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Up-regulation of tissue factor in human pulmonary artery endothelial cells after ultrafine particle exposure.

BACKGROUND: Epidemiology studies have linked exposure to pollutant particles to increased cardiovascular mortality and morbidity, but the mechanisms remain unknown.

OBJECTIVES: We tested the hypothesis that the ultrafine fraction of ambient pollutant particles would cause endothelial endothelial /en·do·the·li·al/ (-the´le-al) pertaining to or made up of endothelium.
A layer of cells that lines the inside of certain body cavities, for example, blood vessels.
 cell dysfunction.

METHODS: We profiled gene expression of human pulmonary artery endothelial cells (HPAEC HPAEC High-Performance Anion-Exchange Chromatography ) exposed to ultrafine particles (UFPs; 100 [micro]g/mL) from Chapel Hill, North Carolina Chapel Hill is a town in North Carolina and the home of the University of North Carolina at Chapel Hill (UNC-CH), the oldest state-supported university in the United States. As of the 2000 census, it had a population of 48,715. As of 2004 its estimated population was 52,440. , or vehicle for 4 hr with Affymetrix HG U133 Plus 2.0 chips (n = 4 each).

RESULTS: We found 320 up-regulated genes and 106 down-regulated genes (p < 0.01, 5% false discovery rate). We noted up-regulation of genes related to coagulation coagulation (kōăg'ylā`shən), the collecting into a mass of minute particles of a solid dispersed throughout a liquid (a sol), usually followed by the precipitation or  [tissue factor (F3) and coagulation factor II coagulation factor II Prothrombin, see there  receptor-like 2 (F2RL2)] and differential regulation of genes related to F3 signaling (FOS FOS
free on steamer
, JUN, and NFKBIA NFKBIA Nuclear Factor of Kappa Light Chain Gene Enhancer in B Cells Inhibitor, Alpha ). Results of quantitative polymerase chain reaction Quantitative polymerase chain reaction (qPCR) is a modification of the polymerase chain reaction used to rapidly measure the quantity of DNA, complementary DNA or ribonucleic acid present in a sample.  show a significant upregulation of F3 after 10 and 100 [micro]g/mL UFP UFP United Federation of Planets (Star Trek)
UFP Union des Forces Progressistes (French: Union of the Forces Progressists, Quebec provincial party)
UFP URL Filtering Protocol
 exposures. Additionally, the water-soluble fractions of UFPs were sufficient to induce the expression of F3, F2RL2, and heme oxygenase 1 (HMOX HMOX Heme Oxygenase 1). Treatment of HPAEC with UFPs for 16 hr increased the release of interleukin (IL)-6 and IL-8. Pretreatment pretreatment,
n the protocols required before beginning therapy, usually of a diagnostic nature; before treatment.

pretreatment estimate,
n See predetermination.
 of HPAEC with a blocking antibody against F3 attenuated Attenuated
Alive but weakened; an attenuated microorganism can no longer produce disease.

Mentioned in: Tuberculin Skin Test


having undergone a process of attenuation.
 IL-6 and IL-8 release by 30 and 70%, respectively.

CONCLUSIONS: Using gene profiling, we discovered that UFPs may induce vascular endothelial cells to express genes related to clotting. These results indicate that PM may cause adverse cardiovascular health effects by activating coagulation-inflammation circuitry.

KEY WORDS: coagulation cascade, human endothelial cells, microarray, particulate matter, tissue factor. Environ Health Perspect 115:535-540 (2007). doi:10.1289/ehp.9556 available via [Online 8 January 2007]


Particulate air pollution remains a threat to public health despite the decline in the ambient concentration in the past decades. It has been estimated by the World Health Organization that particulate matter (PM) exposure accounts for 800,000 deaths per year worldwide (Brook et al. 2004). The increased mortality has been attributed to respiratory and more recently to cardiovascular compromise (Borja-Aburto et al. 1998; Brook et al. 2004; Dockery 2001). Short-term exposure to polluted air for as little as 2 hr increases the risk of myocardial infarction in people at risk of developing cardiovascular disease (Peters et al. 2001).

Various mechanisms have been proposed to explain the cardiovascular health effects of PM. These hypotheses include pulmonary and systemic oxidative stress and inflammation (Gurgueira et al. 2002), direct vasoconstriction vasoconstriction /vaso·con·stric·tion/ (-kon-strik´shun) decrease in the caliber of blood vessels.vasoconstric´tive

 (Huang et al. 2002; Li et al. 2005a), enhanced coagulation pathways (Nemmar et al. 2002b), and altered cardiac autonomic function (Gold et al. 2000). The ultrafine fraction of ambient pollutant particles [diameter < 100 nm, ultrafine particles (UFPs)] may represent a particular concern because these small particles can remain airborne for an extended period of time. Although they represent only a relatively small fraction of the total mass (Peters et al. 1997), UFPs are a substantial component of PM in the number of particles. Once inhaled, UFPs can be deposited in greater numbers in deeper lung zones than larger particles (Nemmar et al. 2002b) and have a greater potential to permeate the alveolar alveolar /al·ve·o·lar/ (al-ve´o-lar) [L. alveolaris ] pertaining to an alveolus.

Relating to an alveolus.
 capillary barrier to be in contact with vascular endothelial cells.

