Romain Pauwels

Guy F Joos

Spartanburg Charles Fogarty

Dr Isidore Faiferman

Dr Sandra Hirschberg

Emiel F M Rupertvessey

Wouters

Dr Rupert Vessey

Dr Emiel F M Wouters

Dr Romain Pauwels#

Dr Guy F Joos#

USA North Carolina

Bronchodilating effect of combined therapy with ipratropium bromide and ondansetron in patients with COPD

Keywords: Drug combination, combination bronchodilator, COPD, bronchodilation, ondansetron, 5-HT3, ipratropium bromide

The objectives of this study were to determine the effect of single and repeat dosing with oral ondansetron, a 5-HT3-specific receptor blocker, on the degree and duration of bronchodilation induced by inhaled ipratropium bromide in patients with COPD. Five clinics and university medical centers in four countries participated in the study; forty seven patients with COPD were randomized A c c e p t e d m a n u s c r i p t 2 bronchodilation induced by ipratropium alone. sGaw was consistently more sensitive than FEV1 in measuring extent and duration of bronchodilation.

to treatment; 44 completed all treatments. Patients had a baseline (pre-bronchodilator) FEV1 > 1 liter and post-bronchodilator (200 mcg salbutamol) FEV1 < 90% of predicted, with FEV1 reversibility (to 80 mcg inhaled ipratropium bromide and 400 mcg salbutamol) of at least 12% or 200 ml over baseline. The study was divided into two parts. In Part A, each patient received in a random order, four-way cross-over manner, single doses of ondansetron placebo (oral) plus ipratropium bromide placebo (inhaled), ondansetron placebo plus ipratropium bromide 40 mcg inhaled via MDI, ondansetron 24 mg oral plus ipratropium bromide placebo and ondansetron 24 mg plus ipratropium bromide 40 mcg. In Part B, each patient received in a random order, two-way crossover manner, ipratropium bromide 40 mcg tid via MDI plus ondansetron 8 mg oral, qid, for two days; on day 3 patients received a single dose of ipratropium bromide 40 mcg plus 8 mg oral ondansetron. Alternatively, patients received ipratropium bromide via MDI and oral ondansetron placebo, as described above. Statistically significant differences in weighted mean FEV1 (0-6 hours), peak FEV1 and FEV1 determined 6 hours post-dose were noted comparing ipratropium bromide to placebo. Similar positive results were observed for sGaw and FVC. Addition of ondansetron to ipratropium bromide did not significantly modify values obtained with ipratropium alone. Ipratropium bromide induced a marked bronchodilation, compared to placebo. Addition of ondansetron (single or repeated doses) did not significanlty increase the degree or duration of 1. Introduction COPD has been recognized as a major health problem for about 50 years [START_REF] Senior | Chronic Obstructive Pulmonary Disease (COPD)[END_REF]. In contrast with other leading chronic diseases, its prevalence and disease-related morbidity and mortality are increasing [START_REF] Thoracic | Standard for the diagnosis and care of patients with Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Siafakas | Optimal assessment and management of chronic obstructive pulmonary disease (COPD)[END_REF][START_REF] British | BTS guidelines for the management of chronic obstructive pulmonary disease[END_REF][START_REF]Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management and prevention of Chronic Obstructive Lung Disease[END_REF][START_REF] Fabbri | Global strategy for the diagnosis, management and prevention of COPD: 2003 update[END_REF]. Bronchodilators are typically prescribed for maintenance therapy of the reversible obstructive component of the disease. In patients with minimally reversible or irreversible obstruction, symptom improvement observed following bronchodilator therapy may be related to the degree of reduction in gas trapping and dynamic hyperinflation, which, in turn, may result in reduction in the overall work of breathing [START_REF] O'donnell | Exertional breathlessness in patients with chronic airflow limitation: the role of hyperinflation[END_REF][START_REF] Calverley | The challenge of providing better care for patients with chronic obstructive pulmonary disease: the poor relation of airway obstruction?[END_REF]. The bronchodilators most commonly used are inhaled beta2adrenoceptor agonists and inhaled anticholinergics [START_REF] Thoracic | Standard for the diagnosis and care of patients with Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Siafakas | Optimal assessment and management of chronic obstructive pulmonary disease (COPD)[END_REF][START_REF] British | BTS guidelines for the management of chronic obstructive pulmonary disease[END_REF][START_REF]Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management and prevention of Chronic Obstructive Lung Disease[END_REF][START_REF] Rennard | Combination bronchodilator therapy in COPD[END_REF]; current international guidelines recommend combination therapy for patients not well controlled by monotherapy [START_REF] Thoracic | Standard for the diagnosis and care of patients with Chronic Obstructive Pulmonary Disease[END_REF][START_REF] Siafakas | Optimal assessment and management of chronic obstructive pulmonary disease (COPD)[END_REF][START_REF] British | BTS guidelines for the management of chronic obstructive pulmonary disease[END_REF][START_REF]Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management and prevention of Chronic Obstructive Lung Disease[END_REF][START_REF] Fabbri | Global strategy for the diagnosis, management and prevention of COPD: 2003 update[END_REF], since anticholinergics and beta2-adrenoceptor antagonists achieve their bronchodilating effects via different mechanisms [START_REF] Rennard | Combination bronchodilator therapy in COPD[END_REF]. A number of studies have indeed demonstrated that, in patients with stable COPD, the combination is more effective than either agent alone [START_REF] Rennard | Combination bronchodilator therapy in COPD[END_REF][START_REF]Combivent Inhalation Aerosol Study Group. In chronic obstructive pulmonary disease, a combination of ipratropium bromide and albuterol is more effective than either agent alone[END_REF][START_REF] Ikeda | Bronchodilating effects of combined therapy with clinical dosages of ipratropium bromide and salbutamol for patients with COPD: comparison with ipratropium bromide alone[END_REF][START_REF] Van Noord | Long-term treatment of chronic obstructive pulmonary disease with salmeterol and the additive effect of ipratropium bromide[END_REF][START_REF] Sichletidis | Bronchodilatory responses to formoterol, ipratropium bromide and their combination in patients with COPD[END_REF]. Dominant autonomic control of the airways is provided by the parasympathetic nerves, which control a variety of airway functions [START_REF] Boushey | Role of the vagus nerves in bronchoconstriction in humans[END_REF][START_REF] Nadel | Autonomic regulation of the airways[END_REF]. Acetylcholine, released from the parasympathetic nerves, stimulates muscarinic receptors, causing bronchoconstriction and mucus secretion. The effect of acetylcholine in the airways is mediated mainly through three muscarinic receptor subtypes, designated M1, M2 and M3 [START_REF] Ramnarine | On muscarinic control of neurogenic mucus secretion in ferret trachea[END_REF]. M1 receptors facilitate neurotransmission through the cholinergic ganglia; M2 receptors are found on pre and postganglionic parasympathetic nerves and inhibit release of acetylcholine, while M3 receptors are present in airway smooth muscle (ASM) and submucosal glands and are responsible for mediating bronchoconstriction and mucus secretion [START_REF] Ramnarine | On muscarinic control of neurogenic mucus secretion in ferret trachea[END_REF][START_REF] Van Nieuwstadt | Characterization of muscarinic receptors in equine tracheal smooth muscle in vitro[END_REF]. Inhibition of acetylcholine release by M2 receptors may be decreased or lost due to M2 receptor dysfunction in asthma and in COPD [START_REF] Schultheis | Ozone-induced airway hyperresponsiveness and loss of neuronal M2 muscarinic receptor function[END_REF][START_REF] Jacoby | Human eosinophil major basic protein is an endogeous allosteric antagonist at the inhibitory muscarinic M2 receptor[END_REF], resulting in unopposed activity at the M3 receptors. This excessive M3 receptor stimulation may contribute to the bronchoconstriction and mucus hypersecretion observed in inflammatory airways diseases.

Serotonin (5-HT) has also been reported to have effects on airway smooth muscle contraction through interactions with multiple receptor subtypes. Most data has been generated in in vitro experiments. In isolated rat bronchi 5-HT induced contraction of airway smooth muscle (ASM) directly, by activating airway smooth muscle 5-HT2 receptors, and indirectly, by activating 5-HT2 receptors on parasympathetic nerve endings, leading to release of acetylcholine [START_REF] Szarek | Mechanisms of 5-Hydroxytryptamine-induced contraction of isolated rat intrapulmonary bronchi[END_REF][START_REF] Germonpre | Modulation by 5-HT 1A receptors of the 5-HT 2 receptor-mediated tachykinin-induced contraction of the rat trachea in vitro[END_REF]. partially by ondansetron, a 5-HT3 receptor blocker [START_REF] Dupont | The effects of 5-HT on cholinergic contraction in human airways in vitro[END_REF]. In subjects with asthma, plasma levels of 5HT are elevated and correlate with clinical severity rating and FEV1 [START_REF] Lechin | Increased levels of free serotonin in plasma of symptomatic asthmatic patients, 1966[END_REF]. One possible source of 5-HT are the endocrine cells of the lung [START_REF] Gosney | Pulmonary endocrine cells in chronic bronchitis and emphysema[END_REF]; an increase in the number of endocrine cells (and presumably 5-HT) has been described in bronchiectatic lungs, lung containing neoplasms and lungs of subjects exposed to hypoxia [START_REF] Gould | Neuroendocrine components of the bronchopulmonary tract: hyperplasias, dysplasias and neoplasms[END_REF]. In a small group of subjects with COPD reversible to albuterol, administration of inhaled or of intravenous ketanserin, a 5-HT2 antagonist, induced a small degree of bronchodilation that was statistically significantly different from placebo, 30 minutes following administration of the drug [START_REF] Cazzola | Acute respiratory and cardiovascular effects of inhaled ketanserin in COPD. A comparative study with intravenously administered ketanserin[END_REF]. Combination therapy has become standard in the treatment of airway disease; this allows the targeting of diverse mechanisms that participate in the disease process. Identification and characterization of these mechanisms utilizing specific inhibitors may improve our understanding of the disease and could help in the development of optimal treatment. The objective of this study was to determine the short term effect of single and repeat dosing with ondansetron, a 5-HT3-specific receptor blocker, on the degree of bronchodilation induced by inhaled ipratropium bromide in patients with stable COPD. Three endpoints were evaluated in the determination of bronchodilation: FEV1 and FVC, measured by spirometry, and sGaw, determined by plethysmography.

