| Objective Angiogenesis is tightly controlled by two counter-balancing systems: angiogenic stimulators such as vascular endothelial growth factor (VEGF) and angiogenic inhibitors such as pigment epithelium-derived factor (PEDF). Hypoxia induces production of VEGF and inhibits generation of PEDF, and causes neovascularization. After onset of hypoxia, glycolytic flux increases, so do lactate and H+. Impairment of vascular cells in diabetic retinopathy includes apoptosis of pericytes and proliferation of endothelia. Retinal accumulation of lactate and H+ also occurs in the condition of diabetes, when glucose intake and glycolysis are increased. In the study, we investigated the role of acidosis or lactate in the regulation of VEGF and PEDF and its relation to oxidative stress by retinal culture, and explored the role of acidosis in the regulation of growth and death of retinal pericytes and endothelia.Methods 1. Isolated rat retinas were cultured in the mediums of pH 7.2, pH 6.8 and pH 6.5, respectively. Measurement of VEGF and PEDF mRNA and protein was conducted by real-time PCR and Western blot. They also were measured when added with antioxidants to the mediums. 2. Made model of diabetic rats by intraperitoneal injection of alloxan. The concentration of lactate in the diabetic retinas was compared with control. By retinal culture, VEGF and PEDF production in the group with 30 mM or 50 mM of lactate was compared with that with 10 mM of lactate, which served as control. 3. Bovine retinal pericytes and endothelia were cultured. Effects of acidosis on the growth of pericytes and endothelia were observed through MTS, uptake of Brdu, measurement of cell cycles and AnnexinⅤ/PI staining.Results 1. After retinas were cultured for 24 hours, VEGF mRNA levels in the retinas of pH 6.8 and pH 6.5 were significantly higher than those in the retinas of pH 7.2. PEDF mRNA levels in the retinas of pH 6.5 were significantly higher than those in the retinas of pH 7.2. VEGF/PEDF between three groups was not significantly different. When retinas were cultured with antioxidants, the increased VEGF mRNA in the retinas of pH 6.8 was completely inhibited, however, in the retinas of pH 6.5, which was partially inhibited, and PEDF mRNA levels in the retinas of pH 6.5 were still significantly higher than those in the retinas of pH 7.2. Western blot analysis demonstrated VEGF and PEDF protein in the retinas of pH 6.8 and pH 6.5 were higher than those in the retinas of pH 7.2. 2. The concentration of lactate in the diabetic retinas (1.97 ug/mg±0.42 ug/mg)was significantly higher than control (1.27 ug/mg±0.19 ug/mg) . After retinas were cultured for 24 hours, VEGF mRNA levels in the retinas of 30 mM and 50 mM of lactate were significantly higher than those in the retinas of 10 mM of lactate, but VEGF mRNA in the 50 mM of lactate was lessen than that in the 30 mM of lactate. Results showed by Western blot were similar to those by real time PCR. By ELISA, the concentration of VEGF in the mediums of 30 mM (21.33 pg/mg±1.67 pg/mg) and 50 mM (16.93 pg/mg±1.89 pg/mg) of lactate was higher than that of 10 mM (12.0 pg/mg±1.59 pg/mg), and VEGF in the 50 mM of lactate was lessen than that in the 30 mM of lactate. PEDF mRNA was not different between each group. 3. Acidosis significantly inhibited the survival and proliferation of pericytes and endothelia. Apoptosis in the pericytes of pH 6.5(34.97%±10.54%)was increased compared with that of pH 6.8(11.80%±4.50%) and pH 7.2(11.30%±3.04%), however, apoptosis in the endothelia of pH 6.5(9.03%±2.08%)was decreased compared with that of pH 6.8(24.67%±8.02%)and pH 7.2(22.67%±4.04%)Conclusions 1. Similar to hypoxia, acidosis induces production of retinal VEGF, which indicates acidosis play a role in the onset of retinal neovascularization caused by hypoxia and high glucose. Acidosis induces production of PEDF in SD rat retinas, which may account for the lack of retinal neovascularization in the SD rats in the ischemic retinal angiogenesis model. 2. Increased production of retinal lactate caused by hypoxia and diabetes induces VEGF expression, and may help to result in retinal vasculopathy. 3. The fact acidosis induces apoptosis of retinal pericytes and inhibits apoptosis of retinal vascular endothelia reveals retinal acidosis may have a role in the pathogenesis of diabetic retinopathy.
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