Gulsev Ozen

Chabha Benyahia

Yasmine Amgoud

Jigisha Patel

Heba Abdelazeem

Amel Bouhadoun

Sonia Yung

Fangfang Li

Youcef Mahieddine

Adam M Silverstein

Heba M Abd Elmone

Yves Castier

Aurélie Cazes

Dan Longrois

Lucie H Clapp

Xavier Norel
email: xavier.norel@inserm.fr

Interaction between PGI2 and ET-1 pathways in vascular smooth muscle from Group-III pulmonary hypertension patients

Keywords: Human pulmonary artery, pulmonary hypertension, PGI2, ET-1, endothelin receptor antagonist

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

Introduction

Pulmonary hypertension (PH) is a chronic and progressive disease defined by a mean pulmonary arterial pressure (mPAP) higher than 20 mmHg and is associated with a high mortality rate [START_REF] Liang | Ambrisentan and tadalafil synergistically relax endothelin-induced contraction of rat pulmonary arteries[END_REF][START_REF] Simonneau | Haemodynamic definitions and updated clinical classification of pulmonary hypertension[END_REF] . Endothelial dysfunction is involved in the pathogenesis of PH and leads to increased production of vasoconstrictor/proliferative mediators [such as endothelin-1 (ET-1), thromboxane (TxA2)] and reduced production of vasodilator/anti-proliferative mediators [such as prostacyclin (PGI2), nitric oxide (NO)] [START_REF] Giaid | Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension[END_REF][START_REF] Adatia | Thromboxane A2 and prostacyclin biosynthesis in children and adolescents with pulmonary vascular disease[END_REF][START_REF] Christman | An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension[END_REF][START_REF] Yoshibayashi | Plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. Evidence for increased production of endothelin in pulmonary circulation[END_REF] . Pharmacologic treatments for PH include PGI2 analogues or mimetics (epoprostenol, iloprost, treprostinil, selexipag), ET-1 receptor antagonists (ERA: bosentan, ambrisentan, macitentan), phosphodiesterase inhibitors (tadalafil, sildenafil) or guanylate cyclase stimulators (riociguat) [START_REF] Hoeper | Pulmonary Hypertension[END_REF] . However, these treatments were primarily tested and found effective in PH Group-I (pulmonary arterial hypertension, PAH) patients according to classification established by the World Health Organization based upon etiology of disease [START_REF] Galie | ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)[END_REF] . On the other hand, PH Group-III is defined as PH due to lung disease (like chronic obstructive pulmonary disease, interstitial lung disease, or overlap syndromes) or conditions that cause hypoxemia (like obstructive sleep apnea, alveolar hypoventilation disorders) [START_REF] Galie | ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)[END_REF] . Although PH Group-III is the most common and lethal form of PH [START_REF] Wijeratne | Increasing Incidence and Prevalence of World Health Organization Groups 1 to 4 Pulmonary Hypertension: A Population-Based Cohort Study in Ontario, Canada[END_REF] , treatment studies performed in these patients are limited, and none of the treatments described above for PH Group-I have been approved for use in PH Group-III [START_REF] Hoeper | Treatment of pulmonary hypertension[END_REF] .

Since PH is associated with enhanced plasma and arterial ET-1 levels, which are correlated with severity of the disease, the suppression of ET-1 activity is one of the therapeutic approaches for the treatment of PH [START_REF] Rubens | Big endothelin-1 and endothelin-1 plasma levels are correlated with the severity of primary pulmonary hypertension[END_REF][START_REF] Stewart | Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease?[END_REF][START_REF] Yamakami | Arterial endothelin-1 level in pulmonary emphysema and interstitial lung disease. Relation with pulmonary hypertension during exercise[END_REF][START_REF] Carratu | Exhaled and arterial levels of endothelin-1 are increased and correlate with pulmonary systolic pressure in COPD with pulmonary hypertension[END_REF][START_REF] Kwon | Plasma C-reactive protein and endothelin-1 level in patients with chronic obstructive pulmonary disease and pulmonary hypertension[END_REF] . Furthermore ERAs have been shown to improve 6-min walking distance (6MWD) and decrease pulmonary artery pressure and vascular resistance in PH Group-III patients [START_REF] Valerio | Effect of bosentan upon pulmonary hypertension in chronic obstructive pulmonary disease[END_REF][START_REF] Tanaka | Potential benefit of bosentan therapy in borderline or less severe pulmonary hypertension secondary to idiopathic pulmonary fibrosis-(18)[END_REF][18] . However, the role of ET-1 in the pathogenesis of PH is complex, owing to the fact that it acts through two different receptor subtypes (ETA and ETB). In humans, these receptors have different roles depending of the cells (endothelial or smooth muscle) and/or type of pulmonary artery (conductance, resistance) where they are expressed [START_REF] Clozel | Endothelin research and the discovery of macitentan for the treatment of pulmonary arterial hypertension[END_REF][20][START_REF] Mcculloch | EndothelinB receptor-mediated contraction in human pulmonary resistance arteries[END_REF][START_REF] Mcculloch | EndothelinB receptor-mediated contraction of human and rat pulmonary resistance arteries and the effect of pulmonary hypertension on endothelin responses in the rat[END_REF] . ETA receptors are expressed predominantly in pulmonary smooth muscle cells and induce vasoconstriction and smooth muscle cell proliferation, which contributes to the progression of PH [START_REF] Zamora | an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells[END_REF] . On the other hand, ETB receptors are found on endothelial cells and to a much lesser extent on smooth muscle cells 24 .

Activation of ETB receptors on endothelial cells releases vasodilator and anti-proliferative mediators (such as PGI2 and NO) and mediates the clearance of ET-1, while ETB receptors on smooth muscle cells induce vasoconstriction [START_REF] Seo | Both ETA and ETB receptors mediate contraction to endothelin-1 in human blood vessels[END_REF][START_REF] Hall | Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment[END_REF] . However, in human pulmonary artery (HPA), the major effect of ETB activation results in a vasoconstriction [START_REF] Mcculloch | EndothelinB receptor-mediated contraction in human pulmonary resistance arteries[END_REF] . Despite the fact that a non-selective ERA such as bosentan leads to clinical improvements of PH, inhibition of ETB receptors on endothelial cells is not desirable since inhibition of ET-1 clearance is unwanted in PH [START_REF] Vachiery | The endothelin system in pulmonary and renal vasculopathy: les liaisons dangereuses[END_REF] . Therefore, the selective ETA receptor antagonist, ambrisentan might be considered the most appropriate ERA therapy for PH Group-I patients [START_REF] Valerio | Effect of bosentan upon pulmonary hypertension in chronic obstructive pulmonary disease[END_REF][START_REF] Tanaka | Potential benefit of bosentan therapy in borderline or less severe pulmonary hypertension secondary to idiopathic pulmonary fibrosis-(18)[END_REF][18] .

Another therapeutic approach for PH is administration of PGI2 mimetics. PGI2 is produced from pulmonary arteries/veins and acts via the IP receptor to cause vasodilation and inhibit smooth muscle cell proliferation [START_REF] Norel | Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone[END_REF][START_REF] Del Pozo | The prostacyclin pathway in pulmonary arterial hypertension: a clinical review[END_REF][START_REF] Clapp | The mechanistic basis of prostacyclin and its stable analogues in pulmonary arterial hypertension: Role of membrane versus nuclear receptors[END_REF] . The production of PGI2 and expression of IP receptor are decreased in PH [START_REF] Christman | An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension[END_REF][START_REF] Lai | Role of the prostanoid EP4 receptor in iloprost-mediated vasodilatation in pulmonary hypertension[END_REF][START_REF] Falcetti | Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension[END_REF] ; therefore, drugs targeting the PGI2 pathway including synthetic PGI2 (epoprostenol), PGI2 analogues (iloprost, treprostinil, beraprost) and selective IP receptor agonists (selexipag) are treatment options for PH [START_REF] Del Pozo | The prostacyclin pathway in pulmonary arterial hypertension: a clinical review[END_REF] . Although these treatments have not been recommended for PH Group-III patients by European Guidelines due to the lack of randomized controlled trials [START_REF] Olschewski | Pulmonary hypertension due to lung diseases: Updated recommendations from the Cologne Consensus Conference 2018[END_REF] , several studies found that they are effective to significantly decrease pulmonary vascular resistance, mPAP, right heart dysfunction and/or to increase 6MWD Group-III patients, mostly with severe PH [START_REF] Saggar | Changes in right heart haemodynamics and echocardiographic function in an advanced phenotype of pulmonary hypertension and right heart dysfunction associated with pulmonary fibrosis[END_REF][START_REF] Reichenberger | Effects of nebulised iloprost on pulmonary function and gas exchange in severe pulmonary hypertension[END_REF][START_REF] Olschewski | Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis[END_REF][START_REF] Wang | Hemodynamic and gas exchange effects of inhaled iloprost in patients with COPD and pulmonary hypertension[END_REF][START_REF] Dernaika | Iloprost improves gas exchange and exercise tolerance in patients with pulmonary hypertension and chronic obstructive pulmonary disease[END_REF] .

