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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.

International audience; no abstract

Background: In pulmonary arterial hypertension (PAH), pathological changes in pulmonary arterioles progressively raise pulmonary artery pressure and increase pulmonary vascular resistance, leading to right heart failure and high mortality rates. Recently, the first potassium channelopathy in PAH, because of mutations in KCNK3 , was identified as a genetic cause and pharmacological target. Methods: Exome sequencing was performed to identify novel genes in a cohort of 99 pediatric and 134 adult-onset group I PAH patients. Novel rare variants in the gene identified were independently identified in a cohort of 680 adult-onset patients. Variants were expressed in COS cells and function assessed by patch-clamp and rubidium flux analysis. Results: We identified a de novo novel heterozygous predicted deleterious missense variant c.G2873A (p.R958H) in ABCC8 in a child with idiopathic PAH. We then evaluated all individuals in the original and a second cohort for rare or novel variants in ABCC8 and identified 11 additional heterozygous predicted damaging ABCC8 variants. ABCC8 encodes SUR1 (sulfonylurea receptor 1)—a regulatory subunit of the ATP-sensitive potassium channel. We observed loss of ATP-sensitive potassium channel function for all ABCC8 variants evaluated and pharmacological rescue of all channel currents in vitro by the SUR1 activator, diazoxide. Conclusions: Novel and rare missense variants in ABCC8 are associated with PAH. Identified ABCC8 mutations decreased ATP-sensitive potassium channel function, which was pharmacologically recovered.

Introduction: Pulmonary hypertension (PH) is a well-recognized complication of idiopathic pulmonary fibrosis (iPF) that is characterized by chronic inflammation and scar tissue formation. Macrophage migration inhibitory factor (MIF) and its signaling through CD74 have been identified as key contributors at the crossroad of inflammation and pulmonary endothelial dysfunction in PH development. Aims of the study: We investigated the activation state of MIF/CD74 axis in iPF patients with or without PH and tested the efficacy of MIF inhibition in a murine model of bleomycin (BLM)-induced lung fibrosis. Methods: We examined the expression and distribution pattern of MIF and CD74 in lungs of patients with iPF with or without PH as well as in lungs of BLM-induced mice. We evaluated pulmonary hemodynamics, vascular remodeling, inflammatory cell infiltration and collagen deposition in mice treated daily during 21 days with two specific MIF antagonists: the (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester34 (ISO-1) and the N-(5-methylphenyl)-benzoxazol-2-one (compound 31). Results: In contrast to lung tissues of control and iPF patients, MIF and CD74 expressions are markedly up-regulated in the dysfunctional pulmonary endothelium of remodeled arteries in lungs of iPF patients with PH. Consistent with these observations in human lung tissues, we found that chronic treatments with the two MIF antagonist ISO-1 or compound 31 partially decreased the collagen deposition and attenuated PH development in mice. Conclusions: These data indicate that MIF and CD74 are markedly increased and activated in iPF patients with PH. MIF antagonists may be interesting agents for investigation in patients with iPF.

Pulmonary hypertension (PH), an increased blood pressure within the pulmonary vasculature, is a devastating and progressive disease. PH is characterized by increased proliferation of the pulmonary artery smooth muscle cells (PASMC) and subsequent remodeling of the pulmonary arteries. Activation of the ADORA2B receptor on smooth muscle cells has previously been implicated in vascular tissue remodeling. We hypothesize that activation of the ADORA2B receptor in PASMC mediates the development of pulmonary vascular remodeling. We evaluated the development of pulmonary vascular remodeling in two distinct mouse models. We used mice that have ADORA2B conditionally deleted from smooth muscle cells and corresponding controls. Mice were exposed to a combination of SUGEN and hypoxia for 28 days or alternatively, a bi-weekly exposure to bleomycin for 33 days. Additionally, we exposed primary human PASMC to an ADORA2B agonist and evaluated hyaluronan synthase 2 (HAS2) and interleukin-6 (IL-6) expression. Our data demonstrates that conditional deletion of ADORA2B from smooth muscle cells protected the mice against the development of PH features in both mouse models. Remarkably, the mice with conditional deletion of ADORA2B exposed to bleomycin, were not protected against the development of fibrosis. Our in vitro studies using PASMC revealed that activation of the ADORA2B receptor resulted in increased expression of HAS2 and IL-6, both important mediators of vascular remodeling. Taken together, our in vivo and in vitro data indicate that ADORA2B on PASMC mediates the development of pulmonary vascular remodeling. Importantly, these studies also point at ADORA2B as a potential therapeutic target to treat PH.

Background We aimed to investigate the efficacy of abatacept in preclinical mouse models of digestive involvement, pulmonary fibrosis, and related pulmonary hypertension (PH), mimicking internal organ involvement in systemic sclerosis (SSc). Methods Abatacept has been evaluated in the chronic graft-versus-host disease (cGvHD) mouse model (abatacept 1 mg/mL for 6 weeks), characterized by liver and intestinal fibrosis and in the Fra-2 mouse model (1 mg/mL or 10 mg/mL for 4 weeks), characterized by interstitial lung disease (ILD) and pulmonary vascular remodeling leading to PH. Results In the cGvHD model, abatacept significantly decreased liver transaminase levels and markedly improved colon inflammation. In the Fra-2 model, abatacept alleviated ILD, with a significant reduction in lung density on chest microcomputed tomography (CT), fibrosis histological score, and lung biochemical markers. Moreover, abatacept reversed PH in Fra-2 mice by improving vessel remodeling and related cardiac hemodynamic impairment. Abatacept significantly reduced fibrogenic marker levels, T-cell proliferation, and M1/M2 macrophage infiltration in lesional lungs of Fra-2 mice. Conclusion Abatacept improves digestive involvement, prevents lung fibrosis, and attenuates PH. These findings suggest that abatacept might be an appealing therapeutic approach beyond skin fibrosis for organ involvement in SSc. Electronic supplementary material The online version of this article (10.1186/s13075-018-1694-9) contains supplementary material, which is available to authorized users.

Pulmonary hypertension (PH) is defined by a mean pulmonary artery pressure of at least 25 mmHg during resting right heart catheterization. PH is not a single disease, but a haemodynamic feature found in a rather large group of diseases that can result from pre-capillary (arterial) or post-capillary (venous) pathophysiological mechanisms. The current PH clinical classification gathers together groups of PH that share similar haemodynamic criteria and types of pulmonary vascular lesions in order to optimize therapeutic approaches, to predict patient outcomes, and facilitate research strategies. However, the five different PH groups exhibit several common points and key regulators are shared, and they include a combination of the following in varying degrees of importance: pulmonary endothelial dysfunction, significant accumulation of pulmonary vascular cells, excessive lung recruitment of inflammatory cells, and the presence of specific genetic mutations and environmental factors. This chapter aims to outline the key cellular and molecular mechanisms contributing to the sustained remodelling of the pulmonary vascular bed in PH and discuss the urgent need of additional therapies directly targeting the pulmonary vascular remodelling in the future management of PH.

