Hélène Le Ribeuz

Lucie To

Maria-Rosa Ghigna

Clémence Martin

Chandran Nagaraj

Elise Dreano

Catherine Rucker-Martin

Barbara Girerd

Jérôme Bouliguan

Christine Pechoux

Mélanie Lambert

Angèle Boet

Justin Issard

Olaf Mercier

Konrad Hoetzenecker

Boris Manoury

Frédéric Becq

Pierre- Régis Burgel

Charles-Henry Cottart

Andrea Olschewski

Isabelle Sermet-Gaudelus

Frédéric Perros

Marc Humbert

David Montani

Fabrice Antigny
email: fabrice.antigny@universite-paris-sacaly.fr

Pierre-Régis Burgel

-Cftr In Pah

Marie Hôpital

Le Lannelongue

France Plessis-Robinson

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension

Keywords: CFTR, pulmonary arterial hypertension, relaxation, hypoxia, monocrotaline

ERSpublications

CFTR expression is lost in the pulmonary arteries of human pulmonary arterial hypertension (PAH) patients and experimental PAH models. We found that pharmacological inhibition of CFTR increases vascular cell proliferation and reduces pulmonary artery relaxation, contributing to PAH pathogenesis.

INTRODUCTION

Pulmonary arterial hypertension (PAH) is a progressive and severe disease with an estimated prevalence of 15 to 50 per million people in the population [1]. When no causative factors are identified, PAH is termed idiopathic PAH (iPAH). However, it can also be heritable; induced by drugs or toxins; or associated with other conditions, such as connective tissue disease, congenital heart disease, portal hypertension, and HIV infection [2]. In the new pulmonary hypertension (PH) classification proposed during the 6 th PH World Symposium in 2018, PH is hemodynamically defined by a mean pulmonary artery (PA) pressure (mPAP) >20 mmHg and a pulmonary vascular resistance (PVR) >3 Wood units at rest [1]. PH is the consequence of an increase in pulmonary vascular resistance due to remodeling of distal PAs and arterioles (diameter <500 µm) that causes adaptive right ventricular (RV) hypertrophy and, when unchecked, right heart failure [3]. PA remodeling is characterized mainly by an increase in PA vasoconstriction, a reduction in PA relaxation and an imbalance between pulmonary vascular cell proliferation and apoptosis in favor of cell proliferation and apoptosis resistance [2]. Remodeling of ion channels in several families contributes to the pathogenesis of PAH, including Cl -channels [4]. Pulmonary arterial smooth muscle cells (PASMCs) have been described to exhibit various Cl -currents, including voltage-sensitive, volume-regulated, Ca 2+ -activated and cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl - currents [4]. The Ca 2+ -activated Cl -channel isoform TMEM16A was recently found to be upregulated in PASMCs from iPAH patients, contributing to vasoconstriction and remodeling of PAs in PAH.

Papp et al also demonstrated that CFTR mRNA levels are severely reduced in PAs isolated from iPAH patients and that pharmacological inhibition of TMEM16A may be a novel therapeutic approach to reduce PA remodeling in PAH [5]. The CFTR channel is a cyclic adenosine monophosphate (cAMP)dependent and ATP-gated Cl -channel expressed at the apical membrane in epithelial cells, and mutations in the CFTR gene are responsible for cystic fibrosis (CF) [6]. CFTR is expressed not only in epithelial cells but also in different cell types, including cardiac muscle, endothelial, tracheal smooth muscle and vascular smooth muscle cells [7].

Robert et al demonstrated that CFTR is also expressed in rat PAs and contributes to endotheliumindependent relaxation of PAs [8]. More recently, Totani et al. demonstrated that CFTR is functionally expressed in human pulmonary arterial endothelial cells (PAECs) [9]. PH is a recognized comorbidity associated with mortality in CF patients [START_REF] Fraser | Pulmonary hypertension and cardiac function in adult cystic fibrosis: role of hypoxemia[END_REF][START_REF] Wells | Pulmonary artery enlargement and cystic fibrosis pulmonary exacerbations: a cohort study[END_REF][START_REF] Selimovic | Growth factors and interleukin-6 across the lung circulation in pulmonary hypertension[END_REF]. The prevalence of PH in CF is largely unknown, but in some cohorts, such as the Florida CF cohort, approximately 60% of CF patients developed PH [START_REF] Ratjen | Pulmonary artery hypertension: an underrated disease manifestation in cystic fibrosis?[END_REF]. Wells et al. found that PA enlargement, measured by computed tomography, is present in approximately half of CF patients and is associated with a risk of pulmonary exacerbation [START_REF] Wells | Pulmonary artery enlargement and cystic fibrosis pulmonary exacerbations: a cohort study[END_REF].

However, the role of CFTR dysfunction in the remodeled pulmonary vascular bed has not yet been investigated. In the present study, we first investigated the expression of CFTR in patients with iPAH.

We also evaluated the role of CFTR in the development of PH using well-established PH rat models induced by monocrotaline (MCT) or chronic hypoxia (CH) exposure. Using organ bath studies, we measured the involvement of CFTR in PA relaxation in humans, pigs and rats. Furthermore, we analyzed the consequences of chronic long-term in vivo inhibition of CFTR in rats using a selective CFTR inhibitor (CFTR inh172 ) at the pulmonary vascular level. Finally, we histologically analyzed PAs from CF patients and F508delCftr homozygous rats. Our overall hypothesis is that loss of function of CFTR in PAH contributes to PAH pathogenesis.

MATERIALS AND METHODS

A detailed description of the Materials and Methods is presented in the Online Data Supplement.

RESULTS

Reduced expression of CFTR in PAs from PAH patients

We analyzed the localization of CFTR via immunohistochemistry of paraffin-embedded lung sections from control and iPAH patients (Figure 1). In both control and iPAH patients, CFTR staining labeled PAECs and PASMCs (red arrow), but the staining intensity was lower in pulmonary vascular cells from iPAH patients than in those from control patients (red arrow) (Figure 1i). CFTR staining was reduced within plexiform lesions, the characteristic morphological hallmarks of advanced PAH (Figure 1ii and iii), but the staining intensity was less affected in PAs from patients with PH associated with COPD (Group 3 PH) (Figure 1iv). CFTR staining confirmed that the protein localized at the apical membranes of airway epithelial cells in control iPAH patients without variations in fluorescence intensity (not shown). Next, we analyzed the expression of CFTR by immunofluorescence staining in frozen lung sections of control and iPAH patients. In PAs from controls, CFTR staining (red) was localized in both PAECs (blue arrow) and PASMCs (blue square) (Figure 2A). Conversely, CFTR staining seemed to be reduced in PAECs and PASMCs in iPAH PAs. Immunofluorescence staining for CFTR in bronchi demonstrated clear CFTR staining at the apical membranes of airway epithelial cells in controls and iPAH patients without any variations in fluorescence intensity (Supplemental Figure 1A). Coimmunostaining of CFTR and von Willebrand factor (VWF, an endothelial cell marker) confirmed the localization of CFTR in PAECs (Supplemental Figure 1B) and confirmed the reduction in CFTR expression in PAECs from iPAH patients (Supplemental Figure 1B).

As shown in Figure 2B, to examine CFTR at the ultrastructural level in PAECs and PASMCs, we analyzed the localization of CFTR using correlative electron microscopy (Figure 2B). As expected, in non-PAH PAs, CFTR was present in the luminal plasma membrane and in the cytosol of PAECs.

