Expression of cellular FLICE-inhibitory protein in human coronary arteries and in a rat vascular injury model.

We previously isolated MACH-related inducer of toxicity (MRIT), a homolog of caspase 8. MRIT, also known as c-FLICE-inhibitory protein (c-FLIP), is an enzymatically inactive homolog of caspase 8 with homology to viral FLIP (v-FLIP). Because of this homology and resemblance to dominant negative proteins, c-FLIP is widely believed to be an antagonist to the death receptor-initiated apoptotic pathways that use caspase 8. We generated a polyclonal antibody, MAG1, and show that this antibody specifically recognizes two splice forms, long form (c-FLIPL) and short form (c-FLIPS). By in situ hybridization and immunohistochemistry, we demonstrate that c-FLIP is expressed in endothelial cells, macrophages, and smooth muscle cells (SMCs) both in human coronary arteries and in cultured cells. In an uninjured rat carotid arteries, c-FLIP protein is abundant in the vascular media. After balloon angioplasty, c-FLIP protein is rapidly down-regulated in medial SMCs for 2 weeks and regains expression by 4 weeks. In contrast, the neointima is strongly immunoreactive to c-FLIP from day 7 after the initial injury and remains strongly immunoreactive until 4 to 6 weeks. Similarly there is strong c-FLIP immunoreactivity in SMCs from nonatherosclerotic diffuse intimal thickening and in the overlying endothelial cells. In contrast, c-FLIP immunoreactivity is uneven and often absent in SMCs within the atherosclerotic plaque. Double labeling with c-FLIP antibody and terminal deoxynucleotidyltransferase-mediated UDP end labeling (TUNEL) in the injured rat common carotid artery show that TUNEL-positive cells in the first 2 days after injury lack detectable c-FLIP, suggested a role for caspase 8 in this form of death. In contrast, there is no correlation of c-FLIP with the spontaneous elevation in death of intima seen at 7 days after injury. For human atherosclerotic plaques, the majority of TUNEL-positive cells lack detectable c-FLIP. The expression pattern of c-FLIP and the relation between c-FLIP and TUNEL suggest a role for c-FLIP- and caspase 8-driven death in control of viability of the cells of the atherosclerotic intima.