Impact/Purpose:

This project will complement the epidemiologic analyses and state-of the art characterization of US city-specific PM and other pollutants by defining the comparative pathobiologic cardiopulmonary responses to inhaled PM exposure.

Description:

(August 1, 2009 – July 31, 2010):

1. In vitro Toxicity Assessment of Baltimore PM.

   Overview and summary: Previously, we have utilized human bronchial epithelium and human lung endothelium to evaluate PM-induced cytokine secretion (GM-CSF, IL-6, IL-8, and IL-1β), generation of ROS, such as H₂O₂ and superoxide, and signaling mechanisms regulating cytokine/ROS production, cytotoxicity, and vascular/epithelial permeability. These in vitro effects of Baltimore PM on lung cell function evidence a “pro-inflammatory lung cell phenotype” with increases in epithelial and endothelial permeability in PM fraction-specific pathways. PM also induces elaboration of ROS, an effect which is partially reversed by the anti-oxidant N-acetyl-L-cysteine (NAC). These findings have been summarized and published (Zhao, et al., 2009; Wang, et al., 2010). In this period, we have further investigated the in vitro effects of PM to mediate vascular hyper-permeability and achieved the following observations/conclusions:

   • Fine PM collected from Ft. McHenry tunnel, Baltimore induced tight junction protein ZO-1 relocation from the cell periphery, as well as a significant reduction in ZO-1 protein levels, while not the adherens junction protein, VE-Cadherin. PM induced significant ROS generation in human lung ECs, abolished by N-acetyl-cysteine (NAC 5 mM), which resulted in prevention of TER decreases, and attenuation of ZO-1 degradation. PM mediated intercellular calcium leakage via ROS transient receptor potential cation channel M2 (TRPM2), which in turn activates calpain. PM-mediated ZO-1 degradation and EC barrier disruption was dependent on calpain activation. These results demonstrate that PM induces ROS generation in vascular ECs, resulting in calcium leakage via impaired TRPM2, and ZO-1 degradation by activated calpain, which results in a significant and delayed EC barrier disruption. These findings support a novel mechanism for PM-induced lung damage and adverse cardiovascular outcomes. These findings have been submitted to Circulation Research for publication.
   • Particulate air pollution is known to be associated with cardiopulmonary morbidity and mortality. PM exposure triggers massive oxidative stress in endothelial cells, further inducing loss of endothelial integrity and lung vascular hyper-permeability. We investigated the protective role of H₂S, an endogenous gaseous molecule in circulation on PM-induced endothelial barrier disruption and pulmonary inflammation. Changes in endothelial monolayer permeability reflected by Transendothelial Electrical Resistance (TER), reactive oxygen species (ROS) generation, and murine pulmonary inflammatory responses were studied after exposure to PM and NaSH, a H₂S donor. Like N-acetyl cysteine (NAC, 5 mM), NaSH (10 µM) significantly attenuated PM-induced ROS in human lung ECs, and inhibited activation of p38 MAP kinase. In parallel studies, NaSH (10 µM) activated Akt, which contributed to protect endothelial integrity. Thus, both of these pathways contribute to the endothelial barrier protection of H₂S against PM-induced barrier disruption. Furthermore, NaSH (20 mg/kg) treatment reduced PM-induced vascular protein leak, leukocyte infiltration, and pro-inflammatory cytokine release in bronchoalveolar lavage (BAL) fluids in a murine model. These data suggest a protective role of H₂S in PM-induced endothelial disruption, pulmonary inflammation and remodeling against. These findings will be submitted to the American Journal of Respiratory Cell and Molecular Biology.
2. In vivo Effects of PM exposure in a Murine Model of Asthma

   Recently published results by our group (Wang and Moreno-Vinasco et al., Environmental Health Perspectives 116: 1500-1508, 2008) demonstrated a dose-dependent effect of Baltimore PM instillation (0.01 – 30mg/kg body weight) on airway hyperresponsiveness (AHR), BAL protein, and BAL inflammatory leukocytes infiltration in both asthmatic and naïve animals. Based on these findings, an optimal dose range (1, 3, and 10 mg/kg body weight) was selected to conduct the following comparative study of fine-PM pairs collected from Maricopa AZ (MAR) and Sacramento CA (SAC), Pinellas FL (PIN) and Harris TX (HAR), or Jefferson KY (JEF) and Hennepin MN (HEN).

