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International audience; Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. The non-receptor tyrosine kinase c-Abelson (cAbl) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is down-activated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from PAH patients (PAH-ECs). We found cAbl and phosphorylated cAbl levels to be lower in the endothelium of remodeled pulmonary vessels in the lungs of PAH patients than controls. Similar observations were obtained for the lungs of sugen+hypoxia (SuHx) and monocrotaline (MCT) rats with established pulmonary hypertension. These in situ abnormalities were also replicated in vitro, with cultured PAH-ECs displaying lower cAbl expression and activity and an altered DNA damage response and capacity of tube formation. Downregulation of cAbl by RNA-interference in Control-ECs or its inhibition with dasatinib resulted in genomic instability and the failure to form tubes, whereas upregulation of cAbl with DPH reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of crosstalk between cAbl and bone morphogenetic protein receptor type II (BMPRII). This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

Systemic sclerosis (SSc) is a debilitating autoimmune disease characterized by severe lung outcomes resulting in reduced life expectancy. Fra-2-transgenic mice offer the opportunity to decipher the relationships between the immune system and lung fibrosis. This study was undertaken to investigate whether the Fra-2-transgenic mouse lung phenotype may result from an imbalance between the effector and regulatory arms in the CD4+ T cell compartment.We first used multicolor flow cytometry to extensively characterize homeostasis and the phenotype of peripheral CD4+ T cells from Fra-2-transgenic mice and control mice. We then tested different treatments for their effectiveness in restoring CD4+ Treg cell homeostasis, including adoptive transfer of Treg cells and treatment with low-dose interleukin-2 (IL-2).Fra-2-transgenic mice demonstrated a marked decrease in the proportion and absolute number of peripheral Treg cells that preceded accumulation of activated, T helper cell type 2-polarized, CD4+ T cells. This defect in Treg cell homeostasis was derived from a combination of mechanisms including impaired generation of these cells in both the thymus and the periphery. The impaired ability of peripheral conventional CD4+ T cells to produce IL-2 may greatly contribute to Treg cell deficiency in Fra-2-transgenic mice. Notably, adoptive transfer of Treg cells, low-dose IL-2 therapy, or combination therapy changed the phenotype of Fra-2-transgenic mice, resulting in a significant reduction in pulmonary parenchymal fibrosis and vascular remodeling in the lungs.Immunotherapies for restoring Treg cell homeostasis could be relevant in SSc. An intervention based on low-dose IL-2 injections, as is already proposed in other autoimmune diseases, could be the most suitable treatment modality for restoring Treg cell homeostasis for future research.

BACKGROUND: Activins are novel therapeutic targets in pulmonary arterial hypertension (PAH). We therefore studied whether key members of the activin pathway could be used as PAH biomarkers. METHODS: Serum levels of activin A, activin B, α-subunit of inhibin A and B proteins, and the antagonists follistatin and follistatin-like 3 (FSTL3) were measured in controls and in patients with newly diagnosed idiopathic, heritable, or anorexigen-associated PAH (n=80) at baseline and 3 to 4 months after treatment initiation. The primary outcome was death or lung transplantation. Expression patterns of the inhibin subunits, follistatin, FSTL3, Bambi, Cripto, and the activin receptors type I (ALK), type II (ACTRII), and betaglycan were analyzed in PAH and control lung tissues. RESULTS: Death or lung transplantation occurred in 26 of 80 patients (32.5%) over a median follow-up of 69 (interquartile range, 50–81) months. Both baseline (hazard ratio, 1.001 [95% CI, 1.000–1.001]; P =0.037 and 1.263 [95% CI, 1.049–1.520]; P =0.014, respectively) and follow-up (hazard ratio, 1.003 [95% CI, 1.001–1.005]; P =0.001 and 1.365 [95% CI, 1.185–1.573]; P P =0.009) and 0.17 (95% CI, 0.06–0.45; P P =0.019) and 0.27 (95% CI, 0.09–0.78, P =0.015), respectively. Prognostic values of activin A and FSTL3 were confirmed in an independent external validation cohort. Histological analyses showed a nuclear accumulation of the phosphorylated form of Smad2/3, higher immunoreactivities for ACTRIIB, ALK2, ALK4, ALK5, ALK7, Cripto, and FSTL3 in vascular endothelial and smooth muscle layers, and lower immunostaining for inhibin-α and follistatin. CONCLUSIONS: These findings offer new insights into the activin signaling system in PAH and show that activin A and FSTL3 are prognostic biomarkers for PAH.

Background: We studied the ability of the nonsteroidal MR (mineralocorticoid receptor) antagonist finerenone to attenuate vascular remodeling and pulmonary hypertension using two complementary preclinical models (the monocrotaline and sugen/hypoxia rat models) of severe pulmonary hypertension. Methods: We first examined the distribution pattern of MR in the lungs of patients with pulmonary arterial hypertension (PAH) and in monocrotaline and sugen/hypoxia rat lungs. Subsequent studies were performed to explore the effect of MR inhibition on proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. To validate the functional importance of MR activation in the pulmonary vascular remodeling characteristic of pulmonary hypertension, mice overexpressing human MR (hMR+) were studied, and curative treatments with finerenone (1 mg/kg per day by gavage), started 2 weeks after monocrotaline injection or 5 weeks after Sugen injection were realized. Results: We demonstrated that MR is overexpressed in experimental and human PAH and that its inhibition following small interfering RNA-mediated MR silencing or finerenone treatment attenuates proliferation of pulmonary artery smooth muscle cells derived from patients with idiopathic PAH. In addition, we obtained evidence that hMR+ mice display increased right ventricular systolic pressure, right ventricular hypertrophy, and remodeling of pulmonary arterioles. Consistent with these observations, curative treatments with finerenone partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and vascular remodeling. Finally, we found that continued finerenone treatment decreases inflammatory cell infiltration and vascular cell proliferation in monocrotaline and sugen/hypoxia rat lungs. Conclusions: Finerenone treatment appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.

Summary Cardiopulmonary complications are a leading cause of death in systemic sclerosis. Pulmonary hypertension in particular carries a high mortality and morbidity burden. Patients with systemic sclerosis can suffer from all of the clinical groups of pulmonary hypertension, particularly pulmonary arterial hypertension and pulmonary hypertension related to interstitial lung disease. Despite a similar pathogenetic background with idiopathic pulmonary arterial hypertension, different mechanisms determine a worse prognostic outcome for patients with systemic sclerosis. In this Viewpoint, we will consider the link between pathogenetic and potential therapeutic targets for the treatment of pulmonary hypertension in the context of systemic sclerosis, with a focus on the current unmet needs, such as the importance of early screening and detection, the absence of agreed criteria to distinguish pulmonary arterial hypertension with interstitial lung disease from pulmonary hypertension due to lung fibrosis, and the need for a holistic treatment approach to target all the vascular, immunological, and inflammatory components of the disease.

Pulmonary arterial hypertension (PAH) is a complex multifactorial disease with poor prognosis characterized by functional and structural alterations of the pulmonary circulation causing marked increase in pulmonary vascular resistance (PVR), ultimately leading to right heart failure and death. Mutations in the gene encoding Bone Morphogenetic Protein Receptor type 2 (BMPR2), a receptor for the transforming growth factor-beta (TGF-β) superfamily, account for over 70% of families with PAH, and approximately 20% of sporadic cases. In recent years, however, less common or rare mutations in other genes have been identified. This review will consider how these newly discovered PAH genes could help to provide a better understanding of the molecular and cellular bases of the maintenance of the pulmonary vascular integrity, as well as their role in the PAH pathogenesis underlying occlusion of arterioles in the lung. We will also discuss how insights into the genetic contributions of these new PAH-related genes may open up new therapeutic targets for this, currently incurable, cardiopulmonary disorder.