Quantification of CFTR expression in PAECs and PASMCs via gold particle density measurement confirmed that CFTR expression was lower in PAs from iPAH patients than in those from non-PAH patients (Figure 2C). To better quantify the expression of CFTR in PAs from iPAH patients, we measured the mRNA levels of CFTR. We found that CFTR mRNA expression was strongly reduced (by 75%) in iPAH PAs compared with non-iPAH PAs (Figure 2D).

Given the prevalence of heterozygous CFTR mutations in the human population, we analyzed the presence of the most frequent CFTR mutations (Supplemental Table 1) in all iPAH patients in the study. No CFTR mutations were found in the iPAH patients, confirming that the changes in CFTR expression observed in these patients were independent of CFTR mutations.

Reduced CFTR expression and CFTR activation-mediated PA relaxation in MCT-exposed rats

Via quantitative PCR, we found that MCT exposure for 2 and 3 weeks progressively reduced CFTR mRNA expression in PAs during the development of PH by 65% and 75%, respectively (Figure 3A), while it did not alter CFTR mRNA expression in bronchi in the same animals (Figure 3B) or in pulmonary veins (Supplemental Figure 2A). Assessment of the Fulton index confirmed the development of RV hypertrophy, indicating that PH induced by MCT exposure was present 2 weeks after exposure (Figure 3C). CFTR localization was examined via immunohistochemistry (Figure 3D) in PAECs in paraffin-embedded lung sections (red circles) and in PASMCs (red arrows) in lung sections from control and MCT-exposed rats (3 weeks post induction). We found reduced staining intensity in pulmonary vascular cells from rats with MCT-induced PH (Figure 3D).

Immunofluorescence experiments on frozen lung sections confirmed that CFTR was localized in PAECs and PASMCs in lung sections from control and MCT-exposed rats. We did not detect any CFTR staining (Supplemental Figure 2B) in the PAs of MCT-exposed rats.

Next, we investigated the consequences of pharmacological modulation (activation or inhibition) of CFTR on PA contractility. As previously shown by Robert et al, CFTR activation induces PA relaxation in rats [8]. Using isolated PAs from control rats, we first assessed the consequence of pharmacological CFTR inhibition with the selective CFTR inhibitor [START_REF] Ma | Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion [Internet[END_REF] CFTR inh172 (10 µmol/L) on the contractile response to different doses of KCl (ranging from 10 to 90 mmol/L). As shown in Figure 3E, the contractile response of PAs in response to increasing doses of KCl was unchanged in the presence of CFTR inh172 (Figure 3E left panel, unchanged EC50 between DMSO-and CFTR inh172treated PAs). However, the amplitude of the contractile response to 60 mM KCl was significantly higher in PAs incubated with CFTR inh172 than in PAs incubated with DMSO (Figure 3E right panel).

Using isolated PAs from MCT-treated rats (after 1, 2 and 3 weeks of MCT exposure), we found that the dose-response curve for KCl-induced contraction was progressively shifted to the left during the course of PH development (Supplemental Figure 3A). We also measured CFTR-dependent PA relaxation using increasing concentrations of the CFTR activator MPB91 [8]. PA rings were preconstricted with 1 µM U46619 (a thromboxane A2 mimetic). Increasing concentrations of MPB91 produced progressive PA relaxation under control conditions (black curve) (Figure 3F). This response was progressively attenuated in PAs from MCT-exposed rats (Figure 3F): the relaxation produced by 300 µmol/L MPB91 was 32%, 60% and 80% lower than that produced by the control treatment after 1, 2 and 3 weeks of MCT exposure, respectively.

We next used a second CFTR activator, CFTRact-A1 (CactA1) [START_REF] Namkung | Novel Amino-Carbonitrile-Pyrazole Identified in a[END_REF]. Similar to MPB91, C act A1 at increasing concentrations produced progressive PA relaxation under control conditions following preconstriction of PA rings with 1 µM U46619 (black curve) (Figure 4A). Importantly, this C act A1dependent relaxation was strongly reduced in the presence of 10 µmol/L or 30 µmol/L CFTR inh172 , suggesting that PA relaxation in response to C act A1 was a consequence of CFTR activation. As shown in Figure 4B, C act A1-induced relaxation was also strongly reduced in PAs from MCT-exposed rats (W3) (70% at 100 µmol/L C act A1, Figure 4B). Next, we investigated the role of CFTR in PH induced by CH in rats (3 weeks at 10% O 2 ). C act A1-dependent relaxation was strongly reduced (by more than 90% at 100 µmol/L C act A1) in isolated PAs from CH-exposed rats compared to PAs from normoxia (Nx)-exposed rats (Figure 4C). MPB91-induced relaxation was 45% lower in isolated PAs from CH rats than in PAs from Nx-exposed rats (Supplemental Figure 3B). As demonstrated by the Fulton index, a strong increase in RV hypertrophy indicated that CH-exposed rats developed PH (Figure 4D).

We found that Cftr mRNA expression was lower in PAs isolated from CH-exposed rats than in PAs isolated from Nx-exposed rats (Figure 4E); in addition, less CFTR staining was observed in PAs from CH-exposed rats (Figure 4F). Moreover, the results showed that increasing concentrations of C act A1 produced progressive relaxation of isolated PAs from healthy pigs (Supplemental Figure 3G).

Finally, increasing concentrations of VX770 (Ivacaftor), a CFTR potentiator approved to treat CF [START_REF] Ramsey | A CFTR Potentiator in Patients with Cystic Fibrosis and the G551D Mutation[END_REF][START_REF] Van Goor | Rescue of CF airway epithelial cell function in vitro by a CFTR potentiator, VX-770[END_REF], also produced progressive rat PA relaxation (black curve) (Supplemental Figure 3C). Importantly, this VX770-dependent PA relaxation was strongly reduced in PAs from MCT-exposed rats (W3) (by 70% at 200 µmol/L VX770, Supplemental Figure 3C).

Reduced CFTR activation-mediated relaxation in PAs from iPAH patients

Confirming the vasoconstrictive effect on PAs in iPAH patients, we found that the dose-response curve of KClinduced contraction was strongly shifted to the left for PAs from iPAH patients compared to PAs isolated from controls (Figure 5A); consistent with this finding, a smaller EC50 value was observed for PAs isolated from iPAH patients than for those isolated from control (Figure 5B).

Given the dose responses to CFTR activators previously obtained in rat and pig PAs (Figures 3 and4), we challenged human control and iPAH-isolated PAs with 100 µmol/L MPB91, VX770 and CactA1 (Figure 5C-F). We found that all three activators produced significant relaxation in control arteries (MPB91, 38%; VX770, 28%; CactA1, 19%) (Figure 5F). (Figure 5C-E). Nevertheless, as shown in Figure 5F, relaxation was severely reduced in PAs from iPAH patients (MPB91, 6.15%; VX770, 5.18%; CactA1, 3%). The same volume of DMSO (vehicle) had a minimal impact on human PA relaxation (Figure 5F). These results demonstrate that CFTR is functionally expressed in human control PAs, where it contributes to the regulation of PA tone. The results also indicated that CFTRmediated PA relaxation was strongly reduced in PAs from iPAH patients. As indicated in Supplemental Table 1, no CFTR mutations were found in iPAH patients.

Chronic in vivo inhibition of CFTR induced PH with exaggerated pulmonary vascular cell proliferation

To evaluate whether loss of function of CFTR can contribute to initiation of PH, we chronically inhibited CFTR in healthy rats using CFTR inh172 (2 mg/kg/d i.p.). After 2 weeks, CFTR inh172 -treated rats displayed significantly increased RV systolic pressure (RVSP) (Figure 6B), but the right ventricle was not hypertrophied (Fulton index, Figure 6A), and neither cardiac output (Figure 6C) nor systemic blood pressure (Figure 6D) was altered. In association with the increase in RVSP in CFTR inh172 -treated rats, we also measured a significant decrease in PA acceleration time (PAAT) by echocardiography (Supplemental Table 2). We did not measure any modifications in RV or left ventricular (LV) thickness, RV ejection time (RVET), cycle length (CL), heart rate (HR), PA velocity time integral (VTI Pa), or RV LV fractional shortening in CFTR inh172 -treated rats (Table 1).

