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. Author manuscript; available in PMC: 2013 Mar 22.
Published in final edited form as: Inhal Toxicol. 2011 Jan;23(1):1–10. doi: 10.3109/08958378.2010.535572

Hypercholesterolemia potentiates aortic endothelial response to inhaled diesel exhaust

J Gregory Maresh 1, Matthew J Campen 2, Matthew D Reed 3, April L Darrow 1, Ralph V Shohet 1
PMCID: PMC3606052  NIHMSID: NIHMS383791  PMID: 21222557

Abstract

Background

Inhalation of diesel exhaust induces vascular effects including impaired endothelial function and increased atherosclerosis.

Objective

To examine the in vivo effects of subchronic diesel exhaust exposure on endothelial cell transcriptional responses in the presence of hypercholesterolemia.

Methods

ApoE (−/−) and ApoE (+/+) mice inhaled diesel exhaust diluted to particulate matter levels of 300 or 1000 μg/m3 vs. filtered air. After 30 days, endothelial cells were harvested from dispersed aortic cells by fluorescent-activated cell sorting (FACS). Relative mRNA abundance was evaluated by microarray analysis to measure strain-specific transcriptional responses in mice exposed to dilute diesel exhaust vs. filtered air.

Results

Forty-nine transcripts were significantly dysregulated by >2.8-fold in the endothelium of ApoE (−/−) mice receiving diesel exhaust at 300 or 1000 μg/m3. These included transcripts with roles in plasminogen activation, endothelial permeability, inflammation, genomic stability, and atherosclerosis; similar responses were not observed in ApoE (+/+) mice.

Conclusions

The potentiation of diesel exhaust-related endothelial gene regulation by hypercholesterolemia helps to explain air pollution-induced vascular effects in animals and humans. The observed regulated transcripts implicate pathways important in the acceleration of atherosclerosis by air pollution.

Keywords: Diesel engine exhaust, endothelium, gene expression, microarray, apolipoprotein E, hypercholesterolemia, air pollution

Introduction

Air pollution contributes to cardiovascular disease, including atherosclerosis (Künzli et al. 2005), acute myocardial infarction (Peters et al. 2004), and vascular endothelial dysfunction (Mills et al. 2005). Traffic-related emissions, as a major contributor to ambient air contamination, have been identified as a likely driver of vascular effects (Hoffmann et al. 2007). Pathophysiology of the endothelium (including impaired vasoregulation and anticoagulation, as well as enhanced cellular adhesivity and permeability) is exacerbated by atherosclerosis, hypertension, and diabetes; emerging evidence now shows a contribution from exposure to air pollution as well (Brook 2008). The proximity of endothelial cells to circulating, inhaled toxicants underscores their potential susceptibility. However, demonstrating the effects of inhaled particulate matter (PM) on endothelial cells has been difficult, owing to the tiny volume occupied by the endothelium in any tissue.

The effects of air pollution on atherosclerosis have been explored in animal models of hypercholesterolemia. These include ApoE (−/−) and LDL (−/−) mice and Watanabe hyper-lipidemic rabbits, which display accelerated atherosclerosis upon exposure to air pollutants, including diesel engine particulates (Yatera et al. 2008), concentrated ambient air nanoparticles (CAPS) (Araujo et al. 2008; Ying et al. 2009). Pollution-related expression of adhesion molecules in the rabbit model reveals a clear effect on the systemic vascular endothelium (Yatera et al. 2008) and the responses of ApoE (−/−) mice to CAPS include dysfunctional endothelial physiology (Ying et al. 2009). Importantly, exposure to CAPS results in augmented atherogenesis in a murine model of hypercholesterolemia (ApoE [−/−]) compared with wild type (C57BL/6) (Sun et al. 2005).

The responses of cultured endothelial cells to specific model particulates have been assessed in microarray studies (Yamawaki and Iwai 2006). However, cultured endothelium exposed to various components of air pollution represents an oversimplified model, given the complex set of responses that are likely to influence the vasculature in vivo. It has been shown that ultrafine PM from the lung is translocated to other organs, including the endothelium (Furuyama et al. 2009). However, the lung also acts to modify and amplify the response to PM by the secondary release of humoral mediators (Arimoto et al. 2007) and by the promotion of oxidative (Li et al. 2008) and nitrosative (Rundell et al. 2008) stress. Furthermore, PM exposures are coincident with exposure to pollutant gases, such as carbon monoxide, oxides of nitrogen, and volatile organic compounds, all of which have differing uptake and disposition. With the growing recognition of a systemic vascular effect of inhaled pollutants, ascertaining endothelial-specific transcriptional responses to complex “real-world” air pollutant mixtures will be useful. Our analysis of freshly isolated endothelial cells from ApoE (−/−) mice exposed to engine exhaust in vivo is a robust approach to discerning authentic vascular responses to air pollution.