Despite the fact that approved PH drugs improve clinical and hemodynamic outcomes, morbidity and mortality remain high [START_REF] Ryerson | Pharmacotherapy in pulmonary arterial hypertension: a systematic review and meta-analysis[END_REF][START_REF] Kang | The PPARgamma ligand rosiglitazone attenuates hypoxia-induced endothelin signaling in vitro and in vivo[END_REF] . The use of combinations of drugs with differential mechanisms of action is a strategy for PH treatment. In this study, we tested the hypothesis that the combining of a PGI2 analogue (iloprost, treprostinil) with an ERA (ETA receptor antagonist: BQ123, ETB receptor antagonist: BQ788) may be more effective than either drug alone to decrease the elevated ET-1 levels, the elevated human pulmonary artery smooth muscle cells (hPASMCs) proliferation and the increased pulmonary vascular tone observed in PH Group-III patients.

Materials and Methods

Human pulmonary vascular preparations and lung parenchyma

After obtaining informed patient consent, the pulmonary preparations (pulmonary arteries, veins and lung parenchyma) were collected in the Department of Thoracic and Vascular Surgery at Bichat-Claude Bernard Hospital (Paris, France). The control pulmonary tissues were obtained from patients who underwent surgery for lung carcinoma (7 females, 6 males aged between 57-79 years old). HPA and human pulmonary veins (HPV) were carefully removed from macroscopically normal regions of the lungs. The PH pulmonary tissues have been obtained from patients who have undergone surgery for lung transplantation (explanted sick lung tissue). The category of PH patients is PH due to lung diseases and/or hypoxia (PH Group-III). The patient characteristics of PH Group-III patients are presented in Table S1. PH lungs used in our study were from patients having catheter-measured mPAP ≥ 20 mmHg. The investigation conforms to the principles outlined in the Declaration of Helsinki. All research programs involving the use of human tissue are approved and supported by the INSERM Ethics Committee and the study (n° 11-045) was approved by the CEERB IRB00006477.

Western blot analysis

Samples of HPA were homogenized in liquid nitrogen, using a porcelain mortar.

Homogenates were diluted (100 mg/ml) in Tris-HCl buffer with a protease inhibitor cocktail.

Proteins were quantified by BCA protein assay kit and a 50 μg of protein sample loaded on a 12% polyacrylamide gel. Proteins were blotted onto nitrocellulose membranes. Membranes were blocked (TBS, 0.1% Tween 20, 5% non-fat dry milk) and incubated overnight at 4°C with a primary antibody specifically targeting ETA or ETB receptors (dilution ratio: 1/700, 1/350, respectively) in TBS/0.1% Tween-20/1% non-fat dry milk. Subsequently, the membranes were incubated with an appropriate alkaline phosphatase-conjugated secondary antibody. Bands were visualized using the ECL prime luminescence system. For quantification, the film was scanned, and the integrated optical density of the bands was estimated with Scion Image (Scion Corporation, NIH, Frederick, MD, USA) and normalized to β-actin. For each sample, both ETA and ETB receptor expression were determined and calculated as a ratio of ETA to ETB receptor expression.

Organ bath and isometric measurements

Vascular preparations (pulmonary arteries and veins), cut as rings of 3 mm in width, were set up in 10 mL organ baths containing Tyrode's solution (concentration mM): NaCl 139.2, KCl 2.7, CaCl2 1.8, MgCl2 0.49, NaHCO3 11.9, NaH2PO4 0.4, glucose 5.5, gassed with 5% CO2 and 95% O2 at 37°C and pH 7.4. Each ring was initially stretched to an optimal load (~1.5 grams). Changes in force were recorded by isometric force displacement transducer (Narco F-60, Biosystems, Houston, TX, USA) and data acquisition system IOX (EMKA, Paris, France). Rings were then equilibrated for 90 min with bath fluid changes taking place every 10 min. After the equilibration period, the viability (contractility) of the vessel specimens was checked with norepinephrine (NE, 10 µM) stimulation and the preparations were washed until the initial resting tone was re-established. Thereafter, the vessels were contracted with increasing concentrations of ET-1 (0.001-0.3 μM) in a cumulative manner to establish the concentration-response curves.

Ex-vivo tissue culture and ET-1 measurements

Samples of HPA and lung parenchyma were dissected and cut into small pieces and placed into 12-well plates (100-200 mg tissue/well) containing RPMI (pH 7.4) supplemented with antibiotics (penicillin, 1000 IU/mL; streptomycin, 100 μg/mL) and an antimycotic agent (amphotericin, 0.25 μg/mL). Fresh pulmonary preparations were incubated in the presence or absence of one selective ET-1 receptor antagonist (ETA receptor antagonist: BQ123, ETB receptor antagonist: BQ788; 1 µM) or/and the PGI2 analogue (treprostinil; 1 µM). The volume of the culture medium was adjusted to 1 mL for 70 mg of tissue. All tissue incubations were performed at 37°C in a humidified atmosphere of 5% CO2 in air using a culture incubator for 12h. Subsequent to this exposure, ET-1 concentrations were measured in culture media using an enzyme immunoassay kit.

Culture of human pulmonary artery smooth muscle cells (hPASMCs)

The culture of hPASMCs and all treatments were carried out in a biosafety level 2 laboratory in a vertical laminar flow hood. The hPASMCs were obtained from HPA samples from control or PH patients. These arteries were removed after dissection of lung lobes or whole lungs. First, proximal arteries were opened, cleaned of any connective tissue (parenchyma) and then rinsed with phosphate-buffered saline (PBS) containing 1/20 penicillin, streptomycin, amphotericin B (PSA). After rinsing, the artery media was isolated and cut with a scalpel into small 1-2 mm pieces. This preparation was placed in a medium containing collagenase (Type 1) and elastase, and then incubated for 30 to 40 minutes at 37°C. After incubation and enzymatic digestion of the extracellular matrix, the preparation was filtered (40 microns filter) and centrifuged at 1000 rpm for 10 minutes at 20°C. After centrifugation, the supernatant was aspirated and the pellet was resuspended in a T25 flask containing Smooth Muscle Cell Growth Medium 2) supplemented with 20% fetal calf serum (FCS), PSA and growth factors [hEGF (epidermal growth factor), hbFGF (fibroblast growth factor), IGF (insulin-like growth factor)] to allow the proliferation of SMCs. .The cells were cultured in an incubator at 37°C in humid atmosphere containing 5 % CO2. When confluence was reached, the hPASMCs were detached from the T25 flask using 1 ml of collagenase (0.3%) then 2 ml trypsin. In this first passage, the cells were diluted in 12 ml of culture medium and transferred to a larger flask (T75). Depending on the extend of cell confluence, passages were performed approximately every 2 weeks. The SMCs were confirmed morphologically; we obtained spindle shaped cells forming the "hill and valley" configuration which is typical of SMC.

Pharmacological treatment of hPASMCs and MTT proliferation assay

At passage 3-4, the hPASMCs were washed twice with PBS (12 ml). After the wash, hPASMCs were detached as previously described and diluted in culture medium containing 20% FCS to a concentration of 3x10 6 cells per 100 ml. Maintaining one cell culture derived from one individual, the cells in a homogeneous suspension were then seeded (200 μL /well) in four 48 well plates. After proliferation of the cells in the 48-well plates (25-50% confluence), the culture medium (with 20% FCS) was aspirated and replaced with a 0% FCS (200 μl) culture medium for 24 h in order to synchronize proliferation of the hPASMCs. After 24 hours of FCS deprivation, the medium was aspirated and replaced with 100 μl of culture medium (15% FCS) in each well in order to restart proliferation. 100 μl of a single pharmacological treatment (iloprost, treprostinil, BQ123 or BQ788) or combination (one analogue of PGI2 + one ERA) were added in this medium to determine the effect of each treatment on the proliferation of hPASMCs. Within the same plate, each treatment was tested in triplicate and the control (no treatment, 100% proliferation) was tested in sextuplicate.

The MTT solution (5 mg/ml) was prepared in the specific culture medium for hPASMCs containing 0% FCS. The mixture was filtered (22 microns) and subsequently stored at 4°C protected from light. 3-4 days after the pharmacological treatment, the medium in each well was removed. The hPASMCs were washed twice with RPMI-1640 (200 μl) and then incubated with the diluted MTT solution in RPMI 1/10 for 4 h at 37°C and 5% CO2.

After incubation (2h), the MTT solution was removed by gentle inversion of the plates. The formazan crystals (purple colored) obtained were visualized under a microscope, and after dissolution with DMSO, the violet coloration was measured using an OPTIMA spectrophotometer (Tokyo, Japan) at a wave length of 540 nm.

