Heterogeneity in lung (18)FDG uptake in pulmonary arterial hypertension: potential of dynamic (18)FDG positron emission tomography with kinetic analysis as a bridging biomarker for pulmonary vascular remodeling targeted treatments.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a disease of progressive vascular remodeling, characterized by dysregulated growth of pulmonary vascular cells and inflammation. A prevailing view is that abnormal cellular metabolism, notably aerobic glycolysis that increases glucose demand, underlies the pathogenesis of PAH. Increased lung glucose uptake has been reported in animal models. Few data exist from patients with PAH. METHODS AND RESULTS: Dynamic positron emission tomography imaging with fluorine-18-labeled 2-fluoro-2-deoxyglucose ((18)FDG) ligand with kinetic analysis demonstrated increased mean lung parenchymal uptake in 20 patients with PAH, 18 with idiopathic PAH (IPAH) (FDG score: 3.27±1.22), and 2 patients with connective tissue disease (5.07 and 7.11) compared with controls (2.02±0.71; P<0.05). Further compartment analysis confirmed increased lung glucose metabolism in IPAH. Lung (18)FDG uptake and metabolism varied within the IPAH population and within the lungs of individual patients, consistent with the recognized heterogeneity of vascular pathology in this disease. The monocrotaline rat PAH model also showed increased lung (18)FDG uptake, which was reduced along with improvements in vascular pathology after treatment with dicholoroacetate and 2 tyrosine kinase inhibitors, imatinib and sunitinib. Hyperproliferative pulmonary vascular fibroblasts isolated from IPAH patients exhibited upregulated glycolytic gene expression, along with increased cellular (18)FDG uptake; both were reduced by dicholoroacetate and imatinib. CONCLUSIONS: Some patients with IPAH exhibit increased lung (18)FDG uptake. (18)FDG positron emission tomography imaging is a tool to investigate the molecular pathology of PAH and its response to treatment.

Structural and functional asymmetry of the neonatal cerebral cortex.

Features of brain asymmetry have been implicated in a broad range of cognitive processes; however, their origins are still poorly understood. Here we investigated cortical asymmetries in 442 healthy term-born neonates using structural and functional magnetic resonance images from the Developing Human Connectome Project. Our results demonstrate that the neonatal cortex is markedly asymmetric in both structure and function. Cortical asymmetries observed in the term cohort were contextualized in two ways: by comparing them against cortical asymmetries observed in 103 preterm neonates scanned at term-equivalent age, and by comparing structural asymmetries against those observed in 1,110 healthy young adults from the Human Connectome Project. While associations with preterm birth and biological sex were minimal, significant differences exist between birth and adulthood.

The Developing Human Connectome Project Neonatal Data Release.

The Developing Human Connectome Project has created a large open science resource which provides researchers with data for investigating typical and atypical brain development across the perinatal period. It has collected 1228 multimodal magnetic resonance imaging (MRI) brain datasets from 1173 fetal and/or neonatal participants, together with collateral demographic, clinical, family, neurocognitive and genomic data from 1173 participants, together with collateral demographic, clinical, family, neurocognitive and genomic data. All subjects were studied in utero and/or soon after birth on a single MRI scanner using specially developed scanning sequences which included novel motion-tolerant imaging methods. Imaging data are complemented by rich demographic, clinical, neurodevelopmental, and genomic information. The project is now releasing a large set of neonatal data; fetal data will be described and released separately. This release includes scans from 783 infants of whom: 583 were healthy infants born at term; as well as preterm infants; and infants at high risk of atypical neurocognitive development. Many infants were imaged more than once to provide longitudinal data, and the total number of datasets being released is 887. We now describe the dHCP image acquisition and processing protocols, summarize the available imaging and collateral data, and provide information on how the data can be accessed.

