Role of AIF in human coronary artery endothelial cell apoptosis

Abstract

Apoptosis-inducing factor (AIF), which exerts its effect via a caspase-independent pathway, has been suggested to be a mediator of cell injury. We have recently identified the expression of AIF in human coronary artery endothelial cells (HCAECs). The present study was designed to determine the pathophysiological role of AIF in oxidized low-density lipoprotein (ox-LDL)-induced apoptosis of HCAECs. The cells were cultured and treated with ox-LDL (40 microg/ml) for 24 h. Ox-LDL increased AIF expression, caused apoptosis of HCAECs (determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining and large-scale DNA fragmentation), and induced translocation of AIF from the cytoplasm to the nucleus (fluorescence immunocytochemistry). Pretreatment of HCAECs with a caspase inhibitor (ZVAD-fmk) did not influence AIF-mediated apoptosis in response to ox-LDL. We developed a specific antisense oligonucleotide targeted to the 5'-TCG CCG AAA TGT TCC GGT GTG GA-3' portion of the human AIF mRNA sequence (AIF-AS) to bind a complementary sequence overlapping the translational start site. Pretreatment of cells with the AIF-AS for 24 h resulted in suppression of ox-LDL-upregulated AIF protein, as measured by immunoblot analysis. AIF-AS also reduced apoptosis and AIF translocation (P < 0.01 vs. ox-LDL alone). Next, we constructed a recombinant AIF plasmid by inserting whole-length AIF cDNA into the expression vector pcDNA3.1 with a cytomegalovirus promoter. HCAECs transfected with plasmid showed a two- to fourfold increase in AIF expression, extensive apoptosis, and translocation of AIF from the cytoplasm to the nucleus. These results from two approaches indicate that AIF plays an important role in ox-LDL-induced endothelial injury.

Two Distinct Calcium-Dependent Mitochondrial Pathways Are Involved in Oxidized LDL-Induced Apoptosis

“…Zhang et al 17 recently reported that oxLDLs enhance the expression of AIF, and thereby promote caspase-independent apoptosis. Therefore, we investigated whether oxLDLinduced AIF release is mediated by the same mechanism as cytochrome C release.…”

“…It was therefore suggested that another caspase-independent apoptotic pathway was activated by oxLDLs. Zhang et al 17 reported recently that oxLDLs enhance the expression of the 57-kDa flavoprotein AIF, but the mechanism of release of AIF from mitochondria is indeterminate. Our data show that AIF release is calciumdependent, but that downstream from calcium, the pathways leading to release of AIF and cytochrome C are distinct because AIF release was not inhibited by calpeptin and cyclosporin-A in contrast to cytochrome C release and caspase-3 activation.…”

“…Cytotoxicity was evaluated using the MTT (dimethylthiazolyl diphenyltetrazolium bromide) test, as previously used [17]. Cell lysis (necrosis) was evaluated by lactate dehydrogenase (LDH) release.…”

“…15 However, whereas Porn-Ares et al 15 found that oxLDLs induce Bid truncation without any activation of caspase-3, Chen et al 12 observed that oxLDLs did not cause Bid truncation but activated caspase-3 in agreement with Dimmeler et al 16 Furthermore, oxLDLs can activate a caspase-independent apoptotic pathway mediated by the release of apoptosis-inducing factor (AIF) from mitochondria. 17 Because data in the literature on mechanisms implicated in oxLDL-induced apoptosis are controversial and because calcium can trigger multiple apoptotic pathways, 18 we presently investigated the potential link between oxLDL-induced cal-…”

CopA3 peptide from Copris tripartitus induces apoptosis in human leukemia cells via a caspase-independent pathway

“…This activity was mediated by activation of AIF (apoptosis inducing factor), which is a regulator of a caspase-independent programmed cell death pathway (6). These results suggest a possible therapeutic use of CopA3 for inducing apoptosis of leukemia cells, in addition to its application as an antibiotic.…”