.3. The level of ES protein expression was increased with glucose dose-dependent manner during short period. Nitric oxide/cGMP pathway may play part of role in modulating endothelial ES release.