Manuela M Bergmann
email: bergmann@dife.de

Madlen Schütze

Annika Steffen

Heiner Boeing

Jytte Halkjaer

Anne Tjonneland

Noémie Travier

Antonio Agudo

Nadia Slimani

Sabina Rinaldi

Teresa Norat

Dora Romaguera

Sabine Rohrmann

Rudolf Kaaks

Marianne Uhre Jakobsen

Kim Overvad

Ulf Ekelund

Elizabeth A Spencer

Laudina Rodríguez

Maria J Sánchez

Miren Dorronsoro

Aurelio Barricarte

María-Dolores Chirlaque

Philippos Orfanos

Androniki Naska

Antonia Trichopoulou

Domenico Palli

Sara Grioni

Paolo Vineis

Salvatore Panico

Rosario Tumino

Elio Riboli

Nicholas J Wareham

Bas Bueno-De-Mesquita

Anne May

Petra Hm Peeters

The association of lifetime alcohol use with measures of abdominal and general adiposity in a large-scale European cohort

Keywords: alcohol, waist circumference, waist-to-hip ratio, BMI, multi-centric cohort study

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Abstract

Background:

The relation between lifetime use of alcohol and measures of abdominal and general adiposity is unknown.

Methods:

Among 99,381 men and 158,796 women of the European Prospective Investigation into Cancer and Nutrition (EPIC) study, means of waist circumference (WC), waist-to-hip-ratio (WHR) and body mass index (BMI), and odds ratios (OR) for a larger WC than predicted for a given BMI (WClp=positive residuals of gender specific linear regression of BMI on WC) across categories of average lifetime use of alcohol (total, from wine and from beer) were calculated, all adjusted for socio-demographic, lifestyle and health factors.

Results:

WC, WHR and BMI in men using lifetime ≤6g/d alcohol were 95.1cm, 0.942 and 27.3kg/m 2 , and 96.2cm, 0.961 and 28.3kg/m 2 when using >96g/d. WC and WHR in women was 83.2cm and 0.813 for ≤6g/d, and 84.6cm and 0.830 for >60g/d while BMI deviated only slightly with the lowest BMI (26.7kg/m 2 ) observed for >6-24g/d. Compared to ≤6g/d, OR for a WClp in both genders increased steadily across categories of alcohol use (up to 1.40 (95% confidence interval 1.32, 1.49) in men using >60g/d and 1.63 (1.54, 1.73) in women using >24g/d); though increase was higher for alcohol from beer than from wine (p for difference between beer and wine<0.001 (men) and =0.002 (women)).

Introduction

Abdominal fat accumulation, independently from general adiposity, increases the risk for adverse health outcomes [START_REF] Pischon | General and Abdominal Adiposity with Risk of Death in Europe[END_REF]. The association of alcohol use with measures of abdominal and general adiposity in cross-sectional and prospective epidemiological studies was so far inconclusive. Abdominal adiposity was more prominent in men or in women who used alcohol heavily [START_REF] Dallongeville | Influence of alcohol consumption and various beverages on waist girth and waist-to-hip ratio in a sample of French men and women[END_REF][START_REF] Duncan | Association of the waist-to-hip ratio is different with wine than with beer or hard liquor consumption. Atherosclerosis Risk in Communities Study Investigators[END_REF][START_REF] Halkjaer | Food and drinking patterns as predictors of 6-year BMI-adjusted changes in waist circumference[END_REF][START_REF] Laws | Behavioral covariates of waist-to-hip ratio in Rancho Bernardo[END_REF][START_REF] Slattery | Associations of body fat and its distribution with dietary intake, physical activity, alcohol, and smoking in blacks and whites[END_REF]. However, some studies did not reveal such a relationship [START_REF] Halkjaer | Intake of macronutrients as predictors of 5-y changes in waist circumference[END_REF][START_REF] Koh-Banerjee | Prospective study of the association of changes in dietary intake, physical activity, alcohol consumption, and smoking with 9-y gain in waist circumference among 16 587 US men[END_REF] or did not show the relationship in both genders [START_REF] Halkjaer | Food and drinking patterns as predictors of 6-year BMI-adjusted changes in waist circumference[END_REF]. Also results on types of beverages and anthropometric measures vary depending on gender and country [START_REF] Dallongeville | Influence of alcohol consumption and various beverages on waist girth and waist-to-hip ratio in a sample of French men and women[END_REF][START_REF] Duncan | Association of the waist-to-hip ratio is different with wine than with beer or hard liquor consumption. Atherosclerosis Risk in Communities Study Investigators[END_REF][START_REF] Halkjaer | Food and drinking patterns as predictors of 6-year BMI-adjusted changes in waist circumference[END_REF][START_REF] Lukasiewicz | Alcohol intake in relation to body mass index and waist-to-hip ratio: the importance of type of alcoholic beverage[END_REF][START_REF] Vadstrup | Waist circumference in relation to history of amount and type of alcohol: results from the Copenhagen City Heart Study[END_REF].

Alcohol-related body measures may also result from metabolic changes due to a long term exposure to alcohol and therefore, information on a long term exposure to alcohol is important. However, most of the previous studies are based on information on use of alcohol over a short period of time, e.g. in the past year, month, or week, and lifetime abstainers were not distinguished from users of alcohol who had quitted at the time of the measurement. In the European Prospective Investigation into Cancer and Nutrition (EPIC) information on alcohol use in the past was collected, offering the opportunity to investigate the role of lifetime alcohol use on measures of abdominal and general adiposity.

Methods

Study Population

The European Prospective Investigation into Cancer and Nutrition (EPIC) is an ongoing multi-centric cohort study. The study has been described in full elsewhere [START_REF] Riboli | European Prospective Investigation into Cancer and Nutrition (EPIC): study populations and data collection[END_REF]. In brief, the EPIC population consists of 521,448 individuals, ages 25 to 70 years, recruited from the general population in 23 centres in 10 European countries during 1992-2000. Persons who accepted the invitation to participate in the study gave informed consent and completed questionnaires on their diet, lifestyle, and medical history, and visited a study centre where a blood sample and anthropometric measurements were taken.

Because we used a common data set created also for prospective data analyses, subjects with unknown vital status during follow-up (n=1,517) had been excluded. Further, whose ratio of energy intake to estimated energy requirement calculated from body weight, height, and age was in the top or bottom 1% (n=10,273) was excluded because alcohol use may have been reported invalidly too [START_REF] Bingham | Comparison of dietary assessment methods in nutritional epidemiology: weighed records v. 24 h recalls, food-frequency questionnaires and estimated-diet records[END_REF]. We further excluded participants who had not filled out the dietary or lifestyle questionnaires (n=6,611), were pregnant at recruitment (n=623), had the anthropometric examination more than one year apart from the recruitment data collection (n=22,084), had extreme or implausible values in body mass index (BMI<16), height (<1.30 m) or waist circumference (WC>160) (n=397), had no anthropometric measures (n=1,429), had missing information (n=4,072) or an implausible high average lifetime alcohol use of >250 g/d (n=158). Information on lifetime use of alcoholic beverages was not available for the study centres Naples (Italy), Bilthoven (The Netherlands), Sweden and Norway (n=116,149). We also excluded the health conscious part of the Oxford cohort (Great Britain) (n=47,651; 85% of the British cohort)) and the French cohort (n=51,267) since anthropometry was self-reported (Park et al., 2011). Therefore, 258,177 (99,381 men and 158,796 women) participants from Denmark (Aarhus, Copenhagen), Germany (Heidelberg, Potsdam), Greece, Italy (Florence, Varese, Ragusa, Turin), the Netherlands (Utrecht), Spain (Asturias, Granada, Murcia, Navarra, San Sebastian), and the United Kingdom (Cambridge, Oxford), representing 50% of the original cohort, were eligible for this analysis.

The study was approved by ethical review boards of the single centres and the International Agency for Research on Cancer in Lyon, France.

Assessment of lifetime alcohol use

Lifetime alcohol use was assessed based on data collected during the recruitment procedure by applying questionnaires on lifestyle and diet during the past 12 month. Glasses alcoholic beverage used at certain ages and during the 12 month prior to the recruitment have been selfreported and transformed into grams alcohol used per day by applying gender specific definitions of a standard drink for each country included in the EPIC study. We favoured this approach, since the application of common standard drinks for the calculation of alcohol intake involve some methodological problems [START_REF] Lemmens | The alcohol content of self-report and 'standard' drinks[END_REF], and specific data in the form of 36,994 24-hour dietary recalls [START_REF] Slimani | Structure of the standardized computerized 24-h diet recall interview used as reference method in the 22 centers participating in the EPIC project. European Prospective Investigation into Cancer and Nutrition[END_REF] had been available. Variations of concentrations and amounts of alcohol used on weekdays vs. weekend days and filling levels were taken into account by calculating weighed averages of alcohol content of one glass wine, beer, and hard liquor (based on a sub-sample of EPIC participants (N=12,443) who reported the use of alcoholic beverages during the 24-hour dietary recall).

The average alcohol use in g/d at ages 20, 30, 40, and 50 years and at recruitment was then calculated as the sum of the number of glasses per week of the respective beverage multiplied with the calculated alcohol content per glass divided by seven. Because time intervals across points in time where information on alcohol use was available were not equal for all participants, lifetime use of alcohol was calculated as the weighted average of intakes at different ages, including recruitment, with weights equal to the total subject-specific time of investigation, i.e. the individual time until recruitment Participants were categorized as 'never users' of alcohol when reported alcohol use was zero at all points in time and as 'former users' when use of alcohol was reported for past points in time but not for the 12 month before recruitment.

