Disrupted BMP-9 Signaling Impairs Pulmonary Vascular Integrity in Hepatopulmonary Syndrome.

RATIONALE: Hepatopulmonary syndrome (HPS) is a severe complication of liver diseases characterized by abnormal dilatation of pulmonary vessels, resulting in impaired oxygenation. Recent research highlights the pivotal role of liver-produced bone morphogenetic protein (BMP)-9 in maintaining pulmonary vascular integrity. OBJECTIVES: This study aimed to investigate the involvement of BMP-9 in human and experimental HPS. METHODS: Circulating BMP-9 levels were measured in 63 healthy controls and 203 cirrhotic patients, with or without HPS. Two animal models of portal hypertension were employed: common bile duct ligation (CBDL) with cirrhosis and long-term partial portal vein ligation (PPVL) without cirrhosis. Additionally, the therapeutic effect of low-dose BMP activator FK506 was investigated, and the pulmonary vascular phenotype of BMP-9 knockout rats was analyzed. MEASUREMENTS AND MAIN RESULTS: Patients with HPS related to compensated cirrhosis demonstrated lower levels of circulating BMP-9 compared to patients without HPS. Severe cirrhosis patients exhibited consistently low levels of BMP-9. In animal models, HPS characteristics, including intrapulmonary vascular dilations (IPVDs) and alveolo-arterial gradient enlargement, were observed. HPS development in both rat models correlated with reduced intrahepatic BMP-9 expression, decreased circulating BMP-9 level and activity, and impaired pulmonary BMP-9 endothelial pathway. Daily treatment with FK506 for 2-weeks restored BMP pathway in the lungs, alleviating IPVDs, and improving gas exchange impairment. Furthermore, BMP-9 knockout rats displayed a pulmonary HPS phenotype, supporting its role in disease progression. CONCLUSION: The study findings suggest that portal hypertension-induced loss of BMP-9 signaling contributes to HPS development.

Serum and Pulmonary Expression Profiles of the Activin Signaling System in Pulmonary Arterial Hypertension.

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<0.001, respectively) serum levels of activin A and FSTL3 were associated with transplant-free survival in a model adjusted for age and sex. Thresholds determined by receiver operating characteristic analyses were 393 pg/mL for activin A and 16.6 ng/mL for FSTL3. When adjusted with New York Heart Association functional class, 6-minute walk distance, and N-terminal pro-B-type natriuretic peptide, the hazard ratios for transplant-free survival for baseline activin A <393 pg/mL and FSTL3 <16.6 ng/mL were, respectively, 0.14 (95% CI, 0.03-0.61; P=0.009) and 0.17 (95% CI, 0.06-0.45; P<0.001), and for follow-up measures, 0.23 (95% CI, 0.07-0.78; 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.

Loss of cAbl Tyrosine Kinase in Pulmonary Arterial Hypertension Causes Dysfunction of Vascular Endothelial Cells.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by the dysfunction of pulmonary endothelial cells (ECs) and obstructive vascular remodeling. cAbl (non-receptor tyrosine kinase c-Abelson) plays central roles in regulating cell-cycle arrest, apoptosis, and senescence after cellular stress. We hypothesized that cAbl is downactivated in experimental and human PAH, thus leading to reduced DNA integrity and angiogenic capacity of pulmonary ECs from patients with PAH (PAH-ECs). We found cAbl and phosphorylated cAbl concentrations to be lower in the endothelium of remodeled pulmonary vessels in the lungs of patients with PAH than in control subjects. Similar observations were obtained for the lungs of Sugen + hypoxia and monocrotaline 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 5-(1,3-diaryl-1H-pyrazol-4-yl) hydantoin reduced DNA damage and apoptosis in PAH-ECs. Finally, we establish the existence of cross-talk between cAbl and bone morphogenetic protein receptor type II. This work identifies the loss of cAbl signaling as a novel contributor to pulmonary EC dysfunction associated with PAH.

Loss of lung microvascular endothelial Piezo2 expression impairs NO synthesis, induces EndMT, and is associated with pulmonary hypertension.