Since the 1960s, several drugs have been linked to the onset or aggravation of pulmonary arterial hypertension (PAH): dasatinib, some amphetamine-like appetite suppressants (aminorex, fenfluramine, dexfenfluramine, benfluorex) and recreational drugs (methamphetamine). Moreover, in numerous cases, the implication of other drugs with PAH have been suggested, but the precise identification of iatrogenic aetiologies of PAH is challenging given the scarcity of this disease and the potential long latency period between drug intake and PAH onset. In this context, we used the World Health Organization's pharmacovigilance database, VigiBase, to generate new hypotheses about drug associated PAH.We used VigiBase, the largest pharmacovigilance database worldwide to generate disproportionality signals through the Bayesian neural network method. All disproportionality signals were further independently reviewed by experts in pulmonary arterial hypertension, pharmacovigilance and vascular pharmacology and their plausibility ranked according to World Health Organization causality categories.We included 2184 idiopathic PAH cases, yielding a total of 93 disproportionality signals. Among them, 25 signals were considered very likely, 15 probable, 28 possible and 25 unlikely. Notably, we identified 4 new protein kinases inhibitors (lapatinib, lorlatinib, ponatinib and ruxolitinib), 1 angiogenesis inhibitor (bevacizumab), and several chemotherapeutics (etoposide, trastuzumab), antimetabolites (cytarabine, fludarabine, fluorouracil, gemcitabine) and immunosuppressants (leflunomide, thalidomide, ciclosporin).Such signals represent plausible adverse drug reactions considering the knowledge of iatrogenic PAH, the drugs' biological and pharmacological activity and the characteristics of the reported case. Although confirmatory studies need to be performed, the signals identified may help clinicians envisage an iatrogenic aetiology when faced with a patient who develops PAH.

Background Platelet‐derived growth factor is a major regulator of the vascular remodeling associated with pulmonary arterial hypertension. We previously showed that protein widely 1 (PW1 + ) vascular progenitor cells participate in early vessel neomuscularization during experimental pulmonary hypertension (PH) and we addressed the role of the platelet‐derived growth factor receptor type α (PDGFRα) pathway in progenitor cell‐dependent vascular remodeling and in PH development. Methods and Results Remodeled pulmonary arteries from patients with idiopathic pulmonary arterial hypertension showed an increased number of perivascular and vascular PW1 + cells expressing PDGFRα. PW1 nLacZ reporter mice were used to follow the fate of pulmonary PW1 + progenitor cells in a model of chronic hypoxia–induced PH development. Under chronic hypoxia, PDGFRα inhibition prevented the increase in PW1 + progenitor cell proliferation and differentiation into vascular smooth muscle cells and reduced pulmonary vessel neomuscularization, but did not prevent an increased right ventricular systolic pressure or the development of right ventricular hypertrophy. Conversely, constitutive PDGFRα activation led to neomuscularization via PW1 + progenitor cell differentiation into new smooth muscle cells and to PH development in male mice without fibrosis. In vitro, PW1 + progenitor cell proliferation, but not differentiation, was dependent on PDGFRα activity. Conclusions These results demonstrate a major role of PDGFRα signaling in progenitor cell–dependent lung vessel neomuscularization and vascular remodeling contributing to PH development, including in idiopathic pulmonary arterial hypertension patients. Our findings suggest that PDGFRα blockers may offer a therapeutic add‐on strategy to combine with current pulmonary arterial hypertension treatments to reduce vascular remodeling. Furthermore, our study highlights constitutive PDGFRα activation as a novel experimental PH model.

Introduction: Activation of the kynurenine pathway (KP) has been reported in patients with pulmonary arterial hypertension (PAH) undergoing PAH therapy. We aimed to determine KP-metabolism in treatment-naïve PAH patients, investigate its prognostic values, evaluate the effect of PAH therapy on KP-metabolites and identify cytokines responsible for altered KP-metabolism. Methods: KP-metabolite levels were determined in plasma from PAH patients (median follow-up 42 months) and in rats with monocrotaline- and Sugen/hypoxia-induced PH. Blood sampling of PAH patients was performed at the time of diagnosis, six months and one year after PAH therapy. Results: KP activation with lower tryptophan, higher kynurenine (Kyn), 3-hydroxykynurenine (3-HK), quinolinic acid (QA), kynurenic acid (KA), and anthranilic acid was observed in treatment-naïve PAH patients compared with controls. A similar KP-metabolite profile was observed in monocrotaline, but not Sugen/hypoxia-induced PAH. Human lung primary cells (microvascular endothelial cells, pulmonary artery smooth muscle cells, and fibroblasts) were exposed to different cytokines in vitro. Following exposure to interleukin-6 (IL-6)/IL-6 receptor a (IL-6Ra) complex, all cell types exhibit a similar KP-metabolite profile as observed in PAH patients. PAH therapy partially normalized this profile in survivors after one year. Increased KP-metabolites correlated with higher pulmonary vascular resistance, shorter six-minute walking distance, and worse functional class. High levels of Kyn, 3-HK, QA, and KA measured at the latest time-point were associated with worse long-term survival. Conclusion: KP-metabolism was activated in treatment-naïve PAH patients, likely mediated through IL-6/IL-6Ra signaling. KP-metabolites predict response to PAH therapy and survival of PAH patients.

IntroductionAlthough the physiological role of the C-terminal hydrolase domain of the soluble epoxide hydrolase (sEH-H) is well investigated, the function of its N-terminal phosphatase activity (sEH-P) remains unknown.ObjectivesThis study aimed to assess in vivo the physiological role of sEH-P.MethodsCRISPR/Cas9 was used to generate a novel knock-in (KI) rat line lacking the sEH-P activity.ResultsThe sEH-P KI rats has a decreased metabolism of lysophosphatidic acids to monoacyglycerols. KI rats grew almost normally but with less weight and fat mass gain while insulin sensitivity was increased compared to wild-type rats. This lean phenotype was more marked in males than in female KI rats and mainly due to decreased food consumption and enhanced energy expenditure. In fact, sEH-P KI rats had an increased lipolysis allowing to supply fatty acids as fuel to potentiate brown adipose thermogenesis under resting condition and upon cold exposure. The potentiation of thermogenesis was abolished when blocking PPARγ, a nuclear receptor activated by intracellular lysophosphatidic acids, but also when inhibiting simultaneously sEH-H, showing a functional interaction between the two domains. Furthermore, sEH-P KI rats fed a high-fat diet did not gain as much weight as the wild-type rats, did not have increased fat mass and did not develop insulin resistance or hepatic steatosis. In addition, sEH-P KI rats exhibited enhanced basal cardiac mitochondrial activity associated with an enhanced left ventricular contractility and were protected against cardiac ischemia-reperfusion injury.ConclusionOur study reveals that sEH-P is a key player in energy and fat metabolism and contributes together with sEH-H to the regulation of cardiometabolic homeostasis. The development of pharmacological inhibitors of sEH-P appears of crucial importance to evaluate the interest of this promising therapeutic strategy in the management of obesity and cardiac ischemic complications.