Materials

Animals

BL/6 ApoE (−/−) and Tie2 GFP mice (strain name B6.129P2-Apoetm1Unc/J [stock #002052] and Tg[TIE2GFP]287Sato [stock #003658], from Jackson Labs, Bar Harbor, ME) were interbred and genetically selected to obtain Tie2 GFP/ApoE (−/−) mice on an approximately 1:1 mixed genetic background of C57Bl/6 and FVB/N. All mice received standard chow. A cholesterol assay (Cayman Chemical, Ann Arbor, MI; cat. #10007640) was used according to the manufacturer’s instructions to measure total cholesterol in the sera of male mice of the strain Tie2 GFP/ApoE (−/−) and both parental strains Tg(TIE2GFP)287Sato and B6.129P2-Apoetm1Unc. Tie2 GFP and Tie2 GFP/ApoE (−/−) mice were designated as ApoE (+/+) and ApoE (−/−) in this study for simplicity of nomenclature.

Diesel engine exhaust exposures

Four-month-old male mice underwent unrestrained (whole body) inhalation exposure in their cages to diesel exhaust generated from a Yanmar Diesel engine under controlled conditions of load. Exhaust particulate concentrations in a flow-through exposure chamber (model H2000; Hazleton Systems, Maywood, NJ) were monitored continuously with a nephelometer (model 8200; TSI Inc., Shoreview, MN) calibrated against determinations made gravimetrically with a Pallflex filter (Pall Inc., New York, NY) and were adjusted to 300 and 1000 μg PM/m3 by dilution with HEPA-filtered air. NOx levels in these atmospheres were 12 and 30 ppm, respectively, and CO levels were 3 and 100 ppm, respectively. Greater detail regarding volatile organic compounds, other pollutants, and uncertainty ranges have been previously published (McDonald et al. 2004). Exposures were performed 4 h/day, 7 days/week for 4 weeks in both ApoE (−/−) and ApoE (+/+) strains; littermates exposed to HEPA-filtered air were designated as controls. Separate groups containing four experimental and four control mice of each strain were utilized for each dose of PM. Mice were euthanized and aortic samples from each group were pooled within 24 h of the last exposure for subsequent processing and analysis.

All studies were approved by the Animal Care and Use Committees of the University of Texas Southwestern, Dallas, TX (the previous institution of JGM and RVS), and the Lovelace Respiratory Research Institute, Albuquerque, NM.

Tissue collection and extraction

Animals were euthanized by CO2 asphyxiation. Aortae were excised by dissection and freed of adherent tissue from the iliac bifurcation to the aortic root. Luminal blood was rinsed and aortas were sliced into 1-mm segments. Segments pooled from four animals were suspended in 5 mL of Dulbecco’s phosphate-buffered saline (PBS) with 2 mg/mL of dextrose. The suspension was combined with 5 mL of prewarmed PBS containing 10 mg/mL type II collagenase (Worthington Biochemical, Lakewood, NJ), and 60 units/mL deoxyribonuclease I, agitated continuously at 37°C on a shaking platform, and triturated 10 times every 10 min for a total digestion period of 40 min to generate a single cell suspension. The cell suspension was maintained at 0–4°C throughout the remainder of the isolation, which lasted a total of 2–3 h. The suspension was then combined with 10 mL of 10% fetal bovine serum (FBS) in Dulbecco’s modified Eagle’s medium (DMEM) and cells were collected by centrifugation and resuspended in 10 mL of PBS followed by filtration through a sterile 40-μm mesh filter to remove undigested tissue fragments. Following centrifugation, the cellular pellet was resuspended in 0.3 mL PBS containing 0.5 mM ethylenediaminetetraacetic acid (EDTA), 30 U/mL deoxyribonuclease I, 3% FBS, and 2 mg/mL dextrose and once again filtered through a 40-μm mesh filter.

