Publications by authors named "Martin Giera"

103 Publications

Metabolic Analysis of Vitreous/Lens and Retina in Wild Type and Retinal Degeneration Mice.

Int J Mol Sci 2021 Feb 26;22(5). Epub 2021 Feb 26.

Department of Ophthalmology, Ludwig-Maximilians-Universität München, Mathildenstraße 8, 80336 Munich, Germany.

Photoreceptors are the light-sensing cells of the retina and the major cell type affected in most inherited retinal degenerations. Different metabolic pathways sustain their high energetic demand in physiological conditions, particularly aerobic glycolysis. The principal metabolome of the mature retina has been studied, but only limited information is available on metabolic adaptations in response to key developmental events, such as eye opening. Moreover, dynamic metabolic changes due to retinal degeneration are not well understood. Here, we aimed to explore and map the ocular metabolic dynamics induced by eye opening in healthy (wild type) or -mutant (retinal degeneration 1, Rd1) mice, in which photoreceptors degenerate shortly after eye opening. To unravel metabolic differences emerging before and after eye opening under physiological and pathophysiological conditions, we performed nuclear magnetic resonance (NMR) spectroscopy-based metabolome analysis of wild type and Rd1 retina and vitreous/lens. We show that eye opening is accompanied by changes in the concentration of selected metabolites in the retina and by alterations in the vitreous/lens composition only in the retinal degeneration context. As such, we identify NAcetylaspartate as a potential novel vitreous/lens marker reflecting progressive retinal degeneration. Thus, our data can help elucidating mechanisms underlying key events in retinal physiology and reveal changes occurring in pathology, while highlighting the importance of the vitreous/lens in the characterization of retinal diseases.
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http://dx.doi.org/10.3390/ijms22052345DOI Listing
February 2021

Sema7A is crucial for resolution of severe inflammation.

Proc Natl Acad Sci U S A 2021 Mar;118(9)

Department of Anesthesiology and Intensive Care Medicine, Molecular Intensive Care Medicine, University Hospital Eberhard-Karls University, 72076 Tübingen, Germany;

Endogenous mediators regulating acute inflammatory responses in both the induction and resolution phases of inflammatory processes are pivotal in host defense and tissue homeostasis. Recent studies have identified neuronal guidance proteins characterized in axonal development that display immunomodulatory functions. Here, we identify the neuroimmune guidance cue Semaphorin 7A (Sema7A), which appears to link macrophage (MΦ) metabolic remodeling to inflammation resolution. Sema7A orchestrated MΦ chemotaxis and chemokinesis, activated MΦ differentiation and polarization toward the proresolving M2 phenotype, and promoted leukocyte clearance. Peritoneal MΦ displayed metabolic reprogramming, characterized by reductions in fatty acid oxidation and oxidative phosphorylation, increases in glycolysis and the pentose phosphate pathway, and truncation of the tricarboxylic acid cycle, which resulted in increased levels of the intermediates succinate and fumarate. The low accumulation of citrate in MΦ correlated with the decreased synthesis of prostaglandins, leading to a reduced impact on lipid-mediator class switching and the generation of specialized pro resolving lipid mediators. Signaling network analysis indicated that Sema7A induced the metabolic reprogramming of MΦ by activating the mTOR- and AKT2-signaling pathways. Administration of Sema7A orchestrated the resolution response to tissue homeostasis by shortening the resolution interval, promoting tissue protection in murine peritonitis, and enhancing survival in polymicrobial sepsis.
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http://dx.doi.org/10.1073/pnas.2017527118DOI Listing
March 2021

Virgin Olive Oil Phenolic Compounds Modulate the HDL Lipidome in Hypercholesterolaemic Subjects: A Lipidomic Analysis of the VOHF Study.

Mol Nutr Food Res 2021 Feb 9:e2001192. Epub 2021 Feb 9.

Universitat Rovira i Virgili, Facultat de Medicina i Ciències de la Salut, Departament de Medicina i Cirurgia, Grup Nutrició Funcional, Oxidació i Malalties Cardiovasculars (NFOC-Salut), Reus, Spain.

Scope: The lipidomic analysis of high-density lipoprotein (HDL) could be a useful way to identify new biomarkers of HDL function.

Methods And Results: A randomized, controlled, double-blind, crossover trial (33 hypercholesterolaemic subjects) was performed with a control virgin olive oil (VOO), VOO enriched with its own phenolic compounds (FVOO) or VOO enriched with additional phenolic compounds from thyme (FVOOT) for 3 weeks. HDL lipidomic analyses were performed using the Lipidyzer platform. VOO and FVOO intake increased the presence of monounsaturated-fatty acids (FAs) and decreased saturated and polyunsaturated FAs in triacylglyceride (TAG) species, among others species. In contrast, FVOOT intake did not induce these FAs changes. The decrease in TAG52:3(FA16:0) after VOO intake and the decrease in TAG52:5(FA18:2) after FVOO intake were inversely associated with changes in HDL resistance to oxidation. After FVOO intake, the decrease in TAG54:6(FA18:2) species in HDL was inversely associated with changes in HDL cholesterol efflux capacity.

Conclusion: VOO and FVOO consumption has an impact on the HDL lipidome, in particular TAG species. Although TAGs are minor components of HDL mass, the observed changes in TAG modulated HDL functionality towards a cardioprotective mode. The assessment of the HDL lipidome is a valuable approach to identify and characterize new biomarkers of HDL function. This article is protected by copyright. All rights reserved.
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http://dx.doi.org/10.1002/mnfr.202001192DOI Listing
February 2021

Reproducibility of Targeted Lipidome Analyses (Lipidyzer) in Plasma and Erythrocytes over a 6-Week Period.

Metabolites 2020 Dec 31;11(1). Epub 2020 Dec 31.

Department of Rheumatology, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands.

