title: Calcium entry mediates hyperglycemia-induced apoptosis through Ca(2+)/calmodulin-dependent kinase II in retinal capillary endothelial cells.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, PR China.
authors.forename: Jun
authors.initials: J
authors.lastname: Li
authors.forename: Peipei
authors.initials: P
authors.lastname: Wang
authors.forename: Songping
authors.initials: S
authors.lastname: Yu
authors.forename: Zhi
authors.initials: Z
authors.lastname: Zheng
authors.forename: Xun
authors.initials: X
authors.lastname: Xu
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000873:Anthracenes / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D001896:Boron Compounds / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002829:Choroid / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D015870:Gene Expression / Q000187:drug effects; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D008251:Macaca; D053078:Membrane Potential, Mitochondrial; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D012160:Retina / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism
chemical_list: D000873:Anthracenes; D001896:Boron Compounds; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D047428:Protein Kinase Inhibitors; D019014:fas Receptor; C432165:pyrazolanthrone; D000255:Adenosine Triphosphate; C109986:2-aminoethoxydiphenyl borate; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D048031:JNK Mitogen-Activated Protein Kinases; D005947:Glucose; D002118:Calcium