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Toxicology 297 (2012) 34–46

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DEP induction of ROS in capillary-like endothelial tubes leads to VEGF-A
Ming Wei Chao a , Iris P. Po a , Robert J. Laumbach b , John Koslosky c , Keith Cooper d , Marion K. Gordon a,∗
Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854, USA
Environmental and Occupational Medicine, UMDNJ, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA
Environmental Sciences, Rutgers University, New Brunswick, NJ 08901, USA
Biochemistry and Microbiology, SEBS, Rutgers University, New Brunswick, NJ 08901, USA

a r t i c l e i n f o a b s t r a c t

Article history: Inhalation of diesel exhaust particles (DEPs) is associated with pulmonary and cardiovascular disease.
Received 7 March 2011 One contributor to pathogenesis is inhaled particles reaching and injuring the lung capillary endothe-
Received in revised form 13 March 2012 lial cells, and possibly gaining access to the blood stream. Using in vitro capillary tubes as a simplified
Accepted 29 March 2012
vascular model system for this process, it was previously shown that DEPs induce the redistribution of
Available online 7 April 2012
vascular endothelial cell-cadherin (VE-Cad) away from the plasma membrane to intracellular locations.
This allowed DEPs into the cell cytoplasm and tube lumen, suggesting the tubes may have become per-
meable (Chao et al., 2011). Here some of the mechanisms responsible for endothelial tube changes after
Diesel exhaust particles
Endothelial tubes
DEP exposure were examined. The results demonstrate that endothelial tube cells mounted an oxidative
Toxicity stress response to DEP exposure. Hydrogen peroxide and oxidized proteins were detected after 24 h of
ROS exposure to DEPs. Particles induced relocalization of Nrf2 from the cytoplasm to the nucleus, upregulating
VEGF-A the expression of the enzyme heme oxygenase-1 (HO-1). Surprisingly, vascular endothelial cell growth
VEGFR2 factor-A (VEGF-A), initially termed “vascular permeability factor” (VPF), was found to be up-regulated
VE-cadherin in response to the HO-1 expression induced by DEPs. Similar to DEPs, applied VEGF-A induced relocal-
Endothelial permeability ization of VE-Cadherin from the cell membrane surface to an intracellular location, and relocalization of
VE-cadherin was associated with permeability. These data suggest that the DEPs may induce or contribute
to the permeability of capillary-like endothelial tube cells via induction of HO-1 and VEGF-A.
© 2012 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ROS (Bai et al., 2001; Becker et al., 2005), initiating inflamma-
tory responses (Salvi et al., 1999); alteration of autonomic balance
Diesel exhaust particles (DEPs) are a major component of atmo- (Pope et al., 1999), and disruption of vascular function (Ikeda et al.,
spheric particulate matter (PM) air pollution, which is associated 1998) are a few. All of these mechanisms may contribute to the
with adverse pulmonary and cardiovascular health effects (Brook, final pathology. Our interest is in endothelial dysfunction induced
2008). In the atmosphere, DEPs contribute to fine PM (particles with by DEPs. Vasoconstriction has been demonstrated in humans who
diameters of 2.5 ␮m or smaller, or PM2.5 ) and ultrafine PM (diame- inhaled ambient PM2.5 and ozone for 2 h (Brook et al., 2002; Brook,
ters of 0.1 ␮m, i.e., 100 nm, or smaller, or PM0.1 ), both of which are 2008), and impairment of vasodilatation was observed 24 h after a
able to reach alveolar regions of the lung. Several mechanisms have 1 h diesel exhaust inhalation study (Tornqvist et al., 2007). Impor-
been proposed to explain how inhalation of DEPs and other PM can tantly, 24 h after intratracheal installation of DEPs or DEP extracts,
cause cardiovascular health effects. Inducing production of harmful the water content of mouse lungs increased (Inoue et al., 2006),
indicating an increase in pulmonary endothelial permeability.
DEPs likely injure alveolar epithelia, and possibly also lung cap-
illaries (Bai et al., 2001; Brook et al., 2002; Salvi et al., 1999). If
Abbreviations: DEPs, diesel exhaust particles; HUVEC, human umbilical vein
endothelial cells; PM2.5 , particulate matter with diameters equal to or less than the effect on epithelial and endothelial cells is indirectly induced
2.5 ␮m; VE-Cad, vascular endothelial cell cadherin; ROS, reactive oxygen species; by DEPs, the inflammatory mediators released by neutrophils and
VEGF-A, vascular endothelial cell growth factor-A; VEGFR2, vascular endothelial cell macrophages in response to DEPs may explain the permeability
growth factor receptor-2.
observed in vivo. In addition, DEPs may have direct effects on capil-
∗ Corresponding author at: EOHSI and Department of Pharmacology and Toxicol-
lary endothelia. Inhaled nanoparticles have been shown to reach
ogy, Ernest Mario School of Pharmacy, Rutgers University, 170 Frelinghuysen Rd,
Piscataway, NJ 08854, USA. Tel.: +1 732 445 3751; fax: +1 732 445 0119. the circulation (Geiser et al., 2005; Kreyling et al., 2002, 2009;
E-mail address: (M.K. Gordon). Nemmar et al., 2001, 2002, 2004, 2005; Oberdorster et al., 2004).

0300-483X/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved.

