Professor Ben-Gary Harvey

Robert J Kaner

Abraham Sanders

Yael Strulovici-Barel

Renat Shaykhiev

Jacqueline Salit

Ruba S Deeb

Anja Krause

Thomas L Vincent

Francisco Agosto-Perez

Guoqing Wang

Charleen Hollman

Vignesh Shanmugam

Ahmad M Almulla

Hisham Sattar

Mai Mahmoud

Jason G Mezey

Steven S Gross

Michelle R Staudt

Matthew S Walters

Ronald G Crystal

QA1 Pulmonary Abnormalities in Young, Light-Use Waterpipe (Hookah) Smokers

Keywords: fumee, pumon waterpipe, smoking, lung ………59 3.1 Article 1: Pulmonary Abnormalities in Young, Light-use Waterpipe (Hookah) Smokers…………………………………………………………………… ...68 4.6 Implications………………………………………………………………………69 References……………………………………………………………………………….70 Appendices………………………………………………………………………………85

who introduced me to the world of lung function

First and foremost, I would like to express my deepest appreciation and thanks to my thesis supervisor, Professor Ronald Crystal, Chairman of the Department of Genetic Medicine at Weill Cornell Medical College, who has been my mentor for over a decade. I would like to thank you for having the faith in me, and always encouraging me to broaden my knowledge and expertise to so many different fields I never even considered possible. Your advice and support in both my research and career have been priceless and the opportunities you have given me were beyond my dreams. Your passion for research has incented me to be a better researcher myself, while enjoying every moment along the way, and for that I will always be grateful to you.

I would like to thank Professor Christian Auclair and Professor Patricia Lemarchand for agreeing to take the time and effort to serve as my jury members and make this journey come to an end. Special thanks to Professor Noel McElvaney and Professor Jeremie Arash Rafii Tabrizi who, in addition to serving as jury members, have also agreed to review my thesis in advance, and comment on it, contributing from their knowledge and expertise to improving the thesis. My deepest gratitude to Professor Jeremie Arash Rafii Tabrizi who helped orchestrate my PhD studies. Thank you for always being available and patiently and willingly advise me throughout this journey.

The studies described in this thesis could not have been made possible without the help of each and every member of the Department of Genetic Medicine at Weill Cornell Medical College. From recruitment of research subjects, to lung function screening, to sample collection, to analysis, every step was conducted by expert scientists who have shared their knowledge and taught me so much about the various aspects of the studies. I am grateful to all my colleagues desirable mommy time. I hope one day you appreciate this effort and understand how difficult it was for me to dedicate time for work, rather than being with you. You amaze me every day with your wisdom and beauty, and I am so fortunate to have you. I promise to have more time for you now. You are, by far, my biggest accomplishments, and I dedicate this degree to you three, my future.

Am Israel Chai

Longue Résumé

La Chicha qui sert à fumer du tabac parfumé est utilisé par des millions de personnes. Il y a peu de données sur les effets du chicha sur la santé, peu de régulation et les utilisateurs pensent que la chicha n'est ni addictif ni nocif. Nous avons émis l'hypothèse que la consommation même occasionnelle de la chicha chez le sujet jeune a des conséquences sur la biologie pulmonaire.

Nous avons ainsi comparé 21 sujets jeunes fumeur occasionnel de Chicha à un groupe de 19 non fumeur apparié pour le sexe et l'ethnicité. Les premières anomalies chez le fumeur de cigarette étant présent au niveau des cellules pulmonaires nous avons évalué plusieurs paramètres : (1) taux plasmatique de carboxyhemoglobine (CO), ( 2 LA capacité de diffusion du monoxyde de carbone qui est un paramètre lié à l'emphysème et aux pathologies des petites voies pulmonaires était affectée par l'utilisation de la chicha. Nos précédentes études avaient montré que chez les sujets fumeurs de cigarette la réduction de la capacité de diffusion malgré une spirométrie normale était associée à un risque de développer un BPCO. Nous avons ainsi évalué le risque de développer une BPCO chez le sujet fumeur avec spirométrie normale par des scanners haute résolution comparant des groupes avec capacité de diffusion diminuée (46) et normale (59). La réduction de la capacité de diffusion était associée à un risque élevé de développer une BPCO dans les 4 ans.

Par ailleurs les niveaux plasmatiques des microparticules endothéliales totales et apoptotiques était élevé dans le groupe d'étude. Dans une étude chez les sujets fumeurs de cigarette, nous avons comparé des non fumeurs (28) à des fumeurs sains (61) et des fumeurs BPCO (49) sur un an. Nous avons montré que le niveau de microparticules endothéliales apoptotiques étaient élevé en continu chez les sujets fumeurs sains et avec BPCO. Un sous groupe des fumeurs sains (17) et BPCO (18) a accepté d'arrêter de fumer. 12 mois après l'arrêt de la cigarette le niveau des microparticules endothéliales totale et apoptotique était retourné à la normale pour les fumeurs sains mais restait élevé chez les fumeurs BPCO. Ainsi le niveau élevé de ces microparticules indiquait des lésions persistantes et irréversibles des capillaires pulmonaires et pourrait servir à évaluer les fumeurs de chicha au long cours. Au total, l'utilisation occasionnelle de chicha chez le sujet jeune a des conséquence clinique et biologique pulmonaire en relation avec une diminution de la capacité de diffusion.

Certaines anomalies mises en évidence dans notre étude (diminution de la capacité de diffusion, Microparticules endothéliales) pourraient prédire la survenue de maladies pulmonaires chroniques obstructives.

Long abstract

Waterpipe, an instrument for smoking fruit-flavored tobacco, is used by millions of people worldwide. There is limited data on the health effects of waterpipe smoking, and no regulations to its use. We hypothesized that even young, light-use waterpipe smokers have abnormalities relevant to lung health. Based on the knowledge that the first abnormalities associated with cigarette smoking are in lung cells long before there are clinical abnormalities, we compared young, light-use waterpipe smokers to nonsmokers, using a variety of lung-related parameters, including: blood carboxyhemoglobin (CO) levels; cough and sputum scores; lung function; metabolites present in lower respiratory tract epithelial lining fluid (ELF); cell differentials and transcriptome of small airway epithelium (SAE); cellular composition of ELF; transcriptome of alveolar macrophages (AM); and levels of total and apoptotic endothelial microparticles (EMPs). Light-use waterpipe smokers displayed abnormalities in all parameters assessed. Compared to nonsmokers, waterpipe smokers had more cough and sputum, higher CO levels, reduced diffusing capacity for carbon monoxide (DLCO), abnormal ELF metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and AM transcriptomes, and elevated levels of total and apoptotic EMPs. DLCO, a lung function parameter linked to emphysema and small airway disease, was affected by light-use waterpipe smoking. The relevance of this comes from our studies that demonstrated, in a separate cohort of cigarette smokers with normal spirometry, that reduced DLCO predicted a high risk for developing chronic obstructive pulmonary disease (COPD), a leading cause of death worldwide. We assessed the risk for developing COPD, a clinical disorder characterized by a mixture of small airway disease and parenchymal destruction (emphysema), with a serial lung function in cigarette smokers with normal spirometry and no emphysema as assessed by HRCT, by comparing smokers with reduced DLCO vs normal DLCO. Despite having normal spirometry, cigarette smokers with reduced DLCO were at significantly higher risk for developing COPD within <4 years compared to those with normal DLCO i.e., the DLCO can be used to identify smokers at high risk for developing COPD, and could be a unique parameter in future studies to assess waterpipe smokers over time.

Plasma levels of total and apoptotic EMPs, indicative of pulmonary capillary endothelial apoptosis, were elevated in light-use waterpipe smokers. The possible importance of this observation was highlighted by a parallel study, where we assessed the stability and reversibility of EMP levels in nonsmokers, healthy cigarette smokers and COPD cigarette smokers at 4 time points over a period of 1 year. The levels of total and apoptotic EMPs remained high with continuous smoking in healthy and COPD cigarette smokers. A subset of the healthy cigarette smokers and COPD cigarette smokers agreed to quit smoking. Following smoking cessation for 1 year, total and apoptotic EMP levels returned to normal nonsmoker levels in healthy cigarette smokers but remained abnormally high in COPD cigarette smokers. High levels of circulating and apoptotic EMPs are indicative of persistent and irreversible destruction of pulmonary capillaries and may be another unique parameter to assess waterpipe smokers over time.

In summary, young, light-use waterpipe smokers have a number of lung clinical and biologic abnormalities compared to nonsmokers, including reduced DLCO, found to predict high risk for developing COPD in cigarette smokers, and elevated plasma levels of total and apoptotic EMPs, a marker of alveolar destruction, shown to be persistent and irreversible in COPD cigarette smokers despite smoking cessation. Together, these studies suggest that even light-use waterpipe smokers may be at risk for developing lung disease. Waterpipe, also known as narghile, hookah, shisha and hubble-bubble, is a device used to smoke tobacco. It dates back over 400 years to the Middle East [1][2][3] . There is regional variation in the size and appearance of the device, the type of tobacco smoked, the heating device, and the frequency the water in the device is changed 2,[4][5] . The waterpipe device that is mostly used is the one originating from the Middle East, consisting of a small bowl where a pre-made tobacco mixture with flavors or spices is placed and burned, by a lamp, coal or wood (Figure 1) 2,5 . The generated smoke is drawn through a pipe connecting the bowl to a base filled with water. The smoke then passes through a hose and mouth piece into the lungs. In the Indian waterpipe (Jajeer), the coal is in direct contact with the tobacco in the waterpipe device head, without the tin or silver foil separator that is used in the Middle Eastern waterpipe device 6 . In the Chinese waterpipe, the tobacco is directly lit without charcoal [7][8] .

List of Abbreviations

Global prevalence of waterpipe smoking

Waterpipe smoking was originally associated with Middle Eastern culture, but since the 1990s, migration, tourism, curiosity and the social media has facilitated its spread among populations in Europe, the United States and South America. The use of waterpipe is now considered a global epidemic, with millions of daily users worldwide, making it the second most popular form of tobacco use, after cigarettes [2][3][4]9 . The raising prevalence of waterpipe smoking is due to its low cost, the lack of waterpipe-specific smoking regulations, and its social acceptance.

Many users believe that waterpipe smoke is far less harmful and addictive than cigarette smoke because the smoke passes through water, which they presume acts as a filter [4][5][8][9][10] . The setting of waterpipe smoking in public places, such as bars or cafes, with one device shared by several smokers during long smoking sessions (45-60 min), makes it a form of socialization and entertainment that attracts youth and teens 2,[4][5] . Added flavors are used to attract smokers to the pleasant aroma and reduce irritation from the tobacco products accompanying the inhaled nicotine; therefore, the tobacco mixture used for waterpipe smoking a flavored tobacco product, a category that is not regulated 5 . Tobacco-related laws that do not explicitly reference waterpipe smoking, do not regulate waterpipe smoking in public, making it more accessible to the general public and specifically to adolescents 5,8 .

The common profile of waterpipe smokers includes adults and adolescents in Middle

Eastern countries, as well as school and university students who are descendants of Middle Eastern countries living in the West 2 . The Global Youth Tobacco Survey summarizing the smoking habits of half a million young teenagers (13 to 15 years old) from 95 countries found that, while cigarette smoking prevalence is stable or in decline, waterpipe smoking shows a rising trend. In Middle Eastern countries, as high as 60% of adolescents have tried waterpipe smoking in their lifetime, with the male gender being dominant, and with more women regularly smoking waterpipe than cigarettes as it is considered more acceptable 11 . In the US it is estimated that 10 to 20% of college student are current waterpipe smokers and that 41 to 48% have tried it.

Eleven to 17% of high-school students and 2 to 10% of middle school students are current waterpipe smokers 5 .

Waterpipe smoke components

There are many similar compounds in waterpipe tobacco and cigarette tobacco. Both contain high amounts of nicotine, tar and heavy metals, such as arsenic, chromium and lead, known carcinogens such as polycyclic aromatic hydrocarbons and naphthylamines, primary aromatic amines and carbon monoxide 1,4,[12][13][14] . Waterpipe smokers are exposed to higher concentrations of toxins than cigarette smokers due to the higher toxin concentration in the smoke itself and/or the mode of smoking, including the frequency of puffing, the depth on inhalation and the length of smoking session 1,4 . Studies assessing waterpipe smoking habits found that during one waterpipe smoking session, the smoker inhales 10 times more puffs and up to 200 times more smoke compared to one cigarette smoked. The waterpipe smoker, and those in the local milieu, are also exposed to high levels of second-hand smoke [3][4][5]9 . Despite the belief that most hazardous components are filtered by the water in the device, analyses of the water following a waterpipe smoking session showed that only 3% of the total metals and 5% of the nicotine are filtered out by the water while the rest passes through the water, as well as other volatile carcinogens and particles. These results did not vary by the flavor or the type of tobacco used [4][5]15 .

The combination of charcoal and tobacco is unique to waterpipe smoking. The charcoal burning temperature is twice as high as needed for cigarettes, exposing the waterpipe smoker to large quantities of combustion-generated toxicants in addition to the inhaled toxicants transferred from the tobacco itself 1,[4][5] . Approximately 90% of the carbon monoxide, >95% of the benzo(a)pyrene, and 75 to 92% of the polycyclic aromatic hydrocarbons compounds measured in the waterpipe smoke originate in the charcoal used to heat the tobacco 16 . Further, the added flavors expose the waterpipe smoker to various allergens and phenol and its derivatives, which are known to promote DNA mutations and cardiovascular diseases, in quantities up to 1,000 times greater than in smoke from a single cigarette. Finally, the use of a shared mouthpiece during smoking sessions can spread infectious diseases 5,[17][18] . Table I summarizes the toxicants of machine-generated waterpipe smoke compared to machine-generated cigarette smoke. Together, these studies indicate that, compared to cigarette smokers, the waterpipe smokers are exposed to high levels of toxins that penetrate the lungs much deeper 1,4,19 . However, a caveat to these is that the levels of toxicants were evaluated in the released smoke or water left in the waterpipe bowl, rather than measuring the levels reaching the smoker's lungs. In addition, the assessments were done on a machine-generated smoke programed to imitate waterpipe and cigarette smoking behavior rather than during real waterpipe and cigarette smoking [15][16][20][21][22] .

Table I. Comparison of Toxicant Yield in Waterpipe Smoke and Cigarette Smoke

Long-term and acute effects of waterpipe smoking on the respiratory system

A number of studies have assessed the long-term effects of waterpipe smoking on pulmonary function, cancer prevalence and other clinical symptoms in older (40 to 60 years old), heavy-use waterpipe smokers (30 to 60 waterpipe-year history), and mostly in waterpipe smokers who already have manifested disease 3,8,[23][24][25][26] . Long-term effects of heavy-use waterpipe smoking include increased symptoms of chronic bronchitis (frequent, productive cough), emphysema, a significant reduction in forced expiratory volume in 1 sec (FEV1) and FEV1/forced vital capacity (FVC) ratio, and a shorter 6-minute walk distance compared to nonsmokers. A few studies of older waterpipe smokers show an association between chronic obstructive pulmonary disease (COPD) and smoking the traditional Middle-Eastern or the Chinese waterpipe. An association with asthma and waterpipe smoking remains inconclusive 8,23 .

Animal models have demonstrated airway resistance, lung inflammation, and oxidative stress as possible mechanisms leading to the impaired lung function with prolonged exposure to waterpipe smoke 8,24 .

Meta-analysis reviews of lung function studies in older, heavy-use waterpipe smokers compared to nonsmokers and cigarette smokers showed reduced FEV1 and a trend to lower FVC and FEV1/FVC in heavy-use waterpipe smokers compared to nonsmokers 8,23,25 . There was no difference in lung function in heavy-use waterpipe smokers compared to cigarette smokers. Both cigarette smokers and heavy-use waterpipe smokers had similar prevalence of cough and sputum symptoms, though the symptoms appeared at an earlier age in the waterpipe smokers than in the cigarette smokers (Table II). However, the reviews concluded the quality of evidence in the studies was low with no standardized measure of frequency and length of waterpipe smoking sessions, the type of tobacco smoked, or the exposure to other toxins. Also, there was no distinction between nonsmokers and passive smokers 23,25 .

Studies conducted in long-term smokers of the Indian waterpipe showed high prevalence of severe respiratory symptoms compared to nonsmokers. Some of the lung function parameters correlated with the duration and amount of waterpipe smoking. Indian waterpipe smoking had a similar effect on the respiratory system as deep inspiration of cigarettes 26,28 .

A study conducted in China assessed the effects of active Chinese waterpipe smoking in men (>40 years old) and of passive Chinese waterpipe smoking in women compared to active and passive cigarette smokers and nonsmokers. The active waterpipe smokers demonstrated a higher prevalence of cough and sputum and a lower FEV1/FVC ratio compared to nonsmokers and cigarette smokers 7 . The passive waterpipe smokers had significantly worse lung function compared to nonsmokers and passive cigarettes smokers. Eighteen % of the active waterpipe smokers and 8% of the passive waterpipe smokers had emphysema on CT, compared to 5% of the cigarette smokers, 1% of the passive cigarette smokers and 0% of the nonsmokers 7 .

In a study of n=110 waterpipe-only smoking men (20 to 60 years old), none of the subjects had normal spirometry and many waterpipe smokers had an estimated lung age significantly higher than the chronological lung age 24 . Restrictive ventilator defects, usually associated with obesity and rare in cigarette smokers, were found in 14% of the waterpipe smokers (Table III) 24 . A recent study comparing young, waterpipe-only male smokers to nonsmokers found a significant reduction in FEV1, FEV1/FVC and forced expiratory flow (Table IV) 35 . There was also a significant reduction in fractional exhaled nitric oxide. While of interest, this study did not include a detailed history of waterpipe smoking (i.e., number of years smoked or number of sessions per week) or past or current exposure to other tobacco products. 35 Acute effects of waterpipe smoking measured in heavy-use waterpipe smokers immediately after a waterpipe session include increased respiratory rate, changes in the FVC, FEV1, FEV1/FVC and diffusion capacity of the lung for carbon monoxide (DLCO), and acute increase in carboxyhemoglobin levels (Table V) 8,27 .

Limitations of current studies

There is limited data on the long term health effects of waterpipe smoking. While cigarette smoking is a well-established risk factor for lung cancer, COPD and other disorders, there is need for more robust, longitudinal, well-designed studies of the potential health risks associated with waterpipe smoking 3 . Despite the growing prevalence of young adults smoking waterpipe, most studies assess the effects of heavy-use, long-term waterpipe smoking in older individuals, disregarding the health effects and clinical and biologic abnormalities associated with light-use, short-term waterpipe smoking.

Many of the studies of waterpipe smoking have limited details of the quantity and type of the tobacco used, exposure to other risk factors such as the use of other tobacco products, second-hand exposures to tobacco or other toxins, the frequency and length of the waterpipe smoking session or the number of years of smoking 5,[8][9] . Most of the spirometry studies of waterpipe smoking have low sample size and the subjects had other diseases affecting lung function 8 . In several studies evaluating the association of waterpipe smoking with cancer, almost all of the waterpipe smokers also smoked cigarettes or other tobacco products, making it difficult to isolate the effect of waterpipe smoking [START_REF] Tageldin | Distribution of COPD-related Symptoms in the Middle East amd North Africa: Results of the BREATHE Study[END_REF] .

Another complication in making conclusions about the health effects of waterpipe smoking is the lack of uniformity in the type of waterpipe smoked. Several studies of waterpipe smoking take place in China and India where the waterpipe devices are unique as the tobacco burns directly on the charcoal (India) or is directly lit (China). In contrast, in most other countries, the Middle Eastern device is used, where the tobacco is indirectly heated by the charcoal. Since the carcinogenic potential of the smoke may be related to the temperature achieved during the smoking session, the Chinese and Indian waterpipe likely have a higher carcinogen potential than the Middle Eastern waterpipe 6 . Another difference is the frequency the water in the bowl is changed: whereas in the Middle Eastern waterpipe the water is changed after each use, in the Indian and Chinese waterpipe, the water is only changed after several days of use, exposing the smoker to higher levels of toxins accumulating from smoking multiple sessions [6][7] .

Despite these limitations, there is enough evidence to suggest that waterpipe smoking has harmful health effects. In this regard, it has been suggested that the current knowledge should be used to educate the public and design intervention and research to help guide regulations to stop the epidemic of waterpipe smoking from spreading, to ban misleading information, and to limit access of youth and minors to waterpipe smoking 4,[8][9] . As recommended by the World Health Organization, there is need to identify and quantify with confidence the health effects associated with short-term and long-term waterpipe smoking and to explore the interaction of waterpipe smoking with other forms of smoking with standardized exposure methods 9,25 . In Turkey, one of the leading countries rectifying smoking-related laws to include waterpipe smoking, the public, and specifically school students, is being educated about the health risks associated with waterpipe smoking, leading to a significant reduction (65%) in waterpipe smoking prevalence within the past few years 10 .

