ABSTRACT
Objective To study the role of autophagy in proliferation,migration and tube formation of retinal vascular endothelial cells (RVECs) under the condition of hypoxia in vitro.Methods Mouse RVECs were isolated from 50 C57BL/6J mice primarily cultured using explant culture method,and the cells were identified by immunofluorescence of CD34.Well cultured RVECs were divided into normal control group,hypoxia control group and hypoxia+ 3-methyladenine (3-MA) group.The cells in the normal control group were cultured in the normal environment for 24 hours,and the cells in the hypoxia control group were cultured 1％ O2 environment for 24 hours,and the cells in the hypoxia+3-MA group were pretreated with 5 mmol/L 3-MA for 4 hours and then exposed to 1％ O2 environment for 24 hours.Microtubule-related protein 1 light chain 3 (LC3-Ⅱ/LC3-Ⅰ) and Beclin-1 protein contents in the cells were detected by Western blot analysis;the ultrastructure of autophagosome was examined under the transmission electron microscope.The proliferation,migration and tube formation of the cells were detected by Click-iTEdU kit,scratch assay and matrigel,respectively.Results Primarily culture cells grew well with the cobblestone-like appearance 5-7 days after culture and showed positive response for CD34.The autophagosome number was increased in the hypoxia control group compared with the hypoxia+3-MA group and normal control group.The LC3-Ⅱ/LC3-Ⅰ ratio was 0.243 ± 0.030,0.658 ± 0.032 and 0.405 ± 0.095;the relative expression of Beclin-1 protein in the cells was 0.260±0.040,0.650±0.071 and 0.461±0.089;the proliferation rate of the cells was (45.93± 6.39) ％,(22.74± 2.35) ％ and (24.12 ± 3.59) ％;the scratch healing rate of the cells was (36.02 ± 5.84) ％,(57.26±11.98) ％ and (18.16±9.73) ％;the number of tube formation was 29.20±6.10,41.40±4.04 and 22.00± 2.92 in the normal control group,hypoxia control group and hypoxia + 3-MA group,respectively,with significant differences among the groups (F =35.86,23.53,34.28,21.12,23.27;all at P＜0.01).Compared with the normal control group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein,scratch healing rate and the number of tube formation were significantly increased,and the proliferation rate was significantly reduced in the hypoxia control group (all at P ＜ 0.05).Compared with the hypoxia control group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression of Beclin-1 protein,scratch healing rate and the number of tube formation were significantly decreased in the hypoxia + 3-MA group.Conclusions Hypoxia environment activates autophagy of mouse RVECs,which enhances the migration and tube formation abilities of the cells.3-MA inhibits the angiogenesis abilities of mouse RVECs.
ABSTRACT
Objective To investigate the effects of adiponectin (APN) on proliferation,migration and tube formation of RF/6A cells,and explore the effects of APN on choroidal and retinal angiogenesis.Methods Well cultured RF/6A cells were randomly divided into the control group and three groups with different concentrations of recombinant adiponectin (5 pg · mL-1,50 pg · mL-1,500 pg · mL-1) for 1 hour.After 24 hours,cell proliferation,migration and tube formation were detected by MTT assay,wound scratch assay and seeding cells in matrigel,respectively.Results The cell proliferation in all APN groups was weaker than that of control group (P ＜ 0.05),the cell migration area (pixels) of all APN groups was significantly smaller than that of the control group (P ＜ 0.05),and the number of tube formation of all APN groups was significantly less than the control group (P ＜ 0.05).Cell proliferation,migration and tube formation decreased along with the increase of APN concentration.Conclusion APN can obviously inhibit the angiogenesis process of RF/6A cells.This inhibitive effect indicates the protective role of APN in choroidal and retinal angiogenesis.
ABSTRACT
Metformin is a kind of biguanide hypoglycemic agent that has been widely used in patients with diabetes mellitus. In clinical practice, whether metformin should be stopped before Fundus fluorescein angiography (FFA) remains largely unclear. Some endocrinologists suggest stop metformin before FFA. However, ophthalmologists do not always adopt this opinion in their practice. This situation may lead to disputes between physicians and patients. This article analyzed contrast-induced nephropathy(CIN) and the related contrast agent, as well as the adverse reactions of fluorescein angiography. It pointed out that the discrepancy may be caused by misunderstanding of contrast agents used in FFA. For angiography using iodine contrast agent, metformin must be stopped because of the increased possibility of CIN, while for FFA using fluorescein sodium, no CIN has been reported yet. Therefore, the authors believe FFA is safe for diabetic patients with oral metformin and it is unnecessary to stop metformin before the examination.