International audience; Background and purpose: Pulmonary hypertension (PH) is a multifactorial chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signaling in PA play a major role in PH. We recently described the involvement of the Rho protein Rac1 in the control of systemic blood pressure through its involvement in NO-mediated relaxation of arterial smooth muscle cell (SMC). The aim of this study was thus to analyze the role of SMC Rac1 in PH.Experimental approach: PH is induced by exposure of control and SMC Rac1-deficient (SM-Rac1-KO) mice to chronic hypoxia (10% O2 , 4 weeks). PH is assessed by the measurement of RV systolic pressure and hypertrophy. PA reactivity is analyzed by isometric tension measurements. PA remodeling is quantified by immunofluorescence in lung sections and ROS are detected using the DHE probe and electronic paramagnetic resonance analysis. Rac1 activity is determined by immunofluorescence.Key results: Rac1 activation is observed in PA of hypoxic mice and patients with idiopathic PH. Hypoxia-induced rise in RV systolic pressure, RV hypertrophy and loss of endothelium-dependent relaxation were significantly decreased in SM-Rac1-KO mice compared to control mice. SMC Rac1 deletion also limited hypoxia-induced PA remodeling and ROS production in PASMCs.Conclusion and implications: Our results provide evidence for a protective effect of SM Rac1 deletion against hypoxic PH. Rac1 activity in PASMCs plays a causal role in PH by favoring ROS-dependent PA remodeling and endothelial dysfunction induced by chronic hypoxia.

BackgroundRisk stratification and assessment of disease progression in patients with pulmonary arterial hypertension (PAH) are challenged by the lack of accurate disease-specific and prognostic biomarkers. To date, brain natriuretic peptide (BNP) and/or its N-terminal fragment (NT-proBNP) are the only markers for right ventricular dysfunction used in clinical practice, in association with echocardiographic and invasive haemodynamic variables to predict outcome in patients with PAH.MethodsThis study was designed to identify an easily measurable biomarker panel in the serum of 80 well-phenotyped PAH patients with idiopathic, heritable or drug-induced PAH at baseline and at first follow-up. The prognostic value of identified cytokines of interest was secondly analysed in an external validation cohort of 125 PAH patients.ResultsAmong the 20 biomarkers studied with the multiplex Ella platform, we identified a three-biomarker panel composed of β-NGF, CXCL9 and TRAIL that were independently associated with prognosis both at the time of PAH diagnosis and at the first follow-up after initiation of PAH therapy. β-NGF and CXCL9 were predictors of death or transplantation, whereas high levels of TRAIL were associated with a better prognosis. Furthermore, the prognostic value of the three cytokines was more powerful for predicting survival than usual non-invasive variables (New York Heart Association Functional Class, 6-min walk distance and BNP/NT-proBNP). The results were validated in a fully independent external validation cohort.ConclusionThe monitoring of β-NGF, CXCL9 and TRAIL levels in serum should be considered in the management and treatment of patients with PAH to objectively guide therapeutic options.

BackgroundUncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterised by generalized fibrosis affecting particularly the lungs and skin. Co-stimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of ALPN-101 (acazicolcept), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. MethodsExpression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, ALPN-101 was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 µg of ALPN-101 or a molar-matched dose of an Fc control protein twice a week for six weeks. After six weeks, skin and lung were evaluated.Results ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc Control and ALPN-101 groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of ALPN-101 treatment. In mice challenged with HOCL, ALPN-101 induced a significant decrease in dermal thickness, collagen content, myofibroblast number and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, ALPN-101 treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of ALPN-101-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, ALPN-101 reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by ALPN-101 was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. Conclusions Our results confirm the importance of co-stimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by ALPN-101 decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Background Uncontrolled immune response with T cell activation has a key role in the pathogenesis of systemic sclerosis (SSc), a disorder that is characterized by generalized fibrosis affecting particularly the lungs and skin. Costimulatory molecules are key players during immune activation, and recent evidence supports a role of CD28 and ICOS in the development of fibrosis. We herein investigated the efficacy of acazicolcept (ALPN-101), a dual ICOS/CD28 antagonist, in two complementary SSc-related mouse models recapitulating skin fibrosis, interstitial lung disease, and pulmonary hypertension. Methods Expression of circulating soluble ICOS and skin-expressed ICOS was investigated in SSc patients. Thereafter, acazicolcept was evaluated in the hypochlorous acid (HOCL)-induced dermal fibrosis mouse model and in the Fra-2 transgenic (Tg) mouse model. In each model, mice received 400 μg of acazicolcept or a molar-matched dose of an Fc control protein twice a week for 6 weeks. After 6 weeks, skin and lung were evaluated. Results ICOS was significantly increased in the sera from SSc patients and in SSc skin biopsies as compared to samples from healthy controls. Similar body weight changes were observed between Fc control and acazicolcept groups in both HOCL and Fra-2 Tg mice suggesting a good tolerance of acazicolcept treatment. In mice challenged with HOCL, acazicolcept induced a significant decrease in dermal thickness, collagen content, myofibroblast number, and inflammatory infiltrates characterized by B cells, T cells, neutrophils, and macrophages. In the Fra-2 Tg mouse model, acazicolcept treatment reduced lung collagen content, fibrillar collagen, histological fibrosis score, and right ventricular systolic pressure (RVSP). A reduction in frequency of CD4+ and T effector memory cells and an increase in the percentage of CD4+ T naïve cells in spleen and lung of acazicolcept-treated Fra-2 Tg mice was observed as compared to Fc control-treated Fra-2 Tg mice. Moreover, acazicolcept reduced CD69 and PD-1 expression on CD4+ T cells from the spleen and the lung. Target engagement by acazicolcept was demonstrated by blockade of CD28 and ICOS detection by flow cytometry in treated mice. Conclusions Our results confirm the importance of costimulatory molecules in inflammatory-driven fibrosis. Our data highlight a key role of ICOS and CD28 in SSc. Using complementary models, we demonstrated that dual ICOS/CD28 blockade by acazicolcept decreased dermal and pulmonary fibrosis and alleviated pulmonary hypertension. These results pave the way for subsequent research on ICOS/CD28-targeted therapies.

Objective Lysyl oxidase (LOX) is an extracellular enzyme that cross-links collagen fibrils. LOX was found to be increased in serum of SSc patients and was suggested to be related to skin fibrosis, yet a vascular source of LOX has been demonstrated in idiopathic pulmonary arterial hypertension (iPAH). We aimed to validate elevated LOX serum levels in SSc and to study its correlation with clinical characteristics and investigate its main source at the tissue level. Methods A total of 86 established SSc patients were compared with 86 patients with very early diagnosis of systemic sclerosis (VEDOSS), 110 patients with primary RP (PRP) and 80 healthy controls. LOX serum levels were determined by ELISA. Five lung and 12 skin biopsies from SSc patients were stained for LOX and compared with controls. Results Serum levels of LOX in SSc were significantly higher than in VEDOSS, PRP and healthy controls (P < 0.001). LOX inversely correlated with the diffusing capacity of the lung for carbon monoxide diffusing capacity (DLCO) in diffuse SSc (r = −0.376, P = 0.02). Patients with moderate to severe estimated systolic PAH had higher LOX levels (P < 0.01). Lung biopsies demonstrated intense LOX staining in SSc patients with PAH that was predominantly located in the endothelium of the remodelled pulmonary vessels. Conclusion Serum LOX levels are increased in established SSc and inversely correlate with the DLCO. LOX is elevated in patients with moderate to severe PAH and is located in the proliferating endothelium in lung arterioles, suggesting a possible role for LOX in SSc-associated PAH.