Mechanical forces are translated into biochemical stimuli by mechanotransduction channels, such as the mechanically activated cation channel Piezo2. Lung Piezo2 expression has recently been shown to be restricted to endothelial cells. Hence, we aimed to investigate the role of Piezo2 in regulation of pulmonary vascular function and structure, as well as its contribution to development of pulmonary arterial hypertension (PAH). The expression of Piezo2 was significantly reduced in pulmonary microvascular endothelial cells (MVECs) from patients with PAH, in lung tissue from mice with a Bmpr2+/R899X knock-in mutation commonly found in patients with pulmonary hypertension, and in lung tissue of monocrotaline (MCT) and sugen-hypoxia-induced PH (SuHx) PAH rat models, as well as from a swine model with pulmonary vein banding. In MVECs, Piezo2 expression was reduced in response to abnormal shear stress, hypoxia, and TGFß stimulation. Functional studies in MVECs exposed to shear stress illustrated that siRNA-mediated Piezo2 knockdown impaired endothelial alignment, calcium influx, phosphorylation of AKT, and nitric oxide production. In addition, siPiezo2 reduced the expression of the endothelial marker PECAM-1 and increased the expression of vascular smooth muscle markers ACTA2, SM22a, and calponin. Thus, Piezo2 acts as a mechanotransduction channel in pulmonary MVECs, stimulating shear-induced production of nitric oxide and is essentially involved in preventing endothelial to mesenchymal transition. Its blunted expression in pulmonary hypertension could impair the vasodilator capacity and stimulate vascular remodeling, indicating that Piezo2 might be an interesting therapeutic target to attenuate progression of the disease.NEW & NOTEWORTHY The mechanosensory ion channel Piezo2 is exclusively expressed in lung microvascular endothelial cells (MVECs). Patient MVECs as well as animal models of pulmonary (arterial) hypertension showed lower expression of Piezo2 in the lung. Mechanistically, Piezo2 is required for calcium influx and NO production in response to shear stress, whereas stimuli known to induce endothelial to mesenchymal transition (EndMT) reduce Piezo2 expression in MVECs, and Piezo2 knockdown induces a gene and protein expression pattern consistent with EndMT.

Serum and pulmonary uric acid in pulmonary arterial hypertension.

Previous studies have suggested an association between uric acid (UA) and the severity of pulmonary arterial hypertension (PAH), but it is unknown whether UA contributes to disease pathogenesis.The aim of this study was to determine the prognostic value of circulating UA in the era of current management of PAH and to investigate the role of UA in pulmonary vascular remodelling.Serum UA levels were determined in idiopathic, heritable or anorexigen PAH at baseline and first re-evaluation in the French Pulmonary Hypertension Network. We studied protein levels of xanthine oxidase (XO) and the voltage-driven urate transporter 1 (URATv1) in lungs of control and PAH patients and of monocrotaline (MCT) and Sugen/hypoxia (SuHx) rats. Functional studies were performed using human pulmonary artery smooth muscle cells (PA-SMCs) and two animal models of pulmonary hypertension (PH).High serum UA levels at first follow-up, but not at baseline, were associated with a poor prognosis. Both the generating enzyme XO and URATv1 were upregulated in the wall of remodelled pulmonary arteries in idiopathic PAH patients and MCT and SuHx rats. High UA concentrations promoted a mild increase in cell growth in idiopathic PAH PA-SMCs, but not in control PA-SMCs. Consistent with these observations, oxonic acid-induced hyperuricaemia did not aggravate MCT-induced PH in rats. Finally, chronic treatment of MCT and SuHx rats with benzbromarone mildly attenuated pulmonary vascular remodelling.UA levels in idiopathic PAH patients were associated with an impaired clinical and haemodynamic profile and might be used as a non-invasive indicator of clinical prognosis during follow-up. Our findings also indicate that UA metabolism is disturbed in remodelled pulmonary vascular walls in both experimental and human PAH.

Selective BMP-9 Inhibition Partially Protects Against Experimental Pulmonary Hypertension.

