P264	Acidosis-induced Ca2+ overload and caspase-12 activation participate in ischemic death of coronary endothelial cells.
1S.Kumar, 2S.Kasseckert, 2Y.Abdallah, 2C.Schafer, 1A.Kaminski, 1G.Steinhoff, 1Y.Ladilov
1Department of Cardiac Surgery, Rostock, DE; 2Gießen, DE & 2Gießen, DE.

Short ischemic insult can jeopardize function of coronary vessels through induction of apoptosis/necrosis of endothelial cells (ECs) leading to endothelial dysfunction. However, the mechanism of the ischemia-induced endothelial injury is still poorly understood. To analyse the pathways of ischemia-induced ECs death the primary culture of rat coronary ECs was used. Cells were exposed to 2 h of simulated ischemia (anoxia, glucose-free, pHo6.4 or 7.4). Apoptosis was detected by caspase-3 / -12 activation (ELISA-Kit, Western blot), DNA-ladder and nuclear condensation (Hoechst-33342), while necrosis by propidium iodide staining. Cytosolic Ca2+ (Ca2+i) and pH (pHi) were analysed with fluorescent dyes Fura-2 and BCECF, respectively. During ischemia at pHo6.4, only a moderate necrosis (10.3±1.4% vs. 2.6±0.8% in control) was detected, while marked apoptosis (24.2±1.8% vs. 3.1±0.3% in control), caspase-3 activation and DNA-fragmentation were observed. Additionally, Ca2+i overload (Fura-2 ratio increased from 1.10±0.02 to 2.81±0.04 a.u.) and acidosis (pHi reduced from 7.16±0.04 to 6.64±0.06) were found at the end of ischemia. Inhibition of ryanodine- and IP3-sensitive channels in endoplasmic reticulum (ER) with 3µM ryanodine and 3µM xestospongin-C completely abolished apoptosis of ECs and prevented Ca2+i overload, indicating a role of ER in ischemia-induced apoptosis. Indeed, a marked increase of the active caspase-12 during ischemia was found, which was prevented by treatment with ryanodine and xestospongin-C. Prevention of cytosolic acidosis by performing ischemia at pHo7.4 significantly reduced apoptosis (11.2±2.3%, p<0,05 vs. ischemia at pHo6.4), abolished activation of caspase-12 and Ca2+i overload. Normoxic incubation of ECs for 2 h at pHo6.4 led to similar apoptosis (23.8±2.6%), Ca2+i overload and acidosis as ischemia at pHo6.4. In conclusion, acidosis leading to Ca2+ release from ER is an important trigger of apoptosis in ECs under ischemia. Ca2+ release induced activation of caspase-12 seems to be a relevant pathway of the ischemia-induced apoptosis.

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