2.1. Diesel exhaust particles and cell culture
Japanese automobile diesel exhaust particles (DEPs) were collected by Dr. Masaru Sagai, who donated them to researchers at UCLA. Particles were a gift from Dr. David Diaz-Sanchez, formerly of UCLA. The effects of these particles have been studied in a number of in vitro
and in vivo
models (Bai et al., 2001
; Inoue et al., 2006
; Ito et al., 2000
; Kumagai et al., 1997
; Sagai et al., 1993
; Singh et al., 2004
). A 10 mg/ml DEP stock solution was made by suspending 0.1 g of DEP in 10 ml in PBS, 0.05% Tween 80. Particles were vortexed for 3 minutes, then sonicated at 60 Hz for 5 minutes, then assessed six times (120 sec/run) by dynamic light scattering using a Zetasizer Nano ZS90. This yielded particles of ~30 nm to ~1050 µm (see Chao et al., 2010), fitting into the commonly used category of PM2.5
(2.5µm diameter and smaller). After vortexing and sonicating, the stock suspension was immediately diluted to 1, 10 or 100 µg/ml in medium, ending up with a final concentration of 1X PBS, 0.05% Tween 80 due to the particle suspension. (1X PBS is 137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic, pH 7.4.) The dilutions were immediately applied to the endothelial tube cultures via pipet.
Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial cell growth medium EBM-2 Bulletkit (Lonza) as previously described (Chao et al., 2011
). Medium was supplemented with phosphate buffered saline and Tween-80 to make a final concentration of 1X PBS, 0.05% Tween-80. The PBS/Tween-80 was always included to minimize differences between non-DEP-exposed controls and DEP-treated samples, since DEPs are suspended in 1X PBS, 0.05% Tween-80. (This Tween-medium with dispersed DEPs was prepared in exactly the same manner as that used in the Zetasizer Nano ZS90 determination of particle size, ensuring the particles were consistent in all experiments.) HUVECs at passage 5 to 15 were used to assemble tubes on 10 mg/ml LDEV-free Matrigel (BD Biosciences), that was solidified onto 2-well chamber slides (120 µl Matrigel/well) or 6-well (150 µl Matrigel/well) plastic tissue culture plates. The Matrigel was solidified for at least 30 min before adding cells. HUVECs were plated at 156 cells/mm2
on both the 2-well chamber slides (6 X 104
cells/well), and on the 6-well plastic dishes (1.5 X 105
cells/well), to ensure that parameterns were equivalent. Endothelial tubes were allowed to form for 12 hr prior to adding Tween/medium containing dispersed DEPs. Medium was changed daily. For monolayer cultures, endothelial cells were treated exactly the same, but were plated at the same density directly on plastic wells without Matrigel. Once confluent, cells were allowed to incubate for 12 hr, the same time allotted for the cells forming tubes on Matrigel. Well characterized DEPs (Bai et al., 2001
; Singh et al., 2004
) were prepared as previously described to create a suspension resulting in sizes of PM2.5
(Chao et al., 2011
). A volume of medium proportional to the well size was added, delivering either medium (medium always with PBS-Tween alone (i.e., 0 µg DEPs/ml), or 1, 10, or 100 µg DEPs/ml to each well in a volume to make exposures always equivalent per cell between the two well sizes. Unless otherwise indicated, analysis was performed after a 24 hr incubation at 37°C. Since Matrigel is a liquid at 4°C, endothelial tubes could always be separated from the Matrigel by putting the culture dishes in the refrigerator, then pipetting out the Matrigel. If samples were at room temperature, cells could be lysed in the culture dishes, then separated from the substratum and cell debris by scraping the entire contents into a centrifuge tube, and spinning it at 10K × g.
N-Acetyl Cysteine was obtained from Sigma-Aldrich (St. Louis, MO). Tin protoporphyrin IX (SnPP) was bought from Frontier Scientific, Inc. (Logan, UT). Other reagents employed are incorporated into the appropriate method sections below.
2.3. Analysis of intracellular ROS accumulation
The ROS detection studies were performed using an Image-iT™ Live Green Reactive Oxygen Species Detection Kit (Molecular Probes/Invitrogen). The method involved analyzing the intracellular accumulation of ROS due to H2O2 generation, based on the conversion of the non-fluorescent probe carboxy-H2DCFDA (2’, 7’-dichlorohihydrofluorescein diacetate) to green-fluorescent carboxy-DCF. Endothelial tubes were treated with 0, 1, 10 and 100 µg DEPs/ml for 24 hr. DEPs were then washed away with 37°C 1X PBS, and the samples were incubated for 25 min at 37°C with fresh medium containing 25 µM carboxy-H2DCFDA, which penetrates cells. After washing again with 37°C 1 X PBS, ROS detection was visualized with epifluorescence microscopy at 200X magnification (emission at 495–529 nm, Olympus IX71 Inverted Microscope), examining the carboxy-DCF which could not be transported out of the cells.