In our study of the health effects associated with light-use, short-term waterpipe smoking in young individuals (Article 1), we assessed various clinical and biologic lung-related parameters of lung health, specifically in cells directly exposed to waterpipe smoke, in waterpipe smokers compared to nonsmokers. The results of the study add to the growing evidence of harmful effects of waterpipe smoking and specifically demonstrate the abnormalities associated with even light-use waterpipe smoking.

Chronic Obstructive Pulmonary Disease (COPD) 1.2.1 Definition

COPD is defined as a progressive airflow limitation that is not fully reversible with bronchodilators [START_REF] Mannino | Lung Function and Mortality in the United States: Data from the First National Health and Nutrition Examination Survey Follow-up Study[END_REF][START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF] . The airflow limitation or obstruction is caused by a mixture of small airway disease, caused by lesions that obstruct the small conductive airways, parenchymal destruction (emphysema), reducing the elastic recoil of the lung available to force air out of the lung, or both. The relative contribution of each characteristic varies among affected individuals [START_REF] Mannino | Lung Function and Mortality in the United States: Data from the First National Health and Nutrition Examination Survey Follow-up Study[END_REF][START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF] . The major cause of COPD is chronic inhalation of tobacco smoke. Prolonged exposure to cigarette smoke leads to a chronic inflammation, parenchymal tissue destruction, and disruption of normal repair and defense mechanisms (Figure 2) [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF] . These changes lead to defective mucociliary clearance and disruption of the epithelial barrier provided by the innate host defense system, causing air trapping and progressive airflow limitation, breathlessness, and excessive cough and sputum production among other symptoms [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF] . The airway obstruction affects the time constant for lung empting, measured by the air that can be expired in one second (FEV1), the forced vital capacity (FVC) and their ratio (FEV1/FVC). The levels of these parameters, measured by pulmonary function test are used to diagnose airway disease [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . COPD is defined by postbronchodilator FEV1/FVC ratio <0.7, while FEV1 % predicted is used to define severity and assess survival rate (Figure 3) [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . The Global Initiative for Chronic Obstructive Lung Disease (GOLD) defines mild COPD as GOLD I (FEV1≥ 80% predicted, moderate COPD as GOLD II (80% > FEV1 ≥ 50% predicted), sever COPD as GOLD III (50% > FEV1 ≥ 30% predicted) and very sever COPD as GOLD IV (FEV1<30% predicted) (Table VIII).

Prevalence of COPD

COPD is a global health issue, the 4 th leading cause of death worldwide and 3 rd in the United States [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . The risk for COPD is related to an interaction between genetic factors and environmental exposure, but is also affected by comorbid diseases [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF] . Tobacco smoke is the main risk factor for COPD and, in general, the longer people smoke, the higher the risk for developing COPD is. However, for unknown reasons, likely associated with genetics, only a minority of smokers has an excessive decline in FEV1 leading to COPD and individuals with similar smoking and exposure histories vary in the severity of their disease and response to intervention [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF] Spurzem | Pathogenesis of COPD[END_REF][START_REF] Rennard | Pathogenesis of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF][START_REF] Macnee | New Paradigms in the Pathogenesis of Chronic ObstructivE Pulmonary Disease I[END_REF][START_REF] Dahl | Markers of Early Disease and Prognosis in COPD[END_REF][START_REF] Rennard | Natural Histories of Chronic Obstructive Pulmonary Disease[END_REF] . Other risk factors include occupational hazards such as exposure to various dusts, chemicals, vapors and fumes, indoor or outdoor air pollution and infections [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF] . In the previous decades, the risk for developing COPD among cigarette smokers was estimated to be 15 to 20%, but recent estimates suggest a much higher proportion of smokers, develop COPD, in part due to the worldwide aging population [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF][START_REF] Lundback | Not 15 but 50% of Smokers Develop COPD?-Report from the Obstructive Lung Disease in Northern Sweden Studies[END_REF] . A longitudinal study following lung function in 8045 individuals from the general population for 25 years, found that at least 25% of the smokers developed COPD GOLD II and above while 30 to 40% of smokers developed COPD GOLD I and above [START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF] .

Treatment for COPD

The development of COPD usually takes decades [START_REF] Rennard | Pathogenesis of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Rennard | Natural Histories of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF] . The small airways, the first site with abnormalities associated with cigarette smoke, account for only 10-15% of total airway resistance, therefore small airway impairment might accumulate for many years with very little effect on lung function 60,71. Once COPD manifests, there is no therapy that modifies the longterm decline in lung function nor reduces mortality, and pharmacologic therapy can only reduce the symptoms, and improve the health status and exercise tolerance [START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF][START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF][START_REF] Rennard | Natural Histories of Chronic Obstructive Pulmonary Disease[END_REF] . Smoking cessation has a beneficial effect on lung function as measured by a reduction in the excessive decline in FEV1, mainly if done at an early age [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Rennard | Natural Histories of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF] . Smoking cessation at an older age usually occurs after COPD has manifested, and only has a minor effect on FEV1 level, with little or no impact on the long term incidence of COPD [START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF] . Regardless of the age of smoking cessation, the rate of decline in FEV1 is not fully reversible to that of never smokers (Figure 4) [START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF] . [START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF] Since even mild COPD is associated with increased mortality, early detection of COPD is important as it can lead to early therapeutic intervention, including smoking cessation, adequate treatment of the asthmatic component in some patients, and modification of risk factor such as exposure or prevention of complications in patients with established disease [START_REF] Mannino | Lung Function and Mortality in the United States: Data from the First National Health and Nutrition Examination Survey Follow-up Study[END_REF][START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF][START_REF] Anthonisen | Smoking and Lung Function of Lung Health Study Participants after 11 Years[END_REF] . Various studies assess clinical and genetic markers for identification of those smokers at high risk for developing COPD.

Our study assessing the risk for developing COPD among smokers with normal spirometry but reduced DLCO compared to smokers with normal spirometry and normal DLCO (Article 2) demonstrates the utility of this parameter as a tool for early detection of smokers at risk for developing COPD that can help contribute to early intervention.

Definition

DLCO is a measurement of the ability of oxygen transfer from the alveoli to the blood.

Carbon monoxide acts as a surrogate for oxygen, and is usually used in a test assessing this ability [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . The DLCO depends on the surface area of the pulmonary microvascular bed available for gas diffusion, the membrane conductivity, the diffusion properties of the alveolar capillary membrane, the binding of carbon monoxide to hemoglobin, the volume of hemoglobin in alveolar capillary blood, and other processes affecting these factors 76 . The DLCO is calculated as the accessible alveolar volume (VA) into which carbon monoxide (CO) is distributed and transferred across the capillary membrane X a rate constant for carbon monoxide removal from alveolar gas (kCO): DLCO = VA x kCO [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF] .

Measuring DLCO

Single-breath determination of DLCO measures the uptake of carbon monoxide from the lung over a breath-holding period evaluating the transfer of gas from the alveoli to the red blood cells [START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF] . The DLCO is a standard noninvasive test to assess the integrity of the alveolar capillary surface area [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF][START_REF] Pellegrino | Interpretative Strategies for Lung Function Tests[END_REF] . However, it is not routinely used in lung function assessment as it is expensive, and without expertise and experience, is difficult to reproduce [START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF][START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . There are big inter-lab differences in reported DLCO levels caused by differences in DLCO interpretation. Different labs use different methods for calculating DLCO based on variable references for predicted values, and some lack the adjustment for factors affecting the DLCO, such as hemoglobin and carboxyhemoglobin levels [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Gaensler | Attachment for automated single breath diffusing Capacity Measurement[END_REF] . In addition to various factors affecting the actual level of DLCO, the measured level can be biased due to sub-optimal testing caused by submaximal inspired volume, breath hold time and inspiration time.

Therefore, the DLCO test should be conducted under standardized conditions and by experienced technicians [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF][START_REF] Graham | Effects of Increasing Carboxyhemoglobin on the Single Breath Carbon Monoxide Diffusing Capacity[END_REF] . To minimize test variability, it is recommended that the maneuver is demonstrated to the subject ahead of time, and that other conditions, such as the subject's position, level of exercise, and room temperature are standardized [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . DLCO predicted values should be derived from reference individuals recruited from a similar population to that of the tested subjects, measured in a similar setting to that of the tested subjects [START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF] . Also, to prevent inaccurate measurements, at least two DLCO tests should be performed. The average of two acceptable measures that meet the repeatability requirement as detailed in the ATS/ERS guidelines should be reported as the result [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] .

Adjustment of DLCO levels for carbon monoxide and carboxyhemoglobin and levels

Most regression values for DLCO are derived from studies of groups of lifetime healthy nonsmokers, estimating the inspired carbon monoxide to be 1 to 2% and the hemoglobin level to be 14.6 gdL -1 in adult and adolescent males and 13.4 gdL -1 in adult females and male and female children <15 years old [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . In smokers, the carboxyhemoglobin level can be as high as 10 to 15% [START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF][START_REF] Wald | Carbon Monoxide in Breath in Relation to Smoking and Carboxyhemoglbin levels[END_REF] . Hemoglobin binds to carbon monoxide in a higher affinity than to oxygen, creating carboxyhemoglobin. In smokers, the hemoglobin will be tightly bounded by the excessive carbon monoxide from the cigarette smoke, reducing the overall amount of hemoglobin available for further binding by the fresh carbon monoxide transferred during the DLCO maneuver, leading to less uptake of carbon monoxide and therefore, reduced DLCO level measured [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Wald | Carbon Monoxide in Breath in Relation to Smoking and Carboxyhemoglbin levels[END_REF] . The effect is similar to that of having a reduced blood hemoglobin level, with less hemoglobin available for binding, and is often termed the "anemia" effect [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF][START_REF] Wald | Carbon Monoxide in Breath in Relation to Smoking and Carboxyhemoglbin levels[END_REF] . In addition, high levels of carboxyhemoglobin increase the backpressure assumed to be 0 during the DLCO maneuver, resulting in overestimation of the driving pressure for carbon monoxide across the air-blood barrier, leading to underestimation of the DLCO level [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . It is estimated that in individuals with carboxyhemoglobin >2%, for each 1% increase in carboxyhemoglobin, the DLCO measured is reduced by 0.8 to 1% [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . The backpressure is responsible for 58.5% and the anemia effect for 41.5% of this reduction in measured DLCO [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Neas | The Determinants of Pulmonary Diffusing Capacity in a National Sample of U.S. Adults[END_REF] .

To reduce carbon monoxide levels from cigarette smoke affecting DLCO levels measured in smokers, smokers are asked to refrain from smoking or from exposure to other carbon monoxide sources on the day of the test. However, since the time of the last cigarette or exposure may still vary, the measured DLCO level should be adjusted for carbon monoxide level resulting from recent and heavy smoking to compensate for both the back pressure and anemia effect [START_REF] Graham | Effects of Increasing Carboxyhemoglobin on the Single Breath Carbon Monoxide Diffusing Capacity[END_REF] . In addition, an adjustment of the measured DLCO level should be made in individuals with low hemoglobin levels [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] .

Factors affecting DLCO levels

The diffusing capacity measures the effectiveness of the alveolar volume and the rate of diffusion per unit volume (kCO); therefore, factors affecting either of these parameters would affect DLCO [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF] . A decreased alveolar volume that is associated with age and smoking status will be associated with a decline in FVC as well as a reduced DLCO level [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF] . In a study of 1635 never smokers, 775 former smokers and 1392 current smokers (healthy 25 to 74 years old men and women, of European and African origin), FVC was found to have the highest correlation with DLCO (r=0.53), followed by height (r=0.45) [START_REF] Martinez | Sex Difference in Sever Pulmonary Emphysema[END_REF] . After adjusting for gender, race and height, DLCO was found to decrease by 0.5% each year in never smokers. DLCO was found to be lower in women but this effect disappeared when adjusting for height, therefore it might only demonstrate a physiologic difference [START_REF] Martinez | Sex Difference in Sever Pulmonary Emphysema[END_REF] . In young adults, the kCO is the same in both genders but declines with age at a slower rate in young women than in young men, until the age of 47, when the rate of decline is similar in both genders [START_REF] Martinez | Sex Difference in Sever Pulmonary Emphysema[END_REF][85] .

In both males and females, former smokers had lower DLCO levels than never smokers, correlating with pack year history (Figure 5). Current smoking status had a smaller effect on DLCO levels in women, correlating with pack year history and packs per day, with DLCO levels reducing more rapidly than FVC levels (Figure 6). However, the decline in DLCO among smokers might be due to carbon monoxide exposure from the latest cigarette smoked and not a physiological change, as no adjustment for carbon monoxide levels was done to the DLCO measured level [START_REF] Martinez | Sex Difference in Sever Pulmonary Emphysema[END_REF] . In comparison to European, Africans had lower DLCO levels but the changes in DLCO levels associated with age and height were similar in both races.

The use of DLCO level as a diagnostic tool

Reduced DLCO leads to inadequate oxygen levels when there is need for more oxygen (for instance during exercise) as the lung will not have sufficient gas exchanging surface to meet the demand [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF] . A DLCO % predicted is calculated to assess the function of the lower respiratory tract by comparing the measured DLCO level to a predicted DLCO level derived from reference individual levels and is used in the differential diagnosis of airway obstruction.

DLCO % predicted between the lower limit of normal and 60% is considered a mild reduction, DLCO % predicted between 40 and 60% a moderate reduction and DLCO % predicted <40% a severe reduction [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF] . A reduction in DLCO levels is observed in a variety of pulmonary disorders affecting the lower respiratory tract, while an elevated DLCO level is associated with asthma, obesity and intrapulmonary hemorrhage (Table IX) [START_REF] Gaensler | Attachment for automated single breath diffusing Capacity Measurement[END_REF] . In patients with long-term smoking history and evidence of airway obstruction, normal DLCO suggests chronic bronchitis and reduced DLCO suggests emphysema [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF] . Importantly, the DLCO is more sensitive than spirometry to

Isolated reduced DLCO

Normal spirometry with a reduced DLCO level in a cigarette smoker is usually associated with early small airway disease and/or emphysema, although the reduction can result from anemia, pulmonary vascular disorder, or early interstitial lung disease [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF] . High resolution chest tomography (HRCT) in patients with isolated reduced DLCO helps discriminate various patterns of complex mixed obstructive and restrictive abnormalities [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF][START_REF] Desai | Quantification of Emphysema: a Composite Physiologic Index Derived from CT Estimation of Disease Extent[END_REF][START_REF] Coxson | A Quantification of the Lung Surface Area in Emphysema using Computed Tomography[END_REF][START_REF] Hogg | Chronic Obstructive Pulmonary Disease -Part 2: Pathology and Biochemistry of Emphysema[END_REF] . In a study of 27 individuals with reduced DLCO (<70% predicted), 48% had emphysema on HRCT. In 85% of those, the reduction was associated with a restrictive lung process. The other 52% had interstitial lung disease, pulmonary vascular disease or other isolated findings [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF] . The importance of a reduced explored, and there are no large studies of subjects with isolated DLCO [START_REF] Crapo | Single-breath Carbon Monoxide Diffusing Capacity[END_REF] .

Our study assessed the risk for developing COPD among smokers with normal spirometry, no emphysema on HRCT, but with isolated reduced DLCO compared to smokers with normal spirometry, no emphysema on HRCT and normal DLCO (Article 2). The data demonstrated the importance of DLCO as a marker for early detection of COPD. While the measurement of DLCO is not routine for technical and financial reasons, these results advocate the need to develop an easier to use technique that will allow to follow DLCO levels in cigarette smokers and waterpipe smokers with normal spirometry who are falsely presumed to be normal and help reduce the prevalence of COPD.

Endothelial Microparticles

Microparticles

Microparticles are small (<1.5 um) vesicular fragments released from the membrane of various cell types in response to injury, activation or apoptosis [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Jimenez | Endothelial Cells Release Phenotypically and Quantitatively Distinct Microparticles in Activation and Apoptosis[END_REF][START_REF] Horstman | Endothelial Microparticles as Markers of Endothelial Dysfunction[END_REF][START_REF] George | Microparticles in Vascular Disease[END_REF] . The release, or vesiculation, of these microparticles is caused when a cell membrane loses its normal phospholipid asymmetry, leading to an increase of phosphorylation on the outer layer and bledding of the membrane causing microparticle formation and shedding by exocytic budding [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Brogan | Endothelial and Platelet Microparticles in Vasculitis of the Young[END_REF] . Though there are several mechanisms suggested (Figure 8), the formation of microparticles is not yet completely understood [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Ayers | Dynamic Microvesicle Release and Clearance within the Cardiovascular System: Triggers and Mechanisms[END_REF][START_REF] Boilard E | The Diversity of Platelet Microparticles[END_REF] . Typically, there is low grade cell activation caused by normal cell turnover leading to low levels of circulating microparticles found in the blood. However, cellular response to a variety of injury stimuli leads to the generation of microparticles, in some circumstances as a form of defense against sub-lethal complement attack, allowing the cells to shed complement components from the surface [START_REF] Jimenez | Endothelial Cells Release Phenotypically and Quantitatively Distinct Microparticles in Activation and Apoptosis[END_REF][START_REF] Piccin | Circulating Microparticles: Pathophysiology and Clinical Implications[END_REF] . This response to stimuli leads to high levels of microparticles circulating in peripheral blood originating from the plasma membrane of diverse activated or apoptotic cells of platelet, leukocyte or endothelial origin. The cell type from which these microparticles are released and the type of stimuli that released them (cell activation or apoptosis) can be traced by the specific phospholipids and oxidized lipids and the diverse proteins expressed on their membrane [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Boilard E | The Diversity of Platelet Microparticles[END_REF][START_REF] Piccin | Circulating Microparticles: Pathophysiology and Clinical Implications[END_REF] .

Endothelial Microparticles

The alveolar tissue is composed of 3 major cell types: type I alveolar epithelial cells, responsible for maintenance of the alveolar structure; type II alveolar cells, the major source of surfactant and the progenitors of type I alveolar epithelial cells; and endothelial cells, lining the capillaries. Together, the type I cells and endothelial cells modulate gas diffusion between the alveoli and blood [START_REF] Morissette | Alveolar Epithelial and Endohtleial Cell Apoptosis in Emphysema: What We Know and What We Need to Know[END_REF][START_REF] Barkauskas | Type 2 Alevolar Cells are Stem Cells in Adult Lung[END_REF] . Maintenance of an intact monolayer endothelial cell barrier is crucial for normal vascular structure [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Townsley | Structure and Composition of Pulmonary Arteries, Capillaries and Veins[END_REF] . An intact endothelium monolayer ensures homeostasis by antiinflammatory, anti-thrombotic and anti-atherogenic properties [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Morissette | Alveolar Epithelial and Endohtleial Cell Apoptosis in Emphysema: What We Know and What We Need to Know[END_REF] . The functional integrity of the vascular endothelium is maintained by continuous regeneration of the endothelial cell layer and the incorporation of endothelial progenitor cells [START_REF] Piccin | Circulating Microparticles: Pathophysiology and Clinical Implications[END_REF][START_REF] Morissette | Alveolar Epithelial and Endohtleial Cell Apoptosis in Emphysema: What We Know and What We Need to Know[END_REF] . Under normal conditions, the basal replication rate is 0.1% per day and the release of endothelial microparticles (EMPs), vesicular fragments shed from the endothelial cell membrane, is low grade, local and reversible [START_REF] Ayers | Dynamic Microvesicle Release and Clearance within the Cardiovascular System: Triggers and Mechanisms[END_REF][START_REF] Piccin | Circulating Microparticles: Pathophysiology and Clinical Implications[END_REF][START_REF] Barkauskas | Type 2 Alevolar Cells are Stem Cells in Adult Lung[END_REF] .

However, in response to stimuli, the endothelial cells transform to an over pro-coagulant and pro-inflammatory state, releasing higher levels of EMPs [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Jimenez | Endothelial Cells Release Phenotypically and Quantitatively Distinct Microparticles in Activation and Apoptosis[END_REF] . Heterogeneous EMPs may be released from endothelium disturbed by different types of injury and thus, high levels of circulating EMPs are a biologic marker of dysfunctional endothelium and quantification of EMPs in plasma can provide useful information on endothelial cell status [START_REF] Ayers | Dynamic Microvesicle Release and Clearance within the Cardiovascular System: Triggers and Mechanisms[END_REF][START_REF] Barkauskas | Type 2 Alevolar Cells are Stem Cells in Adult Lung[END_REF]100 .

