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DETAIL MATERIALS AND METHODS
Chemicals and reagents
HAART drugs d4T, DDI, and IDV were obtained from the AIDS Research and Reference
Reagent Program, Division of AIDS, NIAID, NIH. HAART drugs were dissolved in dimethyl
sulfoxide (DMSO) at the desired concentrations and the final concentration of DMSO in the
experiments was adjusted to less than 0.1% (v/v), which was used in all controls. [1α,2α(n)-3H]Cholesterol was purchased from Amersham (Piscataway, NJ) and human acetylated low density
lipoproteins (acLDL) from Intracel (Frederick, MD). Rabbit polyclonal caveolin-1 antibody and mouse
monoclonal anti-β-actin antibody were obtained from Norvus Biologicals (Littleton, CO). Rabbit
polyclonal p67phox antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
Horseradish peroxidase-conjugated goat anti-rabbit IgG and anti-mouse IgG were purchased from
Jackson Immuno-Research (West Grove, PA). Ginsenosides Rb1 and Rg1, S-allyl cysteine sulphoxide
(SACS), simvastatin (SVT), vitamins C and E, and apolipoprotein A-I (apoA-I) were obtained from
Sigma (St. Louis, MO).
Cell culture
Human THP-1 monocytes (human acute monocytic leukaemia) were obtained from ATCC
(Manassas, Va), and cultured in RPMI 1640, and differentiated into macrophages by the addition of
phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) for 4 days as described.4 Fresh human blood
samples were purchased from Gulf Coast Regional Blood Center (Houston, TX, USA). Peripheral
blood mononuclear cells (PBMCs) were purified by gradient centrifugation using Ficoll-Paque PLUS
(Amersham Biosciences, Uppsala, Sweden). PBMCs were seeded onto 96-well plates at 2×105 cells
per well and cultured at 37°C in a 5% CO2 atmosphere. The cells that adhered to the plate after two
hours were used as monocytes and were allowed to differentiate into monocyte-derived macrophages
for five days in RPMI containing 50 ng/ml M-CSF (macrophage colony stimulating factor) and 1%
FBS.
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acLDL loading and pretreatment
Macrophages were transformed into foam cells by incubation with acLDL (50 μg/ml) and
[1α,2α(n)-3H]-Cholesterol (1 μCi/ml) in the serum-free medium containing 1.5% BSA for 48 hours.
The macrophage-derived foam cells were then incubated for 24 hours in the presence or absence of
d4T (2-8 μM), ddI (5.5-22 μM), IDV (6.25–25 μM), HAART 3-plex (d4T: 4 μM; DDI:11 μM; IDV:
12.5 μM) and antioxidants at 10 μM including Rb1, Rg1, SACS, SVT, vitamins C and E.
Cholesterol efflux
Cholesterol efflux from THP-1 macrophages was initiated by the addition of 50 μg/ml apoA-I
for 24 hours. Samples of both supernatants and cell lysates were applied to UniFilter-96 plates
(PerkinElmer, Boston, MA), respectively. After drying, samples were measured by TopCount-NXT
(Packard) in the presence of 25 μl MicroScint cocktail in each well. Fractional cholesterol efflux was
calculated as [cpm(supernatants)/cpm(supernatants+cells)]×100% as previously described.4
Real-time RT-PCR
Macrophage-derived foam cells were treated with HAART drugs for 48 hours. Total cellular
RNA was then extracted using an Ambion RNAqueous-4PCR kit (Austin, TX). Primers for caveolins,
scavenger receptor B1 (SR-B1), and several enzymes related to superoxide anion production including
NADPH oxidase subunits p22phox, p47phox and p67phox, were designed via the Beacon Designer 2.1
software (Bio-Rad), as shown in our previous publication.4 The iQ SYBR green Supermix Kit and
iCycler iQ Real-time PCR detection system (Bio-Rad) were used in real-time PCR. A house keeping
gene,-actin, was included for comparison. Relative mRNA levels of genes of interest were presented
as 2^[Ct(β-actin)−Ct(gene of interest)].