Assessment of anthropometric data

Weight and height were measured by trained personnel with subjects wearing no shoes.

Depending on study centre, WC (cm) was measured either at the narrowest torso circumference, at the midpoint between the lower ribs and iliac crest, or by a combination of methods, whereby in most centres, WC was measured at the narrowest torso circumference.

Hip circumference (HC; cm) was measured horizontally at the level of the largest lateral extension of the hips, or over the buttocks. Body weight, WC and HC were corrected for differences in clothing worn during measurement. A constant was subtracted from the original measure depending on whether study participants were measured in light underwear (no correction), in normal clothing without shoes (weight minus 1.5 kg; circumference minus 2.0 cm), or in light clothing without shoes (weight minus 1.0 kg) [START_REF] Haftenberger | Overweight, obesity and fat distribution in 50-to 64-year-old participants in the European Prospective Investigation into Cancer and Nutrition (EPIC)[END_REF].

Assessment of data on confounding factors

Standardized self-administered questionnaires were applied at recruitment to collect information on the attained educational level, physical activity during the 12 month prior to the recruitment, smoking status including number of cigarettes and duration of smoking, and in women also reproductive history and hormone replacement therapy. [START_REF] Riboli | European Prospective Investigation into Cancer and Nutrition (EPIC): study populations and data collection[END_REF].

Dietary variables were derived from country specific validated food-frequency questionnaires which covered between 98 and 266 food items. Self-reported frequencies of consumption were transformed into calorie intake per day by applying a standardized food-composition table [START_REF] Slimani | The EPIC nutrient database project (ENDB): a first attempt to standardize nutrient databases across the 10 European countries participating in the EPIC study[END_REF].

Statistical Analysis

Adjusted means of WC, WHR (WC divided by HC), and BMI (weight (kg) divided by squared height (m)) across categories of average lifetime use of alcohol were calculated for men and women separately using generalized linear models. Cut points for categorization of alcohol use were based on the assumption that any glass of alcoholic beverage (drink) contains in average 12 g/d alcohol: never users, former users, and lifetime users of alcohol in g/d of ≤6, >6-12, >12-24, >24-60, >60-96, >96. Means were adjusted for age (years, continuous), study centre, education (no graduation or primary school, technical or professional school, secondary school, university degree, unknown), physical activity (index, combining work activity and leisure time physical activity with gender specific cut points: inactive, moderately inactive, moderately active, active, unknown), non-alcoholic energy intake (kcal, continuous), smoking status (never smokers, ex-smokers for ≥10 or for <10 years, smokers at recruitment of <15 or of ≥15 cigarettes per day, status unknown), smoking duration (<10yrs, 10-<20yrs, 20-<30yrs, 30-<40yrs, 40 or more, unknown), and prevalent diseases (self-reported myocardial infarction, stroke, cancer, diabetes, hypertension, unknown) at the time of recruitment. Models for women additionally included menopausal status (premenopausal, postmenopausal, unknown), and parity (nulliparous, 1-2, >2 live birth, unknown) and models for WC in both genders height and weight. Linear regression was used to investigate the trend of the association of alcohol use and anthropometric measures among lifetime users (per 12 g/d increment) continuously.

Since abdominal adiposity was in the focus of this analysis, we investigated the relative contribution of alcohol to a positively deviating WC from that predicted by the BMI. Firstly, for men and women separately, positive residuals of linear regression analysis with BMI as the independent and WC as the dependent variable were utilized to create a dichotomized variable (yes, no) for a WC that was larger than predicted (WClp) for a given BMI [START_REF] Han | Separate associations of waist and hip circumference with lifestyle factors[END_REF]. Secondly, the relative contribution of each category of lifetime alcohol use, and alcohol from wine and from beer to a larger WC than predicted was investigated by employing logistic regression with the lifetime users of ≤6 g/d alcohol as the reference category, mutually controlling for alcohol from wine or from beer in the respective models.

Models were further adjusted for alcohol from other beverages, and variables listed for the adjusted means. Heterogeneity of odds ratios (OR) for alcohol from wine and alcohol from beer across EPIC-centres was tested calculating Cochran's Q and I 2 [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF]. While Cochran's Q gives the test statistic for the heterogeneity, I 2 describes the percentage of total variation across study centres, which is due to heterogeneity rather than due to chance.

All p-values presented are two-tailed and the level of significance was 0.05. Analyses were performed using SAS 9.1 (SAS Institute, Cary, NC).

Results

Men and women who did use alcohol in the past but not at recruitment (former alcohol users) were older, more likely to be also former smokers (men) or have a prevalent disease (myocardial infarction, stroke, cancer, diabetes, or hypertension) than those who were lifetime alcohol users (table 1). Among lifetime heavy alcohol using men of >96 g/d, proportion of long term smokers (>20 years) and non-alcoholic calorie and dietary fat intake was highest.

The highest proportion of university graduates was seen among men who used ≤24 g/d alcohol. Among women who used lifetime >24 g/d alcohol average age was little younger and the percentage of smokers at the time of recruitment, nulliparity, or university degree was highest.

Lifetime alcohol use was positively associated with WC, WHR and BMI in men (table 2). Men who used lifetime on average >96 g/d had a 1.1 cm larger WC and a 1.0 kg/m 2 higher BMI than those who used ≤6 g/d alcohol. WC, WHR and BMI of men who never used alcohol or who were former users were comparable with that of men who used ≤24 g/d. WC and WHR were highest among heavy alcohol users of >96 g/d alcohol. Differences of means between lowest and highest category of alcohol use for WC, WHR and BMI were greater for beer than for wine.

In women lifetime use of alcohol was positively related to WC and WHR and U-shaped to BMI with the lowest BMI observed among women who used on average >6-24 g/d alcohol.

Conversely, the difference of WC between the lowest and the highest category of lifetime alcohol use was 1.7 cm; and WC and WHR increased steadily across categories of lifetime alcohol use (table 2). Women who never used alcohol during lifetime were heaviest (BMI 27.6 kg/m 2 ), but their WC was comparable to those women who used low amounts of alcohol (≤12 g/d).

While in men differences of means of WC, WHR and BMI between lowest and highest category of alcohol use were greater for beer than for wine it wasn't as clear in women (table 2).

In table 3 the OR of a WClp reflect the relative risk of abdominal adiposity across categories of lifetime alcohol use. In men and in women the OR increased steadily across categories of lifetime alcohol use which was true for alcohol from wine and from beer.

However, the OR for alcohol from beer were higher than that for the same amount of alcohol from wine, and the differences appeared more pronounced in women compared to men. The OR for wine and beer varied across countries (figure 1 and2). Between 50% and 80% of the total variation across study centres was due to heterogeneity rather than due to chance. In general, OR for beer were higher in men and women residing in beer preferring countries (Denmark, Germany, The Netherlands, and UK). In the Mediterranean centres, the role of beer for WClp was not so clear, though a positive relationship for beer was also seen for some of these centres (e.g. Asturias, Navarra (men), and Varese (women). However, Wine was also positively associated with a WClp in men and women of most centres.

Discussion

In men and women participating in this large European cohort study, lifetime alcohol use was related to the risk of abdominal adiposity, whereas alcohol from beer played a prominent role in men and in women as well, though risk was also higher for alcohol from wine.

Lifetime alcohol use was also positively associated with general adiposity in men. Although differences of adjusted means of WC, WHR and BMI appeared small, on the population level they exhibit health relevant differences.

Positive associations between the use of alcohol and abdominal adiposity have consistently been reported for men and frequently for women in cross-sectional [START_REF] Dallongeville | Influence of alcohol consumption and various beverages on waist girth and waist-to-hip ratio in a sample of French men and women[END_REF][START_REF] Han | Separate associations of waist and hip circumference with lifestyle factors[END_REF][START_REF] Marti | Body fat distribution in the Finnish population: environmental determinants and predictive power for cardiovascular risk factor levels[END_REF][START_REF] Randrianjohany | The relationship between behavioural pattern, overall and central adiposity in a population of healthy French men[END_REF][START_REF] Slattery | Associations of body fat and its distribution with dietary intake, physical activity, alcohol, and smoking in blacks and whites[END_REF][START_REF] Troisi | Cigarette smoking, dietary intake, and physical activity: effects on body fat distribution--the Normative Aging Study[END_REF] or prospective studies [START_REF] Grinker | Body habitus changes among adult males from the normative aging study: relations to aging, smoking history and alcohol intake[END_REF][START_REF] Vadstrup | Waist circumference in relation to history of amount and type of alcohol: results from the Copenhagen City Heart Study[END_REF][START_REF] Vernay | Alcohol consumption and insulin resistance syndrome parameters: associations and evolutions in a longitudinal analysis of the French DESIR cohort[END_REF]. Studies showing different results focused on the role of alcohol for the change of anthropometric measures [START_REF] Halkjaer | Food and drinking patterns as predictors of 6-year BMI-adjusted changes in waist circumference[END_REF][START_REF] Halkjaer | Intake of macronutrients as predictors of 5-y changes in waist circumference[END_REF][START_REF] Koh-Banerjee | Prospective study of the association of changes in dietary intake, physical activity, alcohol consumption, and smoking with 9-y gain in waist circumference among 16 587 US men[END_REF].

Alcohol, in distinction to other sources of energy, cannot be stored. This is why it has absolute priority in metabolism using different pathways, e.g. increasing thermogenesis or decreasing lipolysis [START_REF] Buemann | How does the body deal with energy from alcohol?[END_REF][START_REF] Suter | Is alcohol consumption a risk factor for weight gain and obesity?[END_REF][START_REF] Yeomans | Alcohol and food intake[END_REF]. Alcohol metabolism may also trigger endocrine changes, such as increased cortisol secretion or modified steroid metabolism in the liver [START_REF] Bjorntorp | Obesity and diabetes[END_REF][START_REF] Sarkola | The Role of the Liver in the Acute Effect of Alcohol on Androgens in Women[END_REF][START_REF] Suter | Is alcohol consumption a risk factor for weight gain and obesity?[END_REF].

