Endothelial cell apoptosis induced by oxidized LDL is associated with the down-regulation of the cellular caspase inhibitor FLIP.

Fas (CD-95/APO-1) is a death receptor that initiates an apoptotic signal when activated by its ligand, FasL. Normal vascular endothelial cells are resistant to Fas-mediated apoptosis though they express both Fas and FasL. Oxidized low density lipoprotein (OxLDL) or lysophosphatidylcholine (LPC), a major component of OxLDL, induces endothelial cell suicide by sensitizing endothelial cells to Fas-mediated apoptosis. Here, we show that endothelial cell apoptosis by OxLDL and LPC-C16:0 was dose-dependent and correlated with down-regulation of FLICE-inhibitory protein (FLIP), an intracellular caspase inhibitor. FLIP down-regulation also occurred when endothelial cells were treated with toxic doses of LPC-C18:0 or minimally modified low density lipoprotein (LDL). In contrast, FLIP was not down-regulated by native LDL, acetylated LDL, LPC-C12:0, cholesterol, or 7-ketocholesterol, which are not toxic to endothelial cells. The cytotoxicity of oxidized lipids was reversed by transfecting endothelial cells with a FLIP expression plasmid. The results demonstrate, for the first time, FLIP regulation under conditions that lead to pathological tissue destruction.

expressing both Fas and FasL do not undergo suicide or fratricide under normal conditions (4,5), but these cells can become dramatically sensitized to the Fas-mediated apoptosis in response to specific stimuli or injuries (6 -9). The increased sensitization to Fas/FasL-mediated suicide may play a role in regulation of physiological T cell number (4) and in pathological tissue destruction (10). However, the mechanisms by which stimuli sensitize cells to Fas-mediated apoptosis remains to be elucidated.
Caspases are cysteine proteases that play a central role in Fas-mediated apoptosis signaling pathway (11). Upon ligand activation, Fas is oligomerized (3) and recruits Fas-associated death domain protein (FADD) and pro-FLICE (FADD-homologous ICE-like protease), resulting in proteolytic activation of FLICE (12,13). Active FLICE is released into the cytosol and triggers a cascade of caspases (11). FLIP (for FLICE-inhibitory protein) is a recently identified FADD-binding suppressor of apoptosis (14 -16). Multiple isoforms are predicted based upon the analysis of FLIP cDNAs (14 -16), two of which designated FLIP L (long FLIP isoform) and FLIP S (short FLIP isoform) have been isolated from activated human peripheral blood leukocytes (14). All isoforms of FLIP contain FADD binding domain but lack the active-center cysteine residue and may function as dominant negatives for FLICE, thus blocking Fasmediated apoptosis (14 -16). FLIP isoforms are expressed during the early stage of T-cell activation and disappear when T cells become susceptible to Fas ligand-mediated apoptosis (14,17), suggesting that the levels of intracellular FLIP may determine sensitivity of the cells to Fas-mediated apoptosis. FLIP is reported to inhibit Fas-mediated apoptosis (14,18) and an apoptosis-protective role has been found in the viral FLICEinhibitory proteins (19,20). However, the role of FLIP is controversial as in some cell types its overexpression can induce apoptosis (14,15,21,22).
It is firmly established that elevated levels of oxidized low density lipoprotein (LDL) are associated with accelerated atherogenesis (23,24). OxLDL and its lipid constituents have numerous detrimental effects on endothelial cell function, including the induction of apoptosis (25)(26)(27). Vascular endothelial cells express functional FasL and detectable Fas on their cell surface, but they are resistant to Fas-mediated apoptosis under normal conditions (5,28). Recently, we documented that OxLDL induces endothelial cell suicide through Fas-FasL interaction by sensitizing endothelial cells to Fas-mediated apoptosis (29). Here, we show that FLIP may play a role in regulating the sensitivity of endothelial cells to Fas-mediated apoptosis. This is the first report of FLIP regulation outside of the immune system and under conditions of pathological tissue destruction.

EXPERIMENTAL PROCEDURES
Cell Viability Assay and Reagents-Human umbilical vein endothelial cells (HUVECs) were isolated as described (30) and cultured in endothelial growth medium (EGM; Clonetics, San Diego, CA). HUVECs cultured in a 96-well plate at 80% confluency were incubated with OxLDL or LPC at indicated doses for 16 h. Cell viability was measured by means of MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay (31). LDL was isolated by sequential ultracentrifugation (d ϭ 1.019 -1.063) from freshly drawn, citrated normolipidemic human plasma to which EDTA was added (32). LDL was oxidized in the presence of 5 M CuSO 4 for 24 h at 25°C, and the degree of oxidation was assessed by the increase of mobility on 1% agarose gel (1.4 versus native LDL) (32). Minimally modified LDL (MM-LDL) was prepared by dialyzing native LDL against 9 M FeSO 4 in phosphate-buffered saline for 72 h at 4°C as described (33). The electrophoretic mobility increased 1.1 to 1.2 versus native LDL. Acetylation of LDL was performed with excess acetic anhydride. All lipid reagents were purchased from Sigma.
Anti-FLIP Antibody-An antiserum against human FLIP was generated in New Zealand White rabbits against a peptide spanning amino acids 2-27 (SAEVIHQVEEALDTDEKEMLLFLCRD) synthesized using the multiple antigen technology (34). The IgG fraction of the antiserum was isolated using E-Z-SEP kit (Amersham Pharmacia Biotech, Piscataway, NJ) and affinity-purified on the corresponding peptides coupled to Affi-Gel 15 gel (Bio-Rad, Hercules, CA).
Transfection of HUVECs with FLIP Expression Plasmid-HUVECs cultured in 24-well plates were transfected with ␤-galactosidase expression plasmid (1.5 g) and a pCR3-based (Invitrogen, Carlsbad, CA) expression plasmid (0.15 g) for human long form of FLIP (FLIP L ) (a generous gift from Dr. J. Tschopp), Bcl-2, or Bcl-X L using the Superfect procedure (Qiagen). After 48 h, HUVECs were incubated with 60 M LPC-C16:0 for 24 h, fixed in 2% formaldehyde and 0.2% glutaraldehyde for 10 min, and stained with X-gal (Amersham Pharmacia Biotech). The number of viable blue cells attaching to the culture plate was counted as described (35)(36)(37).

