Effect On Diabetic Retinopathy By Blocking The Action Of Vascular Endothelial Growth Factor

The number of patients suffering from diabetes mellitus is constantly rising worldwide, and diabetic retinopathy (DR) has become the most frequent cause of postnatal blindness. Compared with the general population, diabetes sufferers have an 25-fold increase in the risk of blindness. However, more than 90% of diabetes sufferers eventually develop retinopathy, with 60% of type 1 and 20% of type 2 diabetes sufferers developing proliferative retinopathy. Considering the large number of people suffering from diabetes, there is an urgent need to develop new therapies for diabetic retinopathy, as the present treatments, photocoagulation and vitrectomy , are invasive and provide only temporary protection.Numerous studies have showed that Vascular Endothelial Growth Factor (VEGF) is known to play a central role during DR development. Thus, inhibiting the effects of VEGF may hamper the disease progression,and EPA,which is a sealant of VEGF receptor-flk-1, is an attractive approach for this purpose. And also, the lack of suitable animal models of PDR hindered the evaluation of this strategy. Thus , in order to solve the problems above, we established an ideal model for human PDR, And we also observed the therapeutic effect of EPA on inhibiting the progression of PDR. Thus, we designed five sections to enforce our experiment:Part 1:To ascertain the present suppressing effect of EPA on cultured Human Umbilical Vascular Endothelial Cells in vitro and determine its value in clinical application in the future. Furthermore , to approach the mechanism of its suppressing effect on proliferation.Part 2:To estabalish ideal animal model of human PDR. To make sure whether it is successful or not , Retinal changes were monitored by fluorescein fundus angiography , CD105 positive endothelial cells were detected using immunohistochemistry by light microscope , retinal ultramicrostructure lesion was observed by transmitting electronic microscope.Part 3:According to different stage of DR, EPA were injected intravitreously. To make sure the therapeutic effect of EPA , retinal changes were monitored by fluorescein fundus angiography , CD105 positive endothelial cells were detected using immunohistochemistry by light microscope , retinal ultramicrostructure lesion was observed by transmitting electronic microscope.Part 4:To demonstrate the application value of the animal model on molecular and protein level.Part 5:To demonstrate the therapeutic action of EPA application in vivo on molecular and protein level.Part 1:The inhibiting effect of EPA on the proliferation of human vascular endothelial cellsObject To investigate inhibiting effect and the mechanism of EPA on the proliferation of Human Umbilical Vascular Endothelial Cells(HUVEC). Methods Different concentrations of EPA were added to the cultured HUVEC in vitro. The time cause and does response for the inhibition the cells proliferation in all groups were measured by the MTT assay. Light absorption values and cytostasis ratios in all groups were compared. One-way ANOVA in the SPSS13.0 version statistical software was used. The effect of EPA on cell cycle , proliferative index(PI) and apoptosis of HUVEC in vitro were observed by flow cytometry.χ2-test of R×C contingency table was used as a method for statistical analysis. Results When the concentration of EPA was equal to or more than 0.15 g/L, MTT assay showed a significant difference of light absorption value in the cultured cell after EPA exposure compared with control, the suppressing effects enhanced as the treatment time increased. The peak time of the inhibition of the cell proliferation induced by EPA was at 60 hours and the effect was last until 72 hours. The proliferative index in the treatment group was 23.9%, which was lower than that in the control group(26.9%). No apoptosis was found in the cell in each group. Conclusions EPA plays an important role of inhibition of proliferation of cultured HUVEC in vitro. No apoptosis was induced by the exposure HUVEC to EPA, therefore, it suggests a potential application for clinical trial.Part 2: Establishment of PDR animal model—An approach for ideal PDR animal modelObject To establish ideal animal model imitating human PDR. Methods VEGF was injected intravitreously in Diabetic Sprague-Dawley rats at 1 month after receiving intraperitoneal injection of 60mg/kg body weight of Streptozotocin. At 2, 4, 8 weeks after VEGF injection , fluorescence fundus angiographic changes were observed, CD105 positive retinal vascular endothelial cells were observed by microscope, and retinal ultramicrostructural damages were observed by transmission electron microscope. Results were compared with rats those only received intraperitoneal injection of 60mg/kg body weight of Streptozotocin. Results Fluorescence fundus angiography performed on VEGF-injected eyes demonstrated vascular leakages or hemorrhage or intraretinal new vessels that imitating human diabetic retinopathy by fluorescence fundus angiography, appearing at 2–4 weeks postinjection. From 2 weeks postinjection , CD105 positive retinal vascular endothelial cells were observed by microscope, which coincides with intraretinal new vessels by fluorescence fundus angiography at the same time. Macular edema and exudation were also observed at 8 weeks postinjection, which coincides with Lebherz's findings. Transmission electronic microscope observation demonstrated that Ultramicrostructural damages in each retinal tissue layer of VEGF-injected eyes were more severe than that of single STZ-induced diabetic rats significantly. Conclusion This animal model with VEGF injection intravitreously after STZ-induced diabetes can not only develop