Acyl-CoA Dehydrogenase Drives Heat Adaptation by Sequestering Fatty Acids.

Cells adapt to temperature shifts by adjusting levels of lipid desaturation and membrane fluidity. This fundamental process occurs in nearly all forms of life, but its mechanism in eukaryotes is unknown. We discovered that the evolutionarily conserved Caenorhabditis elegans gene acdh-11 (acyl-CoA dehydrogenase [ACDH]) facilitates heat adaptation by regulating the lipid desaturase FAT-7. Human ACDH deficiency causes the most common inherited disorders of fatty acid oxidation, with syndromes that are exacerbated by hyperthermia. Heat upregulates acdh-11 expression to decrease fat-7 expression. We solved the high-resolution crystal structure of ACDH-11 and established the molecular basis of its selective and high-affinity binding to C11/C12-chain fatty acids. ACDH-11 sequesters C11/C12-chain fatty acids and prevents these fatty acids from activating nuclear hormone receptors and driving fat-7 expression. Thus, the ACDH-11 pathway drives heat adaptation by linking temperature shifts to regulation of lipid desaturase levels and membrane fluidity via an unprecedented mode of fatty acid signaling.

Preclinical lentiviral vector-mediated hematopoietic stem and progenitor cell gene therapy corrects Pompe disease-related muscle and neurological manifestations.

Pompe disease, a rare genetic neuromuscular disorder, is caused by a deficiency of acid alpha-glucosidase (GAA), leading to an accumulation of glycogen in lysosomes, and resulting in the progressive development of muscle weakness. The current standard treatment, enzyme replacement therapy (ERT), is not curative and has limitations such as poor penetration into skeletal muscle and both the central and peripheral nervous systems, a risk of immune responses against the recombinant enzyme, and the requirement for high doses and frequent infusions. To overcome these limitations, lentiviral vector-mediated hematopoietic stem and progenitor cell (HSPC) gene therapy has been proposed as a next-generation approach for treating Pompe disease. This study demonstrates the potential of lentiviral HSPC gene therapy to reverse the pathological effects of Pompe disease in a preclinical mouse model. It includes a comprehensive safety assessment via integration site analysis, along with single-cell RNA sequencing analysis of central nervous tissue samples to gain insights into the underlying mechanisms of phenotype correction.

Pharmacological and pre-clinical safety profile of rSIV.F/HN, a hybrid lentiviral vector for cystic fibrosis gene therapy.

RATIONALE AND OBJECTIVE: Cystic fibrosis (CF) is caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. CFTR modulators offer significant improvements, but approximately 10% of patients remain nonresponsive or are intolerant. This study provides an analysis of rSIV.F/HN, a lentiviral vector optimized for lung delivery, including CFTR protein expression, functional correction of CFTR defects and genomic integration site analysis in preparation for a first-in-human clinical trial. METHODS: Air-liquid interface cultures of primary human bronchial epithelial cells (HBEC) from CF patients (F508del/F508del), as well as a CFTR-deficient immortalized human lung epithelial cell line mimicking Class I (CFTR-null) homozygous mutations, were used to assess transduction efficiency. Quantification methods included a novel proximity ligation assay (PLA) for CFTR protein expression. For assessment of CFTR channel activity, Ussing chamber studies were conducted. The safety profile was assessed using integration site analysis and in vitro insertional mutagenesis studies. RESULTS: rSIV.F/HN expressed CFTR and restored CFTR-mediated chloride currents to physiological levels in primary F508del/F508del HBECs as well as in a Class I cells. In contrast, the latter could not be achieved by small-molecule CFTR modulators, underscoring the potential of gene therapy for this mutation class. Combination of rSIV.F/HN-CFTR with the potentiator ivacaftor showed a greater than additive effect. The genomic integration pattern showed no site predominance (frequency of occurrence ≤10%), and a low risk of insertional mutagenesis was observed in an in vitro immortalization assay. CONCLUSIONS: The results underscore rSIV.F/HN as a promising gene therapy vector for CF, providing a mutation-agnostic treatment option.

Tumour ischaemia by interferon-γ resembles physiological blood vessel regression.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models. Although IFNγ can impede tumour growth by acting directly on cancer cells, it must also act on the tumour stroma for effective rejection of large, established tumours. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ-GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Knock-in mice expressing a humanized arachidonic acid 15-lipoxygenase (Alox15) carry a partly dysfunctional erythropoietic system.