1. redistribution tion. HUVECs were plated at 156 cells/mm2 on both the 2-well chamber non-DEP studies. M. who donated them to researchers at UCLA. 2002. 2011). Masaru Sagai. HUVECs at passage 5–15 were used to erature: (1) adherens junctions are organizers of other cell–cell assemble tubes on 10 mg/ml LDEV-free Matrigel (BD Biosciences)... Singh et al. Analysis of intracellular ROS accumulation facilitate permeability by disrupting the endothelial cell–cell junc- The ROS detection studies were performed using an Image-iTTM Live Green tions. incorporating the following observations from the lit. Keck et al.. tion component. PM0. After vortexing assembled endothelial tubes as a model system to visualize the and sonicating. demonstrating that in in vitro capillary-like endothelial tubes. then pipetting out the Matrigel. 2 mM potassium phosphate monobasic. that was solidified junctions. since this fac. For monolayer cultures. MO).1 g of DEP in 10 ml in PBS. 2008).. 1983.05% Tween-80.. and was initially named vascular permeability factor (VPF) indicated. then cadherin has been shown to be associated with interaction with separated from the substratum and cell debris by scraping the entire contents into the VEGF receptor 2 (VEGFR2) at the endothelial plasma membrane a centrifuge tube. 2001). 2000. In addition.. a more recent report used carbon particles fitting the designation of PM0.5 (2. Nemmar 2. 2002. Inc. VE-cadherin and permeability.. 2004). inducing gaps between the endothelial 2. six times (120 s/run) by dynamic light scattering using a Zetasizer Nano ZS90. and traverse the alveolar membrane to enter the 80. or 1. cell growth medium EBM-2 Bulletkit (Lonza) as previously described (Chao et al. yielded particles of ∼30 nm to ∼1050 ␮m (see Chao et al.. The method The goal of the experiments described below was to explore involved analyzing the intracellular accumulation of ROS due to H2 O2 genera- this potential connection between DEP exposure. 2005. First a standard thereby suggesting a possible pathway that might explain one curve was prepared using serial dilutions of H2 O2 . have a direct effect on DEP stock solution was made by suspending 0. Because of the known association between VE- were then washed away with 37 ◦ C 1× PBS. but were plated at the same density directly on plastic wells without Matrigel.. 0. With these issues in mind. Ito et al. Singh on vascular smooth muscle cells.. 1989.. Materials and methods inhalation studies (Geiser et al.5 ␮m.05% Tween the endothelium. moved from cell–cell junctions to 2011). 10 or 100 ␮g/ml effect of PM2. 2002).. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial tube culture experiments demonstrated that the adherens junc. may in part be reagents employed are incorporated into the appropriate method sections below.. and on the 6-well plastic dishes (1. for 12 h prior to adding Tween/medium containing dispersed DEPs. A 10 mg/ml cles are able to reach the alveolar spaces. David Diaz-Sanchez. Sagai et al. Endothelial tubes were treated aspect of how vascular permeability is initiated VE-cadherin’s by with various concentrations of DEPs (0. 2002). The effects of these particles have been blood (Kreyling et al. (3) in non-DEP studies the cells forming tubes on Matrigel. This would thereby 2.W. 10. The Matrigel was solidified for at least 30 min before to become leaky (for reviews see Dejana et al. 0 ␮g DEPs/ml). and 100 ␮g/ml) for 24 h. Chao et al. Well-characterized DEPs (Bai et al. This Our recent data with confocal microscopy used in vitro. Medium was changed daily. 2001. leading to HO-1 expression.. which pen- we asked whether DEPs would induce VEGF-A/VPF. since DEPs are suspended in 1× PBS. then assessed bloodstream. formerly of UCLA.. A volume of medium proportional to the well size was added. Louis. exposures always equivalent per cell between the two well sizes.7 mM KCl. 0. 2011).e. 1997. Kumagai et al. and the VEGFR2 interaction (2 . DEPs may distribute similarly after inhalation. (2) in adding cells.2. 2006. or exposure to. DEPs. they were found in many organs. examining the carboxy-DCF which could not be transported out of the cells. The dilutions were 2. studied in a number of in vitro and in vivo models (Bai et al. particles consisted of labeled 80 nm and 20 nm nanoparticles. the same time allotted for contributing to endothelial permeability.1 . 2001. VEGF expression..5 (Chao et al...). VEGF-A was first discovered func. then sonicated at 60 Hz for 5 min. ROS detection was visualized tor has potential to be a mechanistic cause of internalization of with epifluorescence microscopy at 200× magnification (emission at 495–529 nm. 10.. to make a final concentration of 1× PBS.. (4) the basis for VEGF’s affect on VE- samples were at room temperature.1 ) and about 85% were greater than 100 nm but less than phate dibasic. fitting into the commonly used category of PM2. analysis was performed after a 24 h incubation at 37 ◦ C. and interruption of them causes the endothelial cells onto 2-well chamber slides (120 ␮l Matrigel/well) or 6-well (150 ␮l Matrigel/well) plastic tissue culture plates. which in turn stimulates secretion of VEGF-A. and VE-cadherin is an impor- Tween-medium with dispersed DEPs was prepared in exactly the same manner as tant player in this property. which is an inducer of (i. scraped from wells and collected in pH adjusted SDS-PAGE buffer (25 mM Tris. The PBS/Tween-80 was rangement of cell–cell junctions is associated with permeability always included to minimize differences between non-DEP-exposed controls and DEP-treated samples. 2004). ending up with a final concentration of 1X PBS. that the generation of H2 O2 may be induced by DEP. and the samples were incubated for cadherin and VEGFR2 in the cell membrane (Weis et al. Such rear. 1990). and that the endothelial cells junctions become disrupted. In fact. or 100 ␮g DEPs/ml to each well in a volume to make vascular permeability. 2008). 2. to lial cells treated with hydrogen peroxide (H2 O2 ) caused junctional ensure that parameters were equivalent. Particles were a gift from 24 h after inhalation. DEPs with VE-cadherin. particles were consistent in all experiments).4. a commercially available Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Molecular Probes/Invitrogen).5 ␮m diameter and smaller). 2004). Unless otherwise tionally. Endothelial tubes were allowed to form components such as VE-cadherin to become disorganized. Ferrara and Henzel. 2011). we hypothe- N-acetyl cysteine was obtained from Sigma–Aldrich (St. monolayers of bovine pulmonary artery endothe. (Weis et al. Kreyling et al. 2004) were prepared as previously described to create a suspension resulting (HO-1) resulted in expression of vascular endothelial cell growth in sizes of PM2.000 × g at room temperature. 10 mM sodium phos- (i. DEP induce ROS such as H2 O2 .. Measurement of H2 O2 production effects of inflammatory cells. VE-cadherin.. cells were allowed to incubate for 12 h. Other observed after inhalation of. Medium was supplemented with phosphate buffered saline and Tween-80 intracellular locations after exposure (Chao et al. Reactive Oxygen Species Detection Kit (Molecular Probes/Invitrogen). Tin pro- sized that the human and murine pulmonary permeability that is toporphyrin IX (SnPP) was bought from Frontier Scientific. 25 min at 37 ◦ C with fresh medium containing 25 ␮M carboxy-H2 DCFDA. 0.05% Tween 80 due to number. an a 3 dimensional culture model system. slides (6 × 104 cells/well). 0. By particle in medium..5 DEPs on the capillary-like structures. (Logan. thereby Once confluent. then were inhalation of DEP in vivo. Endothelial tubes were treated with 0. . and spinning it at 10.1. Reagents cells (Weis et al. If Senger et al.. After washing again with 37 ◦ C 1× PBS.. etrated cells. Diesel exhaust particles and cell culture et al. VEGF stimulation transiently dissociates the VE- cadherin-VEGFR2 complex. Particles were vortexed for 3 min.7-dihydroxyphenoxazine using the induced upregulation of HO-1 stimulates VEGF-A secretion. 1989. ensuring the our results. Oberdorster et al. ROS-induced hemeoxygenase-1 et al. delivering either medium (medium always with PBS-Tween alone factor-A (VEGF-A) (Dulak et al. due to DEP-induced generation of ROS.05% Tween-80 (this (for review see Dejana et al. UT). 1995..5 × 105 cells/well). 1. based on the conversion of the non-fluorescent probe carboxy-H2 DCFDA of VE-cadherin. 1993. 1989. Inoue et al. cells could be lysed in the culture dishes. the stock suspension was immediately diluted to 1.3. free of the confounding 2. 2009). pH 7.. endothelial cells were treated exactly the ing in gaps between the cells (Kevil et al. The results of our previously reported in vitro endothelial immediately applied to the endothelial tube cultures via pipet. This suggests that very small parti.e. Our results support this idea in Olympus IX71 Inverted Microscope).. This indicated same.4. is a liquid at 4 ◦ C. These Japanese automobile diesel exhaust particles (DEPs) were collected by Dr. including Dr. with some protocol modifications. A hypothesis was developed from that used in the Zetasizer Nano ZS90 determination of particle size. H2 O2 production was assessed with 10-acetyl-3. 2001. and.. 2004. 2004). about 14% of these DEPs were between 20 and 100 nm the particle suspension (1× PBS is 137 mM NaCl.7 -dichlorohihydrofluorescein diacetate) to green-fluorescent carboxy-DCF. endothelial tubes could always be separated from the Matrigel by putting the culture dishes in the refrigerator. Since Matrigel (Connolly et al. / Toxicology 297 (2012) 34–46 35 While these data are largely based on TiO2 and iridium particle 2. result. 10 and 100 ␮g DEPs/ml for 24 h.