The resulting aortic cell suspensions were sorted using a MoFlo from Dako Cytomation (Carpinteria, CA). Cells were excited by a 488 nm laser and GFP signals collected at 510 to 550 nm. A pressure of up to 30 psi was utilized, generating 5000 to 10,000 events per second. Positive cells were collected directly into Trizol (Invitrogen, Carlsbad, CA) and RNA was isolated (with the addition of glycogen) according to the manufacturer’s protocol.

RNA amplification

RNA obtained from 10,000 endothelial cells obtained by FACS was amplified with the SPIA Aminoallyl™ system (Nugen, CA) according to the manufacturer’s instructions, producing at least 4 μg of cDNA.

cDNA hybridizations

Two-color array hybridizations were performed. For each hybridization, 2 μg of amplified cDNA was labeled with Cy3 and Cy5 with CyDye™ Post-Labeling Reactive Dye Pack (GE Healthcare, Piscataway, NJ) according to the manufacturer’s instructions. cDNA probes were combined, purified, and hybridized for 14 h at 42°C in a total volume of 35 μL of a hybridization solution containing 25% formamide, 3× SSC (0.45 M NaCl, 0.045 M Na citrate, pH 7.0), 0.2% sodium dodecyl sulfate (SDS), 1 μg of murine Cot1 DNA, 4 μg of polyA RNA, and 4 μg of tRNA. Microscopic slides pin-spotted with 32,000 long oligonucleotides of the Operon V3 set representing the entire mouse genome were produced by the UT Southwestern Microarray Core (Dallas, TX). Stringency washing at room temperature was sequentially performed for 10 min in each of the following solutions: 2× SSC containing 0.1% SDS, 0.1× SSC containing 0.1% SDS, and finally, 0.1× SSC. Performance of each experiment by microarray analysis was duplicated with the Cy3 and Cy5 labeling order reversed.

Data analysis

Slides were scanned with a Genepix 4000B dual channel scanner and the resultant images analyzed with Genepix and Acuity software (Molecular Devices, Sunnyvale, CA). Genepix 6.1 default settings were employed: median local background pixil intensity was subtracted, and features found “bad,” “absent,” “not found,” were flagged; features with a ratio of 635/532 <0.1 or >10 were excluded from normalization. Results were normalized by the ratio of medians method and filtered to exclude those with any of the following characteristics: a percentage of saturated pixils >3, a signal/noise ratio <3, a (regression ratio 635/532)2 <0.6, or a Genepix flag. Data passing the filter and exceeding threshold fold changes in replicate, dye-reversed analyses were considered to be dysregulated. For statistical analysis, the one sample T-test, with Benjamini–Hochberg correction, was applied to the combined results obtained at 300 and 1000 μg PM/m3 (4 microarrays per strain).

Immunolocalization

Tissue sections (5 μm) obtained from formalin-fixed, paraffin-embedded aortae were deparaffinized and exposed to the following: 95°C sodium citrate at 100 mM for 20 min, 0.3% (v/v) H2O2 for 5 min, and PBS containing 1% (w/v) bovine serum albumin and 5% (v/v) donkey serum for 20 min. Immunohistological localization was performed with goat anti-Terf1 (Santa Cruz Inc., Santa Cruz, CA) at 4 μg/mL for 16 h followed by biotinylated donkey anti-goat (Jackson Immunoresearch, West Grove, PA) at 1.0 μg/mL for 1 h. Slides were then incubated with ABC Elite™ and visualized with VIP™ substrate (both from Vector Labs, Burlingame, CA) according to the manufacturer’s instructions.

Results

ApoE (−/−) mice were hypercholesterolemic vs. ApoE (+/+), with cholesterol elevation comparable with the ApoE-deficient parental strain BL/6 ApoE (−/−) (see Figure 1). Furthermore, Tie2 GFP/ApoE (−/−) mice developed aortic atheroma (results not shown), as reported for the BL/6 ApoE (−/−) strain (Breslow 1996).

Figure 1.

Figure 1

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Total serum cholesterol levels in ApoE (+/+), ApoE (−/−), and BL/6 ApoE (−/−) mice (n = 3). * indicates a P-value <0.05.