Androgen hormone status was shown to be related to fat distribution, whereas men with lower levels and women with higher levels of androgen sex hormones were at higher risk of abdominal fat accumulation [START_REF] Blouin | Androgens and body fat distribution[END_REF][START_REF] Khaw | Lower endogenous androgens predict central adiposity in men[END_REF]). Alcohol use in other studies was associated with higher levels of androgen hormones in women and lower levels in men [START_REF] Eriksson | Sex hormone response to alcohol[END_REF][START_REF] Sarkola | Acute effect of alcohol on androgens in premenopausal women[END_REF].

There was a role of beverage type for the degree of abdominal adiposity in our study, where alcohol from beer showed a higher risk of having a larger waist circumference than would be expected from the BMI than alcohol from wine. There was heterogeneity across centres of the EPIC study, and the Southern (except most of the Spanish) centres showed wider confidence intervals for alcohol from beer due to lower numbers of beer users in these centres. Conversely, wine use seemed to be common among women and men in almost all centres. This was indicated by the generally smaller confidence intervals for alcohol from wine in men and in women; whereas the confidence intervals for alcohol from beer were small only in countries or centres where beer was more common.

One prospective study in a Danish population found in women, but not men, high intakes of beer to be associated with gain in WC [START_REF] Halkjaer | Food and drinking patterns as predictors of 6-year BMI-adjusted changes in waist circumference[END_REF]. In cross-sectional studies beer intake was either not related to WHR or BMI [START_REF] Bobak | Beer and obesity: a cross-sectional study[END_REF][START_REF] Lukasiewicz | Alcohol intake in relation to body mass index and waist-to-hip ratio: the importance of type of alcoholic beverage[END_REF] or it was related in both genders and all ethnic groups [START_REF] Slattery | Associations of body fat and its distribution with dietary intake, physical activity, alcohol, and smoking in blacks and whites[END_REF][START_REF] Wannamethee | Alcohol and adiposity: effects of quantity and type of drink and time relation with meals[END_REF]. However, comparable to our study, abdominal adiposity was also seen for other types of beverage, independent from BMI [START_REF] Dallongeville | Influence of alcohol consumption and various beverages on waist girth and waist-to-hip ratio in a sample of French men and women[END_REF].

Since there are many possible confounders [START_REF] Suter | Is alcohol consumption a risk factor for weight gain and obesity?[END_REF], it also cannot be ruled out that residual confounding influenced our results. For instance, alcohol use in general and type of beverage in particular may be differentially related to smoking. Smoking has been shown to be associated with abdominal adiposity [START_REF] Travier | Smoking and body fatness measurements: a cross-sectional analysis in the EPIC-PANACEA study[END_REF]. In our analysis, in both genders, among current smokers at recruitment using lifetime alcohol heavily (>60 g/d) the BMI was lower than that of never or ex-smokers using the same amount of alcohol. In contrast, the WC among current smokers who used alcohol lifetime heavily was noticeable higher.

Our study has limitations: We had no information about the onset and times and reasons for cessation of alcohol use. The categories of average lifetime use of alcohol may misclassify never, former or lifetime users of alcohol due to misreporting or due to the design of the question that inquires only about the use of alcohol at defined ages. Furthermore, the retrospective direction of the information collected on lifetime alcohol use is prone to recall bias. This problem was already addressed in the 1958 British Birth Cohort Study where it was shown that a certain proportion of the individuals' retrospective reports about abstention, occasional or regular drinking of alcohol were inconsistent [START_REF] Caldwell | Drinking histories of self-identified lifetime abstainers and occasional drinkers: findings from the 1958 British Birth Cohort Study[END_REF]. However, the opportunities to obtain information on lifetime use of alcohol in a population based cohort study are limited, and the method used in our study was judged to generate acceptable information [START_REF] Friesema | Measurement of lifetime alcohol intake: utility of a selfadministered questionnaire[END_REF][START_REF] Lemmens | Measurement of lifetime exposure to alcohol: data quality of a self-administered questionnaire and impact on risk assessment[END_REF]. In our study, the amount of alcoholic beverages used in history by comparable age cohorts at certain ages was validated by comparison of self-reported data with the respective per capita measures [START_REF] Klipstein-Grobusch | Trends in self-reported past alcoholic beverage consumption and ethanol intake from 1950 to 1995 observed in eight European countries participating in the European Investigation into Cancer and Nutrition (EPIC)[END_REF].

Further, information on lifetime alcohol use was not available in all centres, which is why we had to exclude participants of the respective centres from this analysis. Due to further exclusion of the health conscious part of the UK and the French cohort because anthropometric variables were self-reported, the final data set consisted of about 50% of the original cohort. To test the sensitivity of our results to these exclusions, we rerun the models with the cohorts included, and the associations remained unchanged.

Strengths of our study include a large sample size of men and women throughout Europe, information on the use of alcohol over adult life period, including never and former use of alcoholic beverages, and measured body weight, height, waist and hip circumference. We adjusted the models for dietary, lifestyle and health factors potentially confounding the association and performed a sensitivity analysis by excluding individuals who reported a prevalent myocardial infarction, stroke, cancer, diabetes, or hypertension at recruitment. Future research in this field may investigate biological pathways of alcohol as a possible long term determinant of abdominal adiposity including interaction of sex and stress related hormones, genetic variations and behavioural factors. It also would be important to investigate whether the risk of abdominal adiposity could be reversed by cutting down the alcohol use and how long it would take to see effects.

In conclusion, lifetime use of alcohol in men is positively related to abdominal and general adiposity, and in women it is related to abdominal adiposity. In this context, beer may contribute additionally to abdominal adiposity.

Figure 1

Odds ratio of having a larger waist circumference than expected from BMI for increase of one more alcoholic drink (12 g alcohol) per day across centres of the EPIC study in men (Adjusted for age, centre, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, prevalent chronic diseases) Figure 2 Odds ratio of having a larger waist circumference than expected from BMI for increase of one more alcoholic drink (12 g alcohol) per day across centres of the EPIC study in women (Adjusted for age, centre, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, prevalent chronic diseases, menopausal status and number of live birth) estimated by linear regression in lifetime users only; + positive trend, -negative trend, significant at the 0.05 level 3 adjusted for age, center, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, prevalent chronic diseases, height and weight (women also for menopausal status and number of live birth). 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0 0,0 0,5 1,0 1,5 2,0 2,5 3,0 3,5 4,0 4,5 5,0 5,5 6,0

  2

Table 1 .

 1 Frequency distribution and description of characteristics at recruitment across categories of lifetime use of alcohol in a sub-sample of the EPIC study

		Use of alcohol	Overall	Never	Former	Lifetime
		g/d 1		0	>0	≤6	>6-12		>12-24	>24-60	>60-96	>96
	Men
		N	99381	1715	4792	13843	15971		24919	29258	6006	2877
		Age (mean, SD, in years)	53.9 (8.4)	54.0 (10.8)	55.9 (9.6)	54.0 (9.5)	54.8 (8.4)	54.2 (7.9)	53.3 (7.8)	52.8 (7.9)	52.5 (8.0)
	non-alcoholic calorie intake (mean, SD, kcal 2 )	2269 (632)	2281 (679) 2262 (675)	2269 (648)	2230 (617)	2228 (608)	2284 (630)	2364 (639)	2494 (674)
		Total fat intake (mean, SD)	98.3 (36.0)	98.3 (36.0) 94.4 (35.1)	95.2 (33.4)	93.7 (31.9)	94.6 (31.6)	98.0 (32.9)	105.4 (35.2)	114.5 (38.6)
		University degree (%)	25.0	18.7	12.5	25.4	28.5		29.5	25.2	16.8	8.0
	High/very high physical activity (%)	48.6	41.6	48.3	46.2	49.3		50.8	49.6	45.4	40.9
		Smokers (%)	31.7	27.7	30.6	25.2	26.1		29.5	35.3	44.8	54.5
		Ex-smokers (%)	38.6	24.2	42.5	33.3	38.4		40.9	41.0	35.4	29.3
		>20 years of ever smoking (%) Prevalent chronic diseases 3 (%)	65.4 11.4	66.6 13.9	70.1 22.7	58.5 12.3	60.6 12.2		63.3 10.1	67.2 10.1	76.0 10.2	83.0 12.6
	Women
		N	158796	19863	10906	70769	31045		20533	5495	185
		Age (mean, SD, in years)	52.9 (9.0)	52.5 (10.3)	54.5 (9.7)	52.7 (9.1)	53.2 (8.4)	52.8 (8.1)	52.2 (7.8)	51.7 (8.5)
	non-alcoholic calorie intake (mean, SD, kcal 2 )	1867 (520)	1824 (525) 1863 (536)	1874 (521)	1881 (506)	1868 (515)	1862 (539)	1907 (580)
		Total fat intake (mean, SD)	78.1 (27.2)	79.0 (28.7) 77.2 (27.8)	78.0 (27.3)	77.8 (25.9)	78.2 (26.4)	78.9 (28.2)	80.7 (31.5)
		University degree (%)	14.7	7.3	8.5	14.3	18.0		19.5	22.9	24.3
	High/very high physical activity (%)	63,3	78.6	72.4	63.0	56.9		57.2	53.6	55.1
		Smokers (%)	21.3	15.1	18.4	19.6	22.7		28.1	37.4	43.2
		Ex-smokers (%)	21.7	9.2	18.6	20.3	27.1		29.6	30.5	29.2
		>20 years of ever smoking (%) Prevalent chronic diseases 3 (%)	52.3 9.2	44.7 10.5	54.5 15.2	49.0 8.9	53.3 8.0		57.6 8.0	64.9 8.6	63.5 10.0
		Prämenopausal (%)	31.1	37.3	28.1	31.8	28.3		28.8	30.3	31.9
		Nulliparous (%)	13.0	10.5	13.3	12.0	13.7		15.9	20.9	27.0
	1 2 3	1-2 live birth (%) 12 grams alcohol equivalents about one drink. 59.1 1 kcal = 4.186kJ Self-report of myocardial infarction, stroke, cancer, diabetes, hypertension at the time of enrolment into the study. 53.1 51.4 60.7 61.5	60.0	58.1	51.4

Table 2 .

