Expression of cellular FLICE-inhibitory protein in human coronary arteriesand in a rat vascular injury model

We previously isolated MACH-related inducer of toxicity (MRIT), a homolog o f 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 prote ins, c-FLIP is widely believed to be an antagonist to the death receptor-in itiated apoptotic pathways that use caspase 8, We generated a polyclonal an tibody, MAG1, and show that this antibody specifically recognizes two splic e forms, long form (c-FLIPL) and short form (c-FLIPs). By in situ hybridiza tion and immunohistochemistry, we demonstrate that c-FLIP is expressed in e ndothelial cells, macrophages, and smooth muscle cells (SMCs) both in human coronary arteries and in cultured cells. In an uninjured rat carotid arter ies, c-FLIP protein is abundant in the vascular media. After balloon angiop lasty, 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 i mmunoreactive to c-FLIP from day 7 after the initial injury and remains str ongly immunoreactive until 4 to 6 weeks, Similarly there is strong c-FLIP i mmunoreactivity in SMCs from nonatherosclerotic diffuse intimal thickening and in the overlying endothelial cells. In contrast, c-FLIP immunoreactivit y is uneven and, often absent in SMCs within the atherosclerotic plaque, Do uble labeling with c-FLIP antibody and terminal deoxynucleotidyl-transferas e-mediated UDP end labeling (TUNEL) in the injured rat common carotid arter y show that TUNEL-positive cells in the first 2 days after injury lack dete ctable c-FLIP, suggested a role for caspase 8 in this form of death. In con trast, there is no correlation of c-FLIP with the spontaneous elevation in death of intima seen at 7 days after injury. For human atherosclerotic plaq ues, the majority of TUNEL-positive cells lack detectable c-FLIP, The expre ssion 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.