DNA methyltransferase 3B deficiency unveils a new pathological mechanism of pulmonary hypertension.

DNA methylation plays critical roles in vascular pathology of pulmonary hypertension (PH). The underlying mechanism, however, remains undetermined. Here, we demonstrate that global DNA methylation was elevated in the lungs of PH rat models after monocrotaline administration or hypobaric hypoxia exposure. We showed that DNA methyltransferase 3B (DNMT3B) was up-regulated in both PH patients and rodent models. Furthermore, Dnmt3b -/- rats exhibited more severe pulmonary vascular remodeling. Consistently, inhibition of DNMT3B promoted proliferation/migration of pulmonary artery smooth muscle cells (PASMCs) in response to platelet-derived growth factor-BB (PDGF-BB). In contrast, overexpressing DNMT3B in PASMCs attenuated PDGF-BB-induced proliferation/migration and ameliorated hypoxia-mediated PH and right ventricular hypertrophy in mice. We also showed that DNMT3B transcriptionally regulated inflammatory pathways. Our results reveal that DNMT3B is a previously undefined mediator in the pathogenesis of PH, which couples epigenetic regulations with vascular remodeling and represents a therapeutic target to tackle PH.

Spermine promotes pulmonary vascular remodelling and its synthase is a therapeutic target for pulmonary arterial hypertension.

Pathological mechanisms of pulmonary arterial hypertension (PAH) remain largely unexplored. Effective treatment of PAH remains a challenge. The aim of this study was to discover the underlying mechanism of PAH through functional metabolomics and to help develop new strategies for prevention and treatment of PAH.Metabolomic profiling of plasma in patients with idiopathic PAH was evaluated through high-performance liquid chromatography mass spectrometry, with spermine identified to be the most significant and validated in another independent cohort. The roles of spermine and spermine synthase were examined in pulmonary arterial smooth muscle cells (PASMCs) and rodent models of pulmonary hypertension.Using targeted metabolomics, plasma spermine levels were found to be higher in patients with idiopathic PAH compared to healthy controls. Spermine administration promoted proliferation and migration of PASMCs and exacerbated vascular remodelling in rodent models of pulmonary hypertension. The spermine-mediated deteriorative effect can be attributed to a corresponding upregulation of its synthase in the pathological process. Inhibition of spermine synthase in vitro suppressed platelet-derived growth factor-BB-mediated proliferation of PASMCs, and in vivo attenuated monocrotaline-mediated pulmonary hypertension in rats.Plasma spermine promotes pulmonary vascular remodelling. Inhibiting spermine synthesis could be a therapeutic strategy for PAH.

Circulating Plasma Metabolomic Profiles Differentiate Rodent Models of Pulmonary Hypertension and Idiopathic Pulmonary Arterial Hypertension Patients.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a severe progressive disease with systemic metabolic dysregulation. Monocrotaline (MCT)-induced and hypoxia-induced pulmonary hypertension (PH) rodent models are the most widely used preclinical models, however, whether or not these preclinical models recapitulate metabolomic profiles of PAH patients remain unclear. METHODS: In this study, a targeted metabolomics panel of 126 small molecule metabolites was conducted. We applied it to the plasma of the 2 preclinical rodent models of PH and 30 idiopathic pulmonary arterial hypertension (IPAH) patients as well as 30 healthy controls to comparatively assess the metabolomic profiles of PAH patients and rodent models. RESULTS: Significantly different metabolomics profiling and pathways were shown among the 2 classical rodent models and IPAH patients. Pathway analysis demonstrated that methionine metabolism and urea cycle metabolism were the most significant pathway involved in the pathogenesis of hypoxia-induced PH model and MCT-induced model, respectively, and both of them were also observed in the dysregulated pathways in IPAH patients. CONCLUSIONS: These 2 models may develop PAH through different metabolomic pathways and each of the 2 classical PH model resembles IPAH patients in certain aspects.

3-Bromopyruvate Attenuates Experimental Pulmonary Hypertension via Inhibition of Glycolysis.

BACKGROUND: The shift of metabolism from mitochondrial oxidative phosphorylation to glycolysis and mitochondria binding partner of hexokinase are features common to cancer. These have been seen in pulmonary hypertension (PH) as well. An inhibitor of hexokinase 2 (HK 2), the small molecule 3-bromopyruvate (3-BrPA) is an incredibly powerful and swift-acting anticancer agent. However, whether it could be of potential benefit to PH has still been unknown. METHODS: Sprague-Dawley rats with monocrotaline (MCT)-induced PH were administered 2 oral doses of 3-BrPA (15 and 30 mg/kg/day, respectively) for 14 days. Hemodynamic parameters were obtained by right heart catheterization. Histopathology, immunohistochemistry, transmission electron microscopy, flow cytometry, and assessments of relative protein expressions were conducted. RESULTS: Compared with MCT treatment, 3-BrPA decreased mean pulmonary arterial pressure and pulmonary vascular resistance, and increased cardiac output. 3-BrPA significantly suppressed proliferation in addition to enhancing apoptosis of pulmonary artery smooth muscle cells, attenuating small pulmonary artery remodeling and right ventricular hypertrophy. Treatment with 3-BrPA markedly reduced the mitochondrial membrane potential and restored mitochondrial structure. Furthermore, 3-BrPA significantly inhibited HK 2 expression but not HK 1. The expression of both pyruvate dehydrogenase kinase and lactate dehydrogenase was decreased whereas that of pyruvate dehydrogenase and cytosolic cytochrome c was upregulated with 3-BrPA administration. CONCLUSION: This study demonstrates the reversal of PH by 3-BrPA is related to alteration in glycolysis and improved mitochondria function, indicating the "metabolic targeting" as a rational therapeutic strategy for PH.