 2 Mean body measures according to categories of lifetime alcohol use in a sub-sample of the EPIC study

	Use of alcohol	Never	Former	Lifetime

Table 3 .

 3 Odds ratios for having a waist circumference larger than predicted for a given body mass index across categories of lifetime alcohol use of in a subadjusted for age, centre, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, prevalent chronic diseases 3 adjusted for age, centre, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, prevalent chronic diseases, menopausal status and number of live birth 4 additionally mutually adjusted for alcohol from beer and from wine.

	sample of the EPIC study
	Use of alcohol	Never	Former	Lifetime
		g/d 1	0	>0	≤6	>6-12	>12-24	>24-60	>60
	Men
	Total alcohol	N OR (95% CI) 2	1715 1.02 (0.92-1.14)	4792 1.07 (1.00-1.15)	13843 REF	15971 1.01 (0.96-1.06) 1.06 (1.02-1.11) 1.20 (1.15-1.26) 1.40 (1.32-1.49) 24919 29258 4947
	Alcohol from beer § OR (95% CI) 2,4 N	10431 0.98 (0.93-1.03)	10402 1.03 (0.98-1.09)	40716 REF	16362 1.06 (1.02-1.10) 1.15 (1.10-1.20) 1.36 (1.29-1.44) 1.76 (1.53-2.02) 12968 7522 980
	Alcohol from wine § OR (95% CI) 2,4 N	7493 1.04 (0.99-1.10)	5813 1.04 (0.98-1.11)	43510 REF	14736 1.05 (1.00-1.09) 1.06 (1.01-1.10) 1.13 (1.07-1.19) 1.28 (1.18-1.40) 12307 12248 3274
	P for difference between	0.098	0.798		0.650	0.007	<0.001	<0.001
	wine and beer
	Women
	Total alcohol	N OR (95% CI) 3	19863 1.04 (1.00-1.07)	10906 1.12 (1.08-1.18)	70769 REF	31045 1.10 (1.07-1.13) 1.31 (1.27-1.36) 20533	3257 1.63 (1.54-1.73)
	Alcohol from beer § OR (95% CI) 3,4 N	62331 0.98 (0.95-1.00)	14958 1.00 (0.96-1.04)	76526 REF	3607 1.32 (1.23-1.42) 1.51 (1.33-1.71) 1133	241 2.57 (1.93-3.45)
	Alcohol from wine § OR (95% CI) 3,4 N	30132 1.05 (1.02-1.08)	11335 1.14 (1.09-1.19)	83641 REF	20321 1.13 (1.10-1.17) 1.34 (1.28-1.40) 11129	2238 1.59 (1.45-1.74)
	P for difference between	<0.001	<0.001		<0.001	0.083	0.002
	wine and beer
	OR Odds ratio; 95% CI 95 percent confidence interval 1 12 grams alcohol equivalents about one drink.

2

adjusted for age, center, education, physical activity, smoking status, smoking duration, non-alcoholic calorie intake, and prevalent chronic diseases, (women also for menopausal status and number of live birth).

additionally mutually adjusted for alcohol from beer or from wine

Acknowledgements

This publication arises from the project PANACEA, which has received funding from the European Union, in the framework of the Public Health Programme (project number: 2005328). The work was further financially supported by the European Commission: Public Health and Consumer Protection Directorate 1993-2004; Research Directorate-General 2005; Ligue contre le Cancer, Societé 3M, Mutuelle Générale de l'Education Nationale, Institut National de la Santé et de la Recherche Médicale (INSERM) (France); German Cancer Aid, German Cancer Research Center, Federal Ministry of Education and Research (Germany); Danish Cancer Society (Denmark); Health Research Fund (FIS) of the Spanish Ministry of Health, The participating regional governments and institutions (Spain); Cancer Research UK, Medical Research Council, Stroke Association, British Heart Foundation, Department of Health, Food Standards Agency, the Wellcome Trust (United Kingdom); Greek Ministry of Health and Social Solidarity, Hellenic Health Foundation and Stavros Niarchos Foundation (Greece); Italian Association for Research on Cancer, National Research Council (Italy); Dutch Ministry of Public Health, Welfare and Sports, Dutch Ministry of Health, Dutch Prevention Funds, LK Research Funds, Dutch ZON (Zorg Onderzoek Nederland), World Cancer Research Fund (WCRF) (the Netherlands); Swedish Cancer Society, Swedish Scientific Council, Regional Government of Skane (Sweden); Norwegian Cancer Society (Norway).

Special thank deserve Wolfgang Bernigau for his major support in conducting the statistical analysis.

Conflict of interest.

The authors declare no conflict of interest.


Sotirios Kakavas

Theano Demestiha

Panagiotis Vasileiou

Theodoros Xanthos
email: theodorosxanthos@yahoo.com

Erythropoetin as a novel agent with pleiotropic effects against acute lung injury

Keywords: Erythropoietin, Acute lung Injury, Acute respiratory distress syndrome, ALI, ARDS

Introduction

Clinically, acute lung injury (ALI) and its more severe form, acute respiratory distress syndrome (ARDS), can occur in response to a variety of pulmonary or systemic insults that induce damage of the alveolar-capillary membrane and pulmonary inflammation. This acute condition is characterized by bilateral pulmonary infiltrates, non-cardiogenic pulmonary oedema, impaired gas exchange and need for mechanical ventilation [START_REF] Bernard | The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination[END_REF][START_REF] Matthay | Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management[END_REF][START_REF] Wheeler | Acute lung injury and the acute respiratory distress syndrome: a clinical review[END_REF]. Pulmonary inflammation complicated by a systemic response leads to the release of cytokines, oxidants and other pro-inflammatory mediators that subsequently propel the inflammatory and apoptotic response in the lung [START_REF] Baue | Systemic inflammatory response syndrome (SIRS), multiple organ dysfunction syndrome (MODS), multiple organ failure (MOF): are we winning the battle?[END_REF][START_REF] Boueiz | Regulation of endothelial barrier function by reactive oxygen and nitrogen species[END_REF][START_REF] Crimi | Inflammation and the acute respiratory distress syndrome[END_REF][START_REF] Perl | Epithelial cell apoptosis and neutrophil recruitment in acute lung injury-a unifying hypothesis? What we have learned from small interfering RNAs[END_REF][START_REF] Tang | Acute lung injury and cell death: how many ways can cells die?[END_REF][START_REF] Tasaka | Roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome[END_REF][START_REF] Vincent | Why do patients who have acute lung injury/acute respiratory distress syndrome die from multiple organ dysfunction syndrome? Implications for management[END_REF]. About 55% of ALI/ARDS cases can be attributed to direct causes, such as pneumonia or aspiration, whereas indirect causes such as extra-pulmonary sepsis or trauma, account for 20% of the cases. In about 21% of instances, multiple risk factors are recognized while, in 4% of cases, no underlying pathophysiology can be identified [START_REF] Brun-Buisson | ALIVE Study Group (2004) Epidemiology and outcome of acute lung injury in European intensive care units. Results from the ALIVE study[END_REF]. The annual incidence of ALI and ARDS in the United States of America is estimated to be 79 and 59 cases/100,000 population respectively [START_REF] Wheeler | Acute lung injury and the acute respiratory distress syndrome: a clinical review[END_REF][START_REF] Rubenfeld | Incidence and outcomes of acute lung injury[END_REF], whereas the associated mortality remains high at 30%-50%, despite optimal supportive care [START_REF] Rubenfeld | Incidence and outcomes of acute lung injury[END_REF][START_REF]Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[END_REF]. Patients are likely to have prolonged hospital courses with mechanical ventilation, while those surviving often suffer from cognitive deficits or long term disabilities, such as reduced vital capacity, exercise tolerance and physical strength [START_REF] Brun-Buisson | ALIVE Study Group (2004) Epidemiology and outcome of acute lung injury in European intensive care units. Results from the ALIVE study[END_REF][START_REF] Rubenfeld | Incidence and outcomes of acute lung injury[END_REF][START_REF]Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[END_REF][START_REF] Hopkins | Quality of life, emotional abnormalities, and cognitive dysfunction in survivors of acute lung injury/acute respiratory distress syndrome[END_REF][START_REF] Mchugh | Recovery of function in survivors of the acute respiratory distress syndrome[END_REF].

