Grantee Research Project Results

2007 Progress Report: Endothelial Cell Responses to PM—In Vitro and In Vivo

EPA Grant Number: R832414C002
Subproject: this is subproject number 002 , established and managed by the Center Director under grant R832414
(EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
Center: UC Davis Center for Children's Environmental Health and Disease Prevention
Center Director: Van de Water, Judith
Title: Endothelial Cell Responses to PM—In Vitro and In Vivo
Investigators: Wilson, Dennis , Rutledge, John
Current Investigators: Wilson, Dennis , Barakat, Abdul , Anastasio, Cort , Tablin, Fern , Rutledge, John
Institution: University of California - Davis
EPA Project Officer: Chung, Serena
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
Project Period Covered by this Report: October 1, 2006 through September 30, 2007
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Human Health , Air

Objective:

The overall goal of this project is to determine the relationship between vascular disease and systemic effects of particulate matter.

Progress Summary:

In our first year, we concentrated on signaling responses associated with oxidant stress using laboratory generated particles as well as historically archived (NIST) environmental particles until ambient particulate matter from the San Joaquin Valley are collected. We also performed several experiments evaluating culture exposure methods that will best recapitulate expectations of exposure conditions in vivo. In the current year, we obtained our first SJV derived CAPs and performed experiments evaluating overall gene responses and potential signaling mechanisms induced by direct exposure of human endothelial cell cultures (HAEC) to CAPs. Our initial studies evaluated our original hypothesis that general endothelial cell activation through the TGFβ family of signaling molecules would occur. While previous studies in our laboratory have documented activation of this family of receptors by a variety of stimuli, treatment with CAPs failed to stimulate the translocation of the common transcription factor (SMAD4) associated with this family of growth regulation factors (Figure 1).

Figure 1: Immunostaining for SMAD4 in HAEC treated with 10 μg/ml CAPs. No nuclear translocation indicative of TGFβ family signaling is evident.

1. Gene responses of HAEC to direct CAPs exposure:

We hypothesized that exposure of endothelial cells to PM results in the activation of multiple pro-inflammatory genes. To characterize vascular gene responses, human aortic endothelial cells (HAECs) were exposed to fine and ultrafine concentrated ambient particulate matter (CAP) collected in an urban setting in California’s San Joaquin valley. Organic chemical analysis demonstrated that these particles contained multiple PAH compounds associated with automotive fuel combustion (Table 1). RNA isolated from HAECs exposed for 3 hr to 10 μg/ml CAP was analyzed using high density oligonucleotide arrays (U133A 2.0). CAP exposure resulted in the up-regulation of 30 and the down regulation of 15 genes. Compared with similar experiments from another project examining responses to lipolysis products derived from human blood lipids, this was a modest response (Figure 2).

The altered expression was observed to occur within the following functional classifications: transcription factors, cytokines, xenobiotic metabolism and inflammation-related genes (Table 2). PM exposure resulted in the specific up-regulation of cytochrome P450 1A1 (CYP1A1), aldehyde dehydrogenase 1 A3 (Aldh1a3), TCDD-inducible poly (ADP-ribose) polymerase (Tiparp) and E-selectin. Array data was verified by quantitative real-time RT-PCR (Figure 3).

Table 1. PAH analysis of the Fresno Summer 2006 filter samples.

	Airborne Conc	Stock Conc
	ng/m3	ng/mL
Naphthalene	0.010	0.31
2-Methylnaphthalene	0.009	0.29
1-Methylnaphthalene	0.005	0.14
Biphenyl	0.002	0.06
2,6-Dimethylnaphthalene	0.005	0.16
Acenaphthylene	N.D.	N.D.
Acenaphthene	N.D.	N.D.
2,3,5-Trimethylnaphthalene	N.D.	N.D.
Fluorene	0.006	0.18
Phenanthrene	0.020	0.62
Anthracene	0.002	0.06
1-Methylphenanthrene	0.006	0.19
Fluoranthene	0.025	0.78
Pyrene	0.028	0.87
Benz(a)anthracene	0.009	0.29
Chrysene	0.026	0.81
Benzo(b)fluoranthene	0.041	1.27
Benzo(k)fluoranthene	0.013	0.4
Benzo(e)pyrene	0.032	0.97
Benzo(a)pyrene	0.019	0.57
Perylene	0.011	0.35
Indeno(1,2,3-cd)pyrene	0.037	1.13
Dibenz(a,h)anthracene	0.002	0.08
Benzo(g,h,i)perylene	0.058	1.79

N.D. = Not detected.

Figure 2: Classification of genes altered by treatment with (A) Fresno CAPs vs. (B) alterations with treatment with lipolysis products of human blood triglycerides

Figure 3: Correlation of gene array responses with selected analysis by RT-PCR. Results from array are one result from 3 pooled treatments while PCR analysis is mean SD for 3 replicate experiments

Table 2: Genes upregulated in HAEC in response to treatment with 10μg/ml CAPs for 3 hours: Results from Affymetrix human gene array U133A 2.0.

