Final Report: Air Pollution Role in Hypertension and Heart Attacks

EPA Grant Number: R831952
Title: Air Pollution Role in Hypertension and Heart Attacks
Investigators: Kleinman, Michael T. , Kloner, Robert
Institution: University of California - Irvine , The Heart Institute of Good Samaritan Hospital
EPA Project Officer: Chung, Serena
Project Period: September 1, 2004 through July 31, 2008 (Extended to August 31, 2009)
Project Amount: $1,534,855
RFA: The Role of Air Pollutants in Cardiovascular Disease (2003) RFA Text |  Recipients Lists
Research Category: Air Quality and Air Toxics , Health Effects , Particulate Matter , Air


This project examined adverse effects of particulate air pollution (PM) on the heart using animal models of susceptible human populations to:
  1. Determine if exposure to ultra fine (< 0.18 micrometer) particulate matter (UFP) typical of polluted ambient air causes direct physiologic and pathologic damage (not involving the lung) to hearts of healthy individuals or individuals with increased susceptibility due to heart disease or aging.
  2. Determine whether temporal variations in particle concentrations or chemical compositions induce disturbances of blood pressure, heart rate and heart rate variability (BP, HR and HRV, respectively) in healthy individuals or individuals with increased susceptibility due to heart disease.
  3. Determine if chronic exposure to polluted ambient air adversely affects the physiology, pathology, and gene expression in the hearts of healthy or susceptible individuals exposed to ambient particulate air pollution.

Summary/Accomplishments (Outputs/Outcomes):

To accomplish the project objectives, we conducted chronic inhalation studies with animal models (in vivo) to examine damage caused by PM to the heart and changes in heart functions induced by PM exposures.  To determine if PM that entered the circulation after inhalation could have direct effects on the heart we performed in vitro studies with isolated perfused hearts and intensively evaluated acute changes in heart functions after instilling UFP into a vein leading into hearts from normal rats, aged rats, spontaneously hypertensive rats and rats with induced myocardial infarctions.

For the in vivo studies we exposed rats to PM in Riverside, California, a city with hourly average PM concentrations that ranged from about 30 to a peak of 655 µg/m3 during the July through October 2003 period, a time when photochemical activity in the atmosphere was high. The exposures were performed using a particle concentrator, a device that increased the ambient concentrations of PM by about 10-fold, while removing, or greatly reducing, the concentrations of toxic gases such as nitrogen dioxide or ozone.  Although the main focus of the research was on UFP ( ≤ 0.18 μm), we also included exposures to fine particles (FP; ≤ 2.5 μm) and coarse particles (CP; 2.5 ≤ dp ≤ 10 μm).

During the in vivo studies we monitored and recorded the electrocardiograms (ECGs), blood pressure and activity levels of rats during their exposures to UFP using surgically implanted devices and used radio telemetry to collect the data.  Acute and chronic changes in blood pressure, heart rate, abnormal heart rhythms, and heart rate variability (HRV) were acquired on an hourly and daily basis and related to temporal variations in chemical and physical characteristics of the exposure atmosphere.  After the exposures were completed, the hearts and lungs of these animals were examined for adverse pathological changes, blood plasma was examined for the presence of biomarkers that have been associated with heart diseases and gene expression differences in the hearts of normal and susceptible models were determined to evaluate whether the physiological effects were associated with other clinical findings.


Objective 1:  Determine if exposure to ultra fine (< 0.18 micrometer) particulate matter (UFP) typical of polluted ambient air causes direct physiologic and pathologic damage (not involving the lung) to hearts of healthy individuals or individuals with increased susceptibility due to heart disease or aging.