Pulmonary arterial hypertension (PAH) is a progressive incurable condition that is characterized by extensive remodeling of the pulmonary circulation, leading to severe right-sided heart failure and death. Similar to other vascular contractile cells, pulmonary arterial smooth muscle cells play central roles in physiological and pathologic vascular remodeling because of their remarkable ability to dynamically modulate their phenotype to ensure contractile and synthetic functions. The dysfunction and molecular mechanisms underlying their contribution to the various pulmonary vascular lesions associated with PAH have been a major focus of research. The aim of this review is to describe the medial and nonmedial origins of contractile cells in the pulmonary vascular wall and present evidence of how they contribute to the onset and progression of PAH. We also highlight specific potential target molecules and discuss future directions that are being explored to widen the therapeutic options for the treatment of PAH.

Background Severe asthma is a chronic lung disease characterised by inflammation, airway hyperresponsiveness (AHR) and airway remodelling. The molecular mechanisms underlying uncontrolled airway smooth muscle cell (aSMC) proliferation involved in pulmonary remodelling are still largely unknown. Small G proteins of the Rho family (RhoA, Rac1 and Cdc42) are key regulators of smooth muscle functions and we recently demonstrated that Rac1 is activated in aSMC from allergic mice. The objective of this study was to assess the role of Rac1 in severe asthma-associated airway remodelling. Methods and results Immunofluorescence analysis in human bronchial biopsies revealed an increased Rac1 activity in aSMC from patients with severe asthma compared with control subjects. Inhibition of Rac1 by EHT1864 showed that Rac1 signalling controlled human aSMC proliferation induced by mitogenic stimuli through the signal transducer and activator of transcription 3 (STAT3) signalling pathway. In vivo, specific deletion of Rac1 in SMC or pharmacological inhibition of Rac1 by nebulisation of NSC23766 prevented AHR and aSMC hyperplasia in a mouse model of severe asthma. Moreover, the Rac1 inhibitor prevented goblet cell hyperplasia and epithelial cell hypertrophy whereas treatment with corticosteroids had less effect. Nebulisation of NSC23766 also decreased eosinophil accumulation in the bronchoalveolar lavage of asthmatic mice. Conclusion This study demonstrates that Rac1 is overactive in the airways of patients with severe asthma and is essential for aSMC proliferation. It also provides evidence that Rac1 is causally involved in AHR and airway remodelling. Rac1 may represent as an interesting target for treating both AHR and airway remodelling of patients with severe asthma.

We have shown that the nerve growth factor NGF plays a pathophysiological role in pulmonary hypertension by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. The aim of this study was to determine the mechanisms of NGF-induced PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Expression of NGF and its TrkA receptor were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from controls or from patients suffering from idiopathic pulmonary arterial hypertension (IPAH). Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and cell surface biotinylation, after NGF treatment. Gap junction activity was assessed by FRAP microscopy. Contractions of rat PA were induced by phenylephrine (PHE), with or without NGF. The role of Cx43 in NGF-induced rat PA hyperreactivity was evaluated by use of 43Gap26, a Cx43 blocking peptide, or by an anti-Cx43 siRNA. NGF and TrkA expression were increased in hPASMC from IPAH patients compared to controls. NGF increased Cx43 expression in hPASMC, with a higher sensitivity in IPAH cells compared to controls cells. This increase was abolished with K252a, a TrkA kinase inhibitor. Cx43 plasma membrane localisation and GAP junction activity were also increased after NGF treatment. NGF-induced PA hyperreactivity to PHE was inhibited after ex vivo treatment with 43Gap26 or after in vivo treatment with Cx43 siRNA. In conclusion, NGF pathway is upregulated in IPAH cells. NGF increases Cx43 PA expression through TrkA receptor. Cx43 increased expression at plasma membrane upregulates GAP junction activity, thus promoting PA hyperreactivity.

Introduction: Pulmonary endothelial cells (P-ECs) in idiopathic pulmonary arterial hypertension (iPAH) accumulate DNA lesions during the disease progression; however, the mechanisms underlying this phenomenon remain unclear. Since the non-receptor tyrosine kinase c-Abelson (c-Abl) responds to signals from DNA damage and activates apoptosis, we hypothesized that DNA damage accumulation is due to a loss of c-Abl activity in P-ECs derived from PAH patients. Aims and Objectives: To study the role of c-Abl in the loss of genome integrity in iPAH P-ECs. Methods: Primary cultures of human control and iPAH P-ECs were analyzed by comet assay to determine the amount of DNA strand breaks. P-ECs were treated with a c-Abl inhibitor dasatinib (a specific BCR-ABL tyrosine kinase inhibitor), c-Abl siRNA, or DPH (a c-Abl activator) to analyze DNA lesions by comet assays and the activation of DNA repair pathways by Western blotting. Cleaved caspase-3 expression was also measured to determine the level of apoptosis. Results: iPAH P-ECs display a larger amount of DNA strand breaks compared to control P-ECs along with higher apoptosis and impaired c-Abl expression and activity. Treatment of control P-ECs with dasatinib generates more DNA damage and apoptosis than vehicle conditions and alters the DNA repair pathways. Consistent with these observations, downregulation of c-Abl by RNA-interference in control P-ECs resulted in an increase in DNA damage and apoptosis, whereas upregulation of c-Abl with DPH treatment reduced DNA damage and apoptosis levels in iPAH-ECs. Conclusions: Our results demonstrate a central role of c-Abl in the loss of DNA integrity in iPAH P-ECs.

Introduction: Despite increasing evidence suggesting that the Bone Morphogenetic Protein (BMP)-9 signaling pathway is playing a role in the pulmonary hypertension (PH) pathogenesis, the data in the literature are still controversial. Aims and Objectives: To investigate the role of BMP9 signaling pathway in PH pathophysiology. Methods: Bmp9 KO-/- rats were subjected to 3 distinct well characterized experimental PH models: the chronic hypoxia (CHx), monocrotaline (MCT) and Sugen/hypoxia (SuHx) models. Wild-type littermates were used as controls. We then performed transthoracic echocardiography and right heart catheterization as well as histological analyses in these 3 models: 21 days after CHx and MCT treatment and 8 weeks in the SuHx model. Results: While Bmp9 KO-/- rats did not develop any spontaneous phenotype, they were protected against PH induced in our 3 different PH models, compared with their wild-type littermates, as reflected by lower values of mean pulmonary artery pressure (mPAP), total pulmonary vascular resistance (TPVR), and Fulton index. We also found a less pronounced remodeling of the pulmonary arterioles and collagen accumulation in the right ventricle in these Bmp9 KO-/- rats compared to their wild-type littermates. Finally, we found that the in vitro exposure of human pulmonary endothelial cells (ECs) to BMP9 modulates the secretion of pro- and anti-angiogenic factors. These in vitro observations were confirmed in vivo, with Bmp9 KO-/- rats exhibiting increased serum and lung levels of pro-angiogenic factors. Conclusions: BMP9 deficiency does not lead to spontaneous PH, but attenuates the development of experimental PH.