Comment onLower DHEA-S levels predict disease and worse outcomes in post-menopausal women with idiopathic, connective tissue disease- and congenital heart disease-associated pulmonary arterial hypertension. [Eur Respir J. 2018]; International audience; no abstract

Background Cytotoxic T-lymphocyte associated molecule-4 (CTLA-4) is an immunoregulatory membrane receptor resulting in the down-regulation of T-cell responses. A previous report showed that abatacept (CTLA4-Ig) prevented and induced regression of inflammation-driven dermal fibrosis in two different mouse models of systemic sclerosis (SSc). 1 Objectives We aimed to assess the effects of abatacept in two complementary mouse models reflecting digestive involvement, lung fibrosis and pulmonary hypertension (PH), mimicking severe SSc organ damage. Methods Abatacept was given in the chronic graft-versus-host disease (cGvHD) mouse model, characterised by digestive involvement, and in the Fra-2 mouse model which is characterised by non-specific interstitial pneumonia and pulmonary vascular remodelling leading to PH. Mice were treated by intraperitoneal injections of abatacept (1 mg/mL in the cGvHD model for 6 weeks; 1 mg/mL or 10 mg/mL in the Fra-2 mouse model for 4 weeks) or human IgG1 (100 mg) used as a negative control. Results In the cGvHD model, treatment of allogeneically mice with abatacept led to a significant reduction of alanine aminotransferase (24%, p=0.014) and aspartate aminotransferase levels (61%, p When assessed by chest micro-CT imaging, Fra-2 mice treated with abatacept displayed a 12% decrease in lung density (10 mg/ml, p=0.037) as well as an increase in functional residual capacity as compared to IgG1-treated mice (16% for 1 mg/ml, p=0.001% and 14% for 10 mg/ml, p=0.005). Consistent with these results, abatacept 10 mg/L decreased histological fibrosis score (Ashcroft score) as well as hydroxyproline content by 79% (p=0.009) and 31% (p=0.044) respectively, as compared to IgG1-treated mice. Treatment with abatacept 10 mg/mL markedly reduced protein levels in the lesional lungs of Fra-2 mice of the fibrogenic markers MCP1 by 79% (p=0.043) and osteopontin by 87% (p=0.039). Levels of TGF-b were also reduced with abatacept (61% for 1 mg/mL, p=0.037% and 69% for 10 mg/mL, p=0.013). Further, abatacept decreased M1 and M2 macrophages infiltration as well as T-cell proliferation in the lesional lungs of Fra-2 mice. Upon treatment with abatacept a reduction of right ventricular systolic pressure (28.1±1.5 mmHg vs 36.0±5.1 mmHg, p=0.037 for 10 mg/mL) and right ventricular hypertrophy (0.29±0.01 vs 0.33±0.010, p=0.037 and 29±0.01% vs 0.33±0.01% for 10 mg/mL, p=0.037) was observed compared to IgG1-treated mice. Consistent with these findings, abatacept 10 mg/mL was associated with significant decrease in percent medial wall thickness and numbers of muscularized distal pulmonary arteries. Conclusions We demonstrate that treatment with abatacept improves digestive involvement, prevents lung fibrosis and attenuates PH in SSc pre-clinical mice models. These findings suggest that abatacept might be an appealing therapeutic approach for severe internal organ involvement in SSc beyond its already demonstrated effects on skin fibrosis. Reference [1] Ponsoye M, et al. Ann Rheum Dis2016Dec;75(12):2142–9. Disclosure of Interest None declared

Experimental models are critical for the understanding of lung health and disease and are indispensable for drug development. However, the pathogenetic and clinical relevance of the models is often unclear. Further, the use of animals in biomedical research is controversial from an ethical perspective.The objective of this task force was to issue a statement with research recommendations about lung disease models by facilitating in-depth discussions between respiratory scientists, and to provide an overview of the literature on the available models. Focus was put on their specific benefits and limitations. This will result in more efficient use of resources and greater reduction in the numbers of animals employed, thereby enhancing the ethical standards and translational capacity of experimental research.The task force statement addresses general issues of experimental research (ethics, species, sex, age,ex vivoandin vitromodels, gene editing). The statement also includes research recommendations on modelling asthma, chronic obstructive pulmonary disease, pulmonary fibrosis, lung infections, acute lung injury and pulmonary hypertension.The task force stressed the importance of using multiple models to strengthen validity of results, the need to increase the availability of human tissues and the importance of standard operating procedures and data quality.

Pulmonary arterial hypertension (PAH) is characterized by a progressive accumulation of pulmonary artery smooth muscle cells (PA-SMCs) in pulmonary arterioles leading to the narrowing of the lumen, right heart failure, and death. Although most studies have supported the notion of a role for IL-6/glycoprotein 130 (gp130) signaling in PAH, it remains unclear how this signaling pathway determines the progression of the disease. Here, we identify ectopic upregulation of membrane-bound IL-6 receptor (IL6R) on PA-SMCs in PAH patients and in rodent models of pulmonary hypertension (PH) and demonstrate its key role for PA-SMC accumulation in vitro and in vivo. Using Sm22a-Cre Il6rfl/fl, which lack Il6r in SM22A-expressing cells, we found that these animals are protected against chronic hypoxia-induced PH with reduced PA-SMC accumulation, revealing the potent pro-survival potential of membrane-bound IL6R. Moreover, we determine that treatment with IL6R-specific antagonist reverses experimental PH in two rat models. This therapeutic strategy holds promise for future clinical studies in PAH.

Macrophage migration inhibitory factor (MIF) is a key pleiotropic mediator and a promising therapeutic target in cancer as well as in several inflammatory and cardiovascular diseases including pulmonary arterial hypertension (PAH). Here, a novel series of N-(phenylmethyl)-benzoxazol-2-thiones 5-32 designed to target the MIF tautomerase active site was synthesized and evaluated for its effects on cell survival. Investigation of structure-activity relationship (SAR) particularly at the 5-position of the benzoxazole core led to the identification of 31 that potently inhibits cell survival in DU-145 prostate cancer cells and pulmonary endothelial cells derived from patients with idiopathic PAH (iPAH-ECs), two cell lines for which survival is MIF-dependent. Molecular docking studies helped to interpret initial SAR related to MIF tautomerase inhibition and propose preferred binding mode for 31 within the MIF tautomerase active site. Interestingly, daily treatment with 31 started 2 weeks after a subcutaneous monocrotaline injection regressed established pulmonary hypertension in rats.

An obesity paradox, wherein patients who are obese have lower mortality, has been described in cardiopulmonary diseases, including pulmonary arterial hypertension (PAH). Our objective was to determine whether obesity and BMI are associated with mortality in patients with PAH.We assessed incident patients with idiopathic, drug-induced, and heritable PAH from the French Pulmonary Hypertension Network registry. Cox regression and Kaplan-Meier analysis were used to assess the association between BMI and obesity with all-cause mortality.Of 1,255 patients included, 30% were obese. A higher proportion of women (65.1% vs 53.4%, P .01), drug-induced PAH (28.9% vs 9.2%, P .01), systemic hypertension, diabetes, and hypothyroidism were present in the obese group. More obese patients were in New York Heart Association class III (66.4% vs 57.1%), fewer were class IV (11.8% vs 16.9%, P .01), and 6-min walk distance was lower (276 ± 121 vs 324 ± 146, P .01). Right atrial pressure, pulmonary wedge pressure, and cardiac index were higher, whereas pulmonary vascular resistance was lower in patients who were obese. Neither BMI (hazard ratio [HR], 0.99; 95% CI, 0.97-1.01; P = .41) nor obesity (HR, 1.0; 95% CI, 0.99-1.01; P = .46) were associated with mortality in multivariable analyses. There was a significant interaction between age and obesity such that mortality increased among patients 65 years of age who were morbidly obese (HR, 3.01; 95% CI, 1.56-5.79; P = .001).Obesity was not associated with mortality in the overall population, but there was an age-obesity interaction with increased mortality among young patients who were morbidly obese. These results have implications for active weight management in younger patients who are morbidly obese who are otherwise candidates for lung transplantation.

