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Department of Internal Medicine and Department of Physiology and Biophysics, University of Arkansas for Medical Sciences and Central Arkansas Veterans Healthcare System, Little Rock, Arkansas 72205
Submitted 20 June 2003 ; accepted in final form 19 August 2003
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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antisense; apoptosis-inducing factor; endothelium
In previous studies, we (13, 15) examined the biochemical mechanism of oxidized low-density lipoprotein (ox-LDL)-induced apoptosis of human coronary artery endothelial cells (HCAECs). We found that ox-LDL induces expression and activity of a lectin-like receptor for ox-LDL, LOX-1, which leads to apoptosis (13). We (25) have recently demonstrated that ox-LDL also upregulates AIF expression in HCAECs in a concentration- and time-dependent manner. In the present study, we employed two different approaches, antisense phosphorothioate oligonucleotides (ODNs) directed at AIF mRNA and AIF overexpression, to determine the role of AIF in apoptosis of HCAECs.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Cell culture. An original batch of HCAECs was purchased from Clonetics (East Rutherford, NJ). The cells were cultured in endothelial basal medium-2 (Clonetics), which consisted of 500 ml endothelial cell basal medium, 5 ng human recombinant epidermal growth factor, 0.5 mg hydrocortisone, 25 mg gentamycin, 50 µg amphotericin B, 6 mg bovine brain extract, and 25 ml FBS, as described previously (12, 13, 15, 25). Fourth-generation HCAECs were used in this study. HCAECs were incubated with ox-LDL (10–40 µg protein/ml) for 24 h, and the expression of AIF was determined. Parallel groups of HCAECs were pretreated with the caspase inhibitor ZVAD-fmk (50 µM) for 30 min before the addition of ox-LDL for 24 h. Other groups of cells were treated with antisense ODNs to AIF (AIF-AS) or sense ODNs (AIF-S) (1 µM) for 24 h, and HCAECs were then exposed to ox-LDL (40 µg protein/ml) for 24 h to study the expression of AIF and induction of cell injury. Concentrations of all reagents were based on previous studies (12, 13, 25). Parallel groups of cells were treated with pcDNA-AIF.
Preparation of ox-LDL. LDL was prepared as described previously (12, 13, 15, 25). LDL was oxidized at 37°C in the presence of 5 µmol/l CuSO4 for up to 24 h and then dialyzed against three changes of PBS for 24 h. Ox-LDL samples were filter sterilized and incorporated in the culture medium. The integrity of the lipoproteins was confirmed by agarose gel electrophoresis.
AIF cloning of AIF cDNAs and transfection of HCAECs. AIF cDNAs were cloned by a combination of RT-PCR and rapid sequencing, using primers specific for the 5'- and 3'-untranslated regions of the human AIF sequence. To generate the vector pcDNA-AIF, pcDNA3.1+ (Invitrogen) was cut with EcoRI/XhoI and ligated. PCR-based, site-directed mutagenesis of the AIF cDNA was followed by verification of the entire AIF cDNA sequence. pcDNA3.1 and pcDNA-AIF were purified using Qiagen Plasmid Kits.
The protein-coding region of AIF cDNA was inserted into the expression vector pcDNA (termed pcDNA-AIF). Vector alone (pcDNA) was used as a negative control. HCAECs were transfected with pcDNA plasmid alone or with pcDNA-AIF with the use of 3 µl/ml SuperFect (Qiagen) and mixed with 5 µg/ml plasmid for 48 h. The day before transfection, 106 cells were placed in a 100-mm dish in 10 ml endothelial culture medium. The cell number seeded produced 
80% confluence on the day of transfection. On the day of transfection, 10 µg DNA dissolved in Tris-EDTA buffer and 60 µl SuperFect Transfection Reagent was added to the DNA solution, and the transfection complex was formed as per the manufacturer's instructions. The cells were washed with PBS and then added to the tube containing the transfection complexes. Cells were allowed to incubate with the transfection complexes for 2–3 h. Thereafter, the medium was removed, and cells were washed with PBS. The cells were assayed for expression of the transfected gene. HCAECs were transfected with pcDNA-AIF or pcDNA for 48 h.
AIF-AS design. Antisense phosphorothioate ODNs and sense phosphorothioate ODNs (as controls) directed to the 5'-coding sequence of human AIF mRNA were developed as 23-mers targeted to the 5'-TCG CCG AAA TGT TCC GGT GTG GA-3' portion of the AIF sequence in collaboration with Integrated DNA Technologies. The corresponding control (sense) was also 23-mer.