To evaluate the consequence of CFTR inhibition on in situ pulmonary cell proliferation, we visualized proliferating lung cells by measuring the incorporation of 5-ethynyl-2'-deoxyuridine (EdU; pink nuclei and yellow arrow in Figure 6E) in CFTR inh172 -exposed rats. We detected very few EdU-positive cells in control lung parenchyma (DMSO-treated rats, Figure 6E left), whereas there were areas of intense pulmonary cell proliferation in CFTR inh172 -exposed rat lung parenchyma (Figure 6E right). The quantification results are shown in a bar graph in Figure 6F. CFTR inh172 -exposed rats exhibited increased numbers of lung proliferative cells, including PASMCs, as the numbers of α-smooth muscle actin (αSMA)-and EdU-positive cells were augmented (Figure 6F). As indicated in Figure 6G, greater accumulation of proliferating cells, as measured by immunostaining of Ki67 (a marker of cell proliferation), was observed in the media as well as in the endothelium in PAs from CFTR inh172 -exposed rats compared to PAs from vehicle-exposed rats (Figure 6H-I). However, the percentages of Ki67-positive nuclei in adventitia and bronchi did not differ between CFTR inh172 -exposed rats and vehicle-exposed rats (Figure 6J). Finally, Western blot analysis showed that P-AKT308, CD45 (a pan-leukocyte marker), serum albumin, VE-cadherin and transient receptor potential canonical channel 6 (TRPC6) levels were unchanged in the lungs of CFTR inh172 -exposed rats (Supplemental Figure 4). We also used Western blot analysis to evaluate the expression of CFTR protein in human PASMCs (hPASMCs) from control and iPAH patients. As illustrated in Figure 6K, CFTR protein (175 kDa, detected with an Alomone CFTR antibody) was present in control hPASMCs as well as hPASMCs cultured from iPAH patients. However, it was not present in three iPAH patients, and its levels were unchanged in one iPAH patient. The CFTR protein was strongly expressed in the positive control samples (human lung tissues) and absent in the negative control samples (A549 human adenocarcinomic alveolar basal epithelial cells), demonstrating the specificity of the CFTR bands. A549 cells are known to not express CFTR [START_REF] Hamai | Defective CFTR increases synthesis and mass of sphingolipids that modulate membrane composition and lipid signaling[END_REF][START_REF] Ren | An Optimised Human Cell Culture Model for Alveolar Epithelial Transport[END_REF]. The results indicated that the CFTR protein was weakly expressed in the majority of iPAH hPASMCs (Figure 6K). We obtained similar results using a second CFTR-specific antibody (175 kDa, CFTR antibody #596 from the CF Foundation) (Supplemental Figure 6). Therefore, we evaluated the role of CFTR only in hPASMC proliferation by pharmacologically inhibiting CFTR with 10 µmol/L CFTR inh172 for 24 hours. This treatment enhanced the proliferation of control hPASMCs (Figure 6L).

As shown in Supplemental Figure 7, we performed pathological assessment of the bronchial trees, livers and kidneys of CFTR inh172 -exposed rats. In CFTR inh172 -exposed rats, the large bronchi were patent and were delimited by normally structured walls that included collagen tissue, smooth muscle and cartilage rings. No bronchial inflammation could be detected in any specimen (Supplemental Figure 7i). In CFTR inh172 rats, the liver architecture was maintained, with regular distribution of portal spaces and centrilobular veins. Neither portal fibrosis nor inflammation was identified in any of the samples. Hepatocytes were distributed along thin trabeculae. No centrilobular inflammation was observed (Supplemental Figure 7ii). Finally, the structure of the renal cortex was maintained in all specimens, and glomeruli were well represented within the cortex without hyaline involution.

Glomerular inflammation, interstitial fibrosis and inflammation were absent. Renal tubules were lined by regular cuboidal/cylindrical epithelial cells, and tubule dilation was not observed. The renal medullae did not appear to be altered. Renal vessels were patent without parietal thickening. No thrombi were visible (Supplemental Figure 7 iii and iv).

CFTR dysfunction or inhibition was associated with PA remodeling

Lung distal vessel muscularization was assessed by immunostaining (αSMA = smooth muscle marker, VWF = endothelial cell marker) (Figure 7A). CFTR inh172 -treated rats displayed a significantly increased percentages of small vessel muscularization (Figure 7A and Supplemental Figure 5A). We also found that the percentages of nonmuscularized vessels (VWF-positive vessels) were significantly lower in the lungs of F508delCftr-mutated rats than in those of wild-type (WT) rats, while the percentages of muscularized vessels (αSMA-and VWF-positive vessels) were significantly higher in F508delCftr-mutated rats than in WT rats (Figure 7B, Supplemental Figure 5B). We further analyzed PA thickening in lung samples from 10 CF patients (Supplemental Material). We observed PA wall, bronchial artery, intimal and medial thickening without obstructive thrombotic lesions, while we did not observe any plexiform lesions or pulmonary vein remodeling. As expected, PAs from non-CF and non-PAH patients did not show any morphological alterations (Figure 7C). However, CF patients displayed substantial arterial remodeling in comparison with controls (Figure 7D), as determined by the total wall (media+intima) thickness (Figure 7C) (21.7%±0.7 vs 12.4%±0.4). Echocardiography revealed that 2 CF patients had PH with severe RV dysfunction, while 4 CF patients had moderate PH (not shown). The extent of PA wall thickening in CF patients was mild in comparison with the same parameter in iPAH patients (40%; data not shown). Importantly, CF patients have variable percent predicted forced expiratory volume in 1 second (ppFEV 1 ) values, which could influence PA remodeling. As presented in Figure 7E, we did not find any correlation between the wall thickness percentage and ppFEV 1 , suggesting that the PA remodeling observed in CF patients was partly independent of hypoxia/lung parenchymal destruction.

Chronic in vivo inhibition of CFTR induced exaggerated PH in MCT-exposed rats

As shown in Figure 6, rats with chronic CFTR inhibition displayed modest but significant increases in RVSP. Therefore, we exposed rats with chronic CFTR inhibition and vehicle-exposed rats to MCT (60 mg/kg). One week before MCT exposure, the animals were treated daily with vehicle or CFTR inh172 (2 mg/kg/d i.p.). Then, the animals were exposed to MCT (60 mg/kg) or saline and continuously exposed to vehicle or CFTR inh172 for two weeks (Figure 8A). In vivo CFTR inhibition increased the severity of MCT-induced PH, as demonstrated by elevated RVSP values in CFTR-inhibited rats (Figure 8B) with no modification of RV hypertrophy, total peripheral resistance (TPR) or mean carotid artery pressure (Figure 8C-E). We also found that RV end diastolic pressure (RV EDP) was higher in MCT-and CFTR inh172 -treated animals than in MCT-and DMSO-treated animals (Figure 8F). Finally, we found that the RV contractility index was lower in MCT-and CFTR inh172 -treated animals than in MCT-and DMSO-treated animals (Figure 8G). These results demonstrate that CFTR dysfunction predisposes rats to PH development under MCT exposure.