Pleural effusion is a frequent side-effect of dasatinib, a second-generation tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia. However, the underlying mechanisms remain unknown. We hypothesised that dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion.To test this, we established the first animal model of dasatinib-related pleural effusion, by treating rats with a daily regimen of high doses of dasatinib (10 mg·kg−1·day−1for 8 weeks).Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks. Consistent with thesein vivoobservations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of vascular endothelial cadherin and zonula occludens-1 from cell–cell junctions, and the development of actin stress fibres. These results were replicated using human umbilical vein endothelial cells and confirmed by decreased endothelial resistance. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanismin vitroandin vivousing a cotreatment with an antioxidant agent,N-acetylcysteine.This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent mannerin vitroandin vivoleading to pleural effusion.

More studies are needed to measure nasal decongestant exposure in patients with pulmonary arterial hypertension http://ow.ly/OFxs8

Pulmonary arterial hypertension (PAH) is a disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in mean pulmonary arterial pressure, resulting in a progressive functional decline despite current available therapeutic options. The fundamental pathogenetic mechanisms underlying this disorder include pulmonary vasoconstriction, in situ thrombosis, medial hypertrophy, and intimal proliferation, leading to occlusion of the small to mid-sized pulmonary arterioles and the formation of plexiform lesions. Several predisposing or promoting mechanisms that contribute to excessive pulmonary vascular remodeling in PAH have emerged, such as altered crosstalk between cells within the vascular wall, sustained inflammation and dysimmunity, inhibition of cell death, and excessive activation of signaling pathways, in addition to the impact of systemic hormones, local growth factors, cytokines, transcription factors, and germline mutations. Although the spectrum of therapeutic options for PAH has expanded in the last 20 years, available therapies remain essentially palliative. However, over the past decade, a better understanding of new key regulators of this irreversible pulmonary vascular remodeling has been obtained. This review examines the state-of-the-art potential new targets for innovative research in PAH, focusing on (1) the crosstalk between cells within the pulmonary vascular wall, with particular attention to the role played by dysfunctional endothelial cells; (2) aberrant inflammatory and immune responses; (3) the abnormal extracellular matrix function; and (4) altered BMPRII/KCNK3 signaling systems. A better understanding of novel pathways and therapeutic targets will help in the designing of new and more effective approaches for PAH treatment.

Pulmonary complications of bosutinib therapy and potential cross-intolerance with dasatinibhttp://ow.ly/UKWu303KGhx

Heterozygous germline mutations in the Bone Morphogenetic Protein Receptor type 2 (BMPR2) gene have been identified as critical genetic factors predisposing to pulmonary arterial hypertension (PAH) development. At the endothelial cell surface, BMPRII forms heterocomplexes with its co-receptor activin receptor like kinase 1 (ALK1), which are bound by the ligand BMP9, a promising therapeutic target in PAH. Therefore, a better knowledge on the contribution of BMP9 and its signaling pathway to the pulmonary vascular remodeling is clearly needed. We have used Bmp9-KO mice, that are viable and fertile, and two experimental models of pulmonary hypertension: the chronic hypoxia (CHx) and the monocrotaline pyrrole (MCTp). We found that Bmp9-KO mice developed mild increase in right ventricular systolic pressure (RVSP) in room air when compared with wildtype (WT) littermates. Surprisingly, we found that they are less susceptible to develop CHx- and MCTp-induced PH, with significantly lower values in RVSP, right ventricular hypertrophy (RVH), assessed by the Fulton index and pulmonary-artery muscularization when compared with WT. Consistent with these findings, mice treated with an anti–BMP9 neutralizing antibody and rats treated with a soluble ALK1/extracellular domain (ECD) Fc-fusion protein were protected against the increase in RVSP, RVH and muscularization of pulmonary arteries induced by experimental PAH models. Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, protects rodents against the development of experimental PAH. These findings are apparently counter-intuitive and further studies are needed for a better understanding of BMP9 signaling pathway in PAH.

Introduction: C-X-C motif chemokine 12 (CXCL12) can directly control cell proliferation, migration and differentiation via interaction with either CXCR4 or CXCR7, and represents a promising target in pulmonary hypertension (PH). However, the efficacy of a CXCL12 neutraligand has never been tested in animal models of severe PH. Objective: To test and compare the efficacy of a CXCL12 neutraligand, chalcone-4, versus a CXCR4 antagonist, AMD3100, in the Sugen 5416/ hypoxia (SuHx) rat model, in a curative approach. Methods & Results: Height weeks after the Sugen 5416 administration, a substantial decrease in values of total pulmonary vascular resistance and of Fulton index (assessing right ventricular hypertrophy) were found in SuHx rats treated with chalcone-4 (100mg/kg/day for 2 weeks) and AMD3100 (5mg/kg/day for 2 weeks) when compared with the SuHx rats treated with vehicle. Interestingly, a substantial increase in values of cardiac output in SuHx rats treated with chalcone-4 was found when compared with the SuHx rats treated with AMD3100. Consistent with these observations, chalcone-4 and AMD3100 substantially attenuated the pulmonary vascular remodeling and the progressive narrowing of pulmonary arteries in the SuHx rat model. Conclusions: We report here a greater beneficial effect of chalcone-4 than AMD3100 treatment in the SuHx rat model of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.

Background Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors known to modulate fibrosis. The pan-PPAR agonist IVA337 recently demonstrated efficacy in prevention and treatment of experimental skin fibrosis (1). Objectives Our objective was to evaluate the antifibrotic effects of IVA337 in preclinical mouse models of pulmonary fibrosis and related pulmonary hypertension (PH). Methods IVA337 has been evaluated in the mouse model of bleomycin-induced pulmonary fibrosis and in Fra-2 transgenic mice, this latter being characterized by non-specific interstitial pneumonia and severe vascular remodeling of pulmonary arteries leading to PH. Mice received 2 doses of IVA337 (30 mg/kg or 100 mg/kg) or vehicle administered by daily oral gavage up to 4 weeks. Results Both 30 mg/kg and 100 mg/kg doses of IVA337 were well tolerated in all mouse models. IVA337 demonstrated at a dose of 100 mg/kg a marked protection from the development of lung fibrosis induced by bleomycin compared to mice receiving 30 mg/kg of IVA337 or vehicle. Indeed, IVA337 (100 mg/kg) strongly reduced by 61% and 28% tissue density on histological measurements and total lung hydroxyproline concentrations, respectively, as compared to vehicle. IVA337 at 100 mg/kg also significantly decreased col1, col3 and fibronectin in lesional lungs. Similarly, Fra-2 transgenic mice treated with 100 mg/kg of IVA337 displayed reduced lung density (20% vs. vehicle) and significant increase of functional residual capacity (30% vs. vehicle) when assessed by chest micro-CT imaging. These results were emphasized by a 50% reduction of the Ascroft fibrosis score (Figure 1A) and by a 48% reduction of hydroxyproline concentrations upon IVA337 (100 mg/kg) compared to vehicle treated mice. Successful targeting of the TGF-b signaling axis was observed in both mouse models upon treatment with 100 mg/kg of IVA337. 100 mg/kg IVA337 also significantly reduced T cell and B cell infiltration in lesional lungs of Fra-2 transgenic mice. Regarding vessel remodeling and related pulmonary hypertension, treatment with 100 mg/kg of IVA337 led to a substantial attenuation of right ventricular systolic pressure and right ventricular hypertrophy compared to mice receiving the vehicle (Figure 1B and 1C). Furthermore, IVA337 given at 100 mg/kg markedly reduced medial wall thickness (Figure 1D) and the number of muscularized distal pulmonary arteries. In vitro in primary human lung fibroblasts, IVA337 inhibited in a dose-dependent manner TGFβ-mediated fibroblasts to myofibroblasts transition and PDGF-mediated proliferation. Conclusions We 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 systemic sclerosis both, for skin and key cardiovascular complications. References Ruzehaji et al, Ann Rheum Dis 2016;75(12):2175–2183. Disclosure of Interest J. Avouac: None declared, I. Konstantinova Employee of: INVENTIVA, C. Guignabert: None declared, J. Sadoine: None declared, G. Thomas: None declared, S. Pezet: None declared, A. Cauvet: None declared, L. Tu: None declared, J. M. Luccarini Employee of: INVENTIVA, J. L. Junien Consultant for: INVENTIVA, P. Broqua Employee of: INVENTIVA, Y. Allanore Grant/research support from: Inventiva