2.4. Measurement of H2O2 production
H2O2 production was assessed with 10-acetyl-3,7-dihydroxyphenoxazine using a commercially available Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/ Invitrogen), with some protocol modifications. First a standard curve was prepared using serial dilutions of H2O2. Endothelial tubes were treated with various concentrations of DEPs (0, 1, 10, and 100 µg/ml) for 24 hr, then were scraped from wells and collected in pH adjusted SDS-PAGE buffer (25 mM Tris, 192 mM glycine, pH 7.4, 0.1% SDS) to which 1/1000 volume of Sigma’s protease inhibitor cocktail (cat. # P2714, containing AEBSF, Aprotinin, bestatin hydrochloride, E-64, EDTA and Leupeptin) was added. The buffer contained no azide, so as not to interfere with the Amplex Red. The protein in the lysate was quantitated, and made 4000 µg/ml. A 2.5 µl aliquot of lysate was added to 47.5 µl 1X PBS, then 50 µl of Amplex Red reagent/0.2 U/ml horse radish peroxidase, 0.5 mM NADPH solution were added, and the mixture was incubated at room temperature for 30 min. The absorbance of samples was read on an HTS 7000 Plus Bio Assay Reader (540 nm excitation, 590 nm emission-Perkin Elmer Life Sciences). The amount of H2O2 was determined by comparison to the standard curve.
2.5. Assessment of oxidative modifications of proteins
ROS-induced oxidative alterations in proteins (i.e., amino acid side chains modified with carbonyl groups by ROS) was detected using an OxyBlot Protein Oxidation Detection kit (Chemicon). Briefly, the HUVEC tubes were scraped from wells, sonicated for 1 min with pH adjusted SDS-PAGE buffer (as above) containing a final concentration of 0.1% Sigma’s P2714 protease inhibitor cocktail, then Matrigel and cell debris were removed by centrifugation at 10,000 × g for 10 min. Protein concentrations were determined using the bicinchoninic acid method (BCA Protein Assay, Pierce) at absorbance 540 nm. Lysate proteins (5 µg/sample) were derivatized with 1,3-dinitro-phenyl-hydrazine (DNP) following the manufacturer’s protocol, and subjected to SDS-PAGE on 12% gels. Proteins were electrotransferred to 0.22 µm nitrocellulose membrane blots (Bio-Rad). Blots were incubated for 1 hr at room temperature in blocking buffer (3% BSA with 0.02% NaN3 in TBST [25 mM Trizma base, pH 7.3, 3.0 mM KCl, 140 mM NaCl and 0.05% Tween-20,]), then incubated with the kit’s polyclonal DNP antibody (diluted 1:150) for 1 hr at room temperature. This was followed by incubation with goat anti-rabbit HRP-conjugated IgG secondary antibody (Bio-Rad cat. #170-6515), diluted 1-5000, for 1 hr at room temperature. The nitrocellulose membrane was treated with Super Signal West Pico chemiluminescence reagent (Pierce) for visualizing immunoreactive proteins on X-ray film. The band intensities of the many carbonyl-modified proteins in DEP-exposed sample lanes were visually compared with that of the negative control (i.e., endothelial tubes not exposed to DEPs) to make a qualitative evaluation, but differences were not quantitated.
2.6. Protein Preparation, Immunoprecipitation and Western Blot analysis
For Western blots of cell lysates, HUVEC tubes were collected and lysed by sonication for 1 min in pH adjusted SDS-PAGE buffer supplemented with 0.1% Sigma’s protease inhibitor cocktail, then Matrigel and cell debris were separated out by centrifugation at 10,000 × g for 10 min. The concentration of protein in each sample was measured using the bicinchoninic acid method (BCA Protein Assay, Pierce). Cell lysate proteins were loaded (30 µg per well) onto SDS polyacrylamide gels for electrophoresis.