DISCUSSION

Using a combination of histology, myograph recording, molecular biology, and ex vivo and in vivo approaches, we obtained several major findings. First, CFTR is functionally expressed in the PAs of humans, rats and pigs, contributing to PA tone. Second, CFTR expression and CFTR-mediated PA relaxation are severely reduced in iPAH and decrease progressively during the development of experimental PH. Third, long-term inhibition of CFTR significantly increases RVSP and distal neomuscularization and exacerbates pulmonary vascular cell proliferation. Fourth, lungs from patients with severe CF and recently implemented F508delcftr rat models are characterized by PA remodeling.

Pulmonary vascular disease, particularly PH, is not well characterized in CF. PH has been estimated via right heart catheterization to occur in 26-63% of patients with CF [START_REF] Hayes | Pulmonary hypertension in cystic fibrosis with advanced lung disease[END_REF]. Similarly, in one study, RV catheterization demonstrated that among individuals in the Florida CF cohort who underwent lung transplantation, 63.2% of CF patients had PH [START_REF] Tonelli | Prevalence of pulmonary hypertension in end-stage cystic fibrosis and correlation with survival[END_REF]. In another CF cohort (78 adults with CF), PA enlargement (an indicator of PH) was associated with increases in pulmonary vascular hemodynamics (mPAP, pulmonary capillary wedge pressure (PCWP), PVR) in severe CF and with decreased survival [START_REF] Wells | Pulmonary artery enlargement and cystic fibrosis pulmonary exacerbations: a cohort study[END_REF]. The occurrence of PH in CF patients could reflect other factors, such as lung hyperinflammation, lung destruction or pulmonary vascular matrix alterations [START_REF] Wells | Pulmonary artery enlargement and cystic fibrosis pulmonary exacerbations: a cohort study[END_REF]; it is not necessarily only directly related to CFTR dysfunction in PAs. Here, we found that the PA wall thickness in CF patients (F508delCFTR) was not correlated with ppFEV 1 , suggesting that the PA remodeling observed in CF patients was partly independent of hypoxia/lung parenchymal destruction and was due mostly to CFTR mutation. Indeed, we demonstrated that F508delCftr rats developed pulmonary vessel neomuscularization without an abnormal lung phenotype, and CFTR-dependent Cl -secretion is impaired in the nasal airways of F508delcftr mutant rats [START_REF] Dreano | Characterization of two rat models of cystic fibrosis-KO and F508del CFTR-Generated by Crispr-Cas9[END_REF]. This feature is remarkable, as the concomitant study on the lungs of these animals did not show any CF-related alterations, such as inflammation or bronchial accumulation. We also excluded heterozygous CFTR mutations in our iPAH cohort by genotyping the CFTR gene in all patients included in the study. These results suggest that CFTR dysfunction could predispose patients to or facilitate pulmonary vascular remodeling independently of lung parenchymal destruction.

Exposure to CH has been found to downregulate CFTR in human lung epithelial cells [START_REF] Bartoszewska | miR-200b downregulates CFTR during hypoxia in human lung epithelial cells[END_REF]. The main molecule activated by hypoxia exposure is hypoxia inducible factor-1 alpha (HIF-1α), which regulates the expression of CFTR [START_REF] Zheng | Hypoxia inducible factor-1 (HIF-1)-mediated repression of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium[END_REF]. Chromatin immunoprecipitation has revealed a binding site for HIF-1α in the CFTR gene [START_REF] Zheng | Hypoxia inducible factor-1 (HIF-1)-mediated repression of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium[END_REF]. Consequently, HIF-1α overexpression leads to altered short circuit measurements and changes in transepithelial fluid movement [START_REF] Zheng | Hypoxia inducible factor-1 (HIF-1)-mediated repression of cystic fibrosis transmembrane conductance regulator (CFTR) in the intestinal epithelium[END_REF]. Overexpression of HIF-1α is a hallmark of PAH and experimental PH that contributes to PA remodeling [START_REF] Shimoda | Revisiting the role of hypoxia-inducible factors in pulmonary hypertension[END_REF]. Here, we found that CFTR expression was severely downregulated in PAs from patients with iPAH and an experimental rat model of PH, suggesting that these decreases in CFTR expression could partly be due to overexpression of HIF-1α.

In contrast with the present results, Cftr deficiency in mice has been found to diminish hypoxic vasoconstriction, and cftr -/-mice are protected against hypoxic PH [START_REF] Tabeling | CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction[END_REF]. Such discrepancy among humans, rats and mice has also been observed for the role of the K + channel KCNK3 in the development of PAH. There is strong evidence that KCNK3 deficiency contributes to the development of PH in humans [START_REF] Lr | Implication of Potassium Channels in the Pathophysiology of Pulmonary Arterial Hypertension [Internet[END_REF] and rats [START_REF] Lambert | Characterization of Kcnk3-Mutated Rat, a Novel Model of Pulmonary Hypertension[END_REF]. kcnk3 -/-mice are protected from PH induced by CH exposure [START_REF] Kitagawa | Hemodynamic and Pathologic Characterization of the TASK-1-/-Mouse Does Not Demonstrate Pulmonary Hypertension[END_REF],

indicating that the mouse model does not fully recapitulate the pathogenesis of PH in humans, particularly concerning the role of ion channels.

Robert et al. previously demonstrated that CFTR activation mediates relaxation of PA and aorta

relaxation in a dose-dependent manner [8]. CFTR activation-mediated relaxation (of PAs or the aorta) is independent of the endothelium [8,[START_REF] Robert | Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cltransport of mouse aortic smooth muscle cells: CFTR chloride channels in smooth muscle[END_REF]. Aorta relaxation induced by vasoactive intestinal peptide (VIP) or other cAMP agonists is reduced in aortas from cftr -/-mice, indicating that CFTR could be physiologically activated by cAMP agonists in these vessels [START_REF] Robert | Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cltransport of mouse aortic smooth muscle cells: CFTR chloride channels in smooth muscle[END_REF]. Indeed, in epithelial cells, physiological activation of CFTR requires phosphorylation of the regulatory R domain by protein kinase A [START_REF] Becq | Characterization of cAMP dependent CFTR-chloride channels in human tracheal gland cells[END_REF] and ATP hydrolysis by the nucleotide-binding domains [START_REF] Hanrahan | Revisiting Cystic Fibrosis Transmembrane Conductance Regulator Structure and Function[END_REF]. In aortas from CFTR-F508del pigs, CFTR mutation decreases aortic responsiveness by affecting smooth muscle Ca 2+ handling [START_REF] Guo | Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets[END_REF]. In human airways, TRPC6 is an essential component of the receptor-activated Ca 2+ influx machinery in both smooth muscle and epithelial cells, linking Ca 2+ signaling to CFTR pathobiology [START_REF] Antigny | Transient receptor potential canonical channel 6 links Ca2+ mishandling to cystic fibrosis transmembrane conductance regulator channel dysfunction in cystic fibrosis[END_REF] In mice, cftr deficiency is associated with increased contraction of the aorta, suggesting that loss or dysfunction of CFTR alters the physiological regulation of vascular tone. In the present study, we found that CFTR was downregulated in iPAH patients as well as in two experimental models of PH. This downregulation was correlated with a severe reduction in CFTR-dependent PA relaxation in a model associated with a predisposition to PA vasoconstriction. To explain the role played by CFTR in the regulation of PA tone, we postulate that channel activation is associated with relaxation [8,[START_REF] Robert | Disruption of CFTR chloride channel alters mechanical properties and cAMP-dependent Cltransport of mouse aortic smooth muscle cells: CFTR chloride channels in smooth muscle[END_REF].