Arachidonic acid 15-lipoxygenases (ALOX15) play a role in mammalian erythropoiesis but they have also been implicated in inflammatory processes. Seven intact Alox genes have been detected in the mouse reference genome and the mouse Alox15 gene is structurally similar to the orthologous genes of other mammals. However, mouse and human ALOX15 orthologs have different functional characteristics. Human ALOX15 converts C20 polyenoic fatty acids like arachidonic acid mainly to the n-6 hydroperoxide. In contrast, the n-9 hydroperoxide is the major oxygenation product formed by mouse Alox15. Previous experiments indicated that Leu353Phe exchange in recombinant mouse Alox15 humanized the catalytic properties of the enzyme. To investigate whether this functional humanization might also work in vivo and to characterize the functional consequences of mouse Alox15 humanization we generated Alox15 knock-in mice (Alox15-KI), in which the Alox15 gene was modified in such a way that the animals express the arachidonic acid 15-lipoxygenating Leu353Phe mutant instead of the arachidonic acid 12-lipoxygenating wildtype enzyme. These mice develop normally, they are fully fertile but display modified plasma oxylipidomes. In young individuals, the basic hematological parameters were not different when Alox15-KI mice and outbred wildtype controls were compared. However, when growing older male Alox15-KI mice develop signs of dysfunctional erythropoiesis such as reduced hematocrit, lower erythrocyte counts and attenuated hemoglobin concentration. These differences were paralleled by an improved ex vivo osmotic resistance of the peripheral red blood cells. Interestingly, such differences were not observed in female individuals suggesting gender specific effects. In summary, these data indicated that functional humanization of mouse Alox15 induces defective erythropoiesis in aged male individuals.

Effect of cardiopulmonary bypass on plasma and erythrocytes oxylipins.

BACKGROUND: Oxylipins, the oxidative metabolites of polyunsaturated fatty acids (PUFAs), serve as key mediators of oxidative stress, inflammatory responses, and vasoactive reactions in vivo. Our previous work has established that hemodialysis affects both long chain fatty acids (LCFAs) and oxylipins in plasma and erythrocytes to varying degrees, which may be responsible for excess cardiovascular complications in end-stage renal disease. In this study, we aimed to determine changes in blood oxylipins during cardiopulmonary bypass (CPB) in patients undergoing cardiac surgery to identify novel biomarkers and potential metabolites of CPB-related complications. We tested the hypothesis that CPB would differentially affect plasma oxylipins and erythrocytes oxylipins. METHODS: We conducted a prospective observational study of 12 patients undergoing elective cardiac surgery with expected CPB procedure. We collected venous and arterial blood samples before CPB, 15 and 45 min after the start of CPB, and 60 min after the end of CPB, respectively. Oxylipins profiling in plasma and erythrocytes was achieved using targeted HPLC-MS mass spectrometry. RESULTS: Our results revealed that most venous plasma diols and hydroxy- oxylipins decreased after CPB initiation, with a continuous decline until the termination of CPB. Nevertheless, no statistically significant alterations were detected in erythrocytes oxylipins at all time points. CONCLUSIONS: CPB decreases numerous diols and hydroxy oxylipins in blood plasma, whereas no changes in erythrocytes oxylipins are observed during this procedure in patients undergoing cardiac surgery. As lipid mediators primarily responsive to CPB, plasma diols and hydroxy oxylipins may serve as potential key biomarkers for CPB-related complications.

Bony Fish Arachidonic Acid 15-Lipoxygenases Exhibit Different Catalytic Properties than Their Mammalian Orthologs, Suggesting Functional Enzyme Evolution during Vertebrate Development.