Western blots. HCAECs were homogenized in ice-cold buffer [10 mM Tris·HCl (pH 7.9), 10 mM MgCl2, and 0.5 mM PMSF]. The homogenate was centrifuged at 2,000 g for 10 min. Twenty micrograms of supernatant protein were loaded on a 10% SDS-PAGE and blotted on nitrocellulose filters. Immunoblots were performed with goat antibody against human AIF (Santa Cruz Biotechnology). Details of the methods are provided elsewhere (12, 25).
Immunocytochemistry. Immunocytochemistry was used to identify translocation of AIF from mitochondria to the nucleus during apoptosis, as described previously (25). Briefly, the cells were fixed on slides in cool methanol for 10 min at –20°C. The specimen was washed twice with PBS, blocked with 3% BSA in PBS for 15 min, and then covered with anti-AIF polyclonal antibody (1:20 dilution) in 3% BSA in PBS for 2 h at room temperature. After a wash, FITC-conjugated secondary antibody (1:40 dilution) was applied for 1 h. Cells without anti-AIF antibody served as controls. After several washes, the slides were visualized under fluorescent microscopy.
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Cultured cells were pretreated with AIF-AS or AIF-S (1 µM) for 24 h and then exposed to ox-LDL (40 µg protein/ml) for 24 h. The cells were then examined for apoptosis by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay (Promega). The method for TUNEL staining has been described previously (15, 17).
Pulse-field gel electrophoresis. HCAECs were pretreated with AIF-AS or AIF-S before incubation with ox-LDL as described in Immunocytochemistry. To avoid nonspecific DNA breaks caused by phenol-chloroform extraction, chromosomal DNA samples were prepared in agarose plugs (18) using a CHEF mammalian genomic DNA plug kit (Bio-Rad). Cells (2.5 x 105) were resuspended in 50 µl of PBS, mixed with 50 µl of preheated (50°C) 2% CleanCut agarose, and transferred into agarose plug molds. After solidification at room temperature, the plugs were incubated in 1 mg/ml proteinase-K overnight at 50°C. Deproteinized DNA-containing agarose plugs were washed in buffer. Pulse-field gel electrophoresis (PFGE) was performed on a CHEF-DR III pulsed-field electrophoresis system (Bio-Rad). Fragments were separated on a 1% pulse-field agarose gel at 14°C for 20 h in 0.5x Tris-borate-EDTA. Field strengths were 6 V/cm; initial and final switching time were set at 5–60 s with a 120° angle ramp. The gel was stained with ethidium bromide and visualized under ultraviolet light. DNA size standards (48.5–1,000 kb) were from Bio-Rad.
Caspase activity assay. Caspase activity was assessed by a colorimetric system (Oncogene). After pretreatment with AIF-AS or AIF-S (1 µM) for 24 h, the cells were exposed to ox-LDL (40 µg/ml) for 24 h. HCAECs were then scraped into lysis buffer and incubated for 30 min on ice. Lysates were clarified by centrifugation at 10,000 g for 10 min at 4°C, and protein concentration measured by a protein assay kit (Bio-Rad). The protease reaction utilized cleavage of the chromogenic peptide substrate Ac-DEVD-pNA for caspase-3 activity. The caspase activity assay was carried out in the reaction buffer [100 mM HEPES (pH 7.5), 5 mM DTT, 20% (vol/vol) glycerol, 0.5 mM EDTA, 0.1% (wt/vol) BSA, and 10 mM caspase substrate]. Cleavage of the substrate was measured by spectrophotometric analysis.
Statistical analysis. All data are means ± SD of at least three independent experiments and were subjected to ANOVA. A difference of P < 0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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In preliminary studies, we determined the inhibition of ox-LDL-mediated AIF protein expression by AIF-AS (0.2–2 µM, incubation time 4–48 h) by HCAECs. We found that a 0.2 µM concentration caused only minimal inhibition and 2 µM concentration caused cell death. Maximal inhibition of AIF protein expression occurred with 1 µM AIF-AS with an incubation time of 24 h. The 1 µM concentration of AIF-AS and 24-h incubation time were used for subsequent studies. Others (13) have also observed similar concentration of AS and incubation time (24 h) as optimal. It is noteworthy that there was no significant difference in AIF expression when cells were treated with ox-LDL alone or with AIF-S plus ox-LDL. Figure 1 shows representative examples and summary data from three independent experiments.