/ Toxicology 297 (2012) 34–46 .36 M. Chao et al.W.

followed by incubation with goat anti-rabbit HRP-conjugated IgG secondary anti- body (Bio-Rad cat.. 10. For monolayer cultures of HUVECs. the gel was cut in half. FITC-conjugated dextran (molecular mass 70 kDa. penetrated the confluent monolayers and gained access were then washed and the immunotargeted protein was eluted from the Dynabead to the lower chamber between 1 and 6 h. Assessment of oxidative modifications of proteins immunoprecipitated and medium proteins). nitrocellulose membrane blots (Bio-Rad). Invitrogen) were incubated with primary rabbit monoclonal Sigma) was added and allowed time to penetrate to the lower chamber.02% NaN3 in TBST) to reduce non-specific reactivity of antibodies. Aprotinin. and the VEGFR2 antibody (Abcam) was diluted 1:200 prior to was measured using the bicinchoninic acid method (BCA Protein Assay. 2.5 × 104 cells/well.5% SDS–polyacrylamide gels for elec- ride. rabbit anti-mouse GAPDH antibody centrations were determined using the bicinchoninic acid method (BCA Protein (G9547. 1994). unexposed confluent monolayers were used to see how long it took for gated Dynabeads–antibody was placed on a magnet.5 ␮l 1× PBS. 1 mM EDTA. diluted protease inhibitor cocktail. Antibodies against VEGF-B and C were diluted 1:1000. Stressgen). 1. A parametric ANOVA statistical analysis was performed to determine significance (*p < 0. nuclear. Endothelial tubes were incubated with primary polyclonal antibody against Nrf2 (Abcam ab31163) at a 1:100 dilution for 1 h at room temperature. The protein in the extract was quantitated. 0. and ImmunoResearch) were incubated with cell lysates as negative controls. The green punctuate fluorescence represents conversion of non-fluorescent DCFH-DA (2 . wells were exposed to various concentrations of DEP (0. antibodies were diluted 1:1000 for use unless indicated. 0. then incubated with the incubating blots for 1 h at room temperature. nifications on a wide field (epifluorescence) microscope (Olympus IX71 Inverted cells were collected and sonicated 1 min in a relatively low salt lysis buffer (0. 0. The secondary antibody for each was goat anti-rabbit IgG (H + L) con- perature in blocking buffer (3% BSA with 0. whole cell. Blots were incubated for 1 h at room tem. 420 mM NaCl. and the epifluorescence is shown on the right. 590 nm emission-Perkin Elmer Life Sciences). Briefly.1% Sigma’s P2714 protease inhibitor cocktail. For all of the protein preparations mentioned (i. The protein concentration of the whole cell extract and the super.W.1% SDS) to which 1/1000 volume of Sigma’s protease sample following the manufacturer’s instructions.22 ␮m nitrocellulose membrane.5 mM DTT. ROS detection was accomplished with the Image-iTTM Live Green Reactive Oxygen Species Detection Kit. son.02% NaN3 in PBS for 1 h at room 2. right side). so as not trophoresis. Immunofluorescence microscopy nitrocellulose membrane was treated with Super Signal West Pico chemilumines- cence reagent (Pierce) for visualizing immunoreactive proteins on X-ray film. 10 mM HEPES. The Dynabeads–antibody-extracts tran. The pelleted nuclei were resuspended in a higher salt lysis solution (25% glycerol. A 1 h ROS-induced oxidative alterations in proteins (i. The used directly for SDS–PAGE. indicating oxidization of proteins in endothelial tubes treated with increasing concentrations of DEPs. Protein lysates were made from HUVEC tubes after exposure to DEPs for 24 h.. One half was used for excitation. M. Samples were then electrophoresed and blotted for reaction with a DNP-specific antibody.6% Microscope). rabbit anti-human anti-Nrf2 peptide antibody concentration of 0. (1. incubation at room temperature was performed in blocking buffer (3% BSA with fied with carbonyl groups by ROS) were detected using an OxyBlot Protein Oxidation 0. followed by a 1 h room temperature incubation with goat anti-rabbit secondary anti- ication for 1 min in pH adjusted SDS–PAGE buffer supplemented with 0.4. amino acid side chains modi. for 1 h at room temperature. 0. then 50 ␮l Because endothelial tubes are always plated at the same density and incubated of Amplex Red reagent/0. for secreted products. 150 mM NaCl. Abcam) at 4 ◦ C. The concentration of protein in each sample in Chao et al. but VEGF- Assay. 3–6. then 2. Insoluble nuclear debris was pelleted by a 30 s (6.5 mg/50 ␮l Dynabeads (Immunoprecipitation kit.5 mM NADPH solution in the same relative volume of medium. EDTA and Leupeptin) was added.9. These were: rabbit anti-rat icated for 1 min with pH adjusted SDS–PAGE buffer (as above) containing a final HO-1 antibody (SPA-895. In initial anti-human VE-Cad antibody (1:80 dilution. The supernatant. ture medium volume in each upper chamber was 100 ␮l and in each lower chambers and the samples were frozen in liquid nitrogen and stored at −80 ◦ C until used for was 600 ␮l. after electrophoresis the proteins were electrophoretically transferred to 0.05. The eluates were denatured by inhibitor cocktail (cat. #170-6515).7 -dichlorohihydrofluorescein diacetate) to fluorescent DCF by ROS. Incubations were at 4 ◦ C. and grown to confluence on fibronectin (7 ␮g/ml)-coated filters of transwell units tinin). **p < 0. was next incubated for 5 min on ice.5 mm. Tubes were visualized at 200× magnifications on an Olympus IX71 Inverted Microscope. 3. Cul- natant containing the nuclear extract was determined using a BCA Assay (Pierce). jugated to horseradish peroxidase (HRP) (Bio-Rad cat # 170-6515). Pierce). VEGF-D was 1:250 and VEGFR2 1:200. 10 and 100 ␮g DEPs/ml was assessed by Amplex Red assays.5 mM PMSF. graphed as relative light units (Y axis. . then Matrigel and cell debris were separated out by cen. cells were seeded at 1.4 ␮m pore size. Phase contrast images are shown on the left. (C) H2 O2 production generated in endothelial tube cells in response to 0. any FITC-dextran to reach the medium in the lower chamber. Sigma). diluted 1:5000. but differences were intracellular locations. the cells in the upper SDS–PAGE. 0. The amount of H2 O2 was the Western blot and the other half was stained with Coomassie blue to evaluate determined by comparison to the standard curve. Cell use. / Toxicology 297 (2012) 34–46 37 192 mM glycine. Briefly. (B) ROS were also detected by an increase in carbonyl groups.2 mM EDTA. The respective with 1.5. Nonspecific reactivity was blocked by incubation with 2% normal goat serum plus 0. For Western blots of cell lysates. (2011). the HUVEC tubes were scraped from wells.1% Sigma’s body labeled with Alexa 488 (Molecular Probes/Invitrogen cat # A11008). 1–200. 6–9. E-64. This was West Pico chemiluminescence reagent (Pierce). and this was indeed the case.000 × g for 10 min.2 mM MgCl2 . lysate proteins were loaded (30 ␮g/well) onto SDS polyacrylamide gels for elec. which contained the nuclei. Proteins were electrotransferred to 0.6. immunoprecipitation and Western blot analysis temperature. When the cultures reached confluence (3–5 days). 140 mM NaCl and 0.7. and the mixture was incubated at room temperature for 30 min.01). Chao et al.e. Dynabeads Protein A. or 100 ␮g DEPs/ml for 24 h. 40 ␮l of medium was were added. companies all indicated that the non-human antibodies cross react with the corre- and subjected to SDS–PAGE on 12% gels.22 ␮m sponding human proteins. Permeability assays centrifuged for 5 min at 5000 rpm. A non-confluent monolayer culture centrifugation. The protein in the lysate was quantitated.05% not quantitated. The HUVECs were plated as monolayers or were seeded onto Matrigel-coated 2-well band intensities of the many carbonyl-modified proteins in DEP-exposed sample chamber slides for tube formation. Corning Costar).3.6 cells/mm2 ) served as a control for 100% permeability. heating at 95 ◦ C for 5 min and loaded onto 7. After electrophoresis. and 5 ␮g/ml of apro.02% NaN3 in TBST [25 mM Trizma base. 10. 15. The nmol amount of H2 O2 was experimentally determined by comparison to a standard curve (Y axis. The conju.8. ing them for 24 h at 37 ◦ C.0 mM KCl. and were collected in 3 h intervals (0–3.e. Error bars indicate ± SD (n = 3). and a 250 ␮l aliquot was added to the beads (=1 mg) for a 10 min continuously incubating the cultures at 37 ◦ C. Small quantities of fluorescent dex- incubation with rotation at room temperature. Monolayer Nuclear protein extracts were prepared from the endothelial tube cells by and comparative endothelial tube images were observed at 100× and 400× mag- adapting a 1 h minipreparation technique (Deryckere and Gannon. Jackson to interfere with the Amplex Red. Blots were enhanced with Super Signal kit’s polyclonal DNP antibody (diluted 1:150) for 1 h at room temperature.3-dinitro-phenyl-hydrazine (DNP) following the manufacturer’s protocol. assessed at 490 nm. After DEP treatment for 24 h. overnight. pH 7. Protein preparation. and incubated on ice for 20 min. containing AEBSF. bestatin hydrochlo. Protein con. experiments. # P2714. The VE-Cad antibody was used on monolayer and endothelial tube cells as trifugation at 10. DEPs cause generation of ROS in endothelial tubes. 1. Other endothelial tube images were observed at 630× magnification Nonidet P-40 [NP-40]. rabbit anti-human polyclonal antibody against VEGF-A. 1.5 mM on a Leica TCS SP5 Spectral Confocal Microscope. pH 7. The buffer contained no azide. Pierce) at absorbance 540 nm. then Matrigel and (ab31163. incubat- For immunoprecipitation. pH 7. When this was done. overnight.. All primary Detection kit (Chemicon).5 × 105 cells/well 0. A 2. when 20 ␮g protein per lane was applied to the gels. VEGF- cell debris were removed by centrifugation at 10.5 ␮l aliquot of lysate was added to 47. Prolong Gold anti-fade mounting media including DAPI (Invitrogen) was added trophoresis. to sections. When antibody was required to reach tubes not exposed to DEPs) to make a qualitative evaluation. calculated from a standard curve where assays were run with known nmol amounts of H2 O2 from serial dilutions (not shown). (A) HUVEC tubes were treated with no DEPs. the equivalence of lane loading. VEGF-D and VEGFR2 from Abcam. the HUVECs were lanes were visually compared with that of the negative control (i. the fixed endothelial cells were permeabilized with 0. then centrifuged for 30 s at 2500 rpm. endothelial fixed with 4% paraformaldehyde for 10 min. Aliquots (100 ␮l/well) then cell extracts were added. 100 ␮g/ml). 0.9. 1.e. and slides were covered for an overnight incubation at 4 ◦ C. Since no dextran was obtained in the Fig. Lysate proteins (5 ␮g/sample) were derivatized A required a 1:500 dilution. or 1. and equal amounts of protein from each treatment group were used to derivatize carbonyl groups with dinitro-phenyl hydrazine (DNP). phenylmethylsulfonyl fluoride [PMSF]). 1. Then. The 2. 2 mM benzamidine. Abcam). and exposed to X-ray film. B. VEGF-C.000 × g for 10 min. diluted 1-5000. made 4000 ␮g/ml. and 9–12 h time frames) while made 4 mg/ml. left side). Triton X-100 for 10 min at room temperature. HUVEC tubes were collected and lysed by son.05% Tween-20]). samples were applied in order on absorbance of samples was read on an HTS 7000 Plus Bio Assay Reader (540 nm the gels twice. 20 mM HEPES pH 7.2 U/ml horse radish peroxidase. Lysates were quantitated. the supernatant was removed. Dynabeads conjugated with rabbit IgG (cat # 011-000-003.