Nuclear protein extracts were prepared from the endothelial tube cells by adapting a one hour minipreparation technique (Deryckere and Gannon, 1994
). Briefly, cells were collected and sonicated 1 min in a relatively low salt lysis buffer (0.6 % Nonidet P-40 [NP-40], 150 mM NaCl, 10 mM HEPES pH 7.9, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride [PMSF]), then centrifuged for 30 sec at 2500 rpm. The supernatant, which contained the nuclei, was next incubated for 5 min on ice, then centrifuged for 5 min at 5000 rpm. The pelleted nuclei were resuspended in a higher salt lysis solution (25% glycerol, 20 mM HEPES pH 7.9, 420 mM NaCl, 1.2 mM MgCl2
, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 2 mM benzamidine, and 5 µg/ml of aprotinin), and incubated on ice for 20 min. Insoluble nuclear debris was pelleted by a 30 sec centrifugation. The protein concentration of the whole cell extract and the supernatant containing the nuclear extract was determined using a BCA Assay (Pierce), and the samples were frozen in liquid nitrogen and stored at −80°C until used for SDS-PAGE, when 20 µg protein per lane was applied to the gels.
For immunoprecipitation, 1.5 mg/50 µl Dynabeads (Immunoprecipitation kit-Dynabeads Protein A, Invitrogen) were incubated with primary rabbit monoclonal anti-human VE-Cad antibody (1:80 dilution, Abcam) at 4°C, overnight. The conjugated Dynabeads-antibody were placed on a magnet, the supernatant was removed, then cell extracts were added. The protein in the extract was quantitated, and made 4 mg/ml, and a 250 µl aliquot was added to the beads (= 1 mg) for a 10 min incubation with rotation at room temperature. The Dynabeads-antibody-extracts were then washed and the immunotargeted protein was eluted from the Dynabead sample following the manufacturer’s instructions. The eluates were denatured by heating at 95°C for 5 min and loaded onto 7.5% SDS polyacryamide gels for electrophoresis. Dynabeads conjugated with rabbit IgG (cat # 011-000-003, Jackson ImmunoResearch) were incubated with cell lysates as negative controls.
Because endothelial tubes are always plated at the same density and incubated in the same relative volume of medium, for secreted products, 40 µl of medium was used directly for SDS-PAGE. When this was done, samples were applied in order on the gels twice. After electrophoresis, the gel was cut in half. One half was used for the Western blot and the other half was stained with Coomassie blue to evaluate the equivalence of lane loading.
For all of the protein preparations mentioned (i.e., whole cell, nuclear, immunoprecipitated and medium proteins), after electrophoresis the proteins were electrophoretically transferred to 0.22 µm nitrocellulose membrane. A 1 hr incubation at room temperature was performed in blocking buffer (3% BSA with 0.02% NaN3 in TBST) to reduce non-specific reactivity of antibodies. All primary antibodies were diluted 1 to 1000 for use unless indicated. These were: rabbit anti-rat HO-1 antibody (SPA-895, Stressgen); rabbit anti-human anti-Nrf2 peptide antibody (ab31163, Abcam); rabbit anti-human polyclonal antibody against VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGFR2 from Abcam; rabbit anti-mouse GAPDH antibody (G9547, Sigma). Antibodies against VEGF-B and C were diluted 1to 1000, but VEGF-A required a 1 to 500 dilution, VEGF-D was 1–250 and VEGFR2 1 to 200. The respective companies all indicated that the non-human antibodies cross react with the corresponding human proteins, and this was indeed the case. Incubations were at 4°C, overnight. The secondary antibody for each was goat anti-rabbit IgG (H+L) conjugated to horseradish peroxidase (HRP) (Bio-Rad cat # 170-6515), diluted 1 to 5000, incubating blots for 1 hr at room temperature. Blots were enhanced with Super Signal West Pico chemiluminescence reagent (Pierce), and exposed to X-ray film.
2.7. Immunofluorescence Microscopy
HUVECs were plated as monolayers or were seeded onto Matrigel-coated 2-well chamber slides for tube formation. After DEP treatment for 24 hr, the HUVECs were fixed with 4% paraformaldehyde for 10 min. When antibody was required to reach intracellular locations, the fixed endothelial cells were permeabilized with 0.05% Triton X-100 for 10 min at room temperature. Nonspecific reactivity was blocked by incubation with 2% normal goat serum plus 0.02% NaN3
in PBS for 1 hr at room temperature. Endothelial tubes were incubated with primary polyclonal antibody against Nrf2 (Abcam ab31163) at a 1:100 dilution for 1 hr at room temperature, followed by a 1 hr room temperature incubation with goat anti-rabbit secondary antibody labeled with Alexa 488 (Molecular Probes/ Invitrogen cat # A11008), diluted 1 to 200. The VE-Cad antibody was used on monolayer and endothelial tube cells as in Chao et al. (2011)
, and the VEGFR2 antibody (Abcam) was diluted 1 to 200 prior to use.