Microarray results were submitted to the Gene Expression Omnibus database (US NCBI) as series GSE13160. A total of 176 transcripts were found to be down-regulated (<0.35-fold) or up-regulated (>2.8-fold) in the ApoE (−/−) strain in at least one experimental dose; only two transcripts appeared dys-regulated in the ApoE (+/+) strain. A subset of transcripts possessing known RefSeq identities are shown in Tables A and B of the appendix. Thirty days of exposure to diesel exhaust resulted in >50 transcripts appearing dysregulated in ApoE (−/−) mice at both 300 and 1000 μg PM/m3 doses, with no transcripts appearing commonly dysregulated in ApoE (+/+) mice.

In the combined microarray results of ApoE (−/−) mice exposed to 300 and 1000 μg PM/m3, 49 of the highly dys-regulated transcripts appeared statistically significant (P-value <0.05); no transcripts appeared statistically significant in pollution-exposed ApoE (+/+) mice. These results were subjected to hierarchical cluster analysis, displayed as a heat map shown in Figure 2, which indicates a high level of concordance of responses to either concentration of diesel exhaust in ApoE (−/−) mice. Transcripts lacking RefSeq identity are not displayed.

Figure 2.

Figure 2

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Microarray results of endothelial responses to diesel engine exhaust in vivo. Heat map of aortic endothelial transcripts dysregulated by >2.8- or <0.35-fold in ApoE (−/−) and ApoE (+/+) mice exposed for 30 days to fresh exhaust from a Yanmar diesel engine diluted with air to particulate concentrations of 300 or 1000 μg PM/m3. Transcripts shown were significantly dysregulated in the combined analyses of the ApoE (−/−) strain (n = 4, P < 0.05). Each rectangle represents the average results of two microarray analyses of pooled endothelium from four mice exposed to exhaust at 300 or 1000 μg PM/m3 vs. four control mice exposed to HEPA-filtered air. Red indicates up-regulation, green indicates down-regulation, and black indicates no change.

A literature search revealed important pathophysiological roles for a subset of these transcripts within the vasculature. Supporting references are provided in Table C of the appendix. The highly dysregulated transcripts observed within the ApoE (−/−) strain were subjected to analysis with the DAVID bioinformatics tool according to default settings (Huang da et al. 2009) to detect enrichment of annotation terms: Fisher’s exact t-test with Benjamini correction revealed no evidence of significant enrichment of annotation terms (all P-values >0.05).

As displayed in Figure 3, a subset of transcripts appearing highly dysregulated in ApoE (−/−) mice exposed to diesel exhaust at 300 and 1000 μg PM/m3 possess important and overlapping roles in vascular function. Transcripts responding to diesel exhaust in Apo E (−/−) mice include molecules known to maintain normal vascular homeostasis or contribute to pathology in other models of vascular disease. Pdgfra, Vcam1, Cav1, and Clu/Clusterin appear down-regulated in response to diesel exhaust in our study. The progression of atherosclerosis in ApoE (−/−) mice can be suppressed pharmacologically with an inhibitor of PDGF signaling, Imatinib (Lassila et al. 2004) or by a peptide of Clusterin (Navab et al. 2005); atherogenesis in the ApoE (−/−) can be decreased by genetic deficiency of Cav1 (Frank et al. 2004) or Vcam1 (Dansky et al. 2001). Down-regulated transcripts, Anxa2/Annexin A2 and Srgn/serglycin, possess known roles in plasminogen activation and plasminogen intracellular trafficking, respectively (He et al. 2008; Kolset and Tveit 2008). Endothelial permeability has been shown to be increased by Angptl4 (Cazes et al. 2006) as well as both components of the adrenomedullin receptor complex, Ramp2 (Ichikawa-Shindo et al. 2008) and Calcrl (Temmesfeld-Wollbrück et al. 2007), all of which appear down-regulated by diesel exhaust. Transcripts of lipid metabolic enzymes dysregulated by diesel exhaust included up-regulated Bcdo2 and down-regulated Adipsin; lipid-binding proteins Clu and Fabp6 both appear down-regulated in our study. Transcripts up-regulated upon exposure to diesel exhaust included a glutamate receptor, Gria3, and a glutamate transporter, Slc1a3.

Figure 3.

Figure 3

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Selected transcripts dysregulated by >2.8- or <0.35-fold in ApoE (−/−) mice exposed for 30 days to fresh exhaust from a Yanmar diesel engine diluted with HEPA-filtered air to engine exhaust at 300 or 1000 μg PM/m3. Transcripts shown have recognized roles in vascular pathophysiology. Fold changes are presented on a log2 scale with each bar representing the average results of two microarray determinations of pooled endothelium from four mice exposed to exhaust vs. four control mice exposed to air.