Over the past two decades, a variety of interventions and intensive care strategies have been used in treating patients with ALI/ARDS. Treatment of the underlying cause of injury or disease is essential, along with mechanical ventilation with lung protective strategies, supportive care, and judicious fluid management [START_REF] Wheeler | Acute lung injury and the acute respiratory distress syndrome: a clinical review[END_REF][START_REF]Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[END_REF][START_REF] Raghavendran | Pharmacotherapy of acute lung injury and acute respiratory distress syndrome[END_REF]. Unfortunately, current specific options for treatment are limited, although, the complex pathology of this condition offers an array of potential pharmacological targets such as ventilation/perfusion abnormalities, vascular dysfunction, edema, inflammation, oxidant injury, arterial hypoxemia, surfactant dysfunction and fibrosis [START_REF] Raghavendran | Pharmacotherapy of acute lung injury and acute respiratory distress syndrome[END_REF]. It has been suggested that pleiotropic therapeutic strategies may be more appropriate to restore the disturbed homeostasis during ALI/ARDS. In other words the investigation of new therapeutic approaches should include the use of combinedagent treatments or a distinct substance with multiple actions. In this regard, exogenous erythropoietin (EPO) represents a possible candidate since, over the last years, it has emerged as a multifunctional tissue-protective cytokine with anti-apoptotic, anti-inflammatory and immunomodulatory properties [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF].

EPO is an endogenous 30.4-kDa glycoprotein hormone and a member of the type I cytokine superfamily that stimulates red blood cell production in order to increase oxygen transfer and delivery. The circulatory mature protein has a span of 165 amino acids and contains 4 glycosylated chains including 3 N-linked and 1 Olinked acidic oligosaccharide side chains [START_REF] Jelkmann | Biology of erythropoietin[END_REF]. Reduced arterial oxygen content associated with anaemia or hypoxia is the predominant stimulus for EPO production mainly in the kidneys [START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Jelkmann | Biology of erythropoietin[END_REF][START_REF] Farrell | The erythropoietin receptor and its expression in tumor cells and other tissues[END_REF][START_REF] Lacombe | Erythropoietin: sites of synthesis and regulation of secretion[END_REF]. EPO promotes bone marrow erythroid cell proliferation, differentiation, and survival through its binding to an erythroid progenitor cell surface EPO receptor (EPO-R) [START_REF] Jelkmann | Biology of erythropoietin[END_REF][START_REF] Farrell | The erythropoietin receptor and its expression in tumor cells and other tissues[END_REF][START_REF] Brines | Erythropoietin mediates tissue protection through an erythropoietin and common beta-subunit heteroreceptor[END_REF]. Secondary production of EPO as well as expression of the EPO-R have been also demonstrated on several tissues and organs including brain, heart, lungs, liver, kidneys, vascular endothelium, and lymphoid tissues [START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Farrell | The erythropoietin receptor and its expression in tumor cells and other tissues[END_REF][START_REF] Lacombe | Erythropoietin: sites of synthesis and regulation of secretion[END_REF].

The exogenous administration of recombinant human erythropoietin (rhEPO) is presently approved for the treatment of anaemia as a consequence of a variety of disorders. However, it is well-established that EPO also possesses therapeutic properties beyond mere haemopoiesis and therefore, EPO is now considered to have applicability in a variety of disorders, such as cerebral ischaemia, spinal cord injury, head trauma, epilepsy, kidney damage, myocardial infarction and reperfusion injury, and chronic congestive heart failure [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF]. In agreement with these studies, EPO has been also shown to have significant beneficial effects in experimental models of ALI.The remainder of this article reviews recent advances that have elucidated biological and biochemical activities of EPO which may be potentially applicable for ALI/ARDS management.

ERYTHROPOIETIN AND ALI/ARDS

Around 7% of intensive care patients will eventually develop ALI/ARDS, whereas delivery of critical care to patients with ALI/ARDS accounts for a significant proportion of intensive care unit capacity [START_REF] Brun-Buisson | ALIVE Study Group (2004) Epidemiology and outcome of acute lung injury in European intensive care units. Results from the ALIVE study[END_REF]. In the last decade EPO has emerged as a multifunctional endogenous mediator that participates in a broad array of pathophysiological processes during critical illness. EPO is considered to be relatively deficient in critical illness, since the EPO response to known physiological stimuli appears to be blunted in critically ill patients [START_REF] Rogiers | Erythropoietin response is blunted in critically ill patients[END_REF]. Interestingly, local and circulating mediators of critical illness, including prostaglandin, arachidonic acid, glucocorticoids, reactive oxygen species (ROS) and cytokines such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and interleukin 6 (IL-6), are known to modulate EPO production and the expression of EPO-R [START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Faquin | Effect of inflammatory cytokines on hypoxia-induced erythropoietin production[END_REF]. Furthermore, EPO appears to be a survival negative prognostic factor during sepsis, a major risk factor for ALI/ARDS [START_REF] Abel | Serum erythropoietin levels in patients with sepsis and septic shock[END_REF][START_REF] Tamion | Erythropoietin and renin as biological markers in critically ill patients[END_REF]. In patients suffering from septic shock, high serum EPO levels correlate with the severity of organ injury, hypoxia score (PaO2/FiO2 ratio) and lactate values [START_REF] Abel | Serum erythropoietin levels in patients with sepsis and septic shock[END_REF][START_REF] Tamion | Erythropoietin and renin as biological markers in critically ill patients[END_REF]. Important to the present review, numerous pre-clinical studies have revealed that several tissues are protected by EPO administration before or following various stressors, which also represent risk factors for ALI/ARDS [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF] and clinical data also support this concept [START_REF] Ehrenreich | Erythropoietin therapy for acute stroke is both safe and beneficial[END_REF][START_REF] Lipsic | A single bolus of a long-acting erythropoietin analogue darbepoetin alfa in patients with acute myocardial infarction: a randomized feasibility and safety study[END_REF].

Consistent with these findings, several reports in animal models of ALI have documented that EPO administration decreases the severity of lung injury by various etiological factors. EPO appears to protect the integrity of the pulmonary epithelial and endothelial cells and to attenuate the associated pulmonary oedema and deterioration of pulmonary oxygenation function. Treatment with EPO has been proven beneficial in experimental ALI caused by hyperoxia [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF][START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF], acute necrotizing pancreatitis [START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF], ischaemia/reperfusion [START_REF] Wu | Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats[END_REF][START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF], experimental sepsis induced by cecal ligation and puncture [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF] or LPS [START_REF] Shang | Erythropoietin attenuates lung injury in lipopolysaccharide treated rats[END_REF][START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF], zymosan induced nonseptic shock [START_REF] Cuzzocrea | Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice[END_REF] and tracheobronchial and pulmonary type II epithelial injury, following traumatic brain injury [START_REF] Yildirim | Protective effect of erythropoietin on type II pneumocyte cells after traumatic brain injury in rats[END_REF]. In these studies EPO has been found to interact with its receptor (EPO-R) to induce a range of pleiotropic cytoprotective actions.

Nevertheless, the potential use of EPO could be limited or even halted by the dosedependent risk of associated side effects, especially in high-risk groups such as patients with vascular thrombosis, hypertension, congestive heart failure, and neoplasms [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. Therefore, a careful consideration of appropriate EPO doses is certainly indicated before extending experimental findings to humans or other animal models.

Protection of vascular integrity: Anti-apoptotic and angiogenic action of EPO in ALI/ARDS

The principle mechanism by which EPO may preserve cellular integrity involves the inhibition of cellular apoptosis that seems to represent an early trigger in the pathogenesis of ALI/ARDS [START_REF] Perl | Epithelial cell apoptosis and neutrophil recruitment in acute lung injury-a unifying hypothesis? What we have learned from small interfering RNAs[END_REF][START_REF] Tang | Acute lung injury and cell death: how many ways can cells die?[END_REF]. Thus, in a number of tissues and organs, EPO exhibits anti-apoptotic properties secondary to various insults [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF]. Ιn rats, the pulmonary apoptosis index was markedly reduced as a result of treatment with EPO prior to ischaemia/reperfusion lung injury [START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF]. Moreover, EPO prevents LPS-induced apoptosis and eventually promotes the viability of bovine pulmonary artery endothelial cells [START_REF] Carlini | Effect of recombinant human erythropoietin on endothelial cell apoptosis[END_REF]. Additionally, EPO treatment protects against apoptotic cell death in the lung after induction of experimental endotoxaemia in mice [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF], while repeated administration of EPO reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice [START_REF] Yoshimi | Recombinant human erythropoietin reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice[END_REF]. Fas-mediated apoptosis of respiratory epithelium [START_REF] Perl | Epithelial cell apoptosis and neutrophil recruitment in acute lung injury-a unifying hypothesis? What we have learned from small interfering RNAs[END_REF][START_REF] Tang | Acute lung injury and cell death: how many ways can cells die?[END_REF] was inhibited by EPO in an in vitro model of ALI [START_REF] Macredmond | Erythropoietin inhibits respiratory epithelial cell apoptosis in a model of acute lung injury[END_REF]. This antiapoptotic effect of EPO was associated with induction of a pro-apoptotic Bclx(L)/Bax ratio, that seems to confer protection during ALI by suppressing apoptosis [START_REF] Macredmond | Erythropoietin inhibits respiratory epithelial cell apoptosis in a model of acute lung injury[END_REF]. In the same way, EPO nearly completely restored the marked suppression of the antiapoptotic proteins Bcl-x(L) and Bcl-2 in isolated vessels during rodent endotoxaemia, ultimately resulting in a pronounced improvement of both endothelial vasoconstriction and relaxation [START_REF] Di Villa Bianca | Recombinant human erythropoietin prevents lipopolysaccharide-induced vascular hyporeactivity in the rat[END_REF]. Given the underlying evidence that apoptosis is importantly involved in ALI/ARDS, the fundamental concept emerging is that EPO promotes cell survival and ultimately induces tolerance to the subsequent insult.