Statistical analysis

All results obtained from different patients (n) were expressed as mean ± standard error of the mean (SEM). The concentration-response curve induced by ET-1 was expressed as % of the Emax of the NE (10 µM) control. Statistical analysis was performed by Student's ttest, Mann Whitney-U test or two-way ANOVA and Bonferroni's multiple comparison post hoc tests. The null hypothesis is that there is no difference between PH Group-III versus control patients or there is no difference between measurements with and without treatments in cell proliferation or ET-1 levels. The null hypothesis is rejected if the P value is less than 0.05 and indicates data significantly different. Statistical analyses were performed using SigmaStat version 3.5 (Systat Software, Point Richmond, CA, USA).

Compounds and materials

Protease inhibitor cocktail, NE, antibiotics, antimycotic, trypsin BQ123, BQ788, βactin antibody and MTT colorimetric assay were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iloprost, ET-1 and ET-1 ELISA kits were obtained from Cayman Chemical (Ann Arbor, MI, USA). Treprostinil was a gift from United Therapeutics Corporation (Silver Spring, Maryland, ABD). RPMI, trypsin and collagenase were obtained from Gibco Invitrogen (Paisley, UK). Elastase was purchased from Worthington (Lakewood, NJ, USA).

BCA protein assay kit was from Thermo (Rockford, USA). Nitrocellulose membranes and ECL Plus® system were obtained from Amersham Biosciences (Buckinghamshire, UK).

Antibodies against ETA and ETB were from Abcam (Cambridge, UK). Smooth Muscle Cell Growth Medium 2 was from PromoCell (Heidelberg, Germany).

Results

ETA and ETB receptor expression in human pulmonary arterial preparations from control and PH Group-III patients

The expression of ETA and ETB receptors was determined in HPA preparations derived from control and PH patients. There was no significant difference in ETA receptor expression between samples from control and PH patients (Figure 1A). However, the ETB receptor expression was significantly lower in PH Group-III patients as compared to control patients, resulting in a significantly higher ratio of ETA to ETB (Figures 1A,B).

Contraction induced by ET-1 in human pulmonary artery and vein derived from control and PH Group-III patients

ET-1 induced contraction in a concentration-dependent manner in HPA and HPV preparations, with no differences observed for control or PH Group-III patients (Figure 2). Of note, HPV preparations exhibited greater contractions induced by ET-1 versus HPA in both control and PH patients at concentrations above 10 nM (Figure 2).

The effect of ET-1 receptor antagonists and/or treprostinil on ET-1 levels in PH

Group-III patients

HPA and lung parenchyma derived from PH patients were incubated in the presence or absence of a selective ETA receptor antagonist (BQ123, 1 µM) or ETB receptor antagonist (BQ788, 1 µM) or/and treprostinil (1 µM). Following 12 h of incubation, ET-1 concentrations were measured in the culture medium. Incubation with BQ788 or BQ123 statistically significantly increased ET-1 levels in HPA preparations. BQ788 also increased ET-1 levels in parenchyma from PH Group-III patients (Figures 3A,B). In HPA preparations derived from PH Group-III patients, co-incubation with treprostinil and BQ788 resulted in statistically significant higher concentrations of ET-1 as compared to control incubation or those incubated with treprostinil alone (Figure 3A).

The effect of PGI2 analogues and/or ET-1 receptor antagonists on hPASMCs proliferations in control and PH patients

Globally, when considering each treatment presented in Figure 4, proliferation of hPASMCs derived from PH patients (-18±03%) were significantly less inhibited in comparison with those from control patients (-28±03%; P= 0.014).

In hPASMCs from control patients, single agent treatment with treprostinil (1 µM) or iloprost (1 nM / 1 µM) statistically significantly decreased proliferation to a similar degree (about -25%, Figure 4A). In hPASMCs preparations from PH Group-III patients, single agent treatment with 1 µM, but not 1 nM of treprosinil or iloprost statistically significantly decreased proliferation (-24±06% and -19±06%, respectively; Figure 4B).

In both control and PH patient hPASMCs, neither BQ123 (1 µM) nor BQ788 (1 µM) as single agent treatment caused a statistically significant inhibition of proliferation. In addition, combined treatments with treprostinil (1 µM) +BQ123 (1 µM) or +BQ788 (1 µM) did not increase the inhibition of hPASMCs proliferation observed with treprostinil alone. In contrast, combination treatments including iloprost (1 nM) +BQ123 (1 µM) showed an increased inhibition of proliferation in both control and PH patients (-48±14, -32±06, respectively, P<0.1) versus iloprost (1 nM) alone. These results suggest an additive effect of combining iloprost with the ETA receptor antagonist BQ123 (Figures 4).

Discussion

In the present study, first we characterized the ET-1 pathway for receptor expression and responsiveness in preparations from PH Group-III patients. Our results indicated an increased ETA/ETB ratio in HPA preparations derived from PH Group-III patients, yet vascular contraction induced by ET-1 was not impacted (Figures 1,2). We also showed that mostly incubation with an ETB receptor antagonist significantly increased ET-1 production in pulmonary arteries and parenchyma from PH Group-III patients, an effect that is magnified in pulmonary arteries when co-incubated with treprostinil (Figure 3). In the second part of our study, co-incubation of an ETA receptor antagonist with iloprost suggested additive inhibition of hPASMCs proliferation from both control and PH patients (Figure 4). Under physiological conditions, the release of vasodilator and anti-proliferative mediators by ETB receptor activation balances the vasoconstrictor and proliferative effects of ETA receptor. However, this fine balance between ETA and ETB receptor-mediated effects is disrupted and transformed into detrimental effects in pathological conditions [START_REF] Mazzuca | Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease[END_REF] . Increased ETA/ETB receptor expression ratio results in enhanced proliferative effects mediated by ET-1 and may play a role in PH pathogenesis. Moreover, since ETB receptor is also responsible for ET-1 clearance in the circulation, disruption of functional ETB receptor promotes increased levels of ET-1 and results in enhanced adverse effects on vascular tone and proliferation [START_REF] Mazzuca | Vascular endothelin receptor type B: structure, function and dysregulation in vascular disease[END_REF] . In the present report, we demonstrated decreased ETB receptor expression and greater ratio of ETA/ETB receptor expression in HPA derived from PH Group-III patients (Figure 1). A similar observation was recently published for lung tissue derived from patients with idiopathic pulmonary fibrosis who have a high prevalence of PH Group-III [START_REF] Bellaye | Macitentan reduces progression of TGF-beta1-induced pulmonary fibrosis and pulmonary hypertension[END_REF] . On the other hand, the studies performed on the other types of PH reported that there is an increase of ETB receptor expression or mRNA levels in the lungs, pulmonary artery or hPASMCs of patients with PH Group I and IV, while others demonstrated ETB mRNA levels was decreased in hPASMCs of PH Group-I [START_REF] Hall | Endothelin receptor expression in idiopathic pulmonary arterial hypertension: effect of bosentan and epoprostenol treatment[END_REF][START_REF] Yu | Altered expression and signal transduction of endothelin-1 receptors in heritable and idiopathic pulmonary arterial hypertension[END_REF][START_REF] Bauer | Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension[END_REF][START_REF] Shao | The role of endothelin-1 in the pathogenesis of pulmonary arterial hypertension[END_REF][START_REF] Davie | ET(A) and ET(B) receptors modulate the proliferation of human pulmonary artery smooth muscle cells[END_REF] . These contradictory results could be due to etiology, stage of disease, treatments, age of the patients and different experimental techniques.

Even though we have demonstrated a higher ETA/ETB receptor expression ratio in PH Group-III patients, the contraction induced by ET-1 on HPA and HPV was not different between control and PH Group-III patients (Figure 2). In accordance with our findings, there was no change in ET-1 induced contraction in PH Group-I models of rats with decreased ETB receptor expression in pulmonary arteries [START_REF] Sauvageau | Change in pharmacological effect of endothelin receptor antagonists in rats with pulmonary hypertension: role of ETBreceptor expression levels[END_REF] . Furthermore, the signalling mechanisms of the contractile responses to ET-1 of PH Group-I and control hPASMCs were very similar [START_REF] Wilson | Unraveling endothelin-1 induced hypercontractility of human pulmonary artery smooth muscle cells from patients with pulmonary arterial hypertension[END_REF] . The decreased expression of the ETB receptor appears to not have an impact on vasoconstriction induced by ET-1 in isolated lung vasculature from PH Group-III patients. We also demonstrated that contractions induced by ET-1 were greater for HPV than for HPA, consistent with results previously described by our group in these human vessels [START_REF] Brink | Effects and specific binding sites of endothelin in human lung preparations[END_REF][START_REF] Pussard | Endothelin-1 modulates cyclic GMP production and relaxation in human pulmonary vessels[END_REF] , by other research groups in many mammalian pulmonary vessels and also in human internal mammary arteries/veins [START_REF] Yang | Different effects of endothelin-1 on cAMP-and cGMP-mediated vascular relaxation in human arteries and veins: comparison with norepinephrine[END_REF][START_REF] Gao | Role of veins in regulation of pulmonary circulation[END_REF] .

