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.

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.

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.

Get to Know Your Neighbors: Characterization of Close Bacillus anthracis Isolates and Toxin Profile Diversity in the Bacillus cereus Group.

Unexpected atypical isolates of Bacillus cereus s.l. occasionally challenge conventional microbiology and even the most advanced techniques for anthrax detection. For anticipating and gaining trust, 65 isolates of Bacillus cereus s.l. of diverse origin were sequenced and characterized. The BTyper3 tool was used for assignation to genomospecies B. mosaicus (34), B. cereus s.s (29) and B. toyonensis (2), as well as virulence factors and toxin profiling. None of them carried any capsule or anthrax-toxin genes. All harbored the non-hemolytic toxin nheABC and sphygomyelinase spH genes, whereas 41 (63%), 30 (46%), 11 (17%) and 6 (9%) isolates harbored cytK-2, hblABCD, cesABCD and at least one insecticidal toxin gene, respectively. Matrix-assisted laser desorption ionization-time of flight mass spectrometry confirmed the production of cereulide (ces genes). Phylogeny inferred from single-nucleotide polymorphisms positioned isolates relative to the B. anthracis lineage. One isolate (BC38B) was of particular interest as it appeared to be the closest B. anthracis neighbor described so far. It harbored a large plasmid similar to other previously described B. cereus s.l. megaplasmids and at a lower extent to pXO1. Whereas bacterial collection is enriched, these high-quality public genetic data offer additional knowledge for better risk assessment using future NGS-based technologies of detection.

Additive protective effects of sacubitril/valsartan and bosentan on vascular remodelling in experimental pulmonary hypertension.

AIMS: Although right ventricular (RV) function is an important determinant of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), there is no treatment targeting directly the RV. We evaluate the efficacy of sacubitril/valsartan (LCZ 696) as add-on therapy to bosentan in rats with severe pulmonary hypertension (PH). METHODS AND RESULTS: Combination therapy of LCZ 696 and bosentan has additive vascular protective effects against the pulmonary vascular remodelling and PH in two preclinical models of severe PH. Compared with monotherapy, co-treatment of LCZ 696 (30 or 68 mg/kg/day for 2 weeks, per os) and bosentan (100 mg/kg/day for 2 weeks, per os) started 7 days after monocrotaline (MCT) injection substantially reduces pulmonary pressures, vascular remodelling, and RV hypertrophy and fibrosis in rats. Consistent with these observations, co-treatment of rats with established PH induced by sugen/hypoxia (SuHx) with LCZ 696 (30 mg/kg/day for 3 weeks, per os) and bosentan (100 mg/kg/day for 3 weeks, per os) started 5 weeks after Sugen injection partially attenuate total pulmonary vascular resistance and cardiovascular structures. We also obtained evidence showing that LCZ 696 has anti-proliferative effect on cultured human pulmonary artery smooth muscle cells derived from patients with idiopathic PAH, an effect that is more pronounced in presence of bosentan. Finally, we found that the plasma levels of atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) are higher in rats co-treated with LCZ 696 (30 mg/kg/day) and bosentan (100 mg/kg/day) than in MCT and SuHx rats treated with vehicle. CONCLUSION: Dual therapy with LCZ 696 plus bosentan proved significantly superior beneficial effect to LCZ 696 or bosentan alone on vascular remodelling and severity of experimental PH.

Neutralization of CXCL12 attenuates established pulmonary hypertension in rats.

AIMS: The progressive accumulation of cells in pulmonary vascular walls is a key pathological feature of pulmonary arterial hypertension (PAH) that results in narrowing of the vessel lumen, but treatments targeting this mechanism are lacking. The C-X-C motif chemokine 12 (CXCL12) appears to be crucial in these processes. We investigated the activity of two CXCL12 neutraligands on experimental pulmonary hypertension (PH), using two complementary animal models. METHODS AND RESULTS: Male Wistar rats were injected with monocrotaline (MCT) or were subjected to SU5416 followed by 3-week hypoxia to induce severe PH. After PH establishment, assessed by pulsed-wave Doppler echocardiography, MCT-injected or SU5416 plus chronic hypoxia (SuHx) rats were randomized to receive CXCL12 neutraligands chalcone 4 or LIT-927 (100 mg/kg/day), the C-X-C motif chemokine receptor 4 (CXCR4) antagonist AMD3100 (5 mg/kg/day), or vehicle, for 2 or 3 weeks, respectively. At the end of these treatment periods, echocardiographic and haemodynamic measurements were performed and tissue samples were collected for protein expression and histological analysis. Daily treatment of MCT-injected or SuHx rats with established PH with chalcone 4 or LIT-927 partially reversed established PH, reducing total pulmonary vascular resistance, and remodelling of pulmonary arterioles. Consistent with these observations, we found that neutralization of CXCL12 attenuates right ventricular hypertrophy, pulmonary vascular remodelling, and decreases pulmonary artery smooth muscle cell (PA-SMC) proliferation in lungs of MCT-injected rats and SuHx rats. Importantly, CXCL12 neutralization with either chalcone 4 or LIT-927 inhibited the migration of PA-SMCs and pericytes in vitro with a better efficacy than AMD3100. Finally, we found that CXCL12 neutralization decreases vascular pericyte coverage and macrophage infiltration in lungs of both MCT-injected and SuHx rats. CONCLUSION: We report here a greater beneficial effect of CXCL12 neutralization vs. the conventional CXCR4 blockade with AMD3100 in the MCT and SuHx rat models of severe PH, supporting a role for CXCL12 in the progression of vascular complications in PH and opening to new therapeutic options.