As shown in Figure 3, exposure of ApoE (−/−) mice to diesel exhaust resulted in dysregulation of inflammatory signaling transcripts within the aortic endothelium, including up-regulation of Nfkbie and down-regulation of Vcam1 and Sdcbp. Our observed increase in transcription of Nfkbie potentially reflects a compensatory mechanism to limit ongoing NF-κB signaling associated with responses to diesel engine particulate, similar to those observed in lung epithelium (Shukla et al. 2000).

As shown in Figure 4, immunolocalization revealed up-regulation of Terf1 in the aortic endothelium of ApoE (−/−) mice exposed to diesel exhaust at 1000 μg PM/m3, consistent with the 6-fold up-regulation indicated by our microarray analysis. Terf1 is an inhibitor of telomerase activity; telomere shortening has been observed in circulating endothelial progenitors obtained from patients with coronary artery disease (Satoh et al. 2008).

Figure 4.

Figure 4

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Representative immunolocalization of Terf1, indicated by elevated staining (purple) in the aortic endothelium of ApoE (−/−) mice following exposure to dilute diesel exhaust at 1000 μg PM/m3 for 30 days vs. purified air. Arrows indicate endothelia; L indicates luminal space. Tissue sections lacking primary antibody displayed minimal background staining.

Discussion

We report substantial transcriptional responses in the aortic endothelium of ApoE (−/−) mice exposed to diesel engine exhaust. Importantly, our analysis revealed a much lower degree of dysregulation in identically exposed wild-type mice, suggesting that: (1) healthy endothelial cells are able to maintain homeostasis over a prolonged exposure to diesel emissions and (2) hypercholesterolemia increases the sensitivity of the endothelial transcriptional response to air pollution. The high degree of concordance of the responses of ApoE (−/−) mice exposed to either 300 or 1000 μg PM/m3 implies a threshold of activation below 300 μg PM/m3, a concentration of diesel particulate observed in mining and other occupational settings (Pronk et al. 2009); due to their similarity, these data sets were combined for statistical evaluation.

Interestingly, studies of ApoE (−/−) mice exposed to gasoline or diesel emissions for up to 50 days in the presence of a high-fat diet revealed enhancement of matrix metalloproteinase (MMP), heme oxygenase-1, and endothelin-1 gene expression in the whole aorta, accompanied by evidence of enhanced remodeling and monocyte invasion of the vascular wall (Lund et al. 2007, 2009). The results of the present study of normally fed mice, wherein gene regulation in non-endothelial cell types has been minimized by FACS isolation, did not reveal MMPs or other previously reported transcripts. Furthermore, serum cholesterol levels of ApoE (−/−) mice receiving normal chow used in our study were >2-fold lower than those receiving a high fat (BioServ #S3282, results not shown), thus more closely approximating levels found in clinical hypercholesterolemia. These differences highlight the importance of understanding cell-specific responses that drive whole-organ outcomes.

In an earlier study, acute exposure of rats to diesel exhaust at 300 μg PM/m3 for 5 h resulted in endothelial-dependent changes in coronary responsiveness to endothelin-1 (Cherng et al. 2009). One might expect prolonged exposure of wild-type mice to diesel exhaust at 300 μg PM/m3 would lead to transcriptional changes as well; however, it is possible that biochemical effects in the absence of gene regulation, or homeostatic mechanisms counterbalanced these effects upon 30 days of exposure. In the present study, substantial transcriptional responses in the endothelium to the effects of diesel exhaust were apparent only in the ApoE (−/−) strain. This differential response is attributable to dyslipidemia, impaired macrophage lipid scavenging activity (Atkinson et al. 2008), or other metabolic differences characteristic of the ApoE (−/−) strain. Such susceptibility in the ApoE (−/−) strain further demonstrates diesel exhaust and other forms of air pollution as cardiovascular risk factors, which may become manifest only in the added presence of impaired lipid disposition.