Leptin is a pleotropic hormone known to regulate a wide range of systemic functions, from satiety to inflammation. Increasing evidence has shown that leptin and its receptor (ObR) are not only expressed in adipose tissue but also in several organs, including the lungs. Leptin levels were first believed to be elevated only in the lungs of obese patients, and leptin was suspected to be responsible for obesity-related lung complications. Aside from obesity, leptin displays many faces in the respiratory system, independently of body weight, as this cytokine-like hormone plays important physiological roles, from the embryogenic state to maturation of the lungs and the control of ventilation. The leptin-signaling pathway is also involved in immune modulation and cell proliferation, and its dysregulation can lead to the onset of lung diseases. This review article addresses the thousand faces of leptin and its signaling in the lungs under physiological conditions and in disease.

Impairment of the pulmonary endothelial communications contributes to the development and progression of pulmonary arterial hypertension (PAH). Investigations currently under way are likely to produce novel therapeutic strategies that target pulmonary endothelial dysfunction linked to PAH. This chapter outlines the complex role of the dysfunctional pulmonary endothelium in PAH and its potential value as a target for innovative therapies.

International audience; Heightened pulmonary artery smooth muscle cell (PA-SMC) proliferation and migration and dynamic remodeling of the extracellular matrix are hallmark pathogenic features of pulmonary arterial hypertension (PAH). Pirfenidone (PFD) is an orally bioavailable pyridone derivative with antifibrotic, antiinflammatory, and antioxidative properties currently used in the treatment of idiopathic pulmonary fibrosis. We therefore evaluated the efficacy of curative treatments with PFD in the sugen/hypoxia (SuHx) rat model of severe pulmonary hypertension. Treatment with PFD (30 mg/kg per day by mouth 3 times a day for 3 wk) started 5 wk after sugen injection partially reversed established pulmonary hypertension, reducing total pulmonary vascular resistance and remodeling. Consistent with these observations, we found that continued PFD treatment decreases PA-SMC proliferation and levels of extracellular matrix deposition in lungs and right ventricles in SuHx rats. Importantly, PFD attenuated the proproliferative and promigratory potentials of cultured PA-SMCs from patients with idiopathic PAH and their capacity to produce extracellular matrix components. Finally, we found that PFD dose dependently enhanced forkhead box O1 protein levels and its nuclear translocation in cultured idiopathic PAH PA-SMCs and in PFD-treated SuHx rats. PFD appears to be a potential therapy for PAH worthy of investigation and evaluation for clinical use in conjunction with current PAH treatments.-Poble, P.-B., Phan, C., Quatremare, T., Bordenave, J., Thuillet, R., Cumont, A., Huertas, A., Tu, L., Dorfmüller, P., Humbert, M., Ghigna, M.-R., Savale, L., Guignabert, C. Therapeutic effect of pirfenidone in the sugen/hypoxia rat model of severe pulmonary hypertension.

Spontaneous reporting is the primary method used in pharmacovigilance (PV) to detect drug safety signal. Specific criteria used in pharmacovigilance to prove accountability of a drug are rarely present in rare disease. The low number of alerts also makes it challenging. The aim of this commentary is to raise awareness among pharmacists on issues and opportunities for pharmacovigilance in rare diseases, taking pulmonary arterial hypertension (PAH) as example, from which a subset of cases are drug-induced. It is demonstrated how a dedicated program named VIGIAPATH created to reinforce pharmacovigilance of drug-induced pulmonary arterial hypertension at a national level, led to increase self-reporting and confirm safety signals. Thanks to a specific program such as VIGIAPATH, pharmacists can play an important role in communication with clinicians, patients and regulatory agencies, facilitating the detection of potential safety signals at an early stage in rare disease.

Background: The beneficial effects of parasympathetic stimulation have been reported in left heart failure, but whether it would be beneficial for pulmonary arterial hypertension (PAH) remains to be explored. Here, we investigated the relationship between parasympathetic activity and right ventricular (RV) function in patients with PAH, and the potential therapeutic effects of pyridostigmine (PYR), an oral drug stimulating the parasympathetic activity through acetylcholinesterase inhibition, in experimental pulmonary hypertension (PH). Methods: Heart rate recovery after a maximal cardiopulmonary exercise test was used as a surrogate for parasympathetic activity. RV ejection fraction was assessed in 112 patients with PAH. Expression of nicotinic (α-7 nicotinic acetylcholine receptor) and muscarinic (muscarinic acetylcholine type 2 receptor) receptors, and acetylcholinesterase activity were evaluated in RV (n=11) and lungs (n=7) from patients with PAH undergoing heart/lung transplantation and compared with tissue obtained from controls. In addition, we investigated the effects of PYR (40 mg/kg per day) in experimental PH. PH was induced in male rats by SU5416 (25 mg/kg subcutaneously) injection followed by 4 weeks of hypoxia. In a subgroup, sympathetic/parasympathetic modulation was assessed by power spectral analysis. At week 6, PH status was confirmed by echocardiography, and rats were randomly assigned to vehicle or treatment (both n=12). At the end of the study, echocardiography was repeated, with additional RV pressure-volume measurements, along with lung, RV histological, and protein analyses. Results: Patients with PAH with lower RV ejection fraction ( Conclusions: RV dysfunction is associated with reduced systemic parasympathetic activity in patients with PAH, with an inadequate adaptive response of the cholinergic system in the RV. Enhancing parasympathetic activity by PYR improved survival, RV function, and pulmonary vascular remodeling in experimental PH.

Chronic thromboembolic pulmonary hypertension (CTEPH) is a rare and underdiagnosed complication of acute pulmonary embolism (APE). CTEPH is a common cause of pulmonary hypertension (PH) with distinct management strategy including pulmonary endarterectomy, balloon pulmonary angioplasty, long-term anticoagulation and PH drugs targeting endothelial cell dysfunction. Initially, PH in chronic thromboembolic pulmonary disease (CTEPD) was thought to be due exclusively to the intravascular obstruction of pulmonary arteries by unresolved fibrotic clots. However, it is now well accepted that pulmonary vascular remodelling can include significant pulmonary microvasculopathy, which plays a role in the development of CTEPH. The histological description and clinical consequences of CTEPH microvasculopathy are now better understood. These lesions may involve not only small muscular pulmonary arteries