RATIONALE: Although many familial cases of pulmonary arterial hypertension exhibit an autosomal dominant mode of inheritance with the majority having mutations in essential constituents of the BMP (bone morphogenetic protein) signaling, the specific contribution of the long-term loss of signal transduction triggered by the BMPR2 (type 2 BMP receptor) remains poorly characterized. OBJECTIVE: To investigate the role of BMP9, the main ligand of ALK1 (Activin receptor-like kinase 1)/BMPR2 heterocomplexes, in pulmonary hypertension. METHOD AND RESULTS: The absence of BMP9 in Bmp9-/- mice and its inhibition in C57BL/6 mice using neutralizing anti-BMP9 antibodies substantially prevent against chronic hypoxia-induced pulmonary hypertension judged by right ventricular systolic pressure measurement, right ventricular hypertrophy, and pulmonary distal arterial muscularization. In agreement with these observations, we found that the BMP9/BMP10 ligand trap ALK1ECD administered in monocrotaline or Sugen/hypoxia (SuHx) rats substantially attenuate proliferation of pulmonary vascular cells, inflammatory cell infiltration, and regresses established pulmonary hypertension in rats. Our data obtained in human pulmonary endothelial cells derived from controls and pulmonary arterial hypertension patients indicate that BMP9 can affect the balance between endothelin-1, apelin, and adrenomedullin. We reproduced these in vitro observations in mice chronically exposed to hypoxia, with Bmp9-/- mice exhibiting lower mRNA levels of the vasoconstrictor peptide ET-1 (endothelin-1) and higher levels of the 2 potent vasodilator factors apelin and ADM (adrenomedullin) compared with Bmp9+/+ littermates. CONCLUSIONS: Taken together, our data indicate that the loss of BMP9, by deletion or inhibition, has beneficial effects against pulmonary hypertension onset and progression.

Lineage Tracing Reveals the Dynamic Contribution of Pericytes to the Blood Vessel Remodeling in Pulmonary Hypertension.

OBJECTIVE: Excessive accumulation of resident cells within the pulmonary vascular wall represents the hallmark feature of the remodeling occurring in pulmonary arterial hypertension (PAH). Furthermore, we have previously demonstrated that pulmonary arterioles are excessively covered by pericytes in PAH, but this process is not fully understood. The aim of our study was to investigate the dynamic contribution of pericytes in PAH vascular remodeling. Approach and Results: In this study, we performed in situ, in vivo, and in vitro experiments. We isolated primary cultures of human pericytes from controls and PAH lung specimens then performed functional studies (cell migration, proliferation, and differentiation). In addition, to follow up pericyte number and fate, a genetic fate-mapping approach was used with an NG2CreER;mT/mG transgenic mice in a model of pulmonary arteriole muscularization occurring during chronic hypoxia. We identified phenotypic and functional abnormalities of PAH pericytes in vitro, as they overexpress CXCR (C-X-C motif chemokine receptor)-7 and TGF (transforming growth factor)-ßRII and, thereby, display a higher capacity to migrate, proliferate, and differentiate into smooth muscle-like cells than controls. In an in vivo model of chronic hypoxia, we found an early increase in pericyte number in a CXCL (C-X-C motif chemokine ligand)-12-dependent manner whereas later, from day 7, activation of the canonical TGF-ß signaling pathway induces pericytes to differentiate into smooth muscle-like cells. CONCLUSIONS: Our findings reveal a pivotal role of pulmonary pericytes in PAH and identify CXCR-7 and TGF-ßRII as 2 intrinsic abnormalities in these resident progenitor vascular cells that foster the onset and maintenance of PAH structural changes in blood lung vessels.

Connexin-43 is a promising target for pulmonary hypertension due to hypoxaemic lung disease.

