Publications by authors named "Eckhard Wolf"

390 Publications

The hepatokine fetuin-A disrupts functional maturation of pancreatic beta cells.

Diabetologia 2021 Mar 25. Epub 2021 Mar 25.

Institute for Diabetes Research and Metabolic Diseases of the Helmholtz Center Munich at the Eberhard Karls University of Tuebingen (IDM), Tuebingen, Germany.

Aims/hypothesis: Neonatal beta cells carry out a programme of postnatal functional maturation to achieve full glucose responsiveness. A partial loss of the mature phenotype of adult beta cells may contribute to a reduction of functional beta cell mass and accelerate the onset of type 2 diabetes. We previously found that fetuin-A, a hepatokine increasingly secreted by the fatty liver and a determinant of type 2 diabetes, inhibits glucose-stimulated insulin secretion (GSIS) of human islets. Since fetuin-A is a ubiquitous fetal glycoprotein that declines peripartum, we examined here whether fetuin-A interferes with the functional maturity of beta cells.

Methods: The effects of fetuin-A were assessed during in vitro maturation of porcine neonatal islet cell clusters (NICCs) and in adult human islets. Expression alterations were examined via microarray, RNA sequencing and reverse transcription quantitative real-time PCR (qRT-PCR), proteins were analysed by western blotting and immunostaining, and insulin secretion was quantified in static incubations.

Results: NICC maturation was accompanied by the gain of glucose-responsive insulin secretion (twofold stimulation), backed up by mRNA upregulation of genes governing beta cell identity and function, such as NEUROD1, UCN3, ABCC8 and CASR (Log fold change [LogFC] > 1.6). An active TGFβ receptor (TGFBR)-SMAD2/3 pathway facilitates NICC maturation, since the TGFBR inhibitor SB431542 counteracted the upregulation of aforementioned genes and de-repressed ALDOB, a gene disallowed in mature beta cells. In fetuin-A-treated NICCs, upregulation of beta cell markers and the onset of glucose responsiveness were suppressed. Concomitantly, SMAD2/3 phosphorylation was inhibited. Transcriptome analysis confirmed inhibitory effects of fetuin-A and SB431542 on TGFβ-1- and SMAD2/3-regulated transcription. However, contrary to SB431542 and regardless of cMYC upregulation, fetuin-A inhibited beta cell proliferation (0.27 ± 0.08% vs 1.0  ± 0.1% Ki67-positive cells in control NICCs). This effect was sustained by reduced expression (LogFC ≤ -2.4) of FOXM1, CENPA, CDK1 or TOP2A. In agreement, the number of insulin-positive cells was lower in fetuin-A-treated NICCs than in control NICCs (14.4 ± 1.2% and 22.3 ± 1.1%, respectively). In adult human islets fetuin-A abolished glucose responsiveness, i.e. 1.7- and 1.1-fold change over 2.8 mmol/l glucose in control- and fetuin-A-cultured islets, respectively. In addition, fetuin-A reduced SMAD2/3 phosphorylation and suppressed expression of proliferative genes. Of note, in non-diabetic humans, plasma fetuin-A was negatively correlated (p = 0.013) with islet beta cell area.

Conclusions/interpretation: Our results suggest that the perinatal decline of fetuin-A relieves TGFBR signalling in islets, a process that facilitates functional maturation of neonatal beta cells. Functional maturity remains revocable in later life, and the occurrence of a metabolically unhealthy milieu, such as liver steatosis and elevated plasma fetuin-A, can impair both function and adaptive proliferation of beta cells.

Data Availability: The RNAseq datasets and computer code produced in this study are available in the Gene Expression Omnibus (GEO): GSE144950; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144950.
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http://dx.doi.org/10.1007/s00125-021-05435-1DOI Listing
March 2021

Unbiased analysis of obesity related, fat depot specific changes of adipocyte volumes and numbers using light sheet fluorescence microscopy.

PLoS One 2021 16;16(3):e0248594. Epub 2021 Mar 16.

Research Unit Analytical Pathology, Helmholtz Zentrum München, Neuherberg, Germany.