In the present study, treatments with ERAs significantly increased ET-1 concentrations in HPA preparations. In our results, the selective ETB receptor antagonist (BQ788) significantly increased ET-1 levels in both HPA and lung parenchyma preparations obtained from PH Group-III patients. On the other hand, the selective ETA receptor antagonist (BQ123) only statistically increased ET-1 levels in HPA, this unexpected result and a role in clearance for ETA should be confirmed (Figure 3). Our results may not only involve endothelial cells but also other cells present in the lung preparations, and a previous study suggested that ET-1 clearance occurs in both endothelial cells and hPASMCs [START_REF] Angus | Distortion of KB estimates of endothelin-1 ETA and ETB receptor antagonists in pulmonary arteries: Possible role of an endothelin-1 clearance mechanism[END_REF] . Several studies have shown elevated levels of ET-1 in plasma and HPA derived from PH Group-III patients [START_REF] Yamakami | Arterial endothelin-1 level in pulmonary emphysema and interstitial lung disease. Relation with pulmonary hypertension during exercise[END_REF][START_REF] Carratu | Exhaled and arterial levels of endothelin-1 are increased and correlate with pulmonary systolic pressure in COPD with pulmonary hypertension[END_REF][START_REF] Kwon | Plasma C-reactive protein and endothelin-1 level in patients with chronic obstructive pulmonary disease and pulmonary hypertension[END_REF][START_REF] Celik | Local and peripheral plasma endothelin-1 in pulmonary hypertension secondary to chronic obstructive pulmonary disease[END_REF] . Given our results, ETB receptor antagonism may not desirable in the context of PH Group-III patients who have already increased ET-1 levels. In fact, in accordance with our in vitro results, clinical studies in non-PH Group-III patients demonstrated that treatment with a non-selective ERA (bosentan) increased plasma ET-1 levels, an effect not observed with a selective ETA receptor antagonist (sitaxentan) [START_REF] Givertz | Acute endothelin A receptor blockade causes selective pulmonary vasodilation in patients with chronic heart failure[END_REF][START_REF] Williamson | Hemodynamic effects of Bosentan, an endothelin receptor antagonist, in patients with pulmonary hypertension[END_REF] . Furthermore, a double-blind trial in PH Group-I patients indicated that sitaxentan therapy showed significant benefit over bosentan with respect to discontinuation of monotherapy, clinical worsening and survival rate [START_REF] Langleben | A review of STRIDE-2 and STRIDE-2X: the case for selective endothelin receptor blockade[END_REF] . However, the randomized control trials with ERA conducted in PH Group-III patients were limited and with results depending on severity of disease and duration of treatments. Twelveweeks of treatment with bosentan did not improve the hemodynamic parameters [START_REF] Stolz | A randomised, controlled trial of bosentan in severe COPD[END_REF] , while longer-term treatment increased activity of daily living and 6MWD, overall survival, as well as decreasing pulmonary artery pressure and vascular resistance [START_REF] Valerio | Effect of bosentan upon pulmonary hypertension in chronic obstructive pulmonary disease[END_REF][START_REF] Tanaka | Potential benefit of bosentan therapy in borderline or less severe pulmonary hypertension secondary to idiopathic pulmonary fibrosis-(18)[END_REF] .

Other treatments for PH include PGI2 mimetics because of their vasodilator and antiproliferative properties. Since both PGI2 and ET-1 pathways are involved in the pathogenesis of PH Group-III, combination therapy targeting both pathways is indicated [START_REF] Mclaughlin | Randomized study of adding inhaled iloprost to existing bosentan in pulmonary arterial hypertension[END_REF] . In our study the increased ET-1 level observed in lung preparations incubated with BQ788 was not significantly different from the co-incubation of BQ788 with treprostinil (Figure 3). However, in the presence of this increased ET-1 production in hPASMCs derived from PH patients, the PGI2 analogues were still able to significantly reduce proliferations by 24% (Figure 4B).

In hPASMCs derived from control patients, single agent treatment with treprostinil (1 µM) or iloprost (1 nM or 1 µM) statistically significantly decreased proliferation (Figure 4A).

In our assay, iloprost was slightly more effective than treprostinil when used at a low concentration (1 nM; Figure 4). However, a slightly greater anti-proliferative potency of treprostinil (1 nM) versus iloprost (1 nM) was previously demonstrated [START_REF] Clapp | Differential effects of stable prostacyclin analogs on smooth muscle proliferation and cyclic AMP generation in human pulmonary artery[END_REF] . This discrepancy could be due to experimental variability or to the prior study's use of smaller diameter pulmonary vessels where a greater density of EP2 receptor or PPARγ expression might also contribute to treprostinil's anti-proliferative activity [START_REF] Falcetti | Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension[END_REF][START_REF] Patel | Prostanoid EP(2) Receptors Are Up-Regulated in Human Pulmonary Arterial Hypertension: A Key Anti-Proliferative Target for Treprostinil in Smooth Muscle Cells[END_REF] . In fact, it has been already shown that there is a clear difference in inhibition of hPASMCs proliferation by iloprost and cicaprost (a selective IP agonist) depending on whether distal or proximal pulmonary artery is used [START_REF] Wharton | Prostacyclin analogues differentially inhibit growth of distal and proximal human pulmonary artery smooth muscle cells[END_REF] . Single agent treatment of hPASMCs with ERAs did not statistically inhibit proliferation in our study. However, other published results with higher doses (10 µM) of cicaprost or BQ123 showed anti-proliferative effects on hPASMCs derived from control patients (about -30%) [START_REF] Wort | Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling[END_REF][START_REF] Wort | The prostacyclin-mimetic cicaprost inhibits endogenous endothelin-1 release from human pulmonary artery smooth muscle cells[END_REF] .

Of note, hPASMCs are able to release ET-1 and this release could be stimulated by ET-1 from hPASMCs in a concentration-dependent fashion [START_REF] Wort | Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling[END_REF][START_REF] Wort | The prostacyclin-mimetic cicaprost inhibits endogenous endothelin-1 release from human pulmonary artery smooth muscle cells[END_REF] . In addition, the inhibitor of endothelin converting enzyme (phosphoramidon), which is responsible for ET-1 formation, reduced the proliferation induced by FCS in hPASMCs and that is confirming an autocrine role for ET-1 [START_REF] Wort | Endogenously released endothelin-1 from human pulmonary artery smooth muscle promotes cellular proliferation: relevance to pathogenesis of pulmonary hypertension and vascular remodeling[END_REF] .

In hPASMCs derived from PH Group-III patients, iloprost and treprostinil behaved similarly when comparing their anti-proliferative effects, with only the 1 µM concentrations showing a statistically significant anti-proliferative effect (Figure 4). This finding is similar to the inhibitory effects observed in hPASMCs proliferations derived from children with idiopathic PAH [START_REF] Falcetti | Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension[END_REF] . The decreased anti-proliferative potency of iloprost in PH Group-III versus control patients could be due to a reduced IP receptor expression in the hPASMCs, as was observed in PH Group-I patients [START_REF] Falcetti | Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension[END_REF] . Interestingly, incubating hPASMCs derived from both groups of patients with iloprost (1 nM) and the ETA receptor antagonist BQ123 resulted in a strong tendency toward additive inhibition (Figure 4). In contrast the anti-proliferative effect of treprostinil in hPASMCs was not modified in the presence of either ERA.

In this study, we determined cell proliferation by MTT assay. Even though this technique is widely used, it has some disadvantages. Other cell proliferation techniques such as detection of proliferating nuclear antigen, cyclin D-E or cell counts could be performed to support MTT results. However, insufficient supply of human pulmonary artery is an important factor that limits performing these experiments. This is a limitation of our study and remains to be readdressed in further studies.

Conclusion

Despite the fact that PH Group-III is the most common and lethal form of PH [START_REF] Wijeratne | Increasing Incidence and Prevalence of World Health Organization Groups 1 to 4 Pulmonary Hypertension: A Population-Based Cohort Study in Ontario, Canada[END_REF] , clinical studies on these patients have been limited and no conventional PH therapies are approved for use in this patient population [START_REF] Hensley | Emerging therapeutics in pulmonary hypertension[END_REF] . In the present study, we used several tissues derived from PH Group-III patients including HPA, HPV, lung parenchyma and hPASMCs.

The in vitro results presented support that the increased ETA/ETB receptor expression ratio in HPA may be involved in the pathogenesis of PH Group-III, in ways not impacting ex vivo pulmonary vascular tone induced by ET-1. Using selective ERA antagonists, our results support mostly a role for ETB receptor regulation of ET-1 production in pulmonary tissue; a role in which ETB receptor antagonism or downregulation would lead to a detrimental increase in ET-1 concentrations. In addition, whereas both iloprost and treprostinil individually inhibited proliferation of hPASMCs from either patient group, the combination of the selective ETA receptor antagonist BQ123 and iloprost produced a greater tendency to inhibit hPASMCs proliferation derived from both groups of patients. This combination therapy is currently recommended for the treatment of PH Group-I in the guidelines of ESC/ERS 8 , and our findings will hopefully provide additional mechanistic information to consider when treating (severe) PH Group-III patients with a combination of a PGI2 analogue and a selective ETA receptor antagonist.