The mechanisms underlying pulmonary hypertension (PH) are complex and multifactorial, and involve different cell types that are interconnected through gap junctional channels. Although connexin (Cx)-43 is the most abundant gap junction protein in the heart and lungs, and critically governs intercellular signalling communication, its contribution to PH remains unknown. The focus of the present study is thus to evaluate Cx43 as a potential new target in PH.Expressions of Cx37, Cx40 and Cx43 were studied in lung specimens from patients with idiopathic pulmonary arterial hypertension (IPAH) or PH associated with chronic hypoxaemic lung diseases (chronic hypoxia-induced pulmonary hypertension (CH-PH)). Heterozygous Cx43 knockdown CD1 (Cx43+/-) and wild-type littermate (Cx43+/+) mice at 12 weeks of age were randomly divided into two groups, one of which was maintained in room air and the other exposed to hypoxia (10% oxygen) for 3 weeks. We evaluated pulmonary haemodynamics, remodelling processes in cardiac tissues and pulmonary arteries (PAs), lung inflammation and PA vasoreactivity.Cx43 levels were increased in PAs from CH-PH patients and decreased in PAs from IPAH patients; however, no difference in Cx37 or Cx40 levels was noted. Upon hypoxia treatment, the Cx43+/- mice were partially protected against CH-PH when compared to Cx43+/+ mice, with reduced pulmonary arterial muscularisation and inflammatory infiltration. Interestingly, the adaptive changes in cardiac remodelling in Cx43+/- mice were not affected. PA contraction due to endothelin-1 (ET-1) was increased in Cx43+/- mice under normoxic and hypoxic conditions.Taken together, these results indicate that targeting Cx43 may have beneficial therapeutic effects in PH without affecting compensatory cardiac hypertrophy.

Therapeutic effect of pirfenidone in the sugen/hypoxia rat model of severe pulmonary hypertension.

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.

Dasatinib increases endothelial permeability leading to pleural effusion.

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-1 for 8 weeks).Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks. Consistent with these in vivo observations, 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 mechanism in vitro and in vivo using a cotreatment with an antioxidant agent, N-acetylcysteine.This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.

Macrophage Migration Inhibitory Factor (MIF) Inhibition in a Murine Model of Bleomycin-Induced Pulmonary Fibrosis.

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.

Proinflammatory Signature of the Dysfunctional Endothelium in Pulmonary Hypertension. Role of the Macrophage Migration Inhibitory Factor/CD74 Complex.

RATIONALE: Inflammation and endothelial dysfunction are considered two primary instigators of pulmonary arterial hypertension (PAH). CD74 is a receptor for the proinflammatory cytokine macrophage migration inhibitory factor (MIF). This ligand/receptor complex initiates survival pathways and cell proliferation, and it triggers the synthesis and secretion of major proinflammatory factors and cell adhesion molecules. OBJECTIVES: We hypothesized that the MIF/CD74 signaling pathway is overexpressed in idiopathic PAH (iPAH) and contributes to a proinflammatory endothelial cell (EC) phenotype. METHODS: Primary early passage cultures of human ECs isolated from lung tissues obtained from patients with iPAH and controls were examined for their ability to secrete proinflammatory mediators and bind inflammatory cells with or without modulation of the functional activities of the MIF/CD74 complex. In addition, we tested the efficacies of curative treatments with either the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies on the aberrant proinflammatory EC phenotype in vitro and in vivo and on the progression of monocrotaline-induced pulmonary hypertension. MEASUREMENTS AND MAIN RESULTS: In human lung tissues, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and E-selectin expressions are markedly up-regulated in the endothelium of distal iPAH pulmonary arteries. Circulating MIF levels are increased in the serum of patients with PAH compared with control subjects, and T-cell lymphocytes represent a source of this overabundance. In addition, CD74 is highly expressed in the endothelium of muscularized pulmonary arterioles and in cultured pulmonary ECs from iPAH, contributing to an exaggerated recruitment of peripheral blood mononuclear cells to pulmonary iPAH ECs. Finally, we found that curative treatments with the MIF antagonist ISO-1 or anti-CD74 neutralizing antibodies partially reversed development of pulmonary hypertension in rats and substantially reduced inflammatory cell infiltration. CONCLUSIONS: We report here that CD74 and MIF are markedly increased and activated in patients with iPAH, contributing to the abnormal proinflammatory phenotype of pulmonary ECs in iPAH.

Increased pericyte coverage mediated by endothelial-derived fibroblast growth factor-2 and interleukin-6 is a source of smooth muscle-like cells in pulmonary hypertension.

