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International audience; Abstract :The reported deep learning–based method can be used to evaluate the extent of interstitial lung disease in systemic sclerosis with results comparable to those of radiologists.Purpose :To develop a deep learning algorithm for the automatic assessment of the extent of systemic sclerosis (SSc)–related interstitial lung disease (ILD) on chest CT images.Materials and Methods :This retrospective study included 208 patients with SSc (median age, 57 years; 167 women) evaluated between January 2009 and October 2017. A multicomponent deep neural network (AtlasNet) was trained on 6888 fully annotated CT images (80% for training and 20% for validation) from 17 patients with no, mild, or severe lung disease. The model was tested on a dataset of 400 images from another 20 patients, independently partially annotated by three radiologist readers. The ILD contours from the three readers and the deep learning neural network were compared by using the Dice similarity coefficient (DSC). The correlation between disease extent obtained from the deep learning algorithm and that obtained by using pulmonary function tests (PFTs) was then evaluated in the remaining 171 patients and in an external validation dataset of 31 patients based on the analysis of all slices of the chest CT scan. The Spearman rank correlation coefficient (ρ) was calculated to evaluate the correlation between disease extent and PFT results.Results :The median DSCs between the readers and the deep learning ILD contours ranged from 0.74 to 0.75, whereas the median DSCs between contours from radiologists ranged from 0.68 to 0.71. The disease extent obtained from the algorithm, by analyzing the whole CT scan, correlated with the diffusion lung capacity for carbon monoxide, total lung capacity, and forced vital capacity (ρ = −0.76, −0.70, and −0.62, respectively; P < .001 for all) in the dataset for the correlation with PFT results. The disease extents correlated with diffusion lung capacity for carbon monoxide, total lung capacity, and forced vital capacity were ρ = −0.65, −0.70, and −0.57, respectively, in the external validation dataset (P < .001 for all).Conclusion :The developed algorithm performed similarly to radiologists for disease-extent contouring, which correlated with pulmonary function to assess CT images from patients with SSc-related ILD.

Objective: We introduce a new interpretation method for PFT data. Methods: Our new method consists of 3 steps: To build a diagnostic rule for a specific target from multi-PFT parameters. To estimate the Shapley-score, a game-theory based metric which measures the importance of each PFT index by comparing the model’s predictions with and without that index. To generate the interpretation, indicating the contribution level of each parameter to the positive/negative diagnosis. We applied this method to detect interstitial lung disease (ILD) in patients with systemic sclerosis. A machine learning diagnostic rule was developed from data of 300 patients undergoing 5 PFT techniques. Results: Validated on unseen data (n=100), the rule showed a good clinical performance (Sensitivity=0.86, Specificity=0.89, Positive and negative Likelihood-ratios of 7.86 and 0.16 respectively). At population level, the interpretation revealed that AV, TLCO-NO and FEV1 were the most important contributors to positive diagnosis. Personalized interpretations (Fig. 1) allow to verify the model’s plausibility in difficult cases with false negative/positive diagnosis and identify individual patterns of lung function impairments. Conclusion: Compared to the conventional approaches, our new method offers more advantages including ability of multivariate interpretation and valuable information for personalized treatment plan.

Background and Objective. The concentration of exhaled nitric oxide (eNO), reflecting the activity of inducible NO synthase in airway epithelium, has been found to increase in patients with obstructive sleep apnea (OSA). This study aimed to measure eNO concentration in patients with suspected OSA and to correlate different eNO parameters with clinical and sleep apnea characteristics.Methods. In this cross-sectional study, all patients underwent in-lab overnight polysomnography (PSG) and eNO measurement using a method of multiple flow rates before and after PSG (pre- and post-PSG).Results. According to the result of PSG, 82 persons were divided into two groups: control subjects (n=30;54±14years) and patients with OSA defined as apnea-hypopnea index (AHI) ≥ 5/hour (n=52;53±12years). Body mass index (BMI) and neck and abdomen circumferences of OSA patients were significantly higher than those from control subjects. In OSA group, post-PSG alveolar NO concentration (CANO) (5.3±1.9 ppb) was significantly higher than pre-PSG CANO (4.0±1.7 ppb;P<0.001). Significant correlations have been found between CANO and AHI (P<0.001) and between CANO and nadir SpO2(P<0.05). The daytime CANO value of more than 4.1 ppb can be used to screen symptomatic subjects for the presence of OSA with a high specificity of 93.3%.Conclusion. Our findings indicate CANO as a surrogate marker for OSA in persons with suggestive symptoms.

A recent comprehensive model of oxygen capture (Kang et al., 2015) allows for a quantitative prediction of VO2, PAO2, PaO2 and SaO2 as a function of gaseous (ventilation VE and PIO2) and blood determinants (cardiac output Q and PvO2). Its predictions being made on average healthy lungs were validated against average physiological data in the literature. The aim of the present study was to apply the same theoretical method at the individual level to obtain respiration determinants that are not measured routinely. The method is tested by searching individual Q from individual VO2 and VE measured in clinical tests. Data of 16 patients, none of whom were diagnosed with cardiac or respiratory diseases, were gathered from pulmonary function and cardiopulmonary exercise tests performed at Univ. Hospital of Cochin in Paris. Then, using individual data of VE, VO2, TLC and hemoglobin concentration, numerical computations were made by means of this theoretical method for two cases: rest and peak exercise. We conclude that comparison between predicted Q and experimental Q, which is deduced from the Fick’s principle with measured PaO2, shows a perfect proportionality (R2 ~ 1) with only a small systematic difference of 10%. This difference may be attributed to overestimation of the value of the capillary time used in the numerical implementation of the theory. Reference M.-Y. Kang, I. Katz., B. Sapoval (2015) Respir. Physiol. Neurobiol. 205:109-119

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It has been proposed that oxygen capture by the human lungs depends on four determinants: ventilation, cardiac output, oxygen partial pressure in the inspired air and the venous blood. Indeed, the theoretical-numerical model proposed recently by Kang et al. was able to interpret the known empirical relation between the average of the determinants and the average oxygen capture called VO2. This method is tested here at the individual level in a group of 31 subjects submitted to standard pulmonary function testing and cardiopulmonary exercise testing. For this, an inverse method is used in which individual cardiac output is predicted from the clinical test data. Comparison to the cardiac output deduced from Fick principle confirms that the dynamic model is a “microscopic” justification of the “macroscopic” Fick principle. It shows that in addition to the four determinants, two secondary determinants, namely hemoglobin concentration and Bohr effect, expressed here through P50, play significant roles.