The FITC-dextran adhering to Matrigel samples was averaged and gests that endothelial tube cells would likely mount a detoxification plotted..7 -dichlorodihydrofluorescein diacetate and visualizing endothelial tubes.01 indicated by * and ** respectively. containing AEBSF. 3A.38 M. 192 mM glycine. ROS generation was very abundant. production SDS to which 1/1000 volume of Sigma’s protease inhibitor cocktail (cat. the medium/dextran was removed and discarded. 1. In the samples treated (Fig. but were immunoreactivity.8 times higher (2. however. 2A. Reading was done on an HTS response 7000 Plus Bio Assay Reader at 490 nm-Perkin Elmer Life Sciences. The Heme oxygenase-1 (HO-1) is a detoxification enzyme whose right panels of Fig. In unexposed capillary-like tubes.1. Generation of ROS causes an Nrf2-dependent detoxification aliquot of 200 ␮l of each lysate was loaded into 96-well plates for determination of the relative fluorescence units (RFU) of each sample. but round up and lose cell-cell contacts. and an extreme response was observed at 100 ␮g/ml (Fig.5 × 105 HUVECs per well onto Matrigel-coated 6-well plates. 10 or concentration of 1 mg/ml was added into the culture dishes and incubated for 4 h. with only 5–10 small green of ROS-minimizing enzymes. as Nrf2 (green) was present predominantly in the cytoplasm after had been shown for monolayer HUVEC cultures (Bai et al. 1C. the cells.6 3. The shuttling of the transcription factor from the cytoplasm to the Previous work (Chao et al. The results were expressed as means ± SD and were analyzed using Student’s t-tests and one-way parametric ANOVA.18 nmol) than untreated cells (0. an 3. 2B). HUVEC tube integrity is minimally affected by DEPs at 1 ␮g/ml.W. and mildly affected by 3. some wells were coated with Matrigel only (no cells were plated). Statistics for their expression of Nrf2 in response to DEPs. This factor responds to oxida- value are considered to be representative of the passage of FITC-dextran into the tive stress by translocating from the cytoplasm to the nucleus. The “Matrigel only” samples were treated identically as the test samples. it was 4. upper blot). the 100 ␮g DEPs/ml exposure greatly increased the some wells were coated with Matrigel. including non-confluent control samples. Following a 24 h exposure to 100 ␮g Furuyama et al. Tubes were allowed to form over 12 h. To evaluate treated with 100 ␮g DEPs/ml.8 times higher (0. p < 0. thereby indicating a level of endothelial where it binds to gene promoter regions. left with nearly all the cells fluorescing. At 1 and 10 ␮g/ml DEPs. were also evaluated by phase contrast microscopy (200×. were considered statistically significant. and each One was a whole cell extract of DEP-treated tubes. puncta (via fluorescent carboxy-DCF). 1A). the amount of nuclear Nrf2 was respectively 1. To identify if H2 O2 is among the ROS generated in response or 100 ␮g/ml DEPs for 24 h at 37 ◦ C. for Western blots probed with an antibody against derivatized Since no method exists for evaluating the permeability of in vitro endothelial carbonyls (Fig. respectively. Samples. then subjected to Amplex Red analysis. First endothelial tubes were made. Therefore. tubes At the highest concentration of DEPs. each experiment was performed at least 2 times. it was reasoned that if DEP caused leakiness of the capillary-like structures.4. These data place Nrf2 in the correct location after the fluorescence by microscopy (Fig.9. 0.05) been added. pH 7. These throid 2-related factor 2 (Nrf2) is a transcription factor known to values are indicated on the graph in Fig. This method also allowed DEP exposure to facilitate the expression of detoxification response evaluation of changes in structural integrity of the capillary-like enzymes. tubes. Hsieh et al. / Toxicology 297 (2012) 34–46 7–9 h period after adding the agent (data not shown).7 times higher than that of the non-exposed control (Fig. then cells were added and allowed to form tubes for 12 h before exposure to 1. Merging the Nrf2 and DAPI images clearly shows much of to DEPs was evaluated by labeling reactive oxygen species with the green Nrf2 colocalizing with the blue nuclei in the DEP-exposed carboxy-2 . 2001. the transport of Nrf2 to the nucleus was dramatic (lower (0.. and since >98% of this dextran Endothelial tube extracts were made after DEP exposure to entered the lower chamber in the first 4 of the 6 h preliminary test time. indicating increased levels of oxidized carbonyl never exposed to DEP (0 ␮g/ml DEP). After BCA quantitation. 4D. RFU values greater than this baseline regulate detoxification enzymes. cells of permeable capillary-like structures. E-64. Test sample wells were coated with Matrigel. Results with increasing DEP concentration (Fig.51 nmol. 2006).1% At the 10 ␮g/ml and 100 ␮g/ml concentrations of DEPs. At 100 ␮g/ml the tube cells remain in the same posi- tion on the plate. this blot demon- time.. A separate set of DEP-exposed endothelial tubes was used to isolate and prepare a nuclear pro- tein extract. DEP exposure (Fig.05 and strated that the overall level of Nrf2 does not change in response to p < 0. 1B). 4. from the wells into eppendorfs containing 25 mM Tris. 100 ␮g DEPs/ml for 24 h.05 nmol). 2C. and is indicated as the blank in Fig.6 times greater. although there were no Detection of H2 O2 and oxidative modifications of proteins sug- cells to extract. Immunoblots of Nrf2 derived from two types of protein extracts were performed. where the Nrf2 immunofluores- exposure of DEP is cytotoxic to human umbilical vein endothelial cence pattern of untreated endothelial tubes is compared to those cell (HUVEC) pre-assembled into capillary-like tubes. The RFU value of the second control. Expression of antioxidant protein HO-1 10 ␮g/ml. Chao et al.. HUVEC punctate areas indicating ROS generation. At the end of this time period. bestatin hydrochloride. than controls. N-acetyl cysteine (NAC) was added to medium of one set of . and medium (1 ml/well) containing FITC-conjugated dextran at a final to particles. with 100 ␮g DEPs/ml for 24 h.3. # P2714. Nuclear factor ery- dishes of endothelial tubes receiving no DEPs. endothelial tube cells were exposed to 0. 2A. free DEPs were washed away with PBS. levels of HO-1 were evaluated as a representative used. Samples treated with tube proteins were analyzed for HO-1 levels by Western analysis 10 ␮g/ml DEPs showed more fluorescence. then plated with cells to form tubes.. endothelial tubes were assessed 2. When 1 ␮g DEPs/ml was 2009). p < 0. lower blot). lanes). 2011) demonstrated that a 24 h nucleus is shown in situ in Fig. Zeiss-Axiovert 40 Inverted Microscope). A strong increase was observed at the 10 ␮g/ml exposure. DEPs induce generation of ROS in capillary-like endothelial and 3. groups in endothelial tube cell proteins. The levels of Nrf2 in the nuclear extract increased 3. or 100 ␮g/ml) and assessed. As shown in Fig.16 nmol). To evaluate whether this occurs in the 3 dimensional capillary-like model. The sample was gently pipetted up and down to make an extract of and 12. seeding 1. Equal amounts of FITC-dextran application was chosen as the time to analyze the monolayers for per- protein were loaded in each well of SDS–polyacrylamide gels meability. After incubation. 4D. 1A show DEP-induced ROS as fluorescent green expression is enhanced by Nrf2 (Buckley et al. 2003. Aprotinin. response to enhance their chance of survival. endothelial tubes were exposed to DEP DEPs/ml. 3A). 10. whether cytotoxicity was a consequence of oxidative stress. and is indicated as 0 ␮g/ml DEP in Fig. DEPs caused increased protein oxidation as tubes. samples were all adjusted to 4 mg protein per ml. 1A. assessed by the intensity of the immunoreactivity as compared to the dextran would be able to enter leaky cells of the capillary-like structures due to endothelial tubes not exposed to particles. cells exposed to 1 ␮g/ml DEPs generated only The endothelial tubes were washed twice with PBS and collected by scraping them 1. enhancing transcription tube permeability after DEP exposure. After a 24 h exposure to DEPs. samples were run in triplicate. Oxidative stress in response panels).2. represented the baseline adherence of dextran to unexposed tubes. and other. 4 h after measure oxidative modifications to proteins. ROS production was minimal. 24 h in culture (top panels). For statistical analysis. sures (1 and 10 ␮g/ml) were not significantly different from each As controls.1 times more H2 O2 (0. of oxidative stress genes. As seen in the left panels of Fig. EDTA and Leupeptin) had of H2 O2 in the tube cells was 2. 10. The two lower DEP expo- the lack of blood flow. 1. To ascertain if any FITC label had been able to access the tube structures.