Proteomic analysis of lung tissues from patients with pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension is a disorder of vascular remodeling causing increased resistance to pulmonary blood flow. The expression of proteins in lungs from pulmonary arterial hypertension patients was investigated in an unbiased approach to further understand the pathobiology of this disease. METHODS AND RESULTS: Label-free liquid chromatography tandem mass spectrometry was used to compare protein profiles in surgical samples of lungs from 8 patients with pulmonary arterial hypertension and 8 control subjects. More than 300 proteins were detected. On the basis of robust criteria, the levels of 25 proteins varied between the 2 groups. The majority of upregulated proteins were associated with cell growth, proliferation, and cell metabolism. Novel findings included an increased expression of chloride intracellular channel 4, receptor for advanced glycation end products, and periostin. Increased expression of chloride intracellular channel 4, a multifunctional protein involved in angiogenesis, and several signaling pathways implicated in pulmonary arterial hypertension--transforming growth factor-ß, vascular endothelial growth factor, and bone morphogenetic protein--was confirmed by Western blotting and localized predominantly to endothelial cells in occlusive and plexiform vascular lesions. CONCLUSIONS: Label-free proteomics identified differences in the expression of several proteins in the pulmonary arterial hypertension lung, many of which are relevant to the disease process. Increased expression of chloride intracellular channel 4 may be pertinent to the disorganized angiogenesis of plexiform lesions.

3'-Deoxy-3'-[18F]Fluorothymidine Positron Emission Tomography Depicts Heterogeneous Proliferation Pathology in Idiopathic Pulmonary Arterial Hypertension Patient Lung.

BACKGROUND: Pulmonary vascular cell hyperproliferation is characteristic of pulmonary vascular remodeling in pulmonary arterial hypertension. A noninvasive imaging biomarker is needed to track the pathology and assess the response to novel treatments targeted at resolving the structural changes. Here, we evaluated the application of radioligand 3'-deoxy-3'-[18F]-fluorothymidine (18FLT) using positron emission tomography. METHODS AND RESULTS: We performed dynamic 18FLT positron emission tomography in 8 patients with idiopathic pulmonary arterial hypertension (IPAH) and applied in-depth kinetic analysis with a reversible 2-compartment 4k model. Our results show significantly increased lung 18FLT phosphorylation (k3) in patients with IPAH compared with nonpulmonary arterial hypertension controls (0.086±0.034 versus 0.054±0.009 min-1; P<0.05). There was heterogeneity in the lung 18FLT signal both between patients with IPAH and within the lungs of each patient, compatible with histopathologic reports of lungs from patients with IPAH. Consistent with 18FLT positron emission tomographic data, TK1 (thymidine kinase 1) expression was evident in the remodeled vessels in IPAH patient lung. In addition, hyperproliferative pulmonary vascular fibroblasts isolated from patients with IPAH exhibited upregulated expression of TK1 and the thymidine transporter, ENT1 (equilibrative nucleoside transporter 1). In the monocrotaline and SuHx (Sugen hypoxia) rat pulmonary arterial hypertension models, increased lung 18FLT uptake was strongly associated with peripheral pulmonary vascular muscularization and the proliferation marker, Ki-67 score, together with prominent TK1 expression in remodeled vessels. Importantly, lung 18FLT uptake was attenuated by 2 antiproliferative treatments: dichloroacetate and the tyrosine kinase inhibitor, imatinib. CONCLUSIONS: Dynamic 18FLT positron emission tomography imaging can be used to report hyperproliferation in pulmonary hypertension and merits further study to evaluate response to treatment in patients with IPAH.

Inhibition of pyruvate dehydrogenase kinase improves pulmonary arterial hypertension in genetically susceptible patients.

Pulmonary arterial hypertension (PAH) is a progressive vascular disease with a high mortality rate. It is characterized by an occlusive vascular remodeling due to a pro-proliferative and antiapoptotic environment in the wall of resistance pulmonary arteries (PAs). Proliferating cells exhibit a cancer-like metabolic switch where mitochondrial glucose oxidation is suppressed, whereas glycolysis is up-regulated as the major source of adenosine triphosphate production. This multifactorial mitochondrial suppression leads to inhibition of apoptosis and downstream signaling promoting proliferation. We report an increase in pyruvate dehydrogenase kinase (PDK), an inhibitor of the mitochondrial enzyme pyruvate dehydrogenase (PDH, the gatekeeping enzyme of glucose oxidation) in the PAs of human PAH compared to healthy lungs. Treatment of explanted human PAH lungs with the PDK inhibitor dichloroacetate (DCA) ex vivo activated PDH and increased mitochondrial respiration. In a 4-month, open-label study, DCA (3 to 6.25 mg/kg b.i.d.) administered to patients with idiopathic PAH (iPAH) already on approved iPAH therapies led to reduction in mean PA pressure and pulmonary vascular resistance and improvement in functional capacity, but with a range of individual responses. Lack of ex vivo and clinical response was associated with the presence of functional variants of SIRT3 and UCP2 that predict reduced protein function. Impaired function of these proteins causes PDK-independent mitochondrial suppression and pulmonary hypertension in mice. This first-in-human trial of a mitochondria-targeting drug in iPAH demonstrates that PDK is a druggable target and offers hemodynamic improvement in genetically susceptible patients, paving the way for novel precision medicine approaches in this disease.

