Disruption to the vascular homoeostasis is detrimental in vascular diseases. This study examined how the combination of homocysteine, adenosine and tumor necrosis factor-alpha (TNF-α) influenced endothelial cell survival. In cultured human-derived cardiovascular (EA.hy926) and cerebrovascular (HBEC-5i) endothelial cells, cell death events were initiated by TNF-α (0.1-10 ng/mL) only when both homocysteine (0.5 mM) and adenosine (0.5 mM) were present. The accelerated cell death events induced by

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... he combination were triggered through excessive apoptosis. This was evident by membrane phospholipid phosphatidylserine externalisation, cell shrinkage and DNA fragmentation, as well as an increase in the expressions and occurrence of active caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP) positive cells. Collectively, homocysteine, adenosine and TNF-α are interrelated in the survival of endothelial cells, and this co-existence should be considered in future drug development for cardiovascular and cerebrovascular diseases. Flow cytometric analyses for active caspase-3 and cleaved poly(ADP-ribose) polymerase The cleavage of proteolytic enzyme caspase-3 and poly(ADP-ribose) polymerase (PARP) were determined using PE conjugated anti-active caspase-3 (CPP 32) and FITC conjugated anti-cleaved PARP (Asp 214) antibodies. The experimental procedures were carried out according to the manufacturer's instructions. Brie�ly, both detached cells and attached monolayer were collected by centrifugation at 500 x g for 5 minutes. The pellet was then �ixed on ice for 20 minutes and washed twice with washing buffer. After �ixing, the pellet was resuspended in washing buffer with the antibodies at room temperature for 30 minutes. The pellet was then washed once with washing buffer, and resuspended in appropriate volume of washing buffer for �low cytometric analyses. 10,000 cells were analysed using the FACSCalibur and Weasel software version 3.0.2. The results are presented as mean �luorescence intensity and the percentage of cells with positive staining. Fig. 2 . TNF-α decreased the survival of endothelial cells only with the presence of DL-homocysteine and adenosine. Cell viability, as determined by MTT dye reduction assay, following treatments with TNF-α with or without the presence of homocysteine (DL-Hcy) and adenosine (Aden) in both EA.hy926 and HBEC-5i endothelial cells for 20 hours. Cell viability was expressed as a percentage compared to control. All results were expressed as mean ± S.E.M. from three separate experiments in triplicate. *** P < 0.001 in comparison with TNF-α alone in the respective cell lines. TNFR1 through the initiation and activation of caspase-8 and caspase-3 (extrinsic apoptotic pathway). However, further studies are required to evaluate the cross-talk between hypomethylation and TNF-α. In conclusion, the present study demonstrates that the combination of homocysteineadenosine-TNF-α reduces the survival rate of both cardiovascular and cerebrovascular endothelial cells, at least in part, through promoting excessive apoptosis via activating the caspase-3. Therefore, homocysteine, adenosine and TNF-α are interrelated in the survival of endothelial cells, and this co-existence should be considered in future drug discovery for cardiovascular and cerebrovascular diseases.