Figure 1 ETA

 1 Figure 1 ETA and ETB receptor expression (Figure 1A and 1C) and ETA/ETB receptor expression ratio (Figure 1B) in human pulmonary artery (HPA) derived from control and pulmonary hypertension (PH) Group-III patients. Western blot analyses for endothelin receptors (ETA and ETB) were normalized by β-actin in human preparations and then ETA/ETB receptor expression ratios were calculated. Values are means ± SEM, n=4-7 patients. *Data significantly different between control and PH patient groups (P<0.05). A representative image of Western blot is presented in Figure 1C.

Figure 2 Contraction

 2 Figure 2 Contraction induced by endothelin-1 (ET-1) in human pulmonary arteries (HPA) and human pulmonary veins (HPV) derived from control and pulmonary hypertension (PH) Group-III patients. Concentration-response curves for ET-1-induced contraction. Responses are expressed as a percentage of contraction induced by norepinephrine (NE, 10 µM). Values are means ± SEM, n=3-4 patients in each group. *Data significantly different from HPV for respective patient groups (P<0.05).

Figure 3 Endothelin- 1 (

 31 Figure 3 Endothelin-1 (ET-1) content in human pulmonary arteries (HPA, Figure 3A) and lung parenchyma (Figure 3B) preparations derived from pulmonary hypertension (PH) Group-III patients after different treatments. Human preparations were incubated with PGI2 analogue (TRP, treprostinil, 1 µM) and/or ETA receptor antagonist (BQ123, 1 µM), ETB receptor antagonist (BQ788, 1 µM). Black bars indicate human preparations without any treatment; white bars indicate human preparations with single treatment and lined bars indicate human preparations with combination treatment. The concentration of ET-1 in organ culture supernatant after 12h incubation was expressed as pg/mg of protein (A) or pg/mg of tissue (B). * indicates values significantly different (P<0.05). Values are means ± SEM, n=7-11 (HPA) or 3-4 (parenchyma) patients.

Figure 4 Proliferation

 4 Figure 4 Proliferation of human pulmonary artery smooth muscle cells (hPASMCs) derived from control (Figure 4A) and pulmonary hypertension Group-III patients (PH, Figure 4B) after different treatments. hPASMCs were incubated with PGI2 analogues (TRP:

Figure 1 ETA

 1 Figure 1

Figure 2 Contraction

 2 Figure 2

Figure 3 Endothelin- 1 (

 31 Figure 3

Figure 4 Proliferation

 4 Figure 4

Acknowledgement:

We would like to thank Elisabeth Brunet and Amina El Hilali from the Anapathology laboratory and the secretary of the Department of Anaesthesia and Intensive Care, CHU X. Bichat for their help. We would like to thank United Therapeutics for an educational grant supporting this work.

Conflict of interest:


Fabrice Barbe

Y Carpier

B Vieille
email: benoit.vieille@insa-rouen.fr

A Coppalle

About the tensile mechanical behaviour of carbon fibers fabrics reinforced thermoplastic composites under very high temperature conditions

Keywords: thermoplastic, thermal decomposition, high temperature, mechanical behavior, damage

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

The study of fire resistance aims at understanding and characterizing how and for how long the composite material can bear a mechanical loading when it is subjected to heat fluxes resulting in temperature differences through-the-thickness. Most Polymer Matrix Composite (PMC) materials usually cannot bear significant mechanical loading at temperature higher than 200-250°C. High-performance thermoplasticbased (TP) composites (e.g. PEEK, PPS, PEI…) are promising materials for structural applications in aeronautics as they are resistant to impact. They have a good damage tolerance as well as they have a very good retention of their mechanical properties under fire conditions. Among the most common PMCs used in load-bearing aircraft structures, Carbon/epoxy (thermosetting) composites are flammable and readily decompose when exposed to heat and fire. The literature dealing with the fire behavior of composite materials focuses mainly on thermosetting-based composites [START_REF] Mouritz | Fire properties of polymer composite materials. N° 143 Solid mechanics and its applications[END_REF] and rarely on thermoplastic-based composites [START_REF] Browne | A model for the structural integrity of composite laminates in fire[END_REF] [START_REF] Gibson | High temperature and fire behaviour of continuous glass fibre/polypropylene laminates[END_REF].

Thermal decomposition of PMCs

To better understand the changes in the thermal and mechanical behaviors of polymer-based composites under fire exposure the first step usually is to investigate the influence of thermal heat fluxes, hence temperature, resulting from a fire. Such heat fluxes lead to significant temperature gradients within composite structures. From thermal analyses it is therefore possible to study the decomposition mechanisms as well as their kinetics under different testing conditions (heating rate, isothermal and anisothermal, inert or oxidizing atmosphere). The decomposition mechanisms are closely associated with the chemical nature of the polymer [START_REF] Vieille | Correlation between post fire behavior and microstructure degradation of aeronautical polymer composites[END_REF]. These analyses are extremely useful for determining the primary steps of the decomposition: Td=T5% refers to pyrolysis or decomposition onset and corresponds to the temperature at which 5% of the polymer mass has decomposed; Tmax refers to the maximum peak of decomposition rate. Such analyses can also be considered for studying the thermal decomposition kinetics.

From a general standpoint, the influence of temperature on PMCs properties can be investigated considering two temperature ranges: (i) < and (ii) > .

When < , the first consequence of a temperature increase is the thermal expansion of the specimen [START_REF] Mouritz | Fire properties of polymer composite materials. N° 143 Solid mechanics and its applications[END_REF].

High temperature gradients and the difference in thermal expansion coefficients of the fiber and of the matrix may result in incompatibilities of deformations, leading to the appearance of matrix cracks that contribute to slowing down thermal conduction. The changes in matrix state (glass transition, melting) require energy input which can result in a slowing of the temperature increase by surface or within the sample [START_REF] Atreya | An experimental and theoretical investigation into burning characteristics of PPS-glass fiber composites[END_REF]. These transformations lead to matrix softening and can promote material damage such as porosities which will induce matrix cracks, inter and intra-laminar, leading to delamination. Ancillary mechanisms, such as the formation of water vapour in matrices with high water recovery, can also damage the material via the formation and growth of porosities.

When > , temperature is high enough to induce thermal decomposition and damage within the material is then the most important [START_REF] Mouritz | Fire properties of polymer composite materials. N° 143 Solid mechanics and its applications[END_REF]. Different chemical transformations of the material occur according to the mechanisms described in [START_REF] Drysdale | An introduction to fire dynamics[END_REF]. The issue of fire resistance of polymer matrices with poor temperature resistance is not widely addressed in the literature: most studies primarily focus on matrices with medium (epoxy, polyester, vinylester) or high (phenolic, PPS, PEEK) char yield [START_REF] Vieille | Correlation between post fire behavior and microstructure degradation of aeronautical polymer composites[END_REF].

Thermomechanical coupling at high temperature in PMCs

The influence of high temperatures on both the mechanical properties and the thermomechanical behaviour of PMCs can be considered up to . In thermally stable PMCs, changes in mechanical properties with temperature can be considered reversible up to matrix decomposition [START_REF] Mouritz | Fire properties of polymer composite materials. N° 143 Solid mechanics and its applications[END_REF]. In the case of a fire scenario it is necessary to consider the changes within the laminates occurring at (see section 1.1). In addition, the study of the thermomechanical coupling in PMCs requires specific technical means. Such an issue has been widely addressed in the literature. As for most of the studies dealing with high temperature exposure [START_REF] Mercadé | Modélisation de la dégradation d'un matériau composite carbone-époxy soumis à une sollicitation thermo-mécanique couplée. Application aux réservoirs d'hydrogène de type IV[END_REF], a weak coupling between thermal transfers and mechanics is postulated for our analyses. It means that the thermal state of a given point influences its mechanical behavior but a variation in its mechanical state leaves its temperature unchanged. Weak coupling refers to the influence of temperature on mechanical behaviour but not the influence of mechanical behaviour on thermal one. This section is focused on the tensile behaviour under homogeneous and isothermal temperature conditions. Figure 1 shows the typical influence of temperature on the mechanical properties of PMCs under isothermal conditions (i.e. constant and uniform temperature within the material). Among the classical mechanical properties which are significantly degraded by a temperature increase, the Young's modulus, shear modulus and compressive strength are the most common ones. represents the critical temperature from which the properties start decreasing, the glass transition temperature and the melting temperature. To understand the behaviour of PMCs under critical service conditions, e.g. fire, it is necessary to determine the residual properties at temperatures higher than .