To elucidate the role of gene candidates involved in pulmonary hypertension (PH) associated with systemic sclerosis (SSc).Gene candidates were identified through microarray experiments performed on Affymetrix GeneChip Human Exon 1.0 ST arrays in endothelial progenitor cell (EPC)-derived endothelial cells (ECs) obtained from patients with SSc-associated PH, patients with SSc without PH, and healthy control subjects. Expression of identified gene candidates was assessed by quantitative sandwich enzyme-linked immunosorbent assay in the serum, and by immunohistochemistry in lesional lung tissue. The functional importance of the identified gene candidates was then evaluated in fos-related antigen 2-transgenic (Fra-2-Tg) mice that spontaneously develop SSc-like features associated with an intense pulmonary vascular remodeling.Microarray experiments revealed that the matrix metalloproteinase 10 (MMP-10) gene was the top up-regulated gene in SSc-associated PH EPC-derived ECs. Circulating serum proMMP10 concentrations were markedly increased in patients with SSc-associated PH compared to SSc patients without PH and healthy controls. Consistent with these observations, a strong MMP10 staining of the thickened wall of distal pulmonary arteries was found both in the lungs of patients with SSc-associated PH and in the lungs of Fra-2-Tg mice. Daily treatment of Fra-2-Tg mice with neutralizing anti-MMP10 antibodies did not significantly affect the development and severity of pulmonary fibrosis, but did reverse established PH and markedly reduced pulmonary vascular remodeling by reducing cell proliferation, cell survival, and the platelet-derived growth factor signaling axis.Gene expression profiling of EPC-derived ECs identified MMP10 as a novel candidate gene in SSc-associated PH. MMP10 is overexpressed in the serum and pulmonary arteries of patients with SSc-associated PH, and its blockade alleviates PH in the Fra-2-Tg mouse model. MMP10 appears to be a prospective treatment target for this devastating disorder.

This study aimed to describe the long-term outcomes of pulmonary arterial hypertension (PAH) induced by dasatinib.21 incident, right heart catheterisation-confirmed cases of dasatinib-induced PAH were identified from the French Pulmonary Hypertension Registry. Clinical and haemodynamic variables were compared from baseline to last follow-up (median (range) 24 (1–81) months).Median age was 52 years and 15 patients were female (71%). 19 patients received dasatinib for chronic myelogenous leukaemia for a median (range) duration of 42 (8–74) months before PAH diagnosis. No bone morphogenic protein receptor-2 (BMPR2) mutations were found in the 10 patients tested. Dasatinib was uniformly discontinued and 11 patients received PAH medications. Four patients died during follow-up. New York Heart Association functional class improved from 76% in class III/IV to 90% in class I/II (pDasatinib-induced PAH frequently improves after discontinuation but persisted in over one-third of patients, therefore systematic follow-up is essential.

Pulmonary arterial hypertension (PAH) corresponds to a heterogeneous group of severe clinical conditions characterized by precapillary pulmonary hypertension (PH) diagnosed when mean pulmonary artery pressure equals or exceeds 25 mm Hg at rest with normal pulmonary artery wedge pressure (≤15 mm Hg). According to the current clinical classification, PAH can be idiopathic (IPAH), heritable, drug or toxin induced, or associated with other diseases (eg, connective tissue diseases [CTDs], congenital heart diseases, HIV infection, and portal hypertension).1 PAH has a complex and multifactorial pathogenesis in which excessive migration and proliferation of pulmonary vascular cells (ie, endothelial cells [ECs] and smooth muscle cells [SMCs]) and dysregulated immune responses are critical contributors to the inappropriate pulmonary vascular remodeling. PAH is a fatal condition leading to right heart failure and death within 2 to 3 years after diagnosis if left untreated. During the last decade, therapeutic options for the treatment of this disease have improved exercise capacity, quality of life, and long-term outcomes. However, there is currently no cure available, and further insight into the disease pathophysiology is needed to advance drug development and to improve patient management. It is now widely accepted that altered immune mechanisms play a significant role in PAH by recruiting inflammatory cells, remodeling the pulmonary vasculature, and promoting autoimmune responses.2–4 Inflammation is a general term for the local accumulation of fluid, plasma proteins, and white blood cells that is initiated by physical injury, infection, or a local immune response. These phenomena represent the innate immune response. The innate immune response contributes to the activation of adaptive immunity, which is the response of antigen-specific lymphocytes to antigen, including the development of immunologic memory. The unique features of adaptive immunity, based on clonal selection of lymphocytes bearing antigen-specific receptors, provide the ability to recognize all pathogens specifically …

BACKGROUND: After lung transplantation, increased left ventricular (LV) filling can lead to LV failure, increasing the risk of post-operative complications and mortality. LV dysfunction in pulmonary arterial hypertension (PAH) is characterized by a reduced LV ejection fraction and impaired diastolic function. OBJECTIVES: The pathophysiology of LV dysfunction in PAH is incompletely understood. This study sought to assess the contribution of atrophy and contractility of cardiomyocytes to LV dysfunction in PAH patients. METHODS: LV function was assessed by cardiac magnetic resonance imaging. In addition, LV biopsies were obtained in 9 PAH patients and 10 donors. The cross-sectional area (CSA) and force-generating capacity of isolated single cardiomyocytes was investigated. RESULTS: Magnetic resonance imaging analysis revealed a significant reduction in LV ejection fraction in PAH patients, indicating a reduction in LV contractility. The CSA of LV cardiomyocytes of PAH patients was significantly reduced (∼30%), indicating LV cardiomyocyte atrophy. The maximal force-generating capacity, normalized to cardiomyocyte CSA, was significantly reduced (∼25%). Also, a reduction in the number of available myosin-based cross-bridges was found to cause the contractile weakness of cardiomyocytes. This finding was supported by protein analyses, which showed an ∼30% reduction in the myosin/actin ratio in cardiomyocytes from PAH patients. Finally, the phosphorylation level of sarcomeric proteins was reduced in PAH patients, which was accompanied by increased calcium sensitivity of force generation. CONCLUSIONS: The contractile function and the CSA of LV cardiomyocytes is substantially reduced in PAH patients. We propose that these changes contribute to the reduced in vivo contractility of the LV in PAH patients. © 2014 by the American College of Cardiology Foundation.