To better understand how UFPs may affect vascular endothelial cells, we profiled gene expression in human pulmonary arterial endothelial cells (HPAEC) exposed to UFPs in Chapel Hill, North Carolina. We hypothesize that UFPs would induce transcriptional activation of genes related to the coagulation and inflammatory response which are signs of endothelial cell dysfunction. Cultured HPAEC were treated with Chapel Hill UFPs (100 [micro]g/mL) or control for 4 hr. RNA RNA: see nucleic acid.
 in full ribonucleic acid

One of the two main types of nucleic acid (the other being DNA), which functions in cellular protein synthesis in all living cells and replaces DNA as the carrier of genetic
 from these samples was hybridized to Affymetrix HG U133 Plus 2.0 chips and analyzed for differentially expressed genes. We found that tissue factor (F3, Unigene accession no. 62192;, coagulation factor II receptor-like 2 (F2RL2; Unigene accession no. 42502), interleukin 6 (IL-6, Unigene accession no. 512234) and interleukin 8 (IL-8; Unigene accession no. 624) were up-regulated. We further showed that the release of IL-6 and IL-8 induced by UFP was partially dependent on the activation of the tissue factor pathway.

Materials and Methods

Reagents and chemicals. Molecular mass standards, polyacrylamide pol·y·a·cryl·a·mide  
A white polyamide, (-CH2CHCONH2-), related to acrylic acid.

[poly- + acryl(ic acid) + amide.
, buffers, and protein assay reagents were purchased from Bio-Rad (Richmond, CA). The enhanced chemiluminescent chem·i·lu·mi·nes·cence  
Emission of light as a result of a chemical reaction at environmental temperatures.

 (ECL (Emitter-Coupled Logic) A digital circuit composed of bipolar transistors in which the emitter ends are wired together. ECL gates switch faster than TTL gates, but consume more power. See TTL, I2L and bipolar.

) blotting detection reagents were purchased from Amersham Biosciences Corp. (Piscataway, NJ). TaqMan Universal PCR PCR polymerase chain reaction.

polymerase chain reaction

Polymerase chain reaction (PCR) 
 master mix and Taqman Gene Expression pre-developed assays (reagents concentrated and preoptimized mixes of primers and FAM-labeled TaqMan probes) for tissue factor (F3), coagulation factor II receptor-like 2 (F2RL2), coagulation factor VIII coagulation factor VIII Factor VIII, see there  associated (F8A1, Unigene accession no. 533543;, and heme oxygenase 1 (HMOX1; Unigene accession no. 517581) were obtained from Applied Biosystems (Foster City, CA). All other chemicals and reagents were from Sigma-Aldrich Co. (St. Louis, MO) unless stated otherwise.

Collection and extraction of Chapel Hill UFPs. UFPs were collected in July of 2002 in Chapel Hill, North Carolina, outside the U.S. Environmental Protection Agency Environmental Protection Agency (EPA), independent agency of the U.S. government, with headquarters in Washington, D.C. It was established in 1970 to reduce and control air and water pollution, noise pollution, and radiation and to ensure the safe handling and  Human Studies Facility as described previously (Becker et al. 2005). The elemental composition of the UFPs (nanograms per milligram milligram /mil·li·gram/ (mg) (mil´i-gram) one thousandth (10-3) of a gram.

n. Abbr. mg
A metric unit of mass equal to one thousandth (10-3) of a gram.
) was described previously with aluminum (948), copper (150), iron (879), lead (181), silicon (1098), and zinc (669) representing the most abundant constituents (Becker et al. 2005). UFP were resuspended in sterile [H.sub.2]O (5.0 mg/mL) and stored at -80[degrees]C.

Preparation of the water-soluble and-insoluble fractions of UFP. Water-suspended UFPs were diluted to 25 and 100 [micro]g/mL, centrifuged for 30 min at 20,000 x g, and the supernatants were collected as the water-soluble fractions. Pellets from the centrifugation Centrifugation

A mechanical method of separating immiscible liquids or solids from liquids by the application of centrifugal force. This force can be very great, and separations which proceed slowly by gravity can be speeded up enormously in centrifugal
 were washed with deionized de·i·on·ize  
tr.v. de·i·on·ized, de·i·on·iz·ing, de·i·on·iz·es
To remove ions from (a solution) using an ion-exchange process.

 distilled water 3 times and resuspended in the appropriate volume of water. We designated this as the water-insoluble fraction. Both fractions were stored at -80[degrees]C.

Human pulmonary artery endothelial cells. For microarrays, HPAEC (Cell Applications, Inc., San Diego, CA) were cultured in endothelial growth medium (EGM-2, Clonetics Bio Whittaker Inc., Walkersville, MD), grown to 80% confluency in 100-mm petri dishes, and used during passages 3-5. UFPs were diluted with EGM-2 before experiments. Cells were treated with UFPs (100 [micro]g/mL) or vehicle control for 4 hr at 37[degrees]C. For all other experiments, HPAEC were grown to 80% confluency in 6-well plates.

Two different quantitative polymerase chain reaction (Q-PCR) experiments were conducted with UFPs and water-soluble and -insoluble fractions. HPAEC were treated for 4 hr (0, 1, 10, and 100 [micro]g/mL UFP) for F3 Q-PCR analysis or 2 and 24 hr (0, 25, and 100 [micro]g/mL water-soluble and -insoluble UFP fractions) for all F3, F2RL2, F8A1, and HMOX1.

RNA isolation. RNA was extracted with Trizol reagent (GIBCO GIBCO Grand Island Biological Company (tissue culture media enterprise)  BRL BRL

In currencies, this is the abbreviation for the Brazilian Real.