It is essential to measure lipid biomarkers with a high reproducibility to prevent biased results. We compared the lipid composition and inter-day reproducibility of lipid measurements in plasma and erythrocytes. Samples from 42 individuals (77% women, mean age 65 years, mean body mass index (BMI) 27 kg/m), obtained non-fasted at baseline and after 6 weeks, were used for quantification of up to 1000 lipid species across 13 lipid classes with the Lipidyzer platform. Intraclass correlation coefficients (ICCs) were calculated to investigate the variability of lipid concentrations between timepoints. The ICC distribution of lipids in plasma and erythrocytes were compared using Wilcoxon tests. After data processing, the analyses included 630 lipids in plasma and 286 in erythrocytes. From these, 230 lipids overlapped between sample types. In plasma, 78% of lipid measurements were reproduced well to excellently, compared to 37% in erythrocytes. The ICC score distribution in plasma (median ICC 0.69) was significantly better than in erythrocytes (median ICC 0.51) (-value < 0.001). At the class level, reproducibility in plasma was superior for triacylglycerols and cholesteryl esters while ceramides, diacylglycerols, (lyso)phosphatidylethanolamines, and sphingomyelins showed better reproducibility in erythrocytes. Although in plasma overall reproducibility was superior, differences at individual and class levels may favor the use of erythrocytes.
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http://dx.doi.org/10.3390/metabo11010026DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7823270PMC
December 2020

Disruptions of Anaerobic Gut Bacteria Are Associated with Stroke and Post-stroke Infection: a Prospective Case-Control Study.

Transl Stroke Res 2020 Oct 14. Epub 2020 Oct 14.

Center for Experimental and Molecular Medicine, Amsterdam Infection & Immunity Institute, Amsterdam UMC, location AMC,, University of Amsterdam, Meibergdreef 9, Amsterdam, The Netherlands.

In recent years, preclinical studies have illustrated the potential role of intestinal bacterial composition in the risk of stroke and post-stroke infections. The results of these studies suggest that bacteria capable of producing volatile metabolites, including trimethylamine-N-oxide (TMAO) and butyrate, play opposing, yet important roles in the cascade of events leading to stroke. However, no large-scale studies have been undertaken to determine the abundance of these bacterial communities in stroke patients and to assess the impact of disrupted compositions of the intestinal microbiota on patient outcomes. In this prospective case-control study, rectal swabs from 349 ischemic and hemorrhagic stroke patients (median age, 71 years; IQR: 67-75) were collected within 24 h of hospital admission. Samples were subjected to 16S rRNA amplicon sequencing and subsequently compared with samples obtained from 51 outpatient age- and sex-matched controls (median age, 72 years; IQR, 62-80) with similar cardiovascular risk profiles but without active signs of stroke. Plasma protein biomarkers were analyzed using a combination of nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS). Alpha and beta diversity analyses revealed higher disruption of intestinal communities during ischemic and hemorrhagic stroke compared with non-stroke matched control subjects. Additionally, we observed an enrichment of bacteria implicated in TMAO production and a loss of butyrate-producing bacteria. Stroke patients displayed two-fold lower plasma levels of TMAO than controls (median 1.97 vs 4.03 μM, Wilcoxon p < 0.0001). Finally, lower abundance of butyrate-producing bacteria within 24 h of hospital admission was an independent predictor of enhanced risk of post-stroke infection (odds ratio 0.77, p = 0.005), but not of mortality or functional patient outcome. In conclusion, aberrations in trimethylamine- and butyrate-producing gut bacteria are associated with stroke and stroke-associated infections.
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http://dx.doi.org/10.1007/s12975-020-00863-4DOI Listing
October 2020

Anti-Inflammatory and Proresolving Effects of the Omega-6 Polyunsaturated Fatty Acid Adrenic Acid.

J Immunol 2020 Nov 2;205(10):2840-2849. Epub 2020 Oct 2.

Department of Rheumatology, Leiden University Medical Center, 2333 ZA Leiden, the Netherlands.

Polyunsaturated fatty acids (PUFAs) and their metabolites are potent regulators of inflammation. Generally, omega ()-3 PUFAs are considered proresolving whereas -6 PUFAs are classified as proinflammatory. In this study, we characterized the inflammatory response in murine peritonitis and unexpectedly found the accumulation of adrenic acid (AdA), a poorly studied -6 PUFA. Functional studies revealed that AdA potently inhibited the formation of the chemoattractant leukotriene B (LTB), specifically in human neutrophils, and this correlated with a reduction of its precursor arachidonic acid (AA) in free form. AdA exposure in human monocyte-derived macrophages enhanced efferocytosis of apoptotic human neutrophils. In vivo, AdA treatment significantly alleviated arthritis in an LTB-dependent murine arthritis model. Our findings are, to our knowledge, the first to indicate that the -6 fatty acid AdA effectively blocks production of LTB by neutrophils and could play a role in resolution of inflammation in vivo.
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http://dx.doi.org/10.4049/jimmunol.1801653DOI Listing
November 2020

Results of an explorative clinical evaluation suggest immediate and persistent post-reperfusion metabolic paralysis drives kidney ischemia reperfusion injury.

Kidney Int 2020 12 8;98(6):1476-1488. Epub 2020 Aug 8.

Department of Clinical Chemistry & Laboratory Medicine, Leiden University Medical Centre, Leiden, Netherlands.

Delayed graft function is the manifestation of ischemia reperfusion injury in the context of kidney transplantation. While hundreds of interventions successfully reduce ischemia reperfusion injury in experimental models, all clinical interventions have failed. This explorative clinical evaluation examined possible metabolic origins of clinical ischemia reperfusion injury combining data from 18 pre- and post-reperfusion tissue biopsies with 36 sequential arteriovenous blood samplings over the graft in three study groups. These groups included living and deceased donor grafts with and without delayed graft function. Group allocation was based on clinical outcome. Magic angle NMR was used for tissue analysis and mass spectrometry-based platforms were used for plasma analysis. All kidneys were functional at one-year. Integration of metabolomic data identified a discriminatory profile to recognize future delayed graft function. This profile was characterized by post-reperfusion ATP/GTP catabolism (significantly impaired phosphocreatine recovery and significant persistent (hypo)xanthine production) and significant ongoing tissue damage. Failing high-energy phosphate recovery occurred despite activated glycolysis, fatty-acid oxidation, glutaminolysis and autophagia, and related to a defect at the level of the oxoglutarate dehydrogenase complex in the Krebs cycle. Clinical delayed graft function due to ischemia reperfusion injury associated with a post-reperfusion metabolic collapse. Thus, efforts to quench delayed graft function due to ischemia reperfusion injury should focus on conserving metabolic competence, either by preserving the integrity of the Krebs cycle and/or by recruiting metabolic salvage pathways.
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http://dx.doi.org/10.1016/j.kint.2020.07.026DOI Listing
December 2020

Altered Patterns of Compositional and Functional Disruption of the Gut Microbiota in Typhoid Fever and Nontyphoidal Febrile Illness.

Open Forum Infect Dis 2020 Jul 26;7(7):ofaa251. Epub 2020 Jun 26.