Introduction L’objectif de ce travail est d’etudier les alterations du proteome serique des patients atteints d’hypertension arterielle pulmonaire (HTAP) associee a la clerodermie systemique (SSc), d’identifier les proteines correlees a la severite hemodynamique et determiner leur role potentiel dans la physiopathologie de la maladie. Patients et methodes Les patients eligibles ont ete recrutes dans les centres de reference SSc et/ou HTAP de Boston, Lille, Le Kremlin-Bicetre et Grenoble. Les patients etaient inclus s’ils remplissaient les criteres suivants : diagnostic de SSc selon les criteres ACR/EULAR 2013, forme cutanee limitee, pas de pneumopathie interstitielle diffuse (PID) etendue selon les criteres de Goh, et pas de traitement specifique de l’HTAP. Les patients etaient consideres comme des cas s’ils avaient un diagnostic d’HTAP confirme par catheterisme cardiaque droit (KTD) et un echantillon de serum preleve le meme jour que le KTD. Ils etaient consideres comme des temoins s’ils n’avaient aucun signe evocateur d’HTAP a l’echocardiographie. Resultats Dans une premiere etape exploratoire, l’expression serique de 1129 proteines a ete evaluee chez 15 cas et 16 temoins par analyse proteomique a haut debit (SOMAscan). Nous avons identifie 53 proteines exprimees differentiellement entre les 2 groupes. Parmi ces 53 candidats, seules 2 etaient significativement correlees avec les resistances vasculaires pulmonaires (RVP) : chemerine (p = 0,01, ρ = 0,62) et SET nuclear proto-oncogen (SET) (p = 0,01, ρ = 0,62). Pour valider ces resultats, les taux seriques de chemerine et SET ont ete mesures en ELISA chez 25 cas et 19 controles supplementaires. Les dosages de chemerine etaient de nouveau significativement plus eleves chez les cas (p = 0,01) et correles avec les RVP (p = 0,01, ρ=0,46) dans cette cohorte independante. Dans une deuxieme etape, pour etudier le role physiopathologique potentiel de la chemerine, nous avons realise des marquages en immunofluorescence confocale sur des poumons explantes de temoins sains, de patients SSc-PID sans HTAP et de patients SSc-HTAP. Le marquage du recepteur de chemerine, CMKLR1, etaient significativement augmente sur les cellules musculaires lisses d’artere pulmonaire (CML-AP) de patients SSc-PID et de patients SSc-HTAP. Nous avons ensuite teste l’effet de la chemerine sur la proliferation des CML-AP en stimulant les CML-AP de patients presentant une HTAP idiopathique (HTAPi) avec du serum de patients SSc avec et sans HTAP, en presence ou en l’absence d’un inhibiteur de CMKLR1. On confirmait que les CML-AP d’ HTAPi surexprimaient l’ARNm de CMKLR1 par rapport aux temoins sains (p = 0,03). Le serum des patients SSc-HTAP induisait une proliferation de CML-AP significativement plus elevee (p = 0,005) que le serum des temoins. Cette difference n’etait plus significative (p = 0,69) lors de l’ajout de l’inhibiteur CMKLR1 a-NETA. Conclusion La chemerine semble etre un biomarqueur de substitution valide des RVP dans l’HTAP-SSc. L’elevation de la chemerine circulante et la surexpression de son recepteur, CMKLR1, contribuent a la physiopathologie de l’HTAP-SSc en induisant la proliferation des CML-AP.

Introduction Nerve growth factor (NGF) plays a critical role in pathophysiology of pulmonary hypertension (PH) by promoting pulmonary arterial (PA) inflammation, remodelling and hyperreactivity. Aim To determine the mechanisms of NGF-induced PA hyperreactivity, focusing on Connexin 43 (Cx43), a gap junction protein essential for vascular reactivity. Methods Expression of NGF, its TrkA receptor and Cx43 were evaluated by Western blotting in human PA smooth muscle cells (hPASMC) from control donors or from patients suffering from idiopathic pulmonary arterial hypertension (iPAH). Cx43 expression and localisation were evaluated by Western blotting, immunofluorescence and surface biotinylation, after NGF treatment. Gap junction activity was assessed by FRAP microscopy. Contractions of rat PA were induced by phenylephrine (PHE), with or without NGF pre-treatment. The role of Cx43 in NGF-induced rat PA hyperreactivity was evaluated ex vivo by use of 43Gap26, a Cx43 blocking peptide, or in vivo by use of an anti-Cx43 siRNA. Results TrkA expression was increased in hPASMC from iPAH patients compared to control hPASMC. NGF increased Cx43 expression, plasma membrane localisation and phosphorylation, with a higher sensitivity in iPAH cells compared to controls. In both cell types, this increase was abolished with K252a, a TrkA kinase inhibitor. Activity of Cx43-dependent GAP junctions was also increased after NGF treatment, with iPAH hPASMC being more sensitive to NGF than control cells. NGF-induced PA hyperreactivity to PHE was inhibited after treatment with 43Gap26 or with anti-Cx43 siRNA. Conclusion NGF, through activation of its TrkA receptor, increases Cx43 expression and plasma membrane localisation in hPASMC. This mechanism enhances Cx43-dependent GAP junction activity and contributes to PA hyperreactivity. Moreover, NGF pathway is upregulated in iPAH hPASMC compared to control hPASMC, and iPAH cells are more sensitive to NGF than control cells regarding Cx43 increased expression and Cx43-dependent GAP junction increased activity.

Introduction In pulmonary arterial hypertension (PAH), the current therapies do not target pulmonary vascular remodeling (PVR), the main cause of the disease. Type I cytokine receptors (TypeIR) are known to be overexpressed in PAH pulmonary artery smooth muscle cells (PA-SMCs) and involved in PVR. Aims and objectives To assess whether the regulation of TypeIR intracellular trafficking is altered in PAH, leading to TypeIR overexpression and PVR. Methods We studied TypeIR trafficking through the main regulatory proteins ubiquitin-specific protease 8 (USP8) and ring finger protein (RNF) 41. In PAH patients and in controls, we performed in situ immunostaining in lung samples and western blotting in primary cultures of PA-SMCs isolated from human tissues. We transfected human PA-SMCs with siRNA against USP8 and RNF41 in vitro. In two experimental models, the monocrotaline- and chronic hypoxia-induced PAH, we performed hemodynamic measurements followed by in situ immunostaining and western blotting analysis in lung tissues. Results We showed an increased USP8 and decreased RNF41 expression in PAH lungs compared to controls, and a similar shift in USP8/RNF41 ratio at the protein level in PAH PA-SMCs compared to controls (n = 10). We modulated USP8/RNF41 ratio with siRNAs and restored TypeIR expression and its signalling (n = 5). Consistent with our human in situ and in vitro results, we confirmed the altered USP8/RNF41 ratio in the two in vivo models (n = 8 and n = 5). Conclusions TypeIR intracellular trafficking regulation is altered in PAH patients compared to controls, due to a shift in USP8/RNF41 ratio. Restoring USP8/RNF41 ratio in PA-SMCs may represent a potential target in PAH.

Purpose of review Pulmonary arterial hypertension (PAH) is a hemodynamic state defined by a resting mean pulmonary arterial pressure at or above 25 mmHg with a normal pulmonary capillary wedge pressure, ultimately leading to right heart failure and premature death. Although considerable progress has been made in the development of drug therapies for PAH targeting abnormalities found in the three main pathobiologic pathways (nitric oxide, prostacyclin, and endothelin-1), there is no drug available to specifically stop the progressive cellular accumulation into the pulmonary artery vessel wall. Indeed, this pulmonary vascular remodeling is a key pathological feature in PAH, contributing to the progressive narrowing of the lumen responsible to the functional decline and to the right ventricle hypertrophy and dysfunction. Recent findings Because numerous important discoveries in the PAH pathogenesis have been recently made, our improved understanding of additional pathways in this condition will presumably lead to the development of novel and more powerful therapeutic strategies in the near future. Summary In this review, we highlight some recent biological findings and discuss the opportunities that could lead to the identification of new promising targets in PAH paving the way for future therapeutic strategies.