RESULTS AND DISCUSSION
To study FLIP regulation under conditions of endothelial cell death, we initially determined the parameters influencing oxidized lipid-induced apoptosis. HUVECs were treated with different doses of OxLDL or LPC-C16:0 for 24 h, and mitochondrial function was assessed by the MTT assay, an indicator of cell death (31,38). Consistent with previous reports of cytotoxicity by these agents (26,39), OxLDL at or above 300 g of protein/ml (Fig. 1A) and LPC-C16:0 at or above 60 M (Fig. 1B) markedly decreased mitochondrial function. Lower doses of OxLDL and LPC-C16:0 were slightly stimulatory, consistent with reports that these agents can stimulate growth (40,41).
To study regulation of FLIP under conditions of OxLDL-or LPC-induced endothelial cell apoptosis, a polyclonal antibody was generated against the 26-amino acid sequence of the N terminus of FLIP. This sequence is common among all of the reported isoforms (14 -16). Immunoblotting revealed that untreated HUVECs express a 55-kDa immunoreactive protein that has an identical mobility to the positive control, an extract from COS cells transfected with the FLIP L expression plasmid (Fig. 2, A and B). Treatment with OxLDL ( Fig. 2A) or LPC-C16:0 (Fig. 2B) markedly down-regulated expression of the FLIP-immunoreactive protein in a dose-dependent manner and occurred at doses of OxLDL or LPC that induced endothelial cell death (Fig. 1, A and B). Expression levels of the previously reported FLIP S were very low or undetectable in HUVECs.
The Bcl-2 family of proteins functions as positive and negative regulators of apoptosis (42), including Fas-mediated cell death (43). In contrast to FLIP, OxLDL treatment had no effect on the expression of the pro-apoptotic protein Bax or the antiapoptotic proteins Bcl-x or Bcl-2 (Fig. 2C). Furthermore, LPC did not affect the expression of genes encoding the death-  Other lipid species were examined for their ability to induce cytotoxicity and down-regulate FLIP. Native LDL did not influence HUVEC viability (Fig. 3A). Acetylated LDL, which is internalized by endothelial cells but lacks biological effects (44,45), also did not affect endothelial cell viability. On the other hand, MM-LDL, which promotes atherogenesis by acting as an inflammatory agent (33,46), induced endothelial cell death. LPC-C12:0, which does not inhibit endothelial cell migration (47), also did not affect endothelial cell viability, whereas LPC-C18:0, a component of OxLDL, induced endothelial cell death. Finally, cholesterol and 7-ketocholesterol did not kill endothelial cells. Immunoblotting against FLIP revealed that cytotoxic concentrations of MM-LDL and LPC-C18:0 down-regulated FLIP L expression level, whereas nontoxic lipids did not (Fig.  3B). These results further suggest that FLIP L down-regulation accounts for endothelial cell sensitization to Fas-mediated apoptosis To examine the functional significance of FLIP down-regulation in sensitization of human endothelial cells to Fas-mediated apoptosis, we tested whether ectopic expression of FLIP could protect against LPC-induced apoptosis (Table I). Using a co-transfection cell death assay (35)(36)(37), HUVECs were cotransfected with FLIP expression plasmid and ␤-galactosidase (␤-gal) expression plasmid to mark the transfected cells. As expected, treatment with LPC-C16:0 reduced the number of viable ␤-galactosidase-positive cells. The FLIP expression plasmids significantly increased the number of viable ␤-galactosid-ase-positive cells compared with the pcDNA control plasmid or mock co-transfection, indicating that ectopic expression of FLIP L prevented endothelial cells from LPC-induced apoptosis. Consistent with observations that Bcl-X L but not Bcl-2 protects Fas-mediated apoptosis (43), ectopic expression of Bcl-X L also protected LPC-induced endothelial cell apoptosis, whereas Bcl-2 did not.
Vascular endothelial cells express both Fas and FasL, but they are normally resistant to Fas-mediated apoptosis (5,28). Oxidized lipids sensitize endothelial cells to Fas-mediated apoptosis leading to cell suicide (29). Here, we found that oxidized lipids down-regulate FLIP protein. FLIP down-regulation correlates with a loss of endothelial cell viability, and oxidized lipid-induced cell death was reversed by forced FLIP expression. Previously, FLIP regulation has only been described during T cell activation where it may control Fas-mediated suicide (14,17). The findings described herein suggest that FLIP levels may play a role in determining endothelial cell sensitivity to oxidized lipid-induced apoptosis, and they indicate that FLIP may be a target of agents that cause pathological cell death.