BACKGROUND: Pericytes and their crosstalk with endothelial cells are critical for the development of a functional microvasculature and vascular remodeling. It is also known that pulmonary endothelial dysfunction is intertwined with the initiation and progression of pulmonary arterial hypertension (PAH). We hypothesized that pulmonary endothelial dysfunction, characterized by abnormal fibroblast growth factor-2 and interleukin-6 signaling, leads to abnormal microvascular pericyte coverage causing pulmonary arterial medial thickening. METHODS AND RESULTS: In human lung tissues, numbers of pericytes are substantially increased (up to 2-fold) in distal PAH pulmonary arteries compared with controls. Interestingly, human pulmonary pericytes exhibit, in vitro, an accentuated proliferative and migratory response to conditioned media from human idiopathic PAH endothelial cells compared with conditioned media from control cells. Importantly, by using an anti-fibroblast growth factor-2 neutralizing antibody, we attenuated these proliferative and migratory responses, whereas by using an anti-interleukin-6 neutralizing antibody, we decreased the migratory response without affecting the proliferative response. Furthermore, in our murine retinal angiogenesis model, both fibroblast growth factor-2 and interleukin-6 administration increased pericyte coverage. Finally, using idiopathic PAH human and NG2DsRedBAC mouse lung tissues, we demonstrated that this increased pericyte coverage contributes to pulmonary vascular remodeling as a source of smooth muscle-like cells. Furthermore, we found that transforming growth factor-ß, in contrast to fibroblast growth factor-2 and interleukin-6, promotes human pulmonary pericyte differentiation into contractile smooth muscle-like cells. CONCLUSIONS: To the best of our knowledge, this is the first report of excessive pericyte coverage in distal pulmonary arteries in human PAH. We also show that this phenomenon is directly linked with pulmonary endothelial dysfunction.

Leptin signalling system as a target for pulmonary arterial hypertension therapy.

Excessive proliferation of pulmonary arterial smooth muscle cells (PA-SMCs) and perivascular inflammation lead to pulmonary arterial hypertension (PAH) progression, but they are not specifically targeted by the current therapies. Since leptin (Ob) and its main receptor ObR-b contribute to systemic vascular cell proliferation and inflammation, we questioned whether targeting Ob/ObR-b axis would be an effective antiproliferative and anti-inflammatory strategy against PAH. In idiopathic PAH (iPAH), using human lung tissues and primary cell cultures (early passages ≤5), we demonstrate that pulmonary endothelial cells (P-ECs) over produce Ob and that PA-SMCs overexpress ObR-b. Furthermore, we obtain evidence that Ob enhances proliferation of human PA-SMCs in vitro and increases right ventricular systolic pressure in Ob-treated mice in the chronic hypoxia-induced pulmonary hypertension (PH) model. Using human cells, we also show that Ob leads to monocyte activation and increases cell adhesion molecule expression levels in P-ECs. We also find that Ob/ObR-b axis contributes to PH susceptibility by using ObR-deficient rats, which display less severe hypoxia-induced PH (pulmonary haemodynamics, arterial muscularisation, PA-SMC proliferation and perivascular inflammation). Importantly, we demonstrate the efficacy of two curative strategies using a soluble Ob neutraliser and dichloroacetate in hypoxia-induced PH. We demonstrate here that Ob/ObR-b axis may represent anti-proliferative and anti-inflammatory targets in PAH.

Angiomatoid fibrous histiocytoma of the pulmonary artery: a multidisciplinary discussion.

AIMS: Angiomatoid fibrous histiocytoma (AFH) is a rare neoplastic disease usually occurring in the dermis or subcutis of the extremities of young adults or children. Although sporadic cases in deep soft tissue and visceral organs have been reported, we present here the first description of AFH developing in a large artery. METHODS AND RESULTS: Paraffin sections of the surgical specimen were stained with haematoxylin and eosin, and immunohistochemistry was performed (CKAE1/AE3, EMA, CD34, p63, CD38, smooth muscle actin, and desmin). In addition, FISH and RT-PCR were applied in order to check for EWRS rearrangement. The histomorphological features, and FISH analysis revealing rearrangement of EWSR, indicated the definitive diagnosis of AFH. RT-PCR confirmed EWSR rearrangement, and detected an EWSR1-ATF1 fusion transcript. CONCLUSIONS: A thoracic location of AFH has not been reported until very recently, and shares a differential diagnosis with diverse neoplasms, including spindle cell carcinoma and low-grade sarcoma. We describe the first reported case of thoracic AFH arising in a large vessel, and highlight distinctive histological and molecular features.

