Regulation of vascular endothelial growth factor and pigment epithelium-derived factor in rat retinal explant under the retinal acidification

Abstract

Background Hypoxia and hyperglycemia are the common causes of retinal neovascularization.In these states,H+ accumulates because of the elevated glycolysis and failure of retinal circulation,thus the retinas readily acidified. Objective The present study was to explore whether retinal acidosis independently regulates the production of vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) and whether the regulation is related to oxidative stress. Methods The retinas from 2-week-old male SD rats were cultured with explant method in DMEM modulated by NaHCO3,and culture retinas were randomly divided into pH 7.2,6.8 and 6.5 groups for 24 hours.In addition,after 24 hours of culture as above described,retinas were washed using PBS two times and then followed by again culture in DMEM with pH 7.2 for another 24 hours.Also,antioxidant was added in different pH values of DMEM for culture as above described.The retinal samples were prepared for histopathological examination.The expressions of VEGF and PEDF proteins and their mRNA in retina tissue were detected by Western blot and fluorescence quantitative polymerase chain reaction (PCR) respectively. Results The retina showed the clear structure and morphology in pH 7.2 group and pH 6.8 group,but retinal vacuoles change was seen in pH 6.5 group after culture for 24 hours.No significant difference was seen in the expressing level of VEGF mRNA in retina between normal group and pH 7.2 group( 112％ ±11％ vs 100％ ±7％ ) (P=0.55),but those in pH 6.8 group and pH 6.5 group were significant increased in comparison with pH 7.2 group( 196％ ±43％ vs 100％ ±7％ ;251％ ±29％vs 100％ ±7％ )( P＜0.05 ).The expressing level of PEDF mRNA in retina in normal group was similar to that of pH7.2 group(86％ ±19％ vs 100％ ±33％) (P=0.64),but that in pH 6.5 group was significantly higher than pH 7.2 group( 230％ ±66％ vs 100％ ±33％ ) ( P＜0.05 ).The resemble results were found in the expressions of VEGF and PEDF protein.After pH reversion,the expressing levels of VEGF mRNA were 100％ ±13％,111％ ±9％,113％ ±9％ in pH 7.2 group,pH 6.8 group and pH 6.5 group respectively without significant difference among them (F=2.51,P=0.16).The expressing levels of PEDF mRNA were 100％ ±13％,110％ ±9％,108％ ±11％ in different groups ( F =0.98,P =0.43 ).Under the presence of antioxidant,the expressing level of VEGF mRNA in pH 6.5 group increased in comparison with pH 7.2 group and pH 6.8 group ( P ＜ 0.05 ).The expressing levels of PEDF mRNA were significant different among pH 7.2 group( 100±31 )％,pH 6.8 group( 282±45 )％ and pH 6.5 group(480±117)％ (F=20.73,P=0.00). Conclusions VEGF can be induced by retinal acidification alone,which may be regulated by oxidative stress.Under the retinal acidification,antioxidants promote the expression of PEDF,suggesting that oxidative stress inhibits the production of PEDF. Key words: Retina; Acidosis; Vascular endothelial growth factor; Pigment epithelium-derived factor; Antioxidant