Materials and Methods

Study Design

This was a double-blind, randomized, placebo controlled, crossover, two part study comparing the effect on pulmonary function of inhaled ipratropium bromide (Atrovent ®, Boehringer Ingelheim, 27), 40 mcg (single dose or tid), given by metered-dose inhaler, ondansetron (Zofran ®, GlaxoSmithKline, 28), 24 mg single dose or 8 mg tid, given orally, and their combination.

The study was carried out in five centers in four countries (2 centers in the US, one center each in Belgium, The Netherlands and Germany). The study was approved by the appropriate Ethics Committee/Institutional Review Board at each site and was conducted according to the principles of good clinical practice.

The study was divided into two parts and a screening visit. At screening, baseline measurements were performed and the eligibility of patients to participate in the study was determined. Part A of the study (four-way crossover) was conducted within 2 weeks of the screening visit; patients were randomized to receive, in a cross-over manner, single doses of ondansetron placebo plus ipratropium bromide placebo, ondansetron placebo plus ipratropium bromide 40 mcg via MDI, ondansetron 24 mg oral plus ipratropium placebo, and ondansetron 24 mg oral plus ipratropium A follow-up visit was conducted 7 to 14 days after the last dosing with study medication.

Patients

Inclusion criteria included the following: male and female patients, age > 40 to < 75 years old, who gave written informed consent, with a diagnosis of COPD according to the American Thoracic Society guidelines (2); current or former (discontinued smoking for > 1 yr) smokers (smoking history > 10 packs/year); pre-bronchodilator FEV1 > 1 liter, post-bronchodilator (180 mcg albuterol/200 mcg salbutamol via MDI) FEV1 < 90% of predicted; FEV1/FVC < 0.7; an increase in FEV1 of at least 12% or 200 ml over baseline after 80 mcg inhaled ipratropium bromide and 360 mcg albuterol/400 mcg salbutamol. Entry criteria are similar to those of other publications [START_REF] Sichletidis | Bronchodilatory responses to formoterol, ipratropium bromide and their combination in patients with COPD[END_REF][START_REF] Maesen | Ba 679, a new long-acting antimuscarinic bronchodilator: a pilot dose-escalation study in COPD[END_REF][START_REF] Briggs Dd | Improved daily efficacy of tiotropium compared with salmeterol in patients with COPD[END_REF][START_REF] Littner | Long-acting bronchodilation with once-daily dosing of tiotropium (Spiriva) in stable chronic obstructive pulmonary disease[END_REF][START_REF] Van Noord | A randomized controlled comparison of tiotropium and ipratropium in the treatment of chronic obstructive pulmonary disease[END_REF] except that only one (29) included a reversibility criterion for entry (an increase in FEV1 of 15% over baseline following bronchodilator). The other studies did not include a reversibility requirement; on the other hand, in all studies FEV1 increased by 200 mL or more over baseline following the administration of a single dose of bronchodilator.

A c c e p t e d m a n u s c r i p t 5

Exclusion criteria included: a diagnosis of asthma; an acute COPD exacerbation that required treatment with oral corticosteroids or hospitalization, or upper respiratory tract infection, within one month of screening; need for short or long-term oxygen therapy; use of oral corticosteroids at doses > 10 mg/day; change in the dose of inhaled or oral corticosteroids within 6 weeks of screening.

Efficacy parameters

The primary efficacy variable was the weighted mean FEV1 (0-6 hours) for single doses or the last dose of the repeat-dose period. Secondary efficacy variables were weighted mean sGaw (0-6 hr), peak values for FEV1, FVC, FEV1/FVC ratio and sGaw (peak refers to the maximum value in 0-6 hours), and FEV1, FVC, FEV1/FVC ratio and sGaw determined 6 hours post-dosing. Spirometry and plethysmography were performed following standard procedures [START_REF] Siafakas | Optimal assessment and management of chronic obstructive pulmonary disease (COPD)[END_REF][START_REF] British | BTS guidelines for the management of chronic obstructive pulmonary disease[END_REF][START_REF]Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management and prevention of Chronic Obstructive Lung Disease[END_REF]. Pulmonary function determinations were performed at the same time of day in order to minimize daytime variability of measurements. Rescue inhalations with short-acting bronchodilator were to be avoided for at least 4 hours prior to dosing to at least 6 hours post-dosing. Subjects abstained from long-acting beta2agonists, long-acting anticholinergics (tiotropium) and xanthines for two weeks prior to the first dose to the end of the study.

Safety variables

Safety was assessed using adverse event reporting, electrocardiograms, vital signs and clinical laboratory evaluations.

Statistical Analysis

The primary comparison of interest was weighted mean FEV1 (0-6 hours) in the single dose crossover phase, comparing ondansetron (24mg) and ipratropium (40mcg) vs. ipratropium (40mcg) and ondansetron placebo. This comparison was repeated for weighted mean sGaw (0-6 hours); peak FEV1, FVC, FEV1/FVC ratio and sGaw and for FEV1, FVC, FEV1/FVC ratio and sGaw determined 6 hours post-dosing.

A c c e p t e d m a n u s c r i p t 6

Unless otherwise specified, all analyses were performed using SAS software (version 8.2). This was a multicentre study; for the purpose of this study, centre and/or country differences were not assessed due to the small number of centres in each country and the small number of patients at each site.

Actual sample times were used to calculate the weighted means and peak endpoints. The weighted mean parameters were calculated by dividing the area under the curve (AUC) by time.

The AUC was calculated using the linear trapezoidal method.

In order to evaluate the primary outcome (weighted mean FEV1 0-6 hours) a mixed effects model was fitted, with fixed effects for period, treatment and baseline FEV1 (as a covariant) and random effects for subjects. Baseline FEV1 was fitted as a continuous variable; baseline FEV1 was the pre-dose value of FEV1 on the appropriate dosing day (Study Part A-single dose). Least square (LS) means for each treatment, differences in LS means and the 95% confidence intervals for the difference are presented. The same model was used to evaluate this outcome for Part B of the study (repeat dose) using baseline as the pre-dose value of FEV1 on Day 1. sGaw data was log transformed for analysis., treatment ratios were calculated by taking the difference between least square means and the 95% confidence interval was calculated for the difference and anti-logged.

Information from previous studies comparing ipratropium plus beta2-agonist vs ipratropium , following administration of bronchodilator [START_REF]Combivent Inhalation Aerosol Study Group. In chronic obstructive pulmonary disease, a combination of ipratropium bromide and albuterol is more effective than either agent alone[END_REF][START_REF] Sichletidis | Bronchodilatory responses to formoterol, ipratropium bromide and their combination in patients with COPD[END_REF] has provided estimates of within subject standard deviation for weighted mean FEV1 of 0.15 L and shown differences of 0.06 L and 0.08 L. In order to power the study based on these differences, a sample of more than 60 subjects would have been required. Due to logistics and resources, the subject number was limited to 40: with 40 subjects completing and a within subject standard deviation of 0.15L, differences in FEV1 weighted means of 0.096L and 0.111L can be detected with 80% and 90% power, respectively. All calculations were 2-sided and assumed a significance level of 5%.

Results

Forty seven patients were randomized to treatment; forty four completed all treatments and three were discontinued prematurely. One patient was withdrawn because of a protocol violation, after undergoing all single-dose treatments and one repeat-dose treatment; one patient withdrew consent after receiving one single-dose treatment (placebo), and one patient was withdrawn because of lack of compliance with the protocol, after receiving all single-dose treatments.

A summary of demographic and baseline data is presented in Table 1. Following administration of bronchodilator, 45 patients had an increase in FEV1 of > 200 mL and two had an increase of 200 mL, while 17 patients had increases of > 12% over baseline. There were no differences in the responses to bronchodilator between all patients and those taking corticosteroids. Concomitant Combivent ® (6%). 38.2% of the patients were on inhaled corticosteroids and 2% on oral corticosteroids (7.5 mg/day). The bronchodilator reversibility as percent increase from baseline was 25.0 % (mean value, range from 6.1 to 50.0%). Xanthines, inhaled long-acting beta2-agonists, and tiotropium bromide were discontinued two weeks before administration of the first dose of study medication and for the duration of the study.

The combination of ipratropium and ondansetron appeared to be well tolerated. There were no withdrawals due to adverse experiences. A statistically significant difference in weighted mean FEV 1 (0 -6 hours) between treatments was noted in the comparison of ipratropium bromide vs placebo (Tables 2,3, Figure 1). Treatment with ipratropium bromide (compared to placebo) also resulted in a statistically significant increase in peak FEV1 and in FEV1 determined 6 hours postdose (Table 4). However, dosing with ondansetron and ipratropium together was no more effective than dosing with placebo and ipratropium bromide in increasing the weighted mean of FEV1 (0-6 hours), peak FEV1 and FEV1 6 hr. This was observed for both the single dose and the repeat-dose parts of the study.

