Determinants of systemic vascular function in Patients

0 downloads 0 Views 531KB Size Report
Oct 3, 2008 - Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease ... function (15) and severity of coronary artery disease (16). .... reported findings in patients with COPD were independent of smoking history.
Page 1 of AJRCCM 44 Articles in Press. Published on October 3, 2008 as doi:10.1164/rccm.200709-1412OC

Determinants of systemic vascular function in Patients with Stable COPD

Philipp Eickhoff,1* Arschang Valipour,2* Dora Kiss,2 Martin Schreder,3 Leyla Cekici,2 Kora Geyer,2 Robab Kohansal,2 Otto C Burghuber2

1

St. Anna Childrens Hospital, Vienna, Austria

2

Department of Respiratory and Critical Care Medicine, Ludwig Boltzmann Institute for

COPD, Otto Wagner Hospital, Vienna, Austria 3

1. Department of Medicine, Wilhelminen Hospital, Vienna, Austria

* These authors contributed equally to this work.

Corresponding author:

Otto Chris Burghuber, MD, FCCP Department of Respiratory and Critical Care Medicine, Otto-Wagner-Hospital Sanatoriumstrasse 2, 1140 Wien Vienna, Austria Tel: 0043 1 91060 41008 Fax: 0043 1 91060 49827 E-mail: [email protected]

Running title: Systemic vascular function in COPD

Descriptor number: 53. COPD: pathophysiology

Word count (text): 3620

Copyright (C) 2008 by the American Thoracic Society.

Page 2 of 44

At a glance commentary: We provide evidence of impairment in vascular reactivity in patients with chronic obstructive pulmonary disease due to both endothelium-dependent and endothelium-independent mechanisms. Impaired endothelial function in chronic obstructive pulmonary disease was strongly related to systemic inflammation and airway obstruction, which may help explain the increased cardiovascular morbidity and mortality in this population.

1

Page 3 of 44

Abstract Word count (abstract): 249 Rationale and Objectives: Impaired vascular reactivity is an important factor in the pathogenesis of cardiovascular disease. We sought to assess vascular reactivity in patients with COPD and respective controls, and to investigate the relation between function with airflow obstruction and systemic inflammation. Methods: We studied 60 patients with stable COPD; 20 smokers with normal lung function matched for age, sex, and body weight, and 20 similarly matched nonsmokers. Patients with cardiovascular comorbidities were excluded. The endothelium-dependent and endotheliumindependent function of the vasculature was measured using flow-mediated and nitrogenmediated dilation of the brachial artery, respectively. Systemic inflammatory markers including C-reactive protein, fibrinogen, and interleukin-6 were determined in serum. Measurements and Main Results: Both flow-mediated and nitrogen-mediated dilation of the brachial artery were significantly lower in patients with stable COPD than in smoking and nonsmoking controls. Levels of inflammatory mediators such as interleukin-6 and fibrinogen were higher in patients than they were in controls. In patients with COPD, stepwise multiple regression analysis showed that age, sex, baseline brachial artery diameter, C-reactive protein level, leukocyte count, blood glucose level, and percentage of predicted forced expiratory volume in 1 second were independent predictors of flow-mediated dilation. There was no relation between flow-mediated dilation and pack-years of smoking. Baseline brachial artery diameter was the only independent predictor of nitrogen-mediated dilation in patients with COPD. Conclusions: Both endothelium-dependent and endothelium-independent vasodilation is significantly impaired in patients with stable COPD. Airflow obstruction and systemic inflammation may increase the risk of cardiovascular disease in patients with COPD.

2

Page 4 of 44

Key words: systemic vascular function, chronic obstructive pulmonary disease, cardiovascular disease, systemic inflammation

3

Page 5 of 44

Introduction Chronic obstructive pulmonary disease (COPD) is a chronic inflammatory disease of the lungs; however, there is cumulating data suggesting that the inflammatory reaction associated with COPD is not restricted to the lung (1). Pinto-Plata and coworkers (2) as well as others (35) have demonstrated increased levels of systemic inflammatory markers such as C-reactive protein in patients with stable COPD compared with controls. The degree of systemic inflammation associated with COPD tends to increase over time (5) and increases during acute exacerbations (6, 7), and systemic inflammation contributes to the development of cardiovascular diseases (8). The relation between systemic inflammation and increased risk of cardiovascular morbidity and mortality also has been observed in patients with COPD (9). The underlying biological mechanisms, however, remain unknown. Several authors have reported abnormalities in systemic vascular function in patients with COPD, providing additional support for the link between COPD and cardiovascular disease (10-12). Flow-mediated vasodilation is an endothelium-dependent function that can be measured in forearm human circulation and quantified as an index of vasomotor function (13). Flow-mediated vasodilation is an independent predictor of cardiovascular morbidity and mortality (14) and correlates well with invasive assessment of coronary artery endothelial function (15) and severity of coronary artery disease (16). Several studies have shown impaired endothelial function using this technique in conditions associated with systemic inflammation such as diabetes (17), rheumatoid arthritis (18), and polycystic ovarian syndrome (19). A recently published study furthermore suggests a relation between flowmediated vasodilation and forced expiratory volume in 1 second (FEV1) and emphysema severity in patients with COPD (10). That report, however, did not address the role of systemic inflammation and its relation with endothelial function in COPD. This is of particular importance because there is experimental evidence on a molecular level of impaired endothelial function in response to inflammatory markers, such as C-reactive protein (20, 21). 4

Page 6 of 44

Thus, we sought to use measurements of systemic vascular function in patients with stable COPD and respective controls and to investigate the relation between airflow obstruction, systemic inflammation, and endothelium-dependent as well as endothelium-independent function of the vasculature. Some of the results of this study have been previously reported in the form of an abstract (22).

Methods Participants were explained the study protocol and potential hazards during a personal interview. All subjects gave written informed consent. The study was approved by the Ethics Committee of the Vienna City Council.

Study population The study population consisted of patients with stable COPD; smokers matched for age, sex, and body weight; and matched nonsmoking controls without airflow obstruction. A total of 1074 COPD patients were screened for eligibility from the Otto-WagnerHospital’s outpatient clinic database. Inclusion criteria for patients with stable COPD included an age of 40 to 75 years, a body mass index of less than 30 kg/m2, 20 pack-years or more of cigarette smoking, and evidence of airflow obstruction on spirometry. Our aim was to recruit an equal number of patients with mild (FEV1 predicted 50%-70%), moderate (FEV1 30%50%), and severe COPD (FEV1 < 30%). Stable COPD was defined as no exacerbations during at least 3 visits in the previous 4 months with no changes in respiratory medication and no symptoms of a lower respiratory tract infection. Patients included in the study were not taking oral corticosteroids or other acute or chronic medication that could influence endothelial function (eg, statins or angiotensin-converting enzyme inhibitors). Patients with known cardiovascular comorbidities were excluded. Patients with COPD receiving long-term oxygen therapy were not excluded. Additional details of the exclusion criteria are provided in the 5

Page 7 of 44

online data supplement. Similar criteria were applied to smoking controls, except for the absence of lung disease by clinical evaluation, chest radiograph, and spirometry. Inclusion criteria for nonsmoking controls were the same, except that these participants were life-long nonsmokers. Controls were recruited from the general population during a lung function day organized by the hospital. Smoking controls and current smokers with COPD were asked to refrain from smoking for at least 6 hours before testing (23). Abstinence from smoking was assessed by measuring expiratory carbon monoxide using a cutoff level of less than 10 ppm.