Summary: Using proteomic approaches, we uncovered a DNA damage response (DDR) function for peroxisome proliferator activated receptor γ (PPARγ) through its interaction with the DNA damage sensor MRE11-RAD50-NBS1 (MRN) and the E3 ubiquitin ligase UBR5. We show that PPARγ promotes ATM signaling and is essential for UBR5 activity targeting ATM interactor (ATMIN). PPARγ depletion increases ATMIN protein independent of transcription and suppresses DDR-induced ATM signaling. Blocking ATMIN in this context restores ATM activation and DNA repair. We illustrate the physiological relevance of PPARγ DDR functions by using pulmonary arterial hypertension (PAH) as a model that has impaired PPARγ signaling related to endothelial cell (EC) dysfunction and unresolved DNA damage. In pulmonary arterial ECs (PAECs) from PAH patients, we observed disrupted PPARγ-UBR5 interaction, heightened ATMIN expression, and DNA lesions. Blocking ATMIN in PAH PAEC restores ATM activation. Thus, impaired PPARγ DDR functions may explain the genomic instability and loss of endothelial homeostasis in PAH. : Li et al. identify PPARγ interactions with MRN and UBR5. PPARγ promotes UBR5-mediated ATMIN degradation, necessary for ATM activation upon DNA damage. Pulmonary arterial hypertension (PAH) endothelial cells exhibit genomic instability and disrupted PPARγ-UBR5 interaction. Blocking ATMIN restores ATM signaling in these cells, highlighting the significance of the PPARγ-ATMIN axis. Keywords: PPARγ, DNA damage, vascular biology, pulmonary hypertension, endothelial cells, ATM, MRN

Pulmonary hypertension (PH) is a hemodynamic state defined by a mean pulmonary artery pressure ≥ 25 mm Hg during resting right heart catheterization. PH can result from precapillary (arterial) or postcapillary (venous) pathophysiological mechanisms. Interestingly, recent PH pathology has shown that pulmonary arterial or pulmonary venous remodelling are rarely independent phenomena, but frequently occur in combined fashion in lungs from patients suffering from different forms of PH, including pulmonary arterial hypertension (PAH). In PAH, it is now becoming clear that aberrant signals present in vessel wall microenvironment, which is largely orchestrated by dysfunctional pulmonary endothelial cells, are key contributors of the pulmonary vascular remodeling process, fostering proliferation, and survival and migration of resident pulmonary vascular cells such as smooth muscle cells, myofibroblasts, and pericytes. In addition, both genetic and environmental factors are also critical in the development of pulmonary vascular inflammation and chronic impairment of the pulmonary endothelium. This article outlines the current understanding of this disease from the point of view of pathology and pathobiology.

New treatments for pulmonary arterial hypertension (PAH) are a crucial need. The increased proliferation, migration, and survival of pulmonary vascular cells within the pulmonary artery wall in PAH have allowed successful transposition of pathophysiological elements from oncologic researches. Next steps will require translation of these biological advances in PAH therapeutic arsenal and guidelines. This review synthesizes recent data concerning the role of receptor tyrosine kinases and their inhibitors in PAH, with implications in animal models and humans. Results of clinical trials are now accumulating to establish beneficial role of tyrosine kinase inhibitors (TKIs) in PAH and further findings are expected in the near future. Beside this curative approach, evidences of a possible TKI-induced cardiotoxicity are emerging. These safety issues raise concern about a potential amplified harmful effect in PAH, a pathology characterized by an underlying cardiac dysfunction. In addition, analyses of PAH registries shed light on a selective pulmonary vascular toxicity triggered by TKIs, especially dasatinib. These possible dual effects of the TKIs in PAH need to be taken in account for future pharmacological development of this therapeutic class in PAH.

Toll-like receptors (TLRs) play a crucial role in the innate and adaptive immune responses against microbial infection, tissue injury and cancer. Ligands of TLR9 have been developed as therapy in non-small-cell lung carcinoma (NSCLC). However, phase III clinical trials in metastatic NSCLC were negative. Our objective was to determine whether TLR9 affects tumor growth. We generated a mouse model of lung adenocarcinoma (ADC) mutated for K-ras (K-ras(LA1) ), with and without TLR9 inactivation (K-ras(LA1) TLR9(-/-) and K-ras(LA1) TLR9(+/+) , respectively). TLR9 was functionally expressed only in mononuclear cells of K-ras(LA1) TLR9(+/+) mice. These mice had significantly worse survival and a higher tumor burden than K-ras(LA1) TLR9(-/-) mice. Lung tumors were analyzed for 24 cytokines/growth factors using Bio-Plex multiplex bead-based assays. Factor VIII was assessed by immunochemistry. Tumors from K-ras(LA1) TLR9(+/+) mice were characterized by an angiogenic phenotype with higher concentrations of vascular endothelial growth factor (VEGF) and higher microvessel density than from K-ras(LA1) TLR9(-/-) mice. LKR13 cells, an ADC cell line derived from K-ras(LA1) mice, were subcutaneously injected into TLR9(-/-) and TLR9(+/+) mice. Syngeneic tumors regressed in TLR9(-/-) but not in TLR9(+/+) mice. Peripheral blood mononuclear cells from TLR9(-/-) mice released less VEGF than those from TLR9(+/+) mice. In 61 patients with early-stage NSCLC, TLR9 was expressed in mononuclear cells that infiltrated tumors, as assessed by immunochemistry, and contributed to worse survival. Our results suggest that TLR9 expression in mononuclear cells was associated with an angiogenic phenotype and promoted lung cancer progression. These findings may aid clinical development of TLR9 ligands to treat cancers.

Background— Pulmonary arterial hypertension (PAH) is characterized by dysregulated proliferation of pulmonary artery smooth muscle cells leading to (mal)adaptive vascular remodeling. In the systemic circulation, vascular injury is associated with downregulation of sarcoplasmic reticulum Ca 2+ -ATPase 2a (SERCA2a) and alterations in Ca 2+ homeostasis in vascular smooth muscle cells that stimulate proliferation. We, therefore, hypothesized that downregulation of SERCA2a is permissive for pulmonary vascular remodeling and the development of PAH. Methods and Results— SERCA2a expression was decreased significantly in remodeled pulmonary arteries from patients with PAH and the rat monocrotaline model of PAH in comparison with controls. In human pulmonary artery smooth muscle cells in vitro, SERCA2a overexpression by gene transfer decreased proliferation and migration significantly by inhibiting NFAT/STAT3. Overexpresion of SERCA2a in human pulmonary artery endothelial cells in vitro increased endothelial nitric oxide synthase expression and activation. In monocrotaline rats with established PAH, gene transfer of SERCA2a via intratracheal delivery of aerosolized adeno-associated virus serotype 1 (AAV1) carrying the human SERCA2a gene (AAV1.SERCA2a) decreased pulmonary artery pressure, vascular remodeling, right ventricular hypertrophy, and fibrosis in comparison with monocrotaline-PAH rats treated with a control AAV1 carrying β-galactosidase or saline. In a prevention protocol, aerosolized AAV1.SERCA2a delivered at the time of monocrotaline administration limited adverse hemodynamic profiles and indices of pulmonary and cardiac remodeling in comparison with rats administered AAV1 carrying β-galactosidase or saline. Conclusions— Downregulation of SERCA2a plays a critical role in modulating the vascular and right ventricular pathophenotype associated with PAH. Selective pulmonary SERCA2a gene transfer may offer benefit as a therapeutic intervention in PAH.