Center for Experimental and Molecular Medicine (CEMM), Amsterdam University Medical Centers, Location AMC, Amsterdam, the Netherlands.

Background: Experimental murine models and human challenge studies of Typhi infection have suggested that the gut microbiome plays an important protective role against the development of typhoid fever. Anaerobic bacterial communities have been hypothesized to mediate colonization resistance against species by producing short-chain fatty acids, yet the composition and function of the intestinal microbiota in human patients with typhoid fever remain ill defined.

Methods: We prospectively collected fecal samples from 60 febrile patients admitted to Chittagong Medical College Hospital, Bangladesh, with typhoid fever or nontyphoidal febrile illness and from 36 healthy age-matched controls. The collected fecal samples were subjected to 16s rRNA sequencing followed by targeted metabolomics analysis.

Results: Patients with typhoid fever displayed compositional and functional disruption of the gut microbiota compared with patients with nontyphoidal febrile illness and healthy controls. Specifically, typhoid fever patients had lower microbiota richness and alpha diversity and a higher prevalence of potentially pathogenic bacterial taxa. In addition, a lower abundance of short-chain fatty acid-producing taxa was seen in typhoid fever patients. The differences between typhoid fever and nontyphoidal febrile illness could not be explained by a loss of colonization resistance after antibiotic treatment, as antibiotic exposure in both groups was similar.

Conclusions: his first report on the composition and function of the gut microbiota in patients with typhoid fever suggests that the restoration of these intestinal commensal microorganisms could be targeted using adjunctive, preventive, or therapeutic strategies.
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http://dx.doi.org/10.1093/ofid/ofaa251DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7371416PMC
July 2020

Mild Exercise Does Not Prevent Atherosclerosis in APOE*3-Leiden.CETP Mice or Improve Lipoprotein Profile of Men with Obesity.

Obesity (Silver Spring) 2020 07 17;28 Suppl 1:S93-S103. Epub 2020 Jun 17.

Division of Endocrinology, Department of Medicine, Leiden University Medical Center, Leiden, The Netherlands.

Objective: Exercise has been shown to improve cardiometabolic health, yet neither the molecular connection nor the effects of exercise timing have been elucidated. The aim of this study was to investigate whether ad libitum or time-restricted mild exercise reduces atherosclerosis development in atherosclerosis-prone dyslipidemic APOE*3-Leiden.CETP mice and whether mild exercise training in men with obesity affects lipoprotein levels.

Methods: Mice were group-housed and subjected to ad libitum or time-restricted (first or last 6 hours of the active phase) voluntary wheel running for 16 weeks while on a cholesterol-rich diet, after which atherosclerosis development was assessed in the aortic root. Furthermore, nine men with obesity followed a 12-week mild exercise training program. Lipoprotein levels were measured by nuclear magnetic resonance spectroscopy in plasma collected pre and post exercise training.

Results: Wheel running did not affect plasma lipid levels, uptake of triglyceride-derived fatty acids by tissues, and aortic atherosclerotic lesion size or severity. Markers of training status were unaltered. Exercise training in men with obesity did not alter lipoprotein levels.

Conclusions: Mild exercise training does not reduce dyslipidemia or atherosclerosis development in APOE*3-Leiden.CETP mice or affect lipoprotein levels in humans. Future research on the effects of (time-restricted) exercise on atherosclerosis or lipid metabolism should consider more vigorous exercise protocols.
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http://dx.doi.org/10.1002/oby.22799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7496605PMC
July 2020

Schistosoma haematobium infection is associated with lower serum cholesterol levels and improved lipid profile in overweight/obese individuals.

PLoS Negl Trop Dis 2020 07 2;14(7):e0008464. Epub 2020 Jul 2.

Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands.

Infection with parasitic helminths has been reported to improve insulin sensitivity and glucose homeostasis, lowering the risk for type 2 diabetes. However, little is known about its impact on whole-body lipid homeostasis, especially in obese individuals. For this purpose, a cross-sectional study was carried out in lean and overweight/obese adults residing in the Lambaréné region of Gabon, an area endemic for Schistosoma haematobium. Helminth infection status, peripheral blood immune cell counts, and serum metabolic and lipid/lipoprotein levels were analyzed. We found that urine S. haematobium egg-positive individuals exhibited lower serum total cholesterol (TC; 4.42 vs 4.01 mmol/L, adjusted mean difference [95%CI] -0.30 [-0.68,-0.06]; P = 0.109), high-density lipoprotein (HDL)-C (1.44 vs 1.12 mmol/L, -0.24 [-0.43,-0.06]; P = 0.009) and triglyceride (TG; 0.93 vs 0.72 mmol/L, -0.20 [-0.39,-0.03]; P = 0.022) levels than egg-negative individuals. However, when stratified according to body mass index, these effects were only observed in overweight/obese infected individuals. Similarly, significant negative correlations between the intensity of infection, assessed by serum circulating anodic antigen (CAA) concentrations, and TC (r = -0.555; P<0.001), HDL-C (r = -0.327; P = 0.068), LDL-C (r = -0.396; P = 0.025) and TG (r = -0.381; P = 0.032) levels were found in overweight/obese individuals but not in lean subjects. Quantitative lipidomic analysis showed that circulating levels of some lipid species associated with cholesterol-rich lipoprotein particles were also significantly reduced in overweight/obese infected individuals in an intensity-dependent manner. In conclusion, we reported that infection with S. haematobium is associated with improved lipid profile in overweight/obese individuals, a feature that might contribute reducing the risk of cardiometabolic diseases in such population.
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http://dx.doi.org/10.1371/journal.pntd.0008464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7363109PMC
July 2020

Squalene-Tetrahymanol Cyclase Expression Enables Sterol-Independent Growth of Saccharomyces cerevisiae.

Appl Environ Microbiol 2020 08 18;86(17). Epub 2020 Aug 18.