ObjectiveTo evaluate the antifibrotic effects of the pan-peroxisome proliferator-activated receptor (PPAR) agonist IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH).MethodsIVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterised by non-specific interstitial pneumonia and severe vascular remodelling of pulmonary arteries leading to PH. Mice received two doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks.ResultsIVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis in both mouse models compared with mice receiving 30 mg/kg of IVA337 or vehicle. Histological score was markedly reduced by 61% in the bleomycin model and by 50% in Fra-2 transgenic mice, and total lung hydroxyproline concentrations decreased by 28% and 48%, respectively, as compared with vehicle-treated mice. IVA337 at 100 mg/kg also significantly decreased levels of fibrogenic markers in lesional lungs of both mouse models. In addition, IVA337 substantially alleviated PH in Fra-2 transgenic mice by improving haemodynamic measurements and vascular remodelling. In primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner fibroblast to myofibroblasts transition induced by TGF-β and fibroblast proliferation mediated by PDGF.ConclusionWe demonstrate that treatment with 100 mg/kg IVA337 prevents lung fibrosis in two complementary animal models and substantially attenuates PH in the Fra-2 mouse model. These findings confirm that the pan-PPAR agonist IVA337 is an appealing therapeutic candidate for these cardiopulmonary involvements.

Despite important advances in its therapeutic management, pulmonary arterial hypertension (PAH) remains an incurable disease. Although numerous drugs exhibited beneficial effects in preclinical settings, only few have reached clinical trial phases, highlighting the challenges of translating preclinical investigations into clinical trials. Potential reasons for delayed PAH drug development may include the inherent limitations of the currently available animal and in vitro models, potential lack of appropriate standardization of the experimental design, regulatory agencies requirements, competing clinical trials and insufficient funding. Although this is not unique to PAH, there is urgency for reducing the number of false positive signals in preclinical studies and optimizing the development of innovative therapeutic targets through performance of clinical trials based on more robust experimental data. The current review discusses the challenges and opportunities in preclinical research to foster drug development in PAH.

International audience

Introduction Pulmonary arterial hypertension (PAH) is a chronic disease characterized by an increase in pulmonary artery (PA) resistance leading to right ventricle (RV) failure. Endothelial dysfunction and alteration of NO/cGMP signaling play a major role in PAH. We previously described the major role of smooth muscle cell (SMC) Rac1 in NO-induced dilation of systemic arteries. Objective Here, our aim was to analyze the role of SMC Rac1 in pulmonary circulation and in PAH. Method Rac1 activity was assessed by immunofluorescence in lung biopsies. PAH has been induced by exposure of wild-type (WT) and SMC Rac1-deficient mice (SM-Rac1-KO mice) to chronic hypoxia (10% O2, 4 weeks). In vitro, the proliferation of PASMC in hypoxic condition was measured by EdU staining and contractility of PA rings was analyzed by myography. Oxidative stress was assessed using the DHE probe and by electron paramagnetic resonance (EPR). Results A 3-fold increase of Rac-GTP in PA was observed in PAH patients compared to control (n = 4-6). To determine whether Rac1 activation plays a causal role in PAH, WT and SM-Rac1-KO mice were exposed to hypoxia. As expected, exposure to chronic hypoxia induced an increase in systolic pressure in RV and RV hypertrophy which were reduced by 32.5% and 60% respectively, in SM-Rac1-KO mice compared to WT (n = 5–7). PA remodeling was also significantly reduced in hypoxic SM-Rac1-KO compared to WT mice, while PA contractility was not affected by SMC Rac1 deletion. In vitro, exposure of primary PASMC to hypoxia stimulated proliferation and ROS production that were decreased by 50% and 66%, respectively, by the Rac1 inhibitor, EHT1864. Conclusion Our results show that SMC Rac1 activity is increased in PA of PAH patients and plays a causal role in hypoxia-induced PAH in mice by stimulating ROS-dependent PA remodeling. These data suggest that pharmacological Rac1 inhibition might have a beneficial effect in the treatment of PAH.

Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in pulmonary arterial pressures. The discovery of heterozygous BMPR2 germline mutations as critical predisposing factors together with a remarkable progress in our understanding of the pathogenic mechanisms have helped identify the significant and complex roles of the BMPRII axis in PAH. However, their precise contributions to the condition are still incompletely understood. Areas covered: This review aims to assemble and discuss the cellular actions of BMPs together with the possible clinical applications and prospects for their use in the near future. Expert opinion: Although major advances have been made, several questions remain unanswered regarding development of efficacious therapies targeting the BMPRII axis in PAH. Specifically, the reasons why BMPRII signaling is reduced in PAH and how the alterations influence or even drive the pathogenesis need to be understood. Because the BMPRII axis is ubiquitously expressed and exhibits a wide variety of functions in organ regeneration and homeostasis, a better understanding of the overall risk-benefit ratio of these strategies with long-term follow-up is needed. This knowledge will lay the foundation for discovery of innovative therapeutics for PAH.

Introduction: Heritable PAH due to BMPR2 mutation is an autosomal dominant disease with incomplete penetrance. To date, no strategy has proven its efficacy for early PAH screening in asymptomatic relatives carrying a BMPR2 mutation. Objectives: To evaluate efficacy of PAH screening in this high risk population. Methods: We prospectively followed for 2 years a cohort of asymptomatic subjects carrying a BMPR2 mutation (DELPHI-2 study NCT01600898), continued by an optional long-term extension. Initial and yearly assessment included NYHA functional class (FC), EKG, chest X-ray, 6-min walk distance (6MWD), pulmonary function tests, cardiopulmonary exercise testing, echocardiography and biological testing. Right heart catheterization (RHC) at rest and exercise was proposed at inclusion or at follow-up if PAH was suspected based on predefined criterion. Results: 55 adult subjects (26 males, 29 females) with a median age of 37 yo (min-max 18-78) were included. At inclusion, all subjects were in NYHA FC I with normal 6MWD (median 533 m, min-max 368-693) and normal NT-proBNP. No PAH was suspected based on echocardiography. All 55 subjects accepted RHC at inclusion allowing to identify 2 females (25 and 78yo) with mild PAH (mPAP 25 and 26 mmHg) and 12 subjects (22%) with exercice PH of unclear significance. At follow-up (>200 patient-year), two additional cases were diagnosed among the subjects with exercise PH. In all detected patients, early treatment maintained a low-risk profile with oral PAH therapy. Conclusion: Screening allowed early PAH diagnosis in asymptomatic BMPR2 mutation carriers. This study will describe early biomarkers of PAH in this population.

The pathophysiology of pulmonary arterial hypertension (PAH) induced by protein kinase inhibitors (PKIs) remains unclear. To gain knowledge into this rare and severe pathology we performed a study combining a pharmacovigilance approach and the pharmacodynamic properties of PKIs.A disproportionality analysis on the World Health Organization pharmacovigilance database VigiBase using the reporting odds ratio (ROR) and 95% confidence interval was first performed. Then, we identified the most relevant cellular targets of interest through a systematic literature review and correlated the pharmacovigilance signals with the affinity for the different PKIs. We further performed a hierarchical cluster analysis to assess patterns of binding affinity.A positive disproportionality signal was found for dasatinib, bosutinib, ponatinib, ruxolitinib and nilotinib. Five non-receptor protein kinases significantly correlate with disproportionality signals: c-Src (r=0.79, p=0.00027), c-Yes (r=0.82, p=0.00015), Lck (r=0.81, p=0.00046) and Lyn (r=0.80, p=0.00036), all belonging to the Src protein kinase family, and TEC (r=0.85, p=0.00006). Kinases of the bone morphogenetic protein signalling pathway also seem to play a role in the pathophysiology of PKI-induced PAH. Interestingly, the dasatinib affinity profile seems to be different from that of other PKIs in the cluster analysis.The study highlights the potential role of the Src protein kinase family and TEC in PAH induced by PKIs. This approach combining pharmacovigilance and pharmacodynamics data allowed us to generate some hypotheses about the pathophysiology of the disease; however, the results have to be confirmed by further studies.