Similarly, statistically significant increases in weighted mean sGaw, and in peak values and values determined 6 hours post-dosing for FVC and sGaw were observed after dosing with ipratropium, compared to placebo. As was observed for FEV1, there was no significant increase in FVC and sGaw after dosing with both ipratropium and ondansetron compared with ipratropium alone.

Results for the repeat-dose part of the study were similar to results from single dose administration (Table 4, Figure 2).

The only statistically significant change observed after dosing with ondansetron was an increase in FVC 6 hours post-dosing with a single 24 mg dose, in the presence of ipratropium placebo. When compared with ondansetron placebo/ ipratropium placebo, this treatment resulted in a statistically significant increase in FVC 6 h of 0.132 L (95% confidence interval: 0.021, 0.242 L). This increase was similar to that seen with ipratropium 40 mcg compared with placebo (treatment difference 0.224 L; 95% confidence interval: 0.113, 0.335 L). Following co-administration of ondansetron 24 mg and ipratropium 40 mcg, FVC was not significantly different to FVC after administration of ipratropium 40 mcg alone. In order to compare changes in sGaw and FEV1, effect size was calculated by dividing treatment differences by the standard deviation; sGaw was consistently more sensitive than FEV1, particularly at peak value and 6 hours post-dose (Table 5).

Discussion

The comparison of ipratropium bromide 40 mcg + ondansetron 24 mg with ipratropium bromide 40 mcg + placebo showed that there was no improvement in weighted mean FEV1 (0-6 hours), peak FEV1 or FEV1 determined 6 hours post-dosing when patients received ondansetron, compared Therefore, results of this randomized, double-blind, placebo-controlled, crossover study indicate that dosing with oral ondansetron in addition to ipratroprium is not effective in improving pulmonary function in patients with COPD.

When ipratropium bromide was compared with placebo, statistically significant increases in weighted mean FEV1 (0-6 hours), peak FEV1 and FEV1 6 hours were observed. Similar positive results were determined for FVC and sGaw. The comparison of ondansetron 24 mg with placebo showed a statistically significant improvement in FVC determined 6 hours post-dosing, but no other variable showed a statistically significant change. Results obtained from a limited repeat-dose administration were similar to those observed following single doses. However since we have not corrected for multiple comparisons, this increase could be a false-positive finding.

As expected, ipratropium bromide induced a marked bronchodilation compared to placebo, as determined by spirometry (FEV1). This was accompanied by increases in sGaw (weighted mean, peak value and sGaw determined 6 hours post-dose); sGaw was consistently more sensitive than FEV1, particularly in determinations at peak and 6 hours post-dose. Similar results have been observed with ipratropium bromide [START_REF] Van Noord | Long-term treatment of chronic obstructive pulmonary disease with salmeterol and the additive effect of ipratropium bromide[END_REF] and other bronchodilators [START_REF] Maesen | Ba 679, a new long-acting antimuscarinic bronchodilator: a pilot dose-escalation study in COPD[END_REF][START_REF] Morice | Use of whole-body plethysmography to compare bronchodilator inhaler efficacy[END_REF] in patients with COPD [START_REF] Van Noord | Long-term treatment of chronic obstructive pulmonary disease with salmeterol and the additive effect of ipratropium bromide[END_REF][START_REF] Maesen | Ba 679, a new long-acting antimuscarinic bronchodilator: a pilot dose-escalation study in COPD[END_REF] and in normal healthy volunteers [START_REF] Morice | Use of whole-body plethysmography to compare bronchodilator inhaler efficacy[END_REF]. In a recent publication, spirometry, plethysmography and impulse oscillometry were compared in their ability to measure bronchodilation induced by inhalation of increasing doses of salbutamol in patients with COPD. For the lower doses of salbutamol, plethysmography (sGaw) and impulse oscillometry were more sensitive than FEV1 in detecting bronchodilation; on the other hand, sGaw was the most variable measurement, followed by oscillometry parameters, with spirometry (FEV1) being the least variable [START_REF] Borrill | Measuring bronchodilation in COPD trials[END_REF]. Plethysmography measures airway resistance, conductance and lung volumes while impulse oscillometry measures airway resistance and lung reactance [START_REF] Goldman | Clinical applications of forced oscillation[END_REF]. Unlike spirometry, these tests do not require effort dependent forced expiration. In patients with COPD experiencing bronchodilation induced by increasing doses of salbutamol, the shape of the dose-response curves was different for sGaw compared to other measurements; there was an initial sharp improvement in sGaw at low doses of salbutamol, after which the gradient of the curve was similar to other readings [START_REF] Borrill | Measuring bronchodilation in COPD trials[END_REF]. This intial rapid improvement in airway conductance may explain the increased sensitivity of plethysmography compared with the other methods at low bronchodilator doses. For the purposes of detecting small physiological effects in COPD [START_REF] Borrill | Measuring bronchodilation in COPD trials[END_REF] or asthma [START_REF] Houghton | A comparison of lung function methods for assessing dose-response effects of salbutamol[END_REF], this may be a potential advantage (e.g.

measuring physiological changes at 12 or 24 hours after administration of a bronchodilator .

Serotonin (5-HT) has been reported to have effects on airway smooth muscle contraction through interactions with multiple receptor subtypes. Conversely, blockers of 5-HT receptor subtypes have been shown to reduce constriction or induce bronchodilation. In in vitro studies in human airways, tropisetron, a 5-HT3 and 5-HT4 receptor blocker, and ondansetron, a 5-HT3 receptor blocker, were able to completely or partially, respectively, reduce the facilitation of contraction induced by

A c c e p t e d m a n u s c r i p t

9 electrical stimulation in the presence of 5-HT [START_REF] Dupont | The effects of 5-HT on cholinergic contraction in human airways in vitro[END_REF]. In a small group of subjects with COPD reversible to albuterol, administration of inhaled or of intravenous ketanserin, a 5-HT2 antagonist, induced a small degree of bronchodilation that was statistically significantly different from placebo [START_REF] Cazzola | Acute respiratory and cardiovascular effects of inhaled ketanserin in COPD. A comparative study with intravenously administered ketanserin[END_REF]. In contrast, in this study, addition of a 5HT3 receptor blocker (ondansetron) did not improve the effect of ipratropium in inducing bronchodilation in patients with COPD.

In conclusion, results obtained in this study suggest that the participation of serotonin in establishing pulmonary tone or inducing bronchoconstriction may be mediated through interaction with 5-HT subtype receptors other than 5-HT3. In vitro data suggests that blocking several 5-HT receptors (5-HT3 and 5-HT4; 22) may have been more effective than blocking a single type of receptor. This suggests that tropisetron may have been a better choice than ondansetron. In addition, although both compounds have a high 5-HT3 binding affinity, tropisetron has a considerably longer half life than ondansetron (8-10 hrs and 3.5 hrs, respectively) [START_REF] Houghton | A comparison of lung function methods for assessing dose-response effects of salbutamol[END_REF]. On the other hand, one goal of this study was to identify receptors involved in the disease process; this required specific receptor blockers. As additional mechanisms involved in airway diseases are identified and the number of drugs used in treatment increases, this type of study, conducted in a small number of patients for a short period of time, may be of value in the identification of useful drug combinations (38,39). This study also suggests that determination of sGaw may be a useful The effect size is equal to the treatment difference divided by the standard deviation; treatment differences for sGaw are on the log scale.

A c c e p t e d m a n u s c r i p t 3 Studies

 3 in human airways have shown that cholinergic contraction induced in vitro following electrical stimulation was increased by the presence of 5-HT; this facilitation of cholinergic contraction could be blocked completely by tropisetron, a 5-HT3 and 5-HT4 receptor blocker, and

A c c e p t e d m a n u s c r i p t 4 bromide

 4 40 mcg via MDI. Patients received single doses of each study medication in the clinic; spirometry and plethysmography were performed 30 minutes before and 0.5, 1, 2, 3, 5 and 6 hours following administration of study medication. Dosing days were separated from each other by at least 48 hours. Part B of the study was conducted at least 48 hours following the last administration of study medication in Part A. Part B was a randomized, double-blind, placebo-controlled, two-period crossover study. Patients were randomized to receive ipratropium bromide 40 mcg tid via MDI plus ondansetron placebo qid for two days; on day 3 patients received a single dose of ipratropium bromide 40 mcg via MDI plus ondansetron placebo. Alternatively, patients received ipratropium bromide 40 mcg tid via MDI plus ondansetron 8 mg qid orally, for two days; on day 3 patients received a single dose of ipratropium bromide 40 mcg via MDI plus ondansetron 8 mg orally. The two study periods were separated from each other by at least 48 hours. Patients remained in the clinic for about 7 hours following administration of study medication on day 3 of each crossover period; spirometry and plethysmography were determined as before, 30 minutes prior to and 0.5, 1, 2, 3, 5 and 6 hours following administration of study medication, on day 3 of each crossover period.

A c c e p t e d m a n u s c r i p t 7 medications

 7 at baseline included the following bronchodilators: salbutamol/albuterol (43% of patients), ipratropium bromide (17%), the combination of ipratropium bromide and albuterol,

A c c e p t e d m a n u s c r i p t 8 with

 8 placebo. Similar lack of improvement was observed for FVC, FEV1/FVC ratio and sGaw.