The human genome involves six functional arachidonic acid lipoxygenase (ALOX) genes and the corresponding enzymes (ALOX15, ALOX15B, ALOX12, ALOX12B, ALOXE3, ALOX5) have been implicated in cell differentiation and in the pathogenesis of inflammatory, hyperproliferative, metabolic, and neurological disorders. In other vertebrates, ALOX-isoforms have also been identified, but they occur less frequently. Since bony fish represent the most abundant subclass of vertebrates, we recently expressed and characterized putative ALOX15 orthologs of three different bony fish species (Nothobranchius furzeri, Pundamilia nyererei, Scleropages formosus). To explore whether these enzymes represent functional equivalents of mammalian ALOX15 orthologs, we here compared a number of structural and functional characteristics of these ALOX-isoforms with those of mammalian enzymes. We found that in contrast to mammalian ALOX15 orthologs, which exhibit a broad substrate specificity, a membrane oxygenase activity, and a special type of dual reaction specificity, the putative bony fish ALOX15 orthologs strongly prefer C20 fatty acids, lack any membrane oxygenase activity and exhibit a different type of dual reaction specificity with arachidonic acid. Moreover, mutagenesis studies indicated that the Triad Concept, which explains the reaction specificity of all mammalian ALOX15 orthologs, is not applicable for the putative bony fish enzymes. The observed functional differences between putative bony fish ALOX15 orthologs and corresponding mammalian enzymes suggest a targeted optimization of the catalytic properties of ALOX15 orthologs during vertebrate development.

Functional Characterization of Transgenic Mice Overexpressing Human 15-Lipoxygenase-1 (ALOX15) under the Control of the aP2 Promoter.

Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory, hyperproliferative, neurodegenerative, and metabolic diseases, but the physiological function of ALOX15 still remains a matter of discussion. To contribute to this discussion, we created transgenic mice (aP2-ALOX15 mice) expressing human ALOX15 under the control of the aP2 (adipocyte fatty acid binding protein 2) promoter, which directs expression of the transgene to mesenchymal cells. Fluorescence in situ hybridization and whole-genome sequencing indicated transgene insertion into the E1-2 region of chromosome 2. The transgene was highly expressed in adipocytes, bone marrow cells, and peritoneal macrophages, and ex vivo activity assays proved the catalytic activity of the transgenic enzyme. LC-MS/MS-based plasma oxylipidome analyses of the aP2-ALOX15 mice suggested in vivo activity of the transgenic enzyme. The aP2-ALOX15 mice were viable, could reproduce normally, and did not show major phenotypic alterations when compared with wildtype control animals. However, they exhibited gender-specific differences with wildtype controls when their body-weight kinetics were evaluated during adolescence and early adulthood. The aP2-ALOX15 mice characterized here can now be used for gain-of-function studies evaluating the biological role of ALOX15 in adipose tissue and hematopoietic cells.

Humanization of the Reaction Specificity of Mouse Alox15b Inversely Modified the Susceptibility of Corresponding Knock-In Mice in Two Different Animal Inflammation Models.

Mammalian arachidonic acid lipoxygenases (ALOXs) have been implicated in the pathogenesis of inflammatory diseases, and its pro- and anti-inflammatory effects have been reported for different ALOX-isoforms. Human ALOX15B oxygenates arachidonic acid to its 15-hydroperoxy derivative, whereas the corresponding 8-hydroperoxide is formed by mouse Alox15b (Alox8). This functional difference impacts the biosynthetic capacity of the two enzymes for creating pro- and anti-inflammatory eicosanoids. To explore the functional consequences of the humanization of the reaction specificity of mouse Alox15b in vivo, we tested Alox15b knock-in mice that express the arachidonic acid 15-lipoxygenating Tyr603Asp and His604Val double mutant of Alox15b, instead of the arachidonic acid 8-lipoxygenating wildtype enzyme, in two different animal inflammation models. In the dextran sodium sulfate-induced colitis model, female Alox15b-KI mice lost significantly more bodyweight during the acute phase of inflammation and recovered less rapidly during the resolution phase. Although we observed significant differences in the colonic levels of selected pro- and anti-inflammatory eicosanoids during the time-course of inflammation, there were no differences between the two genotypes at any time-point of the disease. In Freund's complete adjuvant-induced paw edema model, Alox15b-KI mice were less susceptible than outbred wildtype controls, though we did not observe significant differences in pain perception (Hargreaves-test, von Frey-test) when the two genotypes were compared. our data indicate that humanization of the reaction specificity of mouse Alox15b (Alox8) sensitizes mice for dextran sodium sulfate-induced experimental colitis, but partly protects the animals in the complete Freund's adjuvant-induced paw edema model.

Successful gene therapy of Diamond-Blackfan anemia in a mouse model and human CD34+ cord blood hematopoietic stem cells using a clinically applicable lentiviral vector.

