Calcium entry mediates hyperglycemia-induced apoptosis through Ca(2+)/calmodulin-dependent kinase ll in retinal capillary endothelial cells

PURPOSE: Hyperglycemia-induced vascular cell apoptosis is a seminal early event in diabetic retinopathy. Prolonged hyperglycemia is known to increase intracellular cytosolic free calcium ([Ca(2+)]i) in retinal vascular endothelial cells (RECs), suggesting that [Ca(2+)]i is a critical trigger for microvascular degeneration. This study aims to elucidate Ca(2+)-dependent signaling mechanisms that mediate hyperglycemia-induced apoptosis in RECs. METHODS: A cultured macaque choroid-retinal endothelial cell line (RF/6A) was incubated in normal glucose (NG), NG plus the Ca(2+) entry blocker 2-aminoethoxydiphenyl borate (2-APB), high glucose (HG), or HG plus either 2-APB, the c-jun N-terminal kinase (JNK) inhibitor SP600125, or the calcium/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93. Changes in [Ca(2+)]i evoked by adenosine 5′-triphosphate (ATP) were measured in fluo-3/AM-loaded RF/6A cells by confocal microscopy. The mitochondrial membrane potential (ΔΨm) and apoptosis were assessed by flow cytometry. Expression levels of CaMKII, phosphorylated CaMKII (p-CaMKII), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK), the death receptor (Fas), and cytochrome c were detected by western blotting analysis. RESULTS: Prolonged exposure to HG (96 h) potentiated ATP-evoked Ca(2+) entry as well as CaMKII phosphorylation and RF/6A cell apoptosis. Enhanced apoptosis was blocked by 2-APB and KN93. Furthermore, HG increased JNK phosphorylation and Fas expression, and both responses were partially blocked by 2-APB and KN93, while the JNK inhibitor SP600125 partially reduced HG-induced Fas expression. In addition, HG depolarized the ΔΨm and triggered the release of mitochondrial cytochrome c. These early signs of mitochondria-dependent apoptosis were partially reversed by 2-APB and KN93. CONCLUSIONS: HG-induced apoptosis in RF/6A cells depends on Ca(2+) entry and CaMKII activation, leading to the activation of both Fas-dependent and mitochondria-dependent apoptosis pathways. The CaMKII−JNK−Fas pathway is involved in HG-evoked apoptosis of RECs.