Background The present study aimed to develop an automated computed tomography (CT) score based on the CT quantification of high-attenuating lung structures, in order to provide a quantitative assessment of lung structural abnormalities in patients with Primary Ciliary Dyskinesia (PCD). Methods Adult (≥18 years) PCD patients who underwent both chest CT and spirometry within a 6-month period were retrospectively included. Commercially available lung segmentation software was used to isolate the lungs from the mediastinum and chest wall and obtain histograms of lung density. CT-density scores were calculated using fixed and adapted thresholds based on various combinations of histogram characteristics, such as mean lung density (MLD), skewness, and standard deviation (SD). Additionally, visual scoring using the Bhalla score was performed by 2 independent radiologists. Correlations between CT scores, forced expiratory volume in 1 s (FEV1) and forced vital capacity (FVC) were evaluated. Results Sixty-two adult patients with PCD were included. Of all histogram characteristics, those showing good positive or negative correlations to both FEV1 and FVC were SD (R = − 0.63 and − 0.67; p

GLI equations provide new cut-offs for TLCOfor detection of PH in patients with SSc, either with or without ILDhttp://ow.ly/aCpD30l2zch

Introduction Le depistage de l’hypertension pulmonaire (HTP) chez les patients atteints de sclerodermie systemique (ScS) est primordial car l’initiation de traitement adapte permet d’ameliorer la survie. Un des principaux outils utilises pour le depistage sont la mesure du transfert pulmonaire du monoxyde de carbone (TLCO) et de la capacite vitale forcee (CVF). Comme ces mesures sont exprimees en pourcentage de la valeur predite ( % pred), les equations de reference pour calculer les valeurs predites sont d’une importance capitale pour l’interpretation. De nouvelles equations de reference pour la CVF et pour le TLCO, basees sur un large echantillon de sujets normaux, ont recemment ete publiees par le groupe Global Lung Function Initiative (GLI). Objectif comparer les seuils optimaux de TLCO et du ratio CVF/TLCO pour l’identification de l’HTP chez les patients ScS, en utilisant soit les equations de reference utilisees jusqu’a present soit les nouvelles equations GLI. Methodes Au total, 572 patients ScS consecutifs non selectionnes ont ete recrutes retrospectivement dans sept centres francais. Tous les patients ont beneficie d’EFR, d’une echographie cardiaque transthoracique ainsi que d’un catheterisme cardiaque droit lorsqu’une HTP etait suspectee. Resultats Parmi les 572 patients atteints de ScS inclus, 58 presentaient une HTP (35 avaient a la fois une HTP et une pneumopathie interstielle diffuse (PID)) et 514 n’avaient pas d’HTP (313 n’ayant aucune atteinte pulmonaire, 201 avait une PID sans HTP). Chez les patients ScS sans PID, l’analyse des courbes ROC a montre que le seuil optimal de TLCO etait plus eleve (70 % vs 60 % pred) avec l’equation GLI comparee aux anciennes equations pour la detection de l’HTP. Les seuils optimaux etaient cependant similaires pour les patients atteints de SSc avec ILD. Pour le rapport CVF/TLCO nous avons identifie un seuil optimal similaire pour les patients atteints de ScS sans PID, quelles que soient les equations utilisees. Ce seuil etait cependant inferieur d’environ 15 % chez les patients atteints de SSc avec PID avec les equations GLI comparees aux anciennes equations. Conclusion En conclusion, nous fournissons ici les valeurs seuils obtenues avec les nouvelles equations GLI pour le depistage de l’hypertension pulmonaire. Nous suggerons de prendre en compte uniquement la TLCO (et non le ratio CVF/TLCO), avec un seuil de 70 % (ou un score z de −2,00) pour le depistage de l’HTP chez les ScS sans PID et de 50 % (ou un z-score de −3,50) chez les patients atteints de ScS avec PID.

To assess distal/alveolar inflammation in patients with suggestive symptoms of obstructive sleep apnoea (OSA) using exhaled nitric oxide (NO) measured by two-compartment model (2-CM) after correction for axial NO back-diffusion (trumpet model).Ninety five patients suspected for OSA prospectively underwent pulmonary function test, overnight polysomnography (PSG), and exhaled NO measurement. Patients with apnoea-hypopnoea index (AHI) 5/hour were included in non-OSA group. Exhaled NO was repeatedly measured after PSG in 21 OSA patients and 8 non-OSA subjects.Alveolar NO concentration (C(ANO)) was significantly higher in OSA patients (n = 71; 4.07 ± 1.7 ppb) as compared with non-OSA subjects (n = 24; 2.24 ± 1.06 ppb; p0.0001) whilst maximal bronchial NO flux (J'awNO) and fractional exhaled NO (F(ENO)) did not differ between the two groups. C(ANO) was strongly associated to AHI (r = 0.701; p0.0001) and to recording time with SaO290% (ST-90%; r = 0.659; p0.0001) in OSA patients but not in non-OSA persons. The area under ROC curve for screening patients with OSA and significant nocturnal oxygen desaturation (ST-90% 1%) was 0.865 ± 0.036 (95% IC, 0.793-0.937; p0.0001). C(ANO) at 4.5 ppb could detect these patients with specificity of 94% and sensitivity of 46%. Increase of C(ANO) measured after PSG was significantly related to oxygen desaturation index (ST-90%) in OSA patients.Increased alveolar NO concentration was related to the severity of nocturnal oxygen desaturation in patients with OSA, linking the distal airway inflammation to intermittent hypoxia. (250 words).