Diamond-Blackfan anemia (DBA) is an inherited bone marrow failure disorder in which pure red blood cell aplasia is associated with physical malformations and a predisposition to cancer. Twentyfive percent of patients with DBA have mutations in a gene encoding ribosomal protein S19 (RPS19). Our previous proof-of-concept studies demonstrated that DBA phenotype could be successfully treated using lentiviral vectors in Rps19-deficient DBA mice. In our present study, we developed a clinically applicable single gene, self-inactivating lentiviral vector, containing the human RPS19 cDNA driven by the human elongation factor 1a short promoter, which can be used for clinical gene therapy development for RPS19-deficient DBA. We examined the efficacy and safety of the vector in a Rps19-deficient DBA mouse model and in human primary RPS19-deficient CD34+ cord blood cells. We observed that transduced Rps19-deficient bone marrow cells could reconstitute mice long-term and rescue the bone marrow failure and severe anemia observed in Rps19-deficient mice, with a low risk of mutagenesis and a highly polyclonal insertion site pattern. More importantly, the vector can also rescue impaired erythroid differentiation in human primary RPS19-deficient CD34+ cord blood hematopoietic stem cells. Collectively, our results demonstrate the efficacy and safety of using a clinically applicable lentiviral vector for the successful treatment of Rps19-deficient DBA in a mouse model and in human primary CD34+ cord blood cells. These findings show that this vector can be used to develop clinical gene therapy for RPS19-deficient DBA patients.

Phenylalanine hydroxylase contributes to serotonin synthesis in mice.

Serotonin is an important signaling molecule in the periphery and in the brain. The hydroxylation of tryptophan is the first and rate-limiting step of its synthesis. In most vertebrates, two enzymes have been described to catalyze this step, tryptophan hydroxylase (TPH) 1 and 2, with expression localized to peripheral and neuronal cells, respectively. However, animals lacking both TPH isoforms still exhibit about 10% of normal serotonin levels in the blood demanding an additional source of the monoamine. In this study, we provide evidence by the gain and loss of function approaches in in vitro and in vivo systems, including stable-isotope tracing in mice, that phenylalanine hydroxylase (PAH) is a third TPH in mammals. PAH contributes to serotonin levels in the blood, and may be important as a local source of serotonin in organs in which no other TPHs are expressed, such as liver and kidney.

Omega-3 fatty acids protect from colitis via an Alox15-derived eicosanoid.

An increased omega-3 polyunsaturated fatty acid (n-3 PUFA) tissue status can lead to a significant formation of anti-inflammatory lipid mediators and effective reduction in inflammation and tissue injury in murine colitis. Arachidonic acid lipoxygenases (ALOX) have been implicated in the pathogenesis of inflammatory bowel disease as well as in the formation of pro- and anti-inflammatory lipid mediators. To explore the role of Alox15 in the protective response found in fat1 transgenic mice with endogenously increased n-3 PUFA tissue status fat1 transgenic mice were crossed with Alox15-deficient animals and challenged in the dextran sulfate sodium (DSS)- and the 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis model. Transgenic fat1 mice rich in endogenous n-3 PUFAs were protected from colitis. However, additional systemic inactivation of the Alox15 gene counteracted this protective effect. To explore the molecular basis for this effect Alox15 lipid metabolites derived from n-3 PUFA were analyzed in the different mice. Alox15 deficiency suppressed the formation of n-3 PUFA-derived 15-hydroxy eicosapentaenoic acid (15-HEPE). In contrast, treating mice with intraperitoneal injections of 15S-HEPE protected wild-type mice from DSS- and TNBS-induced colitis. These data suggest that the anti-colitis effect of increased n-3 PUFA in the transgenic fat1 mouse model is mediated in part via Alox15-derived 15-HEPE formation.

Predicting genotoxicity of viral vectors for stem cell gene therapy using gene expression-based machine learning.

Hematopoietic stem cell gene therapy is emerging as a promising therapeutic strategy for many diseases of the blood and immune system. However, several individuals who underwent gene therapy in different trials developed hematological malignancies caused by insertional mutagenesis. Preclinical assessment of vector safety remains challenging because there are few reliable assays to screen for potential insertional mutagenesis effects in vitro. Here we demonstrate that genotoxic vectors induce a unique gene expression signature linked to stemness and oncogenesis in transduced murine hematopoietic stem and progenitor cells. Based on this finding, we developed the surrogate assay for genotoxicity assessment (SAGA). SAGA classifies integrating retroviral vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential, SAGA achieved an accuracy of 90.9%. SAGA is more robust and sensitive and faster than previous assays and reliably predicts a mutagenic risk for vectors that led to leukemic severe adverse events in clinical trials. Our work provides a fast and robust tool for preclinical risk assessment of gene therapy vectors, potentially paving the way for safer gene therapy trials.