Metabolic reprogramming of the urea cycle pathway in experimental pulmonary arterial hypertension rats induced by monocrotaline.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a rare systemic disorder associated with considerable metabolic dysfunction. Although enormous metabolomic studies on PAH have been emerging, research remains lacking on metabolic reprogramming in experimental PAH models. We aim to evaluate the metabolic changes in PAH and provide new insight into endogenous metabolic disorders of PAH. METHOD: A single subcutaneous injection of monocrotaline (MCT) (60 mg kg- 1) was used for rats to establish PAH model. Hemodynamics and right ventricular hypertrophy were adopted to evaluate the successful establishment of PAH model. Plasma samples were assessed through targeted metabolomic profiling platform to quantify 126 endogenous metabolites. Orthogonal partial least squares discriminant analysis (OPLS-DA) was used to discriminate between MCT-treated model and control groups. Metabolite Set Enrichment Analysis was adapted to exploit the most disturbed metabolic pathways. RESULTS: Endogenous metabolites of MCT treated PAH model and control group were well profiled using this platform. A total of 13 plasma metabolites were significantly altered between the two groups. Metabolite Set Enrichment Analysis highlighted that a disruption in the urea cycle pathway may contribute to PAH onset. Moreover, five novel potential biomarkers in the urea cycle, adenosine monophosphate, urea, 4-hydroxy-proline, ornithine, N-acetylornithine, and two candidate biomarkers, namely, O-acetylcarnitine and betaine, were found to be highly correlated with PAH. CONCLUSION: The present study suggests a new role of urea cycle disruption in the pathogenesis of PAH. We also found five urea cycle related biomarkers and another two candidate biomarkers to facilitate early diagnosis of PAH in metabolomic profile.

Preparation of well-distributed titania nanopillar arrays on Ti6Al4V surface by induction heating for enhancing osteogenic differentiation of stem cells.

Great effort has recently been devoted to the preparation of nanoscale surfaces on titanium-based implants to achieve clinically fast osteoinduction and osseointegration, which relies on the unique characteristics of the nanostructure. In this work, we used induction heating treatment (IHT) as a rapid oxidation method to fabricate a porous nanoscale oxide layer on the Ti6Al4V surface for better medical application. Well-distributed vertical nanopillars were yielded by IHT for 20-35 s on the alloy surface. The composition of the oxides contained rutile/anatase TiO2 and a small amount of Al2O3 between the TiO2 grain boundaries (GBs). This technology resulted in a reduction and subsequent increase of surface roughness of 26-32 nm when upregulating the heating time, followed by the successive enhancement of the thickness, wettability and adhesion strength of the oxidation layer to the matrix. The surface hardness also distinctly rose to 554 HV in the IHT-35 s group compared with the 350 HV of bare Ti6Al4V. The massive small-angle GBs in the bare alloy promoted the formation of nanosized oxide crystallites. The grain refinement and deformation texture reduction further improved the mechanical properties of the matrix after IHT. Moreover, in vitro experiments on a mesenchymal stem cell (BMSC) culture derived from human bone marrow for 1-7 days indicated that the nanoscale layers did not cause cytotoxicity, and facilitated cell differentiation in osteoblasts by enhancing the gene and osteogenesis-related protein expressions after 1-3 weeks of culturing. The increase of the IHT time slightly advanced the BMSC proliferation and differentiation, especially during long-term culture. Our findings provide strong evidence that IHT oxidation technology is a novel nanosurface modification technology, which is potentially promising for further clinical development.

A bioactive coating with submicron-sized titania crystallites fabricated by induction heating of titanium after tensile deformations.

Thermal oxidation technology was widely investigated as one of effective surface modification method for improving the bioactivity and biocompatibility of titanium and its alloys. In this work, the induction heat oxidization method, a fast, efficient, economical and environmental protective technology, was applied to prepare the submicron-morphological oxide coating with variable rutile TiO2 equiaxed crystallites on the surface of pure Ti substrates after cold-drawing with 10-20% deformations. The results showed the plastic-deformed Ti cylinders recrystallized during induction heating treatment (IHT) for 10-20s which resulted in evolution of microstructures as well as slight improvement of microhardness. The surface characteristics of TiO2 crystallites in oxidation layers were determined by the microstructural evolutions of Ti substrate in terms of the nucleation and growth of TiO2 crystallites. Specially, the oxidized surface with 50-75nm roughness and more uniform and finer equiaxed oxide grains remarkablely improved the apatite deposition after bioactive evaluation in 1.5 × SBF for 7 days. This work provided a potential method to create controlled bioactive oxide coatings with submicro-/nano-scaled TiO2 crystallites on titanium substrate in terms of the role of metallographic microstructure in the formation process of titanium oxides.