Department of Biotechnology, Delft University of Technology, Delft, The Netherlands

Biosynthesis of sterols, which are considered essential components of virtually all eukaryotic membranes, requires molecular oxygen. Anaerobic growth of the yeast therefore strictly depends on sterol supplementation of synthetic growth media. Neocallimastigomycota are a group of strictly anaerobic fungi which, instead of containing sterols, contain the pentacyclic triterpenoid "sterol surrogate" tetrahymanol, which is formed by cyclization of squalene. Here, we demonstrate that expression of the squalene-tetrahymanol cyclase gene from the ciliate enables synthesis of tetrahymanol by Moreover, expression of enabled exponential growth of anaerobic cultures in sterol-free synthetic media. After deletion of the gene from a -expressing strain, native sterol synthesis was abolished and sustained sterol-free growth was demonstrated under anaerobic as well as aerobic conditions. Anaerobic cultures of -expressing on sterol-free medium showed lower specific growth rates and biomass yields than ergosterol-supplemented cultures, while their ethanol yield was higher. This study demonstrated that acquisition of a functional squalene-tetrahymanol cyclase gene offers an immediate growth advantage to under anaerobic, sterol-limited conditions and provides the basis for a metabolic engineering strategy to eliminate the oxygen requirements associated with sterol synthesis in yeasts. The laboratory experiments described in this report simulate a proposed horizontal gene transfer event during the evolution of strictly anaerobic fungi. The demonstration that expression of a single heterologous gene sufficed to eliminate anaerobic sterol requirements in the model eukaryote therefore contributes to our understanding of how sterol-independent eukaryotes evolved in anoxic environments. This report provides a proof of principle for a metabolic engineering strategy to eliminate sterol requirements in yeast strains that are applied in large-scale anaerobic industrial processes. The sterol-independent yeast strains described in this report provide a valuable platform for further studies on the physiological roles and impacts of sterols and sterol surrogates in eukaryotic cells.
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http://dx.doi.org/10.1128/AEM.00672-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7440791PMC
August 2020

Disturbed brain ether lipid metabolism and histology in Sjögren-Larsson syndrome.

J Inherit Metab Dis 2020 11 9;43(6):1265-1278. Epub 2020 Jul 9.

Department of Pediatric Neurology, Radboud university medical center, Amalia Children's Hospital, Donders Institute for Brain Cognition and Behaviour, Nijmegen, Netherlands.

Sjögren-Larsson syndrome (SLS) is a rare neurometabolic syndrome caused by deficient fatty aldehyde dehydrogenase. Patients exhibit intellectual disability, spastic paraplegia, and ichthyosis. The accumulation of fatty alcohols and fatty aldehydes has been demonstrated in plasma and skin but never in brain. Brain magnetic resonance imaging and spectroscopy studies, however, have shown an abundant lipid peak in the white matter of patients with SLS, suggesting lipid accumulation in the brain as well. Using histopathology, mass spectrometry imaging, and lipidomics, we studied the morphology and the lipidome of a postmortem brain of a 65-year-old female patient with genetically confirmed SLS and compared the results with a matched control brain. Histopathological analyses revealed structural white matter abnormalities with the presence of small lipid droplets, deficient myelin, and astrogliosis. Biochemically, severely disturbed lipid profiles were found in both white and gray matter of the SLS brain, with accumulation of fatty alcohols and ether lipids. Particularly, long-chain unsaturated ether lipid species accumulated, most prominently in white matter. Also, there was a striking accumulation of odd-chain fatty alcohols and odd-chain ether(phospho)lipids. Our results suggest that the central nervous system involvement in SLS is caused by the accumulation of fatty alcohols leading to a disbalance between ether lipid and glycero(phospho)lipid metabolism resulting in a profoundly disrupted brain lipidome. Our data show that SLS is not a pure leukoencephalopathy, but also a gray matter disease. Additionally, the histopathological abnormalities suggest that astrocytes and microglia might play a pivotal role in the underlying disease mechanism, possibly contributing to the impairment of myelin maintenance.
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http://dx.doi.org/10.1002/jimd.12275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7689726PMC
November 2020

Metabolic reprogramming related to whole-chromosome instability in models for Hürthle cell carcinoma.

Sci Rep 2020 06 12;10(1):9578. Epub 2020 Jun 12.

Department of Pathology, Leiden University Medical Center, Albinusdreef 2, 2333ZA, Leiden, The Netherlands.

Hürthle cell carcinoma (HCC) is a recurrent subtype of non-medullary thyroid cancer. HCC is characterized by profound whole-chromosome instability (w-CIN), resulting in a near-homozygous genome (NHG), a phenomenon recently attributed to reactive oxygen species (ROS) generated during mitosis by malfunctioning mitochondria. We studied shared metabolic traits during standard and glucose-depleted cell culture in thyroid cancer cell lines (TCCLs), with or without a NHG, using quantitative analysis of extra and intracellular metabolites and ROS production following inhibition of complex III with antimycin A. We found that the XTC.UC1 and FTC-236 cell lines (both NHG) are functionally impaired in complex I and produce significantly more superoxide radicals than SW579 and BHP 2-7 (non-NHG) after challenge with antimycin A. FTC-236 showed the lowest levels of glutathione and SOD2. XTC.UC1 and FTC-236 both exhibited reduced glycolytic activity and utilization of alternative sources to meet energy demands. Both cell lines also shared low levels of α-ketoglutarate and high levels of creatine, phosphocreatine, uridine diphosphate-N-acetylglucosamine, pyruvate and acetylcarnitine. Furthermore, the metabolism of XTC.UC1 was skewed towards the de novo synthesis of aspartate, an effect that persisted even in glucose-free media, pointing to reductive carboxylation. Our data suggests that metabolic reprogramming and a subtle balance between ROS generation and scavenging/conversion of intermediates may be involved in ROS-induced w-CIN in HCC and possibly also in rare cases of follicular thyroid cancer showing a NHG.
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http://dx.doi.org/10.1038/s41598-020-66599-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293353PMC
June 2020

Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease.

Cell Stem Cell 2020 06 26;26(6):862-879.e11. Epub 2020 May 26.

Department of Anatomy and Embryology, Leiden University Medical Center, 2333 Leiden, the Netherlands; Department of Applied Stem Cell Technologies, University of Twente, 7500 Enschede, the Netherlands. Electronic address:

Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.
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http://dx.doi.org/10.1016/j.stem.2020.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7284308PMC
June 2020

Toll-like receptor signaling induces a temporal switch towards a resolving lipid profile in monocyte-derived macrophages.

Biochim Biophys Acta Mol Cell Biol Lipids 2020 09 21;1865(9):158740. Epub 2020 May 21.

Leiden University Medical Center, Department of Rheumatology, Leiden, the Netherlands.