In translational obesity research, objective assessment of adipocyte sizes and numbers is essential to characterize histomorphological alterations linked to obesity, and to evaluate the efficacies of experimental medicinal or dietetic interventions. Design-based quantitative stereological techniques based on the analysis of 2D-histological sections provide unbiased estimates of relevant 3D-parameters of adipocyte morphology, but often involve complex and time-consuming tissue processing and analysis steps. Here we report the application of direct 3D light sheet fluorescence microscopy (LSFM) for effective and accurate analysis of adipocyte volumes and numbers in optically cleared adipose tissue samples from a porcine model of diet-induced obesity (DIO). Subcutaneous and visceral adipose tissue samples from DIO-minipigs and lean controls were systematically randomly sampled, optically cleared with 3DISCO (3-dimensional imaging of solvent cleared organs), stained with eosin, and subjected to LSFM for detection of adipocyte cell membrane autofluorescence. Individual adipocytes were unbiasedly sampled in digital 3D reconstructions of the adipose tissue samples, and their individual cell volumes were directly measured by automated digital image analysis. Adipocyte numbers and mean volumes obtained by LSFM analysis did not significantly differ from the corresponding values obtained by unbiased quantitative stereological analysis techniques performed on the same samples, thus proving the applicability of LSFM for efficient analysis of relevant morphological adipocyte parameters. The results of the present study demonstrate an adipose tissue depot specific plasticity of adipocyte growth responses to nutrient oversupply. This was characterized by an exclusively hypertrophic growth of visceral adipocytes, whereas adipocytes in subcutaneous fat tissue depots also displayed a marked (hyperplastic) increase in cell number. LSFM allows for accurate and efficient determination of relevant quantitative morphological adipocyte parameters. The applied stereological methods and LSFM protocols are described in detail and can serve as a guideline for unbiased quantitative morphological analyses of adipocytes in other studies and species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0248594PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7963095PMC
March 2021

Development of a Sensitive Bioassay for the Analysis of IGF-Related Activation of AKT/mTOR Signaling in Biological Matrices.

Cells 2021 Feb 24;10(3). Epub 2021 Feb 24.

Institute of Genome Biology, Leibniz Institute for Farm Animal Biology (FBN), Wilhelm-Stahl Allee 2, 18196 Dummerstorf, Germany.

The bioactivity of the IGF system is not a function of isolated hormone concentrations in a given biological matrix. Instead, the biological activities of IGFs are regulated by IGFBPs, IGFBP proteases, and inhibitors of IGFBP proteases. Therefore, assays based on IGF-related bioactivity may describe functions of the complete IGF system in a given biological matrix. Of particular interest are the IGF system effects on the AKT/mTOR pathway, as a dominant system for controlling growth, metabolism, and aging. In order to improve the sensitivity of IGF-dependent bioactivity, we made use of the known short-term and enhancing effects of IGFBP2 on the intracellular PI3K pathway. As a specific readout of this pathway, and further as a marker of the mTOR pathway, we assessed the phosphorylation of AKT-Ser473. Preincubation using IGFBP2 enhanced IGF1-dependent AKT-Ser473 phosphorylation in our experimental system. The assay's specificity was demonstrated by inhibition of IGF1 receptors outside or inside the cell, using antiserum or small molecule inhibitors, which reduced AKT phosphorylation in response to exogenous IGF1 ( < 0.05). The maximal response of AKT phosphorylation was recorded 15 to 60 min after the addition of IGF1 to cell monolayers ( < 0.001). In our cellular system, insulin induced AKT phosphorylation only at supra-physiological concentrations (µM). Using this novel assay, we identified the differential biological activity of the IGF system in AKT-Ser473 phosphorylation in serum (mouse, naked mole rat, and human), in cerebrospinal fluid (human), and in colostrum or mature milk samples (dairy cow). We have developed a sensitive and robust bioassay to assess the IGF-related activation of the AKT/mTOR pathway. The assay works efficiently and does not require expensive cell culture systems. By using capillary immuno-electrophoresis, the readout of IGF-related bioactivity is substantially accelerated, requiring a minimum of hands-on time. Importantly, the assay system is useful for studying IGF-related activity in the AKT/mTOR pathway in a broad range of biological matrices.
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http://dx.doi.org/10.3390/cells10030482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7995968PMC
February 2021

Cas9-expressing chickens and pigs as resources for genome editing in livestock.

