2.1 Diesel exhaust particles (DEPs)
Diesel exhaust particles (DEPs) were collected from a Japanese automobile engine by Dr. Masaru Sagai, who subsequently provided them to researchers at UCLA. Our group obtained them as a gift from Dr. David Diaz-Sanchez, formerly of UCLA. The particles have been characterized and used extensively (Bai et al., 2001
; Inoue et al., 2006
; Ito et al., 2000
; Kumagai et al., 1997
; Sagai et al., 1993
; Singh et al., 2004
). DEP powder (0.1 g) was suspended in 10 ml in PBS, 0.05% Tween 80 to make a 10 mg/ml DEP stock solution. Particles were then dispersed to achieve a particle size of PM2.5
(2.5μm diameter and smaller) by vortexing for 3 minutes, then sonicating at 60 Hz for 5 minutes. To determine the range of sizes, an aliquot was fixed with 4% paraformaldehyde for examination at 630X magnification (Leica TCS SP2 Spectral Confocal Microcope). A more accurate assessment was made by dynamic light scattering using a Zetasizer Nano ZS90 with Malvern DTS software version 5.10 (Malvern Instruments, Malvern, MA). With this technique, particles are placed in a laser beam. The intensity of the scattered light fluctuates at a rate that is dependent upon the size of the particles, with smaller particles moving more rapidly. Analysis of the intensity fluctuations yields the velocity of the particles’ Brownian motion. The particle size is then determined using the Stokes-Einstein equation for diffusion of spherical particles though liquid. Specifications were: temperature, 25°C; material refractive index, 1.59; material absorption, 0.01; dispersant refractive index, 1.33; viscosity, 0.8881 centipoise; measurement position, 4.65 (mm). Six runs (120 sec/run) were performed to determine mean particle diameter. For cell exposures, dilutions of the stock suspension to 1, 10 or 100 μg/ml in medium were made immediately after vortexing and sonicating. Additional concentrations of 5 and 50 μg/ml DEPs were prepared prior to modified LDH assays.
2.2 Endothelial cell culture
Medium used was EBM-2 Bulletkit medium (Lonza), an endothelial cell growth medium which contains 2% FBS, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, heparin, and GA-1000 as purchased. In addition, since the DEPs were dissolved in 1x PBS (137 mM NaCl, 2.7 mM KCl, 10 mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic, pH 7.4.), 0.05% Tween-80, the medium was also supplemented to the same concentration with phosphate buffered saline and Tween-80, thereby minimizing differences between non-DEP-exposed controls and DEP-treated samples. In all cases below, the term “medium” refers to medium plus PBS-Tween-80.
Normal human umbilical vein endothelial cells (HUVECs) were obtained from Clonetics (Lonza Walkersville, Inc.) and used at passages 5–15. Cells were always plated at a density of 156 cells per mm2. This translates to 6 × 104 cells per well on the 12 well plates and 1.5 × 105 cells per well on the 6 well plates. Cultures were incubated in a 5% CO2 atmosphere at 37°C in a volume of medium proportional for the cell number, to insure that culturing parameters were always comparable between different well sizes. Medium was changed every day.
For monolayer cultures, HUVECs were plated on plastic tissue culture dishes. When used for assembling tube structures, cells were plated on the basement membrane substratum, Matrigel, a liquid at 4°C which becomes solid at room temperature or above. LDEV-free Matrigel (BD Biosciences) at 10 mg/ml, 4°C, was added to plates to completely coat the bottoms of 12-well (3.8 cm2/well) or 6-well (9.6 cm2/well) culture dishes residing on ice. Matrigel-coated dishes were transferred to the incubator to allow the substratum to solidify at 37°C for 30 minutes before adding cells.
2.3 Preliminary Assessments of Endothelial Tubes
Tube formation time was determined by seeding HUVECs onto Matrigel-coated dishes, and incubating them at 37°C for 1, 2, 4, 6, 12 and 24 hr. Cells were fixed with 4% paraformaldehyde for 10 minutes at room temperature. DAPI (1 ml/well of 300 nM final concentration in PBS) was used to stain nuclei in the samples after fixing. Phase contrast microscopy (Zeiss-Axiovert 40 Inverted Microscope) evaluated tube formation, and showed that by 12 hr tube formation was totally complete, i.e., every DAPI stained nucleus resided in a cell participating in a tubular structure. For DEP exposure experiments, endothelial cells were plated on plastic (to form monolayers) or on Matrigel (to form tubes) at a density of 156 cells/mm2 for 12 hr, allowing the Matrigel samples to complete tube network formation. This 12 hr post-plating time was defined as the “zero” time point in experiments. Endothelial tubes were incubated with either 0 μg/ml DEP (i.e. no DEP), or 1, 10 or 100 μg/ml dispersed DEPs in medium, unless otherwise indicated. Cultures were incubated at 37°C for 24 hr after the zero time point unless otherwise indicated.