Measurements Study participants provided a detailed medical history and were given a physical examination that included blood pressure measurement, electrocardiogram, arterial blood gas analysis, lung function testing, and assessment of endothelial function of the brachial artery after reactive hyperemia. Blood samples were obtained from all participants to analyze complete blood count and systemic inflammatory markers such as interleukin-6 (IL-6), fibrinogen levels, and C-reactive protein levels. Spirometry was done according to the recommendations of the European Respiratory Society (24). Arterial blood gas was analyzed with the patients breathing room air. Endothelium-dependent, flow-mediated dilation after reactive hyperemia, as well as endothelium-independent, nitroglycerine-mediated dilation of the brachial artery were assessed using ultrasound according to the recommended guidelines (13). To account for differences in vessel diameter between the study groups, the ratio of flow mediated dilation and baseline brachial artery diameter was taken as an index of diameter independent impairment of vascular reactivity. Additional details on the method of taking these measurements are provided in the online data supplement.

6

Page 8 of 44

Statistical Analyses All data are expressed as frequencies (percentages) for normally distributed data either as (arithmetic) means ± SD (standard deviation) or medians with interquartile range (Q1=25% Q3=75%) for skewed distributions. Inter-group comparison of normally distributed parameters was performed using the independent t-test. The Mann-Whitney U was performed for comparison of not normally distributed parameters. Depending on data distribution, either a Pearson or Spearman correlation coefficient was calculated to determine relations between ultrasound measurements, clinical characteristics, lung function parameters, and laboratory markers, both in the total sample and in patients with COPD specifically. Multivariate analyses with several potentially confounding factors were done with percentage of flowmediated vasodilation or percentage of nitroglycerine-mediated dilation as the dependent variable, respectively. Values for P less than 0.05 were accepted statistically significant differences, with a Bonferroni correction reported for multiple comparisons. All statistics were analyzed with SPSS software (Statistical Product and Services Solutions, version 13.0, SPSS Inc, Chicago, IL, USA).

Results From the database of 1074 patients with COPD, 190 patients (17.6%) met the inclusion and 884 (82.4%) were excluded (Figure 1). Of the 190 patients, 60 patients were finally included in this study.

Clinical characteristics The clinical characteristics, lung function results, and cardiovascular parameters of the study population are summarized in Table 1. The groups were well matched with respect to age, body weight, and arterial blood pressure measurements. There were statistically significant differences in lung function, heart rate, and arterial PaO2 measurements between patients with 7

Page 9 of 44

COPD, smoking, and nonsmoking controls without airflow obstruction. Patients with COPD had a longer pack-year smoking history than did smoking controls (66 ± 39 pack-years vs 39 ± 23 pack-years, P < 0.001); however, the proportion of current smokers was statistically significantly lower in the patient group than it was in nonsmoking controls. Smoking abstinence before testing was confirmed in all subjects: Similar carbon monoxide expiratory levels were found in smokers with COPD (n=26, expiratory CO 7.0 ± 2.6 ppm) and in smoking controls without airflow obstruction (expiratory CO 7.7 ± 2.6 ppm). Of the patients with COPD, all were prescribed inhaled short-acting and/or long-acting bronchodilators, 45 patients (75%) were receiving inhaled corticosteroids, and 14 patients (23%) were on long term oxygen therapy. Further information on patient characteristics are provided in the online data supplement. (Table E1)

Laboratory markers The study groups were well matched with respect to traditional cardiovascular risk factors, such as cholesterol, triglyceride, and blood glucose levels (Table 2). There were no statistically significant differences in blood leukocyte counts between patients and controls. However, patients with COPD had statistically significantly higher plasma fibrinogen concentrations (median [interquartile range]; 426 mg/dL [range, 354-472 mg/dL]) compared with nonsmoking controls (382 mg/dL [range, 317-428 mg/dL], P < 0.05). Similarly, we observed that IL-6 serum levels were statistically significantly higher in patients with COPD than they were in nonsmokers. There were no statistically significant differences in these laboratory markers between patients with mild, moderate, and severe COPD (Table E2 in the online data supplement).

8

Page 10 of 44

Ultrasound measurements of vascular reactivity of the brachial artery: Ultrasound assessment of vascular reactivity was well tolerated in all subjects, none of whom had evidence of brachial artery atherosclerotic plaque on ultrasound scanning. Baseline brachial artery diameter was statistically significantly higher in patients with COPD than it was in nonsmoking controls (3.64 ± 0.63 mm vs 3.28 ± 0.61 mm, P = 0.031). There were, however, no statistically significant differences among patients with COPD, smokers, and nonsmokers regarding blood flow velocity through the brachial artery at rest (baseline flow), during reactive hyperemia, or after sublingual application of nitroglycerine (Table 3). This indicates that the physical stimulus for flow-mediated vasodilation and nitroglycerinemediated dilation was comparable in all groups. After release of suprasystolic compression, patients with stable COPD had a statistically significantly lower flow-mediated vasodilation response (11% ± 3%), expressed as a percentage of change over the baseline diameter, than did smokers (16% ± 2%, P < 0.005) and nonsmoking controls (19% ± 3%, P < 0.001) without airflow obstruction (Figure 2). Patients with COPD had a significantly lower ratio of flow mediated dilation to baseline brachial artery diameter compared to both smoking and non-smoking controls (P < 0.01 for both). Furthermore, there was a statistically significant difference in the percentage of nitroglycerine-mediated dilation between patients with COPD and nonsmoking controls (22% ± 6% vs 29% ± 7%, P = 0.02), but there was no statistically significant difference compared with smokers without COPD. There were no statistically significant differences in baseline brachial artery diameter, blood flow velocity, percentage of flow-mediated vasodilation (Figure E1 in the online data supplement), and percentage of nitroglycerine-mediated dilation between patients with mild, moderate, and severe COPD (Table E3 in the online data supplement).

9

Page 11 of 44

Relations between vascular reactivity, systemic inflammation, and lung function In the entire study population (n=100), statistically significant relations were observed on univariate analyses between percentage of flow-mediated vasodilation and age, FEV1% predicted, FEV1%FVC (forced vital capacity), leukocyte count, C-reactive protein, IL-6, resting heart rate, percentage of nitroglycerine-mediated dilation, and baseline brachial artery diameter (Table 4). Within the patient group (n=60), FEV1% predicted (Figure 3a), FEV1%FVC, C-reactive protein (Figure 3b), and baseline brachial artery diameter were statistically significantly related with the percentage of flow-mediated vasodilation. Despite statistical significance between percentage of flow-mediated vasodilation and total cholesterol levels in patients with COPD, there were no statistically significant relations between percentage of flow-mediated vasodilation and other cardiovascular risk factors including pack-years of smoking (Figure E2 in the online data supplement), body mass index, triglyceride levels, or blood glucose levels. With respect to measurements of percentage of nitroglycerine-mediated dilation, on univariate analyses there were statistically significant correlations with age (r = –0.376, P < 0.001), blood glucose levels (r = –0.271, P = 0.008), FEV1% (r = 0.243, P = 0.018), and heart rate (r = –0.196, P = 0.058). Associations between percentage of flow-mediated vasodilation and percentage of nitroglycerine-mediated dilation as the dependent variables and age, sex, body mass index, pack-years of smoking, FEV1% predicted, baseline brachial artery diameter, total cholesterol, triglyceride levels, blood glucose levels, leukocyte cell count, blood fibrinogen, C-reactive protein, IL-6, systolic blood pressure, heart rate, inhaled corticosteroid use, and long-term oxygen therapy use were further investigated using stepwise multiple regression analyses in patients with COPD. Table 5 shows the results of multiple regression analyses for the flowmediated vasodilation response. The following factors were independently associated with percentage of flow-mediated vasodilation in the patient group: age (P < 0.001), sex (P = 0.001), brachial artery diameter (P < 0.001), C-reactive protein levels (P = 0.001), leukocyte 10

Page 12 of 44

count (P = 0.004), blood glucose levels (P = 0.014), and FEV1% predicted (P = 0.046). The strongest independent predictor of the postocclusion nitroglycerine-mediated dilation response in the patient group was the baseline brachial artery diameter (Table E4 in the online data supplement).

