Acidic pre-conditioning suppresses apoptosis and increases expression of Bcl-xL in coronary endothelial cells under simulated ischaemia

Abstract

Ischaemic pre-conditioning has a powerful protective potential against ischaemia-induced cell death, and acidosis is an important feature of ischaemia and can lead to apoptosis. Here we tested whether pre-conditioning with acidosis, that is, acidic pre-conditioning (APC), may protect coronary endothelial cells (EC) against apoptosis induced by simulated ischaemia. For pre-conditioning, EC were exposed fo 40 min. to acidosis (pH 6.4) followed by a 14-hrs recovery period (pH 7.4) and finally treated for 2 hrs with simulated ischaemia (glucose-free anoxia at pH 6.4). Cells undergoing apoptosis were visualized by chromatin staining or by determination of caspase-3 activity Simulated ischaemia in untreated EC increased caspase-3 activity and the number of apoptotic cell (31.3 +/- 1.3%versus 3.9 +/- 0.6% in control). APC significantly reduced the rate of apoptosis (14.2 +/- 1.3%) and caspase-3 activity. Western blot analysis exploring the under lying mechanism leading to this protection revealed suppression of the endoplasmic reticulum- (reduced cleavage of caspase-12) and mitochondria-mediated (reduced cytochrome C release) pathways of apoptosis. These effects were associated with an over-expression of the anti-apoptotic protein Bcl-xL 14 hrs after APC, whereas no effect on the expression of Bcl-2, Bax, Bak, procaspase-12, reticulum-localized chaperones (GRP78, calreticulin), HSP70, HSP32 and HSP27 could be detected. Knock-down of Bcl-xL by siRNA-treatment prevented the protective effect of APC. In conclusion, short acidic pre-treatment can protect EC against ischaemic apoptosis. The mechanism of this protection consists of suppression of the endoplasmic reticulum- and mitochondria-mediated pathways. Over-expression of the anti apoptotic protein Bcl-xL is responsible for the increased resistance to apoptosis during ischaemic insult.

1

Effect of acidic pre-conditioning (APC) on caspase-3 activity and the number of apoptotic EC after 2 hrs of simulated ischaemia (SI). (A) Effect of acidic pre-treatment duration: 20–50 corresponds to pre-treatment with acidosis (pH 6.4) for 20–50 min. followed by 14 hrs of recovery period (pH 7.4) and 2 hrs of simulated ischaemia. Caspase-3 activity: *P= 0.001 versus SI; #P= 0.022 versus 20. Apoptotic cells: *P= 0.002 for 30 versus SI, P < 0.001 for 40 versus SI and P= 0.011 for 50 versus SI; §P < 0.001 for 40 versus 20 and P= 0.028 for 40 versus 30. (B) Effect of recovery period: 6–24 corresponds to incubation time for 6–24 hrs after 40-min. pre-treatment with acidosis followed by 2 hrs of simulated ischaemia. Caspase-3 activity: *P= 0.006 versus SI, Apoptotic cells: *P < 0.001 versus SI; #P= 0.002 versus 6. (C) Effect of acidic pre-treatment on caspase-3 activity and the number of apoptotic and necrotic EC during recovery period: 0–14 corresponds to incubation time for 0–14 hrs after 40-min. treatment with acidosis without following simulated ischaemia. Values are mean ± S.E.M., n= 6–11.

2

Western blot analysis of cytosolic cytochrome C (A) and cleaved caspase-12 (B) prepared from extracts of control EC (Con) or EC exposed to simulated ischaemia (SI) without or after 40-min. acidic pre-treatment followed by a 14-hrs recovery period (APC + SI). Data are representative of four independent experiments with similar results.

3

Western blot analysis of procaspase-12, GRP78 and calreticulin prepared from extracts of control EC (Con) or EC after 40-min. acidic pre-treatment followed by a 14-hrs recovery period (APC). Data are representative of three to four independent experiments with similar results.

4

Western blot analysis of HSP27, HSP32 and HSP70 prepared from extracts of control EC (Con) or EC after 40-min. acidic pre-treatment followed by a 14-hrs recovery period (APC). Data are representative of four independent experiments with similar results.

5

Western blot analysis of Bax, Bak, Bcl-2 and Bcl-xL prepared from extracts of control EC (Con) or EC after 40-min. acidic pre-treatment followed by a 14-hrs recovery period (APC). Data are representative of three to four independent experiments with similar results.

6

(A) Western blot analysis of Bcl-xL prepared from extracts of control EC (Con) or EC after treatment with Bcl-xL targeting siRNA (siRNA) for 24, 48 and 72 hrs. Data are representative of three independent experiments with similar results. (B) Effect of pre-treatment for 72 hrs with non-targeting siRNA (nRNA) or targeting siRNA (siRNA) and acidic pre-conditioning (APC) on caspase-3 activity and the number of apoptotic EC after 2 hrs of simulated ischaemia (SI). *P < 0.05 versus SI + nRNA. Values are mean ± S.E.M., n= 4.