Alternatively, CFTR may regulate PA tone independently of its role as a Cl -channel via other mechanisms, including modification of lipid trafficking and Ca 2+ handling in smooth muscle cells [START_REF] Tabeling | CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction[END_REF][START_REF] Guo | Genotype-specific alterations in vascular smooth muscle cell function in cystic fibrosis piglets[END_REF][START_REF] Michoud | Role of the cystic fibrosis transmembrane conductance channel in human airway smooth muscle[END_REF]. In addition to its channel function, the CFTR protein can interact with and regulate a considerable number of proteins because it has a PDZ binding domain in its C-terminal domain [START_REF] Boucherot | Role of CFTR's PDZ1-binding domain, NBF1 and Clconductance in inhibition of epithelial Na+ channels in Xenopus oocytes[END_REF].

Studies have shown that CFTR can interact with NHERF1 (an EB + /H + regulatory factor), ezrinradixin-moesin-binding phosphoprotein 50 (EBP50) and CFTR accessory protein 70 (CAP 70) [START_REF] Wang | Hsp90 cochaperone Aha1 downregulation rescues misfolding of CFTR in cystic fibrosis[END_REF].

CFTR can also interact with and regulate other ion channels, such as epithelial sodium channel (ENaC) [START_REF] Stutts | CFTR as a cAMPdependent regulator of sodium channels[END_REF], TRPC6 [START_REF] Tabeling | CFTR and sphingolipids mediate hypoxic pulmonary vasoconstriction[END_REF][START_REF] Antigny | Transient receptor potential canonical channel 6 links Ca2+ mishandling to cystic fibrosis transmembrane conductance regulator channel dysfunction in cystic fibrosis[END_REF], and renal outer medullary potassium channel 2 (ROMK2) [START_REF] Mcnicholas | A functional CFTR-NBF1 is required for ROMK2-CFTR interaction[END_REF].

In the present study, we found that in vivo CFTR inhibition accelerated the development of PH after MCT exposure in association with increased RV EDP and reduced RV contractility, indicating that CFTR inhibition facilitates RV dysfunction in the context of MCT exposure. Several experimental and clinical studies have reported the occurrence of myocardial dysfunction in CF patients, which affects systolic and diastolic heart function [START_REF] Labombarda | Heart involvement in cystic fibrosis: A specific cystic fibrosis-related myocardial changes?[END_REF]. CF patients may have poorer cardiac and peripheral hemodynamic function than healthy subjects [START_REF] Bisch | Cystic Fibrosis Transmembrane Conductance Regulator Genotype, Not Circulating Catecholamines, Influences Cardiovascular Function in Patients with Cystic Fibrosis[END_REF]. Pallin et al demonstrated that in CF, severity of genotype is associated with cardiac impairment [START_REF] Pallin | Subclinical Left Ventricular Dysfunction is Influenced by Genotype Severity in Patients with Cystic Fibrosis[END_REF]. Via echocardiography, we did not detect alterations in RV or LV function in rats with CFTR inhibition. In a zebrafish model, CFTR inhibition with CFTR inh172 has been found to alter the development of the heart in the blastula stage. Moreover, loss of CFTR impairs cardiac development, resulting in atrial dilation, pericardial edema, and decreased HR [START_REF] Liu | CFTR deficiency causes cardiac dysplasia during zebrafish embryogenesis and is associated with dilated cardiomyopathy[END_REF]. In cftr -/-mice, the lack of CFTR leads to increased myocardial contractility at baseline, which may trigger problematic myocardial remodeling in CF patients independent of lung diseases [START_REF] Jiang | The impact of Cystic Fibrosis Transmembrane Regulator Disruption on cardiac function and stress response[END_REF].

In MCF-7 breast cancer cells, CFTR controls cellular quiescence. Indeed, CFTR inhibition downregulates the Wnt and AURKA signaling pathways and upregulates the p53 signaling pathway, leading to an increase in cell proliferation [START_REF] Li | The chloride channel cystic fibrosis transmembrane conductance regulator (CFTR) controls cellular quiescence by hyperpolarizing the cell membrane during diapause in the crustacean Artemia[END_REF]. In intestinal cells, knockdown of CFTR enhances cell proliferation via inhibition of the hedgehog pathway [START_REF] Liu | CFTR interacts with Hsp90 and regulates the phosphorylation of AKT and ERK1/2 in colorectal cancer cells[END_REF]. In addition, CFTR activation suppresses glioblastoma cell proliferation [START_REF] Zhong | CFTR activation suppresses glioblastoma cell proliferation, migration and invasion[END_REF]. Consistent with the cell proliferative phenotype, CF is associated with an elevated predisposition to cancer [START_REF] Amaral | What Role Does CFTR Play in Development, Differentiation, Regeneration and Cancer?[END_REF] and is not restricted to the digestive tract [START_REF] Maisonneuve | Cancer Risk in Cystic Fibrosis: A 20-Year Nationwide Study From the United States[END_REF]. Many studies have reported that CFTR is downregulated in several cancer types and that CFTR dysfunction promotes the development of cancer in vitro and in vivo, suggesting that CFTR acts as a tumor suppressor protein [START_REF] Amaral | What Role Does CFTR Play in Development, Differentiation, Regeneration and Cancer?[END_REF]. Moreover, CFTR inhibition with CFTR inh172 increases endothelial permeability in human PAECs by reducing VE-cadherin and p120 catenin membrane expression [9]. Here, we found that CFTR inhibition (CFTR inh172 ) enhanced the proliferation rate of control hPASMCs, suggesting that reduced expression of CFTR in PAH PAs also favors PA remodeling. We also found that CFTR protein expression in hPASMCs from individuals with iPAH remained at high levels in some iPAH patients, suggesting that a pharmacological strategy activating CFTR might be of interest for patients with PAH who retain residual levels of CFTR expression, possibly within the frame of personalized therapy.

In conclusion, we provide evidence that the CFTR channel contributes to PA tone in humans, pigs and rats; that PA CFTR expression is reduced in iPAH and in 2 different experimental models of PAH; and that CFTR inhibition increases vascular cell proliferation. Altogether, these alterations may contribute to the development of PH. The data were analyzed using one-way ANOVA followed by Tukey's post hoc test.

SOURCES OF FUNDING

F

  .A. receives funding from the National Funding Agency for Research: ANR-18-CE14-0023. F.A. also receives funding from the Fondation du Souffle et Fonds de Dotation Recherche en Santé Respiratoire, from the Fondation Lefoulon-Delalande and from the Fondation Legs Poix. F.P. is supported by the Legs Poix, Chancellerie des Universités de Paris. M.L. is supported by the Therapeutic Innovation Doctoral School (ED569).

Figure 1 :

 1 Figure 1: Expression of CFTR protein in PAECs and PASMCs in control and iPAH patients.

Figure 2 :

 2 Figure 2: CFTR expression is reduced in iPAH patients. (A) Localization of CFTR by correlative light and electron microscopy in PAs from non-PAH and iPAH patient lungs after CFTR labeling. PAECs and PASMCs exhibited gold particles (white arrows) in the cytosol and small amounts at the plasma membrane. (B-C) Quantification of CFTR expression in PAECs (upper panel) and PASMCs (lower panel) from non-PAH and iPAH patients via gold particle density measurement in PAECs and PASMCs (3 control and 3 iPAH). Scale, 1 μm or 200 nm. EC, endothelial cell; Col, collagen; E, elastic lamina; N, nucleus; PAH, pulmonary arterial hypertension; and PASMC, pulmonary arterial smooth muscle cell. (D) mRNA expression levels of CFTR in PAs isolated from non-PAH patients and patients with iPAH (n=8 for both non-PAH patients and patients with iPAH). *p< 0.05 vs.control. The experimental data were analyzed using the Mann-Whitney test.

