Calcium mediates high glucose-induced HIF-1α and VEGF expression in cultured rat retinal Muller cells through CaMKII-CREB pathway

Aim: To investigate the effects of high glucose (HG) medium on expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha) and vascular endothelial growth factor (VEGF) in cultured rat retinal Muller cells and to determine the signaling pathways mediating the effects. Methods: Primary cultures of retinal Muller cells were prepared from Sprague-Dawley rats, and incubated in a medium containg HG (30 mmol/L) in the presence of the membrane-permeable Ca2+ chelator BAPTA-AM (10 mu mol/L) or the CaMKII inhibitor KN93 (10 mu mol/L). The levels of CaMKII, p-CaMKII, CREB, p-CREB, HIF-1 alpha, and VEGF proteins were measured with Western blotting, while HIF-1a and VEGF mRNA levels were determined using real-time RT-PCR. Results: The stimulation of retinal Muller cell with HG for 24 h remarkably increased the expression levels of HIF-1 alpha and VEGF. These responses were significantly inhibited in the presence of BAPTA-AM or KN93. Both BAPTA-AM and KN93 also significantly inhibited HG-induced phosphorylation of CaMKII and CREB in the cultured retinal Muller cells. Transfection of the cultured retinal Muller cells with antisense CREB oligonucleotide (300 nmol/L) was similarly effective in blocking the HG-induced increase of HIF-1 alpha and VEGF. Conclusion: HG-induced HIF-1 alpha and VEGF expression in cultured rat retinal Muller cells depends on intracellular free Ca2+ and activation of CaMKII-CREB pathway. The activation of CaMKII-CREB pathway by HG may be a possible mechanism underlying the pathogenesis of diabetic retinopathy.