Effects of B Cell Depletion by CD19-Targeted Chimeric Antigen Receptor T Cells in a Murine Model of Systemic Sclerosis.

OBJECTIVE: Our goal was to study the tolerance and efficacy of two B cell depletion strategies, including one with CD19-targeted chimeric antigen receptor (CAR) T cells, in a preclinical model mimicking the severe lung damages observed in systemic sclerosis. METHODS: B cell depletion strategies were evaluated in the Fra-2 transgenic (Tg) mouse model. We considered a first group of 16 untreated mice, a second group of 15 mice receiving a single dose of anti-CD20 monoclonal antibody (mAb), and a third group of 8 mice receiving CD19-targeted CAR-T cells in combination with anti-CD20 monoclonal antibody. After six weeks of clinical evaluation, different validated markers of inflammation, lung fibrosis, and pulmonary vascular remodeling were assessed. RESULTS: CD19-targeted CAR-T cells infusion in combination with anti-CD20 mAb resulted in a deeper B cell depletion than anti-CD20 mAb alone in the peripheral blood and lesional lungs of Fra-2 Tg mice. CAR-T cell infusion worsened the clinical score and increased mortality in Fra-2 Tg mice. In line with the above findings, CAR-T cell infusion significantly increased lung collagen content, the histological fibrosis score, and right ventricular systolic pressure. CAR-T cells accumulated in lesional lungs and promoted T activation and inflammatory cytokine production. Treatment with anti-CD20 mAb in monotherapy had no impact on lung inflammation-driven fibrosis and pulmonary hypertension. CONCLUSION: B cell therapies failed to show efficacy in the Fra2 Tg mice. The exacerbated Fra-2 lung inflammatory burden stimulated accumulation and expansion of activated CD19-targeted CAR-T cells, secondarily inducing T cell activation and systemic inflammation, finally leading to disease worsening.

Central role of ubiquitin-specific protease 8 in leptin signaling pathway in pulmonary arterial hypertension.

BACKGROUND: Leptin receptor (ObR-b) is overexpressed in pulmonary artery smooth muscle cells (PA-SMCs) from patients with pulmonary arterial hypertension (PAH) and is implicated in both mechanisms that contribute to pulmonary vascular remodeling: hyperproliferation and inflammation. Our aim was to investigate the role of ubiquitin-specific peptidase 8 (USP8) in ObR-b overexpression in PAH. METHODS: We performed in situ and in vitro experiments in human lung specimens and isolated PA-SMCs combined with 2 different in vivo models in rodents and we generated a mouse with an inducible USP8 deletion specifically in smooth muscles. RESULTS: Our results showed an upregulation of USP8 in the smooth muscle layer of distal pulmonary arteries from patients with PAH, and upregulation of USP8 expression in PAH PA-SMCs, compared to controls. USP8 inhibition in PAH PA-SMCs significantly blocked both ObR-b protein expression level at the cell surface as well as ObR-b-dependant intracellular signaling pathway as shown by a significant decrease in pSTAT3 expression. USP8 was required for ObR-b activation in PA-SMCs and its inhibition prevented Ob-mediated cell proliferation through STAT3 pathway. USP8 inhibition by the chemical inhibitor DUBs-IN-2 protected against the development of experimental PH in the 2 established experimental models of PH. Targeting USP8 specifically in smooth muscle cells in a transgenic mouse model also protected against the development of experimental PH. CONCLUSIONS: Our findings highlight the role of USP8 in ObR-b overexpression and pulmonary vascular remodeling in PAH.

Arsenic trioxide demonstrates efficacy in a mouse model of preclinical systemic sclerosis.

