Materials
Reagents | Company | Catalog number | Reagents | Company | Catalog number |
[3H]Cholesterol | Perkin Elmer | NET139001 | JNK inhibitor | Sigma-Aldrich | 420118 |
ABCA1 antibody | Santa Cruz Biotechnology | sc-58219 | LDLR antibody | R&D Systems | AF2148 |
Antibiotic-antimycotic | Thermo Fisher Scientific | 15240112 | mHBSS | Sigma-Aldrich | H8264 |
Anti-goat-HRP | R&D Systems | NL001 | NF-kB Inhibitor | Sigma-Aldrich | 481412 |
ApoE antibody | Santa Cruz Biotechnology | sc-13521 | NF-kB Inhibitor | Tocris | 4590 |
ApoE-conjugated agarose | Santa Cruz Biotechnology | sc-13521 AC | Normal goat IgG | R&D Systems | AB-108-C |
ASK 1 inhibitor | Tocris | MSC 2032964A | Normal mouse Ig agarose | Santa Cruz Biotechnology | sc-2343 |
BCA assay kit | Thermo Fisher Scientific | 23227 | p38 inhibitor | Sigma-Aldrich | 506163 |
Bond elut column | Agilent Technologies | 12256060 | PCSK9 | Caymen Chemical | 20631 |
Caspase inhibitor | Tocris | 2163 | pHAECs | ATCC | PCS-100-011 |
Caveolin-1 antibody | Santa Cruz Biotechnology | sc-53564 | Phosphatase inhibitor | Sigma-Aldrich | P0044 |
Centrifugal filter | Pall Corporation | MAP003C36 | Protease inhibitor | Sigma-Aldrich | P8340 |
Cholesterol esterase | MP Biomedicals | 105439 | RAP | Enzo Life Sciences | BML-SE552-0100 |
Cholesterol oxidase | Sigma-Aldrich | C8649 | Sandoz 58 − 035 | Sigma-Aldrich | S9318 |
Desalting columns | Thermo Fisher Scientific | 89890 | Scopoletin | TCI America | S0367 |
Dialysis membrane | Spectrum Labs | 131204 | Sodium heparin | Thermo Fisher Scientific | J16920-EXT |
Dil | Sigma-Aldrich | 468495 | Sodium taurocholate | Beantown Chemicals | 141920 |
EZ-link-Biotin | Thermo Fisher Scientific | A39258 | SR-B1 | Novus Biologicals | NB400-104 |
FAF-BSA | Sigma-Aldrich | A8806 | Strepavidin | GE Healthcare | 28-9872-30 |
FBS | R&D Systems | S11550 | TMTU | Alfa Aesar | L13392 |
Glass bottom dishes | Cellvis | P96-0-N | TNFɑ | ProSci Inc | 96–734 |
HRP | Alfa Aesar | J60026 | VBM | ATCC | PCS-100-030 |
ICAM1 antibody | Santa Cruz Biotechnology | sc-107 | VEGF | ATCC | PCS-100-041 |
Iodination beads | Thermo Fisher Scientific | 28665 | | | |
Cell culture and incubations. pHAECs (passages 3–7) were maintained in VBM with growth factor kit (VEGF) in the presence of 15% FBS, and antibiotic, antimycotic (100 units/mL of penicillin, 100 µg/mL of streptomycin, and 0.25 µg/mL of amphotericin B. Under serum-free conditions, the cells were cultured in VBM, VEGF, with the latter antimicrobials and 0.1% FAF-BSA. Incubations were at 37°C, unless indicated otherwise. Experiments were best started with pHAECs confluent for 5 days or longer. Separate cells from individuals (2–36 years old) were used during the course of the experiments, with similar results. Incubations were 24 ± 5 hours. For cell-associated studies at 37 ºC, cells were washed 3X with mHBSS at 0 ºC. Imaging studies demonstrated that the vast majority of Dil-lipoproteins were within the cell under these conditions. For intracellular LDL levels, pHAECs were incubated at 0°C for 5 minutes with ice cold 400 units/ml sodium heparin in mHBSS, washed twice with the same buffer, then two more times with ice cold mHBSS, to remove surface LDL. In experiments using excess unlabeled lipoproteins, the volume represented 3% or less of the culture medium. Lipids were extracted as described below or the cells were fixed with 4% paraformaldehyde in PBS prior to microscopy.
Lipoprotein purification. All procedures were performed between 0 and 4°C. Centrifugations and dialysis were performed at 4°C. Lipoproteins were isolated from freshly drawn human blood anticoagulated with Na2EDTA. The blood was centrifuged at 4,000 g for 30 minutes to obtain the plasma. To the plasma, butylated hydroxytoluene (BHT) in DMSO was added to 45 µM (0.01%). Then, sequential density ultracentrifugations [57] were performed to obtain d < 1.006 (VLDL), d = 1.019–1.063 (LDL), or d = 1.12–1.21 (HDL3) g/ml. Ultracentrifugations were done using type Ti70 rotor at 50,000 rpm for 20 (VLDL and LDL) or 48 hours (HDL3). The lipoproteins were dialyzed through ~ 4 kD molecular weight cut-off membrane in 3 successions against ~ 180 times dialysis buffer (DB): 10 mM Tris-HCl, 150 mM NaCl, 0.3 mM Na2EDTA, pH 7.5, with deionized water, in the dark, each lasting about 24 hours. After concentration using 3 kD MWCO centrifugal filters, the LDL was filtered through 0.2 µm membrane under sterile conditions. Lipoprotein contents were routinely verified by western blotting and coomassie staining. In addition, the cholesterol and cholesteryl ester contents were verified to be consistent with previous publications [58–60]. Lipoprotein concentrations represent the protein content throughout the manuscript.