Our data indicate that chronic exposure of ApoE (−/−) mice to diesel exhaust affects regulators of endothelial adhesion (Nfkbie and Vcam1), permeability (Angptl4, Calcrl, Ramp2), invasion (Erbb2ip), and genomic stability (Terf1 and Lmna). Not all of these transcripts were up-regulated; the complex response may reflect homeostatic regulation of these processes or selective survival of endothelial cells that exhibit these changes. Our findings of responses to diesel exhaust among modulators of endothelial function are consistent with reports of accelerated atherogenesis in the aortae of ApoE (−/−) mice exposed to air pollution (Sun et al. 2005; Araujo et al. 2008).

A subset of dysregulated transcripts identified in our analysis possesses known vascular function and thus provides a molecular foundation for the epidemiological association of air pollution with accelerated atherogenesis. The present study combines realistic exposure and cell-specific genomic inquiry to obtain robust clues to in vivo mechanisms underlying the augmentation of atherosclerosis by diesel exhaust. Future work using similar methods will enable a greater understanding of the biological outcomes following exposures to the complex mixtures that comprise combustion-source air pollution.

Acknowledgments

This work was supported by the NIEHS ES013395, NCRR RR016453, and NHLBI HL073449 (to RVS).

Appendix

Table A.

Transcripts down-regulated in aortic endothelium in ApoE (+/+) and ApoE (−/−) mice in response to exposure to diesel exhaust for 30 days.

Marker symbol Name Ref Seq Average fold changea
ApoE (+/+)
ApoE (−/−)
300 μg/m3 1000 μg/m3 300 μg/m3 1000 μg/m3
Anxa2 Annexin A2 NM_007585 0.88 1.0 0.29 0.52
Cd93 Cd93 antigen, C1q receptor NM_010740 1.2 0.77 0.34 0.29
Vcam1 Vascular cell adhesion protein 1 NM_011693 0.88 0.91 0.32 0.39
Fxyd5 FXYD domain-containing ion transport regulator 5 NM_008761 1.5 1.12 0.21 0.43
Pdgfra Platelet-derived growth factor receptor, alpha polypeptide NM_011058 1.4 1.9 0.41 0.27
Lmna Lamin A NM_019390 1.2 1.0 0.25 0.34
Irf6 Interferon regulatory factor 6 NM_016851 1.3 0.34 0.30 0.30
Ahr Aryl hydrocarbon receptor NM_013464 0.92 1.1 0.32 0.51
Cxcl2 Chemokine (C-X-C motif) ligand 2 NM_009140 1.2 3.9 0.80 0.22
Apbb2 Amyloid beta (A4) precursor protein-binding, family B, member 2 NM_009686 1.2 1.12 0.31 0.43
Angptl4 Angiopoietin-related protein 4 NM_020581 1.3 1.7 0.33 0.45
Vcl Vinculin NM_009502 1.1 1.2 0.36 0.32
Sdcbp Syntenin 1 NM_016807 1.2 0.87 0.30 0.42
Gdap2 Ganglioside-induced differentiation-associated protein 2 NM_010269 1.0 1.1 0.39 0.34
Srgn Serglycin NM_011157 1.0 1.10 0.33 0.34
Tbc1d15 TBC1 domain family, member 15 NM_025706 0.81 0.91 0.49 0.33
Clic4 Chloride intracellular channel 4 NM_013885 1.1 1.1 0.23 0.48
Cav1 Caveolin 1, caveolae protein NM_007616 0.98 0.95 0.25 0.37
Pls3 Plastin 3 T isoform NM_145629 1.2 0.92 0.32 0.38
Serp1 Stress-associated endoplasmic reticulum protein 1 NM_030685 1.1 1.2 0.47 0.31
Spon1 Spondin 1 extracellular matrix protein NM_145584 1.4 0.81 0.33 0.24
Adamts8 A disintegrin and metalloproteinase with thrombospondin motifs 8 NM_013906 0.75 2.0 0.29 1.1
Clu Clusterin NM_013492 0.73 0.85 0.33 0.40
Ramp2 Receptor activity-modifying protein 2 NM_019444 0.91 0.84 0.33 0.38
Calcrl Calcitonin gene-related peptide type 1 receptor NM_018782 0.70 0.69 0.28 0.34
S100a4 S100 calcium-binding protein A4 NM_011311 0.57 0.85 0.27 0.44
Adn Adipsin NM_013459 0.86 1.8 0.31 0.26
Ptp4a3 Protein tyrosine phosphatase 4a3 NM_008975 1.2 1.1 0.30 0.37
Fstl Follistatin-like 1 NM_008047 1.2 0.64 0.36 0.25
Cd74 Cd74 antigen (invariant polypeptide of major histocompatibility complex, class II antigen-associated) NM_001042605 0.68 1.8 0.29 0.35
P4hb Prolyl 4-hydroxylase, beta-polypeptide NM_011032 0.84 0.89 0.34 0.37
Adfp Adipose differentiation-related protein NM_007408 0.63 2.0 0.34 0.43
Mrpl33 Mitochondrial ribosomal protein L33 NM_025796 0.95 0.98 0.52 0.32
Rbm7 RNA-binding motif protein 7 NM_144948 0.93 0.88 0.37 0.31
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a