Unfortunately, the anti-apoptotic properties of EPO have also been reported to enhance tumor progression under some conditions by blocking tumor cell apoptosis [START_REF] Maiese | Raves and risks for erythropoietin[END_REF].

The signaling cascade of apoptosis inhibition after EPO binding to its receptors has not been fully defined, but several molecular signals are reported to be involved in EPO's anti-apoptotic efficacy. Activation of specific EPO-R, results in the activation of Janus tyrosine kinase 2 (JAK2-kinase) and ultimately in the activation of Akt (protein kinase B) and phosphoinositide-3 kinase (PI-3 kinase) phosphorylation [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. Expression of phosphorylated Akt and Erk, which are thought to mediate the survival signalling pathway induced by EPO, tend to be increased in lung tissues from mice treated with EPO after bleomycin-induced pneumonitis [START_REF] Yoshimi | Recombinant human erythropoietin reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice[END_REF]. In addition, EPO may act at the transcriptional level, at least in part, through Akt-mediated modulation of nuclear factor-κB (NF-κB) [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. However, EPO treatment prevented apoptosis, but did not alter the phosphorylation levels of NFκB in the lung, after LPS administration [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF]. Recent data suggest that JNK (c-Jun NH(2)-terminal kinase) signal pathway may also be involved in the anti-apoptotic of EPO against hyperoxic lung injury in neonatal rats [START_REF] Ding | Effect of erythropoietin on apoptosis following hyperoxic lung injury in neonatal rats[END_REF]. In addition, the cytoprotective effects of EPO have been shown to involve the activation of STAT 3 and STAT 5 as well as the inhibition of caspases 3, 8, and 9 [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. Similarly, EPO treatment significantly decreased the amount of active caspase-3 protein and caspase-3/7 activity in the lung of endotoxaemic mice [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF].

Besides preserving cell integrity, EPO seems to play a dual role in vascular protection by promoting angiogenesis [START_REF] Kertesz | The role of erythropoietin in regulating angiogenesis[END_REF][START_REF] Ribatti | Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo[END_REF]. Over a longer time frame this angiogenesis may also provide indirect cellular protection and promote functional recovery [START_REF] Van Der Meer | Erythropoietin induces neovascularization and improves cardiac function in rats with heart failure after myocardial infarction[END_REF]. Based on this notion, EPO treatment might prevent lung injury seen with hyperoxia by stimulating angiogenesis, since EPO treatment significantly increased vascularization in hyperoxia-exposed rats [START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF]. However, the angiogenic actions of erythropoietin may potentially exacerbate retinopathy or enhance the progression of cancer by assisting with tumor angiogenesis [START_REF] Maiese | Raves and risks for erythropoietin[END_REF].

The angiogenic action of EPO is probably mediated by increased expression of several key molecules in this process such as vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1). EPO seems to interact synergistically with VEGF to potentiate its vascular activity [START_REF] Victoria | Role of vascular endothelial growth factor on erythropoietin-related endothelial cell proliferation[END_REF]. However, VEGF appears to play a complex role in ALI/ARDS and may have diverse beneficial or even deteriorating actions depending on the stage and the initial cause of the disease [START_REF] Lahm | The critical role of vascular endothelial growth factor in pulmonary vascular remodeling after lung injury[END_REF]. Interestingly, the EPO/EPO-R signaling pathway may somehow modulate Ang-1, since EPO stimulation leads to increased Ang-1 expression [START_REF] Kertesz | The role of erythropoietin in regulating angiogenesis[END_REF][START_REF] Keogh | The effect of recombinant human erythropoietin on neurovasculature repair after focal ischemic stroke in neonatal rats[END_REF]. Ang-1, a vascular growth factor involved in later stages of angiogenesis [START_REF] Yancopoulos | Vascular-specific growth factors and blood vessel formation[END_REF], is down-regulated during experimental ALI and has also been shown to attenuate vascular leakage, pulmonary inflammation and apoptosis [START_REF] Karmpaliotis | Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury[END_REF][START_REF] Mccarter | Cell-based angiopoietin-1 gene therapy for acute lung injury[END_REF]. Nevertheless, the effect of EPO administration on lung VEGF or Ang-1 levels during ALI/ARDS has not been studied. In light of the limited data, more work is definitely needed to delineate the putative involvement of VEGF and Ang-1 in EPO-mediated effects in the lung.

It is noteworthy that endogenous EPO is a potent physiologic stimulus of endothelial progenitor cell (EPC) mobilization [START_REF] Heeschen | Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[END_REF][START_REF] Satoh | Important role of endogenous erythropoietin system in recruitment of endothelial progenitor cells in hypoxia-induced pulmonary hypertension in mice[END_REF]. Endothelial cells carry the EPO receptor and can be stimulated by EPO [START_REF] Ribatti | Human erythropoietin induces a pro-angiogenic phenotype in cultured endothelial cells and stimulates neovascularization in vivo[END_REF][START_REF] Heeschen | Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[END_REF]. Stimulation of endothelial cells by EPO increases cell migration and proliferation, endothelin-1 production and release, enhanced vascular sensitivity to nor-epinephrine, and induction of angiogenesis [START_REF] Heeschen | Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[END_REF][START_REF] Satoh | Important role of endogenous erythropoietin system in recruitment of endothelial progenitor cells in hypoxia-induced pulmonary hypertension in mice[END_REF][START_REF] Juul | Nonerythropoietic roles of erythropoietin in the fetus and neonate[END_REF]. Thus, EPO may serve as a cytokine promoting the repair process of the injured pulmonary endothelium through the recruitment of bone marrow-derived EPC, and their incorporation into the pulmonary endothelium as well as the stimulation of preexisting pulmonary endothelial cells [START_REF] Heeschen | Erythropoietin is a potent physiologic stimulus for endothelial progenitor cell mobilization[END_REF][START_REF] Satoh | Important role of endogenous erythropoietin system in recruitment of endothelial progenitor cells in hypoxia-induced pulmonary hypertension in mice[END_REF]. Bone marrowderived EPC are likely to contribute to pulmonary endothelial repair not only by incorporating into the endothelial cells but also by improving the functions of the resident pulmonary endothelial cells through paracrine effects [START_REF] Nagaya | Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells[END_REF][START_REF] Zhao | Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelium-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease[END_REF]. Table 1 summarizes the experimental data suggesting that EPO confers vascular protection in the setting of ALI.

Anti-inflammatory properties of EPO during ALI/ARDS

Although the pathogenesis of ALI/ARDS is incompletely understood and appears multifactorial, over-exuberant inflammation leading to irreversible lung damage and fibrosis is believed to play a pivotal role in the lung injury process both in experimental models and in patients [START_REF] Bernard | The American-European Consensus Conference on ARDS. Definitions, mechanisms, relevant outcomes, and clinical trial coordination[END_REF][START_REF] Matthay | Acute lung injury and the acute respiratory distress syndrome: four decades of inquiry into pathogenesis and rational management[END_REF][START_REF] Bastarache | Development of animal models for the acute respiratory distress syndrome[END_REF]]. Thus, inhibition of the inflammatory response may be one possible pathway of lung injury attenuation by EPO that exhibits anti-inflammatory properties mediated by both hormonal and paracrine modalities [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF][START_REF] Ruscher | Erythropoietin is a paracrine mediator of ischemic tolerance in the brain: evidence from an in vitro model[END_REF]. In addition, since pulmonary inflammation is a key feature in the pathogenesis of fibrosis in ALI/ARDS, the anti-inflammatory activity of EPO might explain the finding that EPO treatment was associated with decreased pulmonary fibrosis in hyperoxia-exposed rats [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF][START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF] or bleomycin-treated mice [START_REF] Yoshimi | Recombinant human erythropoietin reduces epithelial cell apoptosis and attenuates bleomycin-induced pneumonitis in mice[END_REF][START_REF] Sigounas | Erythropoietin ameliorates chemotherapy-induced fibrosis of the lungs in a preclinical murine model[END_REF].

In agreement with this notion, previous studies have demonstrated that EPO attenuated pulmonary neutrophil accumulation in animals experiencing hyperoxic injury [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF][START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF], ischaemia/reperfusion injury [START_REF] Wu | Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats[END_REF][START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF], endotoxemia [START_REF] Shang | Erythropoietin attenuates lung injury in lipopolysaccharide treated rats[END_REF][START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF], nonseptic shock [START_REF] Cuzzocrea | Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice[END_REF], and acute necrotizing pancreatitis [START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF]. Markers of neutrophil infiltration such as malondialdehyde levels and myeloperoxidase activity in the lung tissue are also reduced as a result of EPO administration [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF][START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF][START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF]. Leukocyte adhesion ligands such as P-selectin and intercellular adhesion molecule-1 (ICAM-1) are implicated in the activation of endothelial cells and the adhesion and migration of leukocytes to the sites of inflammation during lung injury [START_REF] Kamochi | P-selectin and ICAM-1 mediate endotoxin-induced neutrophil recruitment and injury to the lung and liver[END_REF]. Modulation of Pselectin expression, possibly via an influence on TNF-α production, appears to represent a major mechanism underlying the anti-inflammatory actions of EPO in the setting of ALI [START_REF] Vachharajani | Modulation of circulating cell-endothelial cell interaction by erythropoietin in lean and obese mice with cecal ligation and puncture[END_REF]. Moreover, EPO has been shown to down-regulate intercellular adhesion molecule-1 (ICAM-1) expression [START_REF] Liu | Mechanism of the cardioprotection of rhEPO pretreatment on suppressing the inflammatory response in ischemia-reperfusion[END_REF]. This observation is consistent with the reported ability of EPO to reduce the number of ICAM1-positive pulmonary endothelial cells in animals with bleomycin-induced pulmonary inflammation [START_REF] Sigounas | Erythropoietin ameliorates chemotherapy-induced fibrosis of the lungs in a preclinical murine model[END_REF].