Elucidation of toxicity pathways in lung epithelial cells induced by silicon dioxide nanoparticles.

A study into the effects of amorphous nano-SiO2 particles on A549 lung epithelial cells was undertaken using proteomics to understand the interactions that occur and the biological consequences of exposure of lung to nanoparticles. Suitable conditions for treatment, where A549 cells remained viable for the exposure period, were established by following changes in cell morphology, flow cytometry, and MTT reduction. Label-free proteomics was used to estimate the relative level of proteins from their component tryptic peptides detected by mass spectrometry. It was found that A549 cells tolerated treatment with 100 µg/ml nano-SiO2 in the presence of 1.25% serum for at least 4 h. After this time detrimental changes in cell morphology, flow cytometry, and MTT reduction were evident. Proteomics performed after 4 h indicated changes in the expression of 47 proteins. Most of the proteins affected fell into four functional groups, indicating that the most prominent cellular changes were those that affected apoptosis regulation (e.g. UCP2 and calpain-12), structural reorganisation and regulation of actin cytoskeleton (e.g. PHACTR1), the unfolded protein response (e.g. HSP 90), and proteins involved in protein synthesis (e.g. ribosomal proteins). Treatment with just 10 µg/ml nano-SiO2 particles in serum-free medium resulted in a rapid deterioration of the cells and in medium containing 10% serum the cells were resistant to up to 1000 µg/ml nano-SiO2 particles, suggesting interaction of serum components with the nanoparticles. A variety of serum proteins were found which bound to nano-SiO2 particles, the most prominent of which were albumin, apolipoprotein A-I, hemoglobin, vitronectin and fibronectin. The use of a proteomics platform, with appropriately designed experimental conditions, enabled the early biological perturbations induced by nano-SiO2 in a model target cell system to be identified. The approach facilitates the design of more focused test systems for use in tiered evaluations of nanomaterials.

Elucidation of xenobiotic metabolism pathways in human skin and human skin models by proteomic profiling.

BACKGROUND: Human skin has the capacity to metabolise foreign chemicals (xenobiotics), but knowledge of the various enzymes involved is incomplete. A broad-based unbiased proteomics approach was used to describe the profile of xenobiotic metabolising enzymes present in human skin and hence indicate principal routes of metabolism of xenobiotic compounds. Several in vitro models of human skin have been developed for the purpose of safety assessment of chemicals. The suitability of these epidermal models for studies involving biotransformation was assessed by comparing their profiles of xenobiotic metabolising enzymes with those of human skin. METHODOLOGY/PRINCIPAL FINDINGS: Label-free proteomic analysis of whole human skin (10 donors) was applied and analysed using custom-built PROTSIFT software. The results showed the presence of enzymes with a capacity for the metabolism of alcohols through dehydrogenation, aldehydes through dehydrogenation and oxidation, amines through oxidation, carbonyls through reduction, epoxides and carboxylesters through hydrolysis and, of many compounds, by conjugation to glutathione. Whereas protein levels of these enzymes in skin were mostly just 4-10 fold lower than those in liver and sufficient to support metabolism, the levels of cytochrome P450 enzymes were at least 300-fold lower indicating they play no significant role. Four epidermal models of human skin had profiles very similar to one another and these overlapped substantially with that of whole skin. CONCLUSIONS/SIGNIFICANCE: The proteomics profiling approach was successful in producing a comprehensive analysis of the biotransformation characteristics of whole human skin and various in vitro skin models. The results show that skin contains a range of defined enzymes capable of metabolising different classes of chemicals. The degree of similarity of the profiles of the in vitro models indicates their suitability for epidermal toxicity testing. Overall, these results provide a rational basis for explaining the fate of xenobiotics in skin and will aid chemical safety testing programmes.