Many factors can injure the endothelium, leading to EMP release, including: inflammation, modification of blood flow, drug toxicity, HIV infection, release of proliferative cytokines and autoimmunity [101][102][103][104] . Elevated levels of circulating EMPs have been reported in vascular diseases, acute coronary syndromes, severe hypertension, metabolic syndrome, type 2 diabetes, end-stage renal disease, pulmonary arterial hypertension, atherosclerosis, heart failure, thrombotic, thrombocytopenic purpura, lupus, multiple sclerosis, sickle cell disease and Other diseases [START_REF] George | Microparticles in Vascular Disease[END_REF][START_REF] Brogan | Endothelial and Platelet Microparticles in Vasculitis of the Young[END_REF][104][105][106][107] . Elevated plasma levels of EMPs have also been reported in multiple pro-inflammatory and pro-thromobotic states in asymptomatic individuals, and the quantification of their levels has been used to predict subclinical atherosclerosis burden in individuals with cardiovascular risk factor, and as a predictor of the recurrence of myocardial infraction or death in patients with acute coronary syndromes [START_REF] Ayers | Dynamic Microvesicle Release and Clearance within the Cardiovascular System: Triggers and Mechanisms[END_REF][START_REF] Boilard E | The Diversity of Platelet Microparticles[END_REF]102 .

Increasing evidence suggest that microparticles not only represent passively released cellular debris, but may also contribute to intercellular signaling mechanisms [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF]108 . EMPs have diverse effects on coagulation, as well as on leukocytes, platelets and endothelium that could contribute to the pathogenesis of an acute vascular injury 103 . EMPs can impair vascular function and initiate atherosclerosis by promoting endothelial dysfunction and arterial wall inflammation and contribute to plaque progression and rupture [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF] ; modulate inflammation via leukocyte activation and transendothelial migration 109 ; decrease release of nitric-oxide by endothelial cells; and increase arterial stiffness 110, suggesting that EMPs may also be mediators of disease, not just a marker of vascular injury [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF]108 .

Endothelial microparticles and smoking

Active smoking is established as a cause of endothelial dysfunction and alteration of the biology of endothelial cells [START_REF] Morissette | Alveolar Epithelial and Endohtleial Cell Apoptosis in Emphysema: What We Know and What We Need to Know[END_REF][111][112][113][114][115] . Second-hand smoke has also been shown to provoke dysfunctional endothelial cells and an increase in EMP levels 116 . This endothelium injury is one of the earliest pathological effects of cigarette smoking [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF]114,117 . Cigarette smoke may affect the endothelium by inducing vascular endothelial growth factor (VEGF), a specific growth factor for endothelial cells which induces cell migration and tube formation 112 . In addition, cigarette smoke reduces endothelial nitric oxide release and surface integrin expression, probably due to excessive generation of reactive oxygen species, affecting normal tube formation and endothelial cell survival. Together, these mechanisms lead to loss of endothelium, resulting in the emphysema observed in COPD 111,114,[118][119] .

In healthy cigarette smokers and in COPD cigarette smokers there is an oxidant/ antioxidant imbalance in favor of oxidants [START_REF] Morissette | Alveolar Epithelial and Endohtleial Cell Apoptosis in Emphysema: What We Know and What We Need to Know[END_REF]120 . In the respiratory system, the pulmonary vascular endothelium detoxifies xenobiotics arriving through the airways such as those released in cigarette smoke 120 . Excessive xenobiotics exceeding the detoxifying capacity of pulmonary vascular endothelial cells will result in cell impairment, compromising the role of endothelial cells in detoxification which will lead to progression to COPD. Inflammation has an important role in the pulmonary vascular abnormalities detected in early stage of COPD 115,120 . In addition to the inflammatory and structural changes in peripheral airways and lung parenchyma, prominent changes also occur in the pulmonary circulation affecting lung microvessels and precapillary arterioles and might be an initiating event that promotes vessel remodeling and pulmonary hypertension in COPD patients 111,112 . Endothelial impairment promotes the progression of COPD and the progression of COPD may exacerbate the damage of endothelium 120 .

In a previous study we assessed plasma levels of total and apoptotic EMPs in nonsmokers, healthy cigarette smokers with normal spirometry and DLCO and healthy cigarette smokers with early lung destruction as assessed by normal spirometry and low DLCO, the physiologic correlate of emphysema 121 . While smokers with normal spirometry and normal DLCO had mild elevated levels of circulating and apoptotic EMPs, smokers with normal spirometry but low DLCO had marked increase of the levels. i.e., there is apoptosis-mediated loss of endothelium before any spirometric evidence of lung disease. Based on the knowledge that smoking is a major cause of COPD and that destruction of alveoli may be initiated, in part, by apoptosis of pulmonary capillaries, we assessed the levels of total and apoptotic EMP levels in a separate cohort of nonsmokers, healthy cigarette smokers with normal spirometry and normal DLCO and COPD cigarette smokers. The consistency of the EMP levels was assessed by measuring the levels in 4 time points over a period of 1 year. Lung function may improve and the rate of decline decrease after a COPD smoker quits smoking, however, airway inflammation persists despite the removal of stimulus [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF]115 . Therefore, we hypothesized that COPD smokers maintain high levels of apoptosis even after smoking cessation and followed the plasma levels of this parameter as an early disease biomarker, we followed plasma levels of total and apoptotic EMPs in nonsmokers (n=29), healthy cigarette smokers (n=61) and cigarette smokers with COPD (n=49) for 1 year at 4 time points (baseline, 3, 6 and 12 months). The effect of cigarette smoking and COPD on EMP levels was assessed by comparing plasma total and apoptotic EMP levels between the groups at each time point. To assess the consistency of the levels, total and apoptotic EMPs were compared at different time points within a group. The reversibility of the elevated total and apoptotic EMP levels measured in the healthy and COPD cigarette smokers compared to nonsmokers was assessed in a subset of the healthy cigarette smokers (n=17) and cigarette smokers with COPD (n=18) who quit smoking for 12 months by comparing the levels in those who quit smoking and those who continued smoking at each time point.

Materials and Methods

Enrollment

Individuals were recruited from the general population of the New York City metropolitan area by posting advertisements in local newspapers and websites. All individuals were evaluated at the Weill Cornell NIH Clinical and Translational Science Center and the Department of Genetic Medicine Clinical Research Facility using Institutional Review Boardapproved clinical protocols after giving informed consent. All individuals had their medical history taken and had a physical exam, complete blood count, biochemical profile, serum α1antitrypsin levels, HIV test, urine analysis, chest X-ray, EKG, and pulmonary function tests, including FVC, FEV1, FEV1/FVC, TLC and DLCO, all carried out under ATS guidelines [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF]122 .

Cough and sputum scores were evaluated based on self-reported history using the St. George's respiratory questionnaire 123 . Smoking assessment included self-reported smoking history including exposure to second-hand smoking or environmental exposure and current smoking status was confirmed using urine nicotine metabolite evaluation (ARUP laboratories, Salt Lake City, UT) 124 .

Pulmonary Function Test

Pulmonary function tests were done as previously described (DLCO paper). Briefly, individuals were instructed to refrain from smoking as of the night before the testing. Pulmonary function test included spirometry (FEV1, FVC, FEV1/FVC) before and after the administration of salbutamol (100 μg, 4 doses) [START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF] , lung volumes and DLCO (Viasys Healthcare, Yorba Linda, CA). The DLCO maneuver was carried out 2 to 4 times; the average of the best 2 trials was used.

The spirometry and DLCO curves of all pulmonary function tests for all individuals were validated based on ATS/ERS guidelines [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . As an additional quality control measure, pulmonary function tests were performed serially in several volunteers during the course of the study. The DLCO % predicted values were calculated using the Gaensler et al equation 124 , and corrected for hemoglobin and carboxyhemoglobin levels using ATS/ERS guidelines [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] .

Sample Collection and Processing

Small airway epithelium, epithelial lining fluid and alveolar macrophages

Small airway epithelium was collected by brushing 10 th to 12 th order bronchi 126 . The cells were removed from the brush by flicking it into 5 ml of ice-cold LHC8 medium (GIBCO, Grand Island, NY). A 0.5 ml aliquot was used to determine the number and types of cells recovered and 4.5 ml were immediately processed for RNA extraction. The origin of the recovered cells was confirmed as the small airway epithelium based on expression of genes encoding surfactant and club (Clara) cell secretory proteins 127 . Alveolar macrophages and epithelial lining fluid were collected by bronchoalveolar lavage and processed as previously described 127 .

RNA processing and quality control

An aliquot of the total RNA extracted from the small airway epithelium and alveolar macrophages was used to determine RNA integrity (Agilent Bioanalyzer, Agilent Technologies, Palo Alto, CA) and concentration (NanoDrop ND-1000 spectrophotometer, NanoDrop Technologies, Wilmington, DE). RNA was hybridized on HG-U133 Plus 2.0 microarrays with probes for >54,000 genome-wide transcripts, using Affymetrix protocols, hardware and software 128 . Microarray quality was verified by signal intensity ratio of GAPDH 3' to 5' probe sets ≤ 3.0 and multi-chip normalization scaling factor ≤ 10.0 129 .

Transcriptome analysis

For the microarray data, the MAS5 algorithm (GeneSpring version 7.3, Affymetrix Microarray Suite Version 5) was used to normalize the data per array to the median expression value of each sample. Genome-wide analysis was used to compare the expression of the small airway epithelium and alveolar macrophages in waterpipe smokers to nonsmokers and define a small airway epithelium and alveolar macrophage waterpipe-responsive genes lists using the following criteria: all probe sets expressed in at least 20% of the samples, an expression level fold change ≥ 1.5 and Benjamini-Hochberg corrected for multiple tests 130 p<0.05. The probe sets found to be differentially expressed between the groups were converted into unique and annotated genes using the Affymetrix site (www.affymetrix.com) and GeneCards (www.genecards.org) and functionally annotated using Gene Ontology and the Human Protein Reference Data Base (www.hprd.org).

Endothelial microparticles

Endothelial microparticles were collected, processed and quantified according to a standard operating procedure to eliminate variability in sample processing as previously described 121 . Briefly, blood was collected, processed within 1 hr and stained for the endothelial markers PECAM (CD31) and E-selectin (CD62E) and the constitutive platelet-specific glycoprotein Ib (CD42b) to differentiate endothelium-originated microparticles from plateletderived microparticles, which also express CD31. EMPs were defined as microparticles <1.5 μm in size, expressing CD31 or CD62E but not CD42b microparticles. Circulating EMPs are present in low levels in plasma of healthy subjects, reflecting normal endothelial turnover 131 . Total EMP levels above the nonsmoker total EMP mean level plus 2 standard deviations were considered abnormally elevated. To assess the presence of relative contribution of pulmonary capillary endothelium to the elevated EMP levels, CD42b‾CD31 + EMPs were co-stained with anti-human angiotensin converting enzyme (ACE) that is abundantly expressed on pulmonary capillary endothelium 117 . EMPs induced by apoptosis express the constitutive CD31 marker, whereas activation-induced EMPs express CD62E. Using this criteria, we assessed the ratio of CD42b‾CD62E + /CD42b‾CD31 + and EMPs with a low CD42b -CD62E + to CD42b -CD31 + ratio were defined as "apoptotic EMPs". The percentage of individuals with apoptotic EMPs with CD42b‾CD62E + /CD42b‾CD31 + ratio below the lowest ratio in healthy nonsmokers was quantified. EMP measurements were performed twice to ensure that the measurements were reproducible.

Metabolite Profiling

Bronchoalveolar lavage fluid was processed as previously described (Article 1).

Suspended metabolite extracts from lung epithelial lining fluid were analyzed by LC-MS in two different detection modes (positive and negative ion-monitoring). A Mass Profiler Professional analysis and the molecular formula generator algorithm were used to generate molecular features and score the molecular formulas.

Chest High Resolution Computed Tomography

HRCT scans were used to determine the percentage of lung affected by emphysema at attenuation -950 Hounsfield Units using the EmphylxJ software application (EmphylxJ, Vancouver, BC, Canada) [START_REF] Coxson | A Quantification of the Lung Surface Area in Emphysema using Computed Tomography[END_REF] . Emphysema was defined as >5% lung volume, a value derived from analyses of HRCT in normal nonsmoking individuals with normal lung function.

Smoking Cessation

All healthy cigarette smokers and COPD cigarette smokers were invited to stop smoking using a combination of varenicline and counseling for 3 months. Smoking status was assessed at baseline and after 3, 6 and 12 months and verified by urine nicotine metabolite levels.

Statistical Analysis

For comparison of numerical data (e.g., age, urine nicotine levels, relative gene expression and lung function) a 2-tailed Student's t-test was used. For comparison of categorical data (e.g., gender and ethnicity) a chi-square test was used, with Yates' correction for low number of subjects when applicable. A pairwise ANOVA was used to compare total and apoptotic plasma EMP levels between groups and at different time points within a group with no correction for multiple test as the number of tests was low (<21). A within-between ANOVA test was used to compare lung function at baseline and at the last visit within the normal spirometry/normal DLCO group and within the normal spirometry/low DLCO group. Gene expression levels were corrected for false discovery rate (Benjamini-Hochberg correction 130 ). An unpaired Student's t-test (targeted analysis) and a 1-way ANOVA (untargeted analysis, corrected for false discovery rate (Benjamini-Hochberg correction 130 ) were used to compare metabolite profile in waterpipe smokers and nonsmokers (Article 1). To assess if DLCO level can predict the development of COPD, a binomial logistic regression model was implemented in which the response was COPD status ("1"=developing COPD, "0" = not developing COPD). In addition, leave-one-out cross-validation was performed in order to assess the predictive accuracy.

Evaluation and fit of the logistic regression model was performed using the "nnet" and "ROCR" packages in the freely available R software 132-133 . Based on the knowledge that the first abnormalities associated with cigarette smoking are in lung cells long before there are abnormalities in clinical parameters such as lung function and lung imaging, we hypothesized that even light-use waterpipe smoking likely mediates abnormalities relevant to lung health. To asses this hypothesis we compared young, light-use waterpipe smokers to nonsmokers, matched for gender and ethnicity, using clinical parameters, including cough and sputum scores, lung function, and chest HRCT, and biologic parameters, including metabolites present in the lung epithelial lining fluid, small airway epithelial cell differentials and transcriptome, alveolar macrophage cellular composition and transcriptome, and plasma microparticle levels derived from pulmonary capillaries undergoing apoptosis. There were abnormalities in all parameters assessed in waterpipe smokers compared to nonsmokers, demonstrating that even light-use waterpipe smoking affects the biology of the human lung with evidence of early disease.

Results

Article 1: Pulmonary Abnormalities in

Objectives: To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers.

Methods: We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles.

Measurements and Main Results: Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles.

Conclusions: Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking 5

.

The waterpipe, also called hookah, shisha, or narghile, an instrument for smoking fruit-flavored tobacco, is used by millions of people worldwide (1-4). The tobacco is placed in a bowl surrounded by burning charcoal; when the smoker inhales, air is pulled through the charcoal and into the bowl holding the tobacco (3, 5). The resulting smoke is bubbled through water, carried through a hose, and inhaled. It includes volatilized and pyrolyzed tobacco products, equivalent in a single bowl waterpipe session over 45-60 minutes to one pack of cigarettes, together with carbon monoxide and charcoal components. In addition to nicotine and its metabolites, urinalyses of waterpipe smokers have identified a variety of compounds that overlap with, but also differ from, those of cigarette smokers (3, 6). While waterpipe smoking is commonly associated with the Middle East, the use of waterpipes is becoming more prevalent in the United States and worldwide (4, 5, 7). In the United States, 9-20% of young adults report that they have used waterpipes (5, 8), and waterpipe "bars" have become common in many U.S. cities, with increasing waterpipe use among young adults (4, 5). Many waterpipe smokers believe that the water filters out "toxins" from the smoke and that therefore the waterpipe is a safer smoking alternative to cigarettes (9, 10). Despite the increasing prevalence of waterpipe smoking, there is a paucity of data on the health effects of waterpipe smoking and there are no federal regulations regarding its use (5, 7, 11).

On the basis of knowledge that the first abnormalities associated with cigarette smoking are found in lung cells long before there are abnormalities in clinical parameters such as lung function and lung imaging (12-17), we hypothesized that even light-use waterpipe smoking for only a few years, exposing the smoker not only to tobacco smoke but also to the flavorings added to the tobacco and the volatile components of the heated charcoal surrounding the tobacco, likely mediates abnormalities relevant to lung health.

To assess this hypothesis, we compared young, light-use waterpipe smokers with nonsmokers matched for sex and ethnicity, using a variety of lung-related parameters, including (1) blood carboxyhemoglobin, cough and sputum scores, lung function, and chest high-resolution computed tomography (HRCT); (2) metabolites present in the lower respiratory tract epithelial lining fluid (ELF); (3) cell differentials and transcriptome of the small airway epithelium (SAE); (4) cellular composition of the ELF of the lower respiratory tract recovered by bronchoalveolar lavage (BAL) and transcriptome of alveolar macrophages (AMs); and (5) plasma levels of circulating endothelial microparticles (EMPs) derived from pulmonary capillaries undergoing apoptosis.

Some of the results presented in this article have been reported previously in the form of abstracts (18, 19).

Methods

Self-reported never smokers ("nonsmokers"; n = 19) and self-reported waterpipe-only smokers ("waterpipe smokers"; n = 21) were recruited from the general population in New York City by posting advertisements in local newspapers, electronic bulletin boards, and waterpipe bars. All subjects were evaluated at the Weill Cornell National Institutes of Health Clinical and Translational Science Center and Department of Genetic Medicine Clinical Research Facility using institutional review board-approved clinical protocols. All subjects were determined to be healthy on the basis of their medical history, physical examination, and detailed laboratory assessments (Table 1; for full inclusion and exclusion criteria, see METHODS section in the online supplement). Urine nicotine and cotinine levels were determined using liquid chromatography-tandem mass spectrometry (ARUP Laboratories, Salt Lake City, UT) (20). All subjects were recruited from the New York metropolitan area. The two study groups had similar environmental exposures; no subject had any industrial exposures; and only one nonsmoker and one waterpipe smoker had a history of exposure to secondhand cigarette smoke. Even though recruitment was open for all waterpipe smokers at least 18 years of age, the waterpipe smokers who volunteered were young and light-use waterpipe smokers, representative of the rise in waterpipe smoking prevalence in the young adult population in the United States.

All subjects underwent pulmonary function tests performed according to American Thoracic Society guidelines (21, 22), and their cough and sputum scores were based on the St. George's Respiratory Questionnaire (23). Chest HRCT was used to quantify emphysema (24). Pulmonary function and HRCT quantification are detailed in the METHODS section in the online supplement. The SAE, AM, and ELF samples were collected using fiberoptic bronchoscopy as previously described (25, 26). The metabolites in the lower respiratory tract ELF of waterpipe smokers and nonsmokers were compared in BAL fluid collected from a random subset of the nonsmokers (n = 5) and waterpipe smokers (n = 8). Total RNA was extracted from the SAE of all subjects, and AM samples were obtained from all nonsmokers and from 19 of the 21 waterpipe smokers (two missing samples due to technical issues during the collection procedure) using TRIzol reagent (Life Technologies, Carlsbad, CA) and RNeasy (RNeasy MinElute RNA Purification Kit; QIAGEN, Valencia, CA) and stored in Ambion RNAsecure reagent (Life Technologies) at 280 8 C. Total RNA processing on Human Genome U133 Plus 2.0 microarrays (Affymetrix, Santa Clara, CA), quality control, and analyses were performed as previously described (16). Processing of plasma EMPs as well as quantification and analysis of total EMPs (CD42b 2 CD31 1 ), pulmonary-derived EMPs (CD42b 2 CD31 1 ACE 1 ), and apoptotic EMPs (ratio of CD42b 2 CD62 1 to CD42b 2 CD31 1 ,2 SD below the average level in nonsmokers) were performed as previously described ( 27) and as detailed in the METHODS section of the online supplement.

Transcriptome Analyses

Transcriptome analyses were performed as detailed in the METHODs section in the online supplement. SAE and AM waterpipe-responsive gene lists were created with all genes differentially expressed in waterpipe smokers versus nonsmokers using the following criteria: genes expressed in at least 20% of subjects in each group with a fold change greater than or equal to 1. 5 Benjamini-Hochberg correction [28]). The number of differentially expressed waterpipe-responsive genes expressed outside the nonsmoker mean expression level (62 SD) divided by the total number of waterpipe-responsive genes was summarized as a percentage and calculated for each subject as a waterpipe transcriptome response score. For both the SAE and AM transcriptomes, the data were depicted (1) using principal component analysis (PCA), collapsing the expression levels of all probe sets present in at least 20% of the subjects' data into a set of linear variables (principal components [PCs]) that summarized the variability between the subjects, with the three components collapsing the largest variability between the groups displayed in a three-dimensional plot; (2) as an SAE and AM waterpipe response score of each subject; and (3) as a fold change of the average expression level in waterpipe smokers compared with nonsmokers of all SAE and AM waterpipe-responsive genes displayed in Gene Ontology functional categories.

