2006 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: San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)
Center Director: Wexler, Anthony S.
Title: Endothelial Cell Responses to PM—In Vitro and In Vivo
Investigators: Wilson, Dennis , Rutledge, John
Current Investigators: Wilson, Dennis , Anastasio, Cort , Barakat, Abdul , Rutledge, John , Tablin, Fern
Institution: University of California - Davis
EPA Project Officer: Hunt, Sherri
Project Period: October 1, 2005 through September 30, 2010 (Extended to September 30, 2011)
Project Period Covered by this Report: October 1, 2005 through September 30, 2006
RFA: Particulate Matter Research Centers (2004) RFA Text |  Recipients Lists
Research Category: Health Effects , Air


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

Progress Summary:

We have 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 oxidative stress experiments, we compared reactive oxygen species (ROS) generation in human aortic endothelial cell cultures (HAEC) in response to laboratory generated particles or NIST standard particles using an intracellular electron accepting compound that fluoresces in direct proportion to cellular redox processes. This compound, 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) is a documented marker of intracellular oxidation status resulting from chemically induced oxidative injury (Liu, 2001). In concert with these and other studies, we have developed assays for nuclear translocation of the ROS second messenger Nrf2.

To quantify ROS generation, we preloaded HAEC cells grown on coverslips with CM-H2DCFDA and exposed them to laboratory generated particles including zinc oxide, iron oxide, yttrium oxide, and archival diesel exhaust samples (NIST). Controls included hydrogen peroxide and soluble iron. After 30 min incubation, cells were fixed and viewed with a fluorescence microscope equipped with a digital camera. Six images per culture were collected under consistent exposure conditions. Images were evaluated for overall intensity using the integrated density function of Image J software. Results were compared with ANOVA using a Fisher’s LSD post hoc test. Findings presented in figure 1 show the most robust response occurred with the NIST archival material. Several problems interfered with the sensitivity of this assay. Chief among them was that any loss of complete monolayer significantly affected the overall result due to the integrated approach inherent in the image analysis. Also challenging was that background levels of fluorescence in control cultures were fairly high making the range of sensitivity limited. We conclude that ROS generation in response to environmental PM may be significant in cultured endothelium but that alternative assays may be necessary to address this problem. We are evaluating electron spin resonance (ESR) approaches to determine whether this technique will offer a more sensitive and specific measurement of cellular oxidation. In experiments supported by other sources, our laboratory has also developed methods of evaluating the ROS sensitive second messenger system associated with the Nrf2 protein. Experiments determining nuclear translocation of Nrf2 in response to particle exposure are currently underway.

Figure 1. ROS Responses to Laboratory Particles in HAEC Cells
Figure 1. ROS Responses to Laboratory Particles in HAEC Cells

Nrf2 Translocation as an Indicator of Oxidative Stress

Nrf2 is a transcription factor involved in the activation of antioxidant response elements (AREs). Under basal conditions, Nrf2 is sequestered in the cytoplasm by Keap1, a cysteine rich regulator protein. Upon insult by electrophiles or ROS , Nrf2 will dissociate from Keap1 and translocate to the nucleus. Since ROS formation is emerging as a key factor in PM mediated injury, we evaluated the specificity of this system in ROS mediated injury relative to other compounds affecting protein sulfhydryls. We compared Nrf2 translocation resulting from a known redox cycle inducing reactive intermediate, napthoquinone (NQ), with a reactive intermediate known to bind sulfhydryl groups, monocrotaline pyrrole (MCTP). We confirmed that NQ treatment elicited a positive ROS response when evaluated by CM-H2DCFDA fluorescence; MCTP treatment was negative in this assay (see Figure 2). Results of Nrf2 translocation experiments demonstrated that both NQ and MCTP stimulated Nrf2 translocation suggesting that the alteration of sulfhydryl groups is the key to Nrf2 signaling activation and that ROS may not be the only mechanism stimulating this response. Given the concerns regarding the sensitivity of the CM-H2DCFDA assay outlined above, however, an alternate explanation could be that MCTP elicits an ROS response below CM-H2DCFDA detection and that Nrf2 translocation is a more sensitive predictor of ROS generation. Distinguishing these alternatives will depend on our success with developing ESR approaches to ROS detection.

Figure 2. Nrf2 Translocation in Pulmonary Artery Endothelium in Response to Treatment With Either Naphthoquinone (1uM) or Monocrotaline Pyrrole (0.1 mM).
Figure 2. Nrf2 Translocation in Pulmonary Artery Endothelium in Response to Treatment With Either Naphthoquinone (1μM) or Monocrotaline Pyrrole (0.1 mM).

Dynamic Light Scattering Evaluation of Particle Suspensions

A key component of both this project and project RD832414C004, Transport Mechanisms and Systemic Fate of Inspired Ultrafine Particles, is the ability to expose cultured endothelial cells to monodisperse particulates as both mechanisms of transport and cellular responses could be significantly influenced by particle size. Our preliminary evaluations by TEM suggested that archival laboratory generated particles formed significant aggregates when resuspended for culture experiments. Subjective examination suggested that using a probe sonicator was more effective in particle dispersion than resuspending particles in with water bath sonication. Given the importance of understanding the reliability of preparing particles from various sources for culture experiments, we addressed this problem more specifically using dynamic light sorting analysis of particle size to determine the relative effectiveness of probe sonication in developing monodisperse particles for culture exposure. Figure 3 shows the results of these studies. Laboratory generated particles with a median size of 0.1 μ m were used (panel A). Resuspension of these particles with water bath sonication resulted in aggregated particles of approximately 0.8 μ m diameter. Probe sonication was much more effective leading to suspensions with median size of 0.1 μm in two replicates (A+B) when expressed on a number of particle basis (panel C). Some aggregates did form and were evident when the data were graphed relative to particle weight (Panel D). We conclude that probe sonication is an effective means of re-suspending archival samples for analysis but that some consideration in dosimetry should be given to particle aggregation.

Figure 3. Size Distribution of Resuspended Fe[2]O[3] Particles
Figure 3. Size Distribution of Resuspended Fe2O3 Particles

Future Activities:

In the next project year we plan to apply our ROS signaling assays to evaluate responses to San Joaquin Valley derived PM in cultured human aortic and pulmonary artery endothelium. We will evaluate potential approaches to ROS detection with ESR as an alternative to dye based assays. We plan to complete the proposed evaluation of TGFb family signaling in response to PM as well as the proposed evaluation of PM effects on endothelial barrier function.

Journal Articles:

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

Supplemental Keywords:

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

Relevant Websites:

http://saherc.ucdavis.edu/ Exit

Progress and Final Reports:

Original Abstract
  • 2007 Progress Report
  • 2008 Progress Report
  • 2009 Progress Report
  • 2010 Progress Report
  • Final Report

  • Main Center Abstract and Reports:

    R832414    San Joaquin Valley Aerosol Health Effects Research Center (SAHERC)

    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