Abstract 3675: Extracellular ATP Protects Human Endothelial Cells From Apoptotic Death During Ischemia by Activating a P2Y2-receptor Mediated MEK/ERK- and PI3K/Akt-signaling
Background: Apoptotic death of endothelial cells (EC) plays a crucial role in the development and progression of cardiovascular disease. Recently it was shown that endothelial apoptosis initiated during ischemia precedes apoptotic death of cardiomyocytes during reperfusion. In the present study we hypothesized that extracellular ATP either released from cells or exogenously applied protects EC from apoptosis during ischemia by activating a P2-receptor mediated signaling.
Methods and Results: Cultured human EC were exposed to 2 hours of hypoxia (Po2<4mmHg) in serum free medium to simulate ischemic conditions (SI). SI led to a significant increase of caspase 3-cleavage (western blot and immunocytochemistry) and apoptotic cell death (annexin V staining) compared to normoxic controls (n=3; P<0.05 for all further parameters). During SI a 2.3±0.64-fold increase of ATP concentration in the supernatant was observed (luciferin-luciferase assay). The addition of the ectonucleotidase-inhibitor ARL 67156 (100μM) during SI reduced caspase 3-cleavage by 35±12%. The protective effect of ARL 67156 was abrogated when the P2-receptor blockers suramin (100μM) or PPADS (5mM) were present. Exogenous addition of the panspecific P2-receptor agonist ATP (100μM) or ATPgammaS (100μM) as well as the addition of the P2Y2-agonist UTP (100μM) showed the same protective effect than ARL 67156. However, the addition of the P2Y1-agonist MRS2365 (100nM) or the P2X-agonist slphabeta-ATP (1mM) failed to reduce caspase 3-cleavage. Activation of P2Y2-receptors by ATP/UTP (100μM each) during SI led to a significant increase of ERK1/2- and Akt phosphorylation, while there was no change in c-Jun phosphorylation. Accordingly the inhibition of MEK/ERK by UO126 (10μM) and PI3K/Akt by LY294002 (10μM) abolished the anti-apoptotic effect of ATP/UTP, while the inhibition of JNK/c-Jun by SP600125 (10μM) had no effect.
Conclusions: The data of the present study show a novel anti-apoptotic mechanism in human endothelial cells that is due to a P2Y2-receptor mediated activation of MEK/ERK- and PI3K/Akt-signaling pathway. Targeted activation of this protective mechanism could represent a new therapeutic strategy in the treatment of endothelial apoptosis during ischemia/reperfusion.