PDR ,but also is short-cycle and more economic. Therefore, it provides an ideal animal model for studies on medication screen clinically.Part 3: Research on treatment of PDR by EPA intravitreouslyObject To identify the utility of EPA on DR in vivo. Methods VEGF was injected intravitreously in Diabetic Sprague-Dawley rats at 1 month after receiving intraperitoneal injection of 60mg/kg body weight of Streptozotocin. Simultaneously, at 2, 4 weeks after VEGF injection ,EPA was injected intravitreously as D,E,F group which represent treating group in background ,preproliferation stage and proliferation stage of DR .Fluorescence fundus angiographic changes were observed, CD105 positive retinal vascular endothelial cells were observed by microscope, and retinal ultramicrostructural damages were observed by transmission electron microscope. Results were compared with diabetic rat models established in part 2. Results Fluorescence fundus angiography demonstrated the best effect in preproliferation stage eyes at 2 weeks after EPA injection, whereas effective and augmented time-dependently in backgroud stage eyes by EPA during 8 weeks after EPA injection. At 6 weeks after EPA injection in preproliferation stage eyes, effect decreased obviously as compared with that at 2,4,8 weeks after EPA injection in background stage eyes. As compared with non-medication group, retinal neovascularization was not detected at the same course of this disease . Retinal neovascularization was also detected and diabetic retinopathy was not relieved in EPA injected eyes at proliferation stage.CD105 immunohistochemistry described that there was no CD105 positive vascular endothelial cells at the same course of disease when treated in background and preproliferation stage .But CD105 positive cells were still observed when treated during proliferation stage . Transmission electronic microscope observation demonstrated that the damages of retinal neurocytes relieved at 6 weeks after EPA injected in preproliferation stage eyes and 4 weeks after EPA injected in background stage eyes which coincides with SanGiovanni JP's research result. Conclusion EPA can not only suppress development of DR by blocking flk-1, but also protect retinal neurocyte which mechanism is still waiting for approach.Part 4:The variation of VEGF,flk-1,bax,bcl-2expression in different DR modelObject To observe The variation of VEGF,flk-1,bax,bcl-2 expression in our established PDR model and definite its superiority.Methods Using factorial design empirical method,we observed the variation of VEGF,flk-1,bax,bcl-2 expression in retinas of diabetic rats established in part 2 by immunohistochemistry and measured the average optical density value of VEGF,flk-1,bax,bcl-2 in retinal ganglion cell layer and inner plexiform layer by MIAS1998 type image analysis system. Results The expression of VEGF and its specific receptor- flk-1 in retinas of PDR models established by symphysial dose regimen increased gradually as DR progressed , this coincides with proceeding researches and our early study. The expression of bax in retinas of this model increased gradually as DR progressed , this coincides with studies of DR model established by single dose regimen.As DR progressing ,bcl-2 expression increased in entoretina of diabetic rats,this is perhaps one of the compensatory conservation mechanisms to retinal cellular damage. Conclusion PDR models established by symphysial dose regimen can better imitate human PDR than DR models established by single dose regimen at molecular and protein level .Part 5:Variation of VEGF,flk-1,bax,bcl-2 in retinas of diabetic rats after treatment by EPAObject To identify the effect of EPA on expression of VEGF,flk-1,bax,bcl-2 in retinas of diabetic rats and elucidate the mechanism of EPA on treating diabetic retinopathy. Method Using complete random design empirical method,we observed the variation of VEGF,flk-1,bax,bcl-2 expression in retinas of diabetic rats established in part 2 and those treated by EPA in Part 3 by immunohistochemistry and measured the average optical density value of VEGF,flk-1,bax,bcl-2 in retinal ganglion cell layer and inner plexiform layer by MIAS1998 type image analysis system. Result 1,VEGF expression was weakened in inner retina layer at 4 weeks after EPA injection during background stage and at 2 weeks after EPA injection during preproliferation stage, thus it can arrest the development of PDR. But it increased when EPA was injected during proliferation stage, this demonstrates that it is ineffective as given EPA during proliferation stage.2,The expression of flk-1 was suppressed effectively as given EPA during background and preproliferation stage, thus the corporation of VEGF and its effective rceptor flk-1 was suppressed to produce a marked effect.3,EPA can weaken expression of bax in retinas and then inhibit advancement of DR during 8 weeks after injection intravitreously. Therapeutic efficacy was better as given during background and preproliferation stage than during proliferation stage, but the specific mechanism is still to be investigated.4,EPA can weaken the higher expression of bcl-2 in retinas by inhibiting advancement of DR, as time went to 8 weeks, the weakening effect disappeared and bcl-2 continued to protect the nerve cells. Conclusion EPA can inhibit advancement of DR not only by suppressing VEGF effect through blocking flk-1 receptor as given during background and preproliferation stages in vivo ,but also by inhibiting apoptosis of retinal nerve cells through downregulating apoptosis trigger gene bax expression. At early stage, it can lessen compensatory higher bcl-2 exprssion. At 8 weeks, this effect disappeared and bax/ bcl-2 ratio decreased,which had the protection to pericyte deficiency.