The role of antiapoptotic Bcl-2 family members in endothelial apoptosis elucidated with antisense oligonucleotides

Abstract

In this study, we utilized potent antisense oligonucleotides to examine the role of two Bcl-2 family members found in human umbilical vein endothelial cells (HUVEC). The first, A1, is thought to be a TNF-α-inducible cytoprotective gene, and the second, Bcl-XL, is constitutively expressed. Inhibition of the constitutive levels of Bcl-XL caused 10-25% of the cell population to undergo apoptosis and increased the susceptibility of cells to treatment with low concentrations of staurosporin or ceramide. The caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)CH2 prevented DNA fragmentation and ΔYm loss caused by Bcl-XL inhibition or Bcl-XL inhibition combined with staurosporin. However, disruption of ΔYm caused by Bcl-XL inhibition combined with ceramide treatment was not inhibited by benzyloxycarbonyl-Val- Ala-Asp(OMe)-CH2, although DNA fragmentation was completely prevented. Taken together, these results demonstrate a direct protective role for Bcl-XL under normal resting conditions and under low level apoptotic challenges to HUVEC. Furthermore, Bcl-XL protects cells from caspase-dependent and -independent mechanisms of ΔYm disruption. In contrast to Bcl-XL, A1 inhibition did not show a marked effect on the susceptibility of HUVEC to undergo apoptosis in response to TNF-α, ceramide, or staurosporin. These results demonstrate that although A1 may be a cytoprotective gene induced by TNF-α, it is not primarily responsible for HUVEC resistance to this cytokine.