G6PD activity contributes to the regulation of histone acetylation and gene expression in smooth muscle cells and to the pathogenesis of vascular diseases.

We aimed to determine 1) the mechanism(s) that enables glucose-6-phosphate dehydrogenase (G6PD) to regulate serum response factor (SRF)- and myocardin (MYOCD)-driven smooth muscle cell (SMC)-restricted gene expression, a process that aids in the differentiation of SMCs, and 2) whether G6PD-mediated metabolic reprogramming contributes to the pathogenesis of vascular diseases in metabolic syndrome (MetS). Inhibition of G6PD activity increased (>30%) expression of SMC-restricted genes and concurrently decreased (40%) the growth of human and rat SMCs ex vivo. Expression of SMC-restricted genes decreased (>100-fold) across successive passages in primary cultures of SMCs isolated from mouse aorta. G6PD inhibition increased Myh11 (47%) while decreasing (>50%) Sca-1, a stem cell marker, in cells passaged seven times. Similarly, CRISPR-Cas9-mediated expression of the loss-of-function Mediterranean variant of G6PD (S188F; G6PDS188F) in rats promoted transcription of SMC-restricted genes. G6PD knockdown or inhibition decreased (48.5%) histone deacetylase (HDAC) activity, enriched (by 3-fold) H3K27ac on the Myocd promoter, and increased Myocd and Myh11 expression. Interestingly, G6PD activity was significantly higher in aortas from JCR rats with MetS than control Sprague-Dawley (SD) rats. Treating JCR rats with epiandrosterone (30 mg/kg/day), a G6PD inhibitor, increased expression of SMC-restricted genes, suppressed Serpine1 and Epha4, and reduced blood pressure. Moreover, feeding SD control (littermates) and G6PDS188F rats a high-fat diet for 4 mo increased Serpine1 and Epha4 expression and mean arterial pressure in SD but not G6PDS188F rats. Our findings demonstrate that G6PD downregulates transcription of SMC-restricted genes through HDAC-dependent deacetylation and potentially augments the severity of vascular diseases associated with MetS.NEW & NOTEWORTHY This study gives detailed mechanistic insight about the regulation of smooth muscle cell (SMC) phenotype by metabolic reprogramming and glucose-6-phosphate dehydrogenase (G6PD) in diabetes and metabolic syndrome. We demonstrate that G6PD controls the chromatin modifications by regulating histone deacetylase (HDAC) activity, which deacetylates histone 3-lysine 9 and 27. Notably, inhibition of G6PD decreases HDAC activity and enriches H3K27ac on myocardin gene promoter to enhance the expression of SMC-restricted genes. Also, we demonstrate for the first time that G6PD inhibitor treatment accentuates metabolic and transcriptomic reprogramming to reduce neointimal formation in coronary artery and large artery elastance in metabolic syndrome rats.

Hypoxic activation of glucose-6-phosphate dehydrogenase controls the expression of genes involved in the pathogenesis of pulmonary hypertension through the regulation of DNA methylation.

Metabolic reprogramming is considered important in the pathogenesis of the occlusive vasculopathy observed in pulmonary hypertension (PH). However, the mechanisms that link reprogrammed metabolism to aberrant expression of genes, which modulate functional phenotypes of cells in PH, remain enigmatic. Herein, we demonstrate that, in mice, hypoxia-induced PH was prevented by glucose-6-phosphate dehydrogenase deficiency (G6PDDef), and further show that established severe PH in Cyp2c44-/- mice was attenuated by knockdown with G6PD shRNA or by G6PD inhibition with an inhibitor (N-ethyl-N'-[(3ß,5α)-17-oxoandrostan-3-yl]urea, NEOU). Mechanistically, G6PDDef, knockdown and inhibition in lungs: 1) reduced hypoxia-induced changes in cytoplasmic and mitochondrial metabolism, 2) increased expression of Tet methylcytosine dioxygenase 2 (Tet2) gene, and 3) upregulated expression of the coding genes and long noncoding (lnc) RNA Pint, which inhibits cell growth, by hypomethylating the promoter flanking region downstream of the transcription start site. These results suggest functional TET2 is required for G6PD inhibition to increase gene expression and to reverse hypoxia-induced PH in mice. Furthermore, the inhibitor of G6PD activity (NEOU) decreased metabolic reprogramming, upregulated TET2 and lncPINT, and inhibited growth of control and diseased smooth muscle cells isolated from pulmonary arteries of normal individuals and idiopathic-PAH patients, respectively. Collectively, these findings demonstrate a previously unrecognized function for G6PD as a regulator of DNA methylation. These findings further suggest that G6PD acts as a link between reprogrammed metabolism and aberrant gene regulation and plays a crucial role in regulating the phenotype of cells implicated in the pathogenesis of PH, a debilitating disorder with a high mortality rate.