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 13 [

 13 complement to the determination of FEV1 in establishing both peak bronchodilatory activity and duration of effect. ACKNOWLEDGMENT : We thank Sheng-Fang Su, MS, of GlaxoSmithKline Research and Development, Ltd., for her contribution to this study. We also thank Vanessa Schelfhout, MD, Ghent University Hospital, Ghent, Belgium and Bruno Dietrich, MD, Clinical Research, Hennigsdorf , Germany for their help in the realisation of this study 37] Wolf H. Preclinical and clinical pharmacology of the 5-HT3 receptor antagonists. Scand J Rheumatol 2000 Suppl 113:37-45 [38] Tonelli, M, Zingoni M, Bacci E et al. Short-term effect of the addition of leukotriene receptor antagonists to the current therapy in severe asthnmatics. Puklm Pharmacol Ther 2003; 16: 237-240. [39] Vatrella A, Ponticiello A, Pelaia G et al. Bronchodilating effects of salmeterol, theophylline and their combination in patients with moderate to severe asthma. Pulm Pharmacol Ther 2005; 18:

wm=weighted mean A c c e p t e d m a n u s c r i p t 19 Figure 1 Figure 2 A c c e p t e d m a n u s c r i p t 20 Figure 1 .Figure 2 .

 19122012 Figure 1

  m g + Ipratropium 40 µg Ondansetron Placebo + Ipratropium 40 µg O ndansetron 8 m g + Ipratropium 40 µg O ndansetron Placebo + Ipratropium Placebo O ndansetron Placebo + Ipratropium 40 µg O ndansetron 24 m g + Ipratropium Placebo

14 Table 1 .

 141

	Table 2
	Summary of Demographic and Baseline Data of ITT Population (n=47) Summary of derived parameters: Weighted Mean 0-6 h FEV1 (L)
	Treatment	N n	Mean (SD)	Median (range)
	V a r i a b l e s			D a t a
	Age, yr; mean (SD), range Single dose (Part A)			59.6 (7.8), 43-75
	Sex, No. (%) Ondansetron pbo + ipratropium pbo	47 47 1.683 (0.4995) 1.608 (0.75-2.96)
	male			42 (89)
	female Race, No. (%) White Black American-Hispanic Concomitant Medications Inhaled corticosteroids Oral Corticosteroids FEV 1 before albuterol/salbutamol (all patients), L FEV1 before albuterol/salbutamol, L (patients taking corticosteroids) FEV1 before albuterol/salbutamol, % Predicted (all patients) FEV1 after albuterol/salbutamol (all patients), L FEV1 after albuterol/salbutamol (patients taking corticosteroids), L Reversibility Delta FEV1: increase over baseline (all patients), L Delta FEV1; increase over baseline (patients taking corticosteroids), L Delta FEV1; % increase over baseline (all patients) Delta FEV1: percent of predicted value (all patients) A c c e p t e d m a n u s c r i p t 5 (11) 42 (89) 4 (9) 1 (2) 18 (38.2%) 1 (2%) 1.70 (0.49) 1.72 (0.53) 49.0 (12.1) 2.09 (0.52) 2.1 (0.51) 0.39 (0.17) 0.38 (0.11) 25.0 (12.1) 11.3 (4.6) Ondansetron pbo + ipratropium 40 mcg 46 46 1.849 (0.5243) 1.794 (0.84-3.18) Ondansetron 24 mg + ipratropium pbo 46 46 1.702 (0.5141) 1.659 (0.72-2.99) Ondansetron 24 mg + ipratropium 40 mcg 46 46 1.858 (0.5288) 1.743 (0.89-3.23) Repeat dose (Part B) Ipratropium 40 mcg qid + Ondansetron pbo tid 45 44 1.795 (0.5245) 1.725 (0.75-3.11) Ipratropium 40 mcg qid + ondansetron 8 mg tid 45 43 1.761 (0.5168) 1.643 (0.81-3.05) A c c e p t e d m a n u s c r i p t
		15 16

Table 3

 3 Summary of Results for Weighted Mean 0 -6 h FEV 1 (L) -Single Dose Difference Between Combinations of Study Medications

	Treatment Comparison	Difference 95 % Confidence Interval
	Ondansetron 24 mg + ipratropium 40 mcg vs 0.001	-0.044, 0.046
	Ondansetron pbo + ipratropium 40 mcg
	Ondansetron 24 mg + ipratropium pbo vs	0.011	-0.033, 0.055
	Ondansetron pbo + ipratropium pbo
	Ondansetron pbo + ipratropium 40 mcg vs	0.149	0.105, 0.194
	Ondansetron pbo + ipratropium pbo

Table 4

 4 Summary of Results for FEV1, FVC and sGaw.

			Ipratropium Bromide vs Placebo -Single Dose
		Differences of Least Square Means for FEV1 and FVC
	Variable		Least Square Means	Test -Reference Treatment
		Test	Reference	Difference	95% CI
		Treatment	Treatment
	Weighted Mean		1.84	1.69	0.149	(0.105, 0.194)
	FEV1 0-6 hr.
	Peak FEV1, L		1.98	1.80	0.173	(0.123, 0.223)
	FEV1 6 hr post-dose 1.73	1.65	0.078	(0.017, 0.138)
	Peak FVC, L		3.70	3.38	0.324	(0.228, 0.420)
	FVC 6 hr post-dose	3.29	3.07	0.224	(0.113, 0.335)
			Ipratropium Bromide vs Placebo -Single Dose
			Ratio of Geometric Least Square Means for sGaw
	Variable	Geometric Least		Ratio of Geometric Means
		Square Means		Test / Reference Treatment
		Test		Reference	Ratio	95% CI
		Treatment	Treatment
	Weighted Mean		0.574	0.420	1.368	(1.179, 1.588)
	sGaw 0-6 hr.
	Peak sGaw		0.721	0.493	1.463	(1.265, 1.692)
	sGaw 6 hr post-dose 0.454	0.383	1.184	(1.008, 1.392)

Table 5

 5 Treatment effect (Treatment Difference / Standard Deviation) -Single Dose

	Parameter	effect size	(treatment difference/
			standard deviation)
	1) FEV1 wm (0-6h)	1.400	(0.149/0.1064)
	2)FEV1 peak (0-6h)	1.455	(0.173/0.1189)
	3)FEV1 6h	0.054	(0.078/0.1447)
	4)sGaw wm(0-6h)	1.436	(0.313/0.2182)
	5)sGaw peak (0-6h)	1.786	(0.381/0.2130)
	6)sGaw 6h	0.717	(0.169/0.2361)

This study was funded by GlaxoSmithKline Pharmaceuticals.


Ingrid De Chazeron

Sandrine Daval

Sylvie Ughetto

Damien Richard

Alain Nicolay

Didier Lemery

Pierre M Llorca

François Coudoré
email: fcoudore@chu-clermontferrand.fr

Pr F Coudoré

GC-MS-determined cotinine in an epidemiological study on smoking status at delivery

Keywords: cotinine, pregnancy, newborn, smoking status

The objective of this study was to measure plasma cotinine levels in pregnant women and their newborns using a gas chromatography-mass spectrometry (GC-MS) method in an epidemiological delivered population with a wide range of tobacco intakes. Nearly one thousand pregnant women from regional maternity wards (n = 1007) were selected for the study. Each patient kept a tobacco diary and underwent a blood test to assess cotinine levels at the same time that newborns' cordonal plasma was taken. These values were then crosschecked.

Cotinine was estimated using a selected-ion monitoring mode with a 1.5 ng/ml quantification limit. Cotinine levels in mothers and newborns were highly correlated, whatever the mother's smoking status with a calculated cut-off for cotinine levels in active smokers of 21.5 ng/ml. Finally, cotinine determined through this GC-MS method offered a sensitive and accurate measure of tobacco exposition of pregnant women and their babies.

Introduction

The association between maternal smoking, retarded maternal condition and birth outcome is well known. Smoking during pregnancy increases the risk of spontaneous abortions, preterm premature membrane ruptures, stillbirths, preterm deliveries, and sudden infant death syndrome. Prenatal exposure to tobacco smoke is a risk factor for respiratory infections, asthma, allergy and childhood cancer [START_REF] Castles | Effects of smoking during pregnancy. Five meta-analyses[END_REF] Risks for most of these conditions have been found to increase with the number of cigarettes smoked [START_REF] Polanska | Influence of smoking during pregnancy on children's health-overview of epidemiologic studies][END_REF]. In order to have a reliable measure of this exposition, a survey was conducted on nearly a thousand pregnant women from regional maternity wards.

Cotinine presents a longer biological half-life than nicotine, and is the best biochemical marker for segregating smokers from non-smokers. Its rate is correlated with the daily cigarettes consumption [START_REF] Blackford | Cotinine concentration in smokers from different countries: relationship with amount smoked and cigarette type[END_REF][START_REF] Dhar | Measuring tobacco smoke exposure: quantifying nicotine/cotinine concentration in biological samples by colorimetry, chromatography and immunoassay methods[END_REF]. Among all biomarkers used for routine diagnosis of in utero tobacco-smoke exposure, cotinine levels in umbilical cords are probably the most reliable and non-invasive for the newborn [START_REF] Diguet | Neonatal management of a child exposed to tobacco in utero and postnatal biomarkers[END_REF]. Cotinine levels were determined in cordonal plasma and blood of new mothers and cross-checked against the mother's answers to a questionnaire on maternal smoking status.

