Study On Cysteine-rich 61 (CYR61) In The Pathogenesis Of Diabetic Retinopathy

Objective1. To observe the effect of CYR61 (cysteine-rich 61; CCN1) in the proliferation, migration and tube formation of choroid-retinal endothelial cells (RF/6A cell line);2. To study the expression of CYR61 in retina of diabetic mouse and normal mouse, and their difference of expression;3. To investigate the CYR61 expression level in proliferative diabetic retinopathy (PDR), and the expression and distribution of CYR61 on epiretinal membranes.Methods1. RF/6A cells were cultured and treatment with CYR61 of different concentration. Effect of CYR61 on cell proliferation, migration and angiogenesis were observed by MTT assay, Transwell assay and tube formation assay.2. Diabetes was induced in C57BL/6 mouse by intraperitoneal injection of streptozotocin (STZ). The expression and distribution of CYR61 on retina layers of diabetic mouse were demonstrated by immunohistochemistry. The expression of Cyr61 mRNA in retina was evaluated by reverse transcription-polymerase chain reaction (RT-PCR).3. Vitreous samples and epiretinal membranes were collected during vitrectomy. Vitreous CYR61 levels of PDR and non-diabetic patients were measured by enzyme linked immunosorbent assay (ELISA). Expression and distribution of CYR61 on epiretinal membrane of PDR, proliferative vitreoretinopathy (PVR) and idiopathic epiretinal membrane were evaluated by immunohistochemistry.Results1. When different concentrations of CYR61 (0,5 ng/mL,10 ng/mL,50 ng/mL,100 ng/mL,500ng/mL) were used to treat RF/6A cells for 72h, A490nm value of the MTT assay increased with the increase of CYR61 concentration, and the difference was significant (p=0.000). when 400ng/mL Cry61 was used to treat RF/6A cells for different time (0,12h,24h,48h,72h), A490nm value increased with the extension of treatment time, and the difference was significant (p=0.000). In Transwell assay, RF/6A cell migration capacity increased with increased concentration of CYR61 (p=0.000). In tube formation assay, RF/6A cell tube formation capacity increased with increased concentration of CYR61 (p=0.000). There were equal effects of 400 ng/ml CYR61 and 80ng/ml VEGF. Anti-CYR61 antibody could inhibit cell migration and tube formation promoted by CYR61.2. In normal mouse, CYR61 was expressed in retinal ganglion cell layer, inner plexiform layer, outer plexiform layer, and photoreceptor layer. Among these layers, outer plexiform layer was stained strongly, and other layers were weakly. In diabetic mouse, CYR61 was expressed in retina layers just as normal mouse, but the staining was stronger than normal in ganglion cell layer and inner plexiform layer. There was cloddy positive staining in the cytoplasm of ganglion cells. The Cyr61 mRNA expression in retina of diabetic mouse was more than that in normal mouse, and the difference was significant (p=0.009).3. Vitreous concentration of CYR61 in patients with PDR and non-diabetic patients were 97.53±34.13 (41.21～154.74)ng/mL and 33.92±18.68 (6.24～70.04)ng/mL respectively, and the difference was significant (p=0.000). PDR patients with plenty neovasculature on retina and epiretinal membranes had higher level of vitreous CYR61 than patients with little neovasculature (p=0.001). No relationship was found between vitreous CYR61 and patients'age, disease duration or preoperative fasting blood-glucose. CYR61 expressed in the cytoplasm of epiretinal membranes in PDR, especially in the wall cells of the tube-like structure. There were sporadic positive staining in the epiretinal membranes of PVR and idiopathic epiretinal membranes.ConclusionCYR61 can promote proliferation, migration and tube formation of choroid-retinal endothelial cells in vitro. Expression of Cyr61 mRNA and protein are higher in retina of diabetic mouse than that of normal comparison. Vitreous levels of CYR61 are elevated in patients with PDR when compared with non-diabetic patients. CYR61 expresses in epiretinal membrane of PDR, especially in the wall cells of the tube-like structure. CYR61 are likely to be involved in the pathogenesis of diabetic retinopathy, and play a role in the course of neovasculation.