Cellular cholesterol determination. Lipids were extracted into hexane: isopropanol (1:1) at room temperature for 3 hours, then the solvent was evaporated at room temperature using centrivap concentrator (Labconco). The extracted lipids were redissolved in isopropanol. To determine the unesterified cholesterol content, the lipids in isopropanol were mixed with 10 times volumes of cholesterol assay buffer (CAB): 0.1% FAF-BSA, 2 mM sodium taurocholate, 50 mM Tris-HCl, pH 7.5, 0.3 mM Na2EDTA, 5% isopropanol, and 250 mM sucrose, with freshly added 0.5 U/ml horseradish peroxidase (HRP) and 0.02 U/ml cholesterol oxidase in the presence of 30–50 µM scopoletin [61] (excitation 360, emission 460). The CAB was kept at room temperature to ensure full solubilization of the lipids. This enzymatic approach is a modification of a previously published procedure [62]. Total cholesterol was determined as above, with the addition of 0.1 U/ml cholesteryl esterase. Readings were obtained after incubation at 37°C for 20 minutes. Cholesteryl esters were determined by subtracting unesterified cholesterol from the total cholesterol. The residual cell matter after lipid extraction was lysed with 0.1 M NaOH, 0.1% SDS, for protein reading using BCA assay kit.
3 H-cholesteryl ester ([ 3 H]CE) generation. 40 µCi of 3H-cholesterol in ethanol was added in 10 µl aliquots to 40 ml of 0.2 µm filtered human serum under sterile conditions. The mixture was incubated at 37°C for 48 hours in the dark. 3H-cholesteryl esters and other lipids were extracted into chloroform: methanol [63]. After evaporation of the solvent, the residue was dissolved in a minimal volume of chloroform. Subsequently, 3H-cholesteryl esters were purified using serial bond elut columns with several hexane passages, as previously described [64].
Western blotting. pHAECs were lysed with lysis buffer (150 mM NaCl, 2% Triton X-100, 20 mM HEPES, pH 7.4, 2% protease inhibitor and 2% phosphatase inhibitor by sonication at 2 setting (Fisher sonic dismembrator model 100) on ice for 10 seconds. After centrifugation at 16,000 g x 10 minutes at 4°C, equal volume of loading buffer (8 M urea, 2% SDS, 125 mM Tris-HCl, pH 7.0, 5% glycerol, 10% beta-mercaptoethanol, 0.06% bromphenol blue) was mixed with the supernatant at room temperature. This was followed by electrophoresis, electrical transfer to PVDF membrane, and immunoblotting.
Dil- and/or [ 3 H]CE-Lipoproteins. Generation of Dil-labeled lipoproteins was obtained essentially as previously described [65], with minor modifications. In brief, a mixture of about 2 mg HDL3, LDL, or VLDL protein and 7 ml of human lipoprotein deficient serum was mixed with ~ 1 mg [3H]CE and/or 0.5 mg Dil in 10 µl DMSO aliquots under sterile conditions. After covering with foil, the mixture was incubated at 37°C for 21 hours. The lipoproteins were repurified as described above, after adjusting to their respective densities with solid KBr. Combined Dil, [3H]CE-LDL radioactivity was 8.1 dpm/µg protein.
LDL iodination with Na 125 I. About 2 mg LDL protein in PBS and 10 iodination beads were incubated with Na125I for 10 minutes at room temperature in glass vials. The transformation was stopped with 50 mM each of unlabeled sodium iodide and sulfite, and 100 µM BHT in DMSO (0.01%). The labeled LDL was washed with desalting columns, then passed through centrifugal filters, 3 kD MWCO, against dialysis buffer.
125 I-LDL cell surface binding. Confluent pHAECs on plastic dishes were cultured with 0 or 5 ng/ml TNFɑ in 15% serum for 48 hours. Treatment continued with serum-free medium in the continued presence of TNFɑ for 3 hours to deplete surface-bound LDL. Subsequently, 125I-LDL was added to 15 µg/ml for 1 hour at 37°C. The cells were then washed two times with PBS at room temperature, then chilled on ice. Afterwards, serum-free medium at 4°C with 0 (buffer) or 300 µg/ml LDL was added. Following additional incubation at 4°C for 1 hour, the radioactivity released to the medium was taken as surface-releasable 125I-LDL.