Proc Natl Acad Sci U S A 2021 Mar;118(10)

Reproductive Biotechnology, Department of Molecular Life Sciences, School of Life Sciences Weihenstephan, Technical University Munich, 85354 Freising, Germany;

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, cross-species comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons.
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http://dx.doi.org/10.1073/pnas.2022562118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7958376PMC
March 2021

Transcriptome dynamics in early in vivo developing and in vitro produced porcine embryos.

BMC Genomics 2021 Feb 27;22(1):139. Epub 2021 Feb 27.

ETH Zurich, Animal Physiology, Institute of Agricultural Sciences, Universitätstrasse 2, CH-8092, Zurich, Switzerland.

Background: The transcriptional changes around the time of embryonic genome activation in pre-implantation embryos indicate that this process is highly dynamic. In vitro produced porcine blastocysts are known to be less competent than in vivo developed blastocysts. To understand the conditions that compromise developmental competence of in vitro embryos, it is crucial to evaluate the transcriptional profile of porcine embryos during pre-implantation stages. In this study, we investigated the transcriptome dynamics in in vivo developed and in vitro produced 4-cell embryos, morulae and hatched blastocysts.

Results: In vivo developed and in vitro produced embryos displayed largely similar transcriptome profiles during development. Enriched canonical pathways from the 4-cell to the morula transition that were shared between in vivo developed and in vitro produced embryos included oxidative phosphorylation and EIF2 signaling. The shared canonical pathways from the morula to the hatched blastocyst transition were 14-3-3-mediated signaling, xenobiotic metabolism general signaling pathway, and NRF2-mediated oxidative stress response. The in vivo developed and in vitro produced hatched blastocysts further were compared to identify molecular signaling pathways indicative of lower developmental competence of in vitro produced hatched blastocysts. A higher metabolic rate and expression of the arginine transporter SLC7A1 were found in in vitro produced hatched blastocysts.

Conclusions: Our findings suggest that embryos with compromised developmental potential are arrested at an early stage of development, while embryos developing to the hatched blastocyst stage display largely similar transcriptome profiles, irrespective of the embryo source. The hatched blastocysts derived from the in vitro fertilization-pipeline showed an enrichment in molecular signaling pathways associated with lower developmental competence, compared to the in vivo developed embryos.
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http://dx.doi.org/10.1186/s12864-021-07430-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7913449PMC
February 2021

Chronic Hyperglycemia Drives Functional Impairment of Lymphocytes in Diabetic Transgenic Pigs.

Front Immunol 2020 22;11:607473. Epub 2021 Jan 22.

Chair of Physiology, Department of Veterinary Sciences, LMU Munich, Munich, Germany.

People with diabetes mellitus have an increased risk for infections, however, there is still a critical gap in precise knowledge about altered immune mechanisms in this disease. Since diabetic transgenic pigs exhibit elevated blood glucose and a stable diabetic phenotype soon after birth, they provide a favorable model to explore functional alterations of immune cells in an early stage of diabetes mellitus . Hence, we investigated peripheral blood mononuclear cells (PBMC) of these diabetic pigs compared to non-diabetic wild-type littermates. We found a 5-fold decreased proliferative response of T cells in tg pigs to polyclonal T cell mitogen phytohemagglutinin (PHA). Using label-free LC-MS/MS, a total of 3,487 proteins were quantified, and distinct changes in protein abundances in CD4 T cells of early-stage diabetic pigs were detectable. Additionally, we found significant increases in mitochondrial oxygen consumption rate (OCR) and higher basal glycolytic activity in PBMC of diabetic tg pigs, indicating an altered metabolic immune cell phenotype. Thus, our study provides new insights into molecular mechanisms of dysregulated immune cells triggered by permanent hyperglycemia.
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http://dx.doi.org/10.3389/fimmu.2020.607473DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862560PMC
January 2021

Genome editing for Duchenne muscular dystrophy: a glimpse of the future?