Inflammation is a tightly regulated process. During the past decade it has become clear that the resolution of inflammation is an active process and its dysregulation can contribute to chronic inflammation. Several cells and soluble mediators, including lipid mediators, regulate the course of inflammation and its resolution. It is, however, unclear which signals and cells are involved in initiating the resolution process. Macrophages are tissue resident cells and key players in regulating tissue inflammation through secretion of soluble mediators, including lipids. We hypothesize that persistent inflammatory stimuli can initiate resolution pathways in macrophages. In this study, we detected 21 lipids in LPS-stimulated human monocyte-derived macrophages by liquid chromatography coupled to tandem mass spectrometry. Cyclooxygenase-derived Prostaglandins were observed in the first six hours of stimulation. Interestingly, a switch towards 15-lipoxygenase products, such as the pro-resolving lipid precursors 15-HEPE and 17-HDHA was observed after 24 h. The RNA and protein expression of cyclooxygenase and 15-lipoxygenase were in line with this trend. Treatment with 17-HDHA increased IL-10 production of monocyte-derived macrophages and decreased LTB production by neutrophils, indicating the anti-inflammatory property of this lipid. These data reveal that monocyte-derived macrophages contribute to the resolution of inflammation in time by the production of pro-resolving lipids after an initial inflammatory stimulus.
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http://dx.doi.org/10.1016/j.bbalip.2020.158740DOI Listing
September 2020

Scientific workflow managers in metabolomics: an overview.

Analyst 2020 Jun 6;145(11):3801-3808. Epub 2020 May 6.

Center for Proteomics and Metabolomics, Leiden University Medical Center, Albinusdreef 2, 2333ZA, Leiden, The Netherlands.

Providing maximum information on the provenance of scientific results in life sciences is getting considerable attention since the widely publicized reproducibility crisis. Improving the reproducibility of data processing and analysis workflows is part of this movement and may help achieve clinical deployment quicker. Scientific workflow managers can be valuable tools towards achieving this goal. Although these platforms are already well established in the field of genomics and other omics fields, in metabolomics scripts and dedicated software packages are still more popular. However, versatile workflows for metabolomics exist in the KNIME and Galaxy platforms. We will here summarize the available options of scientific workflow managers dedicated to metabolomics analysis.
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http://dx.doi.org/10.1039/d0an00272kDOI Listing
June 2020

Lowering the increased intracellular pH of human-induced pluripotent stem cell-derived endothelial cells induces formation of mature Weibel-Palade bodies.

Stem Cells Transl Med 2020 07 12;9(7):758-772. Epub 2020 Mar 12.

The Einthoven Laboratory for Vascular and Regenerative Medicine, Division of Nephrology, Department of Internal Medicine, Leiden University Medical Center, Leiden, The Netherlands.

Differentiation of human-induced pluripotent stem cells (hiPSCs) into vascular endothelium is of great importance to tissue engineering, disease modeling, and use in regenerative medicine. Although differentiation of hiPSCs into endothelial-like cells (hiPSC-derived endothelial cells [hiPSC-ECs]) has been demonstrated before, controversy exists as to what extent these cells faithfully reflect mature endothelium. To address this issue, we investigate hiPSC-ECs maturation by their ability to express von Willebrand factor (VWF) and formation of Weibel-Palade bodies (WPBs). Using multiple hiPSCs lines, hiPSC-ECs failed to form proper VWF and WPBs, essential for angiogenesis, primary and secondary homeostasis. Lowering the increased intracellular pH (pHi) of hiPSC-ECs with acetic acid did result in the formation of elongated WPBs. Nuclear magnetic resonance data showed that the higher pHi in hiPSC-ECs occurred in association with decreased intracellular lactate concentrations. This was explained by decreased glycolytic flux toward pyruvate and lactate in hiPSC-ECs. In addition, decreased expression of monocarboxylate transporter member 1, a member of the solute carrier family (SLC16A1), which regulates lactate and H+ uptake, contributed to the high pHi of hiPSC-EC. Mechanistically, pro-VWF dimers require the lower pH environment of the trans-Golgi network for maturation and tubulation. These data show that while hiPSC-ECs may share many features with mature EC, they are characterized by metabolic immaturity hampering proper EC function.
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http://dx.doi.org/10.1002/sctm.19-0392DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7308639PMC
July 2020

Therapeutic iloprost for the treatment of acute respiratory distress syndrome (ARDS) (the ThIlo trial): a prospective, randomized, multicenter phase II study.

Trials 2020 Mar 4;21(1):242. Epub 2020 Mar 4.

Department of Anesthesiology and Intensive Care Medicine, University Hospital, Universitätsklinikum Tübingen, Universitätsklinikum Tübingen, Hoppe-Seyler-Straße 3, 72076, Tübingen, Germany.

Background: Acute respiratory distress syndrome (ARDS) is caused by rapid-onset (within hours) acute inflammatory processes in lung tissue, and it is a life-threatening condition with high mortality. The treatment of ARDS to date is focused on the prevention of further iatrogenic damage of the lung rather than the treatment of the initial inflammatory process. Several preclinical studies have revealed a beneficial effect of iloprost on the control of pulmonary inflammation, and in a small number of patients with ARDS, iloprost treatment resulted in improved oxygenation. Therefore, we plan to conduct a large multicenter trial to evaluate the effect of iloprost on ARDS.

Methods: The Therapeutic Iloprost during ARDS trial (ThIlo trial) is a multicenter, randomized, single blinded, clinical phase II trial assessing the efficacy of inhaled iloprost for the prevention of the development and progression of ARDS in critically ill patients. One hundred fifty critically ill patients suffering from acute ARDS will be treated either by nebulized iloprost or NaCl 0.9% for 5 days. Blood samples will be drawn at defined time points to elucidate the serum levels of iloprost and inflammatory markers during treatment. Mechanical ventilation will be standardized. In follow-up visits at days 28 and 90 as well as 6 months after enrollment, functional status according to the Barthel Index and a health care-related questionnaire, and frailty (Vulnerable Elders Survey) will be evaluated. The primary endpoint is the improvement of oxygenation, defined as the ratio of PaO/FiO. Secondary endpoints include 90-day all-cause mortality, Sequential Organ Failure Assessment scores during the study period up to day 90, the duration of mechanical ventilation, the length of intensive care unit (ICU) stay, ventilator-associated pneumonia, delirium, ICU-acquired weakness, and discharge localization. The study will be conducted in three university ARDS centers in Germany.

Discussion: The results of the ThIlo trial will highlight the anti-inflammatory effect of iloprost on early inflammatory processes during ARDS, resulting in the improvement of outcome parameters in patients with ARDS.

Trial Registration: EUDRA-CT: 2016-003168-37. Registered on 12 April 2017. ClinicalTrials.gov: NCT03111212. Registered on 4 June 2017.
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http://dx.doi.org/10.1186/s13063-020-4163-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7057516PMC
March 2020

Trans-right ventricle and transpulmonary metabolite gradients in human pulmonary arterial hypertension.