USGS44, a new high-purity calcium carbonate reference material for δ13 C measurements.

RATIONALE: The stable carbon isotopic (δ13 C) reference material (RM) LSVEC Li2 CO3 has been found to be unsuitable for δ13 C standardization work because its δ13 C value increases with exposure to atmospheric CO2 . A new CaCO3 RM, USGS44, has been prepared to alleviate this situation. METHODS: USGS44 was prepared from 8 kg of Merck high-purity CaCO3 . Two sets of δ13 C values of USGS44 were determined. The first set of values was determined by online combustion, continuous-flow (CF) isotope-ratio mass spectrometry (IRMS) of NBS 19 CaCO3 (δ13 CVPDB = +1.95 milliurey (mUr) exactly, where mUr = 0.001 = 1‰), and LSVEC Li2 CO3 (δ13 CVPDB = -46.6 mUr exactly), and normalized to the two-anchor δ13 CVPDB-LSVEC isotope-delta scale. The second set of values was obtained by dual-inlet (DI)-IRMS of CO2 evolved by reaction of H3 PO4 with carbonates, corrected for cross contamination, and normalized to the single-anchor δ13 CVPDB scale. RESULTS: USGS44 is stable and isotopically homogeneous to within 0.02 mUr in 100-µg amounts. It has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr. Single-anchor δ13 CVPDB values of -42.08 ± 0.01 and -41.99 ± 0.02 mUr were determined by DI-IRMS with corrections for cross contamination. CONCLUSIONS: The new high-purity, well-homogenized calcium carbonate isotopic reference material USGS44 is stable and has a δ13 CVPDB-LSVEC value of -42.21 ± 0.05 mUr for both EA/IRMS and DI-IRMS measurements. As a carbonate relatively depleted in 13 C, it is intended for daily use as a secondary isotopic reference material to normalize stable carbon isotope delta measurements to the δ13 CVPDB-LSVEC scale. It is useful in quantifying drift with time, determining mass-dependent isotopic fractionation (linearity correction), and adjusting isotope-ratio-scale contraction. Due to its fine grain size (smaller than 63 µm), it is not suitable as a δ18 O reference material. A δ13 CVPDB-LSVEC value of -29.99 ± 0.05 mUr was determined for NBS 22 oil.

Adipose tissue ATGL modifies the cardiac lipidome in pressure-overload-induced left ventricular failure.

Adipose tissue lipolysis occurs during the development of heart failure as a consequence of chronic adrenergic stimulation. However, the impact of enhanced adipose triacylglycerol hydrolysis mediated by adipose triglyceride lipase (ATGL) on cardiac function is unclear. To investigate the role of adipose tissue lipolysis during heart failure, we generated mice with tissue-specific deletion of ATGL (atATGL-KO). atATGL-KO mice were subjected to transverse aortic constriction (TAC) to induce pressure-mediated cardiac failure. The cardiac mouse lipidome and the human plasma lipidome from healthy controls (n = 10) and patients with systolic heart failure (HFrEF, n = 13) were analyzed by MS-based shotgun lipidomics. TAC-induced increases in left ventricular mass (LVM) and diastolic LV inner diameter were significantly attenuated in atATGL-KO mice compared to wild type (wt) -mice. More importantly, atATGL-KO mice were protected against TAC-induced systolic LV failure. Perturbation of lipolysis in the adipose tissue of atATGL-KO mice resulted in the prevention of the major cardiac lipidome changes observed after TAC in wt-mice. Profound changes occurred in the lipid class of phosphatidylethanolamines (PE) in which multiple PE-species were markedly induced in failing wt-hearts, which was attenuated in atATGL-KO hearts. Moreover, selected heart failure-induced PE species in mouse hearts were also induced in plasma samples from patients with chronic heart failure. TAC-induced cardiac PE induction resulted in decreased PC/ PE-species ratios associated with increased apoptotic marker expression in failing wt-hearts, a process absent in atATGL-KO hearts. Perturbation of adipose tissue lipolysis by ATGL-deficiency ameliorated pressure-induced heart failure and the potentially deleterious cardiac lipidome changes that accompany this pathological process, namely the induction of specific PE species. Non-cardiac ATGL-mediated modulation of the cardiac lipidome may play an important role in the pathogenesis of chronic heart failure.