Persistent elevations of proinflammatory cytokines (TNF-α IL-1β, IL-6, IL-8) are found in the plasma, lavage fluid and lung tissue from patients or animal models with ALI/ARDS and have been shown to be related with similar severity of lung dysfunction scores and to be predictive of poor clinical outcome [1-3, 6, 59, 65, 66].

In several experimental models of ALI, administration of EPO attenuated the accumulation in circulation, BAL and lung tissue of various cytokines, such as TNFα, IL-6 and IL-1β [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF][START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF][START_REF] Wu | Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats[END_REF][START_REF] Juul | Nonerythropoietic roles of erythropoietin in the fetus and neonate[END_REF][START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF][START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF]. Furthermore, in an experimental model of ischaemia/reperfusion ALI, pretreatment with EPO also reduced the over-expression of matrix metalloproteinase (MMP)-9 in lung tissue [START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF], which is considered to be an important factor correlated with the elevation of proinflammatory cytokines and microvascular permeability [START_REF] Yano | Increased matrix metalloproteinase 9 activity and mRNA expression in lung ischemia-reperfusion injury[END_REF]. It is notable that TNF-α can directly inhibit endogenous EPO production [START_REF] Faquin | Effect of inflammatory cytokines on hypoxia-induced erythropoietin production[END_REF]. In this way, the inhibition of endogenous EPO by TNF-α might contribute to the role of this mediator in the pathogenesis of lung injury, and partly explain why exogenously administered EPO is especially beneficial.

Several cellular mechanisms, including the mode of gene regulation and signal transduction, may account for the anti-inflammatory effects of EPO. A growing body of evidence suggests that EPO may act at the transcriptional level, at least in part, through inhibition of NF-κB [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. NF-κB is a transcription factor that has been demonstrated to play a pivotal role in inflammation during ALI by promoting maximal transcription of a wide array of proinflammatory molecules including TNFα, IL-1β and other mediators [START_REF] Abraham | Nuclear factor-κB and its role in sepsis-associated organ failure[END_REF][START_REF] Liu | p38 MAPK inhibition attenuates lipopolysaccharide induced acute lung injury involvement of NF-kappaB pathway[END_REF]. Intrestingly, during LPS induced acute endotoxaemia in rats, pretreatment with EPO attenuated pulmonary inflammation and suppressed TNF-α and IL-1β overproduction, while the enhancement in NF-κB activation by LPS was significantly inhibited [START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF]. It is possible that the mechanism of action of EPO in ALI is via either down-regulation of TNF-α or via modulation of NFκB induced cell adhesion or both [START_REF] Vachharajani | Modulation of circulating cell-endothelial cell interaction by erythropoietin in lean and obese mice with cecal ligation and puncture[END_REF].

As a result, it is reasonable to conclude that EPO may attenuate ALI, partly by decreasing the expression of proinflammatory cytokines, such as TNF-a, and inhibiting the accumulation of neutrophils. Yet, data concerning the effect of EPO administration on serum levels of pro-inflammatory cytokines in LPS induced endotoxemia in animals appear to be contradictory. Low-dose EPO pre-treatment deteriorated LPS-induced pulmonary damage in conscious rats and increased serum TNF-alpha, IL-6, and IL-1beta, along with aggravating endotoxin shock-induced markers of organ injury [START_REF] Wu | Low-dose erythropoietin aggravates endotoxin-induced organ damage in conscious rats[END_REF]. More interestingly, a recent study in healthy humans treated with LPS showed that pretreatment with EPO had no direct suppressive effect on TNF-a and IL-6 plasma levels [START_REF] Hojman | Erythropoietin augments the cytokine response to acute endotoxin-induced inflammation in humans[END_REF]. Furthermore, EPO failed to significantly increase the plasma levels of the anti-inflammatory cytokine IL-10 and to attenuate the increase in circulating neutrophils [START_REF] Hojman | Erythropoietin augments the cytokine response to acute endotoxin-induced inflammation in humans[END_REF]. Accordingly, the beneficial action of EPO administration after LPS administration in rats had no effect on pulmonary inflammation, while the phosphorylation levels of NF-κB in the lung were not affected [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF]. Therefore, it has been proposed that the beneficial effects of EPO may be linked to inhibition of apoptosis rather than to the anti-inflammatory properties of EPO [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF]. Alternatively, EPO might attenuate cytokine production and inflammation in an indirect way by preventing apoptosis and providing increased resistance of cellular targets to the effects of the noxious insult [START_REF] Wu | Low-dose erythropoietin aggravates endotoxin-induced organ damage in conscious rats[END_REF].

Antioxidant properties of EPO during ALI/ARDS

The over-expression and release of reactive oxygen species (ROS), and reactive nitrogen species (RNS) has been implicated as a crucial landmark in the initiation and further development of ALI/ARDS. In response to various inflammatory stimuli, lung endothelial cells, alveolar cells, and airway epithelial cells, as well as activated alveolar macrophages and neutrophils, produce both nitric oxide (NO) and superoxide, which may react to form peroxynitrite, a highly reactive radical [START_REF] Boueiz | Regulation of endothelial barrier function by reactive oxygen and nitrogen species[END_REF][START_REF] Tasaka | Roles of oxidants and redox signaling in the pathogenesis of acute respiratory distress syndrome[END_REF][START_REF] Beckman | Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide[END_REF][START_REF] Lang | Oxidant-antioxidant balance in acute lung injury[END_REF]. In addition to other effects, EPO has been demonstrated in various tissues to be an antioxidant as it can inhibit free radical production and associated lipid peroxidation [START_REF] Coleman | Science review: recombinant human erythropoietin in critical illness: a role beyond anemia?[END_REF][START_REF] Maiese | Raves and risks for erythropoietin[END_REF][START_REF] Thiemermann | Beneficial effects of erythropoietin in preclinical models of shock and organ failure[END_REF]. Likewise, animal studies have shown that EPO administration led to inhibition of the oxidative stress-associated lipid peroxidation in the lung parenchyma of animals after traumatic brain injury, acute necrotizing pancreatitis or endotoxemia [START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF][START_REF] Shang | Erythropoietin attenuates lung injury in lipopolysaccharide treated rats[END_REF][START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF][START_REF] Yildirim | Protective effect of erythropoietin on type II pneumocyte cells after traumatic brain injury in rats[END_REF]. At the same time, EPO has shown potent ability to suppress the inducible NO synthase (iNOS)-mediated NO production and peroxynitrite formation, thereby attenuating cytotoxicity and vascular dysfunction during experimental shock induced by zymosan or LPS [START_REF] Shang | Erythropoietin attenuates lung injury in lipopolysaccharide treated rats[END_REF][START_REF] Cuzzocrea | Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice[END_REF][START_REF] Di Villa Bianca | Recombinant human erythropoietin prevents lipopolysaccharide-induced vascular hyporeactivity in the rat[END_REF]. Besides inhibition of iNOS, EPO is well known to induce expression of endothelial NO synthase (eNOS) [START_REF] Santhanam | In vivo stimulatory effect of erythropoietin on endothelial nitric oxide synthase in cerebral arteries[END_REF]. Thus, recovery of the regulation of the microcirculation by eNOS-mediated nitric oxide may also contribute to the protection achieved by EPO treatment [START_REF] Vollmar | Therapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF]. Interestingly, EPO also attenuated the increase in the activity of pulmonary poly-ADP-ribose polymerase caused by oxidative injury during non-septic shock in zymosan-treated mice [START_REF] Cuzzocrea | Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice[END_REF].

It is believed that EPO exerts its anti-oxidative effect either directly as a potent free radical scavenger by the scavenging actions of its sugar moieties, or indirectly by increasing the activities of anti-oxidant enzymes such as superoxide dismutase, glutathione peroxidase, and catalase [START_REF] Chakraborty | Effect of erythropoietin on membrane lipid-peroxidation, superoxide-dismutase, catalase, and glutathioneperoxidase of rat Rbc[END_REF][START_REF] Genc | Erythropoietin restores glutathione peroxidase activity in 1-methyl-4-phenyl-1,2,5,6tetrahydropyridine-induced neurotoxicity in C57BL mice and stimulates murine astroglial glutathione peroxidase production in vitro[END_REF]. Furthermore, EPO can decrease the plasma iron concentration and increase the ability of plasma to inhibit lipid peroxidation [START_REF] Bany-Mohammed | Recombinant human erythropoietin: possible role as an antioxidant in premature rabbits[END_REF][START_REF] Kaptanoglu | Erythropoietin exerts neuroprotection after acute spinal cord injury in rats: effect on lipid peroxidation and early ultrastructural findings[END_REF]. The inhibitory effect of EPO on iNOS has been shown to require the activation of the phospholipase Cγ1 and protein kinase C pathway [START_REF] Akimoto | The effect of erythropoietin on interleukin-1beta mediated increase in nitric oxide synthesis in vascular smooth muscle cells[END_REF].

Moreover, in LPS-treated rats EPO has been shown to inhibit the pulmonary activation of NF-κB, an important redox-sensitive transcription factor necessary for iNOS gene expression [START_REF] Shang | Reduction of pulmonary inflammatory response by erythropoietin in a rat model of endotoxaemia[END_REF]. Table 2 summarizes the experimental data that suggest an anti-inflammatory and anti-oxidant effect of EPO during ALI.