Heart 2020 Sep 20;106(17):1332-1341. Epub 2020 Feb 20.

Department of Pediatric Cardiology and Critical care, Hannover Medical School, Hannover, Germany

Objective: While metabolic dysfunction occurs in several pulmonary arterial hypertension (PAH) animal models, its role in the human hypertensive right ventricle (RV) and lung is not well characterised. We investigated whether circulating metabolite concentrations differ across the hypertensive RV and/or the pulmonary circulation, and correlate with invasive haemodynamic/echocardiographic variables in patients with PAH.

Methods: Prospective EDTA blood collection during cardiac catheterisation from the superior vena cava (SVC), pulmonary artery (PA) and ascending aorta (AAO) in children with PAH (no shunt) and non-PAH controls (Con), followed by unbiased screens of 427 metabolites and 836 lipid species and fatty acids (FAs) in blood plasma (Metabolon and Lipidyzer platforms). Metabolite concentrations were correlated with echocardiographic and invasive haemodynamic variables.

Results: Metabolomics/lipidomics analysis of differential concentrations (false discovery rate<0.15) revealed several metabolite gradients in the trans-RV (PA vs SVC) setting. Notably, dicarboxylic acids (eg, octadecanedioate: fold change (FC)_Control=0.77, FC_PAH=1.09, p value=0.044) and acylcarnitines (eg, stearoylcarnitine: FC_Control=0.74, FC_PAH=1.21, p value=0.058). Differentially regulated metabolites were also found in the transpulmonary (AAO vs PA) setting and between-group comparisons, that is, in the SVC (PAH-SVC vs Con-SVC), PA and AAO. Importantly, the differential PAH-metabolite concentrations correlated with numerous outcome-relevant variables (e.g., tricuspid annular plane systolic excursion, pulmonary vascular resistance).

Conclusions: In PAH, trans-RV and transpulmonary metabolite gradients exist and correlate with haemodynamic determinants of clinical outcome. The most pronounced differential trans-RV gradients are known to be involved in lipid metabolism/lipotoxicity, that is, accumulation of long chain FAs. The identified accumulation of dicarboxylic acids and acylcarnitines likely indicates impaired β-oxidation in the hypertensive RV and represents emerging biomarkers and therapeutic targets in PAH.
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http://dx.doi.org/10.1136/heartjnl-2019-315900DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7476282PMC
September 2020

Lipid metabolism of leukocytes in the unstimulated and activated states.

Anal Bioanal Chem 2020 Apr 14;412(10):2353-2363. Epub 2020 Feb 14.

Center for Proteomics and Metabolomics, Leiden University Medical Center (LUMC), Albinusdreef 2, 2333 ZA, Leiden, The Netherlands.

Lipidomics has emerged as a powerful technique to study cellular lipid metabolism. As the lipidome contains numerous isomeric and isobaric species resulting in a significant overlap between different lipid classes, cutting-edge analytical technology is necessary for a comprehensive analysis of lipid metabolism. Just recently, differential mobility spectrometry (DMS) has evolved as such a technology, helping to overcome several analytical challenges. We here set out to apply DMS and the Lipidyzer™ platform to obtain a comprehensive overview of leukocyte-related lipid metabolism in the resting and activated states. First, we tested the linearity and repeatability of the platform by using HL60 cells. We obtained good linearities for most of the thirteen analyzed lipid classes (correlation coefficient > 0.95), and good repeatability (%CV < 15). By comparing the lipidome of neutrophils (PMNs), monocytes (CD14+), and lymphocytes (CD4+), we shed light on leukocyte-specific lipid patterns as well as lipidomic changes occurring through differential stimulation. For example, at the resting state, PMNs proved to contain higher amounts of triacylglycerides compared to CD4+ and CD14+ cells. On the other hand, CD4+ and CD14+ cells contained higher levels of phospholipids and ceramides. Upon stimulation, diacylglycerides, hexosylceramides, phosphatidylcholines, phosphoethanolamines, and lysophosphoethanolamines were upregulated in CD4+ cells and PMNs, whereas CD14+ cells did not show significant changes. By exploring the fatty acid content of the significantly upregulated lipid classes, we mainly found increased concentrations of very long and polyunsaturated fatty acids. Our results indicate the usefulness of the Lipidyzer™ platform for studying cellular lipid metabolism. Its application allowed us to explore the lipidome of leukocytes. Graphical abstract.
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http://dx.doi.org/10.1007/s00216-020-02460-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118052PMC
April 2020

Photo-controlled delivery of very long chain fatty acids to cell membranes and modulation of membrane protein function.

Biochim Biophys Acta Biomembr 2020 05 20;1862(5):183200. Epub 2020 Jan 20.

Leiden Institute of Chemistry, Leiden University, 2300 RA Leiden, the Netherlands. Electronic address:

The biophysical properties and biological functions of membranes are highly dependent on lipid composition. Supplementing cellular membranes with very long chain fatty acids (vlcFAs) is notoriously difficult given the extreme insolubility of vlcFAs in aqueous solution. Herein, we report a solvent-free, photochemical approach to enrich target membranes with vlcFA. To prevent aggregation of vlcFA, we created light-sensitive micelles composed exclusively of poly-ethylene-glycol-nervonic acid amphiphiles (NA-PEG), which spontaneously disassemble in the presence of lipid bilayers. Once embedded within a membrane, UV light is used to cleave off PEG, leaving free nervonic acid (NA, i.e. FA24:1) in the target membrane. When applied to living cells, free NA was processed by the cell to generate various species of membrane and other lipids with incorporated vlcFAs. In this way, we were able to alter the membrane lipid composition of cellular membranes and modulate the enzymatic activity of γ-secretase, an intramembrane protease whose dysfunction has been implicated in the onset and progression of Alzheimer's disease.
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http://dx.doi.org/10.1016/j.bbamem.2020.183200DOI Listing
May 2020

The Effect of Initial High vs. Low FiO on Breathing Effort in Preterm Infants at Birth: A Randomized Controlled Trial.

Front Pediatr 2019 12;7:504. Epub 2019 Dec 12.

Department of Neonatology, Leiden University Medical Center, Leiden, Netherlands.