The impact of bone morphogenetic protein receptor 2 (BMPR2) gene mutations on vascular remodelling in pulmonary arterial hypertension (PAH) is unknown. We sought to identify a histological profile of BMPR2 mutation carriers.Clinical data and lung histology from 44 PAH patients were subjected to systematic analysis and morphometry.Bronchial artery hypertrophy/dilatation and bronchial angiogenesis, as well as muscular remodelling of septal veins were significantly increased in PAH lungs carrying BMPR2 mutations. We found that patients displaying increased bronchial artery remodelling and bronchial microvessel density, irrespective of the mutation status, were more likely to suffer from severe haemoptysis. History of substantial haemoptysis (>50 mL) was significantly more frequent in BMPR2 mutation carriers. 43.5% of BMPR2 mutation carriers, as opposed to 9.5% of noncarriers, displayed singular large fibrovascular lesions, which appear to be closely related to the systemic lung vasculature.Our analysis provides evidence for the involvement of the pulmonary systemic circulation in BMPR2 mutation-related PAH. We show that BMPR2 mutation carriers are more prone to haemoptysis and that haemoptysis is closely correlated to bronchial arterial remodelling and angiogenesis; in turn, pronounced changes in the systemic vasculature correlate with increased pulmonary venous remodelling, creating a distinctive profile in PAH patients harbouring a BMPR2 mutation.

Coronavirus disease 2019 (COVID-19) represents a public health crisis of pandemic proportions. Caused by SARS-CoV-2, which stands for severe acute respiratory syndrome (SARS) coronavirus 2, the symptoms most commonly reported include cough, fever, and shortness of breath, but also extra-pulmonary symptoms, such as neurological and gastroenterological manifestations.

Tyrosine kinase inhibitors (TKIs) targeting the Bcr-Abl oncoprotein revolutionised the treatment of chronic myelogenous leukaemia. Following the success of imatinib, second- and third-generation molecules were developed. Different profiles of kinase inhibition and off-target effects vary between TKIs, which leads to a broad spectrum of potential toxicities.Pulmonary complications are most frequently observed with dasatinib but all other Bcr-Abl TKIs have been implicated. Pleural effusions are the most frequent pulmonary complication of TKIs, usually associated with dasatinib and bosutinib. Pulmonary arterial hypertension is an uncommon but serious complication of dasatinib, which is often reversible upon discontinuation. Bosutinib and ponatinib have also been associated with pulmonary arterial hypertension, while imatinib has not. Rarely, interstitial lung disease has been associated with TKIs, predominantly with imatinib.Mechanistically, dasatinib affects maintenance of normal pulmonary endothelial integrity by generating mitochondrial oxidative stress, inducing endothelial apoptosis and impairing vascular permeability in a dose-dependent manner. The mechanisms underlying other TKI-related complications are largely unknown. Awareness and early diagnosis of the pulmonary complications of Bcr-Abl TKIs is essential given their seriousness, potential reversibility, and impact on future treatment options for the underlying chronic myelogenous leukaemia.

Objectives:To investigate alterations in the serum proteome of patients with systemic sclerosis (SSc)-associated pulmonary hypertension (PAH), to identify proteins that correlated with hemodynamic severity and to determine their possible pathogenic role.Methods:Patients were included if they fulfilled the following criteria: diagnosis of limited cutaneous SSc, no extensive interstitial lung disease (ILD), and no treatment with PAH-specific therapy. Patients were classified as cases if they had a definitive diagnosis of PAH confirmed by right heart catheterization (RHC) and a serum sample collected on the same day as RHC. They were classified as controls if they had no sign suggestive of PAH on echocardiography.Results:In a first exploratory step, serum expression of 1129 proteins was assessed in 15 cases and 16 controls by a high-throughput proteomic assay (SOMAscan). We identified 53 proteins differentially expressed between the 2 groups. Among these 53 candidates, only 2 correlated significantly with pulmonary vascular resistance (PVR): chemerin (p=0.01,ρ=0.62) and SET nuclear proto-oncogen (SET) (p=0.01,ρ=0.62).To validate these results, serum levels of chemerin and SET were measured by ELISA assay in 25 additional cases and 19 additional controls. Chemerin levels were confirmed to be significantly higher in cases (p=0.01) and correlated with PVR (p=0.01,ρ=0.46).In a second step, to study the potential pathophysiological role of chemerin, we performed confocal immunofluorescence analyses on explanted lungs of healthy controls, SSc-ILD without PAH and SSc-PAH patients. Chemerin receptor, CMKLR1, was significantly increased on SSc-ILD and SSc-PAH pulmonary artery smooth muscle cells (PA-SMC).We then tested the effect of chemerin on PA-SMC proliferation by stimulating PA-SMCs from idiopathic pulmonary arterial hypertension (iPAH) patients with serum from SSc patients with and without PH, in the presence or absence of a CMKLR1 inhibitor. PA-SMCs from iPAH were confirmed to have higher mRNA expression of CMKLR1 than controls (p=0.03). Serum from SSc-PH patients induced a significantly higher PA-SMC proliferation (p=0.005) than serum from controls. This difference was no longer significant (p=0.69) when adding the CMKLR1 inhibitor α–NETA.Conclusion:Chemerin is a surrogate biomarker for PVR in SSc-PAH. Increased chemerin and its receptor, CMKLR1, contribute to the SSc-PAH pathogenesis by inducing PA-SMC proliferation.Acknowledgments:NoneDisclosure of Interests:Sebastien SANGES: None declared, Lisa Rice: None declared, Ly Tu: None declared, Jean-Luc Cracowski Grant/research support from: JL Cracowski received grants from United Therapeutic, Bioprojet and Topadur, David Montani Grant/research support from: Dr. MONTANI reports grants and personal fees from Actelion, grants and personal fees from Bayer, personal fees from GSK, personal fees from Pfizer, personal fees from MSD, personal fees from Chiesi, outside the submitted work, Julio Mantero: None declared, Camille Ternynck: None declared, Guillemette Marot: None declared, Eric Hachlla: None declared, David Launay Grant/research support from: Dr. Launay reports personal fees from Actelion, grants and personal fees from Takeda, grants and personal fees from CSL Behring, outside the submitted work., Marc Humbert Grant/research support from: Dr. Humbert reports personal fees from Actelion, grants and personal fees from Bayer, grants and personal fees from GSK, personal fees from Merck, from United Therapeutics, personal fees from Acceleron, outside the submitted work., Christophe Guignabert: None declared, Robert Lafyatis Grant/research support from: RL holds research grants from Formation, Elpidera and Kiniksa., Consultant of: R.L. has served as a consultant for Bristol Myers Squibb, Boehringer-Mannheim, Merck, Magenta and Genentech/Roche