The currency market, also known as the Foreign Exchange market, is the largest financial market in the world, with a daily average volume of over US $1 trillion.
 Life Technologies, Gaithersburg, MD) and purified with Qiagen RNeasy mini columns (Qiagen, Valencia, CA) according to manufacturers' protocols. Total RNA was resuspended in RNase-free water and assessed with the Agilent 2100 Bioanalyzer and RNA 6000 Nano assay (Agilent Technologies, Palo Alto, CA). All RNA samples had a 28S/18S ratio [greater than or equal to] 1.8 and were stored at -80[degrees]C before shipment on dry ice to Expression Analysis Inc. (Durham, NC).

Microarray target preparation and hybridization hybridization /hy·brid·iza·tion/ (hi?brid-i-za´shun)
1. crossbreeding; the act or process of producing hybrids.

2. molecular hybridization

. RNA target preparation and hybridization to the Affymetrix HG U133 plus 2.0 GeneChip oligonucleotide microarray (Affymetrix, Inc., Santa Clara, CA) was performed by Expression Analysis, Inc. as described previously (Li et al. 2005b). Fluorescent images were detected in a GeneChip Scanner 3000 and expression data was extracted using the default settings in the Microarray Suite 5.0 software (Affymetrix). All GeneChips were scaled to a median intensity setting of 500. For microarray purposes, four biological replicates were collected each for the treated and control samples.

Q-PCR. Q-PCR was performed for selected genes. cDNAs were synthesized from 0.4 [micro]g of total RNA in 100 [micro]L of buffer containing 5 [micro]M random hexaoligonucleotide primers (Pharmacia, Piscataway, NJ), 10 U/[micro]L Moloney murine leukemia virus The murine leukemia virus belongs to the gammaretroviral genus of the Retroviridae family of viruses, their hosts are vertebrates. It is a Type VI: positive sense ssRNA viruses that replicates through a DNA intermediate, reverse transcriptase.  reverse transcriptase (GIBCO BRL Life Technologies), 1 U/[micro]L RNase inhibitor (RNasin, Promega, Madison, WI), 0.5 mM dNTP (Pharmacia), 50 mM KCl, 3 mM Mg[Cl.sub.2], and 10 mM Tris-HCl (pH 9.3) for 1 hr at 39[degrees]C. Reverse transcriptase was heat inactivated inactivated

rendered inactive; the activity is destroyed.

inactivated viruses
treated so that they are no longer able to produce evidence of growth or damaging effect on tissue.
 at 94[degrees]C for 4 min.

Q-PCR of specimen and standard cDNA was completed using TaqMan predeveloped assay reagents. Quantitative fluorogenic amplification of cDNA was performed using the ABI Abi (ā`bī) [short for Abijah], in the Bible, King Hezekiah's mother.

(Application Binary Interface) A specification for a specific hardware platform combined with the operating system.
 Prism 7500 Sequence Detection System, primers and probes of interest and TaqMan Universal PCR Master Mix (Applied Biosystems). The relative abundance of mRNA levels was determined from standard curves generated from a serially diluted standard pool of cDNA prepared from control HPAEC cultures. The relative abundance of glyceraldehyde-3-phosphate dehydrogenase dehydrogenase /de·hy·dro·gen·ase/ (de-hi´dro-jen-as?) an enzyme that catalyzes the transfer of hydrogen or electrons from a donor, oxidizing it, to an acceptor, reducing it.

 (GAPDH GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase (also seen as G3PDH) , Unigene accession no. 544577; mRNA was used to normalize levels of the mRNAs of interest. For Q-PCR verification experiments, RNA from at least three additional experiments was collected for each timepoint and concentration. RNA samples for microarray and Q-PCR were collected from different experiments and do not represent the same sample.

Experiments with anti-tissue factor antibody. HPAEC were pretreated with a monoclonal blocking antibody against human F3 (product number 4509; American Diagnostica, Inc., Stamford, CT) (0, 50, and 100 ng/mL) for 15 min with gentle rocking before addition of UFPs (100 [micro]g/mL) or control. UFPs were sonicated for 10 min before addition to cultures. Cell cultures were then incubated for 16 hr at 37[degrees]C. Supernatants were removed, centrifuged for 10 min at 150 x g at 4[degrees]C, and stored at -80[degrees]C. IL-6 and IL-8 protein levels in supernatant samples (R & D Systems, Inc., Minneapolis, MN) were determined by immunoassay according to the manufacturer's recommendation. Three biological replicates were run in duplicate for each dose and assay.

Protein isolation and Western blot analysis West·ern blot analysis
An electrophoretic procedure for separating proteins.
. After exposure, HPAEC cells were lysed with 500 [micro]L RIPA RIPA. The bank of a river, or the place beyond which the waters do not in their natural course overflow.
     2. An extraordinary overflow does not change the banks of the river. Poth. Pand. lib. 50, h.t. See Banks of rivers; Riparian proprietors; Rivers.
 buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, and 0.1% SDS 1. (company) SDS - Scientific Data Systems.
2. (tool) SDS - Schema Definition Set.
 in phosphate buffered saline Phosphate buffer saline (abbreviated PBS) is a buffer solution commonly used in biochemistry. It is a salty solution containing sodium chloride, sodium phosphate and potassium phosphate. The buffer helps to maintain a constant pH.  (PBS PBS
 in full Public Broadcasting Service

Private, nonprofit U.S. corporation of public television stations. PBS provides its member stations, which are supported by public funds and private contributions rather than by commercials, with educational, cultural,
), pH 7.4) containing 1 [micro]L protease inhibitor cocktail (Sigma-Aldrich Co.) per 100 [micro]L RIPA (radioimmunoprecipitation) buffer and sheered with a 22-ga needle. Lysates were centrifuged 10 min at 150 x g, 4[degrees]C, and stored at -80[degrees]C. Protein concentration of cell lysates was measured with Bio-Rad protein assay reagents according to manufacturer instructions.

