Tammy Mui

Epidemiologic and animal studies have shown that exposure to particulate matter air pollution (PM) is a risk factor for the development of atherosclerosis. Whether PM-induced lung and systemic inflammation is involved in this process is not clear. We hypothesized that PM exposure causes lung and systemic inflammation, which in turn leads to vascular endothelial dysfunction, a key step in the initiation and progression of atherosclerosis. New Zealand White rabbits were exposed for 5 days (acute, total dose 8 mg) and 4 wk (chronic, total dose 16 mg) to either PM smaller than 10 mum (PM(10)) or saline intratracheally. Lung inflammation was quantified by morphometry; systemic inflammation was assessed by white blood cell and platelet counts and serum interleukin (IL)-6, nitric oxide, and endothelin levels. Endothelial dysfunction was assessed by vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP). PM(10) exposure increased lung macrophages (P<0.02), macrophages containing particles (P<0.001), and activated macrophages (P<0.006). PM(10) increased serum IL-6 levels in the first 2 wk of exposure (P<0.05) but not in weeks 3 or 4. PM(10) exposure reduced ACh-related relaxation of the carotid artery with both acute and chronic exposure, with no effect on SNP-induced vasodilatation. Serum IL-6 levels correlated with macrophages containing particles (P=0.043) and ACh-induced vasodilatation (P=0.014 at week 1, P=0.021 at week 2). Exposure to PM(10) caused lung and systemic inflammation that were both associated with vascular endothelial dysfunction. This suggests that PM-induced lung and systemic inflammatory responses contribute to the adverse vascular events associated with exposure to air pollution.

Some have suggested that chronic obstructive pulmonary disease (COPD) is a disease of accelerated aging. Aging is characterized by shortening of telomeres. The relationship of telomere length to important clinical outcomes such as mortality, disease progression and cancer in COPD is unknown. Using quantitative polymerase chain reaction (qPCR), we measured telomere length of peripheral leukocytes in 4,271 subjects with mild to moderate COPD who participated in the Lung Health Study (LHS). The subjects were followed for approximately 7.5 years during which time their vital status, FEV(1) and smoking status were ascertained. Using multiple regression methods, we determined the relationship of telomere length to cancer and total mortality in these subjects. We also measured telomere length in healthy "mid-life" volunteers and patients with more severe COPD. The LHS subjects had significantly shorter telomeres than those of healthy "mid-life" volunteers (p<.001). Compared to individuals in the 4(th) quartile of relative telomere length (i.e. longest telomere group), the remaining participants had significantly higher risk of cancer mortality (Hazard ratio, HR, 1.48; p = 0.0324) and total mortality (HR, 1.29; p = 0.0425). Smoking status did not make a significant difference in peripheral blood cells telomere length. In conclusion, COPD patients have short leukocyte telomeres, which are in turn associated increased risk of total and cancer mortality. Accelerated aging is of particular relevance to cancer mortality in COPD.

We evaluated a method for performing absolute cell counts of lymphocyte populations with a flow cytometer. In this method, TruCount, test tubes that contain a known number of brightly fluorescent polystyrene beads are provided by the manufacturer. Whole anticoagulated blood is accurately pipetted into the tubes and mixed with fluorochrome-labeled monoclonal antibodies, the erythrocytes are lysed, and this mixture is analyzed on the flow cytometer. Absolute counts of lymphocyte subsets are calculated by determining the ratio of beads to the cell population of interest and then multiplying this ratio by the number of beads in the tube. We found this method to be reproducible. The values we obtained by the TruCount method were 5 to 10% higher than those obtained by conventional methods (flow cytometry and automated hematology) used to determine absolute numbers of cells. We believe that these differences are due to the methods of determining absolute cell counts and not to faulty identification of lymphocyte subsets.

Acute lung injury (ALI) is associated with systemic inflammation and cardiovascular dysfunction. IL-6 is a biomarker of this systemic response and a predictor of cardiovascular events, but its possible causal role is uncertain. Inhaled corticosteroids and long-acting β2 agonists (ICS/LABA) down-regulate the systemic expression of IL-6, but whether they can ameliorate the cardiovascular dysfunction related to ALI is uncertain. We sought to determine whether IL-6 contributes to the cardiovascular dysfunction related to ALI, and whether budesonide/formoterol ameliorates this process. Wild-type mice were pretreated for 3 hours with intratracheal budesonide, formoterol, or both, before LPS was sprayed into their tracheas. IL-6-deficient mice were similarly exposed to LPS. Four hours later, bronchoalveolar lavage fluid (BALF) and serum were collected, and endothelial and cardiac functions were measured, using wire myography of the aortic tissue and echocardiography, respectively. LPS significantly impaired vasodilatory responses to acetylcholine (P < 0.001) and cardiac output (P = 0.002) in wild-type but not IL-6-deficient mice. Intratracheal instillations of exogenous IL-6 into IL-6-deficient mice restored these impairments (vasodilatory responses to acetylcholine, P = 0.005; cardiac output, P = 0.025). Pretreatment with the combination of budesonide and formoterol, but not either alone, ameliorated the vasodilatory responses to acetylcholine (P = 0.018) and cardiac output (P < 0.001). These drugs also attenuated the rise in the systemic expression of IL-6 (P < 0.05) related to LPS. IL-6 contributes to the cardiovascular dysfunction related to LPS, and pretreatment with budesonide/formoterol reduces the systemic expression of IL-6 and improves cardiovascular dysfunction. ICS/LABA may reduce acute cardiovascular events related to ALI.