For temperature testing conditions higher than , polymer matrix decomposition may lead to a high activity of porosity formation and growth in the laminates, which severely degrades their mechanical properties and behaviour [START_REF] Coble | Effect of porosity on the physical properties of sintered alumina[END_REF]. From monotonic tensile tests conducted on different types of orthotropic woven fabric laminates (glass/vinyl ester, glass/polyester and glass/polypropylene) up to 400°C (e.g. lower than ), there is significant tensile strength retention at high temperatures due to the mechanical load borne by the 0° fibers [START_REF] Browne | Fire Behaviour of Composite Laminates[END_REF]. To the authors' best knowledge; there are very few studies in the literature dealing with the mechanical behaviour under fire conditions [START_REF] Browne | Fire Behaviour of Composite Laminates[END_REF][START_REF] Gibson | High temperature and fire behaviour of continuous glass fibre/polypropylene laminates[END_REF]. Gibson et al. showed that the same mechanism appeared to control the failure in tension and compression (associated with the matrix melting)

in TP-based composites (glass fibre reinforced PP laminates). In the case of creep loadings, the evolution of the creep stress at failure as a function of exposure time has been studied in composite materials with TP [START_REF] Browne | Fire Behaviour of Composite Laminates[END_REF][START_REF] Gibson | High temperature and fire behaviour of continuous glass fibre/polypropylene laminates[END_REF] and thermosetting (TS) [START_REF] Feih | Modelling the compression strength of polymer laminates in fire[END_REF] matrices. These studies highlight that damage mechanisms significantly depend on the applied creep stress as well as the testing temperature.

Objectives of the study

Before studying the coupling between thermal and mechanical behaviours in TP-based composites laminates, it is necessary to determine the characteristic temperatures ( , , ). Thus TGA tests were carried out under anisothermal conditions and different atmospheres (inert or oxidizing) to investigate the influence of temperature on the thermal decomposition of C/PPS composite and its constitutive materials.

From the knowledge of the characteristic temperatures, it is therefore possible to conduct mechanical tests (monotonic tension and tensile creep) under isothermal conditions to specifically address the issue of thermomechanical coupling within carbon fibers reinforced PPS laminates for testing temperatures ranging from ambient temperature to temperatures higher than .

Material and methods

Materials and specimens

The studied composite materials are carbon fabric reinforced laminates consisting of a semi-crystalline high-performance PPS supplied by the Ticona company. The woven-ply prepreg, supplied by SOFICAR, consists of 5-harness satin weave carbon fiber fabrics (T300 3K 5HS) whose weight fraction is 58%. The prepreg plates are hot pressed according to a quasi-isotropic [(0,90)/(±45)/(0,90)/(±45)/(0,90)/(±45)/(0,90)] stacking sequence. The glass transition temperature of the material is Tg=98°C and its melting temperature Tm=280°C, as measured by DSC [START_REF] Blond | Correlation between physical properties, microstructure and thermo-mechanical behavior of PPS-based composites processed by stamping[END_REF]. The degree of crystallinity of PPS matrix is close to 30% and the initial porosity ratio is very small (less than 2%).

Experimental set-up

Thermogravimetric Analysis (TGA)

To carry out these experiments a thermo-gravimetric analyzer TA Instruments Discovery is used. Thermal decomposition is characterized under three atmospheres: nitrogen, air and dioxygen. The gas flow rate is set at 20 mL.min -1 .The extent of decomposition is characterized by a degree of progress in the reaction defined by the following expression [START_REF] Flynn | The historical development of applied nonisothermal kinetics[END_REF]:

= (1)
With the initial mass of the sample, is the mass at a time t and is the mass after decomposition.

When the analysis is carried out in an inert atmosphere, corresponds to the degree of pyrolysis and its value reaches 1 for a fully completed pyrolysis. Under oxidizing atmosphere, it corresponds to the degree of decomposition and its value is 1 when the entire solid residue is completely gasified.

The thermal decomposition of studied materials (Plain PPS resin and C/PPS composite) has been conducted using anisothermal heating at 10°C/min from room temperature to 700°C. These experiences are supposed to be representative of the increase in material temperature during the transient phase of its thermal response. However they are not suitable for clearly identifying the involved chemical mechanisms.

These analyses were conducted on samples whose initial mass is about 8mg.

Porosities rate via density measurements

The density of solids can be determined using the buoyancy method. The density is determined using Archimedes' Principle. The upward buoyant force exerted on a body immersed in a fluid is equal to the weight of the fluid the body displaces. Density determination using the buoyancy method is based on the following formula [START_REF] Madsen | Physical and mechanical properties of unidirectional plant fibre composites -an evaluation of the influence of porosity[END_REF]:

= * (2)
Where is the density of sample, is the density of buoyancy liquid, is the weight of sample in air and is the weight of sample in liquid. The density is determined by means of a densitometry balance Sartorius Secura whose accuracy is 1mg.

Finally, the porosity rate or void fraction is therefore defined from the density of sample as follows:

= 1 - (3)
Where is the density of the sample referred to as-received.

Specimens whose dimensions are 25*25*2.2mm where subjected to isothermal temperature conditions (465-500-530°C) for different exposure times (5-10-20-30-40-60 min) in a primary vacuum furnace to evaluate the changes in the residual mass associated with thermal decomposition. Specimens are put into the furnace once the temperature is stabilized.

Tensile mechanical testing under isothermal conditions

The tensile mechanical behaviour under isothermal conditions is investigated using a uniaxial servohydraulic machine MTS, equipped with hydraulic grips and a load cell with a 100kN capacity. Stable and homogeneous temperature conditions are applied by means of a tube furnace composed of 3 zones, which is positioned between the grips of the machine. High temperature testing conditions (e.g. temperatures close to or higher than polymer matrix decomposition temperature) usually requires samples with a specific geometry because temperatures are sometimes beyond the range of testing ovens. In the present case, the dimensions of the tube furnace make it necessary to use specimens with a gage length of 20cm long (Fig. 2). The tensile mechanical properties were determined according to the European standards EN 6035 [START_REF]European Association of Aerospace Industries (AECMA)[END_REF].

The axial modulus (" # ) and ultimate strength (% & ) were calculated from the following definitions:

" # = ∆(
).∆+ , and

% & = ( - ) (1)
Where ∆. is the difference in the tensile loads at (/ # ) 0 = 0.25% and (/ # ) 5 = 0.05%, S is the specimen cross section, ∆/ # = (/ # ) 0 -(/ # ) 5 is the difference in the axial strains obtained from the cross machine displacement,

. & is the maximum force borne by the specimen at failure.

Estimation of the axial stiffness under different isothermal conditions

The evolution of the axial stiffness " # with temperature was measured from the following experimental protocol:

• Heating to a temperature T1 at 50°C/min,

• Two minutes at T1 (to ensure the temperature homogeneity through-the-thickness),

• Mechanical loading (50 MPa) then unloading at 0 MPa,

• Measurement of axial stiffness during unloading (the low viscosity of the matrix can cause the extensometer to slip during loading),

The testing temperatures range from ambient temperature to 520°C.

Tensile response to failure under different isothermal conditions

The monotonic tensile behavior at failure was investigated under different isothermal conditions. As for stiffness measurements, the specimens are heated to 50°C/min and then maintained at the test temperature for 2 minutes to ensure homogeneity of the temperature through-the-thickness. The tensile test is conducted in cross machine displacement-controlled mode at a rate of 2 mm.min-1. The ultimate tensile strength and tensile behaviour have been studied for temperatures representative of four different material states:

• 220°C: rubbery state of the PPS matrix between Tg (Glass transition) and Tm (Melting),

• 270°C: slightly melted material,

• 320°C: completely melted material,

• 470°C: slightly decomposed material.

Tensile creep behavior under isothermal conditions

Finally, creep tests were performed on quasi-isotropic C/PPS specimens according to the following experimental protocol:

• Tensile mechanical loading (loading time = 10 seconds)

• Temperature increase from room temperature to 320°C at a rate of 25°C.min-1. This temperature is selected because the failure behaviour at this temperature has been characterized in the case of monotonic tensile tests.

• Both mechanical loading and temperature level (320°C) are maintained.

It has been chosen to apply the mechanical force first and then the temperature increase because it corresponds to the succession of solicitations an in-service material would have to face in the event of fire.

At the laboratory scale, a mechanically loaded sample is suddenly subjected to a heat flux applied by means of a tube furnace. The creep levels tested vary between 8 and 31% of the ultimate strength % & at ambient temperature). These stress levels are chosen such as to analyze the tensile behavior for tensile loadings lower and higher than the onset of necking.

