Inhibition of Inflammatory Endothelial Responses by a Pathway Involving Caspase Activation and p65 Cleavage

Abstract

Suppression of NFκB activation has been involved in the elimination of survival programs during endothelial cell (EC) apoptosis. We used α-tocopheryl succinate (α-TOS) to trigger apoptosome formation and the subsequent activation of executioner caspases. The level of bcl-2 was reduced by α-TOS, and its downregulation potentiated and its overexpression suppressed pro-apoptotic effects of α-TOS, indicating a mitochondrial role in α-TOS-induced apoptosis in EC. α-TOS treatment was associated with induction of TUNEL-positive apoptosis in EC with a high but not with a low proliferation index. The use of the pan-caspase inhibitor z-VAD.fmk suggested the involvement of caspases in cleavage of p65, and in inhibition of nuclear translocation of p65 and NFκB-dependent transactivation of a gene construct encoding the green fluorescence protein elicited by TNFα in contact-arrested EC. The suppression by α-TOS of inflammatory EC responses induced by TNFα such as VCAM-1 mRNA and surface protein expression and shear-resistant arrest of monocytic cells were also reversed by z-VAD.fmk. NFκB-dependent transactivation was preserved in α-TOS-treated EC stably transfected with a caspase-noncleavable p65 mutant but not with its truncated form, thus establishing a direct link between α-TOS-induced effects and p65 cleavage. Our data infer a pathway by which caspase activation in EC inhibits NFκB-dependent inflammatory activation and monocyte recruitment, and provide evidence for a relationship between pro-apoptotic and anti-inflammatory pathways.