The Inhibition Effect Of FK506 On Neovascularisation In The Mice Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the most common microvascular damage in diabetes. DR is among the leading causes of blindness in people of working age, affecting both the genders equally. Increasing evidence suggests that the pathogenesis of diabetic retinopathy is mediated by inflammatory processes, including leukocyte adhesion and the pro-inflammatory cytokines are increased in the retina. Using selective pharmacologic inhibitors or genetically modified animals, an increasing number of therapeutic approaches have been identified that significantly inhibit development of at least the early stages of diabetic retinopathy, especially occlusion and degeneration of retinal capillaries. A common feature of a number of these therapies is that they inhibit production of inflammatory mediators.Tacrolimus (FK506) is an immunosuppressive drug, which binds FK506 binding protein 12 (FKBP12) and then inhibits the calcium-dependent phosphatase calcineurin, exhibit decreased regulatory T cells, endothelial dysfunction, and has emerged as a potential drug for several inflammatory diseases. FKBP12 were widely expressed in the retina, mechanisms and whether FK506 play a role in the DR are unknown.The mechanisms of retina neovascularization have been investigated and vascular endothelial growth factor (VEGF) is a key mediators involved in this process. VEGF also contributes to the inflammation-driven angiogenesis. Therefore, the purpose of this study was to examine the effect of FK506 on the proliferation and apoptosis of human umbilical vein endothelial cells (EAhy926) in vitro, and on the expression of VEGF and HIF-1 related with the clinical appearance of DR neovascularization in vivo.Sixteen weeks after induction of diabetes by streptozotocin (STZ), animals were treated with a FK506 (0.3mg/kg) or vehicle (0.25% dimethyl sulfoxide [DMSO] in phosphate-buffered saline [PBS]) daily for 1 week. Eyeballs were taken and stained by Griffonia Simplicifolia lectin (Isolectin B4) labeled FITC to detect the retinal capillary. The experssion of HIF-1 proteion was detected by Western-blot and immunohiatochemistry. The VEGF content of retina was detected by enzyme-linked immunosorbent assay (ELISA).EAhy926 cells and human ARPE-19 (ARPE) cells were treated with or without FK506 under hypoxia and normoxia conditions, respectively. The cell proliferation was determined by use of the CCK8 assay, by utilizing Dojindo's highly water-soluble tetrazolium salt (WST-8 [2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt). Apoptotic cells were assessed by Annexin V-FITC apoptosis kit using FACScan according to the manufacturer's instructions. The percentage of early apoptotic (% Annexin V+/PI-) cells and late-stage apoptotic (% Annexin V+/PI+) cells is presented. The expression of proteins related to the VEGF and apoptosis were detected by Westernblot. The secreted VEGF was detected by enzyme-linked immunosorbent assay (ELISA).FK506 at the concentration of 0.3mg/kg for one week exposure significantly inhibited retina neovascularization in STZ induced diabetes mice, accompanied by a decrease in expression of VEGF and HIF-1 . Furthermore, FK506 treatment increased the number of apoptotic EAhy926 cells under hypoxia condition by inhibition of the expression of VEGF and HIF-1 and activation of Akt. FK506 dose not affect the expression of VEGF and apoptosis of ARPE-19 cells. Therefore, FK506 may be useful as an angiogenic regulator in the treatment of retina diseases that manifest with neovascularization.