Values in bold were down-regulated ≤0.35.

Table B.

Transcripts up-regulated in aortic endothelium in ApoE (+/+) and ApoE (−/−) mice in response to exposure to diesel exhaust for 30 days.

Marker symbol Name Ref Seq Average fold changea
ApoE (+/+)
Apo E (−/−)
300 μg/m3 1000 μg/m3 300 μg/m3 1000 μg/m3
Terf1 Telomeric repeat-binding factor 1 NM_009352 1.4 1.1 3.8 6.1
Fabp6 Fatty acid-binding protein 6, ileal (gastrotropin) NM_008375 0.87 0.53 2.61 3.8
Gria3 Glutamate receptor, ionotropic, alpha 3 NM_016886 0.97 0.94 4.3 5.0
Nfkbie NF-κB inhibitor epsilon NM_008690 1.3 0.78 3.2 4.6
Gk2 Glycerol kinase 2 NM_010294 1.1 0.95 3.2 4.4
Bcdo2 Beta-carotene oxygenase 2 NM_133217 0.98 0.55 2.4 4.0
Gcnt3 Glucosaminyl (N-acetyl) transferase 3, mucin type NM_028087 1.2 1.4 2.4 4.3
Dmrt1 Double sex and mab-3-related transcription factor 1 NM_015826 1.1 1.3 2.9 3.1
Hydin Hydrocephalus-inducing NM_172916 1.1 0.88 3.8 5.5
Grb10 Growth factor receptor-bound protein 10 NM_010345 1.1 0.88 3.0 4.6
H2-M10.4 Histocompatibility 2, M region locus 10.4 NM_177634 1.2 1.1 2.8 4.3
Cpne5 Copine V NM_153166 1.5 1.2 3.6 5.0
Rab40c Rab40c, member RAS oncogene family NM_139154 1.0 1.1 2.3 4.4
Inpp4a Inositol polyphosphate-4-phosphatase, type I NM_030266 1.1 0.71 3.3 6.5
Camsap1l1 Calmodulin-regulated spectrin-associated protein 1-like 1 NM_001081360 1.1 1.2 3.5 5.6
Ttn Titin NM_176926 1.3 0.88 3.2 3.6
Erbb2ip Erbb2-interacting protein NM_001005868 1.0 0.87 1.6 3.3
Vtcn1 V-set domain-containing T-cell activation inhibitor 1 NM_178594 1.1 0.98 2.5 3.5
Slc1a3 Solute carrier family 1 (glial high-affinity glutamate transporter), member 3 NM_148938 0.78 1.0 2.7 4.7
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a

Values in bold were up-regulated ≥2.8.

Table C.

Transcripts with known vascular functions, which appear dysregulated in aortic endothelium of ApoE (−/−) mice in response to 30 days of diesel exhaust.