Discussion The present study investigated systemic vascular function of the brachial artery in patients with stable COPD and smokers and nonsmokers without COPD matched for age and body mass index. We found statistically significantly differences in baseline brachial artery diameter, endothelium-dependent and endothelium-independent vasodilation in patients with COPD compared with the respective control groups. We also saw statistically significant relations between flow-mediated dilation with markers of systemic inflammation and severity of airflow obstruction, both in the total sample and in patients with COPD, specifically. The reported findings in patients with COPD were independent of smoking history. The principle of flow-mediated vasodilation is an increase in flow through the brachial artery, which is induced by causing postischemic (nitric-oxide–mediated) vasodilatation in the downstream vascular bed of the distal forearm. Flow-mediated vasodilation has been shown to correlate well with invasive use of acetylcholine infusion to assess coronary artery endothelial function (15). A reduction in the flow-mediated vasodilation response suggests the presence of endothelial dysfunction. Endothelial dysfunction, in turn, is a key early event in atherogenesis and an independent predictor of cardiovascular diseases appearing long before the formation of structural atherosclerotic changes (25). Changes in vessel wall in early atherosclerosis, however, may not be limited to the endothelium and a reduction in vasodilation in response to endothelium-derived and exogenous sources of nitric oxide (NMD) may also be mediated by changes in vascular smooth muscle function. Adams and coworkers have previously shown that smooth muscle dysfunction becomes apparent with 11

Page 13 of 44

increasing number of cardiovascular risk factors, resulting in an impaired NMD response independently from endothelial dysfunction (26). In the present study we observed an impairment in both FMD and NMD response in patients with COPD compared with smoking and non-smoking controls, suggesting an increased cardiovascular risk in this population (25, 26). In fact, a recent health care database cohort study of more than 10 000 patients reported that patients with COPD were 2 to 4 times more likely to die of cardiovascular disease at 3-year follow-up than were age- and sexmatched controls without COPD (27). Furthermore, there is epidemiologic evidence of a link between FEV1 and cardiovascular mortality (28, 29). We have to acknowledge, however, that our findings were largely influenced by differences in baseline brachial artery diameter between the study groups (30). We have no immediate explanation for the differences in vessel size; however, Holubkov and coworkers (31) observed that a large size of the resting brachial artery diameter itself serves as an independent predictor of angiographically verified coronary artery disease. We further observed statistically significant differences in the ratio of flow mediated dilation to baseline brachial artery diameter between the groups, suggesting that there is a diameter-independent impairment of vascular reactivity in COPD patients over and above the impairment related to smoking. It appears, however, that this occurs at smooth muscle rather than endothelial level as the response of the brachial artery to nitroglycerin (NMD) was also impaired, hence that the smooth muscle response to endogenous and exogenous sources of nitric oxide was decreased (26). Few recent studies similarly investigated systemic vascular function as a surrogate of cardiovascular risk in patients with COPD. Sabit and colleagues (11) studied arterial stiffness using pulse wave velocity in 75 clinically stable COPD patients and 43 healthy smoking controls free of cardiovascular disease. The authors observed pronounced arterial stiffness in patients compared with controls and statistically significant relationships between pulse wave 12

Page 14 of 44

velocity and percentage predicted FEV1, suggesting increased cardiovascular risk with increasing airflow obstruction in COPD. McAllister and coworkers (12) extended these findings by demonstrating that in addition to FEV1, emphysema severity based on quantitative CT scans revealed as the most powerful predictor of arterial stiffness. The latter reports support our observations as the underlying pathology of arterial stiffness includes endothelial and vascular smooth muscle dysfunction, as well as elastin loss and thus increased vessel lumen size (32). Barr and colleagues (10) studied flow-mediated dilation in 107 former smokers including 42 patients with mild to severe COPD. Consistent with our findings, Barr and colleagues found a statistically significant relation between flow-mediated vasodilation and FEV1; however, the corresponding mean flow-mediated vasodilation was much lower in Barr and colleagues’ report (flow-mediated vasodilation, 3.8% ± 3.1%) as opposed to the values in our work (flow-mediated vasodilation, 11% ± 3%). Noteworthy, the authors measured the flow-mediated vasodilation response to forearm occlusion in contrast to placing the cuff around the upper arm, as was done in our study. Studies have variably used either upper arm or forearm cuff occlusion, and there is no consensus as to which technique is more accurate or more precise. When the cuff is placed on the upper part of the arm, reactive hyperemia typically elicits a greater percentage of change in diameter compared with the change produced by placement of the cuff on the forearm (13). Because of these methodological differences, it is difficult to directly compare our findings with those by Barr and colleagues (10); however, the consistent relation observed between flow-mediated vasodilation and FEV1 in both studies suggests a link between vasodilator reactivity of the vasculature and COPD pathology. The precise mechanisms for this relation, however, are not clear. Smoking is a major risk factor for endothelial dysfunction and COPD. Studying current and former smokers without evidence of other cardiovascular risk factors, Celermajer and colleagues (33) demonstrated a dose-dependent inverse relation between pack-years of 13

Page 15 of 44

smoking and flow-mediated vasodilation. The authors further observed overall higher flowmediated vasodilation in former smokers compared with current smokers, suggesting some degree of reversibility of the adverse effects of cigarette smoke on the endothelium. This is why we sought to ensure abstinence from acute smoking and to adjust our results for packyears of smoking. Consistent with previous reports, however, we did not find a relation between smoking pack-years and systemic vascular abnormalities in patients with COPD (11,12). We therefore assume that factors other than smoking exert greater influences on systemic vascular function in patients with COPD. Increased systemic inflammation may have an effect on the relation between systemic vascular function and COPD (11). There is cumulating evidence that systemic inflammation in COPD may play an important role in the pathogenesis and prediction of cardiovascular disease (9, 34). In patients with COPD, one mechanism for this may be an increase in proatherogenic markers such as circulating levels of interleukin-6, fibrinogen, and C-reactive protein (1, 2, 7, 35). Among these, C-reactive protein has emerged as one of the most important predictors of myocardial infarction, stroke, and vascular death in several settings (36). C-reactive protein, however, is not only a marker but also a mediator of atherogenesis. C-reactive protein, at concentrations known to predict vascular disease, directly stimulates diverse early atherosclerotic processes including endothelial cell adhesion molecules (37) and macrophage low-density lipoprotein uptake (38). In addition, there have been recent reports of a direct effect of C-reactive protein on endothelial function (20, 21). Verma and colleagues (21) demonstrated downregulation of endothelial nitric oxide synthase in endothelial cells that had been incubated with C-reactive protein, resulting in a statistically significant reduction in basal and stimulated nitric oxide release. A reduction in vascular nitric oxide bioavailability, in turn, is associated with impaired endothelium-dependent vasodilation, one of the earliest detectable vascular changes before atherosclerotic plaque development (39).