Antisense and apoptosis. The number of TUNEL-positive cells increased markedly upon treatment of cells with ox-LDL for 24 h (P < 0.05), in accordance with our previous observations (12, 13, 25). Pretreatment of cells with AIF-AS decreased apoptosis of HCAECs (P < 0.05), whereas pretreatment with AIF-S had no such effect (Fig. 2).
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Antisense and AIF translocation. The immunolocation of AIF protein (green fluorescence) was restricted to out of the nucleus in quiescent cells. After treatment of HCAECs with ox-LDL, AIF protein translocated to the nuclei. Pretreatment with AIF-AS reduced this translocation, whereas AIF-S had no such effect (Fig. 2).
Antisense and large DNA fragmentation. We employed PFGE to examine large DNA fragmentation in HCAECs treated with ox-LDL. PFGE showed that large DNA fragmentation was enhanced by treatment of cells with ox-LDL. Pretreatment of endothelial cells with AIF-AS markedly decreased DNA fragmentation. No significant alterations in DNA fragmentation was observed when cells were pretreated with AIF-S and then treated with ox-LDL or left untreated before exposure to ox-LDL (Fig. 3).
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Antisense and caspase-3 activity. We examined whether the ox-LDL-mediated upregulation of caspase-3 activity was affected by the expression of AIF. In cells treated with ox-LDL alone, there was a marked increase in caspase-3 activity (P < 0.01 vs. control); however, pretreatment with AIF-AS or AIF-S had no significant effect on caspase-3 activity. Note that pretreatment of cells with the caspase inhibitor significantly decreased caspase-3 activity (Fig. 4).
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Recombinant plasmid pcDNA-AIF and apoptosis. As shown in Figs. 5 and 6, transfection of HCAECs with pcDNA-AIF resulted in a marked increase in AIF protein, apoptosis (TUNEL assay), and translocation of AIF from the cytoplasm to the nucleus.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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We observed large-molecular-weight DNA fragmentation when HCAECs were treated with ox-LDL. The large-molecular-weight fragmentation is thought to be characteristic of apoptosis (21). Notably, the pretreatment of cells with the AIF-AS inhibited formation of large DNA fragments. These observations provide strong evidence that ox-LDL induces apoptosis in HCAECs mediated at least in part by expression of AIF.
It is of note that AIF-AS did not affect the activity of caspase-3, which suggests that the effect of AIF-AS was independent of caspase activity. This concept also gains support from the observation that the caspase inhibitor ZVAD-fmk did not affect increased expression of AIF when the cells were treated with ox-LDL, but reduced caspase-3 activity.
We have previously shown an important role of the caspase-dependent pathway in ox-LDL-mediated apoptosis of HCAECs (18). We found that the inhibition of caspase-3 could not completely block ox-LDL-induced apoptosis. The present study suggests that expression and activation of AIF could be a caspase-independent pathway that is responsible for part of the ox-LDL-mediated apoptosis in endothelial cells. In other preliminary studies (data not shown), we examined the effect of the combination of AIF-AS and ZVAD-fmk on ox-LDL-mediated apoptosis and observed that while the combination had more potent effect than either agent alone, there was still significant amount of apoptosis. These data collectively suggest the presence of pathways of apoptosis beyond caspase and AIF.
In other studies, we used pcDNA-AIF and observed that transfection of cells with pcDNA-AIF resulted in an upregulation of AIF expression and a simultaneous increase in apoptosis. These data complement the result from AIF-AS experiments and demonstrate the pathological significance of AIF in HCAECs.
Vascular endothelium becomes activated in the early stages of atherosclerosis (10) and allows attachment of inflammatory cells (17). Programmed cell death or apoptosis is a critical feature of endothelium and other cells, such as monocytes, in the atherosclerotic lesions (11, 16). Apoptosis is also seen in ischemic-reperfused myocardium (14). It is of note that ox-LDL accumulates in significant amount in, as well as around, the ischemic-reperfused regions (4). Significantly, it is important to define different pathways leading to cell injury.
In summary, we have demonstrated by two different approaches, i.e., use of antisense ODNs directed at AIF mRNA to inhibit AIF expression and the use of pcDNA-AIF to upregulate AIF expression, that AIF is a pathological mechanism in the induction of apoptosis in HCAECs. AIF pathway does not negate the importance of the caspases and other pathways of apoptosis in HCAECs.
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ACKNOWLEDGMENTS |
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This study was supported by a Merit Review Grant from the Department of Veteran Affairs and a Scientist Development Grant from the American Heart Association.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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