Background— The role of right ventricular (RV) diastolic stiffness in pulmonary arterial hypertension (PAH) is not well established. Therefore, we investigated the presence and possible underlying mechanisms of RV diastolic stiffness in PAH patients. Methods and Results— Single-beat RV pressure-volume analyses were performed in 21 PAH patients and 7 control subjects to study RV diastolic stiffness. Data are presented as mean±SEM. RV diastolic stiffness (β) was significantly increased in PAH patients (PAH, 0.050±0.005 versus control, 0.029±0.003; P 2 versus control, 218±21 μm 2 ; P P 200%; P interaction P Conclusions— RV diastolic stiffness is significantly increased in PAH patients, with important contributions from increased collagen and intrinsic stiffening of the RV cardiomyocyte sarcomeres.

Pulmonary arterial hypertension (PAH) is a progressive disease defined by a chronic elevation in pulmonary arterial pressure with extensive pulmonary vascular remodeling and perivascular inflammation characterized by an accumulation of macrophages, lymphocytes, dendritic cells, and mast cells. Although the exact etiology of the disease is unknown, clinical as well as preclinical data strongly implicate a role for serotonin (5-HT) in the process. Here, we investigated the chronic effects of pharmacological inhibition of tryptophan hydroxylase 1 (TPH1), the rate-limiting enzyme in peripheral 5-HT biosynthesis, in two preclinical models of pulmonary hypertension (PH), the monocrotaline (MCT) rat and the semaxanib (SUGEN, Medinoah, Suzhou, China)-hypoxia rat. In both PH models, ethyl (S)-8-(2-amino-6-((R)-1-(5-chloro-[1,1'-biphenyl]-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate and ethyl (S)-8-(2-amino-6-((R)-1-(3',4'-dimethyl-3-(3-methyl-1 H-pyrazol-1-yl)-[1,1'-biphenyl]-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)-2,8-diazaspiro[4.5]decane-3-carboxylate, novel orally active TPH1 inhibitors with nanomolar in vitro potency, decreased serum, gut, and lung 5-HT levels in a dose-dependent manner and significantly reduced pulmonary arterial pressure, and pulmonary vessel wall thickness and occlusion in male rats. In the MCT rat model, decreases in lung 5-HT significantly correlated with reductions in histamine levels and mast cell number (P < 0.001, r2 = 0.88). In contrast, neither ambrisentan nor tadalafil, which are vasodilators approved for the treatment of PAH, reduced mast cell number or 5-HT levels, nor were they as effective in treating the vascular remodeling as were the TPH1 inhibitors. When administered in combination with ambrisentan, the TPH1 inhibitors showed an additive effect on pulmonary vascular remodeling and pressures. These data demonstrate that in addition to reducing vascular remodeling, TPH1 inhibition has the added benefit of reducing the perivascular mast cell accumulation associated with PH.

Erythrocyte-endothelial interaction is especially pronounced in the lung microvasculature and altered red blood cell (RBC) membrane structure have been demonstrated to induce endothelial cell (EC) dysfunction in several systemic diseases and to be associated with increased risk of cardiovascular disease development. We are questioning here whether RBCs are altered in PAH and could contribute to P-EC dysfunction leading to PAH development and/or progression. In addition, we are investigating whether changes in RBCs are clinically relevant. To test this hypothesis, we withdrew blood samples from PAH patients (n=40) and controls (n=15) and performed automated blood count analyses to determine RBC distribution width (RDW). Next, we isolated RBCs by centrifugation and identified them by flow cytometry, using the RBC surface marker CD235a. Then, we measured by flow cytometry structural membrane protein expression of CD47 and phosphatidylserine (PS) and the level of advanced glycation end product (AGE) production. We found an increased RDW values in PAH patients, which inversely correlate with the 6 minute walk test distance (r=-0.53, p Our data indicate that RBCs in PAH patients are altered in size, structure and function when compared with controls. Further studies are needed to investigate the mechanisms underlying these RBC abnormalities in PAH and to precise their clinical relevance as well as their contribution in P-EC dysfunction.

Background: Increased levels of uric acid (UA) in serum of patients with pulmonary arterial hypertension (PAH) are associated with severity and clinical outcomes. However, the pathophysiological contribution of UA in pulmonary vascular remodeling associated with PAH remains unknown. Objectives: To study pathophysiological roles of UA in experimental and human PAH and to evaluate the translational potential of benzbromarone, an inhibitor against UA transporter URATv1. Methods: We examined serum UA level and the expression pattern of URATv1 in cultured pulmonary artery smooth muscle cells (PA-SMCs) and in lungs of 10 controls (CTR) and 10 patients with idiopathic PAH (IPAH). In addition, the preventive and curative effects of benzbromarone were tested in two experimental models of pulmonary hypertension (PH). Results: In IPAH, we found elevated levels of serum UA and a strong immunoreactivity for URATv1 in the media of remodeled pulmonary arteries and cultured PA-SMCs of IPAH patients when compared with controls. These findings were also replicated in vivo with high in situ levels of URATv1 in PA-SMCs of PH rats. Consistent with these findings, we also demonstrated in vitro that the upregulation of URATv1 contributes to an increased proliferative response to UA in IPAH PA-SMCs when compared with CTR cells, a phenomenon abolished with benzbromarone. Finally, we found that benzbromarone partially prevented and reversed established PH in the monocrotaline and SU5416+hypoxia rat models. Conclusions: Our results support the possibility that the blockade of UA transporter may represent promising novel approaches for treatment of PAH.

Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, an orally bioavailable dual Src/BCR/ABL tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia (CML). Today, key questions remain regarding the long-term evolution of dasatinib-induced PAH and the mechanism(s) involved. Here, we demonstrate that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents, in contrast to chronic imatinib treatment. We found that rats pretreated with dasatinib, but not with imatinib, exhibit attenuated hypoxic pulmonary vasoconstriction (HPV) responses and increased susceptibility to experimental pulmonary hypertension (PH). Furthermore, in vivo and in vitro observations show that dasatinib treatment induces pulmonary endothelial cell apoptosis and induction of endoplasmic reticulum stress, in a dose dependent manner, in contrast to imatinib. We also demonstrate that dasatinib-induced endothelial cell dysfunction is mediated through production of reactive oxygen species (ROS), independently of Src family kinases. Consistently with our findings, we found significant elevations of markers of endothelial dysfunction or damage in the serum of CML patients treated with dasatinib when compared with CML patients treated with imatinib. Collectively, we report here that dasatinib causes pulmonary endothelial damage and induction of ER stress via ROS production, leading to increased susceptibility to PH development.