For Western blot analysis, cellular proteins were loaded equally, separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The blot was then blocked with 5% milk in PBS with 0.05% Tween-20 for 1 hr at room temperature, washed briefly, then probed with a monoclonal antibody against tissue factor (product number 4509; American Diagnostica, Inc.) overnight at 4[degrees]C followed by incubation with a horseradish horseradish

Hardy perennial plant (Armoracia lapathifolia) of the mustard family, native to Mediterranean lands and grown throughout the temperate zones. Its hotly pungent, fleshy root is used as a condiment and is traditionally considered medicinal.
 peroxidase-conjugated goat antimouse antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Bands were detected with ECL and films according to the manufacturer instructions (Du Pont-New England Nuclear, Boston, MA). Films were scanned on a ScanJet 6200C with Precision Scan Pro 1.02 (Hewlett Packard, Palo Alto, CA).

Microarray data analysis. Affymetrix CEL data files were imported into R, an open-source statistical scripting language (; Ihaka and Gentleman 1996) used in conjunction with the Bioconductor project (; Gentleman et al. 2004). Normalized values with robust multichip analysis background correction, quantile quantile

division of a total into equal subgroups; includes terciles, quartiles, quintiles, deciles, percentiles.
 normalization and median polish were calculated with AffylmGUI (Wettenhall et al. 2006). AffylmGUI allows a graphical user interface graphical user interface (GUI)

Computer display format that allows the user to select commands, call up files, start programs, and do other routine tasks by using a mouse to point to pictorial symbols (icons) or lists of menu choices on the screen as opposed to having to
 for the analysis of Affymetrix microarray GeneChips using the limma package (Smyth 2005) in R. A contrast of treated-control samples was used to generate p-values and M-value [[log.sub.2] (treated/control)]. Probe sets with a p-value (p < 0.01) after an adjustment with a false discovery rate (FDR) 5% were judged by the limma package to be differentially expressed between the treated and control samples. If more than one probe set for the same gene was identified, their M-values (treatment/control ratios) were averaged. The data discussed in this publication have been deposited in the National Center for Biotechnology Information's Gene Expression Omnibus (GEO;; Edgar et al. 2002) and are accessible through GEO series accession number GSE GSE

general somatic efferent system.

Biological pathway and network identification. Biological processes represented by the differentially expressed genes were compiled using the Database for Annotation, Visualization and Integrated Discovery [DAVID David, in the Bible
David, d. c.970 B.C., king of ancient Israel (c.1010–970 B.C.), successor of Saul. The Book of First Samuel introduces him as the youngest of eight sons who is anointed king by Samuel to replace Saul, who had been deemed a failure.
 2.1 (; Dennis et al. 2003]. Biological pathways were compared to all available Homo sapiens pathways specific to the Kyoto Encyclopedia of Genes and Genomes (KEGG KEGG Kyoto Encyclopedia of Genes and Genomes  2007). Only pathways with a p [less than or equal to] 0.05 calculated by DAVID were retained.

Statistical analysis for nonmicroarray data. All nonmicroarray data were presented as mean [+ or -] SE. A paired t-test or a repeated measures analysis of variance (ANOVA anova

see analysis of variance.

ANOVA Analysis of variance, see there
) was used to compare differences between treatment and control where appropriate. A p-value of p < 0.05 was considered to be statistically significant. Statistical analysis was performed with Statview 4.0 (SAS Institute Inc., Cary, NC).


Identification of differentially expressed genes associated with UFP exposure. Using the statistical and filtering algorithms described earlier, we identified 664 differentially expressed Affymetrix probe sets. We removed probe sets that lacked an ENTREZ number or were classified as undefined or hypothetical proteins that reduced our list to 426 unique genes, of which 320 were up-regulated and 106 were down-regulated. Using the pathway analysis software, we found three KEGG biological pathways (p [less than or equal to] 0.05) overrepresented o·ver·rep·re·sent·ed  
Represented in excessive or disproportionately large numbers: "Some groups, and most notably some races, may be overrepresented and others may be underrepresented" 
 from the differentially expressed gene list (Tables 1-3). These pathways were the cytokine-cytokine receptor interaction, Wnt signaling, and MAPK MAPK Mitogen-Activated Protein Kinase
MAPK Map Kinase
 signaling. Of note, the cytokine-cytokine interaction pathway contained the most number of genes, many of which have been implicated in the pathogenesis of vascular disease for example, monocyte monocyte /mono·cyte/ (mon´o-sit) a mononuclear, phagocytic leukocyte, 13µ to 25µ in diameter, with an ovoid or kidney-shaped nucleus, and azurophilic cytoplasmic granules.  chemotactit protein 1 (MCP-1, Unigene accession no. 303649;, IL-8, chemokine chemokine /che·mo·kine/ (ke´mo-kin) any of a group of low molecular weight cytokines identified on the basis of their ability to induce chemotaxis or chemokinesis in leukocytes (or in particular populations of leukocytes) in inflammation.  ligand 1 (CXCL1, Unigene accession no. 789), chemokine ligand 2 (CXCL2, Unigene accession no. 590921), chemokine ligand 3 (CXCL3, Unigene accession no. 89690), and chemokine receptor 4 (CXCR CXCR Chemokine, CXC Motif, Receptor
CXCR Alpha Chemokine Receptor
4, Unigene accession no. 421986).