Global Index Analysis

To summarize the differences observed in waterpipe smokers compared with nonsmokers, a global index was created that included cough and sputum scores, diffusing capacity of the lung for carbon monoxide (DL CO ), SAE PCs, SAE transcriptome response score, AM PCs, AM transcriptome response score, plasma apoptotic EMP levels, and SAE cell differentials. See the METHODS section of the online supplement for index calculations.

Statistical Analysis

For comparison of numerical data (e.g., age, urine nicotine levels, total and apoptotic EMP levels, relative gene expression, lung function, and percentage of emphysema in waterpipe smokers vs. nonsmokers), a two-tailed Student's t test was used. Gene expression levels were corrected for false discovery rate (Benjamini-Hochberg correction [28]). For comparison of categorical data (e.g., sex, ethnicity, and number of subjects with abnormal cough and sputum scores, low DL CO , or apoptotic EMP levels), a x 2 test was used with the Yates correction for low number of subjects when applicable. The differential metabolite profile of the lung ELF samples was assessed using MassHunter Profinder software (Agilent Technologies, Santa Cough and sputum scores were each evaluated on a scale of 0-4, where 0 = not at all; 1 = only with chest infections; 2 = a few days per month; 3 = several days per week; and 4 = most days of the week (23). ‡ Pulmonary function testing parameters are given as percentage of predicted value, with the exception of FEV 1 /FVC, which is reported as percentage observed.

x As a percentage small airway epithelium recovered.

k Alveolar macrophages were purified by adherence before transcriptome analysis (see METHODS section in the online supplement).

Clara, CA) and compared using an unpaired Student's t test (targeted analysis) and Agilent MPP software and one-way analysis of variance (untargeted analysis) corrected for false discovery rate (Benjamini-Hochberg correction [28]), as detailed in the METHODS section of the online supplement.

Results

The study population of nonsmokers and waterpipe smokers was comparable in terms of sex, ethnicity, body mass index, and alpha-1 antitrypsin levels (P . 0.3, all comparisons) (Table 1). The waterpipe smokers were younger than the nonsmokers (mean difference, 8 yr) (Table 1). In prior studies, we observed that there were no age-related modifications to cough and sputum scores, SAE cell differentials, DL CO levels, SAE and AM gene expression, or plasma EMP levels in nonsmokers (r 2 < 0.1, correlation of all parameters with age) (see METHODS section and Figure E1 in the online supplement) (16, 27). Waterpipe smokers smoked an average of 3.5 6 2.5 sessions per week for an average of 4.1 6 2.5 years. Carboxyhemoglobin levels were significantly higher in waterpipe smokers than in nonsmokers (P , 0.02).

Lung-related Clinical Parameters

Cough and sputum scores were significantly higher in waterpipe smokers than in nonsmokers (P , 0.008, both comparisons). Thirty-three percent of waterpipe smokers had an abnormal cough score (>2) compared with 5% of nonsmokers (P , 0.03), and 19% of waterpipe smokers had abnormal sputum production (>2) compared with 0% of nonsmokers (P , 0.04) (Table 1, Figure 1A). DL CO percentage of predicted value, corrected for hemoglobin and carboxyhemoglobin levels, was lower in waterpipe smokers than in nonsmokers (P , 0.04). None of the nonsmokers had a low DL CO level (,80% predicted and below the 95% range of normal DL CO calculated per subject based on sex, age, and height using a dataset comprising 405 healthy nonsmokers [29]).

In contrast, 38% of the waterpipe smokers had a low DL CO level (P , 0.009) (Figure 1B). The HRCT percentage of emphysema was not significantly different between the groups (P . 0.07).

Metabolite Analysis

Metabolic profiling provided quantification for 1,675 features in the lower respiratory tract ELF; of these, 31 features with significantly different abundance in waterpipe smokers versus nonsmokers were structurally identified (P , 0.05) (Table E1; see Figures E2A-E2F for examples).

Small Airway Epithelium

The number of SAE cells recovered and the percentage of total epithelial and inflammatory cells were comparable in waterpipe smokers and nonsmokers (P . 0.6, both comparisons) (Table 1). However, the SAE of waterpipe smokers had an altered cellular composition, with a higher percentage of secretory cells and intermediate cells and a lower percentage of ciliated cells and basal cells (P , 0.005, all comparisons). The SAE transcriptome of waterpipe smokers was significantly modified compared with that of nonsmokers, with a marked segregation of the groups based on the genome-wide PCA (Figure 2A). There were 212 probe sets representing 159 unique, annotated genes significantly different between waterpipe smokers and nonsmokers (Figure E3A). Of those, 35% were downregulated and 65% were upregulated ("SAE waterpipe-responsive genes") (Table E2).

The SAE waterpipe transcriptome response score, a measure of the number of SAE waterpipe-responsive genes differentially expressed in a subject, was significantly higher in waterpipe smokers than in nonsmokers (P , 10 212 ) (Figure 2B). Gene Ontology analysis of the categories of the SAE waterpipe-responsive genes showed a broad distribution dominated by genes related to metabolism, signal transduction, transcription, and transport (Figure 2C). Interestingly, while the SAE transcriptome of cigarette smokers is characterized by upregulation of many oxidative stress-related genes (13-17), very few genes in this category were upregulated in the SAE of waterpipe smokers (categorized as functional category "other" due to the low number of oxidant-related genes) (see DISCUSSION in the online supplement and Table E2).

Alveolar Macrophages

The cell differentials of the lower respiratory tract ELF (AMs, lymphocytes, neutrophils, eosinophils) recovered from the lower respiratory tract by BAL were not statistically different between the groups (P . 0.05, all comparisons), and the number of recovered AM cells was also comparable (P . 0.08) (Table 1). Genome-wide PCA of the AM transcriptome demonstrated a segregation ) and waterpipe smokers (n = 21 SAE and n = 19 AM samples) for all probe sets "present" in at least 20% of the samples in each group. (A-C) SAE gene expression. Differentially expressed probe sets (n = 212, representing 159 unique, annotated genes) identified using criteria of a fold change greater than or equal to 1.5 and P , 0.05 with the Benjamini-Hochberg correction (28) (see Table E2 for the complete SAE waterpipe-responsive gene list). (A) Principal component analysis (PCA). Shown are the first three principal components, representing the greatest variability among the groups. Each circle represents a subject, and all subjects in a of the two groups based on waterpipe smoking status (Figure 2D). Of the probe sets present in at least 20% of samples in each group, 239 probe sets representing 181 unique, annotated genes had significant differential expression between waterpipe smokers and nonsmokers (Figure E3B); 74% were downregulated and 26% were upregulated ("AM waterpipe-responsive genes") (Table E3), an opposite trend to that observed in the SAE.

As with the SAE transcriptome response score, the AM transcriptome response score was significantly higher in waterpipe smokers than in nonsmokers (P , 10 29 ) (Figure 2E). Gene Ontology analysis of the categories of the AM waterpipe-responsive genes showed a broad distribution; that is, as with the SAE, they were dominated by genes related to metabolism, signal transduction, transcription, and transport (Figure 2F). Among these downregulated genes were many linked to lung inflammation and host defense (see Discussion section in the online supplement and Table E3).

Endothelial Microparticles

Waterpipe smokers showed an increase in plasma total EMP levels compared with the nonsmokers (P , 0.04) (Figure 3A). On average, 77 6 8% of the plasma EMPs in the waterpipe smokers were of pulmonary origin (CD42b 2 CD31 1 ACE 1 ), a percentage comparable to that of nonsmokers (P . 0.1) (Figure 3B). The level of EMPs derived from apoptotic cells was increased in the waterpipe smokers compared with nonsmokers (P , 0.05), with 45% of waterpipe smokers having apoptotic EMPs (,2 SD) below the average level in nonsmokers compared with 0% of nonsmokers (P , 0.008) (Figure 3C). For global assessment of all parameters compared in waterpipe smokers and nonsmokers, see the RESULTS section in the online supplement and Figure E4.

Discussion

Despite the assumption among waterpipe users that smoking waterpipe is "safer" than smoking cigarettes (9, 10), evaluation of multiple lung components demonstrated a significant number of lung clinical and biological abnormalities in light-use, waterpipe-only smokers compared with healthy lifelong nonsmokers. The waterpipe smokers had increased cough and sputum scores and lower diffusing capacity, as well as biological abnormalities in several anatomic components in the lung, including (1) in the lower respiratory tract ELF, differentially present metabolites;

(2) in the SAE, the cell population where chronic obstructive pulmonary disease (COPD) and most lung cancers are initiated (30-33), disarray of the proportions of cell types, with increased numbers of secretory and intermediate cells and decreased numbers of ciliated and basal cells and an abnormal transcriptome;

(3) in AMs, the pulmonary representative of the mononuclear phagocyte system, functioning as the scavenger cell in the lower respiratory tract (34, 35), abnormal transcriptome; and (4) in the pulmonary capillary endothelium, an increased proportion of circulating apoptosis-derived EMPs (27, 36).

Clinical Consequences

The use of waterpipes to smoke tobacco is increasing worldwide, mainly among young adults and teens, reaching a global epidemic second only to cigarette smoking. Epidemiological studies suggest that 10-48% of adolescents and young persons in middle school, high school, or universities in the United States, Europe, and other countries admit to ever smoking waterpipes and that 10-35% admit to being current waterpipe smokers (2-5, 7). However, most studies of the long-term effects of waterpipe smoking on pulmonary function, cancer prevalence, and other clinical symptoms have studied older (ages 40-60 yr), heavy-use waterpipe smokers (30-60 waterpipe-year history), mostly in waterpipe smokers who already have disease manifestation (2, 4, 11, 37-39).

Researchers in a number of studies have assessed lung function in older, heavy-use waterpipe users and found evidence of reduced lung function parameters, including reduced FVC, FEV 1 , maximal midexpiratory flow, peak expiratory flow, forced expiratory flow, and midexpiratory phase levels, as well as FEV 1 /FVC, compared with nonsmokers, with a correlation between the duration and quantity of waterpipe smoking and the abnormalities of pulmonary function (3, 11, 37, 38). These older, heavy-use waterpipe smokers have a high frequency of cough and sputum compared with nonsmokers, and these symptoms appear at an earlier age than in cigarette smokers (37, 40, 41). An important observation in the present study is that a significant proportion of young waterpipe smokers with a history of fewer than four waterpipe sessions per week for less than 5 years have clinical abnormalities, including an increase in cough frequency and sputum production, and, strikingly, 38% have reduced diffusing capacity. The subgroup of waterpipe smokers with normal HRCT and normal spirometry but low DL CO are of interest, as we have recently demonstrated that cigarette smokers with the same clinical phenotype (normal HRCT and normal spirometry but low DL CO ) are at a sevenfold greater risk of developing COPD within 4 years than are those with the same phenotype but with normal DL CO (29).

Biological Changes

There have been a number of analyses identifying compounds that are inhaled in waterpipe smoke, likely placing a significant stress on lung biology (3, 6). Compared with one cigarette, one waterpipe session exposes the smoker to 2-4 times the amount of nicotine, 7-11 times the amount of carbon monoxide, 100 times more tar, Figure 2. (Continued). group are linked by a vector to a circle representing the average of the principal components in each group (green = nonsmokers, orange = waterpipe smokers). (B) Waterpipe transcriptome response score calculated on the basis of the percentage of the waterpipe-responsive genes each subject expressed outside the normal expression range, defined as mean (62 SD) expression in nonsmokers. P values were calculated using a two-tailed Student's t test. (C) Gene categories of all waterpipe-responsive genes. Fold change of mean expression of the waterpipe-responsive genes is compared with nonsmokers, presented on a log 2 scale. (D-F) AM gene expression. The AM data for (D-F) were created as described for the SAE. (D) PCA. (E) AM waterpipe transcriptome response score. (F) Gene categories. Differentially expressed probe sets (n = 239, representing 181 unique, annotated genes) were determined using criteria of a fold change greater than or equal to 1.5 and P , 0.05 with the Benjamini-Hochberg correction (28) (see Table E3 for the complete AM waterpipe-responsive gene list).

ORIGINAL ARTICLE 6

American Journal of Respiratory and Critical Care Medicine Volume jj Number jj | jj 2016 2-5 times the amount of high molecular weight carcinogenic polyaromatic hydrocarbons, and 3 times the amount of phenol (3). In addition, high levels of benzene, volatile aldehydes, and other toxins originating from flavoring have been detected in waterpipe smoke (3, 6). Consistent with the concept that at least some components of waterpipe smoke reach the lower respiratory tract, metabolomic profiling of lung epithelial fluid demonstrated a variety of metabolites in the lower respiratory tract ELF of waterpipe smokers, with a differential abundance compared with nonsmokers.

The SAE and AM transcriptomes of waterpipe smokers could easily be differentiated from those of nonsmokers, with hundreds of genes up-and downregulated, indicating potential dysregulation of these lung cell populations in response to waterpipe smoking. Waterpipe transcriptome response scores summarizing the waterpipe-modified gene effect on the SAE and AM transcriptomes distinguished not only light-use waterpipe smokers from nonsmokers but also waterpipe smokers with normal spirometry and normal DL CO from those with normal spirometry but reduced DL CO . For both the SAE and AMs, most of these dysregulated genes were metabolism, transcription, and signal transduction related, some of which were previously associated with the pathogenesis of COPD and/or cancer.

Interestingly, there was little overlap among the SAE genes dysregulated in waterpipe smokers compared with the overlap described for cigarette smokers, suggesting that the SAE pathologic phenotypes may be different from those induced in classic cigarette smoking-induced disorders. In this regard, the SAE of waterpipe smokers had an altered cellular composition with a pattern that combined features both similar to and distinct from those commonly observed in cigarette smokers. Similar to SAE changes in cigarette smokers, there was a decrease in the proportion of ciliated cells, the mediator of mucociliary clearance (42), as well as increased numbers of secretory cells resembling mucous cell hyperplasia, in smokers (43). These morphological alterations may be responsible for higher levels of cough and sputum scores observed in waterpipe smokers. However, in contrast to basal cell hyperplasia commonly observed in the airways of healthy smokers, in the SAE of waterpipe smokers there was a significant decrease in the proportion of basal cells, the stem/progenitor cell population of the airway epithelium (44). This was accompanied by an increased proportion of intermediate undifferentiated cells, which are basal cell-derived precursors of the differentiated cell populations (44). The decreased proportion of basal stem/progenitor cells in the airway epithelium is a rather unique phenotype, previously described only in bronchiolitis obliterans (45) and airway epithelial aging (46). This suggests that waterpipe smoking-induced changes in the SAE transcriptome may have important consequences with regard to the structural organization and maintenance of this anatomic compartment.

While the SAE transcriptome of cigarette smokers is characterized by upregulation of oxidative stress-related genes (13-17), very few genes related to this category were upregulated in the waterpipe smokers, suggesting that passage through water filters out many of the oxidants in waterpipe smoke. Interestingly, while the majority of differentially expressed genes in the SAE were upregulated, the majority of differentially expressed genes in the AMs were downregulated. However, similarly to its effect on the SAE transcriptome, waterpipe smoking induced a unique gene expression pattern in the AMs not previously reported to be evoked by cigarette smoking or other known modulators of the macrophage phenotype (47, 48). Among the downregulated genes were a variety of genes critical for inflammation and host defense functions (see DISCUSSION section in the online supplement for details regarding the specific SAE and AM dysregulated genes). In contrast to cigarette smokers, in whom there is a higher percentage of macrophages recovered compared with nonsmokers (48), there was no significant difference in the proportions of macrophages or other cell types recovered from the BAL of waterpipe smokers compared with nonsmokers, an observation that may be explained in part by the marked difference in the inhaled smoke composition of waterpipe versus cigarette smoke.

Endothelial cells respond to cell activation, injury, and/or apoptosis by shedding submicron membrane vesicles from their plasma membranes, known as EMPs (27, 49). Apoptotic loss of pulmonary capillaries occurs in association with cigarette smoking (50), and analysis of lung sections of individuals with COPD demonstrates increased DNA fragmentation and endothelial apoptosis in the pulmonary capillaries, representing early lung destruction (50, 51). We have previously shown that cigarette smokers undergo pulmonary endothelial apoptosis as measured by high levels of total EMPs and an increased proportion of apoptotic EMPs in their plasma (27). The observation that the total level of circulating EMPs and the proportion of apoptotic EMPs were significantly higher in waterpipe smokers than in nonsmokers suggests the possibility of ongoing lung capillary endothelial apoptosis associated with light-use waterpipe smoking.

Implications

The data we present regarding abnormalities in all clinical and lung-related biological parameters used to compare waterpipe smokers with nonsmokers suggests that even light-use waterpipe smoking in young individuals significantly affects lung biology and health. On the basis of this evidence in the context of the increasing use of waterpipe smoking, together with the accumulating evidence in the literature that older, heavy-use waterpipe smokers have loss of lung function compared with nonsmokers, our findings support efforts to regulate and reduce waterpipe smoking, especially among the young population, and to initiate large epidemiological studies on the harmful effects of waterpipe smoking. n

Article 2: Risk for COPD with Obstruction of Active Smokers with Normal

Spirometry and Reduced Diffusion Capacity

European Respiratory Journal 2015;46:1535

Abstract: Chronic obstructive pulmonary disease (COPD) caused preliminary by cigarette smoking, is defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) using spirometry, with normal post-bronchodilator considered "healthy". In assessing a cohort of 1570 active cigarette smokers, all with normal spirometry, we recognized that a subset had an abnormal diffusion capacity (DLCO), a parameter linked with emphysema and small airway disease. To determine if there is a difference in the risk for developing COPD as defined by the GOLD criteria between the "normal spirometry/low DLCO" and "normal spirometry/normal DLCO" phenotypes, we followed a randomly chosen group with normal spirometry/low DLCO and normal spirometry/normal DLCO with serial lung function over time.

The data reveled that despite appearing normal by GOLD post-bronchodilator spirometry criteria, cigarette smokers with normal spirometry but reduced DLCO are at significantly higher risk for the development of COPD. These results suggest that DLCO measurement could be an additional tool for early detection of smokers at risk for COPD, contributing to early intervention, and that large epidemiologic studies analyzing parameters relevant to "at risk" for COPD should use both spirometry and DLCO to allow for a correct interpretation of their studies.

Article 3: Persistence of Circulating Endothelial Microparticles in COPD Despite

Smoking Cessation

Thorax; In press

Abstract: The levels of plasma circulating endothelial microparticles (EMPs) derived from pulmonary capillaries undergoing apoptosis were previously shown to be elevated in smokers with normal spirometry but reduced diffusion capacity (i.e., can be used as a measure of active alveolar destruction). The levels of total and apoptotic EMPs were assessed in healthy cigarette smokers and cigarette smokers with COPD and the persistence of the EMP levels were followed in nonsmokers, healthy cigarette smokers and cigarette smokers with COPD for one year at 4 time points (0, 3, 6 and 12 months). To ask whether these biologic markers of pulmonary capillary endothelial apoptosis associated with smoking are reversible, we studied a subset of the healthy smokers and COPD cigarette smokers who quit smoking, with sampling before smoking cessation and then again after 3, 6 and 12 months following cessation.

The data replicated our previous observation in healthy cigarette smokers, extends it to COPD cigarette smokers and demonstrated that the elevated levels of total and apoptotic EMPs are stable over a period of 12 months in healthy cigarette smokers and COPD cigarette smokers who continued to smoke. In contrast, in healthy cigarette smokers who quit smoking, the plasma level of total and apoptotic EMPs return to the levels of nonsmokers and remain normal for 12 months following smoking cessation. Interestingly, however, and consistent with epidemiologic data of progression of COPD despite smoking cessation, in COPD cigarette smokers who quit smoking, the plasma levels of total and apoptotic EMPs do not return to the levels of nonsmokers, and remain abnormal even following 12 months of smoking cessation, likely reflecting continued lung endothelial injury.

ORIGINAL ARTICLE

Persistence of circulating endothelial microparticles in COPD despite smoking cessation

Yael Strulovici-Barel, 1 Michelle R Staudt, 1 Anja Krause, 1 Cynthia Gordon, 1 Ann E Tilley, 1 Ben-Gary Harvey, 1,2 Robert J Kaner, 1,2 Charleen Hollmann, 1 Jason G Mezey, 1,3 Hans Bitter, 4,5 Sreekumar G Pillai, 4,6 Holly Hilton, 4,7 Gerhard Wolff, 4,8 Christopher S Stevenson, 4 Sudha Visvanathan, 4,9 Jay S Fine, 4,9 Ronald G Crystal ABSTRACT Introduction Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. Methods Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b -CD31 + ), pulmonary capillary EMP (CD42b -CD31 + ACE + ) and apoptotic EMP (CD42b -CD62E + /CD42b -CD31 + ) levels were quantified by flow cytometry. Results Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. Conclusions Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. Trial registration number NCT00974064; NCT01776398.