. 2.. Nrf2 is found to be translocated to the nucleus. and 9 times higher after response intensifies by 24 h.05). tin protoporphyrin (SnPP). Again. 2001. The positive controls were non-confluent plates (Fig. 3D) (Furuyama et al. but that the higher after exposure to 10 ␮g/ml DEPs.5-fold at 24 h.4. the fold at 12 h. / Toxicology 297 (2012) 34–46 39 Fig. Two Western blots are shown. shows HO-1 was increased over controls by 1. These results suggest that exposure HO-1 expression (Fig. Scale bar = 50 ␮m. (B) The nuclear blot was scanned and the value for the no DEP exposure sample was set to 1. . Chao et al.6- NAC added to the cultures attenuated. Quantitation of the blots in histogram form (Fig. 3A. 3A.8-fold higher than controls. permeability were first performed using confluent monolayers of 10 mM NAC was added to sister cultures at the same time the cells to assess whether permeability resulted after a 24 h DEP 10 ␮g/ml DEPs treatment was started. With exposure to 100 ␮g DEPs/ml for 24 h. or 1. right 4 lanes). 3C). To attenuate ROS. NAC and SnPP significantly reduced the HO-1 response. The magnification (630×) in all panels is the same.8 times between 6 and 12 h to get a 60% increase over controls.. The other nuclear band densities were normalized to this sample (*p < 0. 3B shows a quantitation to 10 ␮g/ml DEPs causes HO-1 induction to begin slowly. DEPs induce translocation of Nrf2 from the cytoplasm to the nucleus. where the 24 h response value was cific inhibitor of HO-1. Since endothelial tube structures do not totally cover the culture HUVEC tubes were treated with 10 ␮g/ml DEPs for 6. tran assay. with the previous experiment. exposure to 100 ␮g/ml DEPs. 10 or 100 ␮g DEPs/ml for 24 h. but did not totally block. with the specific HO-1 inhibitor having more 3.2-fold at 6 h. (A) Capillary-like endothelial tubes were incubated with no DEPs. samples because of its ability to increase endogenous glutathione NAC totally inhibited the induction of HO-1 in tubes exposed to levels and reduce the effects of oxidative stress and cytotoxicity 10 ␮g/ml DEPs. which grew stronger at 12 and at 24 h (Fig. taking of the blots. This was done in a standard 2 chamber FITC-labeled dex- HO-1 by 6 h. DEPs cause endothelial permeability effect at the 100 ␮g/ml DEPs concentration than NAC. experiments to evaluate whether DEPs induce endothelial and HO-1 was evaluated by Western analysis. 4A. totally inhibited 3. DEPs caused an increase in exposure. Nuclei are stained blue with DAPI. Fig. 12 and 24 h. dish. by 1. The more spe.W. Nrf2 immunofluorescence (green) is seen mostly in the cytoplasm with only minor overlapping of the blue fluorescence. Pocernich et al. being 350% higher than controls. and accentuates that HO-1 is approximately 3. M. middle lanes). Olivieri et al. (C) Confocal microscopy of Nrf2 plus or minus DEPs: With no DEPs. 2000). To examine the timing for the increase in HO-1 expression. This is in good agreement induction of HO-1 expression (Fig. 2006. The blots indicate that increasing amounts of DEPs increase the amount of Nrf2 that is translocated to the nucleus. one of Nrf2 from whole cell protein isolates (top) and one of Nrf2 in extracts of isolated nuclei (bottom). and by ∼3.