We also noted altered expression of several genes related to coagulation. These alterations included up-regulation of tissue factor (F3) (2.7-fold) and coagulation factor II receptor-like 2 (F2RL2) (1.5-fold) and down-regulation of coagulation factor VIII-associated (intronic transcript) 1 (F8A1) (0.8-fold). There were also additional genes associated with the tissue factor (TF)-clotting independent signaling pathway (Table 4).

Q-PCR and verification of microarray results. The expression of F3 was verified by Q-PCR. Microarray results showed a 2.7-fold up-regulation in F3, whereas Q-PCR showed a 3.3-fold up-regulation. HPAEC were exposed to 0, 1, 10, and 100 [micro]g/mL UFPs in vitro for 4 hr and showed significant upregulation in a dose-dependent manner (Figure 1).

HPAEC were also exposed to water-soluble and -insoluble fractions of UFPs for 2 and 24 hr. Both water-soluble and -insoluble UFP fractions up-regulated the expression of F3 dose dependently, especially at 24 hr post-exposures (Figure 2A). The water-soluble fraction tended to increase the expression of F2RL2 (Figure 2B). For heme oxygenase (HMOX1), which was upregulated in the microarray (2.5-fold), the water-soluble fraction up-regulated HMOX1 at 24 hr after UFP exposure (Figure 2D).

F8A1, which was down-regulated in the microarray study, showed a significant down-regulation in the Q-PCR results by both soluble and insoluble fractions at 2 hr postexposure (Figure 2C).

Up-regulation of TF protein expression by UFP. To determine if the protein expression of TF was up-regulated, HPAEC were incubated with UFP for 18 hr and TF in the cell lysate ly·sate
The cellular debris and fluid produced by lysis.
 was measured by Western blot analysis. As shown in Figure 3, UFPs increased TF expression by more than 2-fold (Figure 3).

Role of TF in UFP-induced cytokine release. UFP exposure increased the release of IL-8 by HPAEC by 1.7-fold. Pretreatment with a blocking antibody against TF inhibited the UFP-induced IL-8 release by more than 70% at 0.1 [micro]g/mL of anti-TF (Figure 4A). UFP also increased the release of IL-6 by 1.9-fold. Pretreatment with the blocking antibody against TF attenuated the IL-6 release by approximately 30% at 0.1 [micro]g/mL of anti-TF (p = 0.01) (Figure 4B).


Numerous epidemiologic studies have identified a link between PM exposure with adverse cardiovascular outcomes and increased mortality (Borja-Aburto et al. 1998; Dockery et al. 1993). Recent studies have shown a link between PM exposure and oxidative stress, which may impair endothelial-dependent vasodilation vasodilation /vaso·di·la·tion/ (-di-la´shun)
1. increase in caliber of blood vessels.

2. a state of increased caliber of blood vessels.
 as well as increased levels of fibrinogen Fibrinogen

The major clot-forming substrate in the blood plasma of vertebrates. Though fibrinogen represents a small fraction of plasma proteins (normal human plasma has a fibrinogen content of 2–4 mg/ml of a total of 70 mg protein/ml), its conversion
 (an established risk factor for myocardial infarction and stroke), C reactive protein C reactive protein Lab medicine A 120 kD polypeptide of the pentraxin family which is produced by the liver during inflammation and detectable in serum in various conditions particularly during acute immune responses, named for its ability to bind the C , IL-6, and IL-8 (Brook et al. 2003, 2004). Despite ample evidence of a link between PM exposure and cardiovascular morbidity and mortality Morbidity and Mortality can refer to:
  • Morbidity & Mortality, a term used in medicine
  • Morbidity and Mortality Weekly Report, a medical publication
See also
  • Morbidity, a medical term
  • Mortality, a medical term
, our understanding of the biological mechanisms for these adverse outcomes remains incomplete. Our hypothesis in this study was that the ultrafine fraction of PM, which more likely would have direct contact with pulmonary vasculature vasculature /vas·cu·la·ture/ (vas´ku-lah-chur)
1. circulatory system.

2. any part of the circulatory system.

 when inhaled than fine and course PM, is capable of inducing transcriptional changes indicative of endothelial cell dysfunction. Using microarray analysis, we found 426 unique genes differentially expressed after UFP exposure for 4 hr in HPAEC. Among these were genes related to the coagulation-inflammation circuitry, including up-regulation of F3, F2RL2, IL-6, and IL-8 and downregulation of F8A1. There was also a group of genes that were involved in the pathogenesis of vascular disease, including those associated with the clotting independent signaling of F3, which included v-fos FBJ FBJ Finkel-Biskis-Jinkins
FBJ Freiman-Berger-Johnson Bound (constant weight code)
FBJ Final Body Join
 murine osteosarcoma osteosarcoma /os·teo·sar·co·ma/ (os?te-o-sahr-ko´mah) a malignant primary neoplasm of bone composed of a malignant connective tissue stroma with evidence of malignant osteoid, bone, or cartilage formation; it is subclassified as  viral oncogene oncogene

Gene that can cause cancer. It is a sequence of DNA that has been altered or mutated from its original form, the proto-oncogene (see mutation). Proto-oncogenes promote the specialization and division of normal cells.
 homolog hom·o·log  
Variant of homologue.
 (FOS, Unigene accession no. 25647;, v-jun sarcoma virus 17 oncogene homolog (JUN, Unigene accession # 525704), nuclear factor kappa-Bcell, subunit 1 (NFKBIA, Unigene accession no. 81328), and HMOX1. Additionally there also were genes related to the CXC CXC Chandra X-Ray Center
CXC Caribbean Examinations Council
CXC Courage Crew
 family of chemokines (MCP-1, IL-8, CXCL1, CXCL2, CXCL3, and CXCR4).

