Effect And Potential Mechanism Of Hyperglucemia On Choroidal Neovascularization

BACKGROUND Choroidal neovascularization (CNV) is a commonpathological changes of a variety of blinding diseases in which also is one of themain features for age-related macular degeneration (AMD). CNV formation,accompanied by bleeding, exudation and scarring changes on macular usuallypresents in advanced AMD. The disease often results in damage on visionimpairment among people65years of age or older. With the acceleration on theprocess of aging global population, the incidence of AMD is rising, but itspathogenesis is not yet clarified. There are multi-factors in AMD pathogeniccharacteristics, thus studies concerning on the risk factors of AMD, andparticularly CNV to identify susceptible populations and take effective diseaseprevention and treatment measures great significance.Age, genetics, cardiovascular disease and smoking has been proven to bemajor risk factors for AMD incidence. Diabetes, an predisposing factoridentified for cardiovascular disease, has begun to attract scholars concerned about association with CNV related disease progression. But speculation ofdiabetes and CNV are more concentrated in clinical epidemiological studies, andis still lacking of system basic experimental studies in depth, the current resultsare more controversial. After reviewing on the literature, we found that oxidativestress is the basic pathological mechanism of diabetes and related vascularcomplications. Due to the characteristics about the high oxygen consumption,long-term light and multi-unsaturated fatty acid enrichment of macular,oxidative damage tends to occur under pathological conditions,meanwhile,oxidative stress has also been proven to be one of the importantinitiate factors on CNV formation. Signal transduction and activator oftranscription factor3(STAT3) is a key transcription factor integrating a varietyof cell signaling pathways. Through interaction with vascular endothelial growthfactor (VEGF) promoter, STAT3directly regulates VEGF expression. STAT3activation has been proved to play an important role in pathologicalangiogenesis. Based on the correlation of diabetes with CNV status and sharedpathological basis between the two, we speculated that diabetes may have acertain impact on the occurrence of CNV-related disease. But what may involvein the molecular mechanism is still unclear. Exploring possible risk factors andinvolved molecular signaling mechanisms for CNV related diseases, thenfinding for key therapeutic targets will help us better understanding on thepathogenesis of CNV, and also providing new ideas for the control andprevention on development of CNV-related diseases.PURPOSE (1) To analysize the effect of diabetes on CNV severity inlaser-induced mice and detect changes of oxidative stress, phosphorylation ofSTAT3(p-STAT3), and VEGF expression in early stage on CNV formation;(2)To observe the effect ofanti-oxidant N-acetylcysteine (NAC) treatment on CNV in diabetic mice;(3) To study the changes and interactive relationship onthe oxidative stress and STAT3signal transduction pathway in RPE cells underhigh glucose culture conditions and explore the possible molecular mechanismof the impact on angiogenic factors secretion in high-glucose treated RPE cells.METHODS (1) Intraperitoneal injection of streptozotocin (STZ) to inducediabetic model in C57BL/6J mice,2weeks after modeling success,532nm laserphotocoagulation on mice to induce CNV, and then treat mice withintraperitoneal injection of NAC. Fundus fluorescein angiography (FFA)14days after photocoagulation, choroidal flatmout and HE staining were used todetect the severity of each group on day14after photocoagulation;(2)Observing the local expression of oxidative DNA damage marker8-hydroxy-deoxy guanosine (8-OHdG)，p-STAT3and VEGF in CNV in eachgroup on day3after photocoagulation by laser scanning confocal fluorescencemicroscopy, and detect VEGF level changes in mice ocular by ELISA assay;(3) RPE cells culture under high-glucose conditions were treated with NAC andJAK2kinase inhibitor AG490in vitro, RT-PCR and ELISA were processed todetect VEGF expression,2’,7’-dichlorofluorescein diacetate (DCFH-DA)method were used to detect reactive oxygen species (ROS) levels and westernblot to detection p-STAT3protein expression in RPE cells.RESULTS (1)14days after photocoagulation，the leakage of CNVincreased in diabetic mice than in control group，CNV area and thickness alsoenlarged, Antioxidant NAC treatment reduce the severity of CNV in diabeticmice;(2)3days after photocoagulation,8-OHdG, VEGF and p-STAT3highlyexpressed in CNV regions in diabetic mice, secretion of ocular VEGF levels indiabetic mice were significantly higher, NAC partially inhibitedoverexpression of the above factors;(3) VEGF mRNA and protein content increased, ROS levels rised and p-STAT3highly expressed in RPE cells underhigh glucose conditions, NAC reduced ROS levels and inhibited p-STAT3andVEGF overexpression, JAK2/STAT3pathway inhibitor AG490significantlyreduced the expression of VEGF, but have no significant effect on ROS levels.CONCLUSIONS (1) Hyperglycemia severed CNV formation in diabeticmice. This process was accompanied by elevated levels of oxidative stress,p-STAT3and VEGF expression, which maybe one of the reasons promotingCNV formation;(2) Antioxidants had a certain therapeutic effect on theformation of CNV by lightening oxidative damage and downregulatingangiogenic related signal pathway and cytokine expression in diabetic mice;(3)Hyperglycemia increased oxidative stress of RPE cells, and then activate theSTAT3signaling pathway to increase the expression of VEGF may be one of themolecular mechanism on CNV affected by diabetes.