14

Page 16 of 44

Several clinical reports support the observed relation between C-reactive protein and impaired flow-mediated dilation in our report (40-42). Studying 128 smokers without airflow obstruction, Verma and associates (40) observed a statistically significant relation between Creactive protein and vascular endothelial function. Studying 88 patients with peripheral arterial disease, Brevetti and associates (41) similarly observed that flow-mediated vasodilation correlated negatively with circulating concentrations of C-reactive protein and fibrinogen, but the authors found no association with traditional cardiovascular risk factors. These findings may suggest that the influence of systemic inflammation in conditions, including not only COPD but other chronic inflammatory diseases such as diabetes and rheumatoid arthritis, may exceed that of traditional risk factors in affecting endothelial function (43). The absence of a relation between flow-mediated vasodilation with pack-years of smoking, body mass index, and triglyceride and cholesterol levels reported here may support such a view. The lack of a correlation, however, also may be attributed to the study, which had a stringent selection process that basically excluded all patients with clinically or laboratory-diagnosed cardiovascular risk factors. We also identified age and sex as independent predictors of flow-mediated vasodilation, both in the total sample and in patients with COPD. These observations confirm previously reported age- and sex-related differences in the endothelium-dependent vascular response (44). The latter phenomenon may be explained by a steep decline in the female endothelial function commencing at around the time of menopause, thus suggesting a protective effect of estrogens on the vessel wall (45). Another important limitation of our study is the potential confounding effect of pulmonary hypertension in patients with COPD. Peinado and coworkers (46) reported impaired endothelial-dependent relaxation of the pulmonary arteries in resected lung specimens and suggested that this might contribute to the development of pulmonary hypertension in COPD. It is likely that some of our patients had secondary pulmonary 15

Page 17 of 44

hypertension; however, it would have had required catheterization of the right side of the heart to rule out pulmonary hypertension in these patients, because the sensitivity of lessinvasive diagnostic methods such as echocardiography in patients with COPD is rather low (47). In conclusion, we observed impaired endothelial-dependent and endotheliumindependent vascular function in patients with COPD compared with controls. The association between systemic inflammation, airway obstruction, and abnormal systemic vascular function may provide an explanation for the increased risk of cardiovascular disease in patients with COPD.

Acknowledgements: We are grateful to Ralf Zwick for his help in conducting the study. We would like to thank Prof. Celermajer for his helpful comments. We would further like to acknowledge Brigitte Konta and Wilhelm Frank for their assistance in statistical analysis. We appreciate the help by Prof. Bettelheim and the laboratory staff from the Otto-Wagner-Hospital.

16

Page 18 of 44

References 1. Fabbri LM, Rabe KF. From COPD to chronic systemic inflammatory syndrome. Lancet 2007;370:797-799. 2. Pinto-Plata VM, Müllerova H, Toso JF, Feudjo-Tepie M, Soriano JB, Vessey RS, Celli BR. C-reactive protein in patients with COPD, control smokers and nonsmokers. Thorax 2006;61:23-28. 3. Di Francia M, Barbier D, Mege JL, Orehek J. Tumor necrosis factor- levels and weight loss in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1994;150:1453-1455. 4. Schols AM, Buurman WA, Staal-van den Brekel AJ, Dentener MA, Wouters EFM. Evidence for a relation between metabolic derangements and increased levels of inflammatory mediators in a subgroup of patients with chronic obstructive pulmonary disease. Thorax 1996;51:819–824. 5. Donaldson GC, Seemungal TA, Patel IS, Bhowmik A, Wilkinson TM, Hurst JR, Maccallum PK, Wedzicha JA. Airway and systemic inflammation and decline in lung function in patients with COPD. Chest 2005;128:1995-2004. 6. Hurst JR, Perera WR, Wilkinson TMA, Donaldson GC, Wedzicha JA. Systemic, upper and lower airway inflammation at exacerbation of COPD. Am J Respir Crit Care Med 2006;173:71-78 7. Wedzicha JA, Seemungal TA, MacCallum PK, Paul EA, Donaldson GC, Bhowmik A, Jeffries DJ, Meade TW. Acute exacerbations of chronic obstructive pulmonary disease are accompanied by elevations of plasma fibrinogen and serum IL-6 levels. Thromb Haemost 2000;84:210-215. 8. Ross R. Atherosclerosis: an inflammatory disease. N Engl J Med 1999;340:115-126.

17

Page 19 of 44

9. Sin DD, Man SF. Why are patients with chronic obstructive pulmonary disease at increased risk of cardiovascular diseases? The potential role of systemic inflammation in chronic obstructive pulmonary disease. Circulation 2003;107:1514-1519. 10. Barr RG, Mesia-Vela S, Austin JH, Basner RC, Keller BM, Reeves AP, Shimbo D, Stevenson L. Impaired flow-mediated dilation is associated with low pulmonary function and emphysema in ex-smokers: the Emphysema and Cancer Action Project (EMCAP) Study. Am J Respir Crit Care Med 2007;176:1200-1207. 11. Sabit R, Bolton CE, Edwards PH, Pettit RJ, Evans WD, McEniery CM, Wilkinson IB, Cockcroft JR, Shale DJ. Arterial stiffness and osteoporosis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;175:1259-1265. 12. McAllister DA, Maclay JD, Mills NL, Mair G, Miller J, Anderson D, Newby DE, Murchison JT, Macnee W. Arterial stiffness is independently associated with emphysema severity in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;176:1208-14. 13. Corretti MC, Anderson TJ, Benjamin E, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Hermann M, Herrington D, Vallance P, Vita J, Vogel R. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol 2002;39:257-265. 14. Bonetti PO, Lerman LO, Lerman A. Endothelial dysfunction: a marker of atherosclerotic risk. Arterioscler Thromb Vasc Biol 2003;23:168-175. 15. Anderson TJ, Uehata A, Gerhard MD, Meredith IT, Knab S, Delagrange D, Lieberman EH, Ganz P, Creager MA, Yeung AC, et al. Close relation of endothelial function in the human coronary and peripheral circulations. J Am Coll Cardiol 1995;26:1235-1241.

18

Page 20 of 44

16. Neunteufl T, Katzenschlager R, Hassan A, Klaar U, Schwarzacher S, Glogar D, Bauer P, Weidinger F. Systemic endothelial dysfunction is related to the extent and severity of coronary artery disease. Atherosclerosis 1997;129:111-118. 17. Nyström T, Nygren A, Sjöholm A. Persistent endothelial dysfunction is related to elevated C-reactive protein (CRP) levels in Type II diabetic patients after acute myocardial infarction. Clin Sci 2005;108:121-128. 18. Vaudo G, Marchesi S, Gerli R, Allegrucci R, Giordano A, Siepi D, Pirro M, Shoenfeld Y, Schillaci G, Mannarino E. Endothelial dysfunction in young patients with rheumatoid arthritis and low disease activity. Ann Rheum Dis 2004;63:31-35. 19. Tarkun I, Arslan BC, Cantürk Z, Türemen E, Sahin T, Duman C. Endothelial dysfunction in young women with polycystic ovary syndrome: relationship with insulin resistance and low-grade chronic inflammation. Clin Endocrinol Metab 2004;89:5592-5596. 20. Verma S, Kuliszewski MA, Li SH, Szmitko PE, Zucco L, Wang CH, Badiwala MV, Mickle DA, Weisel RD, Fedak PW, Stewart DJ, Kutryk MJ. C-reactive protein attenuates endothelial progenitor cell survival, differentiation, and function: further evidence of a mechanistic link between C-reactive protein and cardiovascular disease. Circulation 2004;109:2058-2067. 21. Verma S, Wang CH, Li SH, Dumont AS, Fedak PW, Badiwala MV, Dhillon B, Weisel RD, Li RK, Mickle DA, Stewart DJ. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 2002;106:913-919. 22. Eickhoff P, Kiss D, Kohansal R, Valipour A, Geyer K, Burghuber OC. Endothelial dysfunction in stable COPD – a link between systemic inflammation and cardiovascular morbidity [abstract]? Eur Respir J 2006;28:A756.