To the Editor: There is mounting evidence that chronic obstructive pulmonary disease (COPD) is a disease of accelerated aging caused by inflammation and oxidative stress.1 COPD has many age-related comorbidities, and the lungs of patients with COPD have been shown to have high levels of senescence markers.1,2 Telomere length is thought to be a biomarker of biological aging, because whenever cells turn over, their telomeres shorten, which eventually results in cellular senescence or apoptosis.3 In humans, telomeres are tandem repeats of the sequence (TTAGGG/CCCTAA)n at chromosome ends, which function in protecting against the loss of genetic information and in maintaining chromosome stability. Studies have found that patients with COPD have shorter telomeres in the lung and in the periperhal circulation than age-matched healthy smoking and nonsmoking controls,4–8 but the relationship between telomere length and COPD severity as assessed according to lung function is unknown. The primary objective of the present study was to determine the relationship between telomere length and disease severity in patients with COPD. Two hundred eighty-three blood samples collected at baseline from participants of the Advair Biomarkers in COPD Study were used. All participants had COPD according to the Global initiative for chronic Obstructive Lung Disease guidelines and were part of the Advair Biomarker in COPD (ABC) Study. Additional criteria and details of the study have been described previously.9 Telomere length was measured in deoxyribonucleic acid extracted from buffy coat from the peripheral circulation using a quantitative polymerase chain reaction protocol modified from Cawthon's method.10 Telomere length was quantified as a relative ratio of telomere to single copy gene (36B4): 2−ΔΔCt, where −ΔΔCt=ΔCtsample−ΔCtreference and ΔCt=Cttelomere−ΔCt36B4. After normalization of the data using natural log transformation, the relationship between telomere length and lung function was determined using univariate and multivariate linear regression modeling in which covariates included age, sex, pack-years of smoking, current smoking status, and presence of cardiovascular disease. SPSS 15.0 (SPSS, Inc., Chicago, IL) was used for statistical analyses, and P<.05 was considered statistically significant. The mean age of the subjects was 69, and mean pack-years of smoking history was 62. There was a negative relationship between log-relative telomere length and age (coefficient of determination=0.03, P=.006), indicating that older subjects had shorter telomeres. Women had slightly longer telomeres than men (P=.003). Neither weight (P=.50) nor body mass index (P=.85) was significantly associated with telomere length, but height was positively associated with higher telomere to single copy ratio (β±standard error of the mean (SEM)=0.007±0.003). Neither smoking status (current vs ex-smokers) (P=.95) nor pack-years of smoking (P=.62) was significantly related to telomere length. There was a significant relationship between airflow obstruction as determined using spirometry according to the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) and telomere length (β±SEM=0.50±0.16, P=.002; Figure 1) and a strong positive trend between predicted FEV1% and telomere length (β±SEM=0.002±0.001, P=.06). Telomere length was inversely related to log-surfactant protein-D levels in serum (β±SEM=−0.10±0.04, P=.02). No significant relationship was found with log-C-reactive protein (CRP) (P=.67) or log-interleukin (IL)-6 concentrations in serum (P=.66). A positive relationship between the ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) and telomere length of circulating leukocytes. Linear multivariate regression: β±standard error of the mean=0.50±0.16, P=.002. This study found a significant relationship between telomere length and airflow obstruction in patients with COPD. Patients with COPD with greater airflow obstruction, determined according to FEV1/FVC ratio, had shorter telomeres in their circulating leukocytes than patients with less-severe airflow obstruction. Two previous studies failed to find a significant relationship between telomere length in circulating leukocytes and lung function measurements, probably owing to small sample sizes and suboptimal statistical power,4,7 although a study of people with emphysema, smokers, and nonsmokers found a positive correlation between telomere signal intensity (proportional to telomere length) and predicted FEV1% in lung endothelial and type II epithelial cells.8 It was also found that the length of telomeres was inversely related to a lung-specific serum inflammatory marker, SP-D, but not to generalized inflammatory serum markers such as CRP or IL-6 levels. Together, the findings from the present study suggest that COPD is a disease of accelerated aging that may be related to lung inflammation. Conflict of Interest: The editor in chief has reviewed the conflict of interest checklist provided by the authors and has determined that the authors have no financial or any other kind of personal conflicts with this letter. Funding was received from the Interdisciplinary Capacity Enhancement: Bridging Excellence in Respiratory Disease and Gender Studies, which is funded by the Canadian Institutes of Health Research, the Canadian Lung Association, and the Heart and Stroke Foundation of Canada and the BC Lung Association and a team grant from the Michael Smith Foundation for Health Research. The ABC Study was funded by GlaxoSmthKline. DDS is a Canada Research Chair in COPD and a senior scholar with the Michael Smith Foundation for Health Research. HOC is a Canadian Institutes of Health Research/British Columbia Lung Association New Investigator. HOC is also supported, in part, by the University of Pittsburgh COPD Specialized Centers of Clinically Oriented Research, National Institutes of Health (NIH), 1P50 HL084948 and R01 HL085096 from the National Heart, Lung, and Blood Institute, NIH, to the University of Pittsburgh. Author Contributions: TSM conducted the telomere measurement and the statistical analysis and wrote the first draft of the manuscript. JMM developed the technique for extracting telomeres from peripheral circulating cells and measuring telomere length. JEM recruited the healthy control subjects and provided input into the statistical analysis. AJS supervised the laboratory measurements and provided input into the write-up of the manuscript. HOC and CLB assisted in the development of the cohort for developing the telomere measurement and in writing the manuscript. YL was involved in blood collection, data storage, and analysis, SFP and DDS are the co-principal investigators of the ABC cohort, helped design the present study, supervised the personnel on this project, and provided input into the writing of the manuscript. Sponsor's Role: These funding agencies had no role in the design and implementation of the project or in the data analysis.