LDL oxidation. LDL was dialysed against PBS at 4°C, passed through 0.2 µm filter, and incubated at 37°C for 22 hours with 5 µM CuSO4 in PBS under sterile conditions. After adding BHT in DMSO (0.005%) to 1 µM and Na2EDTA to 10 mM, the mixture was dialysed against dialysis buffer, and finally filtered through 0.2 µm membrane under sterile conditions.
TBARS Assay. 2-thiobarbituric assay to estimate the extent of LDL oxidation was determined essentially as previously described [66], with some modifications. oxLDL was mixed successively with 3 volumes of 0.67% 2-thiobarbituric acid in 50 mM NaOH and 20% trichloroacetic acid, each containing 1 mM Na2EDTA, respectively. After heating at 55°C for 1 hour, the mixture was centrifuged at 16,000 g x 30 seconds. Fluorescence (530 nm excitation, 590 nm emission) reading in the supernatant was determined. Malonaldehyde bis-(dimethyl acetal) was used as a malondialdehyde (MDA) precursor for determining malondialdehyde standards. Care should be taken when performing this procedure, as volatile products are generated at 100°C. Standards up to 5 nmol/ul were used. oxLDL was measured at 54 nmol MDA equivalents/ mg protein.
Depletion of apoE from Dil-lipoproteins. Normal mouse IgG agarose or apoE3 agarose beads were washed 4X with dialysis buffer under sterile conditions. Subsequently, Dil-HDL3 or Dil-LDL was added to the beads in dialysis buffer. Following 24 hour incubation at 4°C, the unbound lipoproteins were sterile filtered through 0.2 µm membranes.
Cell surface biotinylation. Serum media from confluent pHAECs were replaced with 400 U/ml sodium heparin in mHBSS on ice for 5 minutes, washed 2X with the the same buffer, then 1X with ice cold mHBSS. After 1X wash with ice cold 1X PBS, the cells were treated with EZ Link Sulfo NHS Biotin in 1X PBS for 30 minutes at 4°C. Unreacted biotin reagent was quenched with 100 mM glycine in PBS at 4°C for 5 minutes. Following 1X wash with ice cold mHBSS, the cells were lysed on the plates with lysis buffer as indicated above under Western blotting on ice for 30 minutes, followed by sonication at 2 setting for 10 seconds on ice. Aliquots of the supernatant was mixed with 1 volume of 2X urea loading buffer without heating. The rest of the supernatant was mixed with strepavidin mag sepharose (pre-washed 2X with incubation buffer: 1% BSA, 0.1% NaN3, 0.1% Triton X-100). After 10X dilution with the incubation buffer containing 0.5% each of protease inhibitor and phosphate inhibitor cocktails, the suspension was swirled for 12 hrs at 4°C. The beads were washed 4X with precipitation buffer, then mHBSS at 4°C. After 1X urea loading buffer (1:1 volume of lysis buffer and 2X urea loading buffer), with 1% protease and phosphatase inhibitors, respectively, the samples were heated at 95°C for 5 minutes to solubilize the bound biotinylated proteins.
Dextran-Mn separation of intact and non-intact 125I-LDL. Intact 125I-LDL was separated from non-intact LDL using dextran sulfate, Mn2+ procedure essentially as previously described [67]. To the BL medium, d < 1.21 g/ml FBS was added to 15% to produce a visible precipitate, mixed, then dextran sulfate and MnCl were added to 65 mg/ml and 0.2 M, respectively. After incubation for 20 minutes at room temperature, the mixtures were centrifuged at 5000 g x 5 minutes at 4°C. The pellet was redissolved with 20 mg/ml dextran sulfate before scintillation counting.
Immunofluorescence. Confluent pHAECs in serum medium were incubated with 0 or 100 ng/ml TNFɑ for 24 hours. Subsequently, the media were replaced with serum-free medium containing 10 µg/ml normal goat IgG or LDLR Ab (R&D Systems) in the continued presence of 0 or 100 ng/ml TNFɑ for 0, 5, 30, or 120 minutes at 37 ºC. Afterwards, the cells were chilled on ice and further incubated at 4 ºC for 1 hour. Then the cells were washed 2X with ice-cold mHBSS, fixed with ice-cold methanol at -20 ºC for 10 minutes, washed once with mHBSS at room temperature, then blocked with 50% human serum in mHBSS, 0.1% sodium azide (blocking buffer) for 1 hour at room temperature. Afterwards, it was replaced with 4 µg/ml red antigoat antibody in blocking buffer. Following 30 minute incubation at room temperature, the medium was replaced with mHBSS, washed 3X with blocking buffer (10 minutes each at room temperature), fixed with 4% paraformaldehyde in PBS, followed by fluorescence microscopy.
Fluorescence Microscopy. Fluorescence microscopy was performed using Keyence phase-contrast fluorescence microscope. Images were analyzed using CellProfiler [68]. Confocal microscopy was performed using glass bottom dishes (Cellvis).
STATISTICAL ANALYSIS. Data are reported as averages ± standard deviation. n = number of independent experiments performed on separate days. Most experiments were performed with an average of 3 or greater replicates. Analysis of variance, followed by Tukey post hoc testing was done by using statpages.info website.