INTRODUCTION

COPD, the third leading cause of mortality in the USA, is defined by the Global Initiative for Chronic Obstructive Lung Disease (GOLD) as a chronic lung disorder with airflow limitation that is not fully reversible. 1 There is overwhelming evidence that most cases of COPD are caused by cigarette smoking, with approximately 20% of smokers at risk for COPD if they continue to smoke. 2 The airflow obstruction that characterises COPD is caused by a variable mixture of small airway disease (bronchitis) and parenchymal destruction (emphysema). 1 3 Although the airway and alveolar diseases were classically considered as separate entities, it is now recognised that they usually coexist to variable degrees and are closely linked, with the parenchymal destruction evolving around areas of small airway disease. [3][4][5][6][7] There is increasing evidence that the pathogenesis of COPD is linked, in part, to apoptosis of pulmonary capillaries. [8][9][10][11] Consistent with this concept, we recently demonstrated that smokers and, to a greater extent, smokers with early evidence of lung destruction (normal spirometry, but low diffusing capacity (DLCO)) have elevated levels of circulating endothelial microparticles (EMPs). 12 Importantly, a significant proportion of these EMPs are derived from pulmonary capillaries and have characteristics of apoptotic EMPs, that is, they are derived from lung endothelial cells that have been induced to undergo apoptosis. [12][13][14]

Key messages

What is the key question?

▸ Is pulmonary capillary apoptosis, as measured by plasma levels of microparticles released from apoptotic endothelial cells (EMPs), reversible in healthy smokers and COPD smokers following smoking cessation?

What is the bottom line?

▸ Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but not in COPD smokers despite 12 months of smoking cessation, suggesting that the apoptosis continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation.

Why read on?

▸ Our longitudinal study demonstrates persistent endothelial stress in subjects with COPD despite smoking cessation and provides a biological correlate to the epidemiological data showing that smoking cessation only has a moderate effect on the continuous decline of lung function in COPD smokers, suggesting EMP levels might serve as a useful biomarker to follow smoking-associated endothelial apoptosis.

If elevated levels of circulating apoptotic EMPs are a reflection of active smoking-related injury, to lung endothelium, based on the knowledge that even those COPD smokers who stop smoking continue to have a decline in lung function that is more rapid than that of healthy non-smokers or healthy smokers who quit smoking, 15 we hypothesised that elevated levels of circulating apoptotic EMPs may persist in COPD smokers following smoking cessation, reflecting continuous lung endothelial injury that persists even after the stress of smoking is removed. To assess this hypothesis, we quantified the levels of circulating EMPs, and the fraction represented by apoptotic EMPs, in non-smokers, healthy smokers and smokers with COPD at baseline and at three more intervals over 1 year and then compared those levels with those obtained from a subgroup of healthy smokers and COPD smokers who successfully stopped smoking after baseline assessment. The data demonstrate that circulating EMP levels derived from apoptotic pulmonary capillary endothelial cells remain elevated over 1 year in healthy smokers and COPD smokers who continue smoking. However, while levels of total and apoptotic EMPs return to non-smoker levels in healthy smokers who successfully quit smoking, total and apoptotic EMP levels remain elevated in COPD smokers who quit smoking persisting 12 months of smoking cessation.

METHODS

Human subjects and clinical phenotypes

All subjects were evaluated at the Weill Cornell NIH Clinical and Translational Science Center and Department of Genetic Medicine Clinical Research Facility, under the clinical protocols approved by the Institutional Review Board. Recruitment was from the general population in New York City by posting advertisements in local newspapers and on electronic bulletin boards. All subjects provided written consent prior to enrolment and then underwent thorough medical history, screening and pulmonary function tests. Smoking status was determined based on self-reported history and quantified levels of urine nicotine metabolites. For details and full inclusion/exclusion criteria, see online supplementary methods. A total of 138 subjects were assessed for circulating total and apoptotic EMP levels at baseline, 3, 6 and 12 months (28 non-smokers, 61 healthy smokers and 49 COPD GOLD I/II smokers). See online supplementary figure S1 for study design.

Characterisation of plasma EMPs

EMPs were quantified according to a standard operating procedure as previously described 12 to eliminate variability in sample processing. Briefly, blood was collected, processed within 1 hour and stained for the endothelial markers PECAM (CD31) and E-selectin (CD62E) and the constitutive platelet-specific glycoprotein Ib (CD42b) to differentiate endothelium-originated microparticles from platelet-derived microparticles, which also express CD31. EMPs were defined as microparticles <1.5 μm in size, expressing CD31 + or CD62E + but not CD42b. We have previously shown that staining with annexin V is comparable with CD42b -CD31 + staining, 12 but annexin V was not used because it is not specific for EMPs. 16 Circulating EMPs are present in low levels in plasma of healthy subjects, reflecting normal endothelial turnover, 17 but their levels increase in a variety of vascular-related disorders. As in our previous study, 12 total EMP levels above the non-smoker total EMP mean level plus 2 SDs were considered abnormally elevated. To assess the presence of relative contribution of pulmonary capillary endothelium to the elevated total EMP levels, 1 12 EMPs were co-stained with antihuman ACE inhibitors, which is abundantly expressed on pulmonary capillary endothelium (CD42b -CD31 + ACE + ). 13 To quantify the proportion of EMPs that originated from apoptotic endothelium, we assessed the ratio of CD42b -CD62E + /CD42b -CD31 + EMPs in all groups. EMPs induced by apoptosis express the constitutive CD31 marker, whereas activation-induced EMPs express CD62E. Using these criteria, EMPs with a low CD42b -CD62E + to CD42b -CD31 + ratio were defined as 'apoptotic EMPs', and the percentage of subjects with apoptotic EMPs with CD42b -CD62E + /CD42b -CD31 + ratio below the lowest ratio in healthy non-smokers was quantified. See online supplementary methods for further details on the EMP analysis.

Assessment after smoking cessation

After the baseline levels of total and apoptotic EMPs were determined, all healthy smokers and COPD smokers were invited to stop smoking using a combination of varenicline and counselling for 3 months (see details in online supplementary methods). A total of 17 healthy smokers and 18 COPD smokers successfully quit smoking as confirmed by urine tobacco metabolite level quantification at 3, 6 and 12 months after the baseline; subjects were considered true quitters only if there were no detectable levels of nicotine metabolites in the urine at months 3, 6 and 12. Healthy smokers and smokers with COPD were treated with exact same prescription of varenicline to prevent any effect it might have on EMP levels. All other healthy smokers and COPD smokers were considered current smokers if urine cotinine level was ≥104 ng/mL at each time point, a level based on our previous study of low-level smoke exposure, 18 where 104 ng/mL was calculated as the induction half-maximal level (ID 50 ) at which the small airway epithelium, the initial site of smoking-related pathology, showed an abnormal response. See online supplementary figure S1 for study design.

Statistical analysis

χ 2 test, with a Yates' correction for small sample size, was used for comparing demographic parameters and the number of subjects with high total EMP and apoptotic EMP levels, and pairwise analysis of variance was used to compare total and apoptotic EMP levels between groups and within a group, at different time points with no correction for multiple test, as the number of tests was low (<21). In order to eliminate the effect of diseases known to be associated with elevated EMPs, including diabetes 19 20 and systemic hypertension, 21 or drugs for COPD, including corticosteroids and bronchodilators, subjects with known disease state or drug treatment were removed from statistical analysis. Removal of those subjects did not alter the results.

RESULTS

Study population

Except for minor differences, the study population of nonsmokers was comparable with the healthy smokers and COPD smokers in all demographic parameters (see table 1 for details). At each time point, the non-smokers had undetectable urine nicotine (not shown) and cotinine levels (figure 1). Both the healthy smokers and COPD smokers who continued smoking had urine cotinine levels consistent with tobacco smoking and comparable in both groups at each time point ( p>0.07, all comparisons). In healthy smokers and COPD smokers who quit smoking, urine nicotine and cotinine levels were consistent with smoking at baseline and were undetectable at all time points after baseline (see online supplementary methods for details of urine nicotine metabolite level criteria for smoking/abstinence).

Total EMP levels

Consistent with our prior study with a different cohort, 12 total EMP levels were higher in healthy smokers compared with nonsmokers (figure 2A, p<0.005). In addition, COPD smokers had elevated levels of total EMPs compared with non-smokers (p<0.007), but lower than those of healthy smokers (p<0.02). Twenty-two (36%) healthy smokers and eight (16%) COPD smokers had high levels of total EMPs (p<0.03). There was no correlation between the level of total EMPs and any pulmonary function or demographic parameters (r 2 <0.08, all correlations, see online supplementary figure S2). For the COPD group, total circulating EMP levels were independent of drugs used for treatment, including inhaled and systemic corticosteroids and bronchodilators.

Origin of the circulating EMPs

In our prior study of circulating EMPs, 12 we demonstrated that most of the circulating CD42b -CD31 + EMPs were positive for ACE inhibitors , a surface protein more highly expressed on pulmonary capillary endothelium than in other endothelial beds. 13 In the present study, an average of 75% of the circulating EMPs in all subjects were CD42b -CD31 + ACE + . There were similar levels of ACE + EMPs in the healthy smoker group compared with the non-smokers or COPD smokers (figure 2B, p>0.1, both comparisons), and higher levels of ACE + EMPs in the COPD smoker group compared with the non-smokers (p<0.0001).

EMPs derived from apoptotic endothelium

To quantify the proportion of EMPs originating from apoptotic endothelium, we assessed the ratio of CD42b -CD62E + / CD42b -CD31 + EMPs in all groups. The CD42b -CD62E + / CD42b -CD31 + EMP ratio in non-smokers was distributed around a mean of 0.9, significantly higher than that in healthy smokers (mean 0.6; a lower ratio indicates greater number of apoptotic EMPs) and COPD smokers (mean 0.55, p<0.0001, both groups compared with non-smokers; p>0.6, COPD smokers compared with healthy smokers). CD42b -CD62E + / *Data are presented as mean±SD; all parameters recorded at baseline; health/disease state based on screening and medical history and smoking status based on self-reported history and urine nicotine metabolite levels (detailed in online supplementary methods); non-smokers were comparable with all healthy smokers and all COPD smokers in ethnicity, BMI and all pulmonary function (p>0.1, all comparisons), except for FEV1 and DLCO that were lower in all COPD smokers (p<10 -7 , both comparisons), and FEV1/FVC, that was lower in all healthy smokers and, by definition, in all COPD smokers (p<0.02, both comparisons). Non-smokers were younger than all healthy smokers and all COPD smokers (p<0.002, both comparisons), and there were less female COPD smokers than female non-smokers (p<0.0002). There were more COPD smokers with emphysema compared with non-smokers (p<10 -4 ); All healthy smokers were comparable with all COPD smokers in ethnicity, age, BMI, all smoking history parameters (p>0.3, all comparisons), except for pack-year that was lower in all healthy smokers (p<10 -3 ). FVC and TLC were comparable (p>0.07, both comparisons), but FEV1, DLCO and, by definition, FEV1/FVC were lower in all COPD smokers (p<10 -12 , all comparisons). There were fewer females among all COPD smokers than among all healthy smokers (p<0.04). There were more COPD smokers with emphysema compared with healthy smokers (p<10 -4 ). †Healthy smokers who continued smoking had urine cotinine ≥104 ng/mL (see online supplementary methods for details) at baseline, 3,6 and 12 months. Healthy smokers who quit had undetectable urine nicotine and cotinine levels at 3, 6 and 12 months. The healthy smokers who continued smoking were comparable with those who quit in age, ethnicity, all smoking history (p>0.3, all comparisons), except for pack per day that was lower in those who quit (p<0.03), and comparable in all pulmonary function (p>0.1). There were more females, and the BMI was higher in the healthy smokers who quit group (p<0.04, both comparisons). ‡Gold stage defined by GOLD criteria 1 ; see online supplementary methods for details of subjects on medications; several of those treated were on multiple classes of medications; COPD smokers who continued smoking had urine cotinine ≥104 ng/mL at baseline, 3, 6 and 12 months; COPD smokers who quit had undetectable urine nicotine and cotinine levels at 3, 6 and 12 months; The COPD smokers who continued smoking were comparable with those who quit in age, gender, ethnicity, all smoking history and all pulmonary functions (p>0.3, all comparisons), except for DLCO that was lower in the COPD who continued smoking compared with those who quit (p<0.03). The BMI was lower in the COPD who continued smoking versus those who quit (p<0.002). There was no difference in the number of subjects with emphysema between the COPD smokers who quit smoking and those who continued smoking (p>0.6). §B, black, W, white, O, other. ¶Chest high-resolution CT (HRCT); % emphysema at -950 Hounsfield Units (HU); emphysema defined as >5% lung volume; see online supplementary methods for details. **Pulmonary function testing parameters are given as % of predicted value with the exception of FEV1/FVC, which is reported as % observed. BMI, body mass index; DLCO, diffusing capacity; GOLD; Global Initiative for Chronic Obstructive Lung Disease; TLC, total lung capacity.

CD42b -CD31 + EMPs below the lowest level in non-smokers were defined as apoptotic EMPs with 48% of healthy smokers and 45% of COPD smokers having increased levels of apoptotic EMPs ( p>0.7), that is, even though there are less subjects with total circulating EMPs in COPD smokers compared with healthy smokers (figure 2A), the relative proportion of subjects with apoptotic EMPs was similar (figure 2C), implying that there is active pulmonary capillary apoptosis ongoing in both the healthy smokers and COPD smokers. There was no correlation of CD42b -CD62E + /CD42b -CD31 + EMP ratio with any lung function or demographic parameter (r 2 =0.09, all correlations, see online supplementary figure S3). Within the COPD smoker group, there was no correlation of total CD42b -CD31 + EMP levels or CD42b -CD62E + /CD42b -CD31 + EMP ratio to the DLCO (% predicted) or % emphysema on high-resolution CT (HRCT) (r 2 =0.04, all comparisons; not shown), suggesting these parameters are likely measuring different aspects of the destruction process.

Effect of smoking cessation on total EMP levels

The levels of total EMPs were followed for a period of 1 year at baseline, 3, 6 and 12 months in non-smokers, healthy smokers and COPD smokers (figure 3A,B). In non-smokers, total EMP levels were stably low throughout the duration of the study ( p>0.6, each time point compared with baseline). In healthy smokers who continued smoking, the levels were stably high at each time point ( p>0.1, each time point compared with baseline; p<0.05, each time point compared with the non-smokers at the same time point). In contrast, in the healthy smokers who quit smoking, total EMP levels significantly decreased following smoking cessation to the levels of non-smokers ( p<0.002, each time point compared with baseline; p>0.4, compared with non-smokers at the same time point at 3, 6 and 12 months, figure 3A). At 12 months, the total EMP levels in healthy smokers who continued smoking were significantly higher compared with healthy smokers who quit ( p<10 -3 ). Total EMP levels in COPD smokers who continued smoking were stably elevated compared with non-smokers at each time point (p<0.05, all comparisons). In contrast to the healthy smokers, in COPD smokers who quit smoking, total EMP levels initially decreased following smoking cessation at month 3, but became elevated again at 6 and 12 months (figure 3B). The levels were not significantly different compared with non-smokers at 3 and 6 months ( p>0.1, both comparisons), but were significantly elevated at 12 months ( p<0.05) and similar to those of COPD smokers who continued smoking ( p>0.08).

Effect of smoking cessation on apoptotic EMP levels

The ratio of CD42b -CD62E + /CD42b -CD31 + EMPs was stably high in non-smokers and stably low (ie, EMPs were apoptoticderived) in healthy smokers who continued smoking ( p>0.1, each time point compared with baseline within the non-smoker group and within the healthy smoker group; p<0.01, healthy smokers who continued smoking compared with non-smokers, at baseline, months 6 and 12; figure 3C). Interestingly, in the healthy smokers who quit smoking group, the ratio increased following smoking cessation ( p<10 -3 , within the healthy smokers who quit group, at months 3 and 6 compared with baseline, a p value significant even with correction for multiple tests) to the level of non-smokers at months 3 and 6 ( p>0.1, both comparisons compared with non-smokers at the same time point) and superseded that of non-smokers at month 12 ( p<0.05). The ratio was significantly higher (ie, less apoptotic-derived EMPs) in healthy smokers who quit smoking compared with smokers who continued smoking at month 12 (p<10 -3 , a p value significant even with correction for multiple tests). There were no significant changes in the CD42b -CD62E + /CD42b -CD31 + ratio in COPD smokers who continued smoking ( p>0.1, within the COPD smoker group, each time point compared with baseline), and it remained significantly low compared with non-smokers at each time point (p<0.01, all comparisons, figure 3D). In contrast to the healthy smoker group, in COPD smokers who quit smoking, there was no change in the ratio ( p>0.1, within the COPD who quit group, each time point compared with baseline), and the ratio remained significantly low compared with non-smokers at baseline and month 12 (p<0.05, both comparisons). The ratio at month 12 was similarly low (ie, more apoptotic-derived EMPs) in COPD smokers who continued smoking and in COPD smokers who quit (p>0.4).

DISCUSSION

COPD is a chronic, debilitating disease that is caused primarily by cigarette smoking. 1 3-5 Cigarette smoke is very complex, with 10 14 oxidants and >4000 compounds stressing the lung with each puff. 22 The apoptotic loss of pulmonary capillaries in association with smoking is well recognised, 8-10 although it is not known whether this represents the primary mechanism of lung destruction associated with smoking, a subtype of lung destruction, or is secondary to other mechanisms, such as inflammatory cell-mediated processes. 5 6 9 10 23 Using circulating, total and apoptotic-endothelial cell microparticles as biomarkers for pulmonary capillary apoptosis, the data in the present study document that pulmonary capillary endothelial apoptosis is a persistent process in smokers with and without COPD. In healthy smokers who quit smoking, the levels of total and apoptotic EMPs return to the levels of non-smokers over time. In contrast, in COPD smokers who quit smoking, the levels of total and apoptotic EMPs remain abnormal over 1 year and were still significantly different compared with non-smoker levels at 12 months. The majority of the COPD subjects assessed for EMPs in this study were GOLD I and GOLD II, providing evidence for ongoing pulmonary endothelial apoptosis even in the earliest stages of COPD. Importantly, the FEV1/FVC ratio of the COPD smokers was 0.63±0.06, on average, well below the 0.7 ratio threshold definition of COPD GOLD I. These observations were not altered by the removal of subjects with diseases known to be associated with elevated EMPs, suggesting that smoking has a much stronger effect on EMP levels than hypertension or diabetes.

Endothelial microparticles

Different cell types respond to cell activation, injury and/or apoptosis by shedding submicron membrane vesicles, called microparticles, from their plasma membranes. 16 24 Microparticles detected in plasma are of various cellular origins, predominantly derived from platelets, leucocytes and endothelial cells. 24 Endothelial microparticles, defined as CD42b -CD31 + or CD42b -CD62E + microparticles, can be generally distinguished from microparticles of other cell types by their size (0.1-1.5 mm), constitutive expression of the platelet-endothelial cell adhesion marker CD31 (PECAM) and the absence of the platelet-specific glycoprotein Ib marker CD42b. 24 25 Apoptosis-induced EMPs express CD31, whereas activation-induced EMPs express CD62E. In this regard, a low ratio of CD42b -CD62E + to CD42b -CD31 + EMPs can be used as an index of apoptosis. 24 25 EMPs variably co-express phosphatidylserine (annexin V). 25 26 EMPs can be found in the plasma of healthy subjects; 24 however, increased levels are associated with vascular disease and endothelial dysfunction in atherosclerosis and acute coronary syndrome, 24 27 acute ischaemic stroke, 24 28 failure, 29 pre-eclampsia and gestational hypertension, 30 hypertension, 21 24 29 pulmonary hypertension, 31 metabolic syndrome, 32 venous thromboembolism 33 and obstructive sleep apnoea. 34 Consistent with our prior study 12 and the data in the present study, Heiss and colleagues 35 have shown healthy nonsmokers exposed for 30 min to low levels of cigarette smoke had increased circulating EMP levels.