Harmonized procedures lead to comparable quantification of total oxylipins across laboratories.

Oxylipins are potent lipid mediators involved in a variety of physiological processes. Their profiling has the potential to provide a wealth of information regarding human health and disease and is a promising technology for translation into clinical applications. However, results generated by independent groups are rarely comparable, which increases the need for the implementation of internationally agreed upon protocols. We performed an interlaboratory comparison for the MS-based quantitative analysis of total oxylipins. Five independent laboratories assessed the technical variability and comparability of 133 oxylipins using a harmonized and standardized protocol, common biological materials (i.e., seven quality control plasmas), standard calibration series, and analytical methods. The quantitative analysis was based on a standard calibration series with isotopically labeled internal standards. Using the standardized protocol, the technical variance was within ±15% for 73% of oxylipins; however, most epoxy fatty acids were identified as critical analytes due to high variabilities in concentrations. The comparability of concentrations determined by the laboratories was examined using consensus value estimates and unsupervised/supervised multivariate analysis (i.e., principal component analysis and partial least squares discriminant analysis). Interlaboratory variability was limited and did not interfere with our ability to distinguish the different plasmas. Moreover, all laboratories were able to identify similar differences between plasmas. In summary, we show that by using a standardized protocol for sample preparation, low technical variability can be achieved. Harmonization of all oxylipin extraction and analysis steps led to reliable, reproducible, and comparable oxylipin concentrations in independent laboratories, allowing the generation of biologically meaningful oxylipin patterns.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Apolipoprotein E4 disrupts the neuroprotective action of sortilin in neuronal lipid metabolism and endocannabinoid signaling.

INTRODUCTION: Apolipoprotein E (apoE) is a carrier for brain lipids and the most important genetic risk factor for Alzheimer's disease (AD). ApoE binds the receptor sortilin, which mediates uptake of apoE-bound cargo into neurons. The significance of this uptake route for brain lipid homeostasis and AD risk seen with apoE4, but not apoE3, remains unresolved. METHODS: Combining neurolipidomics in patient specimens with functional studies in mouse models, we interrogated apoE isoform-specific functions for sortilin in brain lipid metabolism and AD. RESULTS: Sortilin directs the uptake and conversion of polyunsaturated fatty acids into endocannabinoids, lipid-based neurotransmitters that act through nuclear receptors to sustain neuroprotective gene expression in the brain. This sortilin function requires apoE3, but is disrupted by binding of apoE4, compromising neuronal endocannabinoid metabolism and action. DISCUSSION: We uncovered the significance of neuronal apoE receptor sortilin in facilitating neuroprotective actions of brain lipids, and its relevance for AD risk seen with apoE4.

Assessment of the Effect of Sorafenib on Omega-6 and Omega-3 Epoxyeicosanoid Formation in Patients with Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a leading cause of cancer death. The multikinase inhibitor sorafenib is widely used for systemic therapy in advanced HCC. Sorafenib might affect epoxyeicosanoids, as it is also a potent inhibitor of the soluble epoxide hydrolase (sEH), which catalyzes the conversion of epoxides derived from long-chain polyunsaturated fatty acids (PUFAs), such as arachidonic acid (AA) and omega-3 docosahexaenoic acid (DHA), into their corresponding diols. Experimental studies with AA-derived epoxyeicosatrienoic acids (EETs) showed that they can promote tumor growth and metastasis, while DHA-derived 19,20-epoxydocosapentaenoic acid (19,20-EDP) was shown to have anti-tumor activity in mice. In this pilot study, we assessed the effect of sorafenib treatment on the presence of lipid mediators, such as EETs, in blood of the patients with HCC using the lipidomics technology. We found a significant increase in 11,12-EET and 14,15-EET levels in HCC patients treated with sorafenib. Furthermore, while not significant in this small sample set, the data presented indicate that sorafenib can also increase the level of omega-3 DHA-derived 19,20-EDP. While the effect on EETs might hamper the anti-tumor effect of sorafenib, we hypothesize that supplementation of DHA in sorafenib-treated HCC patients could increase the level of 19,20-EDP and thereby enhance its anti-tumor effect.