This study was the first to convincingly demonstrate that direct exposure of the heart by instilling small amounts of ambient UFP (~10 μg) and ultrafine diesel particles directly into excised, perfused hearts via a vein would cause adverse changes in heart function, pressures and flows which confirmed changes that we had measured after direct cardiovascular system exposures in surgically prepared rats.
We demonstrated that exposure to ultrafine particles caused abnormal heart rhythms (arrhythmias) and changed the heart’s ejection fraction and diastolic pressure after 30 min of exposure. This effect was absent when the soluble fraction (containing no particles) isolated from the UFIDs was studied. These findings indicate that UFPs can have direct effects on the cardiovascular system that are independent of effects of particles on the lungs and that the effects are driven by the particle fraction and not by the soluble components of the particles (Wold, et al., 2006).
We had hypothesized that older rats would be more susceptible to particle-induced adverse effects.  We showed, however, that ultrafine particles when instilled directly into the cardiac vasculature were equally cardiotoxic in hearts from young adult and old rats
It has been thought that individuals with high blood pressure (hypertension) might be more susceptible to the adverse effects of PM than individuals with normal blood pressures.  We therefore examined the direct effects of UFP on spontaneously hypertensive rats (SHR) vs. their normotensive Wistar-Kyoto counterparts (WKY). After perfusion for 30 minutes cardiac function was reduced in both groups. In fact, the adverse effects were seen to a greater extent in WKY compared to SHR. The results of this study suggest that although UFP depress myocardial contractile response and coronary flow in both SHR and WKY the underlying hypertension does not necessarily worsen the response.
To determine whether oxidative stress was responsible for UFP-induced coronary flow reduction, we exposed Langendorff ­ perfused hearts obtained from normal adult Sprague Dawley rats to a 30-minute infusion of UFP  in the presence or absence of the potent hydroxyl radical scavenger, N ­ (2 ­ mercaptopropionyl) ­ glycine (MPG; l mM).  Thirty minutes of UFP perfusion significantly decreased coronary flow from 7.8 ± 0.5 ml/min to 5.4 ± 0.7 ml/min; a 32% reduction from baseline (p < 0.05); whereas coronary flow in hearts co-treated with MPG was fully preserved.  To the best of our knowledge this is the first study showing that the hydroxyl radical may play a role in the mechanism of the direct and acute toxic effect of UFP on the coronary vasculature and that the direct toxic effects of UFP on coronary flow can be mitigated with an oxygen radical scavenger.
We also addressed the question of whether hearts with a preexisting MI are more susceptible to the direct toxicity of UFPs. We used rats that had surgically-induced MI (produced by occluding a coronary artery) and used a control group consisting of rats that had undergone a sham surgery (SH) and measured the left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP), and rates of change in pressure (dP/dtmin and dP/dtmax). Contractile functions were measured at baseline and at the end of 30-min perfusion with buffer or UFP-buffer and presented as means of three consecutive beats.
Thirty min of UFP perfusion reduced cardiac contractile function in both MI and SH hearts (p<0.05). There was a trend for hemodynamic deterioration after UFP exposure to be worse in the MI group, however the extent of reductions in cardiac function caused by UFPs between MI and SH did not achieve statistical significance (p>0.05 for DP, dP/dtmin and dP/dtmax in MI vs. SH, respectively).  The extent of reductions in coronary flow (CF) although greater in MI hearts did not differ significantly from the extent of reductions in CF of SH hearts.
Although the MI UFP group had larger reductions in function than did the SH UFP group, the differences were not statistically significant.  However, because cardiac function already is reduced in hearts with MI, the direct exposure of these hearts to UFPs with further reduction in cardiac function heightens the potential for UFP exposure to cause or worsen adverse effects in coronary patients.

Objective 2: Determine whether temporal variations in particle concentrations or chemical compositions induce disturbances of blood pressure, heart rate and heart rate variability (BP, HR and HRV, respectively) in healthy individuals or individuals with increased susceptibility due to heart disease.