19

Page 21 of 44

23. Lekakis J, Papamichael C, Vemmos C, Stamatelopoulos K, Voutsas A, StamatelopoulosS. Effects of acute cigarette smoking on endothelium-dependent arterial dilatation in normal subjects. Am J Cardiol 1998;81:1225-1228. 24. Miller MR, Hankinson J, Brusasco V, Burgos F, Casaburi R, Coates A, Crapo R, Enright P, van der Grinten CPM, Gustafsson P, Jensen R, Johnson DC, MacIntyre N, McKay R, Navajas D, Pedersen OF, Pellegrino R, Viegi G, Wanger J. Series „ATS/ERS TASK FORCE“: Standardisation of spirometry. Eur Respir J 2005;26:319-338. 25. Reddy KG, Nair NR, Sheehan HM, Hodgson JM. Evidence that selective endothelial dysfunction may occur in the absence of angiographic or ultrasound atherosclerosis in patients with risk factors for atherosclerosis. J Am Coll Cardiol 1994;23:833-843. 26. Adams MR, Robinson J, McCredie R, Seale JP, Sorensen KE, Deanfield JE, Celermajer DS. Smooth muscle dysfunction occurs independently of impaired endothelium-dependent dilation in adults at risk of atherosclerosis. J Am Coll Cardiol 1998;32:123-127. 27. Curkendall SM, DeLuise C, Jones JK, Lanes S, Stang MR, Goehring E Jr, She D. Cardiovascular disease in patients with chronic obstructive pulmonary disease, Saskatchewan Canada cardiovascular disease in COPD patients. Ann Epidemiol 2006;16:63-70. 28. Sin DD, Wu L, Man SF. The relationship between reduced lung function and cardiovascular mortality: a population-based study and a systematic review of the literature. Chest 2005;127:1952-1959. 29. Anthonisen NR, Skeans MA, Wise RA, Manfreda J, Kanner RE, Connett JE; Lung Health Study Research Group. The effects of a smoking cessation intervention on 14.5-year mortality: a randomized clinical trial. Ann Intern Med 2005;142:233-239.

20

Page 22 of 44

30. Celermajer DS, Sorensen KE, Gooch VM, Spiegelhalter DJ, Miller OI, Sullivan ID, Lloyd JK, Deanfield JE. Non-invasive detection of endothelial dysfunction in children and adults at risk of atherosclerosis. Lancet 1992;340:1111-1115. 31. Holubkov R, Karas RH, Pepine CJ, Rickens CR, Reichek N, Rogers WJ, Sharaf BL, Sopko G, Merz CN, Kelsey SF, McGorray SP, Reis SE. Large brachial artery diameter is associated with angiographic coronary artery disease in women. Am Heart J 2002;143:802-807. 32. Zieman SJ, Melenovsky V, Kass DA. Mechanisms, pathophysiology, and therapy of arterial stiffness. Arterioscler Thromb Vasc Biol 2005;25:932-943. 33. Celermajer DS, Sorensen KE, Georgakopoulos D, Bull C, Thomas O, Robinson J, Deanfield JE. Cigarette smoking is associated with dose-related and potentially reversible impairment of endothelium-dependent dilation in healthy young adults. Circulation 1993;88:2149-2155. 34. Man SF, Connett JE, Anthonisen NR, Wise RA, Tashkin DP, Sin DD. C-reactive protein and mortality in mild to moderate chronic obstructive pulmonary disease. Thorax 2006;61:849-853. 35. de Torres JP, Cordoba-Lanus E, López-Aguilar C, Muros de Fuentes M, Montejo de Garcini A, Aguirre-Jaime A, Celli BR, Casanova C. C-reactive protein levels and clinically important predictive outcomes in stable COPD patients. Eur Respir J 2006;27:902-907. 36. Ridker PM. C-reactive protein and the prediction of cardiovascular events among those at intermediate risk: moving an inflammatory hypothesis toward consensus. J Am Coll Cardiol 2007;49:2129-2138. 37. Pasceri V, Willerson JT, Yeh ET. Direct proinflammatory effect of C-reactive protein on human endothelial cells. Circulation 2000;102:2165-2168.

21

Page 23 of 44

38. Zwaka TP, Hombach V, Torzewski J.C-reactive protein-mediated low density lipoprotein uptake by macrophages: implications for atherosclerosis. Circulation 2001;103:1194-1197. 39. Gewaltig MT, Kojda G. Vasoprotection by nitric oxide: mechanisms and therapeutic potential. Cardiovasc Res 2002;55:250-260. 40. Verma S, Wang CH, Lonn E, Charbonneau F, Buithieu J, Title LM, Fung M, Edworthy S, Robertson AC, Anderson TJ. Cross-sectional evaluation of brachial artery flow-mediated vasodilation and C-reactive protein in healthy individuals. Eur Heart J 2004;25:1754-1760. 41. Brevetti G, Silvestro A, Di Giacomo S, Bucur R, Di Donato A, Schiano V, Scopacasa F. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg 2003;38:374-379. 42. Tan KC, Chow WS, Tam SC, Ai VH, Lam CH, Lam KS. Atorvastatin lowers Creactive protein and improves endothelium-dependent vasodilation in type 2 diabetes mellitus. J Clin Endocrinol Metab 2002;87:563-568. 43. Sattar N. Inflammation and endothelial dyfunction: intimate companions in the pathogenesis of vascular disease? Clin Sci 2004;106:443-445. 44. Benjamin EJ, Larson MG, Keyes MJ, Mitchell GF, Vasan RS, Keaney JF Jr, Lehman BT,Fan S, Osypiuk E, Vita JA. Clinical correlates and heritability of flow-mediated dilation in the community: the Framingham Heart Study. Circulation 2004;109:613619. 45. Celermajer DS, Sorensen KE, Spiegelhalter DJ, Georgakopoulos D, Robinson J, Deanfield JE. Aging is related with endothelial dysfunction in healthy men years before the age-related decline in women. J Am Coll Cardiol 1994;24:471-476.

22

Page 24 of 44

46. Peinado VI, Barbera JA, Ramirez J, Gomez FP, Roca J, Jover L, Gimferrer JM, Rodriguez-Roisin R. Endothelial dysfunction in pulmonary arteries of patients with mild COPD. Am J Physiol 1998;274:L908-913. 47. Arcasoy SM, Christie JD, Ferrari VA, Sutton MS, Zisman DA, Blumenthal NP, Pochettino A, Kotloff RM. Echocardiographic assessment of pulmonary hypertension in patients with advanced lung disease. Am J Respir Crit Care Med 2003;167:735-740.