   Pathophysiology: Instillation of fine PM samples, in a dose-dependent manner, triggered allergic pulmonary inflammatory phenotypes, including AHR, increased BAL white blood cell count (total cell counts, neutrophils and eosinophils) and BAL protein content as compared to control mice. Standard urban-PM (1648a from NIST) also exhibited a significant induction of similar pulmonary inflammatory phenotypes in the murine asthmatic model. Interestingly, fine-PM from PHX, compared to SAC, induced a much higher level of AHR, and infiltration of white blood cells, including neutrophils and eosinophils into the alveolar space. HAR PM also exhibited a significantly higher level of AHR, and infiltration of white blood cell count, including neutrophils and eosinophils. However, no difference in BAL protein (protein leakage) between MAR and SAC PM or between HAR and PIN PM was observed. The preliminary study of fine PM collected from JEF and HEN also demonstrated a trend of dose-dependent effects of PM toxicity in the exacerbation of allergic pulmonary inflammation.

   Overview and Summary: These results suggest a differential effect of fine-PM from multiple cities on asthmatic pulmonary inflammation exacerbation in a murine model of asthma.

3. Cardiomyopathy Model

   A mechanistic link between human exposure to PM pollution and increased cardiovascular morbidity and mortality observed in people with congestive heart failure is poorly defined. Therefore, we have developed a murine model of cardiomyopathy (expressing a cardiac-specific, dominant negative CREB mutant transcription factor, CREB _A133) to investigate PM-mediated cardiac arrhythmias/respiratory dysynchrony.

   • 20 Weeks old CREB, but not CD-1 control, mice exposed to bulk-PM (20 mg/kg body weight) for 36 h exhibited mildly increased PVCs at baseline with a marked increase post-exposure.
   • Analyses of the left ventricles of control- and PM-treated mice utilizing microarray, OntoExpress and Ingenuity Pathway revealed that PM dysregulated 171 genes in CD-1 and 41 genes in CREB mice compared to controls involving critical pathways like complement system, beta-catenins signaling and glycosaminoglycans degradation.
   • Bulk-PM challenge of CREB _A133, but not CD-1, mice displayed respiratory Dysynchrony with waxing and waning patterns of respiration under basal conditions which was pronounced during hypoxia. Such a breathing pattern is often observed in patients with severe congestive heart failure; thus, a prognostic sign for sudden cardiac death and heart failure progression.
   • Mouse carotid body gene expression was dysregulated by PM as determined by quantitative RT-PCR. These included signaling genes (Cd44, Cxcl2, RhoC, and Edg3), inflammatory, oxidative/stress genes (Tgfbr1, Tnfsf9, IL6, Tlr2, Nfe212, Hmox1, Sod1, Sod2, and Cfb), and carotid body function genes (Scn8a, Kcnmb2, Gdnf, Scnn1b, Kcnc1, Kcnd1, Kcnd2, Sstk38l, Slc8a1, and PKCc). A strong role of ion channel dysregulation by PM-induced oxidative stress, which facilitated cardiac arrhythmia, was observed. These data have been incorporated into our newly to be submitted manuscript to JCI revised MS.
   • Comparison of left ventricular tissue samples obtained from Baltimore PM exposed CREB _A133 mice and PBS-challenged CD-1 mice (SAM software) identified 1927 probe sets. Within this probe set, only 60 probe sets displayed significant differential expression (p < 0.05) between PM- and PBS-treated CREB _A133 mice whereas PM alone minimally impacted cardiac gene expression. The magnitude of gene dysregulation in this subset of genes, however, was greater in PM-challenged CREB _A133 mice than in PBS-exposed CREB _A133 mice.
   • In lung tissue, PM-induced greater gene dysregulation in CREB _{A 133} mice (843 probe sets, fold change > 2, FDR < 1.9%) than in CD-1 mice (589 probe sets, fold change > 2, FDR < 2.7%) suggesting that the congestive heart failure phenotype primes the lung for an exaggerated response to PM.
   • A greater number of genes belonging to growth factor signaling, MAPK pathways, cytoskeletal signaling and factors promoting cardiogenesis in vertebrates were down-regulated by PM in CREB _{A 133} lungs (70%, 575/843 probe sets), whereas the majority of PM-induced dysregulated genes in CD-1 lungs were up-regulated (66%, 384/589 probe sets). Interestingly, none of the pathways enriched with PM-down-regulated genes in CREB _{A 133} mice were identified in PM-exposed CD-1 mice.
   • Carotid bodies from CREB _{A 133} mice, but not CD-1 mice, responded to Baltimore PM challenge with marked inflammatory and pro-oxidant gene signatures and dysregulation of carotid body genes. These results suggest that carotid bodies from congestive heart failure mice, similar to lung, are primed for an exaggerated response to PM exposure.
   • The effects of fine-PM from PHX and SAC on cardiac arrhythmias and respiratory Dysynchrony in CD-1 and CREB transgenic mouse model were examined. Both PHX and SAC fine-PM (10 mg/kg, 24 hr, n=3) manifested a reduction in heart rate variability, respiratory dysynchrony and increased frequency of serious ventricular arrhythmias with no significant difference between the two PM samples. Additional experiments with CREB and CD-1 mice (number of animals per group, n=5) are planned to compare the effect of different PM samples from various cities provided by Project 2.

