title: Ischemic acidosis causes apoptosis in coronary endothelial cells through activation of caspase-12.
abstract: OBJECTIVE
  Myocardial ischemia has been shown to induce apoptosis of endothelial cells (EC). However, the mechanism of this endothelial injury is still poorly understood. To analyse the signaling pathway of ischemia-induced EC apoptosis was the aim of the present study.
  
  
  METHODS
  The primary culture of rat coronary EC was exposed to simulated ischemia (glucose-free anoxia at pH(o) 6.4). Apoptosis was defined by staining of nuclei with Hoechst-33342 and TUNEL. Cytosolic Ca2+ and pH were measured with Fura-2 and BCECF, respectively.
  
  
  RESULTS
  Apoptosis (29.2+/-1.7% of cells) induced by exposure to simulated ischemia for 2 h was accompanied by cytosolic Ca2+ overload (1090+/-52 nmol/l) and acidosis (pHi = 6.52+/-0.13). Simulated ischemia had no significant effect on caspase-8 cleavage, but induced cleavage of caspase-3 and caspase-12 and led to a slight release of cytochrome C. Prevention of cytosolic acidosis (anoxia at pH(o) 7.4) had no effect on cytochrome C release, but significantly reduced apoptosis, attenuated cytosolic Ca2+ overload, and prevented cleavage of caspase-12. A similar effect was achieved by inhibition of Ca2+ release channels in the endoplasmic reticulum with ryanodine and xestospongin C. Knock-down of caspase-12 with small interfering RNA suppressed caspase-3 activation and reduced apoptotic cell number by about 70%.
  
  
  CONCLUSIONS
  Acidosis, rather than anoxia, is an important trigger of apoptosis in EC under simulated ischemia. The main pathway of the simulated ischemia-induced apoptosis consists of the Ca2+ leak from the ER followed by activation of caspase-12 and caspase-3.
authors.affiliation: Department of Clinical Pharmacology, Ruhr-Universität Bochum, Germany.
authors.forename: Sanjeev
authors.initials: S
authors.lastname: Kumar
authors.forename: Sascha
authors.initials: S
authors.lastname: Kasseckert
authors.forename: Sawa
authors.initials: S
authors.lastname: Kostin
authors.forename: Yaser
authors.initials: Y
authors.lastname: Abdallah
authors.forename: Claudia
authors.initials: C
authors.lastname: Schafer
authors.forename: Alexander
authors.initials: A
authors.lastname: Kaminski
authors.forename: H Peter
authors.initials: HP
authors.lastname: Reusch
authors.forename: Hans Michael
authors.initials: HM
authors.lastname: Piper
authors.forename: Gustav
authors.initials: G
authors.lastname: Steinhoff
authors.forename: Yury
authors.initials: Y
authors.lastname: Ladilov
mesh_terms: D000138:Acidosis / Q000201:enzymology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western / Q000379:methods; D002118:Calcium / Q000032:analysis / Q000378:metabolism; D053454:Caspase 12 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D020169:Caspases / Q000032:analysis / Q000378:metabolism; D002478:Cells, Cultured; D003331:Coronary Vessels; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000378:metabolism; D042783:Endothelial Cells / Q000201:enzymology* / Q000473:pathology*; D004789:Enzyme Activation; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D008929:Mitochondria, Heart / Q000378:metabolism; D017202:Myocardial Ischemia / Q000201:enzymology* / Q000473:pathology*; D034622:RNA Interference; D051381:Rats; D017208:Rats, Wistar
chemical_list: D045304:Cytochromes c; D053454:Caspase 12; D020169:Caspases; D002118:Calcium