Western blot analysis
Proteins from treated cells were extracted with cell lysis buffer (Cell Signaling Technology,
Inc., Danvers, MA). Equal amount of total proteins (50 µg) were loaded onto 10% SDS-PAGE,
fractionated by electrophoresis, and transferred to PVDF membranes. The membrane was incubated
with the primary antibody at 4C overnight. Dilutions of 1: 200 for p67phox and 1: 1000 for caveolin-1
were used.
Bands were visualized with ECL plus Chemiluminescent Substrate (Amersham
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Biosciences). Densitometric measurement was performed to quantify the relative expression of target
proteins vs. β-actin (AlphaEaseFC software).
Superoxide anion analysis by flow cytometry
Macrophage-derived foam cells were treated with or without HAART 3-plex for 48 hours.
Cells were incubated with 0.5 ml dihydroethidium (DHE, 10 μM) for 20 minutes at room temperature.
DHE is freely permeable to cells. In the presence of superoxide anion, DHE is oxidized to ethidium
bromide (EtBr) with red fluorescence, and it is trapped by intercalating with the DNA. EtBr is excited
at 488 nm with an emission spectrum of 610 nm. Thus, the amount of EtBr detected by fluorescence
measurement instruments such as flow cytometer is well correlated to the level of cellular superoxide
anion. Superoxide anion in the cells was analyzed by FACS Calibure flow cytometry (Becton
Dickinson, San Jose, CA) and presented by the percentage of positive staining cells in macrophagederived foam cells. The negative control for flow cytometery analysis was the sample with no DHE
staining. Based on less 1% positive staining in the negative control in the FL2-H channel, we set the
gate to define the positive populations.
Assessment of mitochondrial membrane potential (∆ψm)
∆ψm was assessed by using flow cytometry analysis of cells stained with 5,5',6,6'-tetrachloro1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1, MitoScreen kit, BD Biosciences). During
the transfer of electrons to molecular oxygen, an estimated 1 to 5% of electrons in the respiratory chain
“leak” to form superoxide radicals. ∆ψm can serve as an indicator for the function of mitochondrial
respiration chain. ∆ψm was assessed by using flow cytometry analysis of cells stained with 5,5',6,6'tetrachloro-1,1',3,3'-tetraethylbenzimidazole-carbocyanide iodine (JC-1). Mitochondria with a normal
∆ψm concentrate JC-1 into aggregates (red fluorescence). Otherwise, cells with low ∆ψm, JC-1 in
monomeric form remains in the cytosol, and shows only green fluorescence. Thus, JC-1 aggregates
(red fluorescence) represent ∆ψm levels. Cells (5x105) were incubated with 10 µg/ml JC-1 for 15
minutes at 37°C and analyzed by FACS Calibure flow cytometry.
Measurement of ATP levels
ATP levels were measured with an ATPLite kit (PerkinElmer, Wellesley, MA) following
manufacturer’s instructions. Briefly, cells were seeded on 96-well plates and cultured with or without
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HAART 3-plex for 48 hours. The lysis solution and substrate solution were added to each well of the
plate, respectively. The luminescence was measured by TopCount-NXT.
Statistical analysis
All data are presented as the mean ± SEM. Inter-group differences were analyzed using oneway ANOVA for comparison of three or more groups. Student’s t-test was used for comparison
between two groups. A P value < 0.05 was regarded as significant.
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Table I. Maximal plasma concentrations of anti-HIV drugs in the clinical dose
Drug
Drug Type
Brand name
Dose
Cmax*
Concentration
Indinavir
PI
Crixivan
800 mg, tid
8.9 μg/ml
12.5 μM
Stavudine (d4T)
NRTI
Zerit
40 mg, bid
0.9 μg/ml
4 μM
Didanosine (ddI)
NRTI
Videx
200 mg, bid
2.6 μg/ml
11 μM
*Cmax: the peak plasma concentration (Physicians’ Desk Reference 2006). PI: Protease inhibitor;
NRTI: Nucleoside Reverse Transcriptase Inhibitor
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