Endothelial cell behavior as monolayers and as tube network cultures was compared. Proliferation was measured using the MTS assay, measuring mitochondrial enzyme activity via conversion of MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and phenazine methosulfate to formazan (MTS kit, Promega). Three plates of cells were assayed at the zero time point (12 hr after plating for both monolayers and endothelial tubes), another set of 3 at the 24 hr post zero time point, and a third set at 48 hr post zero time point. Cells were rinsed 3 times with cold PBS, then a mixture of 60 μl water-soluble kit reagent plus 300 μl fresh medium was added to each well for a 1 hr incubation at 37°C in the dark. Supernatants (100 μl/well) were collected and the absorbance of the generated formazan was measured at 490 nm. This absorbance reflects the total number of cells in each sample, and allows calculation of doubling time.
2.4 Modified LDH cytotoxicity assay to detect cell survival after DEP exposure
DEP cytotoxicity to endothelial tubes was evaluated using the CytoTox-Homogeneous Integrity Assay Kit (Promega), a method that measures cytosolic lactate dehydrogenase (LDH) released into medium when cells are lysed. The method was adapted to remove particles and dead cell-derived-LDH from the living cells after DEP exposure, to ensure the DEPs would minimally interfere with the assay. Plates of formed endothelial tubes were exposed to medium alone (the no DEPs control) or medium containing DEPs (1, 5, 10, 50, and 100 μg/ml medium) for a 24 hr 37°C incubation. Next, medium (containing LDH from dead cells and floating cell debris) was aspirated from the cultures. Endothelial tube cells were then washed 3 times in cold PBS. Cells were collected by centrifugation and lysed for 1 hr in 200 μl lysis solution (Promega) following the manufacturer’s instructions. The relative fluorescence (described in RFUs, relative fluorescence units) of LDH was measured at 490 nm. Unexposed cultures were used as the positive control, defining the maximum amount of LDH potentially released. This value was defined as 100%. With the adaptation described, the absorbance levels of DEP-exposed samples represent cells surviving the exposure, and were expressed as a percentage of the control unexposed sample ().
Cytotoxicity of DEP on HUVEC tubes
2.5 VE-Cadherin Immunofluorescence
HUVECs (6 × 104 cells/well, plated at 156 cell/mm2 density) were seeded onto Matrigel-coated 2-well chamber slides for tube formation prior to DEP exposures for 24 hr. After exposure, endothelial tubes were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Nonspecific reactivity of HUVECs was blocked by addition of 2% normal goat serum with 0.02% sodium azide (NaN3) in PBS for 1 hr at room temperature. The endothelial tube cells were then incubated with primary anti-human VE-cadherin (BD Biosciences) monoclonal antibody at a 1:50 dilution (20 μl in 1 ml blocking buffer, i.e., 2% normal goat serum) for 1 hr at room temperature. Goat anti-mouse secondary antibody labeled with Alexa 488 (green color, Jackson Immuno Research) was used at a 1:100 dilution (10 μl in 1 ml PBS) for 1 hr at room temperature. When wide field (epifluorescence) microscopy was used, nuclei were stained by incubating endothelial tubes in 300 nM DAPI for 5 min at room temp, followed by washing with PBS/Tween for 5 min. When confocal microscopy was used, nuclei were stained by adding 1 ml 20 μM DRAQ5 (Alexis) for 10 min at room temperature to each well. Slides were covered with Prolong Gold (Invitrogen) anti-fade mounting media and incubated at 4°C overnight. All images were observed at 100X and 400X magnifications on an epifluorescence microscope (Olympus IX71 Inverted Microscope) or at 630X magnification (water lens, N.A. 1.3) on a Leica TCS SP2 Spectral Confocal Microscope.
