The Role Of ADMA In Diabetic Retinopathy Blood-retinal Barrier Dysfunction And Its Mechanisms

Background:Diabetic retinopathy (DR), a most common microvascular complication of diabetes, is the main cause of blindness in the working age population. Blood-retinal barrier dysfunction and retinal neovascularization are the main pathological characteristics of DR. Blood-retinal barrier dysfunction is not only the main pathological features of non-proliferative diabetic retinopathy (DR divided into non-proliferative DR and proliferative DR), but also the key step from non-proliferative DR to proliferative DR. Therefore, it is important to reduce or inhibit blood-retinal barrier dysfunction for DR. However, there is still no effective measure to intervene or delay the development of DR blood-retinal barrier dysfunction.Asymmetric dimethylarginine (ADMA), an endogenous nitric oxide synthase inhibitor, is mainly metabolized by dimethylarginine dimethylaminohydrolase (DDAH). Our laboratory and other scholars have proven that elevated ADMA levels are closely associated with endothelial dysfunction and vascular disease in diabetes. However, more researches about the roles of DDAH/ADMA in diabetic vascular complications are mainly focused on peripheral vascular, while diabetic microvascular complications are rarely reported. Clinical researches showed that ADMA concentrations in plasma and aqueous humor in DR patients were significantly increased, which indicated ADMA may play an important role in the development of DR. Gap junction intercellular communication (GJIC) dysfunction is the main mechanism of DR blood-retinal barrier dysfunction. Cx43is the important component of gap junctions. Studies have shown that high glucose could inhibit the expression of Cx43in retinal endothelial cells, which leads to GJIC dysfunction. Moreover, our lab reported that in culutured endothelial cells, exogenous ADMA could directly inhibit expression of Cx43, leading to GJIC dysfunction. These results suggested that ADMA may cause GJIC dysfunction by inhibiting the expression of Cx43in RPCs, which leads to blood-retinal barrier dysfunction in DR.Eph receptor is a newly discovered member of tyrosine kinase receptors family. Researches have shown that EphA2receptor was involved in regulating cell permeability. In cultured human brain microvascular endothelial cells, vascular endothelial growth factor (VEGF) increased cell permeability through upregulating EphA2. Moreover, VEGF is a downstream target of DDAH. These results suggested that the roles of DDAH/ADMA on retinal microvascular barrier may be involved in EphA2This proposal observed the roles of ADMA in retinal microvascular barrier in type I diabetic rat (at different periods), trying to explore the relationship between ADMA and DR blood-retinal barrier dysfunction. We also in RPCs observed the effects of high glucose on blood-retinal barrier, and tested whether endogenous and exogenous ADMA could exert similar effects, and further explored whether ADMA induced RPCs blood-retinal barrier dysfunction was involved in ADMA/Cx43/GJIC and or ADMA/EphA2pathways.Methods:A single intraperitoneal injection (60mg/kg) STZ established type I diabetic rat model. Rats were randomly divided into six groups (two weeks, four weeks or eight weeks group of control, and two weeks, four weeks or eight weeks group of model rats). We tested weight, blood glucose and insulin levels; and measured the blood-retinal barrier permeability by Evans blue assay; and observed the structure of blood-retinal barrier by electron microscopy; and detected the concentrations of ADMA in plasma and aqueous humor by HPLC; and determined the mRNA and protein expression of Cx43and EphA2in retinal tissue by Real-time PCR and Western blotting.RPCs were isolated from rat retinal, and the experiment was divided into three parts:①RPCs were treated with glucose (30mM) for1,3,5,7days;②RPCs were treated with exogenous ADMA (30μM), L-NMMA (30μM) and sodium nitroprusside (0.1mM) for48h;③RPCs were treated with endogenous ADMA (DDAH1siRNA, DDAH2siRNA, DDAH1+2siRNA) for48h. The permeability of RPCs was detected by Transwell assay; the apoptosis of RPCs was determined by Hoechst and Caspase-3activity assay; the function of GJIC was detected by SDLT assay; the concentrations of ADMA were measured by HPLC; the mRNA expression of DDAH, Cx43and EphA2were analyzed by Real-time PCR; the protein expression of Cx43and p-Cx43were detected by Western Blotting and cell immunofluorescence.Results:The blood glucose levels of type I diabetic rats were steadily increased, accompanied by obvious symptoms (polydipsia, polyuria and polyphagia), and the weight, plasma insulin levels of diabetic groups were significantly decreased than that of control groups at different periods, which indicated the success of model. The retinal permeability of diabetic rats was significantly increased as the disease progress, and reached a maximum peak in the eighth week. The ultrastructure of blood-retinal barrier of diabetic rats had changed in initial peroid of diabetes, appeared edema, damaged cell junctions, apoptosis of RPCs and disrupted barrier structure. The levels of ADMA in aqueous humor and plasma were significantly increased, accompanied by decreased Cx43expression and upregulated EphA2mRNA expression in diabetic ratsIn primary RPCs:①High glucose increased RPCs permeability and promoted RPCs apoptosis in a time-dependent manner, accompanied by elevated ADMA, decreased Cx43mRNA and protein expression, and upregulated EphA2mRNA expression;②Exogenous ADMA increased RPCs permeability and promoted RPCs apoptosis in a time and dose-dependent manner, accompanied by elevated ADMA, decreased Cx43mRNA and protein expression, and upregulated EphA2mRNA expression, and L-NMMA also exerted similar effects, and NO donor partly reversed the effects of ADMA;③DDAH siRNA (increased endogenous ADMA levels) were consistent with the effects of exogenous ADMA, and DDAH siRNA enhanced the effects of high glucose on RPCs.Conclusions:1. ADMA is involved in DR blood-retinal barrier dysfunction.2. ADMA is involved in blood-retinal barrier dysfunction of RPCs induced by high glucose via regulation of ADMA/Cx43/GJIC and ADMA/EphA2pathways.