In higher organisms, the coagulation cascade is activated to stop the loss of blood after vascular injury by forming a fibrin fibrin: see blood clotting.  clot. Inappropriate activation of the coagulation pathways, however, can promote intravascular intravascular /in·tra·vas·cu·lar/ (in?trah-vas´ku-lar) within a vessel.

Within one or more blood vessels.
 thrombosis (Chu 2006; Mackman 2005). The blood coagulation cascade is composed of intrinsic and extrinsic pathways. The extrinsic pathway is an inducible signaling cascade that is triggered by up-regulation of TF upon inflammation or endothelial injury (Chu 2006). This initiation process exposes TF to factor VII and forms the tightly bound TF-factor VII-activated complex, which then activates factor X and generates small amounts of thrombin thrombin: see blood clotting.  (Busch et al. 2005). The production of thrombin by the extrinsic pathway is critical because it not only generates fibrin from fibrinogen (Davie et al. 1991; Gailani and Broze 1991) but also amplifies the intrinsic pathway. Our study showed that UFPs or their water-soluble fractions induced the gene and protein expression of TF. Together with differential expression of other coagulation-related genes, our in vitro study raised the possibility that ambient UFP exposure may activate the coagulation cascade. Preliminary results from our UFP-controlled human exposure study showed a 10-25% increase in indicators of coagulation: pro-thrombin fragment F1 +2 and D-dimers (unpublished observation).

F3 expression is markedly increased within arterial atherosclerotic plaques. Spontaneous plaque rupture may trigger intravascular clotting (Day et al. 2005) and may be a primary cause of thrombus thrombus /throm·bus/ (throm´bus) pl. throm´bi   a stationary blood clot along the wall of a blood vessel, frequently causing vascular obstruction.  formation in the onset of acute coronary syndromes (Ishibashi et al. 2003). Studies with an F3-deficient mouse model suggest that thrombosis after arterial injury is driven primarily by vascular wall F3 expression (Day et al. 2005; Ishibashi et al. 2003). Inactivation inactivation /in·ac·ti·va·tion/ (in-ak?ti-va´shun) the destruction of biological activity, as of a virus, by the action of heat or other agent.  of the F3 factor VIIa complex by F3 pathway-inhibitor or active site-blocked factor VIIa has been shown to reduce thrombus weight and increase patency pa·ten·cy
The state or quality of being open, expanded, or unblocked.


the condition of being open.
 in a rabbit vein injury model (Himber et al. 2002). In our study, UFPs altered the expression of several coagulation-related genes in favor of thrombus formation, that is, up-regulation of F3, F2RL2, and down-regulation of thrombomodulin (THBD THBD Thrombomodulin , Unigene accession no. 2030; These changes provide biologically plausible mechanisms by which UFPs may promote thrombus formation, namely, activating the TF-mediated extrinsic coagulation pathway and inhibiting the anticoagulant anticoagulant (ăn'tēkōăg`yələnt), any of several substances that inhibit blood clot formation (see blood clotting).  effects of protein C through suppression of thrombomodulin (Nemerson 1988; Van der Meeren et al. 2003).

TF may also produce biological effects via clotting-independent mechanisms (Rickles et al. 2003; Versteeg et al. 2003). After cell activation, the F3 gene is induced early as a result of the binding of transcription factors, for example, specificity protein-1 (SP-1), activator protein-1 (AP-1), and nuclear factor-kB, to the promoter region of the F3 gene. The functions of clotting-independent TF pathways are not entirely clear, but regulation of angiogenesis appears to be a major one. TF is known to induce vascular endothelial growth factor Vascular endothelial growth factor (VEGF) is an important signaling protein involved in both vasculogenesis (the de novo formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature).  (VEGF VEGF vascular endothelial growth factor. ), one of the most potent regulators of angiogenesis, which in turn up-regulates the expression of TF by activating the early growth response-1 gene (Mechtcheriakova et al. 1999). In tumor-related endothelial cells, decreased PI3-K activity concurrent with increased p38 and ERK ERK Extracellular Signal-Regulated Kinase
ERK Electronic Records Keeping
ERK Externally Regulated Kinases
1/2 MAPK activity enhances F3 expression by VEGF (Blum et al. 2001). In our study, UFPs altered the expression of many of the genes in these processes, including FOS (1.6-fold), JUN (1.3-fold), NFKBIA (1.3-fold), and early growth response-1 (EGR-1, Unigene accession no. 326035; (1.2-fold), indicating potential involvement in activation of clotting-independent mechanisms of TF by UFPs.

