Purpose To investigate the status of proliferation and activation of vascular endothelial cells in preretinal neovascular membranes from patients with insulin dependent diabetetes mellitus(IDDM)by means of immunohistochemical techniques. Methods Status of vascular endothelial cells in 18 preretinal neovascular membranes from 18 patients with IDDM was studied by double-immunofluorescence technique and the alkaline phosphataes-anti-alkaline phosphatase(APAAP)technique and compared the findings with the main clinical features of the patients. Results Of 18 vascularized membranes,16(88.9%)contained proliferating endothelial cells (positive for proliferating vascular endothelial cell marker EN 7/44) and 14 (77.8%) were positive for endothelial cell activation marker anti-VCAM-1;furthermore,by using a double staining technique,we found that in 14 of the 16 cases(87.5%) the proliferating vascular endothelial cells were activated (expressing VCAM-1). Conclusion The proliferation and activation of the vascular endothelial cells of the newly formed vessels in preretinal neovascular membranes suggests the significance of the vascular endothelial cells in the pathophysiology and the progress of proliferative diabetic retinopathy. (Chin J Ocul Fundus Dis,1998,14:141-143)
Objective To study the effect of down-regulation of Claudin-3 mediated by adeno-associated virus (AAV) of shRNA on the cultured retinal ganglion cells (RGCs) in vitro. Methods RGCs isolated from mouse eyes were divided into normal control group, AAV-shScramble group, and AAV-shClaudin-3 group. The RGCs in AAV-shScramble group and AAV-shClaudin3 group were treated with AAV-shScramble and AAV-shClaudin-3 respectively 24 hours after cell seeding. Dynamic live cell fluorescence microscopy was used to observe the transfection efficiency 96 hours after transfection. Immunofluorescent staining of β-tubulin was used to measure the length of RGCs′ axon. 4′, 6-diamidino-2-phenylindole staining was used to observe the nuclei of apoptotic cells. The mRNA level of Claudin-3 and VEGF was measured by real-time polymerase chain reaction. The protein levels of Claudin-3, vascular endothelial growth factor (VEGF), Bcl-2 and Caspase-3 was determined by Western blot. Results The positive transfection rate was more than 50% in both AAV-shScramble group and AAV-shClaudin-3 group. The length of RGCs' axon in AAV-shClaudin-3 group was shorter than that in normal control group and AAV-shScramble group (F=22 363.274,P＜0.05). Down-regulation of Claudin-3 accelerated RGCs' apoptosis with nuclei shrinkage, tapering, and nucleolus formation of apoptotic bodies. The mRNA levels of Claudin-3 and VEGF in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=257.408, 160.533;P＜0.05). The protein levels of Claudin-3, VEGF and Bcl-2 in AAV-shClaudin-3 group were lower than those in normal control group and AAV-shScramble group (F=129.671, 420.552, 62.669;P＜0.05), while the protein level of Caspase-3 in AAV-shClaudin-3 group was higher than that in normal control group and AAV-shScramble group (F=231.348,P＜0.05). Conclusion Down-regulation of Claudin-3 increases the expression of Caspase-3, reduces the expression of VEGF and Bcl-2, accelerates RGCs' apoptosis and inhibit the RGCs' axon growth.