To describe the features of obstructive sleep apnoea (OSA) and its association with arterial hypertension (HT), coronary artery disease (CAD), and arrhythmias in elderly (≥65 years) versus younger patients. All adult patients referred to our Sleep Research Unit for suspected OSA were included and underwent a thorough medical examination and an in-laboratory polysomnography. The severity of OSA was defined by the apnoea–hypopnoea index (AHI) as mild [5–15/h), moderate [15–30/h), and severe (≥30/h). Elderly patients (n = 136) and really old patients (>75 years) had higher prevalence of OSA (89 %) and severe OSA (36.8 %) as compared to younger patients (n = 439; 79.5 and 27.6 %, respectively, p

Interstitial lung disease (ILD) is becoming one of the main causes of death of patients with systemic sclerosis (SSc). The prevalence of ILD associated with SSc (SSc-ILD) varies from 33% to 100% according to diagnostic methods. Clinical features such as dyspnea on exertion, dry cough, and chest pains are not specific and usually late-appearing, implying more specific tests in the diagnostic, prognosis, and follow-up of ILD in patients with SSc. High resolution thoracic CT scanner (HRCT) is more sensitive than chest X-ray in the detection of SSc-ILD. Pulmonary function tests (PFT) are non-invasive and periodically used to assess the impacts of SSc on respiratory function. Diagnostic values of bronchoalveolar lavage and histological examination on lung biopsy are controversial. However, these techniques are essential for studying cellular and molecular mechanisms underlying the pathophysiology of SSc-ILD. Several biomarkers such as surfactant-A (SP-A), -D (SP-D), mucin-like high molecular weight glycoprotein (KL-6), and chemokine CCL-18 have been implicated in SSc-PID. Serum levels of these proteins are correlated with the severity of SSc-ILD, as assessed by HRCT and/or PFT. Finally, alveolar concentration of exhaled nitric oxide can be used to screen SSc patients with high risk of deterioration of respiratory function, in whom immunosuppressant treatment could be useful in preventing the evolution to irreversible lung fibrosis.

Objective: Our study aims to apply a Machine learning (ML) approach to detect the presence of Interstitial Lung disease (ILD) in patients with systemic sclerosis (SSc), using combined data from multiple pulmonary function tests (PFT). Methods: PFT data (Spirometry, Plethysmography and DLNO-CO measurements, all converted into Z-score scale) were obtained in 273 patients with confirmed diagnosis of SSc and 50 healthy non-smokers. The main outcome is ILD, as determined by HRCT. An exhaustive feature selection was performed in R statistical programming language. This process consists of building many classification models (based on support vector machine algorithm) from every possible combination between the PFT indices, then voting for the one that shows the best performance (highest accuracy and lowest error) through a repeated cross-validation (10x10 iterations). Results: The best decision rule implies 7 parameters: DLNO, DLCO, VA, Vc, KCO, TLC and FVC (Figure) This model showed steady and good classification performance through 100 resampled iterations: AUC=0.85, balanced-accuracy=0.76, true positive rate=0.62, true negative rate=0.89, mean misclassification error=0.2, false negative rate=0.38 and false positive rate=0.11. Conclusion: Our results suggest that applying ML technique and multiple PFT data could improve the likelihood to detect early SSc patients with higher risk of ILD.

Objective.Clara cell secretory protein (CC16) is a sensitive marker of bronchial epithelial cell damage. The CC16 serum level is elevated in patients with pulmonary fibrosis, but its predictive value on lung disease progression has not yet been studied. We aimed to assess the value of serum CC16 concentration in predicting lung disease deterioration in patients with systemic sclerosis (SSc).Methods.We prospectively analyzed and followed 106 patients with SSc during a 4-year period for the risk of developing combined deleterious event, defined as a 10% decrease in total lung capacity or forced vital capacity from baseline, or death, according to serum CC16 at inclusion. Receiver-operating characteristic (ROC) curve analysis was performed for prediction of events during the first 2 years after inclusion. Cumulative risks of combined events were computed by Kaplan-Meier analysis.Results.The best cutoff level of serum CC16 for prediction of a combined event was 33 ng/ml, with 76% sensitivity and 65% specificity (area under the ROC curve: 0.71, 95% CI 0.61–0.81, p < 0.0001). Progression of lung disease evaluated by a mean time-to-event differed between patients with high baseline serum CC16 (42.8 mos, 36.3–49.3) and those with low serum CC16 (56.3 mos, 50.9–61.7; log-rank test, p < 0.001). After adjustment for age, duration of disease, clinical and lung function measures, the risk of combined event occurrence in patients with high serum CC16 was significantly higher than in those with low CC16 (HR 2.9, 1.2–6.75, p < 0.05).Conclusion.High baseline serum CC16 predicts lung disease worsening in patients with SSc.

Screening is important to determine whether patients with systemic sclerosis (SSc) have pulmonary hypertension because earlier pulmonary hypertension treatment can improve survival in these patients. Although decreased transfer factor of the lung for carbon monoxide (TLCO) is currently considered the best pulmonary function test for screening for pulmonary hypertension in SSc, small series have suggested that partitioning TLCO into membrane conductance (diffusing capacity) for carbon monoxide (DMCO) and alveolar capillary blood volume (VC) through combined measurement of TLCO and transfer factor of the lung for nitric oxide (TLNO) is more effective to identify pulmonary hypertension in SSc patients compared with TLCO alone. Here, the objective was to determine whether combined TLCO–TLNO partitioned with recently refined equations could more accurately detect pulmonary hypertension than TLCO alone in SSc.For that purpose, 572 unselected consecutive SSc patients were retrospectively recruited in seven French centres.Pulmonary hypertension was diagnosed with right heart catheterisation in 58 patients. TLCO, TLNO and VC were all lower in SSc patients with pulmonary hypertension than in SSc patients without pulmonary hypertension. The area under the receiver operating characteristic curve for the presence of pulmonary hypertension was equivalent for TLCO (0.82, 95% CI 0.79–0.85) and TLNO (0.80, 95% CI 0.76–0.83), but lower for VC (0.75, 95% CI 0.71–0.78) and DMCO (0.66, 95% CI 0.62–0.70).Compared with TLCO alone, combined TLCO–TLNO does not add capability to detect pulmonary hypertension in unselected SSc patients.

