Loss of SMAD3 Promotes Vascular Remodeling in Pulmonary Arterial Hypertension via MRTF Disinhibition.

RATIONALE: Vascular remodeling in pulmonary arterial hypertension (PAH) results from smooth muscle cell hypertrophy and proliferation of vascular cells. Loss of BMPR-II (bone morphogenetic protein receptor 2) signaling and increased signaling via TGF-β (transforming growth factor β) and its downstream mediators SMAD (small body size [a C. elegans protein] mothers against decapentaplegic [a Drosophila protein family])-2/3 has been proposed to drive lung vascular remodeling; yet, proteomic analyses indicate a loss of SMAD3 in PAH. OBJECTIVES: We proposed that SMAD3 may be dysregulated in PAH and that loss of SMAD3 may present a pathophysiological master switch by disinhibiting its interaction partner, MRTF (myocardin-related transcription factor), which drives muscle protein expression. METHODS: SMAD3 levels were measured in lungs from PAH patients, rats treated either with Sugen/hypoxia or monocrotaline (MCT), and in mice carrying a BMPR2 mutation. In vitro, effects of SMAD3 or BMPR2 silencing or SMAD3 overexpression on cell proliferation or smooth muscle hypertrophy were assessed. In vivo, the therapeutic and prophylactic potential of CCG1423, an inhibitor of MRTF, was investigated in Sugen/hypoxia rats. MEASUREMENTS AND MAIN RESULTS: SMAD3 was downregulated in lungs of patients with PAH and in pulmonary arteries of three independent PAH animal models. TGF-β treatment replicated the loss of SMAD3 in human pulmonary artery smooth muscle cells (huPASMCs) and human pulmonary artery endothelial cells. SMAD3 silencing increased proliferation and migration in huPASMCs and human pulmonary artery endothelial cells. Coimmunoprecipitation revealed reduced interaction of MRTF with SMAD3 in TGF-β-treated huPASMCs and pulmonary arteries of PAH animal models. In huPASMCs, loss of SMAD3 or BMPR-II increased smooth muscle actin expression, which was attenuated by MRTF inhibition. Conversely, SMAD3 overexpression prevented TGF-β-induced activation of an MRTF reporter and reduced actin stress fibers in BMPR2-silenced huPASMCs. MRTF inhibition attenuated PAH and lung vascular remodeling in Sugen/hypoxia rats. CONCLUSIONS: Loss of SMAD3 presents a novel pathomechanism in PAH that promotes vascular cell proliferation and-via MRTF disinhibition-hypertrophy of huPASMCs, thereby reconciling the parallel induction of a synthetic and contractile huPASMC phenotype

Bacterial Cyclic Diguanylate Signaling Networks Sense Temperature

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria

A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine, Sphingosine-1-Phosphate, and Heme of Human Lung.

Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH

Macitentan reduces progression of TGF-β1-induced pulmonary fibrosis and pulmonary hypertension

IF 12.242International audienceIdiopathic pulmonary fibrosis (IPF) is a progressive disease with an unknown cause. Two drugs, nintedanib and pirfenidone, have been shown to slow, but not stop, disease progression. Pulmonary hypertension (PH) is a frequent complication in IPF patients and is associated with poor prognosis. Macitentan is a dual endothelin receptor antagonist that is approved for pulmonary arterial hypertension treatment. We hypothesised that using macitentan to treat animals with pulmonary fibrosis induced by adenoviral vector encoding biologically active transforming growth factor-β1 (AdTGF-β1) would improve the PH caused by chronic lung disease and would limit the progression of fibrosis.Rats (Sprague Dawley) which received AdTGF-β1 were treated by daily gavage of macitentan (100 mg·kg-1·day-1), pirfenidone (0.5% food admix) or a combination from day 14 to day 28. Pulmonary artery pressure (PAP) was measured before the rats were killed, and fibrosis was subsequently evaluated by morphometric measurements and hydroxyproline analysis.AdTGF-β1 induced pulmonary fibrosis associated with significant PH. Macitentan reduced the increase in PAP and both macitentan and pirfenidone stopped fibrosis progression from day 14 to day 28. Macitentan protected endothelial cells from myofibroblast differentiation and apoptosis whereas pirfenidone only protected against fibroblast-to-myofibroblast differentiation. Both drugs induced apoptosis of differentiated myofibroblasts in vitro and in vivoOur results demonstrate that dual endothelin receptor antagonism was effective in both PH and lung fibrosis whereas pirfenidone only affected fibrosis

Metabolomic profiling of pulmonary hypertension.

<p>Analysis of the dataset total 376 named biochemical was detected. Statistical comparisons using Welch's Two-Sample t-Test show significantly altered biochemicals in PAH samples (N = 8) compared with biochemical profiling in normal samples (N = 8). The biochemical profiles of PAH tissue exhibited higher levels of 93 altered metabolites compared with the normal lung (p ≤ 0.05).</p

Enhanced Sphingolipids metabolism.

<p>a) Increasing metabolites of galactosylsphingosine, sphinganine, sphingosine and palmitoyl sphingomyelin were found in PAH. Gene array shows that expression of genes coding for the key enzymes that catalyze particular steps in the pathway are significant increased, including serine palmitoyltransferase, long chain base subunit 1(SPTLC1), serine palmitoyltransferase, long chain base subunit 3 (SPTLC3), neutral sphingomyelinase activation associated factor (NSMAF), sphingomyelin synthase 2 (sgms2), N-acylsphingosine amidohydrolase (non-lysosomal ceramidase) 2 (ASAH2) (P<0.01). In all graphs, metabolic data for normal lung are shown in filled green boxes for control, data for PAH lung are represented in filled pink boxes, and the genes encoding key enzyme are shown in open blue boxes for control and open green boxes data for PAH lung (NL = control, PAH = pulmonary hypertension). P<0.05 for the group labeled with red frame.</p

Patient characteristics.

<p>Notes: IPH: Idiopathic pulmonary arterial hypertension; SLE: systemic lupus erythematosus; CHD: congenital heart disease; sPAP: systolic pulmonary artery pressure; mPAP: Mean pulmonary artery pressure; MvO2:Mixed venous oxygen saturation</p><p>Patient characteristics.</p

Polyamines and intermediates were changed in PAH Arginine metabolism.

<p>a) In all graphs, metabolic data for normal lungs are shown in filled green boxes for control, data for PAH lungs are represented in filled pink boxes, and the genes encoding key enzyme are shown in open blue boxes for control and open green boxes data for PAH lung (NL = control, PAH = pulmonary hypertension); P<0.05 for the group labeled with red frame. Quantities are in arbitrary units specific to the internal standards for each quantified metabolite and normalized to protein concentration (N = 8 for each box). PAH patient samples exhibited lower level of arginine and higher levels of creatine, putrescine, tran-4-hydroxyproline and pro-hydroxy. This metabolic disruption can contribute to the formation of advanced arginine end products and to the severity of PAH. The middle panel shows classical arginine pathways. The genes encoding arginase1 (ARG1), nitric oxide synthase 1, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), collagen type XIV alpha 1 (COL14A1) and collagen type III alpha 1 (COL3A1), were significantly changed in PAH lung compared with NL, as shown in left panel with colorless box (p<0.01).</p