Pulmonary arterial hypertension (PAH) is a life-threatening disease that can be induced by dasatinib, a dual Src and BCR-ABL tyrosine kinase inhibitor that is used to treat chronic myelogenous leukemia (CML). Today, key questions remain regarding the mechanisms involved in the long-term development of dasatinib-induced PAH. Here, we demonstrated that chronic dasatinib therapy causes pulmonary endothelial damage in humans and rodents. We found that dasatinib treatment attenuated hypoxic pulmonary vasoconstriction responses and increased susceptibility to experimental pulmonary hypertension (PH) in rats, but these effects were absent in rats treated with imatinib, another BCR-ABL tyrosine kinase inhibitor. Furthermore, dasatinib treatment induced pulmonary endothelial cell apoptosis in a dose-dependent manner, while imatinib did not. Dasatinib treatment mediated endothelial cell dysfunction via increased production of ROS that was independent of Src family kinases. Consistent with these findings, we observed elevations in markers of endothelial dysfunction and vascular damage in the serum of CML patients who were treated with dasatinib, compared with CML patients treated with imatinib. Taken together, our findings indicate that dasatinib causes pulmonary vascular damage, induction of ER stress, and mitochondrial ROS production, which leads to increased susceptibility to PH development.

Item does not contain fulltext RATIONALE: Altered pulmonary hemodynamics and fluid flow-induced high shear stress (HSS) are characteristic hallmarks in the pathogenesis of pulmonary arterial hypertension (PAH). However, the contribution of HSS to cellular and vascular alterations in PAH is unclear. OBJECTIVES: We hypothesize that failing shear adaptation is an essential part of the endothelial dysfunction in all forms of PAH and tested whether microvascular endothelial cells (MVECs) or pulmonary arterial endothelial cells (PAECs) from lungs of patients with PAH adapt to HSS and if the shear defect partakes in vascular remodeling in vivo. METHODS: PAH MVEC (n = 7) and PAH PAEC (n = 3) morphology, function, protein, and gene expressions were compared with control MVEC (n = 8) under static culture conditions and after 24, 72, and 120 hours of HSS. MEASUREMENTS AND MAIN RESULTS: PAH MVEC showed a significantly delayed morphological shear adaptation (P = 0.03) and evidence of cell injury at sites of nonuniform shear profiles that are critical loci for vascular remodeling in PAH. In clear contrast, PAEC isolated from the same PAH lungs showed no impairments. PAH MVEC gene expression and transcriptional shear activation were not altered but showed significant decreased protein levels (P = 0.02) and disturbed interendothelial localization of the shear sensor platelet endothelial cell adhesion molecule-1 (PECAM-1). The decreased PECAM-1 levels were caused by caspase-mediated cytoplasmic cleavage but not increased cell apoptosis. Caspase blockade stabilized PECAM-1 levels, restored endothelial shear responsiveness in vitro, and attenuated occlusive vascular remodeling in chronically hypoxic Sugen5416-treated rats modeling severe PAH. CONCLUSIONS: Delayed shear adaptation, which promotes shear-induced endothelial injury, is a newly identified dysfunction specific to the microvascular endothelium in PAH. The shear response is normalized on stabilization of PECAM-1, which reverses intimal remodeling in vivo.

Pulmonary arterial hypertension (PAH) is a disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in mean pulmonary arterial pressure (mPAP), resulting in a progressive functional decline despite current available therapeutic options. There are multiple mechanisms predisposing to and/or promoting the aberrant pulmonary vascular remodeling in PAH, and these involve not only altered crosstalk between cells within the vascular wall but also sustained inflammation and dysimmunity, cell accumulation in the vascular wall and excessive activation of some growth factor-stimulated signaling pathways, in addition to the interaction of systemic hormones, local growth factors, cytokines, and transcription factors. Heterozygous germline mutations in the bone morphogenetic protein receptor, type-2 (BMPR2) gene, a gene encoding a receptor for the transforming growth factor (TGF)-β superfamily, can predispose to the disease. Although the spectrum of therapeutic options for PAH has expanded in the last 20 years, available therapies remain essentially palliative. Over the past decade, however, a better understanding of key regulators of this irreversible remodeling of the pulmonary vasculature has been obtained. New and more effective approaches are likely to emerge. The present article profiles the innovative research into novel pathways and therapeutic targets that may lead to the development of targeted agents in PAH.

Pulmonary arterial hypertension (PAH) is a disorder in which mechanical obstruction of the pulmonary vascular bed is largely responsible for the rise in mean pulmonary arterial pressure (mPAP), resulting in a progressive functional decline despite current available therapeutic options. There are multiple mechanisms predisposing to and/or promoting the aberrant pulmonary vascular remodeling in PAH, and these involve not only altered crosstalk between cells within the vascular wall but also sustained inflammation and dysimmunity, cell accumulation in the vascular wall and excessive activation of some growth factor-stimulated signaling pathways, in addition to the interaction of systemic hormones, local growth factors, cytokines, and transcription factors. Heterozygous germline mutations in the bone morphogenetic protein receptor, type-2 (BMPR2) gene, a gene encoding a receptor for the transforming growth factor (TGF)-β superfamily, can predispose to the disease. Although the spectrum of therapeutic options for PAH has expanded in the last 20 years, available therapies remain essentially palliative. Over the past decade, however, a better understanding of key regulators of this irreversible remodeling of the pulmonary vasculature has been obtained. New and more effective approaches are likely to emerge. The present article profiles the innovative research into novel pathways and therapeutic targets that may lead to the development of targeted agents in PAH.

Pulmonary hypertension (PH) is a fatal disease with no treatment options, characterised by elevated pulmonary vascular resistanzce and secondary right ventricular failure. The aetiology of pulmonary arterial hypertension is multiple and its pathogenesis is complex. Although the exact role of cellular microparticles remains partially understood, there is increasing evidence to suggest an active role for microparticles in PH pathophysiology. Patients with PH exhibited higher circulating levels of microparticles compared to control subjects and in vitro or in vivo generated microparticles can induce endothelial dysfunction, interfere with coagulation pathways or modulate inflammatory phenomenon. Whether or not these new conveyors of biological information contribute to the acquisition and/or maintenance of the altered endothelial phenotype is unexplored in PH and requires further study.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130(Cas) is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation, and survival.We hypothesized that in experimental and human PAH p130(Cas) signaling is overactivated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).In patients with PAH, levels of p130(Cas) protein and/or activity are higher in the serum, in the walls of distal pulmonary arteries, in cultured smooth muscle cells (PA-SMCs), and in pulmonary endothelial cells (P-ECs) than in control subjects. These abnormalities in the p130(Cas) signaling were also found in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We obtained evidence for the convergence and amplification of the growth-stimulating effect of the EGF-, FGF2-, and PDGF-signaling pathways via the p130(Cas) signaling pathway. We found that daily treatment with the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib, and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuated the abnormal increase in p130(Cas) and ERK1/2 activation and regressed established pulmonary hypertension.Our findings demonstrate that p130(Cas) signaling plays a critical role in experimental and idiopathic PAH by modulating pulmonary vascular cell migration and proliferation and by acting as an amplifier of RTK downstream signals.