Heterogeneous Response in Rabbit Fetal Diaphragmatic Hernia Lungs After Tracheal Occlusion.

BACKGROUND: Fetal tracheal occlusion (TO) is an experimental therapeutic approach to stimulate lung growth in the most severe congenital diaphragmatic hernia (CDH) cases. We have previously demonstrated a heterogeneous response of normal fetal rabbit lungs after TO with the appearance of at least two distinct zones. The aim of this study was to examine the fetal lung response after TO in a left CDH fetal rabbit model. METHODS: Fetal rabbits at 25 d gestation underwent surgical creation of CDH followed by TO at 27 d and harvest on day 30. Morphometric analysis, global metabolomics, and fluorescence lifetime imaging microscopy (FLIM) were performed to evaluate structural and metabolic changes in control, CDH, and CDH + TO lungs. RESULTS: Right and left lungs were different at the baseline and had a heterogeneous pulmonary growth response in CDH and after TO. The relative percent growth of the right lungs in CDH + TO was higher than the left lungs. Morphometric analyses revealed heterogeneous tissue-to-airspace ratios, in addition to size and number of airspaces within and between the lungs in the different groups. Global metabolomics demonstrated a slower rate of metabolism in the CDH group with the left lungs being less metabolically active. TO stimulated metabolic activity in both lungs to different degrees. FLIM analysis demonstrated local heterogeneity in glycolysis, oxidative phosphorylation (OXPHOS), and FLIM "lipid-surfactant" signal within and between the right and left lungs in all groups. CONCLUSIONS: We demonstrate that TO leads to a heterogeneous morphologic and metabolic response within and between the right and left lungs in a left CDH rabbit model.

Untargeted and Semi-targeted Lipid Analysis of Biological Samples Using Mass Spectrometry-Based Metabolomics.

Liquid chromatography coupled to mass spectrometry (LC-MS)-based metabolomics and lipidomics offers invaluable tools to qualitatively and quantitatively study biological systems. Historically, unbiased (or discovery) analysis has been performed independently of targeted, quantitative analysis such as multiple reaction monitoring (MRM). These practices have been aptly carried out based on technical limitations of each assay. The wide mass scanning ranges typical of discovery approaches limit assay sensitivity, while targeted methods that improve analyte detection do not acquire data on ions not included in the targeted assay design. Recent improvements to quadrupole-Orbitrap technology have improved both scan speed as well as sensitivity, thus making these instruments more robust. By combining the improved robustness and coverage with stable isotope dilution (SID) techniques, advantages of the separate assays can now be realized in a single run, thereby improving the throughput of this type of analysis.

Heterogeneous Pulmonary Response After Tracheal Occlusion: Clues to Fetal Lung Growth.

BACKGROUND: Understanding inconsistent clinical outcomes in infants with severe congenital diaphragmatic hernia (CDH) after tracheal occlusion (TO) is a crucial step for advancing neonatal care. The objective of this study is to explore the heterogeneous airspace morphometry and the metabolic landscape changes in fetal lungs after TO. METHODS: Fetal lungs on days 1 and 4 after TO were examined using mass spectrometry-based metabolomics, fluorescence lifetime imaging microscopy (FLIM), the number of airspaces, and tissue-to-airspace ratio (TAR). RESULTS: Two morphometric areas were identified in TO lungs compared with controls (more small airspaces at day 1 and a higher number of enlarged airspaces at day 4). Global metabolomics analysis revealed a significant upregulation of glycolysis and a suppression of the tricarboxylic acid cycle in day 4 TO lungs compared with day 1 TO lungs. In addition, there was a significant increase in polyamines involved in cell growth and proliferation. Locally, FLIM analysis on day 1 TO lungs demonstrated two types of heterogeneous zones-similar to control and with increased oxidative phosphorylation. FLIM on day 4 TO lungs demonstrated appearance of zones with enlarged airspaces and a metabolic shift toward glycolysis, accompanied by a decrease in the FLIM "lipid-surfactant" signal. CONCLUSIONS: In normal fetal lungs, we report a novel temporal pattern of varied morphometric and metabolic changes. Initially, there is formation of zones with small airspaces, followed by airspace enlargement over time. Metabolically day 1 TO lungs have zones with increased oxidative phosphorylation, whereas day 4 TO lungs have a shift toward glycolysis in the enlarged airspaces. Based on our observations, we speculate that the "best responders" to tracheal occlusion should have bigger lungs with small airspaces and normal surfactant production.