   Overview and Summary: In summary, our findings in a murine model of dilated cardiomyopathy are consistent with results of population-based studies showing that short-term exposure to PM is a risk factor for acute cardiovascular morbidity and mortality and provide novel mechanistic insights regarding complex PM pathophysiology in susceptible individuals with CHF. Utilizing complementary physiological and genomic approaches with direct measurements of carotid body neural activity, our current findings significantly extend earlier in vitro and in vivo studies and suggest that PM-mediated serious cardiac arrhythmias, decreases in HRV, and respiratory dysynchrony represent multi-organ pathobiology within the CREB_A133 CHF phenotype which is driven by heightened sensitivity of carotid body function and augmented peripheral chemoreceptor reflexes

URLs/Downloads:

2007 Progress Report

2006 Progress Report

2008 Progress Report

2009 Progress Report

Final Progress Report

Record Details:

Record Type:PROJECT( ABSTRACT )

Start Date:10/01/2005

Completion Date:09/30/2010

Record ID: 201063

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Related Organizations:

Role :OWNER

Organization Name :JOHNS HOPKINS UNIVERSITY

Organization Name :JHPMRC

Mailing Address :3400 N Charles St

Citation :Baltimore

State :MD

Zip Code :21218



Role :OWNER

Organization Name :UNIVERSITY OF CHICAGO

Organization Name :JHPMRC

Mailing Address :3400 N Charles St

Citation :Baltimore

State :MD

Zip Code :21218

Project Information:

Approach :

We will utilize standard toxicologic assessment and high through-put toxicogenomic approaches, to gauge the relative toxicity of PM collected in regions where particles have greater and lesser effects on health outcomes. Project 3 will address potential limitations by integrating state-of-the-art genomic technologies with traditional and complementary toxicologic assessment. To exploit comparisons of pathobiologic cardiopulmonary responses to respirable airborne PM, in Phase I, we will optimize a battery of well-developed in vitro (human) and in vivo (murine) bioassays including multiple cytokines, indicators of reactive oxygen/nitrogen species burden (ROS/RNS), and biomarkers of vascular and cardiac dysfunction for assessing particle toxicity. For this purpose, we will use a set of particles having differing characteristics: standard NIOSH urban PM, Baltimore tunnel PM, ambient Baltimore air PM, New York City air PM. We will use oligonucleotide-based microarrays to address the hypothesis that gene expression profiles in human lung epithelium, murine lung and cardiac tissues, induced by respirable PM, are dependent on the fraction of pollutants bound to the respirable PM. In Phase II, we will survey PM fractions from a number of US sites with this battery, selected according to the sampling plan developed in Project 1, and apply the screening methodologies optimized in Phase I to identify sites with the greatest pathobiologic effects and relate these responses to observed adverse human health effects. Phase III will focus on a smaller number of sites showing the greatest PM toxicity and initiate mechanistic studies using transgenic prototypes, in vitro molecular dissection of signal transduction pathways, and extensive gene profiling.

Cost :$2,015,508.00

Research Component :Health Effects

Project IDs:

ID Code :R832417C003

Project type :Center