Prolong Gold anti-fade mounting media including DAPI (Invitrogen) was added to sections, and slides were covered for an overnight incubation at 4°C. Monolayer and comparative endothelial tube images were observed at 100X and 400X magnifications on a wide field (epifluorescence) microscope (Olympus IX71 Inverted Microscope). Other endothelial tube images were observed at 630X magnification on a Leica TCS SP5 Spectral Confocal Microscope.
2.8. Permeability Assays
For monolayer cultures of HUVECs, cells were seeded at 1.5 X 105 cells per well and grown to confluence on fibronectin (7 µg/ml)-coated filters of transwell units (6.5 mm, 0.4 µm pore size, Corning Costar). A non-confluent monolayer culture (1.5 X 104 cells per well, 15.6 cells/mm2) served as a control for 100% permeability. Culture medium volume in each upper chamber was 100 µl and in each lower chambers was 600 µl. When the cultures reached confluence (3 to 5 days), the cells in the upper wells were exposed to various concentrations of DEP (0, 1, 10, 100 µg/ml), incubating them for 24 hours at 37°C. Then, FITC-conjugated dextran (molecular mass 70 kDa, Sigma) was added and allowed time to penetrate to the lower chamber. In initial experiments, unexposed confluent monolayers were used to see how long it took for any FITC-dextran to reach the medium in the lower chamber. Aliquots (100 µl/well) were collected in 3 hours intervals (0–3, 3–6, 6–9, and 9–12 hours time frames) while continuously incubating the cultures at 37°C. Small quantities of fluorescent dextran, assessed at 490 nm, penetrated the confluent monolayers and gained access to the lower chamber between 1–6 hrs. Since no dextran was obtained in the 7–9 hour period after adding the agent (data not shown), and since >98% of this dextran entered the lower chamber in the first 4 of the 6 hr preliminary test time, 4 hr after FITC-dextran application was chosen as the time to analyze the monolayers for permeability. Samples, including non-confluent control samples, were also evaluated by phase contrast microscopy (200X, Zeiss-Axiovert 40 Inverted Microscope).
Since no method exists for evalulating the permeability of in vitro endothelial tubes, it was reasoned that if DEP caused leakiness of the capillary-like structures, the dextran would be able to enter leaky cells of the capillary-like structures due to the lack of blood flow. First endothelial tubes were made, seeding 1.5 X 105 HUVECs per well onto Matrigel-coated 6 well plates. Tubes were allowed to form over 12 hr. As controls, some wells were coated with Matrigel only (no cells were plated), and some wells were coated with Matrigel, then plated with cells to form tubes, but were never exposed to DEP (0 µg/ml DEP). Test sample wells were coated with Matrigel, then cells were added and allowed to form tubes for 12 hr before exposure to 1, 10, or 100 µg/ml DEPs for 24 hours at 37°C. After incubation, free DEPs were washed away with PBS, and medium (1 ml per well) containing FITC conjugated-dextran at a final concentration of 1 mg/ml was added into the culture dishes and incubated for 4 hours. At the end of this time period, the medium/dextran was removed and discarded. The endothelial tubes were washed twice with PBS and collected by scraping them from the wells into eppendorfs containing 25 mM Tris, 192 mM glycine, pH 7.4, 0.1% SDS to which 1/1000 volume of Sigma’s protease inhibitor cocktail (cat. # P2714, containing AEBSF, Aprotinin, bestatin hydrochloride, E-64, EDTA and Leupeptin) had been added. The sample was gently pipetted up and down to make an extract of the cells. After BCA quantitation, samples were all adjusted to 4 mg protein per ml. To ascertain if any FITC label had been able to access the tube structures, an aliquot of 200 µl of each lysate was loaded into 96-well plates for determination of the relative fluorescence units (RFU) of each sample. Reading was done on an HTS 7000 Plus Bio Assay Reader at 490 nm-Perkin Elmer Life Sciences. The “Matrigel only” samples were treated identically as the test samples, although there were no cells to extract. The FITC-dextran adhering to Matrigel samples was averaged and plotted, and is indicated as the blank in Figure 4D. The RFU value of the second control, dishes of endothelial tubes receiving no DEPs, represented the baseline adherence of dextran to unexposed tubes, and is indicated as 0 µg/ml DEP in Figure 4D. These values are indicated on the graph in Figure 4. RFU values greater than this baseline value are considered to be representative of the passage of FITC-dextran into the cells of permeable capillary-like structures, thereby indicating a level of endothelial tube permeability after DEP exposure.
For statistical analysis, each experiment was performed at least 2 times, and each time, samples were run in triplicate. The results were expressed as means ± SD and were analyzed using Student t-tests and one way parametric ANOVA. p < 0.05 and p < 0.01 indicated by * and ** respectively, were considered statistically significant.