BACKGROUND: Uncontrolled T-cell activation plays a key role in systemic sclerosis (SSc). Arsenic trioxide (ATO) has immunological effects and has demonstrated potential in preclinical SSc models. In this study, we assessed the efficacy of ATO in Fra2 transgenic (Fra2TG) mice, which develop severe vascular remodeling of pulmonary arterioles and nonspecific interstitial pneumonia-like lung disease, closely resembling human SSc-associated pulmonary hypertension, therefore partially resembling to the SSc human disease. METHODS: The efficacy of ATO in Fra2TG mice was evaluated through histological scoring and determination of cell infiltration. Fibrotic changes in the lungs were assessed by measuring collagen content biochemically, using second harmonic generation to measure fibrillar collagen, and imaging via computed tomography. Cardiovascular effects were determined by measuring right ventricular systolic pressure and vessel remodeling. The mechanism of action of ATO was then investigated by analyzing lung cell infiltrates using flow cytometry and bulk RNA with sequencing techniques. RESULTS: After ATO treatment, the Ashcroft histological score was substantially decreased by 33% in ATO-treated mice compared to control mice. Other investigations of fibrotic markers showed a trend of reduction in various measurements of fibrosis, but the differences did not reach significance. Further cardiovascular investigations revealed convergent findings supporting a beneficial effect of ATO, with reduced right ventricular systolic pressure and medial wall thickness, and a significant decrease in the number of muscularized distal pulmonary arteries in ATO-treated Fra2TG mice compared to untreated Fra2TG mice. Additionally, inflammatory cell infiltration was also markedly reduced in lesioned lungs. A reduction in the frequency of CD4 + and T effector memory cells, and an increase in the percentage of CD4 + T naive cells in the lungs of ATO-treated Fra-2TG mice, was observed when compared to PBS group Fra-2Tg mice. RNA-seq analysis of ATO-treated mouse lungs revealed a downregulation of biological pathways associated with immune activity and inflammation, such as T-cell activation, regulation of leucocyte activation, leucocyte cell-cell adhesion, and regulation of lymphocyte activation. CONCLUSIONS: Our results suggest the clinical relevance of ATO treatment in SSc. Using the Fra2TG mouse model, we observed significant lung histological changes, a trend towards a decrease in various fibrotic makers, and a strong reduction in vascular remodeling. The mechanism of action of ATO appears to involve a marked counteraction of the immune activation characteristic of SSc, particularly T-cell involvement. These findings pave the way for further studies in SSc.

Inhibition of Soluble Epoxide Hydrolase Does Not Promote or Aggravate Pulmonary Hypertension in Rats.

Inhibitors of soluble epoxide hydrolase (sEH), which catalyzes the hydrolysis of various natural epoxides to their corresponding diols, present an opportunity for developing oral drugs for a range of human cardiovascular and inflammatory diseases, including, among others, diabetes and neuropathic pain. However, some evidence suggests that their administration may precipitate the development of pulmonary hypertension (PH). We thus evaluated the impact of chronic oral administration of the sEH inhibitor TPPU (N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]-urea) on hemodynamics, pulmonary vascular reactivity, and remodeling, as well as on right ventricular (RV) dimension and function at baseline and in the Sugen (SU5416) + hypoxia (SuHx) rat model of severe PH. Treatment with TPPU started 5 weeks after SU5416 injection for 3 weeks. No differences regarding the increase in pulmonary vascular resistance, remodeling, and inflammation, nor the abolishment of phenylephrine-induced pulmonary artery constriction, were noted in SuHx rats. In addition, TPPU did not modify the development of RV dysfunction, hypertrophy, and fibrosis in SuHx rats. Similarly, none of these parameters were affected by TPPU in normoxic rats. Complementary in vitro data demonstrated that TPPU reduced the proliferation of cultured human pulmonary artery-smooth muscle cells (PA-SMCs). This study demonstrates that inhibition of sEH does not induce nor aggravate the development of PH and RV dysfunction in SuHx rats. In contrast, a potential beneficial effect against pulmonary artery remodeling in humans is suggested.