DISCUSSION

Taken together, the results of the aforementioned studies demonstrate that the pleiotropic action of EPO at multiple levels apparently contributes to the amelioration of crucial pathophysiological aspects of experimental ALI. Unfortunately, no single animal model of ARDS replicates the complex pathophysiological changes seen in patients [START_REF] Bastarache | Development of animal models for the acute respiratory distress syndrome[END_REF]. However, these findings provide the rationale for the potential use of EPO as a therapeutic agent in the treatment or prevention of ALI/ARDS. Benefits in survival in human studies of ALI/ARDS have not yet been seen, but a recent multicenter, placebo controlled, double blind study has shown that when administered in critically ill patients with anemia, EPO decreased mortality in trauma patients in spite of failure to decrease the need for red cell transfusions [START_REF] Corwin | EPO Critical Care Trials Group (2007) Efficacy and safety of epoetin alfa in critically ill patients[END_REF]. Promising results have also been reported in a randomized, double-blind, placebo-controlled, two-center trial, which comprised the administration of weekly rhEPO in critically ill and in patients receiving long-term mechanical ventilation [START_REF] Silver | Efficacy of recombinant human erythropoietin in critically ill patients admitted to a long-term acute care facility: a randomized, double-blind, placebocontrolled trial[END_REF]. Although not significant, the mortality rates in patients treated with EPO (12%) were lower than in the patients treated with placebo (23%). Thus, larger and more definitive studies are needed in order to establish whether EPO treatment affects clinical outcomes in ALI/ARDS patients. In addition to the attempts to investigate its pharmacodynamics, pharmacokinetics, administration routes and doses, future in vivo animal experiments and clinical strategies must also seek to reduce potential adverse effects during EPO treatment.

Experimental findings appear to differ according to the animal model of ALI and the timing of EPO administration. The results in a murine model of endotoxemia indicate that the greatest survival was achieved when EPO was administered 1 hour after the LPS challenge, and that the survival benefit was lost when EPO was administered 1 hour before the LPS challenge or 3 hours or more after the LPS challenge [START_REF] Aoshiba | Τherapeutic effects of erythropoietin in murine models of endotoxin shock[END_REF]. By contrast, in an ischaemia/reperfusion model of ALI, the best therapeutic time was a short time (2 h hours) before the procedure, while the induction of ischemic tolerance by EPO seems to be relatively rapid after injection [START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF]. EPO seems less protective when administered at a same dosage after the onset of reperfusion [START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF]. Based on this, in the case of lung transplantation, it has been postulated that it would be more beneficial to preservation of lung tissue and lung function if EPO was given to donors [START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF][START_REF] Yildirim | Protective effect of erythropoietin on type II pneumocyte cells after traumatic brain injury in rats[END_REF]. Nevertheless, the previous findings may limit the clinical use of EPO because it appears to have a narrow therapeutic window, and that administration must be started before or very soon after the onset of the initial cause of ALI/ARDS. Different doses of EPO have been reported to protect lung against variable challenges. Low doses of EPO (400 U/kg; i.p.) have been found to improve hyperoxia-induced lung injury in neonatal rats [START_REF] Ozer | Effects of erythropoietin on hyperoxic lung injury in neonatal rats[END_REF]. The same dose of EPO administered 18 h after cecal ligation and puncture improved the microcirculatory perfusion and tissue energy balance in murine sepsis [START_REF] Kao | Erythropoietin improves skeletal muscle microcirculation and tissue bioenergetics in a mouse sepsis model[END_REF]. However, EPO 500 IU/kg failed to protect from ischaemia/reperfusion induced ALI in rats [START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF] whereas in the case of LPS (20mg/kg) induced endotoxemia lower doses of EPO (300 U/kg; i.v.) have been reported to aggravate pulmonary damage in rats [START_REF] Wu | Low-dose erythropoietin aggravates endotoxin-induced organ damage in conscious rats[END_REF]. Higher doses of EPO than that normally used in the treatment of anemia attenuated lung injuries induced by acute necrotizing pancreatitis (1000 IU/kg; i.m.) [START_REF] Tascilar | Protective effects of erythropoietin against acute lung injury in a rat model of acute necrotizing pancreatitis[END_REF] or nonseptic shock caused by zymosan in mouse (1000 IU/kg; s.c.) [START_REF] Cuzzocrea | Erythropoietin reduces the development of nonseptic shock induced by zymosan in mice[END_REF]. Contrasting with a lack of effect at low doses, the use of markedly high doses of EPO (3000 U/kg; i.p.), was found to confer lung protection in endotoxic rats after LPS challenge [START_REF] Shang | Erythropoietin attenuates lung injury in lipopolysaccharide treated rats[END_REF]. Likewise, high EPO doses (3000 U/kg; i.p. repeatedly) have been reported to inhibit hyperoxia-induced ALI in neonatal rats [START_REF] Lee | Erythropoietin attenuates hyperoxia-induced lung injury by down-modulating inflammation in neonatal rats[END_REF]. In an experimental model of I/R induced ALI in rats the same dose of EPO (3000 IU/kg i.p.) has been determined as the optimal therapeutic dosage, although doses ≥2000 IU/kg appeared to be beneficial [START_REF] Wu | Pretreatment with recombined human erythropoietin attenuates ischemiareperfusion-induced lung injury in rats[END_REF][START_REF] Wu | Erythropoietin attenuates ischemia-reperfusion induced lung injury by inhibiting tumor necrosis factoralpha and matrix metalloproteinase-9 expression[END_REF].

Considering the above findings, clearly, most studies reporting beneficial effects used high doses of EPO (1000-4000 IU/kg), that exceed the dose (100-200 IU/kg/week) routinely used for patients with anaemia. Long-term clinical experience in anemic patients with chronic renal failure has shown that EPO is relatively safe and well tolerated, but adverse effects, including hypertension, thrombosis, and blood hyperviscosity are concerns for patients receiving large doses [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. Furthermore, the potential enhancement of tumor progression has been another significant concern of prolonged EPO treatment, while pure red cell aplasia represents a rare but serious side effect [START_REF] Maiese | Raves and risks for erythropoietin[END_REF]. However, the recent phase II trial of human stroke treated with EPO used three daily doses of 33,000 IU/patient (approximately 500 IU/kg), and EPO was shown to be safe and did not raise the hemoglobin concentration [START_REF] Ehrenreich | Erythropoietin therapy for acute stroke is both safe and beneficial[END_REF]. Furthermore, EPO at weekly doses of 40,000 IU/patient (approximately 600 IU/kg) was used in intensive care units for critically ill patients to reduce the need for transfusion, and it appears to be safe although mortality was not changed [START_REF] Silver | Efficacy of recombinant human erythropoietin in critically ill patients admitted to a long-term acute care facility: a randomized, double-blind, placebocontrolled trial[END_REF]. However, in a more recent clinical trial, EPO at weekly doses of 40,000 IU/patient was associated with an increase in the incidence of thrombosis in patients with trauma, although the mortality was reduced [START_REF] Corwin | EPO Critical Care Trials Group (2007) Efficacy and safety of epoetin alfa in critically ill patients[END_REF]. Taking into account the devastating consequences of ALI/ARDS, it is reasonable to speculate that the beneficial effects of EPO treatment may prove to override the potential harmful effects. Nonetheless, due to increased risks of high doses future strategies must also seek to reduce potential toxicity. The feasibility of a large dose of erythropoietin may be enhanced by the use of carbamylated EPO that lacks erythropoietic activity yet retains the full cytoprotective actions of EPO [START_REF] Leist | Derivatives of erythropoietin that are tissue protective but not erythropoietic[END_REF].

In summary, a number of preclinical studies have revealed a pleiotropic beneficial effect of EPO administration in various experimental models of ALI suggesting that EPO may be valuable as a clinical candidate for ALI/ARDS. However, no clinical studies exist to date examining EPO in ALI/ARDS patients to demonstrate a significant effect on survival and thus more experimental and clinical studies are required for the clarification of the efficacy and potentially cytoprotective mechanisms of EPO action. Moreover, the optimal dose has not been clearly determined yet, while specific time constraints for efficacious administration of exogenous EPO as a cytoprotectant may exist. Importantly, future studies should also include attempts to minimize any potential toxicity, which is essential for EPO to fulfill the role as a potential candidate for the treatment of ALI in routine clinical practice.

Table 1 .Table 2 .

 12 Summary of EPO actions considered to be beneficial in the preservation of the alveolar-capillary barrier integrity during experimental ALIAttenuation of lung edemaPreservation of pulmonary epithelial and vascular endothelial cell integrityInhibition of pulmonary apoptosisRestoration of suppressed antiapoptotic proteins.Modulation of pro-apoptotic and anti-apoptotic signalling pathways.Promotion of pulmonary angiogenesisDirect angiogenic effectRecruitment and incorporation of bone marrow-derived endothelial progenitor cells into the pulmonary endothelium Modulation of angiogenic factors (VEGF, Ang/Tie system) Summary of anti-inflammatory and anti-oxidative actions of EPO considered to be beneficial during experimental ALIAnti-inflammatory effectsAttenuated pulmonary neutrophil infiltrationModulation of leukocyte adhesion ligands (P-selectin, ICAM-1) expression Decrease of various cytokines (TNF-α, IL-6 and IL-1β) in BAL and lung tissue Decrease of various cytokines (TNF-α, IL-6 and IL-1β) in circulation Attenuation of the over-expression of matrix metalloproteinase (MMP)-9 in lung tissue Anti-oxidative effects Modulation of NFκB induced cell adhesion and cytokine production Inhibition of the oxidative stress-associated lipid peroxidation in the lung parenchyma Attenuation of the increased activity of pulmonary PARP Inhibition of iNOS expression and peroxynitrite formation in the lung