Other findings in our study further support the role of PM in the pathogenesis of vascular diseases. For example, we found that UFPs up-regulated genes in the CXC family of chemokines that have been implicated in the pathogenesis of vascular disease. These genes include MCP-1, IL-8, CXCL1, CXCL2, CXCL3, and CXCR4. MCP-1, which was up-regulated 2.6-fold, has been implicated in the development of vascular disease ranging from arterial injury to the formation of atherosclerosis (Charo and Taubman 2004). Overexpression of MCP-1 in vessel-wall macrophages Macrophages
White blood cells whose job is to destroy invading microorganisms. Listeria monocytogenes avoids being killed and can multiply within the macrophage.
 led to increased foam cells formation and increased atherosclerosis (Aiello et al. 1999). Deletion of MCP-1 in low-density lipoprotein receptor-null mice and mice expressing human apolipoprotein B attenuated the progression of dietary-induced atherosclerosis (Gosling et al. 1999; Gu et al. 1998). Acute coronary syndrome patients with the highest levels of MCP-1 had a significantly increased risk of death or myocardial infarction over 2 months of follow-up (de Lemos et al. 2003). IL-8 also has been linked to the development of early atherosclerotic lesions in the vessels. IL-8, a monocyte agonist, is present in macrophage-rich atherosclerotic plaques (Gerszten et al. 1998, 1999). The expression of these chemokines may enhance the adhesion of circulating monocytes monocytes, the largest of the white blood cells. They have one nucleus and a large amount of grayish-blue cytoplasm. Develop into macrophages and both consume foreign material and alert T cells to its presence.
 on vascular endothelial cells and participate in atherogenesis atherogenesis /ath·ero·gen·e·sis/ (-jen´e-sis) formation of atheromatous lesions in arterial walls.atherogen´ic

 (Hagiwara et al. 1998).

It is also well established that there is cross-talk between the coagulation pathways and inflammation in sepsis (Hack 2000; Levi and van der Poll 2005). Infusion of site-inactivated factor VIIa reduced production of cytokines, including IL-6, IL-8 and soluble TNF TNF
tumor necrosis factor

n an abbreviation for tumor
 (tumor necrosis factor tumor necrosis factor
n. Abbr. TNF
A protein that is produced in the presence of an endotoxin, especially by monocytes and macrophages, is able to attack and destroy tumor cells, and exacerbates chronic inflammatory diseases.
) receptor 1 in a baboon baboon, any of the large, powerful, ground-living monkeys of the genus Papio, also called dog-faced monkeys. Five subspecies live in Africa, with one species extending into the Arabian peninsula.  model of sepsis (Miller et al. 2002; Welty-Wolf et al. 2001). Genetically modified mice expressing low levels of F3 exhibited reduced IL-6 expression and increased survival during endotoxemia (Pawlinski and Mackman 2004). We now show that there may also be a link between the F3 pathways and inflammation after UFP exposure. This was supported by the findings that the UFP-induced production of IL-6 and IL-8 was attenuated by a blocking antibody against TF. Conversely, TF expression can be induced by inflammatory mediators such as IL-6, IL-8, and MCP-1 (Busch et al. 2005), all of which were up-regulated in our microarray results by 2.30, 2.50, and 2.6-fold, respectively.

It remains controversial whether PM can have direct contact with pulmonary vascular endothelial cells when inhaled. Recent studies have shown that ultrafine carbon black particles may be able to pass from the lung into the systemic circulation (Nemmar et al. 2001, 2002a; Oberdorster et al. 2002), although the mechanisms for the transport of these insoluble particles are unclear. For the UFPs derived from the natural environment, such as those used in our study, the components in the water-soluble fraction may more easily permeate the alveolar capillary barrier via paracellular pathways or metal transporters. Indeed, the water-soluble fractions of UFPs were sufficient to induce the expression of F3. The active components in the water-soluble fractions of UFPs, however, are unknown and would require future studies.

In this study, we showed that UFPs at concentrations of 10 [micro]g/mL were sufficient to induce the gene expression of F3. These concentrations are higher than the usual ambient level. In Chapel Hill, the ambient UFP concentration is approximately 5,000 particles/[cm.sup.3] (1.6 [micro]g/[m.sup.3]) (unpublished observations). The concentration of UFPs, however, may increase 50- to 100-fold during indoor smoking and cooking (Afshari et al. 2005; Dennekamp et al. 2001). If a person works in the latter environment for 4 hr with an average ventilation of 10 L/min, the concentration of UFPs to which alveolar epithelial cells may be exposed in vivo may be as high as 10-20 [micro]g/mL, assuming the volume of epithelial lining fluid is 20-40 mL (Rennard et al. 1986). Once UFPs deposit on the lung epithelial cells, the water-soluble fractions of UFPs may reach the endothelial cells and stimulate them to express F3.

In summary, using gene profiling, we found that UFPs induced genes that have been implicated in the pathogenesis of vascular disease, including those linked to clotting-dependent and -independent signaling pathways mediated by TF as well as by the CXC family of chemokines. These results support our hypotheses that UFP exposure may induce endothelial cell dysfunction, leading to vascular changes, and suggest that exposure to high concentrations of UFP may be considered a risk factor for the development of cardiovascular disease.


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Edward D. Karoly, (1) Zhuowei Li, (2) Lisa A. Dailey, (1) Xhevahire Hyseni, (1) and Yuh-Chin T. Huang (1,3)

(1) National Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park Research Triangle Park, research, business, medical, and educational complex situated in central North Carolina. It has an area of 6,900 acres (2,795 hectares) and is 8 × 2 mi (13 × 3 km) in size. Named for the triangle formed by Duke Univ. , North Carolina, USA; (2) Center for Environmental Medicine, Asthma and Lung Biology, University of North Carolina, Chapel Hill, North Carolina, USA; (3) Department of Medicine, Duke University Medical Center, Durham, North Carolina Durham is a city in the U.S. state of North Carolina. It is the county seat of Durham CountyGR6 and is the fourth-largest city in the state by population. , USA

Address correspondence to E.D. Karoly, MD-58D, U.S. EPA EPA eicosapentaenoic acid.

eicosapentaenoic acid

EPA, See acid, eicosapentaenoic.

, Research Triangle Park, NC 27711 USA. Telephone: (919) 843-8031. Fax: (919) 966-6271. E-mail:

The research described in this article has been reviewed by the U.S. EPA National Health Effects and Environmental Research Laboratory and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the U.S. EPA, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use.