Figure 3 :

 3 Figure 3: CFTR expression and relaxation of PAs evoked by CFTR activation are reduced in MCT-

Figure 4 :

 4 Figure 4: CFTR activation by C act A1 induces concentration-dependent relaxation of rat and pig PAs. (A) Relaxant response to increasing concentrations of the CFTR activator C act A1 (1 µmol/L to 200 µmol/L) in rat PA segments precontracted with U46619 (1 µM) in the presence of vehicle (DMSO) or CFTR inh172 used at 10 µmol/L or 30 µmol/L. (B) Left panel, relaxant response to increasing concentrations of C act -A1 (C act A1, 1 µmol/L to 200 µmol/L) in control and MCT-exposed rats (3 weeks). Right panel, corresponding graph showing the percent relaxation in response to C act A1 at 100 µmol/L. (C) Left panel relaxant response to increasing concentrations of C act A1 (C act A1, 1 µmol/L to 200 µmol/L) in PA segments isolated from rats exposed to Nx or CH (3 weeks). Right panel, corresponding graph showing the percent relaxation in response to 100 µmol/L C act A1. (D) Corresponding plot showing the Fulton index. (E) mRNA expression levels of Cftr in PAs isolated from controls and CH-exposed rats (at week 3) (n=4-5 rats). (F) CFTR immunostaining in paraffinembedded lung sections from control and MCT-PH rats: PAEC and PASMC staining is visible. Scale, 50 μm. ns: nonsignificant. *p< 0.05 **p<0.01 vs. control. The data were analyzed using the Mann-Whitney test.

Figure 5 :

 5 Figure 5: CFTR is functionally expressed in human control PAs. (A) Concentration-response curve (normalized to K100) established by applying increasing concentrations of K + to PA rings isolated from human control patients and patients with iPAH. (B) EC50 quantification. In human control and iPAH PA rings precontracted with U46619 (1 µM), the CFTR activator MPB91 (100 µmol/L) (C), the CFTR activator C act A1 (100 µmol/L) (D) and the CFTR potentiator VX770 (100 µmol/L) (E) separately produced PA relaxation. (F) Corresponding graph showing the percent relaxation in response to these CFTR activators 10 min after CFTR activation (n=7 controls vs 6 iPAH samples).

Figure 6 :

 6 Figure 6: In vivo inhibition of CFTR-induced early hemodynamic signs of PH and exaggerated

Figure 7 :

 7 Figure 7: CFTR dysfunction or inhibition promotes PA remodeling. (A) Immunofluorescence labeling and confocal imaging of lungs from DMSO-and CFTR inh172-treated rats stained for α-SMA (green) and VWF (red). Right panel, percentage of muscularized vessels (100 vessels per rat, n=7 rats) as measured by immunostaining against αSMA and VWF. (B) Immunofluorescence labeling and confocal imaging of lungs from WT and homozygous F508delCftr rats stained for α-SMA (green) and vWF (red). Right panel, percentage of muscularized vessels (100 vessels per rat, n=7 rats) as measured by immunostaining against αSMA and VWF. (C) PAs from CF patients showing a thickening of the wall in connection with hypertrophy of smooth muscle cells (i) as well as irregular remodeling of the wall (ii). CF patients also presented with thickened bronchial arteries with increased thickness of the intima and the media (iii) without obstructive thrombotic lesions. Non-CF and non-PH pulmonary arteries without morphological alterations are also shown (iv). (D) Quantification of the PA wall thickness (media+intima) (9 non-CF and control patients (control), 10 CF patients). (E) Correlation

Figure 8 :

 8 Figure 8: In vivo chronic inhibition of CFTR predisposes rats to develop PH under MCT exposure.

Supplemental Material

Involvement of CFTR in the pathogenesis of pulmonary arterial hypertension

Hélène Le Ribeuz 1,2,3 , Lucie To 1,2,3 , Maria-Rosa Ghigna 1,2,3 , Clémence Martin 4,5 , Chandran Nagaraj 6 , Elise Dreano 7 Catherine Rucker-Martin 1,2,3 , Barbara Girerd 1,2,3 , Jérôme Bouliguan 8 , Christine Pechoux 9 , Mélanie Lambert 1,2,3 , Angèle Boet 1,2,3 , Justin Issard 1,2,3 , Olaf Mercier 1,2,3 , Konrad Hoetzenecker 10 , Boris Manoury 11 , Frédéric Becq 12 , Pierre-Régis Burgel 4, 5 , Charles-Henry Cottart 7 , Andrea Olschewski 5,13 Isabelle Sermet-Gaudelus 7 , Frédéric Perros 1,2,3 , Marc Humbert 1,2,3 , David Montani 1,2,3 and Fabrice Antigny 1,2,3 Le Ribeuz et al. -CFTR in PAH Affiliations 1 Université Paris-Saclay, Faculté de Médecine, Le Kremlin-Bicêtre, France (H. L.-R., L. T., M.-R. G., C. R.-M., B. G., J. B., M. L., A. B., O. M., F. P., D. M., M. H., F. A.) 2 INSERM UMR_S 999 « Hypertension pulmonaire: Physiopathologie et Innovation Thérapeutique », Hôpital Marie Lannelongue, Le Plessis-Robinson, France (H. L.-R., L. T., M.-R. G., C. R.-M., B. G., M. L., A. B., O. M., F. P., D. M., M. H., F. A.) 3 Assistance Publique -Hôpitaux de Paris (AP-HP), Service de Pneumologie et Soins Intensifs Respiratoires, Centre de Référence de l'Hypertension Pulmonaire, Hôpital Bicêtre, Le Kremlin-Bicêtre, France. (H. L.-R., L. T., M.-R. G., C. R.-M., B. G., M. L., A. B., O. M., F. P., D. M., M. H., F. A.) 4 Assistance Publique -Hôpitaux de Paris (AP-HP), Dept of Respiratory Medicine, Centre de Référence Maladie Rare Mucoviscidose, ERN-Lung, Cochin Hospital, 27 rue du Faubourg Saint Jacques, 75014 Paris, France (C. M., P.-R. B.) 5 Inserm U1016, Institut Cochin, Université de Paris, Paris, France (C. M., P.-R. B.) 6 Ludwig Boltzmann Institute for Lung Vascular Research, Graz, Austria (C. N., A. O.) 7 Inserm U1151 -CNRS UMR 8253 -Institut Necker Enfants Malades, Centre Maladie Rare Mucoviscidose, ERN Lung, Université de Paris, Paris, France (E. D., C.-H. C., I. S.-G.) 8 Laboratoire de Génétique Moléculaire, Pharmacogénétique et Hormonologie, Assistance Publique-Hôpitaux de Paris (AP-HP), Hôpital Bicêtre, Le Kremlin-Bicêtre, France; INSERM UMR-1185, Université Paris Saclay, Faculté de Médecine, Le Kremlin Bicêtre, France (J. B). 9 GABI, INRA, AgroParisTech, Université Paris-Saclay, 78350 Jouy-en-Josas, France (C. P.) 10 Department of Thoracic Surgery, Medical University of Vienna, Vienna, Austria (K.H.) 11 Signalisation et Physiopathologie Cardiovasculaire -UMR_S 1180, Univ. Paris-Sud, INSERM, Université Paris-Saclay, Châtenay-Malabry, France (B. M.) 12 Laboratoire Signalisation et Transports Ioniques Membranaires, Université de Poitiers, 86073 Poitiers Cedex 9, France (F. B.) 13 Experimental Anesthesiology, Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Graz, Austria (A. O.). Corresponding author: Fabrice Antigny, INSERM UMR_S 999, Hôpital Marie Lannelongue, 133, Avenue de la Résistance, F-92350 Le Plessis Robinson, France. Fax: (33) 1 40 94 25 22, Tel.: (33) 1 40 94 22 99, e-mail: fabrice.antigny@universite-paris-sacaly.fr

CONFLICT OF INTEREST/DISCLOSURES

M.H. has relationships with drug companies, including Actelion, Bayer, GSK, Novartis, and Pfizer. In addition to being an investigator in trials involving these companies, M.H. has other relationships with consultancy services and is a member of scientific advisory boards. The other authors have no conflicts of interest.