23

Page 25 of 44

Figure legends:

Figure 1. Selection flow chart for patients with stable COPD

Figure 2. Flow mediated dilation in patients with stable COPD, smoking and non-smoking controls without airflow obstruction

Figure 3a. Linear regression analysis between flow mediated dilation response and percent predicted forced expiratory volume in one second in patients with COPD (n = 60)

Figure 3b. Linear regression analysis between flow mediated dilation response and circulating C-reactive protein in patients with COPD (n = 60)

Page 26 of 44

Table 1. Clinical characteristics, lung function results, arterial blood gas analysis, and cardiovascular measurements of the study population Non-smoking Smoking

Patients

controls

controls

with COPD

n=20

n=20

n = 60

62 ± 11

59 ± 9

62 ± 8

35

40

55

25 ± 3

26 ± 3

25 ± 4

Current smokers, %

0

100

43*

Smoking history, pack-years

0

39 ± 23‡

66 ± 39*†

4.8 ± 2.1

7.7 ± 3.6‡

7.0 ± 2.6†

VC, % predicted

103 ± 16

104 ± 13

79 ± 17*†

FEV1, % predicted

101 ± 16

99 ± 12

41 ± 18*†

FEV1%VC, % predicted

84 ± 10

82 ± 8

43 ± 15*†

Arterial pO2, mmHg

84 ± 10

83 ± 7

66 ± 10*†

Arterial pCO2, mmHg

37 ± 4

36 ± 4

39 ± 5

Systolic blood pressure, mmHg

126 ± 10

121 ± 12

125 ± 13

Diastolic blood pressure, mmHg

75 ± 6

73 ± 9

76 ± 6

Mean blood pressure, mmHg

92 ± 6

89 ± 8

92 ± 8

Heart rate, bpm

72 ± 13

73 ± 16

87 ± 18*†

Clinical characteristics Age, yrs Male sex, % Body mass index, kg/m2

Expiratory carbon monoxide levels, ppm Lung function

Arterial blood gas analysis

Cardiovascular measurements

Data are shown as mean ± standard deviation. * p < 0.005 (p < 0.01 after Bonferroni correction) in comparisons of subjects with COPD versus smoking controls ; † p < 0.005 (p < 0.01 after Bonferroni correction) in comparisons of subjects with COPD versus non-smoking controls; ‡ p < 0.01 (p < 0.05 after Bonferroni correction) in comparisons of smoking versus non-smoking controls

Page 27 of 44

Table 2. Laboratory cardiovascular risk factors and systemic inflammatory markers in the study population

Non-smoking

Smoking

Patients with

controls

controls

COPD

n=20

n=20

n = 60

Total cholesterol, mg/dl

187 ± 23

196 ± 20

187 ± 26

Triglycerides, mg/dl

92 ± 38

105 ± 39

103 ± 33

HDL, mg/dl

82 ± 18

72 ± 21

77 ± 26

LDL, mg/dl

113 ± 37

118 ± 28

107 ± 34

90 ± 9

88 ± 9

92 ± 13

5.4 ± 0.2

5.3 ± 0.2

5.5 ± 0.4

7.2 (5.4 – 8.2) 382 (317 – 428) 2.0 (1.0 – 4.8) 1.0 (0.7 – 1.8)

7.5 (6.2 – 9.3) 367 (342 – 411) 2.0 (1.0 – 4.0) 2.1 (1.5 – 3.4)

7.9 (6.2 – 9.1) 426† (354 – 472) 4.0 (2.0 – 7.0) 2.5* (1.6 – 5.3)

Traditional cardiovascular risk factors

Glucose, mg/dl HbA1c, % Systemic inflammatory markers Leukocytes, x 103/UL Fibrinogen, mg/dl C-reactive protein, mg/l IL-6, pg/ml

Traditional cardiovascular risk factors are shown as mean ± standard deviation Markers of systemic inflammation are shown as median (interquartile range) Definition of abbreviations: HDL = high-density lipoprotein; LDL = low-density lipoprotein; HbA1c = hemoglobin A1c; CRP: C-reactive protein; IL-6: serum levels of interleukin-6 * p < 0.005 (p < 0.01 after Bonferroni correction) and



p < 0.05 (p = n.s. after Bonferroni

correction) in comparisons of subjects with COPD versus non-smoking controls

Page 28 of 44

Table 3. Vascular reactivity of the brachial artery in the study population Non-smoking

Smoking

Patients with

controls

controls

COPD

n=20

n=20

n = 60

Flow mediated dilation, %

19 ± 3

16 ± 2‡

11 ± 3*†

Nitroglycerine mediated dilation, %

29 ± 7

26 ± 7

22 ± 6**

Baseline brachial artery diameter, mm

3.28 ± 0.61

3.49 ± 0.57

3.64 ± 0.63**

FMD%/Baseline brachial artery diameter

0.21 ± 0.03

0.18 ± 0.02‡

0.15 ± 0.04*†

Baseline flow, m/sec

0.8 ± 0.2

0.8 ± 0.3

0.8 ± 0.2

Hyperemic flow, m/sec

1.7 ± 0.3

1.7 ± 0.4

1.6 ± 0.4

Nitrogen induced flow, m/sec

0.8 ± 0.2

0.8 ± 0.3

0.8 ± 0.2

Hyperemia, %

116 ± 34

98 ± 41

107 ± 47

* p < 0.005 (p < 0.01 after Bonferroni correction) in comparisons of subjects with COPD versus smoking controls; † p < 0.005 (p < 0.01 after Bonferroni correction) in comparisons of subjects with COPD versus non-smoking controls; ** p < 0.05 (p = n.s. after Bonferroni correction) in comparisons of subjects with COPD versus non-smoking controls; ‡ p < 0.01 (p < 0.05 after Bonferroni correction) in comparisons of smoking versus non-smoking controls

Page 29 of 44

Table 4. Correlation coefficients between flow-mediated dilation (%) with clinical characteristics, lung function, laboratory markers, and cardiovascular parameters in the study population All subjects (n = 100) r-value p-value

COPD subjects (n = 60) r-value p-value

Age, yrs

-0.261

0.008

-0.195

0.136

Body mass index, kg/m2

-0.002

0.986

-0.048

0.716

Smoking, pack-years (n = 80)

-0.163

0.149

-0.013

0.919

Inhaled corticosteroid use, yes/no

n.a.

n.a.

-0.183

0.161

Inhaled corticosteroid use, puffs/day

n.a.

n.a.

-0.064

0.625

FEV1, % predicted

0.609

< 0.001

0.302

0.024

FEV1%FVC

0.631

< 0.001

0.261

0.044

Leukocyte count (G/L)

-0.194

0.051

-0.233

0.075

C-reactive protein, mg/l

-0.383

< 0.005

-0.376

0.003

Interleukin-6, pg/ml

-0.294

< 0.005

-0.128

0.337

Fibrinogen, mg/dl

-0.170

0.096

-0.099

0.453

Cholesterol, mg/dl

0.078

0.444

0.247

0.059

Triglycerides, mg/dl

-0.140

0.168

0.023

0.861

Glucose, mg/dl

-0.138

0.174

0.053

0.691

Heart rate, beats/min

-0.270

0.005

-0.168

0.215

Systolic blood pressure, mmHg

-0.136

0.177

-0.198

0.129

Nitroglycerine mediated dilation, %

0.700

< 0.001

0.636

< 0.001

Brachial artery diameter, mm

-0.435

< 0.001

-0.494

< 0.001

Patient characteristics

Lung function parameters

Laboratory markers

Cardiovascular measurements

n.a. = not applicable

Page 30 of 44

Table 5. Multiple regression analysis of flow mediated dilation in patients with COPD n = 60 r2 = 0.699, adjusted r2 = 0.553 p < 0.001 Age, yrs

ß Coefficient -0.501

Standard Error 0.059

p-value < 0.001

Male sex

-0.533

1.097

0.001

Body mass index, kg/m

0.014

0.108

0.899

Brachial artery diameter, mm

-0.530

0.832

0.001

Smoking, pack-years

-0.096

0.011

0.386

FEV1, % predicted

0.225

0.022

0.046

Leukocyte count, G/L

-0.310

0.186

0.004

C-reactive protein, mg/l

-0.402

0.088

0.001

Interleukin 6, pg/ml

0.167

0.161

0.226

Fibrinogen, mg/dl

0.180

0.004

0.130

Cholesterol, mg/dl

0.195

0.019

0.128

Triglycerides, mg/dl

0.102

0.012

0.318

Blood glucose, mg/dl

0.306

0.035

0.014

Heart rate, beats/min

-0.218

0.025

0.079

Systolic blood pressure, mmHg

0.001

0.031

0.993

Inhaled corticosteroid use

-0.097

1.039

0.416

Long term oxygen therapy use

-0.116

0.925

0.320

2

Page 31 of 44

Figure 1.