Since the GC-MS method is recognized as the standard of reference in cotinine analysis, particularly when concentration levels are close to 1 ng/ml, we have examined serum cotinine in pregnant women with a wide range of cigarette intakes and in their newborns, to determine fully its value in assessing tobacco consumption in specific populations.

Methods

Epidemiological study and sample collection

A c c e p t e d m a n u s c r i p t

The study was conducted from July 2003 to June 2004 in all the delivery units (n = 16) of a French administrative area in Central France (Auvergne). This multicenter study was approved by the local ethics committee in 2003. Eligible women (n = 1007) were selfquestioned on their smoking status at delivery, and maternal and cord blood-serum samples were collected in heparinised tubes. Samples were then centrifuged at 3,000 g for 10 min to remove plasma, then frozen until assay.

Gas chromatography and mass spectrometric detection

Cotinine analyses were performed on an HP5973 MS, with an HP6890 series GC (Agilent Technologies, Atlanta, GA). Injections were performed using an HP6890 autosampler.

The glass liner was equipped with a Siltek deactivated inlet glass liner (Restek, Bellefonte, PA). The GC was operated in splitless injection mode, with a constant flow of 0.5 ml/min of helium through the HP-5MS column (30 m x 0.25 mm i.d. with a 0.25-µm film thickness (J&W, Folsom, CA). Splitless injection (1 µl) was performed at 260°C. The transfer line temperature was 290°C. The GC oven temperature was programmed to start at 80°C for 0.5 min, then increase to 285°C at a rate of 20°C/min, and remain at 285°C for a further 5 min. MS detector parameters were: transfer line temperature 290°C; solvent delay 3 min; electron energy 70 eV. The MS was run in Selected-Ion Monitoring mode (SIM) for quantitative analysis. HP Chemstation® software controls the equipment and processes the data.

Sample preparation

To extract cotinine, 0.6ml of potassium hydroxide solution (1M) was added to 1 ml of plasma, 10 µL of the stock solution of deuterated cotinine (10 µg/ml), and 5 ml of dichloromethane in a 10 ml screw-capped glass centrifuge tube. The centrifuge tube was capped and shaken on a mechanical shaker for 15 min, then centrifuged for 10 min at 3,000 rpm. The organic layer was removed and put into a new glass tube containing 50 µL HCl

A c c e p t e d m a n u s c r i p t

0.25M. This tube was shaken on a mechanical shaker for 15 min then centrifuged for 10 min at 3,000 rpm. The organic layer was evaporated to dryness at ambient temperature, and the residue was reconstituted with 60 µL of ethyl acetate and transferred for GC-MS analysis.

Calibration and QC standards were from separate stock solutions and extracted before each set of dosages by mixing various known amounts of unlabeled cotinine with a fixed amount of deuterated cotinine (100 ng) in 1 ml of human blank plasma. Standard concentrations of 0, 10, 25, 50, 100 and 250 ng/ml were used. QC sample concentrations were 5, 30 and 75 ng/ml.

Validation of the GC-MS method

The validation program included linearity, limit of quantification (LOQ), precision and accuracy studies of cotinine in human plasma. The LOQ was defined as the lowest concentration of cotinine that could be measured with acceptable precision and accuracy.

Recovery after the extraction procedure was determined by comparing the areas of extracted cotinine with that of the nonextracted standard samples representing 100% recovery. Standard samples were checked for linearity over ten days. To determine intra-and inter-day precision and accuracy of the assay, a replicate set of spiked samples containing known cotinine concentrations (5, 150 and 300 ng/ml) were analyzed.

Statistical analysis

Data analysis was performed using the SAS® statistical software package. Statistical tests used a 5-percent two-sided risk α. Given the distribution of the variables, the results of the corresponding statistical analyses are generally expressed as means ± standard deviation (SD).

Minimum and maximum values are also given. Spearman correlation coefficients (rs) were used as nonparametric measures to analyze relationships between i) smoking status/mother

Results

GC-MS analysis

Cotinine and deuterated cotinine had a retention time of 8.3 min. Drug-free plasma samples were tested to confirm the absence of cotinine. In SIM mode, the molecular ion of m/z 176 was the quantification ion for cotinine and the confirmation ions were the molecular ions of m/z 98, 104 and 118. For cotinine-D3, the molecular ion of m/z 179 was used as quantification ion and the m/z 101 and 121 ions were the confirmation ions. Limit of quantification was 1.5 ng/ml. The assay was linear over the range of 10-250 ng/ml. Extraction recoveries were between 49 and 63%. Correlation coefficient and y-intercept of the calibration curves for plasma (n = 10) were 0.9996 +/-0.0003 and 0.0049 +/-0.0178, respectively.

Epidemiological application

Mean age of the recruited mother population was 29.5 years (SD = 4.3). Cotinine levels of newborns and their mothers in smoking and non-smoking subgroups appear in Table 1.

Cotinine concentration is significantly higher (p < 0.001) in the smokers subgroup, as expected.

The same results were obtained with the corresponding babies. Mother and paired-newborn plasma cotinine concentrations were highly correlated (rs = 0.95, p < 0.0001, n = 961). For non-smoking mothers, this correlation was 0.70 (p < 0.0001), thus confirming a high cotinine exposure of the foetus. Moreover, a dose-response relationship was found between maternal cotinine levels and number of cigarettes smoked per day (Fig 1).

Women were then classified as non-smokers or active smokers, depending on cotinine levels (mean cotininemia = 101.3 ng/ml; SD = 78.4) (non-smokers: cotinine levels below 15

A c c e p t e d m a n u s c r i p t

ng/ml; n = 766 with n = 600 samples < LOQ, active smokers: cotinine levels above 15 ng/ml; n = 195). A receiver operator characteristic (ROC) curve (Fig. 2) and a sensitivity plot (true positive rate) against l-specificity (false positive rate) was used to allow the selection of the best possible cut-off point between passive/active smokers, i.e. the one which maximizes the sensitivity and specificity of the test. The calculated cut-off for cotinine levels in an active smoker was 21.5 ng/ml. This value showed a specificity of 95.3% and a sensitivity of 83.2%.

Discussion

Plasma was a specimen of choice for cotinine measurement, since it is not subject to bacterial degradation or sample concentration. Admittedly, assay in urine presents some advantages, such as the reliability of the results and weak variations during a 24-hour cycle, but this matrix was not used, as it was difficult to collect during delivery.

Numerous methods have been reported for cotinine analysis in biological samples. To summarise, these methods include radioimmunoassay (6), high-performance liquid chromatography [START_REF] Ghosheh | A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers[END_REF][START_REF] Stolker | Determination of nicotine and cotinine in rat plasma by liquid chromatography-tandem mass spectrometry[END_REF], as well as gas chromatography-mass spectrometry (GC-MS) [START_REF] Cognard | Determination of nicotine and its major metabolite cotinine in plasma or serum by gas chromatography-mass spectrometry using ion-trap detection[END_REF][START_REF] Jacob | Simultaneous determination of mecamylamine, nicotine, and cotinine in plasma by gas chromatography-mass spectrometry[END_REF][START_REF] Jung | Simultaneous GC-MS determination of nicotine and cotinine in plasma for the pharmacokinetic characterization of nicotine in rats[END_REF][START_REF] Man | Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry[END_REF][START_REF] Shin | Sensitive and simple method for the determination of nicotine and cotinine in human urine, plasma and saliva by gas chromatographymass spectrometry[END_REF][START_REF] Torano | Simultaneous determination of the tobacco smoke uptake parameters nicotine, cotinine and thiocyanate in urine, saliva and hair, using gas chromatography-mass spectrometry for characterisation of smoking status of recently exposed subjects[END_REF].

Since the GC-MS method is recognized as the standard of reference in drug analyses, it was used in this study. Limit of quantification was 1.5 ng/ml, which was lower than values obtained with the HPLC/UV method, e.g. 10 ng/ml with Ghosheh et al. 2000 [START_REF] Ghosheh | A simple high performance liquid chromatographic method for the quantification of total cotinine, total 3'-hydroxycotinine and caffeine in the plasma of smokers[END_REF]. It bears stressing that previously published values in GC-MS methods with a liquid-liquid extraction were higher than ours: 2 and 5 ng/ml (9, 10). However, using the same detection, [START_REF] Man | Simple, rapid and sensitive assay method for simultaneous quantification of urinary nicotine and cotinine using gas chromatography-mass spectrometry[END_REF] There is still no real consensus on a precise target value, with ranges from 10 ng/ml (17) to 15 ng/ml [START_REF]Tobacco smoking and pregnancy[END_REF][START_REF] Twardella | Short-term benefit of smoking cessation in patients with coronary heart disease: estimates based on self-reported smoking data and serum cotinine measurements[END_REF], or between 10-15 [START_REF] Bernert | Comparison of serum and salivary cotinine measurements by a sensitive high-performance liquid chromatography-tandem mass spectrometry method as an indicator of exposure to tobacco smoke among smokers and nonsmokers[END_REF], and up to the 10-20 range [START_REF] Vartiainen | Validation of self reported smoking by serum cotinine measurement in a community-based study[END_REF]. To conclude, we believe the GC-MS is a reliable and sensitive method, useful in epidemiological studies to assess maternal and newborn tobacco-smoke exposure.