Online Methods

Material and methods

Chemicals-Monocrotaline (MCT), and Sildenafil were obtained from Sigma. U46619 were obtained from R&D Systems. CFTRAct-A1 (C act A1) was obtained from Calbiochem CFTR inh172 was obtained from Tebu-Bio. MPB-91 was synthesized as previously described [START_REF] Hayes | Pulmonary hypertension in cystic fibrosis with advanced lung disease[END_REF].

Animals-

The animal facility is licensed by the French Ministry of Agriculture (agreement N° C92-019-01). This study was approved by the Committee on the Ethics of Animal Experiments (CEEA26 CAP Sud). The animal experiments were performed conforming to the guidelines from Directive 2010/63/EU on 22 September 2010 of the European Parliament on the protection of animals used for scientific purposes and complied with the French institution's guidelines for animal care and handling.

Male Wistar rats (4 weeks old) were used in four experimental protocols:

1. PH was induced by a single MCT injection (60 mg/kg, s.c.). MCT was dissolved in 1 N HCl and neutralized with 1 N NaOH. Control animals received the same volume of saline solution.

2. For chronic hypoxia (CH) model, Wistars rats were placed 3 weeks in hypoxia (10 % O2) in normobaric condition. Normaxia Wistar rats were placed in the same room during 3 weeks.

3. 5. Daily exposure (2 mg/kg, i.p.) during one month to the selective CFTR blocker CFTR inh172 . CFTR inh172 compound was dissolved in DMSO. Control animals received the same volume of DMSO.

Patients-Human lung specimens were obtained at the time of lung transplantation in 7 patients with PAH and at the time of lobectomy or pneumonectomy for localized lung cancer from 10 control subjects. PAH patients underwent genetic counseling and signed written informed consent for genetic analysis. No mutation was identified in 7 patients. These 7 patients were included in the GWAS analysis of Ma and collaborators [1], and were not carriers of KCNK3, BMPR2 or other mutation.

These patients correspond to idiopathic PAH (iPAH). In the lung specimens from control subjects, pulmonary arteries were studied at distance from tumor areas. Human PASMC and PAEC culture-Human lung specimens were obtained at the time of a lobectomy or pneumonectomy for localized lung cancer from control subjects. Pulmonary arteries were studied at a distance from tumor areas. Human PAECs and PASMCs were cultured as previously described [2] and were used for the study between passages 3 and 5. PASMC or PAEC were isolated from pulmonary arteries obtained during lobectomy or pneumonectomy of localized lung cancer from control subjects. Pulmonary arteries were excised at distance from tumor areas. Patients studied were part of a program approved by our institutional Ethics Committee, and had given written informed consent (ID RCB: 2018-A01252-53, approved on June 18, 2006) CF patients-Human lung tissues containing non-cartilaginous airways were obtained at lung transplantation from 10 nonsmoking CF adults. Seven patients were homozygous for the Phe508del-CFTR mutation and the other three were Phe508del/minimal function (G542X, Q890X or N1303K).

Pre-lung-transplant-percent predicted forced expiratory volume in 1 second (ppFEV 1 ranged from 14 % to 29%, and all patients were under long-term oxygen therapy for chronic hypoxemia. All patients were chronically infected with P. aeruginosa. Lung tissue samples obtained at transplantation were fixed in 10% neutral buffered formalin by inflation-immersion and embedded in paraffin. To reduce the potential sampling bias related to irregular distribution of morphological abnormalities in CF patients, tissues were chosen randomly among two to four blocks (depending on tissue availability) from each patient. The study obtained agreement of the local ethics committee (CPP Ile de France II; number 1072) for the use of samples of human origin.

F508del-Cftr rats were generated using CRISPR-Cas9 technology and genome editing techniques for the introduction into the genome of single-stranded oligo donor sequences by microinjection into rat zygotes, as described [3]. The target sites were based on exon 12; donor DNA generated a codon deletion at F508. F508delCftr heterozygous rats were inbred to generate F508del homozygous rats and WT littermates. Laxative (PEG 3350 -KleanPrep®) was added to the drinking water. To reduce mortality, F508delCftr rats were fed with a liquid diet composed of DietGel 31M and Boost (Clear H2O®, Ssniff-Spezialdiäten GmbH; Soest, Germany) as their respective WT littermates. Then, chow mixed with laxative-containing water (DietGel + laxative) was progressively introduced in the diet.

Both male (n= 5) and female (n= 9) rats were used for histological study. All animal care performed in the Paris facility were approved by the Animal Experimentation Ethics Committee of Paris Descartes University.

Control pig-We used seven Landrace SHAM male piglets for other surgery protocols to minimize the number of scarified animal. Sham pigs just had left thoracotomy. Pulmonary artery from SHAM animal used were isolated and then used for organ bath study.

CFTR genotyping

Screening of frequent CFTR mutations (50 mutations and poly-T repeats polymorphism intron 8) was performed by fluorescent ARMS (Amplification Refractory Mutation System) PCR using the kit CF-EU2v1 (Elucigene®, Manchester, UK) following the manufacturer recommendations. Amplified sequences were separated by capillary electrophoresis using an ABI PRISM 3130XL Genetic Analyzer (Applied BiosystemsTM, Foster City, CA). Data analysis and interpretation were performed with the version 5 of GeneMapper software (Applied BiosystemsTM, Foster City, CA). A sensibility > 87 % is estimated when this test is used for CFTR mutations screening in French Population [4].

Hemodynamic measurements, and collection of tissues-Rats were placed under general anesthesia

and spontaneous breathing with an Isoflurane Rodent Anesthesia System (Minerve, Esternay, France) (maintenance: isoflurane 2% at room air). Hemodynamic measurements such as right ventricular systolic pressure (RVSP; mmHg), cardiac output (CO; mL/min) and mean carotid pressure (mCP; mmHg) were performed prior to tissue collection, as previously described [5]. Hemodynamics values were analyzed using LabChart software. LabChart allow us to calculate several ventricular wave form parameters including; End diastolic pressure (EDP), Contractility index which correspond to Max dP/dt divided by the pressure (P) at the time of Max dP/dt. For Fulton's index of right ventricular (RV) hypertrophy, the ratio of RV weight to left ventricular (LV) plus septal (S) weight (RV/LV+S) was calculated.

Echocardiographic measurement-Evaluation by Trans-thoracic echocardiography (TTE) was

performed with a digital ultrasound system (Vivid E9, GE Healthcare) by using a high-frequency phased array transducer (12 S-D 4-12MHz, GE Healthcare). Echocardiographic evaluation procedure was performed under general anesthesia and spontaneous breathing with an Isoflurane Rodent Anesthesia System (Minerve, Esternay, France) (maintenance: isoflurane 2% at room air). Rats were shaved and body temperature was controlled during experiments. All analyses were performed in a blinded fashion way: experimental conditions were unknown by the operator during TTE examination and data interpretation. Measurement of pulmonary artery acceleration time (PAAT), heart rate (HR), pulmonary artery velocity time integral (VTI), RV ejection time (RVET) velocity time integral (VTI), were performed as previously described [6,7]. In the 4-cavity view performed in the subcostal way, we measured RV and LV thickness, RV or LV end diastolic diameter (RV EDd, LV EDd, respectively) and systolic diameter (RV EDs, LV EDd). Western blot analyses-Total proteins from rat lung-tissue samples were prepared as described previously [2]. All antibodies used are listed in supplemental table 3.