Gene Ther 2021 Feb 2. Epub 2021 Feb 2.

Friedrich Baur Institute, Department of Neurology, LMU Munich, Munich, Germany.

Mutations in Dystrophin, one of the largest proteins in the mammalian body, are causative for a severe form of muscle disease, Duchenne Muscular Dystrophy (DMD), affecting not only skeletal muscle, but also the heart. In particular, exons 45-52 constitute a hotspot for DMD mutations. A variety of molecular therapies have been developed, comprising vectors encoding micro- and minidystrophins as well as utrophin, a protein with partially overlapping functions. With the advent of the CRISPR-Cas9-nuclease, genome editing offers a novel option of correction of the disease-cuasing mutations. Full restoration of the healthy gene by homology directed repair is a rare event. However, non-homologous end-joining (NHEJ) may restore the reading frame by causing exon excision. This approach has first been demonstrated in mice and then translated to large animals (dogs, pigs). This review discusses the potential opportunities and limitations of genome editing in DMD, including the generation of appropriate animal models as well as new developments in genome editing tools.
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http://dx.doi.org/10.1038/s41434-021-00222-4DOI Listing
February 2021

Mammalian VPS45 orchestrates trafficking through the endosomal system.

Blood 2021 Apr;137(14):1932-1944

Department of Pediatrics, Dr von Hauner Children's Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

Vacuolar protein sorting 45 homolog (VPS45), a member of the Sec1/Munc18 (SM) family, has been implicated in the regulation of endosomal trafficking. VPS45 deficiency in human patients results in congenital neutropenia, bone marrow fibrosis, and extramedullary renal hematopoiesis. Detailed mechanisms of the VPS45 function are unknown. Here, we show an essential role of mammalian VPS45 in maintaining the intracellular organization of endolysosomal vesicles and promoting recycling of cell-surface receptors. Loss of VPS45 causes defective Rab5-to-Rab7 conversion resulting in trapping of cargos in early endosomes and impaired delivery to lysosomes. In this context, we demonstrate aberrant trafficking of the granulocyte colony-stimulating factor receptor in the absence of VPS45. Furthermore, we find that lack of VPS45 in mice is not compatible with embryonic development. Thus, we identify mammalian VPS45 as a critical regulator of trafficking through the endosomal system and early embryogenesis of mice.
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http://dx.doi.org/10.1182/blood.2020006871DOI Listing
April 2021

Manipulating the Epigenome in Nuclear Transfer Cloning: Where, When and How.

Int J Mol Sci 2020 Dec 28;22(1). Epub 2020 Dec 28.

Institute of Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, 81377 Munich, Germany.

The nucleus of a differentiated cell can be reprogrammed to a totipotent state by exposure to the cytoplasm of an enucleated oocyte, and the reconstructed nuclear transfer embryo can give rise to an entire organism. Somatic cell nuclear transfer (SCNT) has important implications in animal biotechnology and provides a unique model for studying epigenetic barriers to successful nuclear reprogramming and for testing novel concepts to overcome them. While initial strategies aimed at modulating the global DNA methylation level and states of various histone protein modifications, recent studies use evidence-based approaches to influence specific epigenetic mechanisms in a targeted manner. In this review, we describe-based on the growing number of reports published during recent decades-in detail where, when, and how manipulations of the epigenome of donor cells and reconstructed SCNT embryos can be performed to optimize the process of molecular reprogramming and the outcome of nuclear transfer cloning.
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http://dx.doi.org/10.3390/ijms22010236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7794987PMC
December 2020

Pathways to Clinical Cardiac Xenotransplantation.

Transplantation 2020 Dec 21;Publish Ahead of Print. Epub 2020 Dec 21.