4A. the addition of exposed samples plus or minus 10 mM NAC or 25 ␮M SnPP. the levels of dextran able to tern of cells exposed to 10 ␮g/ml DEP. 10 or Fig. it seemed reasonable to expect that the HO-1 induced by DEPs might ultimately induce secretion of endothelial VEGF-A. 1. To investigate whether HO-1 is manner. As seen in Fig. the expression of VEGF isoforms in HUVEC monolayers and capillary-like tubes was com- pared. monolayer cultures also secreted VEGF-C. suggesting permeability. 2008). The HO-1 band intensity for the effect of DEP on VE-cadherin in endothelial monolayers is shown in 0 time point was defined as one. 6C. This suggests the possibility reach the lower chamber increased. and that HO-1 expression 24 h exposure to DEPs. This strongly suggests that GAPDH immunoreactivity. n = 3. In both types of culture VEGF-D was constant. While the design of the endothelial tube experiment cannot be made to yield data as clear as monolayer cultures.).05 and **p < 0. Chao et al. fluorescent cadherin previously reported (Chao et al. was explored with respect to the changes in VE- for 24 h to medium or DEP. 1. 4B. DEPs stimulate secretion of VEGF-A into the culture medium In experiments with monolayer cultures of HUVECs not involv- ing DEP exposure. 4E). since HO-1 levels are reduced specifically responsible for VEGF-A secretion. Relative expression of the VEGF-A addition of 10 ␮g DEPs/ml to tubes. Most of the cells had separated from each other. 6D). 2011). 6A indicates that. 5C and D. Equal protein loading was levels from these experiments is shown graphically in Fig. (C) is one of the major effectors of DEP-induced VEGF-A secretion. Equal protein loading was confirmed with totally diminished by SnPP treatment. Bars are the relative fold change compared to the control with no added DEPs. Western blotting was performed to determine the timing of HO-1 expression after although not likely the only one. / Toxicology 297 (2012) 34–46 uptake. (A) After a 100 ␮g/ml concentrations of DEP for 24 h with or without 10 mM 24 h exposure to no DEPs or 1. Lin et al. represented the minimum permeability possible.40 M. Therefore. right) that had not been exposed to DEP. The negative controls were fully confluent plates of that were not exposed to DEP were treated for 24 h with 100 ng/ml cells (Fig. Lacking a standard method as does DEP exposure. To evaluate whether VEGF-A had the left). Asterisks indicate statistical significance (*p < 0. 2003). and all other time points are shown as the fold- change relative to this point. were set up and exposed to DEP (Fig. and by parametric ANOVA and asterisks indicate significance (p < 0. 10 or 100 ␮g/ml concentrations of DEP. The same was done with unexposed and A increases with increasing DEP concentrations. increasing concentration of DEPs.W. where cell contact was infrequent and dextran would have ability to rearrange the VE-cadherin pattern of monolayers. After the 24 h incubation. with the exception of the 100 ␮g/ml DEP sample (Fig. VE- confirmed by GAPDH immunoreactivity. Cultures of endothelial tubes that VEGF-A contributes to the permeability of the cell junctions. HO-1 expression had been shown to induce secretion of vascular endothelial cell growth factor-A (VEGF-A) (Dulak et al. SnPP totally inhibits the HO-1 SnPP was employed. 3. 4B). Yellow arrows point out VE-cadherin internalization. While both capillary-like tubes and monolayer cultures secreted VEGF-A and VEGF-D into the medium. to assess the permeability of capillary-like tubes. The Western blot in Fig. indicate that increasing amounts of DEP cause increasing levels of VE-cadherin to be pulled from the cell surface into globules under the cell membrane (Fig. but the secretion of VEGF-A increased with increasing DEP concentration.. 3. internalizing VE-cadherin concentrations of DEP correlated with small increments of dextran away from the cell surface and allowing for disruption of cell–cell . 6B. was induced in a dose dependent manner in DEP-treated HUVECs plated as monolayers and as capillary-like tubes. plus and minus NAC. but not expression induced by DEPs (last 4 lanes). The first 4 lanes of the immunoblot demonstrate that HO-1 is induced by DEPs in a dose dependent NAC totally inhibited its secretion. Values are means ± SD. the major whether dextran was taken up by the endothelial tubes exposed VEGF-A receptor. cadherin is a major player in preventing permeability of cells. and 24 h. Compar- ative densities from the Western bands are graphed in Fig. derived from the Western blots. Furthermore. 1999. ROS is an intermediary for HO-1 induction. 12. then medium was iso- lated for Western analysis (Fig. VEGF-A. As seen in Fig. 5A and B).5. the HO-1 inhibitor when NAC is added (4 middle lanes). 4C. cells free passage. cells have rounded Binding of VEGF-A to VEGFR2 has been implicated in interrupting up. 10 or 100 ␮g/ml.. 2011).. 2011). This is likely because this exposure causes ∼50% of the cells to die by 24 h (Chao et al. endothelial tubes were exposed to 0.. Since the indications relating HO- 1 and VEGF came from data using HUVECs in monolayer culture. To evaluate whether DEP-induced ROS are responsible for the VEGF-A secretion. and the globules of submembrane VE-cadherin resembled the pat- with increasing concentrations of DEPs. 2002. although VEGF- proteins extracted for Western analysis. and differences between the behaviors of monolayer and tube cul- tures had been observed (Chao et al.. Lampugnani et al. DEP exposure and its impact on VEGFR2. and thus VEGF-A (Fig.05).. This data verified that the key VEGF isoform known to be involved in endothelial permeability. At 100 ␮g/ml. DEPs induce endothelial capillary-like tube expression of HO-1. pulling and thinning the cytoplasm between them. VEGF-A secretion was attenuated. This was examined dextran was added to the washed cultures and remained there for because VE-cadherin has been shown to associate and interact 4 h. The 6. endothelial tube structures are altered with with VEGFR2 (Carmeliet et al. (D) Quantitation of HO-1 expression at 0.01). we evaluated Lastly. there was a correlative trend between DEP exposure of endothelial tubes and how much fluorescence they were able to retain. Endothelial tubes and monolayers were treated for 24 h with 0.. Increasing the VE-cadherin–VEGFR2 interaction. (B) Histograms show a quantitation of HO-1 levels after the effect of DEPs is mediated by ROS. 6C. endothelial tubes were lysed and NAC. Statistical analysis was Fig.

It is assumed this is why they do not take up the dextran. At 100 ␮g/ml about 50% of the cells are dead. in VEGF-A is a factor causing endothelial permeability (Esser et al. For these exper. and cells treated with 100 ␮g/ml DEPs. 1983. / Toxicology 297 (2012) 34–46 41 Fig. VE-cadherin are associated. junctions and permeability (Carmeliet et al. There are several reports in the literature indicating that Immunofluorescence analysis (Fig. a pull down assay is shown. 4. 2006. 7A. Chao et al. The 100 ␮g/ml exposure.. and the remaining cells look unhealthy.. the 24 h DEP exposures. it is clear that the two molecules are largely disassociated DEP concentration disrupted VE-cadherin too extremely and killed by the distinct green and red visible in the merged image panel.e. (D) FITC-dextran was added to endothelial tubes for 4 h post a 24 h exposure to different concentrations of DEP.. VE-cadherin (red) and VEGFR2 1998. The endothelial cells. M. In response to matically with increasing concentrations of DEP. Kevil et al.. The RFU of FITC-dextran that was not removed by washing the endothelial tubes suggests the fluorescent dextran has entered inside the tube structures. ∼50% of the cells. DEPs induced oxidative stress in was cut in half for reaction against each of the antibodies. nearly all the VEGFR2 and showed both molecules were present. in the absence of DEP. 1998. Cell lysates were prepared after The colocalization of VE-cadherin with VEGFR2 is greatly reduced. 10 or 100 ␮g/ml DEPs.. non-confluent cells. unexposed endothelial tubes. right. (C) Cell–cell junctions of monolayer cultures are disrupted by DEP exposure as determined by dextran gaining access to the lower chamber (i. The blot To summarize our work. 7B) demonstrated that. Strugar et al. (green) co-localize in areas of the membrane. the interaction ies is unknown.. Weis and Cheresh. Roberts and Palade. meable. Heme- amount of VEGFR2 pulled down with VE-cadherin decreased dra. antibodies against VE-cadherin and VEGFR2 (Fig. 4. and.e. VE-cadherin had first been immunoprecipitated from the unex. 1 ␮g/ml and 10 ␮g/ml DEP exposures the 0 ␮g/ml DEP image. left). In the merged image of the 10 ␮g/ml DEP were employed in cell lysate pull down assays. Discussion posed and exposed cell lysates. then blotted. Hydrogen peroxide was generated. so the bands cannot truly connote a quantitative between the two molecules is attenuated. DEPs induces endothelial permeability in monolayer and tube cultures. oxygenase-1 was a defense enzyme induced by Nrf2. In Fig. cells responded by transporting Nrf2 to the nucleus same since it was the initial pull down epitope. This showing that. 2005). all green overlaps with red) for iments. (B) Bright field microscopy showing endothelial tubes either not treated with DEPs or exposed to 1. The avidity of the antibod. With increasing levels of DEPs. 7A. endothelial tube cells are increasingly pulling away from each other and rounding up. The immunoprecipitates were run in duplicate on an SDS–polyacrylamide gel. only the 0 ␮g/ml. indicated by the Senger et al. ratio between the amount of VE-cadherin and VEGFR2 in the cell membrane. 1999). as VE-cadherin levels at these DEP concentrations was mostly the expected. endothelial cells expressed VEGF-A and became per- associated with VE-cadherin as the DEP concentration increased. Western analysis was performed using The immunofluorescence analysis supports the pull down assays. Nagy et al. 1994. 1995. . (A) Bright field microscopy showing monolayers of confluent endothelia 0 ␮g/ml (no DEP). Less VEGFR2 was DEP exposure.. However. Here.W. but after DEP exposure. the to facilitate transcription of genes to defend against ROS. the culture is permeabilized).. merged orange–yellow color (i. so was not used.