Gene symbol	Gene Title	Fold change
Cyp1a1	cytochrome P450 1a1	3.7
Aldh1a3	aldehyde dehydrogenase 1 family, member A3	2
TIPARP	TCDD-inducible poly(ADP-ribose) polymerase	2
PLA2G4A	phospholipase A2, group IVA	2.1
COL1A1	collagen, type I, alpha 1	2.5
COL1A2	collagen, type I, alpha 2	2
COL6A3	collagen, type 6, alpha 3	2.1
RND1	Rho family GTPase 1	2.3
CCL2	Chemokine (C-C motif) ligand 2	2.3
CXCL2	Chemokine (C-X-C motif) ligand 2	2.5
SELE	Selectin E	2.8
ATF3	Activating transcription factor 3	3
F3	Coagulation factor III	4
PTGS2	prostaglandin-endoperoxide synthase 2	2.8

Induction of CYP1A1 by PM is potentially mediated through the ligand-activated aryl hydrocarbon receptor (AhR). Activation of this receptor results in nuclear translocation with sunsequent association with the nuclear translocation factor ARNT. Immunofluorescence microscopy demonstrated nuclear translocation of AhR and co-localization with ARNT at 3 hr after exposure to CAPs. Similar results were obtained in positive control experiments with Napthalene. (Figure 4) Among the documented responses to PAH exposure is downregulation of the AHR receptor. We compared changes in expression of AHR in response to CAPs using TCDD treatment as a positive control and in comparison with an ROS environment induced by H2O2 treatment. At 24 hours, there was significant downregulation of AHR in response to TCDD and moderate downregulation in response to CAPs treatment but no change in response to H2O2 (Figure 5). To our knowledge this is the first report of altered expression of CYP1A1 by HAECs exposed to PM. Activation of the AHR element may be a signature of PAH containing PM and the role of resultant gene responses in vascular inflammation, injury and cardiovascular disease and important new area of investigation.

Figure 4: Immunofluorescence stain for AhR and Arnt in HAEC treated with CAPs (PM) Napthalene or ethanol vehicle control.

Figure 5: Western Blot for AhR 24 hours after treatment with TCDD or CAPs

2. Gene responses in tissues of mice exposed to urban SJV CAPs

We collected tissues from mice exposed in the HRV protocol in project 3 during the February 2007 Fresno exposure project. Heart and lung were flash frozen and RNA extracted using Quiagen kits and standard methodology. These produced sufficient high purity extracts and are presently being analyzed by RT-PCR for altered expression of the same panel of homologous genes as found in the HAEC array studies. Another group of mice from the same experiment had tissues from all visceral organs harvested and embedded for immunohistochemical analysis. These will be immunostained for vascular proinflammatory molecules including E-selectin and ICAM-1. Additional immunostains will evaluate expression of CYP 1a1 as a possible correlate of the increased expression of this gene in HAEC cultures treated with collected CAPs from the fall exposure at the same site. Current activity in this project is directed towards optimizing the immunostaining protocols using lungs from rats treated with monocrotaline, an endothelial activating and proinflammatory pulmonary toxin.

Future Activities:

In the next project year we plan the following experiments:

• Compare gene responses of endothelium to CAPs with that in airway epithelial cells
• Compare specificity of AhR responses with ROS/Nrf-Keap
• Complete evaluation of mouse exposure to PM collected in Winter with histopathology, immunohistochemistry, and gene expression analysis
• Compare gene responses to PM from urban sources to agricultural origin PM

Journal Articles:

No journal articles submitted with this report: View all 15 publications for this subproject

Supplemental Keywords:

Health, RFA, Scientific Discipline, Air, Health Risk Assessment, Risk Assessments, particulate matter, Environmental Chemistry, Epidemiology, ambient aerosol, lung disease, human exposure, long term exposure, airborne particulate matter, concentrated air particles, ambient particle health effects, epidemiological studies, PM, inhalation toxicology, toxicology, vascular dysfunction, acute cardiovascular effects, cardiovascular disease, human health risk, lung injury, microarray studies, airway disease

Progress and Final Reports:

Original Abstract

2006 Progress Report

2008 Progress Report

2009 Progress Report

2010 Progress Report

Final Report

Main Center Abstract and Reports:

R832414    UC Davis Center for Children's Environmental Health and Disease Prevention

Subprojects under this Center: (EPA does not fund or establish subprojects; EPA awards and manages the overall grant for this center).
R832414C001 Project 1 -- Pulmonary Metabolic Response
R832414C002 Endothelial Cell Responses to PM—In Vitro and In Vivo
R832414C003 Project 3 -- Inhalation Exposure Assessment of San Joaquin Valley Aerosol
R832414C004 Project 4 -- Transport and Fate Particles
R832414C005 Project 5 -- Architecture Development and Particle Deposition