Sy Duong-Quy,1–3 Thuc Vu-Minh,4 Thong Hua-Huy,1 Tram Tang-Thi-Thao,3 Khiet Le-Quang,3 Dinh Tran-Thanh,3 Nhu Doan-Thi-Quynh,3 Nhat-Nam Le-Dong,5 Timothy J Craig,2 Anh-Tuan Dinh-Xuan1 1Department of Respiratory Physiology, Cochin Hospital, Paris Descartes University, Sorbonne Paris Cité, Paris, France; 2Department of Medicine, Penn State University, Hershey, PA, USA; 3Bio-Medical Research Center, Lam Dong Medical College, Dalat, 4Department of Immuno-Allergology, ENT National Institute, Hanoi, Vietnam; 5Department of Pulmonology, St Elisabeth Hospital, Namur, Belgium Background: The measure of fractional exhaled nitric oxide (FENO) in the airways is a useful tool to guide the diagnosis and titration of inhaled corticosteroids in patients with asthma. However, its role in diagnosis of allergic rhinitis (AR), especially in subjects with asthma, is not well established. Objective: To study the cutoff of nasal FENO in the diagnosis of subjects with AR and AR-asthma compared to age-matched subjects without AR or asthma and its correlations with the clinical and functional characteristics. Methods: The study was cross sectional and descriptive. Subjects were grouped into control subjects, AR, and AR-asthma, based on the inclusion criteria. Exhaled NO (nasal FENO, bronchial FENO, and alveolar concentration of NO) was measured by multiple flow electroluminescence device. Results: Six hundred twenty-eight subjects were included: 217 control subjects (children: n=98, 10±4 years; adults: n=119, 50±16 years), 168 subjects with AR (children: n=54, 10±3 years; adults: n=114, 49±15 years), and 243 subjects with AR-asthma (children: n=115, 10±3 years; adults: n=128, 51±14 years). Nasal peak inspiratory flow and peak expiratory flow were lower in subjects with AR and AR-asthma than in control subjects (P

Systemic sclerosis (SSc) is a chronic disease of the connective tissue with an estimated prevalence of 5.8 per 100 000 in a multi-ethnic population living in Europe [1]. The disease mechanisms are highly complex, affecting multiple organs including the pulmonary system. Severe pulmonary complications include systemic sclerosis associated interstitial lung disease (SSc-ILD) and pulmonary hypertension (SSc-PH), which are also associated with premature mortality [2, 3]. Early diagnosis and monitoring of pulmonary complications by spirometry, bodyplethysmography, and pulmonary diffusing capacity are critical to lung health in SSc patients [4, 5]. Pulmonary diffusing capacity for carbon monoxide ( D LCO) is a well-established and frequently applied test in SSc and is used as a surrogate of SSc-ILD and SSc-PH progression [4, 6]. Footnotes This manuscript has recently been accepted for publication in the European Respiratory Journal . It is published here in its accepted form prior to copyediting and typesetting by our production team. After these production processes are complete and the authors have approved the resulting proofs, the article will move to the latest issue of the ERJ online. Please open or download the PDF to view this article. Conflict of interest: Dr. Dressel has nothing to disclose. Conflict of interest: Dr. Dinh Xuan has nothing to disclose. Conflict of interest: Dr. Hua has nothing to disclose. Conflict of interest: Dr. Radtke has nothing to disclose.

Background: Pulmonary hypertension (PH) is a common complication of idiopathic pulmonary fibrosis (IPF) that significantly contributes to morbidity and mortality. Macrophage migration inhibitory factor (MIF) is a critical factor in vascular remodeling of the pulmonary circulation. Objectives: We tested the effects of two small molecules targeting MIF on bleomycin (BLM)-induced collagen deposition, PH, and vascular remodeling in mouse lungs. Methods: We examined the distribution pattern of MIF, CD74, and CXCR4 in the lungs of patients with IPF-PH and the lungs of BLM-injected mice. Then, treatments were realized with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) and N-(3-hydroxy-4-fluorobenzyl)-5 trifluoromethylbenzoxazol-2-thione 31 (20 mg/kg/day per os for 3 weeks) started 24 h after an intratracheal BLM administration. Results: More intense immunoreactivity was noted for MIF, CD74, and CXCR4 in lungs from IPF-PH patients and BLM-injected mice. Furthermore, we found that treatments of BLM-injected mice with ISO-1 or compound 31 attenuated lung collagen deposition and right ventricular systolic pressure increase. Additionally, reduced pulmonary inflammatory infiltration and pulmonary arterial muscularization were observed in the lungs of BLM-injected mice treated with ISO-1 or compound 31. Conclusions: Treatments with ISO-1 or compound 31 attenuates BLM-induced inflammation and fibrosis in lung, and prevents PH development in mice, suggesting that MIF is an important factor for IPF-PH development.

Objective Systemic sclerosis (SSc) is characterized by microvascular damage, fibrosis of skin and visceral organs, and autoimmunity. Previous studies have shown that angiotensin II is involved in the synthesis of type I collagen. We investigated whether the blockade of angiotensin II receptor type I (AT1) by irbesartan reduces skin and lung fibrosis in 2 murine models of SSc. Methods SSc was induced by daily intradermal injection of HOCl into the backs of BALB/c mice (HOCl-induced SSc). Mice were treated daily with irbesartan by oral gavage. Results Irbesartan reduced dermal thickness, collagen concentration, Smad2/3, and α-smooth muscle actin expression, as well as fibroblast proliferation and H-Ras expression in the skin of mice with HOCl-induced SSc. Mice treated with irbesartan also displayed less lung fibrosis, less inflammation, and a lower concentration of collagen in the lungs than untreated mice. Exhaled nitric oxide, inducible nitric oxide synthase, and 3-nitrotyrosine expression in the lungs were decreased following irbesartan treatment. Moreover, irbesartan reduced the number and the proliferation of splenic B and T cells and the serum levels of anti–DNA topoisomerase I autoantibodies. Conclusion Irbesartan, an AT1 antagonist, prevents fibrosis and inflammation and inhibits nitric oxide production in HOCl-induced models of systemic fibrosis. Our findings extend the indication of an AT1 antagonist to SSc patients with diffuse fibrosis, especially those with lung involvement.

The age dependence of NO parameters has not been addressed. We hypothesize that alveolar NO (C A NO) is affected by age and that F E NO 50 and airway flux (J aw NO) reflect airway surface. The lung matures up to the age of 20, hence age groups; young Healthy subjects (n=284) were investigated, see Table. There was only a gender difference in the middle age group for both J aw NO (p=0.001) and F E NO 50 (p=0.001). There was a correlation of J aw NO to both age (r=0.30, p=0.007) and height (r=0.38, p=0.002) in the young group, correlations were not found in the middle age group, but in the older group J aw NO correlated with age (r=0.23, p=0.024). C A NO was correlated to age in the middle group (r=0.32, p=0.001) as well as in the old group (r=0.48, p In the middle and older age groups pulmonary aging seems to increase C A NO possibly reflecting decreased diffusivity of gases in the acinar area. In the growing lung F E NO 50 and J aw NO were related to age and even more to height, possibly reflecting increasing bronchial mucosal surface area with increasing height. Further studies are needed before we can give age related reference values for the NO parameters.