Medium was collected from each exposure sample. and the data was used to compare the relative levels of each VEGF isoform to the respective DEP concentrations. 5. However. Chao et al. 1995). Another angiogenesis. This allowed ing been used in more than three hundreds publications studying the observation of direct effects of DEP on endothelial cells. 2001. First. known to induce vascular permeability. each gel was cut in half. / Toxicology 297 (2012) 34–46 Fig. were absent. endothelia. Sumanasekera et al.. by the particles themselves versus the materials that may be sol- cells in HUVEC tubes are proliferation prohibited (Chao et al. ubilized from them. to ascertain equal loading of samples by comparing the intensity of the medium proteins across the gel. also in vitro endothelial capillary tubes share many limitation of the model system is that it can only evaluate what similarities with in vivo capillary structures (Donovan et al. 1989. However. 1985). (C and D) Densitometric scans were made of the blots. key regulators of permeability. do indeed reach the . literature indicating that particles in size ranges that overlap with HUVEC tube adherens junctions. sis (Schmelz and Franke. the size range of the DEPs employed here. One half was used for Western analysis with the VEGF-A and the VEGF-B antibodies. which subsequently release com- used previously by others to study the effects of particulate expo. 10 or 100 ␮g/ml for 24 h.. 2006). Yamawaki an advantage that the confounding effects of inflammatory cells. 1991. Zimrin et al. 2011). and DEPs might have on lung capillaries. The drawback of the in vitro model The work was begun to gain an understanding of the effects system is that it cannot target any particular area of the lung. and Iwai.. the in vitro morphol. 2007. while our data is consistent with this interpretation.. The other gel halves were stained with Coomassie blue. Increasing DEP concentrations induce increasing secretion of VEGF-A by endothelial tube cells. Third. may occur if inhaled DEPs reach the alveolar or other vascular Grant et al. ponents injurious to the endothelium. it have been demonstrated to have the same junctional components cannot rule out the possibility that other aspects of oxidative stress and structure as in in vivo capillaries by electron microscopic analy- cause the endothelial permeability. and 40 ␮l of each were applied to the wells of an SDS–polyacrylamide gel. HUVECs were chosen for the it is lacking other cell types adversely influenced by DEPs.. 1993). The cells have been extensively characterized.W.. However. there is significant evidence in the as are endothelia in in vivo capillaries (Hadley et al. (A and B) Monolayers of endothelial cell cultures and endotheial tubes were exposed to no DEPs or 1. we considered it sures (Garcia et al. It also does not differentiate what might be caused ogy of the tubes approximates that of in vivo capillaries. such as experiments for three reasons: This endothelial cell type has been neutrophils and macrophages. second.42 M.. This half was then stripped and reprobed with the VEGF C and VEGF D antibodies. After electrophoresis. hav.

VEGF-A inhibitors are VEGF-A bands from the Western was graphed. indicating that DEP-induced HO-1 is a factor contributing to expose the endothelial tubes are already well characterized (Bai to endothelial VEGF-A secretion. HUVEC tubes were exposed to no DEPs or 1. Thus. Strugar et al. The results demonstrating that DEPs induce ROS agree with those using monolayer cultures of endothelia... cated that VEGF-A produced by the endothelial tubes in response to Sagai et al. due to the DEP-induction of VEGF-A. 1998). reaching endothelial surfaces have not been directly measured or reliably estimated. HO-1 expression pattern most resembles that of endothelial cells treated with 10 ␮g/ml DEP. The link between HO-1 and VEGF-A secretion was investigated by exposing endothe- lial tubes to DEP in the presence and absence of NAC and the HO-1 capillaries (Geiser et al. and endothelial tubes exposed to DEPs transported Nrf2 tion. Arrows point to areas where VE-cadherin has moved from the cell membrane to more interior. Confluent endothelial employing DEPs (Cisowski et al.. 2004) and were dispersed to biologi.. and the levels were attenuated by the addition of NAC and effects of DEPs on the vasculature.. with the value of the 0 ␮g/ml DEP used to decrease edema accompanying certain cancers (Gerstner treatment sample set as 1. With no DEP exposure. 2002. Chao et al. 1994).. 1993). Hsieh et al. The experiments performed indi- et al. secretion compared to each DEP exposure concentration.3 to 1. Increasing concentrations of DEPs result in cell–cell separations DEP exposure is. especially since the DEPs used SnPP. Senger et al. VEGF-A has also been shown to parametric ANOVA (*p < 0. Nemmar et al. To date. in part.. 2002. Medium was collected and run on gels for immunoblotting. (for monolayers were treated for 24 h with and without DEP.. Statistical analysis was by et al. causing cell–cell gaps that 100 ␮g DEPs/ml plus or minus NAC (10 mM) or plus or minus SnPP for 24 h. 6. efficiency and rate of pulmonary clearance mechanisms. the cell membrane (yellow arrows). Jozkowicz et al.W. the site and rate of DEP deposition. n = 3. The resulting VE-cadherin from the cytoplasm to the nucleus. 2000. Keck et al. In these in vitro experiments. DEPs does not induce angiogenesis. the DEP-induced reorganization of VE-cadherin demon- secretion. and SnPP mostly blocked it. and were considered significant.. 2011) is. 10 or in endothelial cell monolayer cultures.. Particles of this size would have the ability to penetrate deeper and in our system as well as in vivo this may be how DEPs induce into the respiratory tract (Kreyling et al. Without NAC and SnPP. and the observation that N-acetyl cysteine attenuated DEP cytotoxicity have all been previously shown in different systems. 1983). 2001. real- world exposure to DEP occurs with co-exposure to other gas-phase components of diesel engine emissions that may modify the tox- icological effects of DEP. (A) Secretion of VEGF-A from capillary endothelial tubes is related to because it has been shown to induce VE-cadherin internalization DEP-induced HO-1 expression. 2003. and/or soluble components. and this turned out to be true. since there was no sprouting cally relevant sizes. 2004. protein were loaded in each lane. Ito et al. as well as with ani- mal models (Bai et al. likely contribute to the induction of VEGF-A secretion. Comparisons between biological effects of exposure to DEP in our in vitro system and ‘real-world’ inhalation exposure to DEP are limited. It and in globules of VE-cadherin accumulating intracellularly near to. review see Dulak et al. with a bimodal size distribution that included lial cell proliferation when assessed by MTS assays (not shown). 2005. there was a DEP-induced generation of H2 O2 . was induced within 6–12 h of initial exposure. DEPs induce VEGF-A secretion. in part. To assure that equal amounts of due to the generation of H2 O2 . was stained with Coomassie blue (loading control). indicating that factors other than HO-1 may strated in endothelial tubes (Chao et al. These globules have a more interior local. and both VEGF-A and DEPs disrupt membrane The importance of H2 O2 generation cannot be underestimated VE-cadherin. Thus. in vivo concentrations of inhaled DEP. In addition. 2008). Furthermore. The other major function of VEGF-A is induction of permeability. Values are mean ± SD... 1997... the data must be considered for what it is: simply a model for investigation what may occur in vivo. M. (C) The membrane localization of VE-cadherin is disrupted be induced in endothelia by HO-1 in studies unrelated to. Nor did the VEGF-A induce endothe- from 0.. Oberdorster et al. Kumagai et al. 2002). 2009. the lowest DEP concentration at which observed significant increases in production of hydro- gen peroxide and expression of HO-1 and Nrf2 was 10 ␮g/ml in the culture media.. The majority of our dispersed particles ranged of tubes after DEP addition.. permeability.05 and **p < 0. 2009. Nrf2 is a factor with 100 ng/ml VEGF-A. Thus. Fig. the distribution of particle sizes reaching the lung periphery.. then reacted with VE-cadherin antibody.. was our goal to determine whether DEP induced HO-1. 2003).. particles may exert a direct effect on capillary endothelia. loaded in the same order with the same amount of protein.05 ␮m.. but no longer at. Protein oxidation was increased by DEP. and rates of transmigration of the parti- cles and/or soluble components through the alveolar membranes to the pulmonary capillary endothelial cells. followed by measuring the amount of VEGF-A in et al. Inoue et al. 2006. half of each gel was used for the blot and the VEGF-A is also known to cause weakening of cell–cell junctions. (B) Densitimetric quantitation of VEGF-A increasing permeability (Connolly et al. VEGF- make the monolayers measurably permeable (Kevil et al. . VEGF-A treatment controlling expression of HO-1 (Buckley et al. Singh et al. other half. 2005. Ferrara and Henzel. The relative intensity of 1989. 2001. 2001. sub-membrane intracellular locations.. (D) A confluent monolayer culture of endothelial cells was treated for 24 h mately VEGF-A. Oberdorster et al. and ulti- ization. a distribution in the range of 30–120 nm with a mode of 80 nm. Kreyling et al.01) compared with the control. 2009. NAC totally blocked VEGF-A Thus. caused dissociation of cells from each other and resulted in VE-cadherin disrup- 2009).. 1993. A secretion increased in a dose dependent manner. where capillaries may be covered by only the thin membrane of alveolar type I cells. 1989. we suggest the culture medium after 24 h. Sagai et al.. VEGF-A secretion was induced by that HUVEC tubes can be effectively employed to model potential DEP. 1994). Thus. deep in the respiratory tract. Nemmar inhibitor SnPP. 2002.. and not by DEP exposure as assessed by epifluorescence microscopy... 1989. / Toxicology 297 (2012) 34–46 43 2006. a mechanism warranting con- cells remain in a monolayer with VE-cadherin appearing continuously around the sideration is the possibility that the permeability observed after cells’ membrane. Comparative dosime- try would require assumptions about pulmonary ventilation rate.