1074 patients with COPD Age (< 40 or > 75 y) 900 BMI > 30kg/m2 Co-existing pulmonary disease(s)

734 700 Cardiovascular diseases 424

History of malignancy

Refusion, Death, Unavailable, Exacerbation < 4 months

217 190 60

Co-existing Diabetes a/o Chronic Renal Failure

Page 32 of 44

Figure 2.

Page 33 of 44

Figure 3a.

Figure 3b.

Definition of abbreviations: CI = confidence interval, PI = Prediction interval

Page 34 of 44

Determinants of systemic vascular function in Patients with Stable COPD Philipp Eickhoff, Arschang Valipour, Dora Kiss, Martin Schreder, Leyla Cekici, Kora Geyer, Robab Kohansal, and Otto Chris Burghuber

ONLINE DATA SUPPLEMENT

Page 35 of 44

Exclusion criteria: Exclusion criteria included a diagnosis and/or a history of conditions that could potentially affect measurements of endothelial function such as (listed in alphabetical order): anemia (hemoglobin < 11 g/dL), atopy, autoimmune diseases, cerebrovascular disease, chest pain on exertion, congestive heart failure, coronary heart disease, chronic renal failure (serum creatinine > 1.5mg/dl), diabetes, hypertension, liver disease, lung diseases (other than COPD), lung volume reduction procedures, lung transplantation, malignancy within the past 5 years, obstructive sleep apnea, oral corticosteroid use, peripheral artery occlusive disease, acute pulmonary embolism or revascularization within the past 24 months, and rheumatoid diseases. Subject with a previous diagnosis of asthma and/or evidence of reversible airflow obstruction, defined as an increase in the post-bronchodilator FEV1 > 200ml and > 15%, were excluded. Patients with COPD receiving long term oxygen therapy were not excluded. Long term oxygen therapy was prescribed in accordance with the Austrian guidelines of COPD management (E1).

Laboratory analysis Venous blood samples were taken for measuring total cholesterol, glucose levels, triglyceride, low- and high density lipoproteins, hemoglobin 1Ac, serum creatinine, full blood count, and systemic inflammatory markers such as C-reactive protein (CRP), fibrinogen and interleukin6 (IL-6). Blood counts were performed with an automated hematology analyzer (SE-9500, Sysmex Europe, Norderstedt, Germany). Plasma fibrinogen was determined according to the Clauss method (STA-R® Hemostasis System, Diagnostica Stago, Inc., Parsippany, USA) and results were expressed as mg/dl. CRP levels were assessed using an automated analyzer (Hitachi 917, Boehringer Mannheim, Germany) and results were expressed as mg/l. IL-6 was measured using high-sensitivity quantitative ELISA kits (Quantikine® HS Human IL-6 Immunoassay, range 0–10 pg/ml, intra- and inter-assay variation 6.9 and 9.6%, respectively, 1

Page 36 of 44

R&D Systems, Abingdon, UK) and results were expressed as pg/ml. Laboratory staff was blinded to clinical data.

Endothelium function of the brachial artery Endothelium-dependent flow-mediated dilation (FMD) following reactive hyperemia and the endothelium-independent nitroglycerine-mediated dilation (NMD) of the brachial artery were assessed using ultrasound according to the recommended guidelines (E2). Subjects were asked to refrain from alcohol and/or caffeine-containing beverages and strenuous exercise for at least 12 hours prior to the measurements. Smokers were asked to refrain from smoking for at least six hours prior to the study. Patients with COPD were asked to omit inhaled short-acting and/or long-acting beta-2-agonists for at least 6 and 12 hours prior to FMD measurements, respectively. Studies were always performed between 11 am and 2 pm, at least 4 hours after a light meal. All ultrasound examinations were performed in a quiet semi-darkened room, always by the same investigator who was blinded to clinical data (PE). Ultrasound measurements were performed with a 11.0 MHz linear array transducer with simultaneous ECG recording. Subjects rested on a comfortable bed in supine position for at least 15 min to stabilize cardiovascular parameters before starting the measurements. A sphygmanometer cuff was placed around the right upper arm and the arm was placed in a comfortable position for imaging the brachial artery. The brachial artery was imaged above the antecubital fossa in the longitudinal plane on a segment with clear vision of the vessel wall. The transducer was then put in a probe holding device after optimal positioning on a gel pad. The diameter of the brachial artery from the anterior to the posterior intima was recorded. A baseline rest image was acquired, and blood flow was estimated by time-averaging the pulsed Doppler velocity signal obtained from a midartery sample volume. Thereafter, arterial occlusion was created by cuff inflation to suprasystolic pressure. The cuff was inflated to at least 50 mm Hg above systolic pressure to occlude arterial inflow for 5 minutes. This 2

Page 37 of 44

causes ischemia and consequent dilation of downstream resistance vessels via autoregulatory mechanisms. Subsequently the cuff was deflated, inducing a brief high-flow state through the brachial artery (reactive hyperemia) to accommodate the dilated resistance vessels. The resulting increase in shear stress causes the brachial artery to dilate. The longitudinal image of the artery was recorded continuously from 30 seconds before to 2 min after cuff deflation. A midartery pulsed Doppler signal was obtained upon immediate cuff release and within 15 seconds after cuff deflation to assess hyperemic velocity. After a rest of at least 10 minutes, another image was obtained to reflect reestablished baseline conditions. Then a single dose of sublingual nitroglycerine (0.8mg) was given to obtain the maximum vasodilator response, which serves as a measure of endothelium-independent vasodilation reflecting vascular smooth muscle function. Previous studies have suggested that the maximal increase in lumen diameter occurs approximately 45 to 60 s after peak reactive hyperemic blood flow (E3). Lumen diameters were therefore measured from one media-adventitia interface to the other at least 3 times at baseline, every 20 seconds after reactive hyperemia, and subsequent to the administration of nitroglycerin (E4). The maximum FMD and NMD diameters were taken as the average of the 3 consecutive maximum diameter measurements. Vasodilation was then calculated as the percent change in diameter over the baseline value. Changes in blood flow were quantified as the percent change in flow during hyperemia compared to baseline. To further account for differences in vessel diameter between the study groups, the ratio of flow mediated dilation and baseline brachial artery diameter was taken as an index of diameter independent impairment of vascular reactivity. Intra-observer variability for measurements of baseline diameter was 0.8 ± 0.1 %, 2.8 ± 2.1 % for FMD, and 2.5 ± 0.9 % for NMD.

References:

3

Page 38 of 44

E1. Block LH, Burghuber OC, Hartl S, Zwick H., Austrian Society for Pulmonary Diseases and Tuberculosis. Consensus concerning the management of chronic obstructive pulmonary diseases (COPD). Wien Klin Wochenschr 2004;116:268-78.