The authors declare they have no competing financial interests.

Received 27 July 2006; accepted 8 January 2007.
Table 1. Cytokine-cytokine receptor interaction KEGG pathway (p =
0.001) identified with DAVID 2.1 from the list of differentially
expressed unique genes (p < 0.01) (FDR = 0.05).

Accession no.  Gene symbol  Gene name                            change

303649         MCP1         chemokine (C-C motif) ligand 2       2.6
624            IL8          interleukin 8                        2.5
89690          CXCL3        chemokine (C-X-C motif) ligand 3     2.3
512234         IL6          interleukin 6                        2.3
396530         HGF          hepatocyte growth factor             2.2
590921         CXCL2        chemokine (C-X-C motif) ligand 2     2.0
204044         TNFRSF11A    tumor necrosis factor receptor,      1.8
                            superfamily member 11A
389874         TSLP         thymic stromal lymphopoietin         1.6
557403         IL1R1        interleukin-1 receptor, type I       1.6
513457         IL4R         interleukin 4 receptor               1.5
2250           LIF          leukemia inhibitory factor           1.5
789            CXCL1        chemokine (C-X-C motif) ligand 1     1.4
28792          INHBA        inhibin, beta a                      1.4
479756         KDR          kinase insert domain receptor        1.3
438918         ACVR1B       activin A receptor, type IB          1.3
421986         CXCR4        chemokine (C-X-C motif) receptor 4   1.3
1048           KITLG        kit ligand                           1.3
1976           PDGFB        platelet-derived growth factor beta  0.8
256278         TNFRSF1B     tumor necrosis factor receptor,      0.8
                            superfamily member 1B

Gene annotations are from Unigene (

Table 2. Wnt signaling KEGG pathway (p = 0.02) identified with DAVID 2.1
from the list of differentially expressed unique genes (p < 0.01) (FDR =

Accession no.  Gene symbol  Gene name                             change

211869         DKK2         dickkopf homolog 2                    1.9
592141         NFATC2       nuclear factor of activated T-cells,  1.8
                            cytoplasmic, calcineurin-dependent 2
599590         SOX17        SRY (sex determining region Y)-box    1.4
6347           LRP5         low density lipoprotein receptor-     1.3
                            related protein 5
525704         JUN          v-jun sarcoma virus 17 oncogene       1.3
283565         FOSL1        fos-like antigen 1                    1.2
351887         CAMK2B       calcium/calmodulin-dependent protein  1.2
                            kinase II beta
149413         PPP3CC       protein phosphatase 3, catalytic      1.2
                            subunit, gamma isoforms (calcineurin
                            A gamma)
94234          FZD1         frizzled homolog 1                    0.8
591968         FZD4         frizzled homolog 4                    0.8
524348         PRICKLE1     prickle-like 1                        0.8

Gene annotations are from Unigene (

Table 3. MAPK signaling KEGG pathway (p = 0.05) identified with DAVID
2.1 from the list of differentially expressed unique genes (p < 0.01)
(FDR = 0.05).

Accession no.  Gene symbol  Gene name                            change

417962         DUSP4        dual specificity phosphatase 4       1.7
510225         RPS6KA5      ribosomal protein S6 kinase,         1.6
                            polypeptide 5
25647          FOS          v-fos FBJ murine osteosarcoma viral  1.6
                            oncogene homolog
557403         IL1R1        interleukin-1 receptor, type I       1.6
432453         MAP3K8       mitogen-activated protein kinase     1.5
                            kinase kinase 8
497200         PLA2G4A      phospholipase A2, group IVA          1.5
278733         SOS1         son of sevenless homolog 1           1.3
438918         ACVR1B       activin A receptor, type IB          1.3
525704         JUN          v-jun sarcoma virus 17 oncogene      1.3
149413         PPP3CC       protein phosphatase 5 catalytic      1.2
                            subunit, gama isoform
390428         MAP3K4       mitogen-activated protein kinase     0.9
                            kinase kinase 4
1976           PDGFB        viral (v-sis) oncogene homolog       0.8
186486         MAP3K5       mitogen-activated protein kinase     0.8
                            kinase kinase 5

Gene annotations are from Unigene (

Table 4. Tissue factor clotting-independent mechanism associated genes
from the list of differentially expressed unique genes (p < 0.01)
(FDR = 0.05).

Accession no.  Gene symbol  Gene name                          change

517617         MAFF         v-maf musculoaponeurotic
                              fibrosarcoma oncogene homolog F  1.7
25292          JUNB         jun B proto-oncogene               1.7
590958         FOSB         FBJ murine osteosarcoma viral      1.5
                              oncogene homolog B
132225         PIK3R1       phosphoinositide-3-kinase,         1.5
                              regulatory subunit 1
155396         NRF2         nuclear factor (erythroid-derived  1.3
                              2)-like 2
534313         EGR3         early growth response 3            1.3
81328          NFKBIA       nuclear factor of kappa light      1.3
                              polypeptide gene enhancer in
                              B-cells inhibitor, alpha
252229         MAFG         v-maf musculoaponeurotic           1.2
                              fibrosarcoma oncogene homolog G
326035         EGR1         early growth response 1            1.2

Gene annotations are from Unigene (
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Title Annotation:Research
Author:Karoly, Edward D.; Li, Zhuowei; Dailey, Lisa A.; Hyseni, Xhevahire; Huang, Yuh-Chin T.
Publication:Environmental Health Perspectives
Date:Apr 1, 2007
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