The goal of this aim was to determine whether a preexisting cardiovascular disease makes the heart more vulnerable to chronic UFP exposure, leading to more pronounced cardiac dysfunction. For this purpose, spontaneously hypertensive rats (SHR) vs. their normotensive Wistar-Kyoto (WKY) counterparts were employed. SHR have phenotypic changes that are observed in patients with arterial hypertension including elevated blood pressure, vascular resistance and stiffness, and hypertrophied vascular walls.  We hypothesized that the hearts of hypertensive rats would show greater deterioration in cardiac function when exposed to ultrafine particles.
We did not observe any significant exposure-related pathology in the hearts of either the WKY or SHR animals.  Histological analysis revealed normal myocardium with thickened blood vessels in the SHR group. UFP-exposed SHR and WKY groups hearts demonstrated characteristic linear stacking of mononuclear cells in the blood vessels, but picrosirius red staining revealed no differences between air and UFP-exposed hearts in either the SHR or WKY rats; the scores were 1.75±0.16 and 1.50±0.19 in WKY-FA and WKY-UFP groups and 1.83±0.17 and 1.88±0.23 in the SHR-FA and SHR-UFP rats respectively (p>0.05).

The heart rate (HR), blood pressure (BP) and heart rate variability (HRV) results demonstrated that exposure exacerbated hypertension in SHR rats (measured as mean arterial pressure) but the WKY rats actually demonstrated lower mean arterial BP after 3 months of exposure compared to rats exposed to purified air.   At the beginning of exposures the CAPs-exposed SHR rats showed a normal pattern of heart rate compensation for the elevated BP but by the end of the exposure experiment there was evidence that compensation was compromised.

A second cohort of rats was exposed separately and submitted for echocardiogrphic analysis. Left ventricle (LV) end-systolic (ESD) and end-diastolic (EDD) diameters were measured at the level of the papillary muscle using two-dimensional guided M-mode imaging, and fractional shortening (FS) was calculated as:
The rats were exposed for 3 months to UFP vs. filtered air. After completing the exposure protocol, a second echocardiogram was obtained. Systolic blood pressure (SBP) was increased and fractional shortening (FS) was decreased in all groups at the end of the FA and UFP exposures (p ≤ 0.05).  There was also an indication (Figure 1) that effects were worsened in the SHR group exposed to UFP which approached significance (p ≤ 0.1).
Figure 1.  Fractional shortening was decreased and systolic blood pressure was increased in SHR exposed to UFP.  The trend approached significance (p ≤ 0.10).
As shown in Figure 2 a wildfire in California during the exposures presented an opportunity to observe acute effects of wood smoke particles on cardiac physiology.  The data presented in Figures 3 and 4 demonstrate that exposure to the wood smoke caused an acute and significant decrease in blood pressure and these is an indication that this adverse effect is linked to the ability of the PM to induce formation of free radicals, as indicated by the results of in vitro tests that examined the ability of particles collected during the smoke exposures to deplete dithiothreitol (DTT) in a solution. 
Figure 2.  Smoke from wildfires was initially blown towards the Pacific Ocean (and away from our exposure site) by Santa Ana winds but over the course of the week the Santa Ana conditions weakened and on 10/25 and 10/26 smoke from the fires was present at our exposure site.
Blood pressure measurements on 10/25 and 10/26 demonstrated a significant decrease (p ≤ 0.05) in both SHR and WKY UFP-exposed groups but this was not observed in the FA-exposed rats (Figure 3).
Figure 3.  There was a significant drop in blood pressure in UFP-exposed SHR and WKY groups that coincided with the incursion of smoke from California wildfires.  This pattern was not observed in rats exposed to FA.
Air samples were collected and tested for free radical formation potential using a dithiothreitol (DTT) depletion assay by Arthur Cho (UCLA).  As shown in Figure 4, the samples collected during October, the DTT depletion activity was greater than during previous months and blood pressure was negatively correlated with DTT depletion potential.
Figure 4.  Blood pressure was decreased during exposures characterized by particles with high DTT depletion potential (which corresponds to potential for free radical formation).

Objective 3.  Determine if chronic exposure to polluted ambient air adversely affects the physiology, pathology, and gene expression in the hearts of healthy or susceptible individuals exposed to ambient particulate air pollution.