Results and discussion

TGA tests

Before considering mechanical testing under different isothermal conditions, it is utmost important to investigate the thermal decomposition of plain PPS matrix and C/PPS laminates by means of TGA tests under anisothermal conditions and different atmospheres (inert and oxidizing), with a particular attention paid to the determination of decomposition temperature [START_REF] Feih | Tensile properties of carbon fibres and carbon fibre-polymer composites in fire[END_REF][START_REF] Tadini | Thermal degradation analysis of innovative PEKK-based carbon composites for high-temperature aeronautical components[END_REF]. An inert atmosphere is representative of what specimens undergoes at core with respect to what specimens experience in an oxidizing atmosphere at the outer surface.

Under dioxygen, the thermal decomposition of PPS begins at 502°C, approximately the same temperature as under an inert atmosphere (see Table 1). The curve representing the Mass Loss Rate (MLR) of plain PPS resin as a function of temperature MLR = f(T) is characterized by 3 peaks (see Figure 3). The first peak corresponds to a mass loss of about 15%. Thus it may be associated with the first pyrolysis mechanism, depolymerization (Figure 4b). Following this reasoning, the second peak, which extends to ∆7 = 89%, may be associated with the second pyrolysis mechanism. Even before the pyrolysis ends a new mechanism starts and may be associated with the simultaneous oxidation of PPS matrix and the pyrolysis residue as it extends until the entire initial material is carbonated (Figure 4a). The maximum oxidation rate is virtually 3 times higher than the pyrolysis rate.

The decomposition of carbon fibre-reinforced composites under oxidizing atmosphere is faster than the one of other fibers-reinforced composites (e.g. glass fibers), because carbon fibres are sensitive to oxidation phenomena just like polymeric matrices. For practical reasons decomposition under air has been little studied, as the oxidation phenomena are mainly characterized with TGA analyses under dioxygen. In C/PPS, thermal decomposition under air begins at 517°C compared to 483 and 515°C under N2 and O2 respectively (see Table 1). Thus the mechanisms of crosslinking and improvement of thermal resistance observed under O2 do not appear to occur under air. Oxidation appears to start faster under O2 simply because there are more reactive species and the atmosphere is more oxidizing. under air [START_REF] Ma | Studies on Thermogravimetric Properties of Polyphenylene Sulfide and Polyetherether Ketone Resins and Composites[END_REF]. When heated in an oxidizing atmosphere at temperatures higher than its melting temperature, PPS can crosslink like a thermosetting polymer thus having a higher thermal stability. If the delay in the onset of decomposition is only observed in C/PPS composite, this suggests that these phenomena are promoted by the presence of fibres. As for plain PPS resin pyrolysis and char oxidation overlap in the C/PPS. Nevertheless the fact that the second peak of MLR ends with a mass loss of 43% equivalent to the matrix mass in the composite, it tends to indicate that oxidation of char and fibres occurs separately and not simultaneously (Figure 4).

Under air, the thermal decomposition of PPS begins at 508°C. For the same heating rate, decomposition begins at 503°C and 502°C respectively under N2 and O2 (see Table 1). Pyrolysis results in a mass loss of 39%, which is lower than that observed under inert atmosphere (Figure 5a), which is consistent with the observations made in the case of a PEEK resin by Patel et al [START_REF] Patel | Mechanism of thermal decomposition of poly(ether ether ketone) (PEEK) from a review of decomposition studies[END_REF][START_REF] Patel | Investigation of the thermal decomposition and flammability of PEEK and its carbon and glass-fibre composites[END_REF]. As under dioxygen, oxidation begins before the pyrolysis ends. Nevertheless the appearance of the characteristic peak of pyrolysis on the MLR curve MLR = f(T) indicates that pyrolysis is almost completed when oxidation begins under air (Figure 5b) while it did not even reach its maximum rate when this transition occurs under O2.

Determination of porosities rate as a function of the degree of decomposition

C/PPS laminates specimens undergo different degrees of thermal decomposition when they are subjected to isothermal conditions for different exposure times in a primary vacuum furnace (oxidizing atmosphere).

Three temperature levels (465-500-530°C) and 6 exposure times (5-10-20-30-40-60 min) were considered to induce changes in the residual mass associated with thermal decomposition. From the evaluation of the residual mass it is therefore possible to assess the changes in the degree of decomposition vs time according to Eq. ( 1), as well as the porosity rate vs time with Eq. ( 2) and (3). Figure 6a represents the evolution of specimens' residual mass as a function of the exposure time at given temperatures. The initial decrease in the residual mass is all the more significant than the testing temperature is elevated. As a consequence, the degree of decomposition ranges from 50 to 80% after a 5 minutes exposure (Fig. 6b). The transient stage lasts about 10 minutes before the mass loss and the degree of decomposition reach a stationary state (Fig. 6). During the transient stage, the higher the test temperature, the higher the thermal decomposition rate is contrary to what is observed during the stationary stage. After a 40 minutes exposure, all the curves representing the degree of decomposition vs time converge, suggesting that regardless the testing temperatures, the differences in the thermal decomposition kinetics ultimately result in the same degree of decomposition. Finally, figure 7 represents the porosity rate as a function of the degree of decomposition for the different testing temperatures. It appears that all the experimental points virtually follow the same evolution to reach a plateau corresponding to about 35% of porosities within the specimens. From these curves, one can assume that the porosities rate is very well correlated with the degree of decomposition. The knowledge of the porosities rate is important as porosities within the laminates are stress concentrators contributing to the degradation of the mechanical properties.

Tensile mechanical testing under isothermal conditions: monotonic loading 3.3.1 Evolution of axial stiffness with temperature

The evolution of the axial stiffness under different isothermal conditions is shown in Figure 8. The material initially loses 14% of its axial stiffness when testing temperature becomes higher than the glass transition temperature of the PPS matrix (about 98°C, see section 2.1). This decrease may be attributed to the decrease in the shear modulus of the ±45° plies during softening of the matrix (-67% at Tg [START_REF] Vieille | Influence of temperature on the behavior of carbon fiber fabrics reinforced PPS laminates[END_REF]). Above the glass transition temperature, the axial stiffness of the laminate decreases gradually up to 220°C. This phenomenon is associated with the decrease in shear strength of the PPS matrix and the fibre-matrix interface as the viscosity of the matrix increases.

There is a significant drop in axial stiffness for a temperature taken between 220 and 270°C. This can be linked to the melting of the matrix (Tm=280°C, see section 2.1) and the significant degradation of the fibermatrix interface. Indeed, according to the results of DSC [START_REF] Blond | Correlation between physical properties, microstructure and thermo-mechanical behavior of PPS-based composites processed by stamping[END_REF], the melting occurs between 220 and 295°C

(even if the shape of the melting peak suggests that most of the crystalline zones are destroyed around 280°C). After melting, the ±45° plies no longer play a mechanical role because their behaviour is driven by the matrix. Beyond that temperature, the material has a residual stiffness equal to 58% which comes solely from the contribution of 0/90° oriented plies. The latter initially bear 73% of the load (according to laminate theory), the decrease in their stiffness after melting can be estimated at 19%. These observations are in agreement with the results obtained by Mahieux el al. on a C/PPS laminate [START_REF] Mahieux | Property modeling across transition temperatures in polymers : application to thermoplastic systems[END_REF]. Thus, the stiffness reductions of constitutive elements considered individually do not explain the decrease with temperature observed in the C/PPS composite laminates. The PPS has a stiffness 100 times lower than that of the carbon fibres, which virtually retain their initial stiffness at this temperature level. The decrease in the stiffness of the 0/90° plies may result from other damage mechanisms such as the appearance of transverse cracks at the interface between strands and matrix. These mechanisms are suspected to take place mainly at the overlap areas of the longitudinal and transverse strands as a consequence of the stress concentration induced by longitudinal strands (mechanically loaded) on the transverse strands (Fig. 9), as well as metadelamination.

Above the melting temperature, the axial stiffness no longer changes, at least until 520°C (maximum temperature studied). When the ±45° plies have lost all rigidity after PPS melting, the axial stiffness of the material is mainly due to the stiffness carbon fibres parallel to the loading direction, which is virtually unchanged up to 1000-1400°C (depending on the fibres) in an inert atmosphere [START_REF] Sauder | Relation microstructure/propriétés à haute température dans les fibres et matrices de carbone[END_REF]. Then the longitudinal stiffness of the composite is assumed to be virtually unchanged up to these temperatures, unless fibres undergo oxidation. However these assumptions cannot be confirmed here because the behaviour of C/PPS could not be characterized for temperatures higher than 520°C due to the ignition of pyrolysis gases into the tube furnace.

Tensile response to failure under different isothermal conditions

Figure 10 shows the influence of different isothermal conditions on the tensile response of quasi-isotropic C/PPS laminates whose mechanical behavior is fibre-dominated. The ultimate tensile strength dramatically decreases (about 25% of its initial value -see Table 2) between room temperature to 470°C (lower than the onset decomposition temperature). At 220°C, the material exhibits a quasi-linear behaviour with a bending at around 180 MPa (referred to as knee point in the literature [START_REF] Osada | Initial micro fracture behavior of woven fabric composites[END_REF]) characteristic of matrix cracking, which occurs most frequently in overlapping areas of strands (Figure 9a). The catastrophic failure is driven by the 0° fibres whose elongation at failure is significantly lower than the PPS matrix one. The observation of specimens surfaces show a womb type failure surface resulting from the poor adhesion of the fibre-matrix interface, which is often observed in composites with thermoplastic matrices. This poor adhesion is promoted by the high temperatures and low viscosity of the matrix (Fig. 11).