Kawasaki disease (KD) is an acute inflammatory illness marked by coronary arteritis. However, the factors increasing susceptibility to coronary artery lesions are unknown. Because transforming growth factor (TGF) β increases elastin synthesis and suppresses proteolysis, we hypothesized that, in contrast to the benefit observed in aneurysms forming in those with Marfan syndrome, inhibition of TGF-β would worsen inflammatory-induced coronary artery lesions. By using a murine model of KD in which injection of Lactobacillus casei wall extract (LCWE) induces coronary arteritis, we show that LCWE increased TGF-β signaling in the coronary smooth muscle cells beginning at 2 days and continuing through 14 days, the point of peak coronary inflammation. By 42 days, LCWE caused fragmentation of the internal and external elastic lamina. Blocking TGF-β by administration of a neutralizing antibody accentuated the LCWE-mediated fragmentation of elastin and induced an overall loss of medial elastin without increasing the inflammatory response. We attributed these increased pathological characteristics to a reduction in the proteolytic inhibitor, plasminogen activator inhibitor-1, and an associated threefold increase in matrix metalloproteinase 9 activity compared with LCWE alone. Therefore, our data demonstrate that in the coronary arteritis associated with KD, TGF-β suppresses elastin degradation by inhibiting plasmin-mediated matrix metalloproteinase 9 activation. Thus, strategies to block TGF-β, used in those with Marfan syndrome, are unlikely to be beneficial and could be detrimental.

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

The European Respiratory Society (ERS) Research Seminar entitled “Pulmonary vascular endothelium: orchestra conductor in respiratory diseases - highlights from basic research to therapy” brought together international experts in dysfunctional pulmonary endothelium, from basic science to translational medicine, to discuss several important aspects in acute and chronic lung diseases. This review will briefly sum up the different topics of discussion from this meeting which was held in Paris, France on October 27–28, 2016. It is important to consider that this paper does not address all aspects of endothelial dysfunction but focuses on specific themes such as: 1) the complex role of the pulmonary endothelium in orchestrating the host response in both health and disease (acute lung injury, chronic obstructive pulmonary disease, high-altitude pulmonary oedema and pulmonary hypertension); and 2) the potential value of dysfunctional pulmonary endothelium as a target for innovative therapies.

Rationale: S100A4/Mts1 is implicated in motility of human pulmonary artery smooth muscle cells (hPASMCs), through an interaction with the RAGE (receptor for advanced glycation end products). Objective: We hypothesized that S100A4/Mts1-mediated hPASMC motility might be enhanced by loss of function of bone morphogenetic protein (BMP) receptor (BMPR)II, observed in pulmonary arterial hypertension. Methods and Results: Both S100A4/Mts1 (500 ng/mL) and BMP-2 (10 ng/mL) induce migration of hPASMCs in a novel codependent manner, in that the response to either ligand is lost with anti-RAGE or BMPRII short interference (si)RNA. Phosphorylation of extracellular signal-regulated kinase is induced by both ligands and is required for motility by inducing matrix metalloproteinase 2 activity, but phospho–extracellular signal-regulated kinase 1/2 is blocked by anti-RAGE and not by BMPRII short interference RNA. In contrast, BMPRII short interference RNA, but not anti-RAGE, reduces expression of intracellular chloride channel (CLIC)4, a scaffolding molecule necessary for motility in response to S100A4/Mts1 or BMP-2. Reduced CLIC4 expression does not interfere with S100A4/Mts1 internalization or its interaction with myosin heavy chain IIA, but does alter alignment of myosin heavy chain IIA and actin filaments creating the appearance of vacuoles. This abnormality is associated with reduced peripheral distribution and/or delayed activation of RhoA and Rac1, small GTPases required for retraction and extension of lamellipodia in motile cells. Conclusions: Our studies demonstrate how a single ligand (BMP-2 or S100A4/Mts1) can recruit multiple cell surface receptors to relay signals that coordinate events culminating in a functional response, ie, cell motility. We speculate that this carefully controlled process limits signals from multiple ligands, but could be subverted in disease.

The complex and multifactorial pathogenesis of pulmonary hypertension (PH) involves constriction, remodeling, and in situ thrombosis of pulmonary vessels. Both serotonin (5-HT) and Rho kinase signaling may contribute to these alterations.To investigate possible links between the 5-HT transporter (5-HTT) and RhoA/Rho kinase pathways, as well as their involvement in the progression of human and experimental PH.Biochemical and functional analyses of lungs, platelets, and pulmonary artery smooth muscle cells (PA-SMCs) from patients with idiopathic PH (iPH) and 5-HTT overexpressing mice.Lungs, platelets, and PA-SMCs from patients with iPH were characterized by marked elevation in RhoA and Rho kinase activities and a strong increase in 5-HT binding to RhoA indicating RhoA serotonylation. The 5-HTT inhibitor fluoxetine and the type 2 transglutaminase inhibitor monodansylcadaverin prevented 5-HT-induced RhoA serotonylation and RhoA/Rho kinase activation, as well as 5-HT-induced proliferation of PA-SMCs from iPH patients that was also inhibited by the Rho kinase inhibitor fasudil. Increased Rho kinase activity, RhoA activation, and RhoA serotonylation were also observed in lungs from SM22-5-HTT(+)mice, which overexpress 5-HTT in smooth muscle and spontaneously develop PH. Treatment of SM22-5-HTT(+) mice with either fasudil or fluoxetine limited PH progression and RhoA/Rho kinase activation.RhoA and Rho kinase activities are increased in iPH, in association with enhanced RhoA serotonylation. Direct involvement of the 5-HTT/RhoA/Rho kinase signaling pathway in 5-HT-mediated PA-SMC proliferation and platelet activation during PH progression identify RhoA/Rho kinase signaling as a promising target for new treatments against PH.

Expression of bone morphogenetic protein receptor 1A (BMPR1A) is attenuated in the lung vessels of patients with pulmonary arterial hypertension, but the functional impact of this abnormality is unknown. We ablated Bmpr1a in cardiomyocytes and vascular smooth muscle cells (VSMCs) by breeding mice possessing a loxP allele of Bmpr1a (Bmpr1aflox) expressing R26R with SM22alpha-Cre mice. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice died soon after embryonic day 11 (E11) with massive vascular and pericardial hemorrhage and impaired brain development. At E10.5, SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos showed thinning of the myocardium associated with reduced cell proliferation. These embryos also had severe dilatation of the aorta and large vessels with impaired investment of SMCs that was also related to reduced proliferation. SM22alpha-Cre;R26R;Bmpr1aflox/flox mice showed collapsed telencephalon in association with impaired clearing of brain microvessels in areas where reduced apoptosis was observed. Transcript and protein levels of matrix metalloproteinase (MMP) 2 and 9 were reduced in E9.5 and E10.5 SM22alpha-Cre;R26R;Bmpr1aflox/flox embryos, respectively. Knock-down of BMPR1A by RNA interference in human pulmonary artery SMCs reduced MMP2 and MMP9 activity, attenuated serum-induced proliferation, and impaired PDGF-BB-directed migration. RNA interference of MMP2 or MMP9 recapitulated these abnormalities, supporting a functional interaction between BMP signaling and MMP expression. In human brain microvascular pericytes, knock-down of BMPR1A reduced MMP2 activity and knock-down of either BMPR1A or MMP2 caused resistance to apoptosis. Thus, loss of Bmpr1a, by decreasing MMP2 and/or MMP9 activity, can account for vascular dilatation and persistence of brain microvessels, leading to the impaired organogenesis documented in the brain.