Systemic sclerosis (SSc) is a connective-tissue disease characterized by vascular injury, immune-system disorders, and excessive fibrosis of the skin and multiple internal organs. Recent reports found that RhoA/Rho-kinase (ROCK) pathway is implicated in various fibrogenic diseases. Intradermal injection of hypochlorous acid (HOCl)-generating solution induced inflammation, autoimmune activation, and fibrosis, mimicking the cutaneous diffuse form of SSc in humans. Our study aimed firstly to describe pulmonary inflammation and fibrosis induced by HOCl in mice, and secondly to determine whether fasudil, a selective inhibitor of ROCK, could prevent lung and skin fibroses in HOCl-injected mice.Female C57BL/6 mice received daily intradermal injection of hypochlorous acid (HOCl) for 6 weeks to induce SSc, with and without daily treatment with fasudil (30 mg·kg(-1)·day(-1)) by oral gavage.HOCl intoxication induced significant lung inflammation (macrophages and neutrophils infiltration), and fibrosis. These modifications were prevented by fasudil treatment. Simultaneously, HOCl enhanced ROCK activity in lung and skin tissues. Inhibition of ROCK reduced skin fibrosis, expression of α-smooth-muscle actin and 3-nitrotyrosine, as well as the activity of ROCK in the fibrotic skin of HOCl-treated mice, through inhibition of phosphorylation of Smad2/3 and ERK1/2. Fasudil significantly decreased the serum levels of anti-DNA-topoisomerase-1 antibodies in mice with HOCl-induced SSc.Our findings confirm HOCl-induced pulmonary inflammation and fibrosis in mice, and provide further evidence for a key role of RhoA/ROCK pathway in several pathological processes of experimental SSc. Fasudil could be a promising therapeutic approach for the treatment of SSc.

Inhaled nitric oxide (iNO) is commonly used in the treatment of very ill pre-term newborns. Previous studies showed that exogenous NO could affect endothelial NO synthase (eNOS) activity and expression in vascular endothelial cell cultures or adult rat models, but this has never been fully described in newborn rat lungs. We therefore aimed to assess the effects of iNO on eNOS expression and activity in newborn rats. Rat pups, post-natal day (P) 0 to P7, and their dams were placed in a chamber containing NO at 5 ppm (iNO-5 ppm group) or 20 ppm (iNO-20 ppm group), or in room air (control group). Rat pups were sacrificed at P7 and P14 for evaluation of lung eNOS expression and activity. At P7, eNOS protein expression in total lung lysates, in bronchial and arterial sections, was significantly decreased in the iNO-20 ppm versus control group. At P14, eNOS expression was comparable among all three groups. The amounts of eNOS mRNA significantly differed at P7 between the iNO-20 ppm and control groups. NOS activity decreased in the iNO-20 ppm group at P7 and returned to normal levels at P14. There was an imbalance between superoxide dismutase and NOS activities in the iNO-20 ppm group at P7. Inhalation of NO at 20 ppm early after birth decreases eNOS gene transcription, protein expression and enzyme activity. This decrease might account for the rebound phenomenon observed in patients treated with iNO., Inhaled NO decreases endogenous NO synthesis and favours rebound pulmonary hypertension after inhaled NO withdrawal http://ow.ly/WNDq2

Pulmonary hypertension (PH) is a complex disorder resulting from many etiologies that cause disturbances of normal pulmonary haemodynamics. Recent breakthroughs have led to a better understanding of the pathophysiology of the disease. In PH, haemodynamic disturbances are closely linked to structural changes and excessive remodeling of pulmonary vessels, leading to progressive narrowing of the pulmonary vascular lumen. Imbalances between pulmonary vasoconstrictors and vasodilators on the one hand, and factors favoring cell proliferation and apoptosis on the other hand, probably account for most cases of PH. This review aims to update readers with the current knowledge on the molecular physiopathology of PH and how this can progress the therapeutic of this disorder.

Nitric oxide metabolites (NOx) in serum, and alveolar concentration of NO (CA(NO)), are markers of inflammation and alveolitis, respectively, in systemic sclerosis (SSc). We prospectively evaluated the usefulness of both NOx and CA(NO) to assess lung involvement and skin fibrosis in SSc. Serum NOx, and CA(NO) measured by two different methods, namely the two-compartment (2CM) and the "trumpet" models (TM), were concomitantly assessed in 65 patients with SSc and 17 healthy controls. Whilst serum NOx remained comparable between groups, CA(NO) were significantly higher in SSc patients (n=65, 6.7ppb; 4.8-9.7 and 5.9ppb; 3.9-8.9) as compared with controls (n=17, 3.0ppb; 2.0-3.8 and 1.8ppb; 1.1-2.9, p

Introduction Les etudes epidemiologiques portant sur la bronchopneumopathie chronique obstructive (BPCO) sont rares dans les pays en developpement, en particulier au Viet Nam ou la consommation tabagique ne cesse d’augmenter. Le but de cette etude est d’evaluer la faisabilite d’un depistage precoce de l’obstruction bronchique par l’appareil Piko-6 chez les fumeurs. Materiel et methodes L’inclusion a porte sur tout sujet fumeur de plus de 40 ans ayant fume plus de 10 PA. Les sujets ont ete classes en 3 groupes selon le degre de l’obstruction bronchique mesure par le Piko-6 (groupe 1 : VEMS/VEM6 > 0,80 ; groupe 2 : 0,07 ð VEMS/VEM6 ð 0,80 ; groupe 3 : VEMS/VEM6 Resultats Deux mille trois cent quatre-vingt-dix-sept fumeurs ont ete inclus comprenant 2 130 fumeurs actifs et 267 anciens fumeurs. L’âge moyen etait de 52 ± 13 ans. Le tabagisme moyen etait de 24 ± 13 PA. 267 fumeurs des 3 groupes ont repondu a la convocation pour le bilan complet. La prevalence de la BPCO depistee par le Piko-6 chez la population etudiee etait de 13,5 %. Pour le seuil du rapport VEMS/VEM6 Conclusions Le Piko-6 est un moyen utile pour le depistage precoce de la BPCO chez les fumeurs dans un pays en developpement ou la prevalence de cette pathologie semble sous-estimee.