Reverse transcription quantitative PCR (RT-qPCR) -

For CFTR protein: Human PASMC were grown and harvested using RIPA buffer (Sigma) supplemented with Protease and Phosphatase inhibitor cocktail (Roche) with EDTA. The cell lysate was sonicated and centrifuged for 10 min at 5000 g. Protein concentration of the supernatant was determined using the Pierce BCA Protein Assay Kit (Thermo Scientific), then 5 μg protein from each sample was mixed with 10x Laemmli sample buffer and ran on 8% SDS-PAGE. Followed by electrotransfer to a PVDF membrane (GE Healthcare, Little Chalfont, UK). After blocking the membrane with 5 % non-fat milk in TBS-T (5 mM Tris-Cl, 150 mM NaCl, 0.1% Tween 20, pH 7.5), the membrane was labeled for CFTR (Alomone or CF foundation) , followed by incubation with horseradish peroxidase conjugated secondary antibody. Final detection of the proteins was performed using an ECL Plus Kit (GE Healthcare, Little Chalfont, UK) and a ChemiDocTM Touch Imaging System hPASMCs proliferation measurement-To evaluate cell proliferation, we measured BrDu incorporation to identify cell undergoing DNA replication, using a DELFIA cell proliferation kit (AD0200, PerkinElmer). Experiments were performed according to the kit recommendations. To evaluate the proliferation state of the cells, different treatment were used: CFTR inhibitor (CFTR inh172 at 10µmol/L) or DMSO. To stimulate the cell proliferation, the medium contained 10 percent of serum.

Isometric tension measurement-Contractile responses of rat PA was measured using a small-vessel wire myograph (Danish Myotechnology, Aarhus, Denmark), as reported previously [8]. Briefly, firstorder intrapulmonary arterial segments (length: 1.4-2.0 mm) were mounted on the jaws of the myograph using a 40 μm diameter tungsten wire. Data acquisition was performed using PowerLab 8/30 and LabChart® Pro v7.1 software (AD instrument).

Human and Pig PA were mounted on an emkaBATH4 modular tissue bath system (EMKA Technologie, Paris, France) coupled to IOX software (EMKA Technologie). As determined in preliminary experiments, human PA were set at optimal length by equilibration against a passive load of 0.6g. Vessels were bathed in Krebs solution containing (in mmol/L): 119 NaCl, 4.7 KCl, 2.5 CaCl 2 , 1.17 MgSO 4 , 1.18 KH 2 PO 4 , 25 NaHCO 3 , and 11 glucose at 37°C, and continuously aerated with a mixture of CO 2 /O 2 (5%/95%). After stretching the PA at 0.9 x C 30mmHg , basal tone was measured as the drop of tension resulting from the replacement of Krebs bath solution with a Ca 2+ -free solution. After adding Krebs solution again, vessels were contracted with a solution containing 100 mol/L K + (K100). Once a plateau was reached, the vessels were washed with Krebs solution for 30 min. DMSO or CFTRAct-A1 (C act -A1) (100µmol/L) were used as pretreatments before the dose response to KCl (10-90 mmol/L) whereas MPB91 (10µmol/L to 300µmol/L) and C act -A1 (1µmol/L to 200µmol/L) were used to induce relaxation after vessel contraction was obtained using thromboxane A2 analogue U46619 (1µmol/L).

All modified Krebs solutions prepared with various KCl concentrations (K10 to K100) had equimolar substitution of NaCl in order to keep constant osmolarity and [Cl -] compared to normal Krebs solution.

The contractile responses were expressed relative to the response to K100 challenge. The relaxation responses were expressed as the percentage of the precontractile response obtained with U46619.

Assessment of pulmonary arterial remodeling-

The wall thickness was assessed on pulmonary arteries <500µm and >100µm. Measurements were performed on 4µm thick sections of lung tissue stained with hematoxylin and eosin. Sections were examined on light microscope at 200 x magnification. The percentage of wall thickness of muscularized arteries [(2xmedial wall thickness /external diameter) x100] was performed.

Immunofluorescence staining-Frozen lungs were prepared as described previously [9]. All antibodies used are listed in supplemental table 4.

Immunohistochemistry -Paraffin-embedded 5µm thick sections of lung samples were mounted on SuperFrostPlus slides (Thermoscientific, Villebon sur Yvette, France). The immunostainings using rabbit polyclonal antibody (CFTR 1/100 MAB25031 from R&D system) Ki67 (RBK027; Ztomed Systems) were performed according the facility automated protocol (benchmark GX autostainer, Ventana Medical System, Roche).

Correlative light and electron microscopy (CLEM) -

To determine the ultrastructural localization of CFTR, correlative light and electron microscopy (CLEM) was used after immunolabelling. This approach allows pre-selection of area with features of interest for detailed ultrastructural study in transmission electron microscopy (TEM). Samples of human lung specimens (2 cm 3 ) were embedded and frozen after fixation in 4% paraformaldehyde and immersion in 10% then 40% sucrose solution.

Indirect immunofluorescence was performed on frozen sections (10 µm) collected onto correlative microscopy coverslips® (Delta Microscopies, France). After confocal microscopy acquisition, correlative slides were detached in SPB and returned upside down. Selected thick cryo-sections were fixed 1% glutaraldehyde in SPB for 5 min at room temperature, quenched with glycine 50mM and rinsed before processing to gold amplification. One drop of gold reagent (Gold enhance EM/blot -Nanoprobes -LGF Distribution Ste Consorce -France) was applied onto sections for 10 min. After rinses, the sections were post fixed with 0.5% osmium tetroxide, gradually dehydrated in ethanol (50% to 100%) and embedded in Epon. Ultrathin sections (70 nm) were collected onto 150 mesh cooper grids, and counterstained with lead citrate before examination.

All the electron microscope examination were proceeded on Hitachi HT7700 electron microscope operated at 80 k, microphotographs were acquired with a camera AMT (Milexia, France),(MIMA2 Facility, Université Paris-Saclay, INRAE, AgroParisTech, GABI, 78350, Jouy-en-Josas, France).

After an overnight incubation at 4°C, rabbit polyclonal antibody (CFTR 1/200 MAB25031 from R&D system) was revealed by the goat anti-rabbit Alexa Fluor® 488 FluoroNanogold™ (-Nanoprobes -

LGF Distribution Echirolles -France), slides were examined with a confocal microscope (LSM700; Carl Zeiss, Le Pecq, France) and images were acquired with the software Zen (Centre Chirurgical Marie Lannelongue Microscopy and Imaging Facility, Le-Plessis-Robinson, France).

Statistical analyses-All statistical tests were performed using GraphPad Prism software (GraphPad, version 6.0 for Windows). All data were verified for normal distribution using Shapiro-wilk normality test. All values are reported as mean ± S.E.M. For all experiments, the difference between two groups was assessed by T test. The difference between three groups was assessed with one-way ANOVA completed by Tukey's multiple comparisons test for post hoc analyses. Differences were considered statistically significant at p-values < 0.05.

Supplemental references

Supplemental tables

Supplemental figures

Supplemental Figure 1