Marker symbola Ref Seq Role in vasculature
Anxa2 (↓) NM_007585 Annexin A2 heterotetramer with S100A10/p11 is endothelial locus for tPA and plasmin activation (He et al. 2008)
Cd93 (↓) NM_010740 Cd93 is prominently expressed on endothelium and contributes to removal of apoptotic cells (Fonseca et al. 2001; Norsworthy et al. 2004)
Vcam1 (↓) NM_011693 Vcam1 domain 4-deficient (D4D) +/+ mice crossed with Apo E (−/−) mice exhibit decreased atherogenesis (Dansky et al. 2001)
Pdgfra (↓) NM_011058 Imatinib/Gleevec (blockade of PDGFr) down-regulates atherogenesis induced by type I diabetes model in Apo E mice (Lassila et al. 2004)
Terf1 (↑) NM_009352 Terf1 regulates telomere length. Endothelial progenitor cell telomere length is shorter in CAD patients and in vitro EPC treated with oxidative treatments (Okamoto et al. 2008; Satoh et al. 2008)
Ahr (↓) NM_013464 Benzopyrene leads to up-regulation of ICAM-1, which is disrupted by Cav1 deficiency (Oesterling et al. 2008)
Apbb2 (↓) NM_009686 Amyloid precursor cleavage by gamma secretase is inhibited by Apo E (Irizarry et al. 2004)
Angptl4 (↓) NM_020581 Angiopoietin-like 4 inhibits endothelial permeabilization (Cazes et al. 2006)
Sdcbp (↓) NM_016807 TNF-inducible in cultured endothelium (Stier et al. 2000)
Srgn (↓) NM_011157 Serglycin binds plasminogen activator in endothelium (Kolset and Tveit 2008)
Cav1 (↓) NM_007616 Knockout of Cav1 confers resistance to atherogenesis in the Apo E (−/−) mouse (Frank et al. 2004)
Spon1 (↓) NM_145584 F-spondin (an ECM protein) affects amyloid precursor processing by interacting with Apo E receptor (Hoe et al. 2005)
Fabp6 (↑) NM_008375 Fabp6 levels are regulated by sterol receptors FXR, LXR, and SREBP1c (Besnard et al. 2004)
Clu (↓) NM_013492 Potentially protects against the effects of modified LDL. Clu/ApoJ peptide mimetic suppresses atherosclerosis in Apo E mice (Navab et al. 2005)
Ramp2 (↓) NM_019444 Ramp2 underexpression is associated with increased vascular permeability (Ichikawa-Shindo et al. 2008)
Gria3 (↑) NM_016886 Vasodilation by glutamate is mediated by carbon monoxide produced by heme oxygenase (Parfenova et al. 2003)
Nfkbie (↑) NM_008690 I kappa B epsilon is involved in c-Rel-associated ICAM-1 induction in endothelium (Spiecker et al. 2000)
Bcdo2 (↑) NM_133217 Asymmetric cleavage of beta-carotene
Calcrl (↓) NM_018782 Calcrl knockout resembles adrenomedullin knockout; adrenomedullin protects against endothelial barrier breakdown; AM AMBP instillation protects against endothelial dysfunction in rat model of sepsis (Dackor et al. 2006; Temmesfeld-Wollbrück et al. 2007; Zhou et al. 2007)
S100a4 (↓) NM_011311 S100a is required for metastasis (Grigorian et al. 2008)
Adn (↓) NM_013459 Up-regulated in endothelium of model of Type I diabetes (Maresh and Shohet 2008)
Grb10 (↑) NM_010345 Grb10 inhibits Vegf-r2 internalization (Murdaca et al. 2004)
Ptp4a3 (↓) NM_008975 Expression in endothelium of tumors (Bardelli et al. 2003)
Fstl (↓) NM_008047 Fstl/Tsc-36 promotes endothelial function in ischemic tissue (Ouchi et al. 2008)
H2-M10.4 (↑) NM_177634 MHC Class II, M region locus 10.4
Cpne5 (↑) NM_153166 Ca2+-dependent phospholipid-binding protein (Tomsig and Creutz 2002)
Cd74 (↓) NM_001042605 MHC Class II invariant chain
Inpp4a (↑) NM_030266 PI3 kinase signal regulation (Ivetac et al. 2005)
Adfp (↓) NM_007408 Adfp antisense oligo decreases hepatic insulin resistance (Varela et al. 2008)
Mrpl33 (↓) NM_025796 Mitochondrial ribosomal protein with nuclear code
Erbb2ip (↑) NM_001005868 Endothelium from aged mice susceptible to tumor cell invasion in in vitro assay and this process requires Erbb EGF signaling cross talk (Price et al. 2004)
Vtcn1 (↑) NM_178594 Negative modulator of T-cell response (Suh et al. 2006)
Lmna (↓) NM_001002011 Mutant lamin A (progerin) accumulation in dermal vascular cells of Hutchinson–Gilford progeria (McClintock et al. 2006)
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a

Arrows indicate direction of dysregulation in the Apo E (−/−) strain; n.c. indicates no change upon exposure to exhaust at 300 μg/m3.

Footnotes

Declaration of interest

The authors report no declaration of interest.

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