Introduction: Measurement of FENO is a useful tool for assessment of asthma patients who are treated withinhaled corticosteroids (ICS). Aims of study: This study was planned to assess the use of FENO measurement in therapeuticdecision making, disease control, and cost effectiveness for management of patients with asthma. Method: Patients with uncontrolled asthma with FENO> 25ppb, were included in this study. They were classified into 3 groups according to the intervention used to determine therapy: group 1 (Gr1): used GINA guidelines, group 2 (Gr2): used GINA plus FENO, andgroup 3 (Gr3): used only FENO. Result: 162 patients were included and randomly divided into 3 groups: Gr1: 48 patients (FeNO = 52 ± 12ppb), Gr2: 64 patients (FeNO = 54 ± 18 ppb), Gr3: 50 patients (FeNO = 53 ±16 ppb). Asthma control was achieved after 3 months for all 3 groups. At 9 months the percentage of patients on high-dose ICS in Gr1 > Gr3 > Gr2 (24%, 17%, 12%; respectively; p th month and -95 USD at 18 th month). Conclusion: The use of FENO for titration of ICS dose in asthma patients helps to titrate down the dose of ICS and reduce the cost of treatment and thus provides a useful tool for the management of asthma in developing countries.

Exhaled nitric oxide (NO) is increased as a result of lung inflammation, which in turn causes subsequent interstitial lung disease in patients with systemic sclerosis (SSc). However, the exact time course of inflammatory and fibrotic changes in the SSc lung has not yet been described. Our objective was to assess the chronological evolution of lung inflammatory and fibrotic processes in mice pre-treated with hypochlorous acid (HOCl) or bleomycin. C57BL/6 mice were randomized into three groups receiving subcutaneous injections of HOCl, bleomycin, or PBS for 2, 4 or 6 weeks. Exhaled NO (eNO) was measured at the end of each injection period and after 2 resting weeks without injection (8 week group). Mice were then sacrificed to obtain skin and lung tissues to measure fibrotic changes and NO synthases (NOS) expression. Increased eNO, inducible NOS and nitrotyrosine expression in bronchial epithelium, lung neutrophils and macrophages were observed at early phases in both HOCl- and bleomycin-treated mice. Conversely, lung vascular endothelial NOS expression decreased significantly at 6th and 8th weeks. Skin fibrosis was significantly increased from the 4th week and lung fibrosis from 6th week. We conclude that lung inflammation occurs early after injury as reflected by increased exhaled NO and inducible NOS expression, and precedes fibrotic changes in skin and lungs of mice pre-treated with bleomycin and HOCl. Early detection and treatment of pulmonary inflammation might be useful in preventing subsequent occurrence of lung fibrosis in SSc patients.

Background: Pulmonary hypertension secondary to COPD is characterized principally by impairment of vasoreactivity and vascular remodeling. While the structural changes have been described in all stages of COPD, the impairment of vasoreactivity in response to ET-1, a potent vasoconstrictor, has not been well understood. Materials and methods: Proximal pulmonary arteries were obtained from non-smokers, smokers with normal lung function, and COPD patients who underwent lung resection for other diseases (n=6 in each group). Pulmonary arterial contraction induced by ET-1 was assessed without or with the presence of ET-1 receptor antagonists (BQ- 123 and BQ-788). Expressions of ET-1 receptors were measured by immunohistochemistry, western blot, and qRTPCR. Results: ET-1-induced pulmonary arterial contraction increased significantly in COPD patients in comparison with control subjects and non-COPD smokers (P

Fibrosis is characterized by disproportionate accumulation of collagens and other extracellular matrix substances, resulting in organ dysfunction and failure. In systemic sclerosis, cellular and molecular mechanisms involved in the pathophysiology of fibrosis are highly complex and yet barely understood. Anatomopathological findings showed the coexistence of patchy inflammatory cell infiltration, microvascular injuries, and fibrotic foci. One of the most commonly accepted hypotheses considers endothelial activation as the triggering phenomenon inducing inflammatory and autoimmunity activation. The resulting cytokines and autoantibodies production accelerates the proliferating rate of normal fibroblasts and their transformation into myofibroblasts, leading to diffuse fibrosis. This review aims to focus on cellular and molecular mechanisms implicated in the fibrogenesis of systemic sclerosis.

Inhibition of angiogenesis is an important therapeutic strategy for advanced stage prostate cancer (PCa). RhoA/Rho-associated protein kinases (ROCK) are key regulators of the cytoskeleton and have been implicated in PCa angiogenesis. We investigated the anti-angiogenic effects of fasudil, a ROCK inhibitor, on PCa-induced angiogenesis in vitro. Proliferation of PCa-conditioned human umbilical vein endothelial cells (HUVECs) was assessed using a bromodeoxyuridine (BrdU) assay, and migration was assessed with a wound healing assay. In vitro angiogenesis of PCa-conditioned HUVECs was evaluated by tube formation and a spheroid sprouting assay. Fasudil inhibited PCa-induced endothelial cell proliferation at a concentration of 100 µM, and also decreased PCa-induced endothelial cell migration at a concentration of 30 µM. In the in vitro angiogenesis assay, fasudil exerted a more significant effect. Tube formation was significantly inhibited at fasudil concentrations exceeding 3 µM, and spheroid sprouts were significantly thinner and shorter (at fasudil concentrations of 10 and 30 µM, respectively). Western blotting results showed that expression of phosphorylated myosin phosphatase target subunit 1 (MYPT-1) was significantly lower after fasudil treatment, confirming that fasudil inhibited ROCK activity in these model systems. These data suggest that fasudil may be a useful anti-angiogenic agent for PCa.

Cyclophosphamide (CYC) is not always effective in patients with scleroderma-related interstitial lung disease (SSc-ILD), hence the need for biomarkers able to predict beneficial responses to CYC therapy. We therefore assessed whether baseline alveolar concentration of nitric oxide (CANO) could predict the favourable response to CYC therapy in patients with SSc-ILD. Nineteen non-smoker patients with SSc-ILD, were enrolled and treated with 6 courses of CYC (0.75 g/m2/monthly) for lung function decline the year before inclusion, and followed-up for 2 years period. We assessed the proportion of favourable response to CYC, defined as improvement of forced vital capacity (FVC) or total pulmonary capacity (TLC) more than 10% between the inclusion and each following visit, according to the validated cut-off of CANO at 8.5 ppb identifying progressive SSc-ILD subset. At inclusion, 7 patients out of 19 had CANO >8.5 ppb. Clinical parameters were comparable between patients with high (>8.5 ppb) and low level of CANO (≤8.5 ppb). After CYC therapy, and during the follow-up, 9 out of 19 patients had favourable response to CYC therapy, 10 did not meet responder's criteria, from whom 4 patients died from respiratory failure. Six out of 7 patients with CANO >8.5 ppb at inclusion had favourable response to CYC therapy, while only 3 out of 12 patients with CANO ≤8.5 ppb responded favourably to CYC therapy (p=0.001). High level of CANO >8.5 ppb reflecting alveolar inflammation identify SSc patients with a greater chance to benefit from CYC treatment with a significant lung function improvement.