E2. Corretti MC, Anderson TJ, Benjamin E, Celermajer D, Charbonneau F, Creager MA, Deanfield J, Drexler H, Gerhard-Hermann M, Herrington D, Vallance P, Vita J, Vogel R. Guidelines for the ultrasound assessment of endothelial-dependent flow-mediated vasodilation of the brachial artery. J Am Coll Cardiol 2002;39:257-265.

E3. Corretti MC, Plotnick GD, Vogel RA. Technical aspects of evaluating brachial artery vasodilatation using high-frequency ultrasound. Am J Physiol 1995;268:H1397-H1404.

E4. Berger R, Stanek B, Hülsmann M, Frey B, Heher S, Pacher R, Neunteufl T. Effects of endothelin a receptor blockade on endothelial function in patients with chronic heart failure. Circulation 2001;103:981-986.

4

Page 39 of 44

Table E1. Clinical characteristics, lung function, arterial blood gas results, and cardiovascular measurements of patients with COPD according to GOLD stage of disease severity Patients with COPD GOLD stage (FEV1% predicted) II III IV (50 – 80%) (30 – 50%) (< 30%) n = 20 n = 20 n = 20

All patients with COPD n = 60

Clinical characteristics Age, yrs

61 ± 9

63 ± 7

63 ± 7

62 ± 8

35

50

60

55

Body mass index, kg/m

26 ± 3

24 ± 4

24 ± 4

25 ± 4

Pack years of smoking

65 ± 48

68 ± 39

66 ± 33

66 ± 39

Long term oxygen treatment, n

1/20

3/20

10/20*

14/60

Inhaled corticosteroid use, n

10/20

17/20

18/20*

45/60

20

20

20

60/60

534 ± 116

372 ± 136

297 ± 100*

380 ± 169

VC, % predicted

93 ± 16

74 ± 12

70 ± 14*

79 ± 17

FEV1, % predicted

63 ± 10

38 ± 5

23 ± 5*†

41 ± 18

FEV1%VC, % predicted

59 ± 10

43 ± 10

29 ± 6*†

43 ± 15

Arterial pO2, mmHg

68 ± 6

66 ± 9

64 ± 13

66 ± 10

Arterial pCO2, mmHg

37 ± 2

38 ± 5

40 ± 7

39 ± 5

Systolic blood pressure, mmHg

122 ± 13

127 ± 11

126 ± 15

125 ± 13

Diastolic blood pressure, mmHg

74 ± 7

78 ± 45

75 ± 7

76 ± 6

Mean blood pressure, mmHg

90 ± 8

94 ± 5

92 ± 9

92 ± 8

Heart rate, bpm

82 ± 16

89 ± 18

91 ± 18

87 ± 18

Male sex, % 2

Inhaled beta-agonist use, n 6-Minute-Walking-Distance, m Lung function

Arterial blood gas analysis

Cardiovascular measurements

Data are shown as mean ± standard deviation. * p < 0.01 in comparisons of subjects with COPD stage IV versus COPD stage II †

p < 0.01 in comparisons of subjects with COPD stage IVversus COPD stage III

5

Page 40 of 44

Table E2. Laboratory cardiovascular risk factors and systemic inflammatory markers in patients with COPD according to GOLD stage of disease severity Patients with COPD GOLD stage (range FEV1% predicted) II III IV (50 – 80%) (30 – 50%) (< 30%) n=20 n=20 n=20 Traditional cardiovascular risk factors Total cholesterol, mg/dl

195 ± 21

179 ± 33

187 ± 22

Triglycerides, mg/dl

103 ± 36

110 ± 32

96 ± 30

HDL, mg/dl

75 ± 25

76 ± 28

80 ± 26

LDL, mg/dl

115 ± 25

103 ± 48

102 ± 27

Glucose, mg/dl

94 ± 14

90 ± 14

94 ± 13

HbA1c, %

5.5 ± 0.4

5.5 ± 0.3

5.4 ± 0.4

8.4

7.8

7.5

(6.4 - 9.0)

(6.4 - 9.2)

(5.9 - 9.3)

389

435

427

Systemic inflammatory markers Leukocytes, x 103/UL

Fibrinogen, mg/dl

CRP, mg/l

IL-6, pg/ml

(327 – 497) (361 – 483) (355 – 472) 3.0

3.5

4.5

(1.3 - 7.0)

(2.0 - 6.0)

(2.3 - 8.0)

2.1

2.9

3.2

(1.0 - 4.0)

(1.8 - 4.6)

(1.9 - 7.7)

Traditional cardiovascular risk factors are shown as mean ± standard deviation Markers of systemic inflammation are shown as median (interquartile range)

Definition of abbreviations: HDL = high-density lipoprotein; LDL = low-density lipoprotein; HbA1c = hemoglobin A1c; CRP: C-reactive protein levels; IL-6: serum levels of interleukin-6

6

Page 41 of 44

Table E3. Vascular reactivity of the brachial artery in patients with COPD according to GOLD stage of disease severity Patients with COPD GOLD stage (range FEV1% predicted) II

III

(50 – 80%) (30 – 50%)

IV (< 30%)

n=20

n=20

n=20

FMD, %

13 ± 4

11 ± 4

11 ± 3

NMD, %

23 ± 6

21 ± 6

22 ± 6

Baseline brachialis diameter, mm

3.52 ± 0.63 3.71 ± 0.74 3.71 ± 0.54

Baseline flow, m/sec

0.8 ± 0.2

0.7 ± 0.2

0.8 ± 0.2

Hyperemic flow, m/sec

1.8 ± 0.5

1.5 ± 0.3

1.6 ± 0.3

Nitrogen induced flow, m/sec

0.8 ± 0.2

0.8 ± 0.2

0.7 ± 0.1

Hyperemia, %

95 ± 45

113 ± 50

115 ± 46

Definition of abbreviations: FMD = flow-mediated dilation; NMD = nitroglycerine-mediated dilation

7

Page 42 of 44

Table E4. Full enter model of multiple linear regression of Nitrogen Mediated Dilation (%) on different variables in patients with COPD

All subjects, n = 60 adjusted r2 = 0.340 for the full model p = 0.010 Age, yrs

ß Coefficient -0.258

Standard Error

p-value

0.116

0.106

-0.138

2.117

0.457

0.206

0.211

0.129

Baseline brachial artery diameter

-0.670

1.618

0.001

Smoking, packyears

0.055

0.022

0.694

FEV1% predicted

-0.062

0.042

0.649

Leukocyte count, G/L

-0.086

0.359

0.500

C-reactive protein, mg/dl

-0.142

0.174

0.346

Interleukin-6, pg/ml

0.268

0.314

0.118

Fibrinogen, mg/dl

-0.023

0.008

0.874

Cholesterol, mg/dl

0.120

0.038

0.446

Triglycerides, mg/dl

0.027

0.023

0.833

Blood Glucose, mg/dl

-0.078

0.069

0.600

Heart Rate, bpm

-0.134

0.049

0.379

Systolic blood pressure, mmHg

0.107

0.060

0.446

Inhaled corticosteroid use

-0.152

2.051

0.298

Long term oxygen treatment use

0.033

1.783

0.817

Male sex Body mass index, kg/m2

8

Page 43 of 44

Figure E1. Flow mediated dilation in patients with COPD according to disease severity based on GOLD criteria

9

Page 44 of 44

Figure E2. Linear regression analysis between flow mediated dilation response (%) and smoking history (pack-years) in patients with COPD (n = 60)

10