Clinical measures of alveolar capillary destruction

The observation of endothelial apoptosis in the lungs of humans with emphysema is well documented. There is increased DNA fragmentation in the pulmonary capillaries and arteriolar endothelium of subjects with COPD, and increased alveolar endothelial and epithelial cell death in human emphysematous lungs compared with lungs of non-smokers or smokers without emphysema. 9 10 Lung levels of alveolar epithelial-derived vascular endothelial growth factor are decreased in emphysema, contributing to the complex mechanisms of pulmonary capillary endothelial destruction. 10 The data in the present study add total and apoptotic EMP levels to a growing list of biomarkers that may be useful in assessing active destruction and defining subclinical molecular phenotypes of lung disease. 17 36 37 As described in the editorial by Chandra and colleagues, 14 accompanying our study of apoptotic EMPs in healthy smokers and smokers with normal spirometry but low DLCO, the observation of elevated levels of apoptotic EMPs may represent early lung destruction and a subphenotype of subjects with lung destruction. The observation in the present study that, on average, a fraction of smokers with COPD also have elevated levels of total and apoptotic EMPs is consistent with the discussion of the concept of vascular subphenotypes of COPD by Chandra et al. 14 In this context, the data on circulating and apoptotic EMPs support the idea that, while the global concept of COPD as an FEV1-defined disorder is useful for epidemiologic studies and as a paradigm for routine clinical care, it is likely masking the concept that there are several subphenotypes of COPD. [38][39][40][41][42] Consistent with this concept, correlation of the total EMP levels and apoptotic EMP levels with the conventional measures of lung destruction (DLCO and HRCT) was, at best, very weak. This may imply that, while overlapping, each parameter is measuring a somewhat different aspect of the same process and/or that each parameter is assessing a different subpopulation of what is globally referred to as 'lung destruction'. Our longitudinal study demonstrates that total and apoptotic EMP levels remain stable over a period of 1 year in subjects with no change to their smoking habits. Intervention with smoking cessation can normalise the levels of total and apoptotic EMPs in healthy smokers, but in contrast, cessation did not lead to significant changes in total and apoptotic EMP levels in COPD smokers in our study. This observation provides a biological correlate for epidemiological data showing that smoking cessation only has a moderate effect to slow the decline of lung function in COPD smokers, that is, we believe that these data show that EMP levels might serve as a useful biomarker to follow smoking-associated endothelial apoptosis. The longitudinal aspect of this study demonstrates persistent endothelial stress in subjects with COPD, despite smoking cessation, and may help to explain the irreversible lung destruction associated with most cases of COPD, as evidenced by lung function of COPD smokers following smoking cessation that does not return to normal, 15 and may serve as a basis for additional studies of the mechanisms of the continuous pulmonary damage leading to this persistent EMP release.

Discussion

Waterpipe is currently the second most popular tobacco product after cigarettes worldwide 3,5,9,25 . Only limited data is available on the health effects of waterpipe smoking, particularly in light-use, young waterpipe smokers [3][4][5]9 . Many users believe it is safer and less addictive than cigarette smoking [134][135] . Our observation that even young (25±4 years old), lightuse (<4 sessions/week for <5 years) waterpipe-only smokers have abnormalities in several clinical and biologic lung-related parameters demonstrates that even light-use waterpipe smoking likely has significant effects on lung health. In comparison to gender and ethnicity matched nonsmokers, the light-use waterpipe smokers had (1) increased cough and sputum; (2) increased carboxyhemoglobin; (3) lower diffusing capacity; (4) an abnormal epithelial lining fluid metabolite profile; (6) an abnormal cell composition and transcriptome of the small airway epithelium, the cell population where COPD and most lung cancers are initiated [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][136][137][138] ; (7) an abnormal transcriptome of the alveolar macrophage, the pulmonary representative of the mononuclear phagocyte system, functioning as the scavenger cell in the lower respiratory tract [139][140] ; and (8) increased plasma levels of total and apoptotic capillary endothelial microparticles, indicative of pulmonary capillary endothelial apoptosis 117,121 .

1. Lung Function

Millions of people worldwide use waterpipe to smoke tobacco on a daily basis. Most studies assessing the health effects of waterpipe smoking have studied older, heavy-use waterpipe smokers, usually in waterpipe smokers who already have manifested lung disease [2][3]8,[23][24]26 . Several studies assessing the effect of heavy-use waterpipe smoking on the lungs found reduced lung function parameters, including FVC, FEV1, maximum mid expiratory flow, peak expiratory flow, FEF and FEV1/FVC and increased cough and sputum symptoms in is no therapy for the long-term decline in lung function and increased mortality associated with COPD [START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF][START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . Smoking cessation taking place at an early age slows down the decline in FEV1, but has little or no impact on the long term progression of COPD [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Rennard | Pathogenesis of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF] . Since even mild COPD is associated with increased mortality, detection of cigarette smokers at risk for developing COPD, can help with prevention by modification of risk factors and early therapeutic intervention [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF][START_REF] Graham | Effects of Increasing Carboxyhemoglobin on the Single Breath Carbon Monoxide Diffusing Capacity[END_REF]142 .

The DLCO assesses the potential of the lung for gas exchange [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF] and a reduction in DLCO suggests alveolar destruction, i.e., emphysema [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Gelb | Pseudophysiologic Emphysema Resulting from Sever Small Airways Disease[END_REF][START_REF] Gaensler | Attachment for automated single breath diffusing Capacity Measurement[END_REF] . Decreased DLCO has also been correlated with small airway disease and high levels of circulating EMPs derived from apoptotic pulmonary capillary endothelium [START_REF] Pellegrino | Interpretative Strategies for Lung Function Tests[END_REF][START_REF] Wald | Carbon Monoxide in Breath in Relation to Smoking and Carboxyhemoglbin levels[END_REF] . However, the gold standard to screen cigarette smokers for COPD is using only spirometry levels with bronchodilators, defining smokers with FEV1/FVC ratio ≥0.7 as healthy [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . Even though the phenotype of smokers with normal spirometry but low DLCO is recognized, the DLCO parameter is not routinely measured for technical and financial reasons and there are no data regarding what happens to lung function over time in these individuals [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF] .

In our study (Article 2) we focused on evaluating the addition of the DLCO parameter to the growing list of biomarkers used to identify smokers at risk for the development of COPD. Our observation that cigarette smokers with normal spirometry and no emphysema on HRCT, but with low DLCO are at significantly higher risk for developing COPD than a comparable group of cigarette smokers with normal DLCO suggests that a normal spirometry post-bronchodilators may give a false sense of "normal". Different risk factors, including advanced age, gender and cough and sputum, have been related to the development of COPD [START_REF] Lundback | Not 15 but 50% of Smokers Develop COPD?-Report from the Obstructive Lung Disease in Northern Sweden Studies[END_REF][START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF][START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF]125,[142][143][144][145][146] . However, in our study there were no differences in age, gender or cough and sputum scores between the cigarette smokers with

Metabolite Profile

A number of studies have analyzed compounds in waterpipe smoke, finding high levels of nicotine, carbon monoxide, tar, polycyclic aromatic hydrocarbons, heavy metals and other toxins originating from the tobacco, the flavoring and the charcoal 1,[12][13][14] . Consistent with the concept that at least some components of waterpipe smoke reach the lower respiratory tract, the light-use waterpipe smokers demonstrated an abnormal lower respiratory tract epithelial lining fluid metabolite profile, with a variety of metabolites in the waterpipe smokers with a differential abundance compared to nonsmokers.

Small Airway Epithelium and Alveolar Macrophage Transcriptome and Cellular

Composition

Compared to nonsmokers, the light-use waterpipe smokers displayed an altered small airway epithelial cellular composition. These individuals had less ciliated cells, the mediator of mucociliary clearance 151 , and basal cells, the stem/progenitor cell population of the airway epithelium 152 . In addition, they had more secretory cells 153 and intermediate, undifferentiated cells, the basal cell-derived precursors of the differentiated cell populations 152 . The light-use waterpipe smokers also demonstrated an abnormal small airway epithelium transcriptome compared to the nonsmokers, with hundreds of genes up-and down-regulated. Among the upregulated small airway epithelium genes in the light-use waterpipe smokers compared to nonsmokers were genes previously associated with different types of cancer, including: CGG triplet repeat binding protein 1 (CGGBP1), a cell cycle regulatory protein associated with the growth of lung and cervical cancer [154][155] ; pre-mRNA processing factor 4B (PRPF4B), a CDKlike kinase, with homology to mitogen-activated protein kinases, involved in pre-mRNA splicing, signal transduction, cell cycle progression and hepatocarcinogenesis [156][157] ; and integrin, beta 1 (ITGB1), a membrane receptor involved in cancer progression that mediates interactions of cells with extracellular matrix [158][159] . Among the down-regulated genes in the small airway epithelium of the waterpipe smokers compared to nonsmokers was ankyrin repeat domain 12 (ANKRD12), a gene encoding a member of the ankyrin repeats-containing cofactor family, with low expression linked to poor survival of colorectal cancer 160 . Together, these abnormalities suggest that waterpipe smoking-induced changes in the small airway epithelium cell composition and transcriptome may have important consequences with regard to the health of this anatomic compartment.

In contrast to cigarette smokers, where there is a higher % of macrophages recovered compared to nonsmokers 161 , there was no significant difference in the proportions of macrophage or other cell types recovered from the epithelial lining fluid of waterpipe smokers compared to nonsmokers. The waterpipe smokers also displayed a dysregulated transcriptome of the alveolar macrophages, with hundreds of genes up-and down-regulated in comparison to the nonsmokers. Among the dysregulated genes were genes previously associated with pathogenesis of COPD and/or cancer, including: echinoderm microtubule associated protein like 4 (EML4), a protein found to be involved in lung adenocarcinoma, sarcomas, non-small cell lung cancer and congenital pulmonary airway malformation [162][163] ; ubiquitin protein ligase E3B (UBE3B), with overexpression shown to effect skin carcinogenesis, lung adenocarcinoma, neuroblastoma and risk for coronary artery disease 164 ; epoxide hydrolase 1, microsomal (EPHX1), a gene encoding a protein that activates and detoxifies toxins in response to environmental carcinogens and is associated with risk for hepatocellular carcinoma and esophageal cancer [165][166] ; and cell cycle progression 1 (CCPG1), that regulates Rho-mediated signaling events involved in lung cancer [167][168] . These gene expression patterns and cell compositions are unique, not previously reported to be evoked by cigarette smoking 161,169 .

Endothelial Microparticles

Increased levels of endothelial microparticles have been associated with vascular disease and endothelial dysfunction in various diseases 111,113 . Endothelial microparticles are present in the low levels in plasma of healthy individuals [START_REF] Horstman | Endothelial Microparticles as Markers of Endothelial Dysfunction[END_REF] . In response to cell activation, injury and/or apoptosis the endothelial cells shed EMPs, submicron membrane vesicles from their plasma membranes [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF]117 , therefore high levels of total and apoptotic EMPs in plasma represent early lung destruction [START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF][START_REF] Horstman | Endothelial Microparticles as Markers of Endothelial Dysfunction[END_REF]103,111,113,[130][131][170][171] . The observation that the total level of circulating EMPs and the proportion of apoptotic EMPs are significantly higher in waterpipe smokers compared to nonsmokers suggests an ongoing lung capillary endothelial apoptosis associated with light-use waterpipe smoking.

We have previously shown that cigarette smokers are undergoing pulmonary endothelial apoptosis as measured by high plasma levels of total EMPs and an increased proportion of apoptotic EMPs 121 . Cigarette smokers with normal spirometry and low DLCO demonstrated significantly higher levels of total and apoptotic EMPs compared to nonsmokers and cigarette smokers with normal spirometry and DLCO. Using circulating, total and apoptotic endothelial cell microparticles as biomarkers for pulmonary capillary apoptosis, the data in the present study (Article 3) demonstrated that pulmonary capillary endothelial apoptosis is a persistent process in cigarette smokers with and without COPD. Our longitudinal study demonstrates that total and apoptotic EMP levels remain stable over a period of 1 year in individuals with no change to their smoking habits. Intervention with smoking cessation can normalize the levels of total and apoptotic EMPs in healthy cigarette smokers, but in contrast, cessation did not lead to significant changes in total and apoptotic EMP levels in COPD cigarette smokers. The majority of the COPD individuals assessed for EMP levels in this study were GOLD I and GOLD II, providing evidence for ongoing pulmonary endothelial apoptosis even in the earliest stages of COPD (Article 3).

This observation provides a biologic correlate for epidemiologic data showing that smoking cessation only has a moderate effect on the rate of decline of lung function in COPD smokers, but also adds total and apoptotic EMP levels to a growing list of biomarkers that may be useful in helping to assess active destruction in light-use waterpipe smokers.

6. Implications

Light-use waterpipe smokers demonstrate abnormalities in various clinical-and biologiclung related parameters. Together, the data suggest that even light-use waterpipe smoking in young individuals significantly affects lung biology and health even before any clinical abnormalities are detected. It is likely that these changes are the earliest biologic correlates of epidemiologic studies linking waterpipe smoking to lung health risk. Some of these abnormalities are similar to those seen in cigarette smokers, but most are unique to waterpipe smokers, suggesting that waterpipe smoking is associated with lung pathology and that it may be different from that associated with cigarette smoking. Based on these evidence, in the context of the increasing use of waterpipe smoking, our findings support the efforts to regulate and reduce waterpipe smoking, especially among the young population, and to use DLCO level and total and apoptotic EMP levels as biomarkers for early detection of disease in waterpipe smokers.

 Not pregnant (females)  No history of allergies to medications used in the bronchoscopy procedure  Not taking any medications relevant to lung disease or having an effect on the airway epithelium or alveolar macrophages  Willingness to participate in the study  Self-reported waterpipe-only smokers Exclusion criteria  Unable to meet the inclusion criteria  Current active infection or acute illness of any kind  Evidence of malignancy within the past 5 years  Alcohol or drug abuse within the past 6 months Healthy cigarette smokers with normal spirometry and normal diffusion capacity (DLCO) Inclusion criteria  Males and females, at least 18 years old  Provide informed consent  Normal physical examination  Normal routine laboratory evaluation, including general hematologic studies, general serologic/immunologic studies, general biochemical analyses, and urine analysis  HIV negative  Good health without history of chronic lung disease, including asthma, and without recurrent or recent (within 3 months) acute pulmonary disease  Normal PA and lateral chest X-ray  Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)  Not pregnant (females)  No history of allergies to medications used in the bronchoscopy procedure  Not taking any medications relevant to lung disease or having an effect on the airway epithelium or alveolar macrophages  Willingness to participate in the study  Normal serum α1-antitrypsin level  Self-reported current daily smokers with >5 pack-yr, validated by urine nicotine >30 ng/ml and/or cotinine >50 ng/ml  Normal FEV1 (≥ 80% predicted), FVC (≥ 80% predicted), FEV1/FVC (≥ 0.7) based on postbronchodilator spirometry, TLC (≥ 80% predicted)  DLCO ≥ 80% predicted Exclusion criteria  Unable to meet the inclusion criteria  Current active infection or acute illness of any kind  Evidence of malignancy within the past 5 years  Alcohol or drug abuse within the past 6 months

Healthy cigarette smokers with normal spirometry, but low diffusion capacity (DLCO)

Inclusion criteria  Males and females, at least 18 years old  Provide informed consent  Normal physical examination  Normal routine laboratory evaluation, including general hematologic studies, general serologic/immunologic studies, general biochemical analyses, and urine analysis  HIV negative  Good health without history of chronic lung disease, including asthma, and without recurrent or recent (within 3 months) acute pulmonary disease  Normal PA and lateral chest X-ray  Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)  Not pregnant (females)  No history of allergies to medications used in the bronchoscopy procedure  Not taking any medications relevant to lung disease or having an effect on the airway epithelium or alveolar macrophages  Willingness to participate in the study  Normal serum α1-antitrypsin level  Self-reported current daily smokers with >5 pack-yr, validated by urine nicotine >30 ng/ml and/or cotinine >50 ng/ml  Normal FEV1 (≥ 80% predicted), FVC (≥ 80% predicted), FEV1/FVC (≥ 0.7) based on postbronchodilator spirometry, TLC (≥ 80% predicted)  DLCO <80% predicted and below the 95% range of normal DLCO calculated separately for each individual based on gender and ethnicity Exclusion criteria  Unable to meet the inclusion criteria  Current active infection or acute illness of any kind  Evidence of malignancy within the past 5 years  Alcohol or drug abuse within the past 6 months

  ) Score de toux et d'expectoration; (3) fonction pulmonaire; (4) Métabolites présent dans les fluides des voies respiratoires basses (ELF); (5) différences cellulaires et de transcriptome des petites voies aériennes (6) composition cellulaire des lavages broncho-alvéolaires (7) le transcriptome et (9) niveau des microparticules endothéliales circulantes. Le groupe d'étude montrait des anomalies dans tous les paramètres étudiés. Comparé au groupe contrôle les fumeurs avaient plus de toux et d'expectoration, un niveau de CO plus élevé, une diminution de la capacité de diffusion du CO, des anomalies du profil métabolique des fluides alvéolaires, une augmentation des cellules sécrétoires et intermédiaires et une diminution des cellules ciliées et basales, des anomalies du transcriptome des cellules pulmonaires et de macrophages alvéolaires et une augmentation des microparticules endothéliales.

:

  AM -alveolar macrophages COPD -chronic obstructive pulmonary disease DLCO -diffusion capacity of the lung for carbon monoxide EMPs -endothelial microparticles GOLD -global initiative for chronic obstructive pulmonary disease HRCT -high resolution chest tomography FEF -forced expiratory flow FEV1 -forced expiratory volume in 1 sec FRC -functional residual capacity FVC -forced vital capacity SAE -small airway epithelium List of Tables:

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Middle Eastern waterpipe device Figure 2. Mechanisms of airflow limitation in COPD Figure 3. Survival curves, stratified by lung function at baseline

Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .

 45678 Figure 4. Rate of decline in FEV1 with age Figure 5. Expected change in DLCO based on smoking history Figure 6. Expected change in DLCO based on pack year history Figure 7. Assessment of lung function in clinical practice Figure 8. Possible pathways leading to endothelial microparticle release

Figure 1 .

 1 Figure 1. Middle Eastern waterpipe device. Adapted from Akl et al.2

Figure 2 .

 2 Figure 2. Mechanisms of airflow limitation in COPD. The airway in normal individuals is distended by alveolar attachments during expiration, allowing alveolar emptying and lung deflating.In COPD patients, these attachments are disrupted because of emphysema, trapping gas in the alveoli, contributing to airway closure during expiration, and resulting in hyperinflation. Peripheral airways are also obstructed and distorted by airway inflammation and fibrosis (chronic obstructive bronchiolitis) and by occlusion of the airway lumen by mucus secretions, which may be trapped in the airways because of poor mucociliary clearance. Adapted from Barnes et al.[START_REF] Barnes | Mediators of Chronic Obstructive Pulmonary Disease[END_REF]

Figure 3 .

 3 Figure 3. Survival curves, stratified by lung function at baseline. Adapted from Maninno et al.[START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF]

Figure 4 .

 4 Figure 4. Rate of decline in forced expiratory volume in 1 sec (FEV1) with age. Adapted from Fletcher et al.[START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF]

FEV

Figure 5 . 84 ChangeFigure 6 .

 5846 Figure 5. Expected change in diffusion capacity for carbon monoxide (DLCO) based on smoking history (pack year history) and current smoking status (number of cigarettes per day).Data adjusted for gender, race, height, age, weight, and hemoglobin level. Adapted from Neas el al.[START_REF] Martinez | Sex Difference in Sever Pulmonary Emphysema[END_REF]

Figure 7 .

 7 Figure 7. Assessment of lung function in clinical practice. The algorithm presents classic patterns for various pulmonary disorders. Patients may or may not present the classic patterns, depending on their illness, severity or lung function prior to the disease onset.The algorithm includes DLCO measurements with the predicted value adjusted for hemoglobin level. In the mixed defect group, the DLCO patterns are the same as those for restriction and obstruction. The algorithm is not suitable for assessing upper airway obstruction. FEV1 -forced expiratory volume in 1 sec; VC -vital capacity; LLN -lower limit of normal; TLC -total lung capacity; DLCO -diffusion capacity for carbon monoxide; ILD -interstitial lung disease. Adapted from Pellegrino et al.[START_REF] Gaensler | Attachment for automated single breath diffusing Capacity Measurement[END_REF]

Figure 8 .

 8 Figure 8. Possible pathways leading to endothelial microparticle release.A resting cell membrane is characterized by its phospholipid distribution, with phosphatidylcholine and sphingomyelin located on its external layer and phosphatidylethanolamine and phosphatidylserine (PSer) on its inner layer. This phospholipid asymmetry is maintained by a transmembrane enzymatic balance of flippase, floppase and scramblase. Cell activation or apoptosis is associated with a release of intracellular calcium by the endoplasmic reticulum that changes the transmembrane steady state. This release leads to PSer externalization and activation of cystolic enzymes including calpain, leading to the cleavage of cytoskeleton filaments. This pathway results in bledding and shedding of membrane-derived microparticles into the extracellular fluid. Adapted from Chironi et al.[START_REF] Chironi | Endothelial Microparticles in Diseases[END_REF]

  Young, Light-use Waterpipe (Hookah) Smokers American Journal of Respiratory and Critical Care Medicine; In Press Abstract: Waterpipe, an instrument for smoking fruit-flavored tobacco, commonly associated with the Middle East, is becoming a global phenomenon currently used by millions of people worldwide. Many waterpipe smokers believe waterpipe smoking is a safer alternative to cigarette smoking because the smoke is bubbled through water before inhalation. Despite the widespread use, there is only limited data available on the health risks associated with waterpipe smoking, particularly in young, light-use waterpipe smokers. There are no governmental regulations regarding waterpipe use.