Infants are currently stabilized at birth with initial low FiO which increases the risk of hypoxia and suppression of breathing in the first minutes after birth. We hypothesized that initiating stabilization at birth with a high O concentration, followed by titration, would improve breathing effort when compared to a low O concentration, followed by titration. In a bi-center randomized controlled trial, infants <30 weeks gestation were stabilized at birth with an initial O concentration of 30 or 100%, followed by oxygen titration. Primary outcome was minute volume of spontaneous breathing. We also assessed tidal volumes, mean inspiratory flow rate (MIFR) and respiratory rate with a respiratory function monitor in the first 5 min after birth, and evaluated the duration of mask ventilation in the first 10 min after birth. Pulse oximetry was used to measure heart rate and SpO values in the first 10 min. Hypoxemia was defined as SpO < 25th percentile and hyperoxemia as SpO >95%. 8-iso-prostaglandin F2α (8iPGF2α) was measured to assess oxidative stress in cord blood and 1 and 24 h after birth. Fifty-two infants were randomized and recordings were obtained in 44 infants (100% O-group: = 20, 30% O-group: = 24). Minute volumes were significantly higher in the 100% O-group (146.34 ± 112.68 mL/kg/min) compared to the 30% O-group (74.43 ± 52.19 mL/kg/min), = 0.014. Tidal volumes and MIFR were significantly higher in the 100% O-group, while the duration of mask ventilation given was significantly shorter. Oxygenation in the first 5 min after birth was significantly higher in infants in the 100% O-group [85 (64-93)%] compared to the 30% O-group [58 (46-67)%], < 0.001. The duration of hypoxemia was significantly shorter in the 100% O-group, while the duration of hyperoxemia was not different between groups. There was no difference in oxidative stress marker 8iPGF2α between the groups. Initiating stabilization of preterm infants at birth with 100% O led to higher breathing effort, improved oxygenation, and a shorter duration of mask ventilation as compared to 30% O, without increasing the risk for hyperoxia or oxidative stress. This study was registered in www.trialregister.nl, with registration number NTR6878.
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http://dx.doi.org/10.3389/fped.2019.00504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6927294PMC
December 2019

Short-Chain Fatty Acids Improve Poststroke Recovery via Immunological Mechanisms.

J Neurosci 2020 01 30;40(5):1162-1173. Epub 2019 Dec 30.

Institute for Stroke and Dementia Research, University Hospital, Ludwig-Maximilians-University LMU, 81377, Munich, Germany,

Recovery after stroke is a multicellular process encompassing neurons, resident immune cells, and brain-invading cells. Stroke alters the gut microbiome, which in turn has considerable impact on stroke outcome. However, the mechanisms underlying gut-brain interaction and implications for long-term recovery are largely elusive. Here, we tested the hypothesis that short-chain fatty acids (SCFAs), key bioactive microbial metabolites, are the missing link along the gut-brain axis and might be able to modulate recovery after experimental stroke. SCFA supplementation in the drinking water of male mice significantly improved recovery of affected limb motor function. Using wide-field calcium imaging, we observed that SCFAs induced altered contralesional cortex connectivity. This was associated with SCFA-dependent changes in spine and synapse densities. RNA sequencing of the forebrain cortex indicated a potential involvement of microglial cells in contributing to the structural and functional remodeling. Further analyses confirmed a substantial impact of SCFAs on microglial activation, which depended on the recruitment of T cells to the infarcted brain. Our findings identified that microbiota-derived SCFAs modulate poststroke recovery via effects on systemic and brain resident immune cells. Previous studies have shown a bidirectional communication along the gut-brain axis after stroke. Stroke alters the gut microbiota composition, and in turn, microbiota dysbiosis has a substantial impact on stroke outcome by modulating the immune response. However, until now, the mediators derived from the gut microbiome affecting the gut-immune-brain axis and the molecular mechanisms involved in this process were unknown. Here, we demonstrate that short-chain fatty acids, fermentation products of the gut microbiome, are potent and proregenerative modulators of poststroke neuronal plasticity at various structural levels. We identified that this effect was mediated via circulating lymphocytes on microglial activation. These results identify short-chain fatty acids as a missing link along the gut-brain axis and as a potential therapeutic to improve recovery after stroke.
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http://dx.doi.org/10.1523/JNEUROSCI.1359-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6989004PMC
January 2020

Shear Stress Regulation of Endothelial Glycocalyx Structure Is Determined by Glucobiosynthesis.

Arterioscler Thromb Vasc Biol 2020 02 12;40(2):350-364. Epub 2019 Dec 12.

From the Division of Nephrology, Department of Internal Medicine (G.W., G.L.T., W.M.P.J.S., B.M.v.d.B., T.J.R.), The Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, the Netherlands.

Objective: Endothelial cells exposed to laminar shear stress express a thick glycocalyx on their surface that plays an important role in reducing vascular permeability and endothelial anti-inflammatory, antithrombotic, and antiangiogenic properties. Production and maintenance of this glycocalyx layer is dependent on cellular carbohydrate synthesis, but its regulation is still unknown. Approach and Results: Here, we show that biosynthesis of the major structural component of the endothelial glycocalyx, hyaluronan, is regulated by shear. Both in vitro as well as in in vivo, hyaluronan expression on the endothelial surface is increased on laminar shear and reduced when exposed to oscillatory flow, which is regulated by KLF2 (Krüppel-like Factor 2). Using a CRISPR-CAS9 edited small tetracysteine tag to endogenous HAS2 (hyaluronan synthase 2), we demonstrated increased translocation of HAS2 to the endothelial cell membrane during laminar shear. Hyaluronan production by HAS2 was shown to be further driven by availability of the hyaluronan substrates UDP-glucosamine and UDP-glucuronic acid. KLF2 inhibits endothelial glycolysis and allows for glucose intermediates to shuttle into the hexosamine- and glucuronic acid biosynthesis pathways, as measured using nuclear magnetic resonance analysis in combination with C-labeled glucose.

Conclusions: These data demonstrate how endothelial glycocalyx function and functional adaptation to shear is coupled to KLF2-mediated regulation of endothelial glycolysis.
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http://dx.doi.org/10.1161/ATVBAHA.119.313399DOI Listing
February 2020

TGFβ-induced metabolic reprogramming during epithelial-to-mesenchymal transition in cancer.

Cell Mol Life Sci 2020 Jun 10;77(11):2103-2123. Epub 2019 Dec 10.

Department of Cell and Chemical Biology and Oncode Institute, Leiden University Medical Center, Einthovenweg 20, 2300 RC, Leiden, The Netherlands.