A c c e p t e d m a n u s c r i p t

  cotinine levels, ii) mother/newborn cotinine-level test results for nicotine dependence and other variables.

Table 1 :Figure 1 :Figure 2 :

 112 Figure 1:Relationship between cotinine levels and number of cigarettes smoked per day t test, p<0.0001)

  Stolker et al. in 2003 (8) reported either lower or similar LOQ values, i.e. 0.05 and 1 ng/ml, respectively. The results of this technique provide a satisfactory reflection of the reality of maternal smoking status due to the low LOQ.The dose-response relationship found between maternal cotinine levels and the number of cigarettes smoked by the mother each day is consistent with results presented byMcDonald et al. in 2005 (16). Moreover, our results confirm the high accuracy of self-reported smoking status during pregnancy.Our cut-off value for passive/active smokers, 21.5 ng/ml, has been validated by a good specificity, with only moderate loss of sensitivity, and is consistent with published results.

	A c c e p t e d	m a n u s c r i p t
		,(12) Shin
	et al., 2003,(13) and Cognard and Staub, in 2003,(9) presented lower or similar LOQ values
	than us, 0.5, 1 and 1 ng/ml, respectively, although in the last article, extraction was performed
	using the Toxitubes® system, making it more expensive and not realistically employable in our
	large-scale clinical study. Using HPLC with MS or MS/MS detection, Gunter in 1997 (15) or


Carsten Wunderlich
email: carstenwunderlich@gmx.de

Alexander Schmeisser

Christian Heerwagen

Bernd Ebner

Kristin Schober

Ruediger C Braun-Dullaeus

Carsten Schwencke

Michael Kasper

Henning Morawietz

Ruth H Strasser

Chronic NOS inhibition prevents adverse lung remodelling and pulmonary arterial hypertension in caveolin-1 knockout mice

Keywords: eNOS-endothelial nitric oxide synthase, wt-wild type, cav-1 ko-caveolin- caveolin, eNOS, nitric oxide, superoxide, lung fibrosis

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

Introduction

Caveolae are small invaginations of the plasma membrane abundantly present in most cells of the cardiopulmonary system. In these cells caveolae function in protein trafficking, cholesterol homeostasis and signal transduction. Three family members of caveolins are identified to date. Caveolin-1 (cav-1) and caveolin-2 (cav-2) are coexpressed in adipocytes, fibroblasts and endothelial cells while caveolin-3 (cav-3) expression is confined to muscle cells. [START_REF] Razani | Caveolin-deficient mice: insights into caveolar function human disease[END_REF] Cav-1 and cav-3 are essential for the biogenesis of caveolae in cells that normally express these isoforms [START_REF] Razani | Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities[END_REF][START_REF] Drab | Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice[END_REF][START_REF] Galbiati | Caveolin-3 null mice show a loss of caveolae, changes in the microdomain distribution of the dystrophin-glycoprotein complex, and t-tubule abnormalities[END_REF] , whereas cav-2 is not absolutely required. [START_REF] Razani | Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae[END_REF] Recently, the caveolin proteins have been implicated in structural lung remodelling and development of pulmonary arterial hypertension (PAH). [START_REF] Razani | Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae[END_REF][START_REF] Gratton | Caveolae and caveolins in the cardiovascular system[END_REF][START_REF] Murata | Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice[END_REF][START_REF] Jasmin | Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression[END_REF] Indeed, one prominent feature of caveolin-1 knockout mice (cav-1 ko) is an adverse lung phenotype with thickened alveolar septa, smaller alveolar spaces and an interstitial hypercellularity. 2;3 Cav-1 ko are further characterized by a substantial pulmonary arterial hypertension (PAH) which is accompanied by a hypertrophied right ventricle (RV). [START_REF] Zhao | Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice[END_REF] Moreover, cav-1 ko were shown to suffer from a dramatic reduction in life span. [START_REF] Park | Caveolin-1 null (-/-) mice show dramatic reductions in life span[END_REF] Of note, cav-1 ko also display a near complete ablation of cav-2 expression. These findings provide the basis for an intense debate on the functional implications of cav-1 on lung remodelling. [START_REF] Jasmin | Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression[END_REF] However, the most direct evidence for a independent role of cav-1 on pulmonary function versus alveolar thickening is supported by data documenting that recently generated cav-2 deficient mice (with retained cav-1 expression) do not have PAH, but do exhibit pulmonary hyperplasia. [START_REF] Razani | Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae[END_REF] Moreover, several papers reported a decreased expression of pulmonary caveolins in various animal models of PAH. 8;11 These findings also appear to be relevant to human PAH in that decreases in cav-1 gene and protein expression have been recently demonstrated in plexiform lesions of patients with severe PAH. [START_REF] Achcar | Loss of caveolin and heme oxygenase expression in severe pulmonary hypertension[END_REF] Altogether these studies validate the importance of caveolins in the development of lung pathologies but the specific role of cav-1 in this process awaits further clarification.

A c c e p t e d m a n u s c r i p t 4

Within the vascular endothelium endothelial nitric oxide synthase (eNOS) targets to caveolae of the plasma membrane where it is tonically inhibited by interaction with cav-1 resulting in a constitutive hyperactivation of the nitric oxide (NO) pathway in cav-1 ko. 3;9;13 There is mounting evidence that eNOS can be converted to an important 'reactive oxygen species' generator within the vessel wall. 14;15 Within the so-called "uncoupled" eNOS enzyme electrons flow to molecular oxygen and generate superoxide (O 2 -) instead of NO. This scenario results in a reduced NO production by eNOS while this enzyme additionally produces O 2 -. The superoxide anion avidly reacts with vasodilating NO to form the oxidant species peroxynitrite. [START_REF] Szabo | Peroxynitrite: biochemistry, pathophysiology and development of therapeutics[END_REF] Accordingly, the NO bioavailability is further reduced with concurrent increased oxidant stress.

PAH is a common finding in human lung fibrosis. [START_REF] Humbert | Treatment of pulmonary arterial hypertension[END_REF] Despite recent advances in symptomatic treatment this disease is characterized by a progressive malfunction associated with a poor prognosis of affected individuals. Unfortunately, no current treatment option cures this devastating condition. Various molecules have been implicated as putative candidates of its pathogenesis including endothelial dysfunction due to impaired NO signalling. [START_REF] Humbert | Treatment of pulmonary arterial hypertension[END_REF] In this context recently generated cav-1 ko may serve as an useful model of this disorder and help to further unveil the molecular mechanism of this disease and to uncover potential targets for future therapies.

Given the adverse pulmonary phenotype of cav-1 ko and the close regulatory interaction between cav-1 and eNOS in normal physiology we questioned whether a perturbation of NO signalling with consecutive enhanced oxidative stress may be involved in the genesis of the pathological lung phenotype in cav-1 ko. Therefore we treated respective animals with the NO synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) for 2 month and studied the exercise capacity, pulmonary function, lung morphology and the O 2 -production in related tissues thereafter.

A c c e p t e d m a n u s c r i p t

Materials and Methods

Germany) after retrograde passage of the aortic valve. We applied the following formula for calculation of the transpulmonary gradient (TPG=PAPmean-LVEDP). All data were measured continuously by using a PC with dedicated software (ADInstruments, Chart, Spechbach, Germany).

Blood analysis

After anesthesia and injection with Heparin (250 units i.p.), the mice were ventilated in a pressure-controlled manner at a rate of 110 breaths/min only with room air. A midline skin incision at the lower xiphoid was performed, and the skin was retracted laterally. Afterwards, a substernal abdominal midline incision was done to approach the abdominal surface of the diaphragm, which then was incised to visualize the hearts apex. The oxygenated blood was collected from left ventricle with a heparin-covered syringe. The hemoglobin and hematocrit content and the oxygen saturation were measured with a Micros 60 blood analyzer (Horiba Diagnostics, Irvine, California, USA).

Lung and liver weight

For determination of the lung weight, fresh lungs were flushed in saline to remove contaminating blood and weighed immediately (wet weight). The lungs were then placed in a drying oven at 80 °C until a constant weight was obtained (dry weight). Tissue water content was determined according to the following formula [(wet wt-dry wt/ wet wt) x 100]. For measurement of the liver weight the organ was harvested, flushed in saline to remove contaminating blood and weighed immediately (liver weight). To normalize differences in lungs/liver weight which can be attributed to the overall size of the mouse we then choose to express the results in relation to the total body weight.

A c c e p t e d m a n u s c r i p t

Assessment of right ventricular hypertrophy

The heart was isolated and placed under a dissecting microscope. Attached vessels and both atria were dissected and removed. The right ventricular wall was cut out, blotted, and weighed; then the left ventricular wall and septum were treated the same way and weighed.

Measurement of plasma NO metabolites

After anesthesia and injection with Heparin (250 units i.p.), mouse blood was collected by direct cardiac puncture. The plasma total NO was quantitatively determined based on the enzymatic conversion of nitrate to nitrite by nitrate reductase according with the manufactures instructions and as described previously. 18

Tissue histology

Lungs from untreated and L-NAME treated animals were fixed and stained with hematoxylin and eosin, essentially as described elsewhere. [START_REF] Wunderlich | Disruption of caveolin-1 leads to enhanced nitrosative stress and severe systolic and diastolic heart failure[END_REF] All images were taken using a computerized analyzing system (Leica DMRB microscope, Wetzlar, Germany; Sony Power HAD camera, Tokyo, Japan).