At 270°C, the behaviour remains generally elastic-fragile, with a noticeable inflection around 25 MPa. This non-linearity is associated with the localized crushing of the strands in the overlap area, which first results in: (i) deformation localization in resin-rich areas and (ii), only then, in matrix cracking. On the front and rear faces of the specimens, the surface crack densities is more important than at 220°C (Fig. 9b). The ultimate stress is between 2 and 3 times lower than at 220°C and the elongation at failure is about 20% lower, which means that damage can also be explained by the higher density of inter-and intra-laminar cracks than at 220°C (Fig. 12).

For temperatures higher than Tm, significant changes in the mechanical response are observed. Before melting, the fibres of the same strand are held by the matrix and elongate evenly within the strand. After melting, the cohesion of the strand is no longer insured by the matrix, allowing significant transverse contraction of the fibres and a more significant elongation. Thus, for stresses higher than 70 MPa, the elongation of the 0° strands is accompanied by the generalization of the necking along the 0° direction.

This necking ultimately results in a generalized necking of the 90° oriented plies, which greatly increases the bending force at the crimp (where the warp strands undulate over the weft strands) and leads to the misalignment of the strands (Fig. 13a).

Beyond a certain elongation, the generalization of necking is such that the transverse strands are strongly crushed by the longitudinal strands. Such crushing leads to significant out-of-plane displacements resulting in surface bending effects and extensive delamination (Fig. 14). In ±45° plies, the fusion of the matrix is accompanied by a reorientation of the strands in the direction of solicitation. They then undergo a shear stress at the level of their overlapping areas. The space between the strands decreases until they enter the contact, causing the rotation to be blocked and their transverse contraction to begin [START_REF] Lomov | Full-field strain measurements in textile deformability studies[END_REF]. The transition between the two phenomena (reorientation and compaction) is characterized as follows by a "shear locking" angle. When the specimen is elongated, the compaction increases and results in the stiffening of rotation of ±45° plies leading to the increase in laminates axial stiffness.

For temperatures higher than Tm, the actual failure of the specimens could not be observed. Indeed, the necking of 0°strands extends along the 0° direction until it reaches the area of the specimen taken in the machine grips. Necking generalization is associated with an elongation of approximately 40% and a significant reduction (about 25%) in their initial diameter (Fig. 13b). Tensile tests suggest that both deformation and damage mechanisms significantly change in C/PPS laminates subjected to monotonic loading at temperatures higher than PPS matrix melting temperature. However, creep loadings are more representative of the critical service conditions met by mechanically loaded composite parts exposed to a fire scenario. From the tensile failure behaviour of C/PPS laminates investigated in this section, significant changes in the monotonic thermomechanical behaviors are observed at temperatures higher than Tm. In quasi-isotropic C/PPS laminates whose mechanical behaviour is driven by 0° plies, the rate-dependent behaviour is very limited at temperatures lower than Tm [START_REF] Albouy | Experimental and numerical investigations on the time-dependent behavior of woven-ply PPS thermoplastic laminates at temperatures higher than glass transition temperature[END_REF], but it is expected to be much more important at temperatures higher than Tm.

Tensile mechanical testing under isothermal conditions: creep loading

The evolution of time to failure as a function of tensile creep stress is shown in Figure 15. The time to failure corresponds to the time elapsed since the application of mechanical loading. The time to failure significantly decreases as creep stress increases. As a result, three specific failure mechanisms have been identified in C/PPS laminates under creep loading depending on the applied creep stress:

• For a creep stress higher than 0.25% & , the failure occurs even before the material reaches its melting temperature. On the one hand, the failure therefore results from the decrease in the mechanical strength of the ±45° plies (related to matrix softening and degradation of the fiber/matrix interface). On the other hand, it is associated with the appearance of transverse cracks and meta-delamination in 0/90° oriented plies (Fig. 9a and12a). The failure mechanism is similar to that observed in figure 11, i.e. a transverse break of the fibres at 0°.

• For intermediate creep stresses (0.11% & ≤ % ;;< ≤ 0.15% & ), failure occurs at the beginning of the creep test or in some cases shortly before the material has reaches 320°C. Thus, the failure can be associated with matrix melting and damage mechanisms similar to those observed during tensile tests at 320°C. The creep stress is then approximately equivalent to the one for which necking starts at 320°C (Fig. 10). The creep stress being higher than the stress levels where necking occurs in monotonous tensile tests, the deformation mechanisms that occur at that time (elongation of the longitudinal strands and crushing of transverse strands in 0/90° plies, reorientation of ±45° plies) will be initiated and developed within a few seconds, resulting in a deformation of the specimen without reaching ultimate failure, in agreement with the mechanisms observed during monotonous tension (Fig. 13b).

• For a stress lower than 60 MPa, there is an increase in the elongation of the specimen as temperature increases, but once 320°C is reached, the deformation no longer evolves. Indeed, despite matrix melting, the applied stress is too low to initiate necking.

Conclusion

In the literature, there are very few studies dealing with the mechanical behaviour of thermoplastic matrix composite materials beyond matrix melting temperature Tm. Through the results and discussion proposed in the present work, melting and thermal decomposition are instrumental to rule the tensile behavior of quasiisotropic C/PPS laminates. Between the ambient temperature and Tm, the laminates axial stiffness decreases with increasing temperature, mainly due to the loss of stiffness of the ±45°oriented plies and the degradation of the fibre-matrix interface. Beyond Tm, the axial stiffness no longer changes, as long as no oxidation takes place. Conversely, matrix melting significantly influences the damage mechanisms within the laminate. The bonding of carbon fibres within the strands is no longer ensured, and the 0° strands experience necking resulting in a much higher elongation than the one observed before matrix melting.

List of figure

10 Similar

 10 observations have already been made by Ma et al. in the study of the decomposition of C/PPS

Fig. 1 -Fig. 2 -Fig. 3 -Fig. 4 -Fig. 5 -Fig. 6 -Fig. 7 -Fig. 8 -Fig. 9 -Fig. 10 -Fig. 11 -Fig. 12 -Fig. 13 -Fig. 14 -Fig. 15 -

 123456789101112131415 Fig. 1 -Influence of isothermal heating on the mechanical property of PMC laminates (Adapted from [1]) Fig. 2 -Tensile specimens used for characterizing the mechanical behaviour of C/PPS laminates under homogeneous and isothermal temperature conditions Fig. 3 -TGA tests under O2: evolution of the residual mass and the decomposition rate (MLR) of plain PPS resin and C/PPS as a function of temperature Fig. 4 -Evolution of the decomposition rate under O2 as a function of the mass loss Fig. 5 -Thermal decomposition of the plain PPS resin and C/PPS composites: (a) Residual mass and decomposition rate (MLR) under air in as a function of temperature (b) Decomposition rate as a function of degree of decomposition Fig. 6 -Study of the thermal decomposition in C/PPS laminates subjected to isothermal conditions: Evolution of the residual mass (a) and the degree of decomposition (b) vs time Fig. 7 -Evolution of the porosities rate vs the degree of decomposition in C/PPS laminates subjected to isothermal conditions Fig. 8 -Evolution of the axial stiffness of quasi-isotropic C/PPS laminates under isothermal conditions Fig. 9 -C/PPS laminates front view observations, before failure at: (a) 220°C (b) 270°C Fig. 10 -Influence of temperature on the tensile response of quasi-isotropic C/PPS laminates Fig. 11 -Fractographic observations of tensile failure surfaces at 220°C Fig. 12 -Quasi-isotropic C/PPS laminates edge after failure: (a) 220°C -(b) 270°C Fig. 13 -(a) Disalignment of transverse strands coming along with the necking along the 0° direction (b) Generalization of necking and crushing of transverse strands (for T > Tm) Fig. 14 -Quasi-isotropic C/PPS laminates edge after failure at 470°C Fig. 15 -Creep test at 320°C: time to failure as a function of creep stress

Table 1 -

 1 Temperature at the onset of decomposition of plain PPS and C/PPS composites under different atmospheres

			RT	220°C	270°C	320°C	470°C
	(	)	514±8	286±1	131±38	68±3	80±6

Table 2 -

 2 Evolution of the ultimate tensile strength with temperature

This elongation comes along with other phenomena such as the reorientation of the ±45° plies and the crushing of transverse strands that are not observed before melting. Finally, for testing temperatures close to the onset decomposition temperature, porosities appear and grow within the laminates. The porosities rate is very well correlated with the degree of decomposition. The knowledge of porosities rate is utmost important as they are stress concentrators contributing to the degradation of the mechanical properties.