Inhaled nitric oxide (NO) is frequently administered to full term and preterm newborns in various clinical settings in order to alleviate pulmonary hypertension whilst improving oxygenation. However, the physiological effect of NO on early postnatal lung development has not yet been clearly described. We therefore investigated whether NO administered by inhalation affects lung development at early postnatal life.Pregnant rats were placed in a chamber containing 5 ppm (iNO-5 ppm group) and 20 ppm NO (iNO-20 ppm group), started from the last day of their pregnancy in order to keep rat pups under ambient NO from birth to 7 days postnatal. Control animals were kept at room air and all rat pups were sacrificed at postnatal day 7 and day 14.Lung-to-body weight and wet-to-dry lung weight ratios did not significantly differ among 3 groups at postnatal day 7 and day 14. Vascular volume densities (Vv) in both NO groups (5 and 20 ppm) were higher than controls (P0.05; P0.001). Pulmonary vessel number was significantly increased in iNO-20 ppm group. Radial alveolar counts (RAC) and mean linear intercepts (MLI) markedly increased (consistent with increased alveolarization) in iNO-20 ppm group. This was associated with upregulation of VEGF/VEGFR-2, MT1-MMP/MMP2 and HO-1 protein expression in iNO-20 ppm group.We concluded that inhaled NO at 20 ppm enhanced lung development possibly through increased expression of HO-1, VEGF/VEGFR-2, and MMP2 at early stage of postnatal rat life.

Recent evidence suggests that activation of RhoA/Rho-kinase accounts for systemic and pulmonary endothelial dysfunction in smokers with normal lung function. However, its role in patients with chronic obstructive pulmonary disease (COPD) has not yet been investigated. The aim of this study was to evaluate the regulation of RhoA/Rho-kinase pathway and pulmonary endothelial dysfunction in patients with COPD. Pulmonary arteries were obtained from nonsmokers (control subjects) and patients with nonhypoxemic and hypoxemic COPD (n = 6-7/group). Endothelium-dependent and -independent relaxations were evaluated by acetylcholine and sodium nitroprusside, respectively. Gene and protein expressions of endothelial nitric oxide synthase (eNOS) were measured by RT-PCR, Western blot, and immunohistochemistry. Nitrate, cGMP, and endothelin-1 (ET-1) concentrations, as well as Rho-kinase activity were measured by ELISA. Protein expressions of total RhoA and GTP-RhoA were measured by Western blot and pull-down assay, respectively. Endothelium-dependent relaxation, and nitrate and cGMP levels were significantly reduced in pulmonary arteries of COPD patients as compared with control subjects. Conversely, activity of RhoA/Rho-kinase was increased in pulmonary arteries of COPD patients as compared with control subjects. In patients with COPD, pulmonary endothelial dysfunction was related to the downregulation of eNOS activity and upregulation of RhoA/Rho-kinase activity.

Pulmonary hypertension (PH) associated with pulmonary fibrosis (PF) considerably worsens prognosis of interstitial lung diseases (ILD). RhoA/Rho-kinases (ROCK) pathway is implicated in high pulmonary vascular tone and pulmonary fibrosis but the effect of ROCK inhibitors on PH associated with PF is not known. We therefore aimed to determine whether long-term treatment with fasudil, a selective ROCK inhibitor, could attenuate PF and PH induced by bleomycin in mice. Male C57BL/6 mice received a single dose of intratracheal bleomycin (3.3 U/kg) to induce PF. Treatment with fasudil (30 mg kg(-1) day(-1)) was given intraperitoneally for 7, 14 or 21 days until mice underwent hemodynamic measurements. Right ventricular systolic pressure (RVSP) and RV/(LV + S) ratio were assessed. Lung inflammatory cells profiles, including macrophages, neutrophils, lymphocytes B and lymphocytes T were assessed by immunohistochemistry. Lung fibrosis was evaluated by histological and biochemical methods. Pulmonary arteriole muscularization and medial wall thickness (MWT) were evaluated by immunohistochemical staining for α-SMA. Bleomycin induced severe PF and PH in mice, associated with an increased RhoA/ROCK activity in the lung. Fasudil reduced lung inflammation and lung collagen content, and attenuated the increased RVSP, RV hypertrophy, and pulmonary vascular remodeling in bleomycin-intoxicated mice. Fasudil inhibited the increased activity of RhoA/ROCK pathway, and partly altered bleomycin-associated activation of TGF-β1/Smad pathway, via inhibition of Smad2/3 phosphorylation. The efficacy of long-term treatment with fasudil suggests that the blockade of RhoA/ROCK pathway may be a promising therapy for patients with ILD-associated PH.

Alveolar concentration of nitric oxide (C A NO) is a non invasive prognostic marker of systemic sclerosis (SSc) lung disease. There is, however, as yet no direct evidence showing concomitant increase of C A NO and the presence of inflammatory cells in alveoli. We have therefore measured C A NO and performed broncho-alveolar lavage (BAL) in SSc patients. Exhaled NO was measured, by the means of two different models, the two-compartment model (2CM) and the trumpet model with axial diffusion (TMAD), in 22 SSc patients and compared with 15 healthy controls. BAL was performed in all SSc patients. Alveolitis was defined as lymphocytes >14%, polymorphonuclears >4%, or eosinophils >3% on cell count in BAL fluid. Comparisons of C A NO levels were made between SSc patients with, and without, alveolitis. Levels of C A NO were significantly higher in SSc patients as compared with controls ( p A NO was significantly higher in SSc patients with alveolitis as compared with SSc patients without alveolitis (8.4 ppb; 1st and 3rd interquartile range: 6.0–10.5 vs 3.3 ppb; 2.2–3.5; p = 0.004 for 2CM and 5.4 ppb; 3.2–9.2 vs 3.2 ppb; 1.4–3.3, p = 0.02 for TMAD), while bronchial airway output of NO ( J′ awNO, p = 0.19), and fractional exhaled NO (F E NO, p = 0.12) were comparable. C A NO was consistently high in SSc patients with alveolitis irrespective of the methods chosen (TMAD or 2CM). Our findings showed that increased C A NO was associated with alveolitis in patients with SSc. We submit that C A NO could be used as a reliable non-invasive surrogate biomarker of alveolitis in scleroderma lung disease.