Assessment of exercise tolerance

A 4-liter beaker filled with water (34°C) was used as a swimming pool to assess the exercise tolerance of mice. The mouse was gently placed in the water and carefully observed. The time at which the mouse was initially unable to maintain complete buoyancy was recorded, and the mouse was immediately removed from the pool. No mice were injured in these experiments.

Measurement of superoxide production

Superoxide production was measured using the superoxide dismutase-inhibitable (SODinhibitable) cytochrome c reduction assay. Animals were sacrificed by cervical dislocation.

Materials

All compounds used in this study were either analytical grade or of the highest purity available. Heparin was obtained from ratiopharm (Ulm, Germany), Ketamin from CuraMED Pharma (Karlsruhe, Germany) and xylazine from medistar (Holzwickede, Germany). All other reagents were obtained from Merck (Darmstadt, Germany).

Data Analysis

All results are expressed as means S.D. For multiple comparisons, ANOVA was applied, followed by the Bonferroni correction. Differences were assumed to be significant with a probability value of less than 0.05.

Results

Lowered systemic nitric oxide in L-NAME treated cav-1 ko

Previous studies have unequivocally shown that cav-1 can bind and negatively regulate eNOS activity. We therefore assayed systemic levels the two major oxidation products of nitric oxide (NO), nitrate and nitrite (=NO x ) in untreated and treated mice of both strains. Systemic NO x is generally regarded as a surrogate marker of systemic NO levels. In plasma obtained from untreated cav-1 ko NO x concentration was significantly higher than in WT mice (cav-1 ko:

8.85±1.43 vs. WT: 3.99±0.77 µM, n=10, P<0.01). Several groups have recently shown by western blot analysis that no overt change in expression levels of the three major NO synthases (i.e., inducible, endothelial and neuronal NO synthases) was evident in cav-1 ko as compared to WT animals. Particularly, the inducible NO synthase was undetectable in cav-1 ko. 9;13 Given the close regulatory interaction between cav-1 and eNOS these findings suggest that the high NO x levels are most likely caused by an elevated eNOS activity in cav-1 ko.

After L-NAME application systemic NO x levels fell significantly in both strains (cav-1 ko:

3.81±1.33 vs. WT: 2.31±0.86 µM, n=10, P<0.01 vs. untreated). Notably, NO x concentration in L-NAME treated cav-1 ko approached the level of untreated WT mice.

Normalized lung structure in L-NAME treated cav-1 ko

One prominent feature of cav-1 ko is the adverse lung phenotype with thickened alveolar septa, smaller alveolar spaces and an interstitial hypercellularity. 2;3 All these findings are highly reminiscent of a human disorder named lung fibrosis. [START_REF] Razani | Caveolin-deficient mice: insights into caveolar function human disease[END_REF] Given the hyperactivated NO pathway in cav-1 ko we studied the influence of chronic NOS inhibition by L-NAME on lung structure of cav-1 ko. In L-NAME-treated cav-1 ko we observed improvements of the lung architecture since the alveolar spaces appeared significantly wider and alveolar septa were thinner as compared with untreated cav-1 ko (Figure 1 In view of the adverse lung fibrosis in cav-1 ko we looked in detail on the oxygen saturation with and without L-NAME administration. Without chronic NOS inhibition cav-1 ko displayed a significantly reduced oxygen saturation which was completely normalized after L-NAME treatment (Table 1). As compared to WT littermates hemoglobin and hematocrit levels revealed a polyglobic blood count in untreated cav-1 ko which is most probably a sequel of hypoxic erythropoetin stimulation. As depicted in Table 1 L-NAME application substantially lowered these pathological values although no complete normalization was achieved.

Reversal of the pulmonary hypertension, right ventricular hypertrophy and right ventricular heart failure in L-NAME treated cav-1 ko

Cav-1 ko are additionally characterized by markedly hypertrophied right ventricles (RV). The RV to body weight ratio was significantly increased in untreated cav-1 ko (1.44±0.2, n=10) as compared to WT littermates (1.06±0.2, n=12, P<0.05) and was accompanied by a profound pulmonary hypertension (Figure 3 A). The left ventricular end diastolic pressure (LVEDP)

was elevated in untreated cav-1 ko (15.5±1.9 mmHg) as compared to WT mice (9.0±1.9 mmHg, n=10-12, P<0.05). The same was true for the transpulmonary gradient (TPG -Figure 3 B). Of note, the left ventricular (LV) to RV ratio was dramatically reduced (Figure 2 B) and similar to that seen in other pulmonary hypertension models. Additionally, the central venous pressure was significantly elevated in cav-1 ko as compared to WT counterparts (cav-1 ko:

L-NAME treatment in cav-1 ko results in improved exercise capacity

Cav-1 ko have been found to exhibit a profound exercise intolerance. [START_REF] Drab | Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice[END_REF] This observation may be attributed to the severe lung fibrosis and the pulmonary arterial hypertension. Since we observed substantial regressions in both findings after L-NAME administration we grossly assessed the possible physical consequences of L-NAME treatment by subjecting these animals to a swimming test. In this way, we could directly compare the exercise tolerance of WT, cav-1 ko, and L-NAME treated mice of both strains. As displayed in figure 4 WT animals were able to swim for up to 35 min, while cav-1 ko were exhausted after 13 min. This is contrasted by significant improvements in the exercise capacity of L-NAME treated cav-1 ko (Figure 4).

Enhanced superoxide generation in cav-1 ko is reduced after L-NAME treatment

A possible explanation for the adverse pulmonary phenotype is enhanced oxidant stress in cav-1 ko. To test this hypothesis we harvested aortas, pulmonary arteries and lung tissue from

Discussion

Several groups have independently generated cav-1 ko, and these animals were repeatedly shown to suffer from clear and reproducible vascular and pulmonary phenotypes including a severe lung fibrosis with pulmonary arterial hypertension (PAH). Recent insights into the physiological roles of caveolae and caveolins have been dissected in genetically modified mice. It was revealed that cav-1 is essential for the formation of caveolae in fibroblasts, endothelial cells, adipocytes. 2;3 Additionally, it was shown that the loss of cav-1, which is the primary isoform in the vascular system, results in multiple alterations with a

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Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 13 dysregulation in NO synthesis 3;9 , vascular permeability 6 , mechanotransduction [START_REF] Yu | Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels[END_REF] and pulmonary function. [START_REF] Zhao | Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice[END_REF] These studies unequivocally validate the in vivo importance of cav-1 beyond cells based studies which are hampered by operational definitions of biochemical fractions containing caveolins and the lack of specificity inherent in reagents that extract cholesterol from cells. [START_REF] Gratton | Caveolae and caveolins in the cardiovascular system[END_REF] PAH is a common finding in hypoxic lung disorders like lung fibrosis. Pulmonary hypertension arises when vasoconstriction and adverse remodelling of pulmonary arterioles results in increased pulmonary vascular resistance. The subsequent rise in pressure load promotes right ventricular hypertrophy, which often progresses to premature death from right heart failure. [START_REF] Humbert | Treatment of pulmonary arterial hypertension[END_REF] Cav-1 has been suggested to function as a key regulator of adverse lung remodelling and the development of PAH. [START_REF] Razani | Caveolin-2deficient mice show evidence of severe pulmonary dysfunction without disruption of caveolae[END_REF][START_REF] Gratton | Caveolae and caveolins in the cardiovascular system[END_REF][START_REF] Murata | Reexpression of caveolin-1 in endothelium rescues the vascular, cardiac, and pulmonary defects in global caveolin-1 knockout mice[END_REF][START_REF] Jasmin | Lung remodeling and pulmonary hypertension after myocardial infarction: pathogenic role of reduced caveolin expression[END_REF] Indeed, cav-1 ko were shown to suffer from a severe lung fibrosis with thickened alveolar septa, smaller alveolar spaces and an interstitial hypercellularity. 2;3 Our findings demonstrate that loss of cav-1 in lung parenchyma also results in PAH and right ventricular hypertrophy which validates former findings. [START_REF] Zhao | Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice[END_REF] All these features are highly reminiscent of the human lung fibrosis which is frequently associated with PAH. Moreover, we found signs of right ventricular heart failure as evidenced by dramatically increased liver to body weight ratio in cav-1 ko. To the best of our knowledge this study is the first to show that cav-1 ko additionally display a significantly reduced arterial oxygen saturation accompanied by a polyglobic blood count with increased hemoglobin and hematocrit values. Elevated hemoglobin and hematocrit levels are a common finding in chronic lung disorders and are regarded as a sequel of enhanced hypoxic erythropoetin stimulation. Given these characteristics evident in untreated cav-1 ko these animals may be viewed as an useful model of PAH in lung fibrosis.

In normal lung tissue, endothelial cells and type I pneumocytes, abundantly express cav-1.

Therefore it seems easily conceivable that histological and functional abnormalities develop in lungs of cav-1 ko. Nevertheless the specific role of cav-1 and caveolae in this process is In view of the close interaction of cav-1 and eNOS in normal physiology we questioned whether a perturbation in NO signalling is involved in adverse lung remodelling in cav-1 ko.