Collen.. We thank Linda Everett and Rita Hahn for their help with Lampugnani et al. less VEGFR2 was immunoprecipitated with VE-cadherin. J.. DEP interrupt interactions between VE-cadherin and VEGFR2.. allowing it to be pulled down with the molecule by the VE-cadherin antibody. The VE-cadherin immunoreactivity indicates that the protein was cleanly isolated by the antibody. Chao et al. The References endothelial tube model represents a simplistic view of endothe- lial DEP exposure. Bono. L. R. V. the red color localizing VE-cadherin. R. Right.. Lupu. F. F. our results are consistent with observations from the the UMDNJ/Rutgers Center for Environmental Exposures and Dis- literature showing VE-cadherin internalization. R. may illuminate differ.. Dejana. and the green localizing VEGFR2 are largely distinct. Balconi. D.G... B. 555–562. Wallez and Huber.C. 1999.. Cardiovascular effects of air pollution. A. F. Acknowledgments lial cell membranes has been demonstrated.J. B. .. despite these pitfalls... Nitric oxide stimulates Nrf2 nuclear translocation in vascular endothelium. Lutgens. Biophys. A. S... J. Z. 2008. actual in vivo exposure levels. Since VE-cadherin is internalized in a tis and Musculoskeletal and Skin Disease (NIAMS) [U54AR055073] dose dependent manner by DEP exposure (Chao et al. The immunoprecipitated product was applied to SDS–polyacrylamide gels.. since there are no gaseous components of diesel Bai. Free Radic. Compernolle. (Lond.. 2011). Biochem. (A) Left. M. Silverman. M. such as in this work. / Toxicology 297 (2012) 34–46 Fig.M. Sci. detected by green fluorescence. (B) Confocal immunofluorescence microscopy with VEGFR2.K. Brook. or 10 ␮g/ml DEPs. R. be due to this VE-cadherin internalization. Western analysis showed VE-cadherin and VEGFR2 in endothelial tube cell lysates. Rajagopalan. Marshall. Circulation 105. 2005.. Huang. nor is there any decent correlation with species.. Angellilo. exposed to diesel exhaust. 7.. Lampugnani.R... The association of VEGFR2 and VE-cadherin in the endothe.. G. F. 307. J. However.. Devlin. While we did not examine VE-cadherin phos. M. 1534–1536. S. 2001. Clin. 147–157.. de Ruiter. 1998). The cytotoxic effects of diesel exhaust particles on human pulmonary artery endothelial cells in vitro: role of active oxygen exhaust being considered. DEPs induce VEGF. Environ. 2003.G. that can be obtained. B. Instead. who then provided them to Robert phorylation of VE-cadherin. 2000. Breviario. The VEGFR2 immunoreactivity indicates that VEGFR2 was associated with VE-cadherin. Vincent. and VE-cadherin. David Diaz-Sanchez.M. 30.. S.D. Conflict of interest statement Mattot. VEGF-mediated endothelial survival and angiogenesis. Y. ease and K08 ES013520 to R.. D. Cell 98. 175–187..) 115. E.. Poelmann. the data Becker. V.D.. Dewerchin. Med. R. Dailey.... Grambow. Herbert. by the National Eye Institute (NEI) A. Buckley.. pull down assays were performed by first using VE-cadherin antibody to immunoprecipitate the adherens junction protein from the cell lysates of samples exposed to 0. in part.A. 113.. 2008).M. Res. Spagnuolo.44 M. there is a loss of association of VE-cadherin and VEGFR2. Zanetti... Brook. 973–979.. Carmeliet. A.. data suggest that the vascular permeability found after DEP expo- sure may. M. There is a wealth of information on Inhalation of fine particulate air pollution and ozone causes acute arterial vaso- endothelial cell biology that should be used to uncover potential constriction in healthy adults. A. Nuyens. Biol. mechanisms of DEP toxicity. Clotman. Soukup. Environmental Health Sciences (NIEHS) [P30ES005022] award to phorylation. Sagai.. thereby loosening cell–cell contacts Laumbach. Suzuki. then Western analysis was performed.C. little orange–yellow is seen in the merged image. R. VEGFR2 is co-localized with VE-cadherin (see orange–yellow merged color). and Dr. indicating disassociation of the molecules.J. Tar- geted deficiency or cytosolic truncation of the VE-cadherin gene in mice impairs All authors declare no conflicts of interest. Y. Moons. detected by red fluorescence: The data show that..B. Gittenberger-de Groot. ent ideas and approaches for investigations of humans or animals Brook. L.W. Health Perspect.. in the absence of DEP exposure. our award to the UMDNJ/Rutgers CounterACT Center of Excellence. F.. 1032–1038... VEGF-A has been this manuscript. 2002. suggesting that with increasing concentrations of DEPs. E. The work was supported by the National Institute of (Esser et al. Seasonal variations in air pollution particle-induced inflammatory mediator release and oxidative stress. 1. to Dr... Still. although it is not fully characterized and understood (Carmeliet and Collen..R.K.L. Oosthuyse. 2003. Masaru Sagai for providing the DEP shown to modulate vascular permeability by inducing the phos. Commun. 24 h after 10 ␮g/ml DEP exposure. P. With increasing DEP concentrations.C. Whorton.. and it is likely that its binding to VEGFR2 causes the receptor to award EY009056 to M. Urch. A. and by the National Institute of Arthri- dissociate from VE-cadherin.

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