Unique Heterogeneous Topological Pattern of the Metabolic Landscape in Rabbit Fetal Lungs following Tracheal Occlusion.

INTRODUCTION: Fetal tracheal occlusion (TO) is currently an experimental approach to drive accelerated lung growth. It is stimulated by mechanotransduction that results in increased cellular proliferation and growth. However, it is currently unknown how TO affects the metabolic landscape of fetal lungs. MATERIALS AND METHODS: TO or sham was performed on fetal rabbits at 26 days followed by lung harvest on day 30. Mass spectrometry was performed to evaluate global metabolic changes. Fluorescence lifetime intensity microscopy (FLIM) was performed to estimate local free/bound NADH relative ratio as an indicator of aerobic glycolysis versus oxidative phosphorylation (glycolysis/OXPHOS). RESULTS: TO results in a metabolic shift from tricarboxylic acid cycle towards glycolysis. FLIM reveals uniform structures in control lungs characterized by similar ratios of free/bound NADH indicating a homogenous topological pattern. Similar uniform structures are observed in shams with some variability in the glycolysis/OXPHOS ratio. In contrast, lungs following TO demonstrate different types of unique distinct topological zones: one with enlarged alveoli and a shift towards glycolysis; the other maintains balance between glycolysis/OXPHOS similar to control lungs. CONCLUSION: We demonstrate for the first time a unique variable topological pattern of metabolism in fetal lungs following TO with a wide variation of metabolism between zones.

Heterogeneity of blood processing and storage additives in different centers impacts stored red blood cell metabolism as much as storage time: lessons from REDS-III-Omics.

BACKGROUND: Biological and technical variability has been increasingly appreciated as a key factor impacting red blood cell (RBC) storability and, potentially, transfusion outcomes. Here, we performed metabolomics analyses to investigate the impact of factors other than storage duration on the metabolic phenotypes of stored RBC in a multicenter study. STUDY DESIGN AND METHODS: Within the framework of the REDS-III (Recipient Epidemiology and Donor Evaluation Study-III) RBC-Omics study, 13,403 donors were enrolled from four blood centers across the United States and tested for the propensity of their RBCs to hemolyze after 42 days of storage. Extreme hemolyzers were recalled and donated a second unit of blood. Units were stored for 10, 23, and 42 days prior to sample acquisition for metabolomics analyses. RESULTS: Unsupervised analyses of metabolomics data from 599 selected samples revealed a strong impact (14.2% of variance) of storage duration on metabolic phenotypes of RBCs. The blood center collecting and processing the units explained an additional 12.2% of the total variance, a difference primarily attributable to the storage additive (additive solution 1 vs. additive solution 3) used in the different hubs. Samples stored in mannitol-free/citrate-loaded AS-3 were characterized by elevated levels of high-energy compounds, improved glycolysis, and glutathione homeostasis. Increased methionine metabolism and activation of the transsulfuration pathway was noted in samples processed in the center using additive solution 1. CONCLUSION: Blood processing impacts the metabolic heterogeneity of stored RBCs from the largest multicenter metabolomics study in transfusion medicine to date. Studies are needed to understand if these metabolic differences influenced by processing/storage strategies impact the effectiveness of transfusions clinically.

Clonal expansion of vaccine-elicited T cells is independent of aerobic glycolysis.