There is mounting evidence that validates the negative regulatory influence of cav-1 on eNOS function. 3;7;9;13 For instance, enhanced NO-mediated vascular relaxation in isolated aortic rings [START_REF] Drab | Loss of caveolae, vascular dysfunction, and pulmonary defects in caveolin-1 gene-disrupted mice[END_REF] together with increased systemic NO x levels [START_REF] Zhao | Defects in caveolin-1 cause dilated cardiomyopathy and pulmonary hypertension in knockout mice[END_REF] and enhanced eNOS activity in vitro was reported. [START_REF] Wunderlich | Disruption of caveolin-1 leads to enhanced nitrosative stress and severe systolic and diastolic heart failure[END_REF] Since it was repeatedly demonstrated by western blots that no change in expression level of the major NO synthases (i.e., eNOS, inducible NOS, neuronal NOS) in vessels and lungs of cav-1 ko occurred 7;9;13 these findings suggest that the high NO levels we and others have measured are most likely caused by an elevated eNOS activity. Since eNOS derived NO, through potent vasodilator and antiproliferative effects on vascular smooth muscle cells is critical in maintaining normal pulmonary vascular tone and structure it seems difficult to relate eNOS activity to the adverse pulmonary phenotype of cav-1 ko. But accumulating evidence indicates that in some circumstances eNOS can be converted into a important source of radicals within the vessel wall. 14;15 In line with this concept we measured a markedly increased O 2 -generation in vessels and lung tissue harvested from untreated cav-1 ko. The biological sequelae of the so-called eNOS "uncoupling" on NO bioavailability may be profound, because NO production is reduced and O 2 -generation is increased. Since superoxide is known to act as a potent scavenger of bioactive NO this scenario favours significant reductions in the bioavailability of vasodilating NO. Of note, increased O 2 generation has been observed in other experimental models of PAH 20 and markers of oxidative stress are increased in patients with PAH. [START_REF] Humbert | Treatment of pulmonary arterial hypertension[END_REF]

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A c c e p t e d m a n u s c r i p t 8 Aortas

 8 , pulmonary arteries and lungs were rapidly removed, cleaned of adventitial tissue and placed into chilled modified Krebs-buffer (composition in mmol/l: 117.48 NaCl, 4.74 KCl, 2.50 CaCl 2 , 1.19 MgSO 4 , 1.19 KH 2 PO 4 , 24.76 NaHCO 3 and 10 glucose [pH 7.4]). After cyotchrom c (50 µM; SIGMA C-4186, Sigma-Aldrich, St. Louis, Missouri, USA) were added, the samples were incubated at 37°C for 60 minutes with and without SOD (125 U/ml). Cytochrome c reduction was calculated using absorbance at 550 nm corrected for background readings at 540 and 560 nm. Superoxide production was quantified in picomoles per milligram of the respective tissue (dry weight) from the difference between absorbance with or without SOD.

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  Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 9

  C+D). These findings are further corroborated by marked reductions in lung weight to body weight ratios in L-NAME treated cav-1 ko (Figure 2 A). No differences were evident between untreated and L-NAME treated WT mice.A c c e p t e d m a n u s c r i p t Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 10 Improved lung function in L-NAME treated cav-1 ko

17 .A c c e p t e d m a n u s c r i p t

 17 4±3.5 vs. WT: 4.36±2.4 mmHg, n=7, P<0.01). The liver to body weight ratio was dramatically increased in cav-1 ko as compared to WT mice (cav-1 ko: 57.77±5.52 vs. WT: 44.36±5.65 mg/g, n=8-12, P<0.01).In view of these findings we studied the influence of L-NAME treatment on these distinct parameters. While the RV to body weight ratio in L-NAME treated cav-1 ko significantly Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 11 decreased (0.96±0.1, n=9, P<0.05 vs. untreated cav-1-ko) it did not change in L-NAME treated WT animals (0.94±0.04, n=13). As shown in Figure 2 B L-NAME donation in cav-1ko resulted in an increased LV/RV ratio. Furthermore, after L-NAME treatment LVEDP decreased in both strains mice (cav-1 ko: 6.8±1.9 P<0.01 vs. untreated cav-1 -/-, WT: 7.0±2.8 mmHg, n=8-12). Likewise, as depicted in Figure3, we observed a reduction of both the pulmonary artery hypertension and the TPG after chronic NOS inhibition with L-NAME. As a sign of the reduced right ventricular congestion both the central venous pressure (cav-1 ko: 7.2±4.3 P<0.01 vs. untreated cav-1 ko , WT: 5.7±3.6 mmHg, n=6, P<0.01) and the liver/body weight ratio dramatically decreased after L-NAME treatment (cav-1 ko: 52.22±2.26 P<0.01 vs. untreated cav-1 -/-, WT: 46.37±1.54 mg/g, n=8-12).

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 2 untreated animals and performed a cytochrome c reduction assay for measurement of O Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 12 release. We observed striking disparities between WT and cav-1 ko in aorta, pulmonary artery (data not shown, but almost identical as compared to aortic tissue) and lung tissue with a markedly increased O 2 -production in knockout animals (Figure 5 A, B). Given the aforementioned hyperactivity of the eNOS-NO pathway in cav-1 ko we next treated animals of both strains with oral L-NAME and measured ROS generation in these animals. As shown in Figure 5 L-NAME-supplementation resulted in a decreased O 2 -production in lungs and aortas taken from cav-1 ko.

2 ; 3 ; 9

 239 These phenotypes, driven by the global loss of cav-1, may be caused by the deficiency of the gene in cells that normally express this protein, including endothelial cells, adipocytes, fibroblasts, epithelial cells and smooth muscle cells. A plethora of biochemical and genetic data support the role of cav-1 as a negative regulator of agonist-mediated eNOS activation. 3;7;9;13 Accordingly, constitutive eNOS hyperactivation was observed in cav-1 ko. The present study argues that major lung pathologies are caused at least in part by dysregulated NO synthases resulting in enhanced oxidative stress since chronic treatment with the NO synthase inhibitor L-NAME prevented the evolution of the pulmonary abnormalities and alleviated the oxidative stress in cav-1 ko. These findings are inasmuch of paramount importance since they underline the central role of a balanced NO signalling in normal pulmonary physiology.

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  Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 14 still a matter of discussion. Cav-1 is also regarded as a candidate tumor suppressor. In line with this notion, Razani et al. found dramatically increased markers of cellular proliferation in lungs of cav-1 ko.[START_REF] Razani | Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities[END_REF] Therefore they suggested that disruption of cav-1 leads to profound aberrations in cells cycle checkpoints resulting in the abovementioned pathologies.

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  Superoxide mediates pulmonary vasoconstriction and stimulates pulmonary smooth muscle proliferation.21 In view of our data Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 15 it is tempting to speculate that L-NAME donation resulted in a decreased O 2 -generation by dysfunctional eNOS in the pulmonary endothelium thereby preventing the detrimental effects of O 2 -on pulmonary smooth muscle cells and NO bioavailability. Notably, a recent paper reports that a reduction in oxidative stress by gene transfer of extracellular SOD results in a amelioration of PAH in rat model. 22 In agreement with this concept we could additionally demonstrate that chronic NOS inhibition resulted in a reduced O 2 -generation in cav-1 ko and prevented adverse lung remodelling in treated knockout animals. Improvements of the lung architecture with wider alveolar spaces and thinner alveolar septa (Figure 1 A-D) were accompanied by an improved lung function as demonstrated by normalized oxygen saturation and improved blood cell count in treated knockout animals. Moreover, we observed a normalisation of the pulmonary artery pressure in conjunction with an improved blood oxygenation and a regression of both the right ventricular hypertrophy and right ventricular heart failure in L-NAME treated cav-1 ko. Moreover, L-NAME treatment resulted in an enhanced exercise capacity. The pathogenesis of PAH is complex and involves a blend of structural, functional and biological changes leading to alterations in the pulmonary circulation. In cav-1 ko PAH could be caused by the interstitial lung disease due to cav-1 ablation or it could be secondary to left ventricular impairment. Although we and others observed signs of left ventricular dysfunction the pronounced increase in the TPG in cav-1 ko indicates that intrinsic defects of the pulmonary vessels leading to enhanced vasoconstriction is at least partially involved in the development of PAH. Indeed, recent advances in the understanding of the molecular mechanism involved in the evolution of human PAH suggest endothelial dysfunction as a hallmark of the disease. Impaired production of vasodilating mediators, such as nitric oxide and prostacyclin, along with sustained overexpression of vasoconstrictors like endothelin-1 promote both vasoconstriction and vascular remodelling. Thus, these substances have emerged as important targets of pharmacological therapy of PAH in humans.

17

 17

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  Wunderlich et al. NOS-inhibition prevents lung remodelling in caveolin-1 knockout mice 16 In summary, we suggest a pivotal role of dysfunctional NO signalling in the pathogenesis of the lung fibrosis and severe pulmonary arterial hypertension in cav-1 ko. The present study underlines the utmost importance of a balanced cav-1 eNOS interaction for normal lung physiology and demonstrate that perturbations of normal NOS function may have devastating consequences on lung function. Whether the same holds true in human disorders remains to be established in future studies. However, such a perspective would open a completely new therapeutic strategy to pulmonary diseases.

Figure 1 .

 1 Figure 1. The pronounced pathomorphological lung defects of cav-1 ko are prevented by

Figure 2 .

 2 Figure 2. The beneficial effects of chronic L-NAME treatment on lung structure are further

Figure 3 .

 3 Figure 3. In vivo cardiac catheterization revealed a marked pulmonary hypertension in

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Figure 4 .

 4 Figure 4. As shown by swimming chamber tests untreated cav-1 ko display a physical

Figure 5 .

 5 Figure 5. The enhanced oxidative stress in lung tissue and aortas (as estimated by SOD

Table 1 .

 1 Figure 1

  Figure 4