Metastasis is the most frequent cause of death in cancer patients. Epithelial-to-mesenchymal transition (EMT) is the process in which cells lose epithelial integrity and become motile, a critical step for cancer cell invasion, drug resistance and immune evasion. The transforming growth factor-β (TGFβ) signaling pathway is a major driver of EMT. Increasing evidence demonstrates that metabolic reprogramming is a hallmark of cancer and extensive metabolic changes are observed during EMT. The aim of this review is to summarize and interconnect recent findings that illustrate how changes in glycolysis, mitochondrial, lipid and choline metabolism coincide and functionally contribute to TGFβ-induced EMT. We describe TGFβ signaling is involved in stimulating both glycolysis and mitochondrial respiration. Interestingly, the subsequent metabolic consequences for the redox state and lipid metabolism in cancer cells are found to be in favor of EMT as well. Combined we illustrate that a better understanding of the mechanistic links between TGFβ signaling, cancer metabolism and EMT holds promising strategies for cancer therapy, some of which are already actively being explored in the clinic.
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http://dx.doi.org/10.1007/s00018-019-03398-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7256023PMC
June 2020

Platelet Acetyl-CoA Carboxylase Phosphorylation: A Risk Stratification Marker That Reveals Platelet-Lipid Interplay in Coronary Artery Disease Patients.

JACC Basic Transl Sci 2019 Sep 11;4(5):596-610. Epub 2019 Sep 11.

Pôle de Recherche Cardiovasculaire, Institut de Recherche Expérimentale et Clinique, Université Catholique de Louvain, Brussels, Belgium.

Adenosine monophosphate-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK-induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK-induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function.
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http://dx.doi.org/10.1016/j.jacbts.2019.04.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6872775PMC
September 2019

Bis-allylic Deuterated DHA Alleviates Oxidative Stress in Retinal Epithelial Cells.

Antioxidants (Basel) 2019 Oct 1;8(10). Epub 2019 Oct 1.

IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, 34093, France.

Oxidative stress plays a crucial role in developing and accelerating retinal diseases including age-related macular degeneration (AMD). Docosahexaenoic acid (DHA, C22:6, n-3), the main lipid constituent of retinal epithelial cell membranes, is highly prone to radical and enzymatic oxidation leading to deleterious or beneficial metabolites for retinal tissue. To inhibit radical oxidation while preserving enzymatic metabolism, deuterium was incorporated at specific positions of DHA, resulting in D-DHA when incorporated at position 6 and D-DHA when incorporated at the 6,9 -allylic positions. Both derivatives were able to decrease DHAs' toxicity and free radical processes involved in lipid peroxidation, in ARPE-19 cells (Adult Retinal Pigment Epithelial cell line), under pro-oxidant conditions. Our positive results encouraged us to prepare lipophenolic-deuterated-DHA conjugates as possible drug candidates for AMD treatment. These novel derivatives proved efficient in limiting lipid peroxidation in ARPE-19 cells. Finally, we evaluated the underlying mechanisms and the enzymatic conversion of both deuterated DHA. While radical abstraction was affected at the deuterium incorporation sites, enzymatic conversion by the lipoxygenase 15s-LOX was not impacted. Our results suggest that site-specifically deuterated DHA could be used in the development of DHA conjugates for treatment of oxidative stress driven diseases, or as biological tools to study the roles, activities and mechanisms of DHA metabolites.
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http://dx.doi.org/10.3390/antiox8100447DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6826779PMC
October 2019

Inhibition of Δ24-dehydrocholesterol reductase activates pro-resolving lipid mediator biosynthesis and inflammation resolution.

Proc Natl Acad Sci U S A 2019 10 23;116(41):20623-20634. Epub 2019 Sep 23.

Center for Proteomics and Metabolomics, Leiden University Medical Center, 2333ZA Leiden, The Netherlands

Targeting metabolism through bioactive key metabolites is an upcoming future therapeutic strategy. We questioned how modifying intracellular lipid metabolism could be a possible means for alleviating inflammation. Using a recently developed chemical probe (SH42), we inhibited distal cholesterol biosynthesis through selective inhibition of Δ-dehydrocholesterol reductase (DHCR24). Inhibition of DHCR24 led to an antiinflammatory/proresolving phenotype in a murine peritonitis model. Subsequently, we investigated several omics layers in order to link our phenotypic observations with key metabolic alterations. Lipidomic analysis revealed a significant increase in endogenous polyunsaturated fatty acid (PUFA) biosynthesis. These data integrated with gene expression analysis, revealing increased expression of the desaturase and the key proresolving enzyme Protein array analysis, as well as immune cell phenotype and functional analysis, substantiated these results confirming the antiinflammatory/proresolving phenotype. Ultimately, lipid mediator (LM) analysis revealed the increased production of bioactive lipids, channeling the observed metabolic alterations into a key class of metabolites known for their capacity to change the inflammatory phenotype.
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http://dx.doi.org/10.1073/pnas.1911992116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789642PMC
October 2019

New insights into energy and protein homeostasis by the kidney.

Nat Rev Nephrol 2019 10;15(10):596-598

Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, Netherlands.

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http://dx.doi.org/10.1038/s41581-019-0192-xDOI Listing
October 2019

A gas chromatography-mass spectrometry-based whole-cell screening assay for target identification in distal cholesterol biosynthesis.

Nat Protoc 2019 08 24;14(8):2546-2570. Epub 2019 Jul 24.

Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians University Munich, Munich, Germany.

Distal cholesterol biosynthesis (CB) has recently taken center stage as a promising drug target in several diseases previously not linked to this biochemical pathway, including cardiovascular disease, cancer, multiple sclerosis and Alzheimer's disease. Most enzymes involved in this pathway are hard to isolate, warranting dedicated analytical tools for biochemical screening. We describe the use of gas chromatography-electron ionization mass spectrometry (GC-MS) in a whole-cell screening assay aimed at monitoring interactions with all enzymes of distal CB in a single experiment. Following cell culture and lipid extraction, the trimethylsilyl ethers of sterols are analyzed by GC-MS. Analytical data for 23 relevant sterols (intermediates) are provided, allowing their unambiguous identification. Sterol pattern analysis reveals the target enzyme on the basis of characteristic marker sterols, whereas quantification of 2-C-acetate incorporation correlates with the inhibitory activity of drug candidates. The protocol can be used by both experienced scientists and newcomers to the field, allowing detection and quantification of small molecule-enzyme interactions in distal CB. The entire protocol can be carried out within two working days.
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http://dx.doi.org/10.1038/s41596-019-0193-zDOI Listing
August 2019