In contrast to responses against infectious challenge, T cell responses induced via adjuvanted subunit vaccination are dependent on interleukin-27 (IL-27). We show that subunit vaccine-elicited cellular responses are also dependent on IL-15, again in contrast to the infectious response. Early expression of interferon regulatory factor 4 (IRF4) was compromised in either IL-27- or IL-15-deficient environments after vaccination but not infection. Because IRF4 facilitates metabolic support of proliferating cells via aerobic glycolysis, we expected this form of metabolic activity to be reduced in the absence of IL-27 or IL-15 signaling after vaccination. Instead, metabolic flux analysis indicated that vaccine-elicited T cells used only mitochondrial function to support their clonal expansion. Loss of IL-27 or IL-15 signaling during vaccination resulted in a reduction in mitochondrial function, with no corresponding increase in aerobic glycolysis. Consistent with these observations, the T cell response to vaccination was unaffected by in vivo treatment with the glycolytic inhibitor 2-deoxyglucose, whereas the response to viral challenge was markedly lowered. Collectively, our data identify IL-27 and IL-15 as critical to vaccine-elicited T cell responses because of their capacity to fuel clonal expansion through a mitochondrial metabolic program previously thought only capable of supporting quiescent naïve and memory T cells.

Red blood cell metabolism in Down syndrome: hints on metabolic derangements in aging.

Red blood cells (RBCs) are the most abundant cell in the human body. During their ∼120-day life span in the circulatory system, RBCs release oxygen to all human tissues while being exposed to tissue metabolic activity. Owing to the relative simplicity of their intrinsic metabolism and the abundance of metabolite transporters in RBC membranes, the metabolism of mature erythrocytes indirectly mirrors systemic metabolic homeostasis and its alterations as a function of physiological factors, such as aging. Trisomy 21 (T21), the etiological factor of Down syndrome (DS), has been shown to cause chronic autoinflammation, promoting alterations in RBC life span, size (macrocytosis), and redox homeostasis. Here, we provide the first mass spectrometry-based relative and absolute quantitative metabolomic description of human RBCs from volunteer disomic and trisomic donors (n = 97). The results indicate a widespread deregulation of T21 RBC metabolism, including significant intracellular accumulation of lactate, amino acids (except methionine), purine catabolites, glutathione metabolites, carboxylic acids, bile acids (especially conjugated ones), and acyl-conjugated carnitines. These changes may underlie some of the well-established comorbidities in DS. Finally, we identify sex- and/or T21-specific metabolic signatures of aging.

Global analysis of Arabidopsis/downy mildew interactions reveals prevalence of incomplete resistance and rapid evolution of pathogen recognition.

Interactions between Arabidopsis thaliana and its native obligate oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) represent a model system to study evolution of natural variation in a host/pathogen interaction. Both Arabidopsis and Hpa genomes are sequenced and collections of different sub-species are available. We analyzed ∼400 interactions between different Arabidopsis accessions and five strains of Hpa. We examined the pathogen's overall ability to reproduce on a given host, and performed detailed cytological staining to assay for pathogen growth and hypersensitive cell death response in the host. We demonstrate that intermediate levels of resistance are prevalent among Arabidopsis populations and correlate strongly with host developmental stage. In addition to looking at plant responses to challenge by whole pathogen inoculations, we investigated the Arabidopsis resistance attributed to recognition of the individual Hpa effectors, ATR1 and ATR13. Our results suggest that recognition of these effectors is evolutionarily dynamic and does not form a single clade in overall Arabidopsis phylogeny for either effector. Furthermore, we show that the ultimate outcome of the interactions can be modified by the pathogen, despite a defined gene-for-gene resistance in the host. These data indicate that the outcome of disease and disease resistance depends on genome-for-genome interactions between the host and its pathogen, rather than single gene pairs as thought previously.

Managing unused pharmaceuticals in a hospice setting: a pilot study.

With the escalating use of pharmaceuticals in health care, there has been increasing anxiety over the potential health risks associated with pharmaceutical waste accumulating in the environment. This research provided nurses in a hospice care facility in Concord, California, with education and training to offer patients a medication disposal service through the use of mail-back envelopes. Over the 6-month study period, 160 of the 400 distributed mailers were returned for disposal. The total weight of pharmaceuticals diverted for incineration was 107 pounds, with an average weight of 0.66 pounds per mailer. This study suggests that the mailer system and proper education of medical staff have the potential to improve medical waste management, but alternative approaches may be necessary to increase the rate of envelop return.