Score ≥ 2 Figure 1 . 4 American

 214 Figure 1. Clinical abnormalities of light-use, young waterpipe smokers compared with healthy nonsmokers. (A) Cough and sputum scores. Shown are the percentages of subjects with abnormal cough and sputum scores (>2 on 0-4 scale). P values were calculated using a x 2 test. *None. (B) Diffusing capacity of the lung for carbon monoxide. P value was calculated using a two-tailed Student's t test. Dashed line indicates the lower limit of normal.

Figure 2 .

 2 Figure2. Differential gene expression in the small airway epithelium (SAE) and alveolar macrophages (AMs) of waterpipe smokers compared with nonsmokers. For all panels, the data, normalized by array, were compared in nonsmokers (n = 19

10

 10

Figure 3 .

 3 Figure 3. Levels of plasma total endothelial microparticles (EMPs), pulmonary-derived EMPs, and the proportion of apoptotic EMPs. Shown are data for nonsmokers (n = 19; green circles) and waterpipe smokers (n = 20; orange circles). Each data point represents one subject. Dashed line in each group indicates the group mean. (A) Total CD42b 2 CD31 1 EMPs. (B) Proportion of CD42 2 CD31 1 EMPs that express angiotensin-converting enzyme (ACE 1 ), a gene highly expressed in the pulmonary capillary endothelium (52). (C) Ratio of circulating activated CD42b 2 CD62 1 EMPs to CD42b 2 CD31 1 apoptotic EMPs. The dashed line indicates the level of 2 SD below the mean of CD42b 2 CD31 1 /CD42b2 CD62 1 EMPs in nonsmokers. Values below this line represent elevated levels of apoptotic EMPs. P values were calculated using a two-tailed Student's t test.

Figure 1

 1 Figure1Urine cotinine levels (ng/mL) as a measure of smoking status at baseline and at 3, 6 and 12 months in non-smokers, healthy smokers and smokers with COPD (COPD smokers). Shown are data for non-smokers (n=28, green circles), healthy smokers who continue to smoke (n=44, yellow circles), healthy smokers who quit smoking following baseline (n=17, light blue circles), COPD smokers who continue to smoke (n=31, red circles) and COPD smokers who quit smoking following baseline (n=18, tan circles). Data represent mean ±SE. Dashed lines indicate urine cotinine detection level of ≤5 ng/mL and urine cotinine level of ≥104 ng/mL for active smoking (see online supplementary methods). EMP, endothelial microparticles.

Figure 3

 3 Figure 3 Total circulating CD42b -CD31 + endothelial microparticles (EMPs) and ratio of CD42b -CD62E + to CD42b -CD31 + EMPs over time in non-smokers (n=28, green circles), healthy smokers who continue to smoke (n=44, yellow circles), healthy smokers who quit smoking following baseline (n=17, light blue circles), smokers with COPD (COPD smokers) who continue smoking (n=31, red circles) and COPD smokers who quit smoking following baseline (n=18, tan circles). (A and B) Total CD42b -CD31 + EMPs. (C and D) Ratio of CD42b -CD62E + to CD42b -CD31 + EMPs. (A and C). Healthy smokers who continue to smoke and healthy smokers who quit smoking versus non-smokers. (B and D) COPD smokers who continue to smoke and COPD smokers who quit smoking versus non-smokers. (A-D) Data represent mean±SE. p Values comparing each time point to baseline within the same group are shown at the top of the panel (for the group, ie, above the non-smokers at month 12) and at the bottom of the panel (for the group, ie, below the non-smokers at month 12). p Values comparing each time point in a smoker group to the same time point in the non-smoker group are shown above the group, if the group is above the non-smokers at month 12 and below the group if the group is below the non-smokers at month 12. p Values comparing the subjects who continue to smoke with those who quit smoking at month 12 are to the right of the panel. NS, not significant; *, **, ***, **** indicate p<0.05, p<0.01, p<0.001 and p<0.0001, respectively.

Table I .

 I Comparison of Toxicant Yield in Waterpipe Smoke and Cigarette Smoke Table II. Long-term Effects of Waterpipe Smoking on Pulmonary Function Table III. Spirometric Profile of Waterpipe Smokers Table IV. Comparison of Lung Function and Fractional Exhaled Nitric Oxide (FeNO) in Waterpipe Smokers and A Matched Control Group Table V. Acute Respiratory Effects of Waterpipe Smoking Table VI. Adverse Health Effects Associated with Waterpipe Smoking

Table VII .

 VII Studies on the Association of Waterpipe Smoking and Cancer

Table VIII .

 VIII Classification of COPD Severity

Table IX .

 IX Physiological and Pathological Changes that Affect the DLCO

Table X .

 X Progression to COPD in Smokers with Normal Spirometry/Low DLCO vs Smokers with Normal Spirometry/Normal DLCO Using Different Methods to Determine

	List of Figures:
	Normal Levels (Appendix V)

Table II . Long-term effects of waterpipe smoking on pulmonary function 1,*

 II

	FEV1/FVC	difference 3	-13.8	-13.0	N/A	Decreased (NS)	N/A		N/A	-7.2 (NS)		-8.63 (NS)		+5.08	N/A	N/A		N/A	N/A	N/A	N/A	N/A	N/A	N/A
	% predicted difference 3	FEF 25-75 FVC FEV1	N/A -21.9 -14.6	N/A -7.03 -3.8 (NS)	+13.0 +14.0 +24.0	Decreased Decreased (NS) Decreased	+0.5 4 (NS) N/A -1.1 (NS)	-2.5 4 (NS) N/A -12.2 (NS)	-5.6 4 +0.9 (NS) -2.5 (NS)	-3.02 4 -5.86 (NS) -6.5		+4.49 4 -0.5 (NS) +3.01	+0.1 (NS) N/A +5.3 (NS)	-12.1 +6.1 -9.4		-8.0 +7.1 -4.0	-4.5 -6.6 -9.0	-3.0 -5.5 -6.9	-4.6 -0.43 L -0.54 L	-11.42 -0.19 L -0.41 L	+5.56 -9.1 -4.4 (NS)	+4.28 -2.28 (NS) +1.63 (NS)
		Comparison	WP vs nonsmokers	WP vs cigarette smokers (normal inhalation)	WP vs cigarette smokers	WP vs reference values		WP vs cigarette smokers		WP vs nonsmokers	WP vs passive cigarette smokers		WP vs nonsmokers		WP vs cigarette smokers	WP vs nonsmokers and cigarette smokers	WP vs nonsmokers and cigarette smokers	WP vs cigarette smokers	WP passive vs neversmokers	WP passive vs cigarette-passive	WP smokers vs nonsmokers	Males	Females	WP vs nonsmokers	WP vs cigarette smokers
	All heal-thy?	Yes		Yes	Yes		Yes		Yes				No		No		Yes			Yes	No
	Tobacco type	N/A		Tabamel	N/A		Moassal		N/A				Jurak		N/A		Chinese	WP tobacco		Jurak	Moassal
		WP quantity 2	1.17 ± 0.53 WP	smoked/week	36 ± 22 WP years	Median 14 WP	years	N/A		23.7 ± 8.3 years	smoking 1-2	times/day	37 ± 42 Jurak-	years	N/A		28 ± 11.2 years	of 17.9 ± 8.9 g	tobacco/day	N/A	11.12 ± 17.27	WP/week
		Population	371 men, 301 women average, ages in 30s and 40s	142 men age 35-60 years	110 men, age 20-60 years	139 men, 13 women, age 24-65 years	25 persons average age 49 ±12.2 years		397 men, age 18-85 years	788 women, age 44+ years		1238, mostly men, age 40+ years	441 men (20-59 years old), 154 women (17-59 years old)	87 men, 45 women, average 33.4± 13.29 years
	Refe-rence	28		29	24		25		30				31		32		7			33	34

1

WP= waterpipe; NS = not significant. 2 Data is average ± standard deviation, unless otherwise specified.

3

All pulmonary function values are differences (WP value compared to group value). Units are % predicted, except FEV1/FVC which is % ratio, or otherwise specified; FEV1 = forced expiratory volume in 1 second; FVC = forced vital capacity; FEF = forced expiratory flow between 25% and 75% of the FVC. 4 Percent predicted value .* Adapted from El-Zaatari et al.

8

Table III . Spirometric Profile of Waterpipe Smokers 1,2,*

 III

	Parameter	n (%)
		≤ N
	FEV1 (L)	14 (13%)
	FVC (L)	7 (6%)
	SVC (L)	40 (36%)
	FRC (L)	14 (13%)
	TLC (L)	15 (14%)
	RV (L) PEFR (Ls -1 ) PEF25% (Ls -1 ) PEF50% (Ls -1 ) PEF75% (Ls -1 ) MMEF (Ls -1 )	9 (8%) 31 (28%) 8 (7%) 9 (8%) 9 (8%) 18 (16%)
	FEV1/FVC	6 (6%)
	FEV1/SVC	2 (2%)
		≥ N
	FRC (L)	36 (33%)
	TLC (L)	23 (21%)
	RV (L)	40 (36%)
	RV/TLC	33 (30%)
	FRC/TLC	16 (14%)
	Normal FVC and
	PEF25% or PEF50% or PEF75% or MMEF <N	15 (14%)
	PEF25% <N	8 (7%)
	PEF50% <N	9 (8%)
	PEF75% <N	9 (8%)
	MMEF <N	15 (14%)
	Normal FEV1/FVC and TLC and
	FVC <N or FEV1 <N	5 (5%)
	FVC <N	3 (3%)
	FEV1 <N	5 (5%)
	Adapted from Ben Saad H et al. 24

1

Data is presented as number (%) of subjects with ventilator variables outside the normal range or with clinical obstructive ventilatory defect. 2 N -normal corresponding to the confidence interval of 95%; FEV1 -forced expiratory volume in 1 sec; PEFR -peak expiratory flow rate; PEF -peak expiratory flow; MMEF -maximum mid expiratory flow; FVC -forced vital capacity; SVC -slow vital capacity; RV -residual volume; FRC -functional residual capacity; TLC -total lung capacity. *

Table IV . Comparison of Lung Function and Fractional Exhaled Nitric Oxide (FeNO) Parameters in Waterpipe Smokers and a Matched Control Group 1,*

 IV

	Parameter	Waterpipe (n=73)	Control (n=73)	p value
	Age (years)	21.54 ± 0.41	21.36 ± 0.19	>0.6
	Height (cm)	172.68 ± 0.76	173.71 ± 1.03	>0.4
	Weight (kg)	76.26 ± 2.39	72.84 ± 1.48	>0.2
	FVC (L)	5.76 ± 0.21	5.54 ± 0.11	>0.3
	FEV1 (L)	3.80 ± 0.12	4.49 ± 0.07	<0.0002
	FEV1/FVC (%)	69.34 ± 1.87	82.83 ± 1.29	<0.0002
	FEF25% (L/sec)	6.04 ± 0.25	6.93 ± 0.18	<0.006
	FEF50% (L/sec)	3.13 ± 0.19	5.25 ± 0.17	<0.0002
	FEF75% (L/sec)	1.13 ± 0.13	2.56 ± 0.13	<0.0002
	FEF25-75% (L/sec)	6.86 ± 2.23	4.53 ± 0.19	>0.2
	FEF75-85% (L/sec)	1.21 ± 0.24	1.91 ± 0.11	<0.02
	PEF (L/sec)	7.29 ± 0.22	7.32 ± 0.18	>0.08
	FeNO (ppb)	23.97 ± 2.12	31.38 ± 2.38	<0.03
	1 Data is presented as mean ± standard deviation. FVC -forced vital capacity; FEV1 -forced
	expiratory flow in 1 sec; FEF -forced expiratory Flow; PEF -peak expiratory flow.
	*Adapted from Meo et al.

Table V Acute Respiratory Effects of Waterpipe Smoking* FEV1/FVC difference 3

	1	% predicted difference 3 RR All Tobacco type and FEF 25-75 PEFR FVC FEV1 bpm 2 healthy? amount Smoking session duration Population 1 Reference	N/A N/A N/A N/A N/A +2 No N/A 45 min 202 men, >17 years old 36	-1 (NS) -5 -8 0 -1 (NS) +2.3 Yes 10g 30 min 30 men, 15 women, 18+ 37	Moassal years old, ave age 23.4 ±	23.4 years old, range 18-	65 years old	N/A -0. 22 L/sec N/A -0.05 L/sec -0.08 +2 Yes N/A 45 min 24 men, 18-26 years old 38	(NS) (NS) L/sec	+1.0 (NS) -0.1 (NS) -3.6 -0.7 (NS) +0.1 +3.5 Yes 10g 30 min 39 men, 23 women, 47 39	(NS) Moasaal active smokers, 18+ years	old, ave age 24.9 ± 6.2	-2.28 4 N/A N/A +1.69 4 -1.21 4 N/A No 20g 45 min 87 men, 45 women, ave 34	Moassal age 33.4 ± 13.29 years,	WP exclusive smokers	1 WP = waterpipe, ave=average.	2 All pulmonary function values are changes (WP value after-WP value before). The units are % predicted, except FEV1/FVC, which is % ratio; FEV1 =	forced expiratory volume in 1 second; FVC = forced vital capacity; PEFR = peak expiratory flow rate; FEF25-75 = forced expiratory flow between	25% and 75% of the FVC; N/A = not evaluated; NS = not significant.	3 RR bpm= resting heart rate (beats per min).	4 Statistical significance unspecified.	* Adapted from El-Zaatari et al. 8

.1.5 Additional health effects of waterpipe smoking

	Studies assessing the health effects associated with heavy-use waterpipe smoking show
	many acute and long-term effects (Table VI). Several studies have found a significant correlation
	of waterpipe smoking to lung cancer, and a non-significant increased risk to various types of
	other carcinomas, including bladder, nasopharyngeal and oesphageal cancers (Table VII) 3-5,8,40 .
	Heavy-use waterpipe smoking was significantly associated with various respiratory diseases,
	heart rate variations, hyperglycemia and hypertriglyceridemia, low birth rate, periodontal disease
	and chromosomal aberrations.

Table VI. Adverse Health Effects Associated with Waterpipe Smoking 1,* Acute effects

	not significant after	adjustment for	OR for male, >45 year old	heavy smokers	increased risk with	cumulative exposure	did not control for Chinese	long-stem pipe smoking	did not control for Chinese	long-stem pipe smoking	higher risk with greater	intensity, duration and	cumulative waterpipe	smoking	OR for >32 waterpipe-	years smoking	statistically significant	association was observed	also non-significant for	cumulative waterpipe use;	n of gastric cancer cases also included a small % of	was too small for	OR for smoking >153 significance; most	waterpipe-years; OR also	insignificant for lesser	waterpipe smoking was	also non-significant (but	waterpipe smoking was	had small number of also not exclusive of other	waterpipe smokers
	Increased heart rate Increased blood pressure Carbon monoxide intoxication Impaired pulmonary function (FEF25-75%, PEFR) Decreased exercise capacity Larynx and voice changes Long-term effects Ischemic heart disease Impaired pulmonary function (FEV1, FVC, FEV1/FVC, FEF25-75%, PEF, FRC, RV) Chronic obstructive lung disease Chronic bronchitis Emphysema Lung cancer Oesphageal cancer Gastric cancer Low birth rate Pulmonary problems at birth Periodental disease Larynx and voice changes Lower bone density and increased fracture risk 1 FEF -forced expiratory flow; PEFR -peak expiratory flow rate; FEV1 -forced expiratory rate adjusted=0.8 (0.2 to 4.0) adjusted=1.1 (0.7 to 1.9) for urothelial cancer; 0.5 (0.2 to 1.0) for squamous cancer not calculated (x2=2.646, p<0.05) adjusted=1.1 (0.3 to 3.3) N/A adjusted=3.4 (1.7 to 7.1) adjusted=1.66 (0.65 to 4.22) adjusted=21.4 (11.6 to 39.5) adjusted=1.85 (1.41 to 2.44) 1 adjusted=1.8 (0.8 to 4.2) 1 adjusted=4.44 (1.2 to 16.44) 5.8 (3.9 to 8.6) 6.0 (1.78 to 20.26) adjusted=2.5 (1.08 to 5.82) OR (	OR=7.0 (0.9 to 56.9)		adjusted=1.6 (0.9 to 2.8)	adjusted=0.49 (0.2 to 1.43)	1 A single OR was not reported, but there was an increased risk based on mathematical modelling, which was beyond the scope of the paper. * Adapted	from El-Zaatari et al. 8
	in 1 sec; FVC -forced vital capacity; PEF -peak expiratory rate; FRC -functional residual
	capacity; RV -residual volume. * Adapted from El-Zaatari et al. 8

95% CI) Table VII. Studies on Association of Waterpipe Smoking and Cancer*

		Adjusted for other	cofounders?	yes	yes		no		yes		no	yes	yes	yes		yes		yes		yes		N/A	yes		no		yes	yes	yes	yes	yes
	Controlled for	cigarette	smoking?	no	yes		no		yes		no	yes	yes	yes		no		yes		yes		N/A	yes		N/A		yes	yes	yes	no	no
			Study type	prospective	case-control community -based		case-control		case-control		case-control	case-control	case-control	case-control		case-control		case-control		prospective cohort		case-control	case-control		cross-sectional		case-control	case-control	case-control	case-control	case-control
			Population	20033 Bangladeshi	150 Labanese individuals	individuals	751 Indian	individuals	265 Indian	individuals	148 Chinese men	1438 Chinese men	12011 Chinese men	2365 Indian	individuals	330 Indian	individuals	871 Iranian	individuals	928 Iranian	individuals	92 Iranian individuals	922 Iranian	individuals	3064 Yemeni	individuals	1134 Egyptian men	308 Egyptian men	274 Iranian men	388 Egyptian	1251 North African individuals	individuals
			Cancer type	All cancer death	Lung		Lung		Lung		Lung	Lung	Lung	Oesophageal		Oesophageal		Oesophageal		Gastric		Gastric	Gastric		Gastric and	oesophageal	Bladder	Bladder	Prostate	Pancreatic	Nasopharyngeal
		Refe-	rence	41	40		6		42		43	44	45	46		47		48		49		50	51		52		53	54	55	56	57

Table VIII. Classification of COPD Severity 1,* Classification based on post bronchodilator lung function GOLD

	I (mild)	FEV1/FVC <0.7 and FEV1 ≥ 80% predicted
	GOLD II (moderate)	FEV1/FVC <0.7 and 80% > FEV1 ≥ 50% predicted
	GOLD III (severe)	FEV1/FVC <0.7 and 50%> FEV1 ≥ 30% predicted
	FEV1/FVC <0.7 and FEV1 < 30% predicted or FEV1 <50% predicted plus chronic respiratory failure 1 COPD -chronic obstructive pulmonary disease; GOLD -global initiative for chronic GOLD IV (very severe)
	obstructive pulmonary disease; FEV1 -forced expiratory volume in 1 sec, FVC -forced vital
	capacity. * Adapted from GOLD 2015 63 .

normal COPD Disrupted alveolar attachments (emphysema) Mucosal inflammation and fibrosis Mucosal hypertension Airway obstructed by:  Loss of attachments  Mucosal inflammation and fibrosis  Mucus obstruction of lumenalveolar attachments Airway held open by alveolar attachments

Table IX . Physiological and Pathological Changes that Affect the DLCO 1,* Extrapulmonary reduction in lung inflation (reduced VA) producing changes in DM or

 IX Reduced effort or respiratory muscle weakness Thoracic deformity preventing full inflation Diseases

that reduce ɵVc and thus reduce DLCO

	Anemia
	Pulmonary emboli
	Other conditions that reduce ɵVc and thus reduce DLCO
	Hemoglobin binding changes
	Valsalva maneuver
	Diseases

that reduce (in varying degrees) DM and ɵVc and thus reduce DLCO

	Lung resection
	Emphysema
	Interstitial lung disease
	Pulmonary oedema
	Pulmonary vasculitis
	Pulmonary hypertension
	Diseases that increase ɵVc and thus increase DLCO
	Polycythaemia
	Left to right shunt
	Pulmonary haemorrhage
	Asthma
	Other

conditions that increase ɵVc and thus increase DLCO

Hemoglobin binding changes

Muller maneuver Exercise Supine position (in addition, possibly a slight increase in DM) Obesity (in addition, a possible DM component)

1

DLCO -diffusion capacity for carbon monoxide; VA -alveolar volume; DM -membrane conductivity; ɵ -carbon monoxide-hemoglobin chemical reaction rate; Vc -volume of pulmonary capillary blood. * Adapted from Maclntyre et al.