To evaluate the extent of the VE-cadherin disruptions in the plasma membrane induced by DEP exposure, confocal images of a random selection of cells in endothelial tube networks were examined where 30 μm or more of plasma membrane was found in the plane of focus. Magnification of these 30 μm stretches of plasma membrane showed that interruptions of ~4 μm were not uncommon in unexposed endothelial tube cells, therefore these were considered to represent regions where the cell membrane moved out of the plane of focus. However, 3 μm interruptions in the fluorescent pattern were increasingly predominant after exposure to increasing concentrations of DEP, therefore they appeared to represent DEP-induced disruptions in VE-cadherin, and were tallied. This size criterion was defined to unbias the hand counting of interruptions as much as possible, and yields an analysis that represents a trend, and is referred to as a semi-quantitation. About 200 cells from 12 different control samples, as well as 12 different samples of each exposure condition, were examined. The endothelial membranes of the 100 μg/ml DEP-exposed samples were too disrupted to assess for discontinuities. Internalized globules of VE-cadherin were also assessed: Areas where immunofluorescent VE-cadherin pulled away from the membrane toward an intracellular position were counted. Globules were scored as equal to or under10 μm or greater than 10 μm, and were assessed in ~200 cells from each set of samples.
2.6 Western Analyses
After 24 hr DEP exposure, the endothelial tube cells were collected with the Matrigel at room temperature and sonicated for 1 min in lysis buffer (20 mM Tris-HCl, 0.5% deoxycholate, 0.5% SDS, 1% Triton X-100, 1% Nonidet P-40, 1 mM Na3VO4 and 0.1% protease inhibitor). The solid Matrigel and cell debris was removed by centrifugation at 10,000 rpm for 10 min at room temperature. The protein concentration of the supernatant was measured by absorbance at 540 nm using the bicinchoninic acid method (BCA Protein Assay, Pierce). Twenty μg/well were loaded onto SDS polyacrylamide gels for electrophoresis. Proteins were transferred to 0.45 μ PVDF membrane using electrophoretic transfer (Bio-Rad). Nonspecific reactivity was blocked for 1 hr at room temperature with standard 1x TBST buffer containing 3% BSA and 0.02% NaN3. Primary antibody against VE-cadherin (BD Biosciences), diluted 1 to 1000, or against β-catenin (Abcam), diluted 1 to 2000, or against actin (Sigma), diluted 1 to 1000, or against glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Sigma), diluted 1 to 5000 with blocking buffer, was added to a blot and incubated overnight at 4°C. After washing in 1x TBST, secondary antibodies conjugated with horseradish peroxidase (HRP) were diluted 1 to 5000 in 5% milk, 1x TBST, and applied to the blots for a 1 hr incubation at room temperature. Blots were then reacted with ECL reagent (Pierce) containing luminol, a substrate of horse radish peroxidase (HRP), and exposed to X-ray film.
For HUVEC proliferation, the data included 3 replicates from a single experiment. Thus, a one-way analysis of variance (ANOVA) was used to examine differences in time with Dunnett’s procedure to compare 6, 24 and 48 hr to baseline (0 hr).
For the LDH values at 24 hr, a mixed model analysis evaluated the differences between DEP concentrations of 0, 1, 5, 10, 50 and 100 μg/ml. Three experiments were conducted on different days with three replicates at each concentration for each experiment. The average relative fluorescent unit per dose, with the background average subtracted off, was used as the unit of experiment. As such, analysis was conducted using a mixed model with dose as a fixed effect and a random effect for experiment/day. Dunnett’s procedure, creating p-values that are adjusted for multiple testing, was used to compare all non-zero doses to the zero exposure.
For discontinuities and globules, binomial regression was used to compare the probability of discontinuity/globularity for each nucleus (i.e., per cell). In this approach, the number of cells on the slide per nucleus is considered as the number of trials with the number of discontinuities/globules out of that number modeled as a binomial response variable, with dose (0, 1, or 10 μg/ml of DEP) predicting the probability of discontinuity or globularity within each cell. A Wald Chi-square test was used to evaluate the effect of dose, with a Bonferroni correction applied (individual significance level of 0.025 applied to each comparison in order to maintain a family-wise error rate of 0.05) when comparing each non-zero dose to the zero dose. A value of p < 0.05 was considered statistically significant. Statistical inference was not appropriate for the 100 μg/ml DEP-exposed samples. Observations have been described as best as possible, but statistical significance cannot be calculated.