We expanded the goals of our study to include coarse and fine particulate matter to test the hypothesis that some components of PM can affect cardiac gene expression patterns. Fischer 344 rats were  exposed for 3 months to filtered air (FA), or coarse (CP; 2.5 ≤ dp ≤ 10 μm), fine (FP; dp ≤ 2.5 μm) and quasi-ultrafine (UFP; dp ≤0.18 μm) components of PM.  Exposures to concentrated ambient particles (n=8 per group) were performed in Riverside, California, which is, as mentioned earlier, an area with high ambient levels of photochemically derived gaseous and particulate pollutants. Rats were exposed to concentrated ambient particles for 5 hours per day, 4 days per week for a period of 3 months (1/10/07 through 4/10/07). Detailed descriptions of the exposure methodology (Kleinman et al., 2005; Simkhovich et al., 2008) and concentrator (Kim et al., 2001a; Kim et al., 2001b) used in our study (Versatile Air Concentrator Enrichment System, VACES)  have been previously published.
Particle characteristics and their deposition in lungs (Table 1)
     Mean particle mass, elemental carbon and organic carbon concentrations as well as the particle number of the ultrafine, fine and coarse concentrated ambient particles (CAPs) to which 4-week-old(at the beginning of the exposures) male F334 rats were exposed for 3 months are shown in Table 3.  Control rats were exposed to filtered air.  The mass concentration of the FP exposure aerosol was significantly (p ≤ 0.05) greater than that for either the UFP or CP aerosols.  The percentage of elemental carbon in the UFP was, however, approximately twice that in the FP aerosol, and 2.8 times higher than in the CP. The percentages of the organic carbon in the UFP and CP fractions were significantly (p ≤ 0.05) higher as compared to the FP fraction (27.5%, and 20.3% vs. 12.8% respectively). The fraction of particles deposited in the respiratory tract (deposited fraction, DF) was highest for the coarse particles, intermediate for the UFP, and lowest for the fine particles. With regard to the elemental carbon, its amount deposited in the respiratory tract was reduced in the following order: UFP>FP>CP, while the amount of organic carbon deposited in the respiratory tract was highest in the CP group, intermediate in the UFP, and lowest in the FP group. It is noteworthy that DF of the particles and deposited amount of organic carbon were lowest for the fine particles.
Table 1.  Mass, number, and carbon speciation of UFP, FP, and CP CAPs downwind of a heavily trafficked roadway in Riverside, CA (mean ± SE).
Mass (mg/m3)
63 ± 8
149 ± 24
58 ± 7
Number (particles/cm3 x 103)
65 ± 5
67 ± 6
EC Mass (μg/m3)
5.5 ± 1.1
6.3 ± 1.1
2.3 ± 0.7
OC Mass (μg/m3)
35.8 ± 2.1
38.3 ± 2.6
30.0 ± 1.6
Ambient average particle count:  8.9 ± 1 particles/cm3 x103.
*Aerosol particles >3 mm are inefficiently detected by the TSI 3022 particle counter.
Our results indicate that chronic exposure to concentrated ambient particulate matter can affect gene expression patterns in the hearts of rats. The total number of genes that were affected varied as a function of particle size. This is not entirely unexpected because the source contributions and chemical compositions of the three different particle size ranges studied are not the same.  We did not find any significant differences in gene expression patterns in the hearts of rats exposed to FP as compared to rats exposed to FA.  We found that exposure to UFP affected a greater number of genes than did CP exposure (8 vs. 3 respectively). It is known that CPs are retained in the upper respiratory track and only soluble components from the CP could translocate to the cardiovascular system, whereas as larger fraction of soluble material from the FP and UFP could be more bioavailable.  The most important changes that we observed were those related to thioredoxin oxidative stress defenses (Txnip) and to the xenobiotic-metabolizing cytochrome P450 systems (Cyp2e1). Up-regulation of Cyp2e1 and Txnip is consistent with pro-oxidative and pro-injury-related effects of exposure to ambient PM.