[START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF]

  (P , 0.05 with

	ORIGINAL ARTICLE
	At a Glance Commentary
	Scientific Knowledge on the
	Subject: Waterpipe smoking is
	increasing worldwide, mainly among
	young adults. It is second only to
	cigarette smoking. Researchers in most
	studies have assessed older, heavy-use
	waterpipe smokers with disease
	manifestation and not young, light-use
	waterpipe smokers.
	What This Study Adds to the
	Field: We evaluated multiple lung
	components, including clinical and
	biologic abnormalities, in several
	anatomic components in the lungs of
	young, light-use waterpipe smokers
	with no clinical manifestation of
	disease.

2 American Journal of Respiratory and Critical Care Medicine Volume jj Number jj | jj 2016

Table 1 .

 1 Demographics and Biologic Samples

			Waterpipe
	Parameter	Nonsmokers	Smokers	P Value
	Number of patients	19	21
	Sex, male/female, n Age, yr	9/10 33 6 9	13/8 2 5 6 4	.0.3 ,10 23
	Race, black/white/other, n BMI, kg/m 2	6/5/8 25 6 5	8/2/11 2 5 6 4	.0.3 .0.7
	Alpha-1 antitrypsin, mg/dl	152 6 27	137 6 39	.0.1
	HIV status	Negative	Negative	NA
	IgE, IU/ml	228 6 526	119 6 104	.0.3
	Blood pressure, systolic/diastolic,	115 6 8/71 6 12	115 6 9/65 6 8 .0.8/.0.1
	mm Hg
	Heart rate, beats/min	70 6 11	70 6 10	.0.9
	Smoking history
	Age of initiation, yr	NA	21 6 5	N A
	Duration of smoking, yr	NA	4.1 6 2.5	NA
	Sessions/wk	NA	3.5 6 2.5	NA
	Urinary nicotine,* ng/ml	0	67 6 193	NA
	Urinary cotinine,* ng/ml	0	99 6 205	NA
	Carboxyhemoglobin, % Cough score † Sputum score † Pulmonary function parameters ‡	0 6 0.7 0.5 6 0.6 0.4 6 0.5	2.1 6 1.7 1.3 6 1.1 1.2 6 1.1	,0.02 ,0.008 ,0.007
	FVC, % predicted	106 6 12	98 6 15	.0.06
	FEV 1 , % predicted	105 6 11	98 6 13	.0.1
	FEV 1 /FVC, % observed	84 6 3	8 6 6 5	.0.06
	FEF 25-75% , % predicted	93 6 16	97 6 15	.0.4
	PEF, % predicted	101 6 15	103 6 15	.0.6
	TLC, % predicted	95 6 15	94 6 14	.0.8
	DL CO , % predicted	90 6 10	82 6 14	,0.04
	Percentage of emphysema,	1.5 6 1.8	0.6 6 0.6	.0.07
	2950 HU
	Small airway epithelium Number of cells recovered, 310 6	4.3 6 2.2	4.8 6 4.3	.0.6
	Percentage of inflammatory cells Percentage of epithelial cells x	1.0 6 0.7 98.9 6 0.8	1.0 6 1.0 99.1 6 0.8	.0.6 .0.9
	Percentage of ciliated cells	70.8 6 4.6	62.6 6 8.9	,0.005
	Percentage of secretory cells	9.6 6 4.6	14.5 6 5.6	,0.005
	Percentage of basal cells Percentage of intermediate cells BAL cells k Number of cells recovered, 310 6	11.2 6 7.5 8.6 6 4.4 12.6 6 7.5	4.5 6 4.1 17.9 6 6.3 8.6 6 5.2	,0.002 ,10 25 .0.08
	Percentage of macrophages	85.9 6 10.4	91.8 6 10.0	.0.05
	Percentage of neutrophils	2.7 6 2.4	1.4 6 2.3	.0.08
	Percentage of lymphocytes	8.9 6 8.1	5.7 6 8.1	.0.2
	Percentage of eosinophils	0.5 6 0.7	0.8 6 1.7	.0.6
	Definition of abbreviations: BAL = cells removed by bronchoalveolar lavage; BMI = body mass
	index; DL CO = diffusing capacity of the lung for carbon monoxide; FEF = forced expiratory flow;
	FEF 25-75% = forced expiratory flow, midexpiratory phase; HU = Hounsfield units; NA = not applicable;
	PEF = peak expiratory flow; TLC = total lung capacity.
	Data are presented as average 6 SD. P values of numeric parameters were calculated using a two-tailed Student's t test. P values of categorical parameters were calculated using a x 2 test.
	Values represent prebronchodilator measurements.
	*Undetectable urine nicotine was defined as less than 2 ng/ml, undetectable cotinine as less than
	5 ng/ml.
	†

  1

	▸ Additional material is
	published online only. To view
	please visit the journal online
	(http://dx.doi.org/10.1136/
	thoraxjnl-2015-208274).
	For numbered affiliations see
	end of article.
	Correspondence to
	Dr Ronald G Crystal,
	Department of Genetic
	Medicine, Weill Cornell
	Medical College, 1300 York
	Avenue, Box 164, New York,
	NY 10065, USA;
	geneticmedicine@med.cornell.
	edu
	Received 4 January 2016
	Revised 21 April 2016
	Accepted 27 April 2016

To cite: Strulovici-Barel Y, Staudt MR, Krause A, et al. Thorax Published Online First: [ please include Day Month Year] doi:10.1136/ thoraxjnl-2015-208274

Table 1

 1 Study Population*

			Healthy smokers †		COPD smokers ‡
				Who continued			Who continued
	Parameter	Non-smokers	All	smoking	Who quit	All	smoking	Who quit
	n	2 8	6 1	4 4	1 7	4 9	3 1	1 8
	Gender (M/F)	15/13	47/14	37/7	10/7	46/3	30/1	16/2
	Ethnicity (B/W/O) §	10/7/11	33/9/19	23/7/14	10/2/5	27/12/10	18/6/7	9/6/3
	Age	37±11	44±9	44±9	45±10	53±8	53±7	53±9
	BMI	27±5	28±5	27±4	30±4	27±4	25±3	29±5
	Smoking history
	Pack-year	-	23±12	24±12	20±8	32±14	32±15	34±12
	Pack per day	-	0.8±0.6	1.0±0.6	0.6±0.2	0.8±0.4	0.8±0.5	0.8±0.3
	Age of initiation	-	16±3	16±3	16±3	16±3	16±3	16±3
	Urine cotinine (ng/mL)	-	1693±961	1828±930	1323±979	1747±980	1953±959	1393±938
	Subjects with emphysema (n, %) ¶	1 (4%)	0 (0%)	0 (0%)	0 (0%)	13 (27%)	9 (29%)	4 (22%)
	Pulmonary function**
	FEV1	106±11	109±11	109±10	107±13	85±16	87±16	82±17
	FVC	107±11	111±10	110±10	111±11	108±16	109±17	105±15
	FEV1/FVC	83±5	80±5	81±4	79±6	63±6	64±6	63±7
	TLC	99±16	96±12	95±12	96±10	99±12	100±13	99±10
	DLCO	91±11	89±8	89±9	90±6	71±14	68±13	77±15
	GOLD stage (I/II)	-	-	-	-	31/18	20/11	11/7

  end-stage renal

	Figure 2 Levels of total circulating
	endothelium microparticles (EMPs), ACE + EMPs and apoptotic EMPs in
	plasma at baseline. Shown are data for
	non-smokers (n=28, green circles),
	healthy smokers (n=61, yellow circles)
	and smokers with COPD (n=49, red circles). (A) Total CD42b -CD31 + EMPs.
	The grey shaded area indicates the
	non-smoker mean±2 SDs. The % value
	above the smoker populations
	represents the proportion of smokers
	with EMP levels above that mean. (B) CD42b -CD31 + ACE + EMPs. Proportion of total CD42b -CD31 + EMPs in plasma that express ACE + .
	The grey shaded area represents the
	non-smoker mean±2 SDs. (C) Ratio of CD42b -CD62E + to CD42b -CD31 +
	EMPs. The dashed line indicates the lowest ratio of CD42b -CD62E + / CD42b -CD31 + EMPs in non-smokers.
	The % value below the smoker
	populations represents the proportion
	of smokers with a ratio below that
	level. (A-C) Bold dashed lines
	represent the mean for each group.
	Symbols inside the dots: A horizontal
	line indicates subjects with systemic
	hypertension; a vertical line indicates
	subjects with type 2 diabetes mellitus;
	a star indicates subjects with type 1
	diabetes.

Strulovici-Barel, Shaykhiev, Salit, et al.: Effects of Waterpipe Smoking

American Journal of Respiratory and Critical Care Medicine Volume jj Number jj | jj 2016

Strulovici-Barel Y, et al. Thorax 2016;0:1-8. doi:10.1136/thoraxjnl-2015-208274

Acknowledgments

Acknowledgements The authors thank M Elnashar, A Rogalski, E Blass, S Mootoo and T Wilson for their help in processing blood samples; the Clinical Operations and Regulatory Affairs, Department of Genetic Medicine for help with these studies; and N Mohamed for help in preparing this manuscript. These studies were supported, in part, by P50HL084936, R01HL107882, U01HL121828, UL1 TR000457, UL1 RR02414 and Hoffmann-La Roche.

)

These studies were supported in part by National Institutes of Health (NIH) grants R01 HL107882, 3R01 HL107882-2S1, and P20 HL113443 (R.G.C.); NIH Clinical and Translational Science Center grant UL1 TR000457 and NIH grant UL1 RR024143; and Qatar National Research Fund NPRP 09-742-3-194. The research reported in this publication was supported by the NIH and the Family Smoking Prevention and Tobacco Control Act. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the U.S. Food and Drug Administration.

Funding F. Hoffman-La Roche, National Institutes of Health (P50HL084936), (R01HL107882), (U01HL121828) (UL1 RR02414) and (UL1 TR000457).

Competing interests Hans Bitter, Sriram Sridhar, Sreekumar G. Pillai, Holly Hilton, Gerhard Wolff, Christopher S. Stevenson, Sudha Visvanathan and Jay S. Fine were all former employees of Hoffmann-La Roche.

impairment in gas transfer due to parenchymal destruction and is therefore used for the diagnosis of emphysema [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF]76 .

Reduction in DLCO can be produced by several combinations of reduction in the rate of carbon monoxide removal (kCO) and the accessible alveolar volume. An assessment of both components is essential in the interpretation of DLCO % predicted and can suggest a specific pathophysiological mechanism responsible for the reduction [START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF] . The single breath estimate alveolar volume should approach the total lung capacity minus the anatomic dead space (~200 ml). When the alveolar volume is low but the alveolar volume/total lung capacity ratio is normal (0.94 ± 0.07), the reduction in DLCO is caused by restrictive lung disease, but when the total lung capacity is normal or increased, and the alveolar volume/total lung capacity ratio is low, the reduction is typically secondary to obstructive lung disease (Figure 7) [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF][START_REF] Macintyre | Standardization of the Single-breath Determination of Carbon Monoxide Uptake in the Lung[END_REF][START_REF] Gaensler | Attachment for automated single breath diffusing Capacity Measurement[END_REF] . A low kCO can be caused by emphysema, diffused alveolar-capillary damage associated with connective tissue/autoimmune disease or reduced hemoglobin level [START_REF] Hughes | Historical Review: the Carbon Monoxide Diffusion Capacity (DLCO) and its Membrane (DM) and Red Cell (ɵVc) Components[END_REF] .

total and apoptotic EMPs in healthy cigarette smokers and COPD smokers who quit smoking for 12 months.

Study Design and Aims

We hypothesized that even light-use waterpipe smoking is associated with abnormalities in various clinical and biologic lung-related parameters. Based on the knowledge that the first abnormalities associated with cigarette smoking are in lung cells long before there are abnormalities in clinical parameters, we compared young (25±4 years), light-use (3.5±2.5 sessions/week, for an average of 4.1±2.5 years) waterpipe-only smokers (n=21) to nonsmokers (n=19) matched for gender and ethnicity, using a variety of lung-related parameters, including:

(1) blood carboxyhemoglobin levels; (2) cough and sputum scores, assessed using the St. A reduction in DLCO, a lung function test associated with emphysema and small airway disease, was observed in the young, light-use waterpipe smokers compared to nonsmokers. To assess this parameter as a marker of early disease, we followed a separate cohort of cigarette smokers with normal spirometry, no emphysema, as assessed on HRCT, and normal DLCO (n=59) and a group of cigarette smokers with normal spirometry, no emphysema and low DLCO (n=46) with a serial lung function for an average of 3.5 years for the risk of developing COPD. Levels of total and apoptotic plasma endothelial microparticles were found to be elevated in the young, light-use waterpipe smokers compared to nonsmokers. Elevated levels of EMPs, a marker of alveolar capillary destruction, have been previously shown to be associated with a reduction in DLCO in cigarette smokers, probably measuring early alveolar disease. To evaluate

Smoking

waterpipe smokers compared to nonsmokers 1,8,[23][24]26 . In addition, the manifestation of the cough and sputum symptoms was at an earlier age in waterpipe smokers than typically observed in cigarette smokers [25][26]141 . Our study (Article 1) assessed the effect of waterpipe smoking on lung health in young, light-use waterpipe-only smokers compared to nonsmokers. All individuals had normal spirometry and no emphysema observed on HRCT. Other than waterpipe smoking, the groups did not differ in industrial exposure or second-hand exposure to cigarette smoke.

Compared to the nonsmokers, the light-use waterpipe smokers had increased levels of carbon monoxide and an increase in cough frequency and sputum production. Despite having normal spirometry, the DLCO level was reduced in the light-use waterpipe smokers in comparison to the nonsmokers and 38% of the waterpipe smokers had an isolated reduction of DLCO. This observation of a reduction in DLCO level in, otherwise healthy, young, light-use waterpipe smokers is of interest as in a separate study (Article 2) assessing the risk for developing COPD in cigarette smokers, we observed that cigarette smokers with normal spirometry, but low DLCO were at 7-fold greater risk for developing COPD within <4 years than cigarette smokers with normal spirometry and normal DLCO.

Reduction in DLCO level

COPD is a global health issue and a leading cause of death worldwide [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF] . A major risk factor for developing COPD is cigarette smoking, but only a subset of cigarette smokers will develop COPD, a tendency likely associated with genetics [START_REF] Mannino | Global Burden of COPD: Risk Factors, Prevalence, and Future Trends[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF][START_REF] Lundback | Not 15 but 50% of Smokers Develop COPD?-Report from the Obstructive Lung Disease in Northern Sweden Studies[END_REF][START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF][START_REF] Fletcher | The Natural History of Chronic Airflow Obstruction[END_REF] . The prevalence of COPD among cigarette smokers is estimated to be at least 20%, but is projected to increase as the population ages [START_REF]The Global Strategy for the Diagnosis, Management and Prevention of COPD, Global Initiative for Chronic Obstructive Lung Disease[END_REF][START_REF] Rabe | Global Strategy for the Diagnosis, Management, and Prevention of Chronic Obstructive Pulmonary Disease: GOLD Executive Summary[END_REF] . The development of COPD usually takes decades as the impairment caused by the chronic exposure to toxins, such as those existing in cigarette smoke, accumulate for many years with very little effect on lung function [START_REF] Hogg | Pathophysiology of Airflow Limitation in Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Rennard | Natural Histories of Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Lokke | Developing COPD: a 25 Year Follow up Study of the General Population[END_REF] . However, once COPD manifests, there normal spirometry and normal DLCO and those with normal spirometry, but low DLCO. Further, the individuals followed over time with normal spirometry and low DLCO who developed COPD did not differ in these parameters from those with low DLCO who did not develop COPD.

Several studies have raised concerns about the use of a set cutoff for the definition of COPD (FEV1/FVC <0.7), and for the definition of low DLCO (<80% predicted) rather than using cutoff values based on a lower limit of normal calculated for each individual based on their age, gender and height [147][148][149][150] . In addition to using set values for the definitions of COPD and low DLCO we used several definitions of normal DLCO and FEV1/FVC to evaluate the risk for developing COPD. These included: (1) a gender and ethnicity-based lower limit of normal for DLCO % predicated and FEV1/FVC ratio calculated using an internal database of 405 healthy nonsmokers recruited from the general population, comprised of similar gender and ethnicity composition as in our study groups (Article 2); and (2) a lower limit of normal for the FEV1/FVC ratio based on gender, ethnicity, height and age calculated based on 74187 individuals 147 . The results of all analyses, using either cutoff of the FEV1/FVC ratio to define COPD and/or either cutoff of DLCO % predicted to define normal/low DLCO, were similar. Independent of the method used to determine the normal levels of DLCO, the data demonstrated that cigarette smokers with normal spirometry but low DLCO are at significantly higher risk for developing COPD than a comparable group of cigarette smokers with normal spirometry and normal DLCO (Table X, appendix V). These results advocate the need to assess DLCO levels in cigarette smokers and waterpipe smokers with normal spirometry who are falsely presumed to be normal.

Appendices Appendix I -Inclusion and Exclusion Criteria

Appendix II -Article 1 Supplemental Methods and Supplemental References Appendix III -Article 2 Supplemental Methods and Supplemental References Appendix IV -Article 3 Supplemental Methods and Supplemental References Appendix V -Table X 86

Appendix I -Inclusion and Exclusion Criteria

Nonsmokers

Inclusion criteria  Males and females, at least 18 years old  Provide informed consent  Normal physical examination  Normal routine laboratory evaluation, including general hematologic studies, general serologic/immunologic studies, general biochemical analyses, and urine analysis  HIV negative  Good health without history of chronic lung disease, including asthma, and without recurrent or recent (within 3 months) acute pulmonary disease  Normal PA and lateral chest X-ray  Normal electrocardiogram (sinus bradycardia, premature atrial contractions are permissible)  Not pregnant (females)  No history of allergies to medications used in the bronchoscopy procedure  Not taking any medications relevant to lung disease or having an effect on the airway epithelium or alveolar macrophages  Willingness to participate in the study  Self-reported never-smokers, with smoking status validated by the absence of nicotine and cotinine in urine (nicotine <2 ng/ml and cotinine < 5ng/ml) Exclusion criteria 172 1 COPD -chronic obstructive pulmonary disease; DLCO -diffusion capacity for carbon monoxide; FEV1 -forced expiratory rate for 1 sec; FVC -forced vital capacity; GOLD -global initiative lung disease. 2 Results detailed or summarized in the published manuscript (Article 2). 3 One normal spirometry/normal DLCO excluded from the study due to baseline FEV1/FVC ratio below the lower limit of normal. 4 One normal spirometry/low DLCO excluded from the study due to baseline FEV1 % predicted below the lower limit of normal. * Quanjer et al. 147 Titre : Effets de la Fumee de Nargile sur la Sante du Poumon Mots clés : nargile, fumee, pumon Résumé : La Chicha qui sert à fumer du tabac parfumé est utilisé par des millions de personnes. Il y a peu de données sur les effets du chicha sur la santé, peu de régulation et les utilisateurs pensent que la chicha n'est ni addictif ni nocif. Pour évaluer les effets précoces de la chicha sur les poumons nous avons comparé des fumeurs de chicha occasionnels et des non fumeurs pour les paramètres cliniques et biologiques. L'utilisation de la chicha augmentait la toux et les expectorations ainsi que le niveau sanguin de carboxyhemoglobine. Ces modifications étaient associées à des modifications du métabolome des secrétions pulmonaires, ainsi que de la modification de l'épithelium pulmonaire dans sa composition et son transcriptome. Les fumeurs présentaient une diminiution de la capacité de diffusion qui et un marqueur prédictif du développement de la BPCO. Ils avaient également une augmentation du niveau plasmatique des microparticules endothéliales qui sont un marqueur de la destruction alvéolaire. Notre étude démontre que l'utilisation occasionnelle chez les jeunes de la chicha peut avoir des conséquences sur les maladies pulmonaires.

Title : Effects of Waterpipe Smoking on the Human Lung

Keywords : waterpipe, smoking, lung Abstract : Waterpipe, an instrument for smoking fruit-flavored tobacco, is used by millions worldwide. There is limited data on its health effects, no regulations to its use, and users believe smoking it is not as harmful or addictive as cigarette smoking. To assess the early effects of waterpipe smoking on lung health, light-use waterpipe smokers with normal spirometry were assessed for lung clinical and biologic abnormalities compared to nonsmokers. Waterpipe smokers had increased cough and sputum, increased blood carboxyhemoglobin levels, abnormal lung epithelial lining fluid metabolome profile, abnormal small airway epithelium (SAE) cell composition, and markedly abnormal SAE and alveolar macrophage transcriptomes. They also had reduced diffusion capacity, a lung function marker of high risk for developing COPD in cigarette smokers, and high plasma levels of total and apoptotic endothelial microparticles, biomarkers of alveolar capillary destruction in COPD cigarette smokers that persists despite smoking cessation. These studies suggest that even young, light-use waterpipe smokers are likely at risk for developing lung disease.