Respiratory failure is a life-threatening and unpredictable complication of systemic sclerosis (SSc). A study was undertaken to assess the value of alveolar nitric oxide (NO) in predicting the risk of lung function deterioration leading to respiratory failure or death in patients with SSc.105 patients with SSc were enrolled in this prospective cohort and were followed longitudinally over a 3-year period during which the risk of occurrence of deleterious events was analysed according to alveolar concentration (C(A)NO), conducting airway output (J'(aw)NO) and fractional concentration (F(E)NO(0.05)) of exhaled NO measured at inclusion. Comparison was made between each NO parameter to predict the occurrence of deleterious events, defined as a 10% decrease in total lung capacity or forced vital capacity from baseline, or death.The area under the receiver operating characteristic curve of C(A)NO to predict the occurrence of the combined events was 0.84 (95% CI 0.76 to 0.92; p0.001), which was significantly higher than those of J'(aw)NO and F(E)NO(0.05) (p0.001). A cut-off of C(A)NO of 5.3 ppb had a sensitivity of 88% and a specificity of 62% for the prediction of the occurrence of combined events during follow-up, and was validated in an independent cohort of patients with SSc. Combined events occurred more frequently in patients whose C(A)NO was5.3 ppb. The adjusted HR for patients with C(A)NO5.3 ppb was 6.06 (95% CI 2.36 to 15.53; p0.001). C(A)NO accurately predicted the occurrence of combined events irrespective of forced vital capacity values or the presence of interstitial lung disease at baseline.Increased C(A)NO accurately identifies patients with SSc with a high risk of developing lung function deterioration and may help to initiate early appropriate treatment.

Elevated serum CC chemokine ligand (CCL)18 reflects lung fibrosis activity in systemic sclerosis (SSc) and could be an early marker of lung function worsening. Therefore, we sought to evaluate whether serum CCL18 levels at baseline could predict worsening of lung disease in SSc. In this prospective study, 83 SSc patients were analysed longitudinally over a 4-yr observation period for the risk of occurrence of combined deleterious events, defined as a 10% decrease from baseline of total lung capacity or forced vital capacity % predicted, or death, according to serum CCL18 at inclusion. Receiver operating characteristic (ROC) curve analysis was performed for prediction of events during the first year after inclusion. The best cut-off level of serum CCL18 for prediction of a combined event within the follow-up period was 187 ng · mL(-1), with 53% sensitivity and 96% specificity (area under the ROC curve 0.86; p0.001). After a mean ± SD follow-up of 33.7 ± 10.8 months, a higher rate of disease progression occurred in the group with serum CCL18 levels187 ng · mL(-1). The adjusted hazard ratio was 5.36 (95% CI 2.44-11.75; p0.001). In summary, serum CCL18 is an accurate predictive biomarker for the identification of patients with a higher risk of subsequent scleroderma lung disease worsening.

Objective.Lung inflammation is present in patients with systemic sclerosis (SSc) and interstitial lung disease (ILD), but the mechanisms linking inflammatory and fibrotic processes in ILD are unknown. Our aim was to investigate whether alveolar inflammation, reflected by increased alveolar concentration of exhaled nitric oxide (CANO), is related to the ability of serum from patients with SSc to induce pulmonary fibroblast proliferation (PFP) and myofibroblast conversion.Methods.CANO was measured in all subjects (37 patients with SSc and 10 healthy controls) whose sera were used to stimulate PFP (assessed by BrdU labeling index) and myofibroblast conversion (detected by α-smooth muscle actin expression). The PFP index in patients with SSc was compared to control values, and between patients with SSc who had elevated (> 4.3 ppb) and normal (≤ 4.3 ppb) CANO values.Results.Both CANO and the PFP index were significantly greater in patients with SSc compared to controls. In patients with SSc, the PFP index was directly related to CANO levels (r = 0.48; p = 0.002). The median PFP index was significantly higher in patients with SSc who had elevated CANO (> 4.3 ppb; n = 25, median 1.1, range 0.98–1.23) than in patients with SSc who had normal CANO (≤ 4.3 ppb; n = 12, median 0.93, range 0.82–1.08; p = 0.01). Similarly, myofibroblast conversion induced by SSc serum was significantly greater in patients with CANO > 4.3 ppb than in patients whose CANO was ≤ 4.3 ppb (p < 0.001) and controls (p < 0.001).Conclusion.Alveolar inflammation reflected by increased nitric oxide production was related to serum-induced PFP and myofibroblast conversion, linking the active alveolitis process to cell proliferation and lung fibrosis in patients with SSc.

Endothelial dysfunction is one of the main consequences of the toxic effects of cigarette smoke on the vascular system. Increasing evidence suggests that the small G-protein RhoA and its downstream effectors, the Rho-kinases (ROCKs), are involved in systemic endothelial dysfunction induced by cigarette smoke. This study aimed to evaluate the role of the RhoA/ROCKs pathway in pulmonary artery endothelial function in current smokers with normal lung function. Lung tissues were obtained from nonsmokers and smokers who underwent lobectomy for lung carcinoma. Arterial relaxation in response to acetylcholine (ACh) was assessed in isolated pulmonary arterial rings. Protein expressions and activities of endothelial nitric oxide synthase (eNOS), ROCKs and the myosin phosphatase subunit 1 (MYPT-1) were sought. Relaxation in response to ACh was significantly lower in smokers as compared with nonsmokers (n = 8 in each group), consistent with reduced eNOS activity in the former compared with the latter. eNOS protein expression remained, however, the same in both groups. Expression of ROCKs, guanosine triphosphate-RhoA and phosphorylated MYPT-1 were significantly increased in smokers compared with controls. Pulmonary endothelial dysfunction is present in smokers whose lung function has not yet been impaired. Reduced activity of eNOS accounts at least in part for this endothelial dysfunction. Increased expression and activity of ROCKs accounts for another part through direct or indirect inhibition of the Rho-A/ROCKs pathway on nitric oxide synthesis and sustained pulmonary vasoconstriction through inhibition of myosin phosphatase.