Journal Articles on this Report : 8 Displayed | Download in RIS Format

Other project views: All 18 publications 8 publications in selected types All 8 journal articles
Type Citation Project Document Sources
Journal Article Hwang H, Kloner RA, Kleinman MT, Simkhovich BZ. Direct and acute cardiotoxic effects of ultrafine air pollutants in spontaneously hypertensive rats and Wistar-Kyoto rats. Journal of Cardiovascular Pharmacology and Therapeutics 2008;13(3):189-198. R831952 (Final)
  • Abstract from PubMed
  • Abstract: Sage Journals-Abstract
  • Journal Article Kleinman MT, Araujo JA, Nel A, Sioutas C, Campbell A, Cong PQ, Li H, Bondy SC. Inhaled ultrafine particulate matter affects CNS inflammatory processes and may act via MAP kinase signaling pathways. Toxicology Letters 2008;178(2):127-130. R831952 (Final)
    R832413 (2007)
    R832413 (2008)
    R832413 (2009)
    R832413 (Final)
    R832413C001 (2007)
    R832413C001 (2008)
    R832413C001 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Associated PubMed link
  • Full-text: ScienceDirect-Full Text HTML
  • Abstract: ScienceDirect-Abstract
  • Other: ScienceDirect-Full Text PDF
  • Journal Article Simkhovich BZ, Marjoram P, Kleinman MT, Kloner RA. Direct and acute cardiotoxicity of ultrafine particles in young adult and old rat hearts. Basic Research in Cardiology 2007;102(6):467-475. R831952 (Final)
  • Abstract from PubMed
  • Abstract: SpringerLink-Abstract
  • Journal Article Simkhovich BZ, Kleinman MT, Kloner RA. Air pollution and cardiovascular injury epidemiology, toxicology, and mechanisms. Journal of the American College of Cardiology 2008;52(9):719-726. R831952 (2005)
    R831952 (Final)
  • Abstract from PubMed
  • Full-text: Science Direct-Full Text HTML
  • Abstract: Science Direct-Abstract
  • Other: Science Direct-Full Text PDF
  • Journal Article Simkhovich BZ, Kleinman MT, Kloner RA. Particulate air pollution and coronary heart disease. Current Opinion in Cardiology 2009;24(6):604-609. R831952 (Final)
  • Abstract from PubMed
  • Abstract: LWW-Abstract
  • Journal Article Simkhovich BZ, Kleinman MT, Willet P, Gookin G, Salazar K, Keebaugh A, Kloner RA. Chronically inhaled ambient particles cause cardiac inflammation in normal, diseased, and elderly rat hearts. Air Quality Atmosphere and Health 2011;4(1):27-36. R831952 (Final)
  • Abstract: Springerlink-Abstract
  • Journal Article Simkhovich BZ, Kleinman MT, Mehrian-Shai R, Hsu YH, Meacher D, Gookin G, Kinnon MM, Salazar K, Willet P, Feng G, Lin SM, Kloner RA. Chronic exposure to ambient particulate matter alters cardiac gene expression patterns and markers of oxidative stress in rats. Air Quality Atmosphere and Health 2011;4(1):15-25. R831952 (Final)
  • Abstract: Springerlink-Abstract
  • Journal Article Wold LE, Simkhovich BZ, Kleinman MT, Nordlie MA, Dow JS, Sioutas C, Kloner RA. In vivo and in vitro models to test the hypothesis of particle-induced effects on cardiac function and arrhythmias. Cardiovascular Toxicology 2006;6(1):69-78. R831952 (2005)
    R831952 (Final)
    R827352 (Final)
    R832413 (2008)
    R832413 (Final)
    R832413C001 (2007)
    R832413C001 (Final)
  • Abstract from PubMed
  • Abstract: Springer-Abstract
  • Supplemental Keywords:

    Particulate Matter, PM10, PM2.5, ultrafine particles, cardiovascular response.
    , RFA, Health, Scientific Discipline, Air, particulate matter, Toxicology, Environmental Chemistry, Health Risk Assessment, Risk Assessments, Biochemistry, ambient aerosol, lung injury, hypertension, long term exposure, lung disease, acute cardiovascular effects, airway disease, cardiovascular vulnerability, airborne particulate matter, ambient particle health effects, heart attacks, cardiovascular disease

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    Progress and Final Reports:

    Original Abstract
  • 2005 Progress Report
  • 2006
  • 2007
  • 2008