Abstract
BackgroundPublished work suggests that some types of endothelial cells undergo apoptosis in response to ligation of the receptor Fas (CD95, APO1) but other types are resistant. Because heterogeneity among endothelial cells from different tissues, has been demonstrated, the purpose of this study was to determine, if Fas ligation and/or activation by human Fas ligand induces apoptosis and caspase activities, in cultured human coronary artery endothelial cells, and the differences between TNF-a and FAS induced apoptosis in these cells.ResultsCultured human coronary artery endothelial cells (HCAEC) were exposed to the monoclonal Fas-activating antibody CH-11, to purified recombinant human Fas ligand, to the Fas-neutralizing antibody ZB4, or to purified recombinant human TNF-α. Apoptosis was detected by assessment of chromatin condensation and nuclear fragmentation and by assay of the enzymatic activities of Caspase 1 and Caspase 3 with membrane-permeable substrates applied to intact cells. Fas protein was detected by immunoblotting of HCAEC lysates. Apoptosis was induced in HCAEC by purified Fas ligand or by the monoclonal activating antibody CH-11 at concentrations of 25 or 200 ng/ml, but not by nonspecific isotype-matched immunoglobulins. The apoptotic index elicited by either Fas activator was equal to that induced by TNF-a (3.0-3.6-fold versus control, p < 0.01). The Fas-neutralizing antibody ZB4 abrogated HCAEC apoptosis induced by CH-11, but had no inhibitory effect on apoptosis in response to TNF-a. Fas ligation significantly increased the activities of both Caspase 1 and Caspase 3 at 20 hours of stimulation (1.7- and 2.0-fold versus control, both p < 0.05); in contrast, purified TNF-a increased the activity of Caspase 3 but not Caspase 1 (2.1-fold, p < 0.05). Western blotting of HCAEC lysates with antibody CH-11 identified a single immunoreactive protein of 90 kDa.ConclusionsCultured human coronary artery endothelial cells express functional Fas capable of inducing apoptosis in response to either purified Fas ligand or receptor-activating monoclonal antibodies, at levels equal to those inducible by purified TNF-α. Immunologic studies and differential kinetics of caspase activation suggest that Fas and TNF-α induce apoptosis in HCAEC by signaling pathways that are distinct but equal in potency.
Highlights
Published work suggests that some types of endothelial cells undergo apoptosis in response to ligation of the receptor Fasactivating monoclonal antibody CH-11 (Fas) (CD95, APO1) but other types are resistant
This concept is supported by the findings of increased expression of Caspase 1/interleukin-1β converting enzyme (ICE) and Caspase 3/ CPP-32 in atherosclerotic tissues [4,5,6,7]
We report evidence that the activation of Fas in cultured human coronary artery endothelial cells induces apoptosis through signaling mechanisms distinct from those induced by TNF-α
Summary
Published work suggests that some types of endothelial cells undergo apoptosis in response to ligation of the receptor Fas (CD95, APO1) but other types are resistant. Apoptosis of endothelial cells has been observed as a prominent feature of advanced atherosclerosis, and has been implicated in the pathophysiology of acute coronary syndromes [4,5,6] This concept is supported by the findings of increased expression of Caspase 1/interleukin-1β converting enzyme (ICE) and Caspase 3/ CPP-32 in atherosclerotic tissues [4,5,6,7]. It was shown [8] that foam cells within coronary arteries of patients with chronic ischemic heart disease express Fas (Apo, CD95), a member of the tumor necrosis factor/ nerve growth factor receptor family that induces apoptosis independent of TNF-α [9]. In vitro observations suggest that the regulation of apoptosis in a vessel may be dependent on the cell type but on the local environment [12,18]
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