Walter Brendel Center for Experimental Medicine, LMU Munich, Marchioninistr. 27, 81377 Munich, Germany Department of Anaesthesiology, University Hospital, LMU Munich, Munich, Germany Robert Koch Institute, Nordufer 20, 13353 Berlin, Germany Transplantationslabor, Klinik für Allgemein-, Viszeral-, und Transplantationschirurgie, Medizinische Hochschule Hannover, Carl-Neuberg-Str. 1, 30623 Hannover, Germany Immunology Research Centre, St. Vincent's Hospital Melbourne, and Department of Medicine, University of Melbourne, Victoria, Australia Institute of Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, and Center for Innovative Medical Models (CiMM), LMU Munich, Feodor-Lynen-Str. 25, 81377 Munich, Germany.

Heart transplantation is the only long-lasting life-saving option for patients with terminal cardiac failure. The number of available human organs is however far below the actual need, resulting in substantial mortality of patients while waiting for a human heart. Mechanical assist devices are used to support cardiac function, but are associated with a high risk of severe complications and poor quality of life for the patients. Consistent success in orthotopic transplantation of genetically modified pig hearts into baboons indicates that cardiac xenotransplantation may become a clinically applicable option for heart failure patients who cannot get a human heart transplant. In this overview, we project potential paths to clinical cardiac xenotransplantation, including the choice of genetically modified source pigs, associated requirements of microbiological, including virological, safety, optimized matching of source pig and recipient, and specific treatments of the donor heart after explantation and of the recipients. Moreover, selection of patients and the regulatory framework will be discussed.
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http://dx.doi.org/10.1097/TP.0000000000003588DOI Listing
December 2020

Growth hormone receptor knockout to reduce the size of donor pigs for preclinical xenotransplantation studies.

Xenotransplantation 2021 Mar 25;28(2):e12664. Epub 2020 Nov 25.

Department of Veterinary Sciences, Chair for Molecular Animal Breeding and Biotechnology, Gene Center, LMU Munich, Munich, Germany.

Background: Many genetically multi-modified donor lines for xenotransplantation have a background of domestic pigs with rapid body and organ growth. The intrinsic growth potential of porcine xeno-organs may impair their long-term function after orthotopic transplantation in non-human primate models. Since growth hormone is a major stimulator of postnatal growth, we deleted its receptor (GHR-KO) to reduce the size of donor pigs in one step.

Methods: Heart weight and proteome profile of myocardium were investigated in GHR-KO and control pigs. GHR-KO mutations were introduced using CRISPR/Cas9 in an α1,3-galactosyltransferase (GGTA1)-deficient background expressing the human cluster of differentiation (hCD46) and human thrombomodulin (hTHBD) to generate quadruple-modified (4GM) pigs.

Results: At age 6 months, GHR-KO pigs had a 61% reduced body weight and a 63% reduced heart weight compared with controls. The mean minimal diameter of cardiomyocytes was 28% reduced. A holistic proteome study of myocardium samples from the two groups did not reveal prominent differences. Two 4GM founder sows had low serum insulin-like growth factor 1 (IGF1) levels (24 ± 1 ng/mL) and reached body weights of 70.3 and 73.4 kg at 9 months. Control pigs with IGF1 levels of 228 ± 24 ng/mL reached this weight range three months earlier. The 4GM sows showed normal sexual development and were mated with genetically multi-modified boars. Offspring revealed the expected Mendelian transmission of the genetic modifications and consistent expression of the transgenes.

Conclusion: GHR-KO donor pigs can be used at an age beyond the steepest phase of their growth curve, potentially reducing the problem of xeno-organ overgrowth in preclinical studies.
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http://dx.doi.org/10.1111/xen.12664DOI Listing
March 2021

PAX6 mutation alters circadian rhythm and β cell function in mice without affecting glucose tolerance.

Commun Biol 2020 Oct 30;3(1):628. Epub 2020 Oct 30.

Helmholtz Zentrum München, Institute of Experimental Genetics and German Mouse Clinic, Neuherberg, Germany.

The transcription factor PAX6 is involved in the development of the eye and pancreatic islets, besides being associated with sleep-wake cycles. Here, we investigated a point mutation in the RED subdomain of PAX6, previously described in a human patient, to present a comprehensive study of a homozygous Pax6 mutation in the context of adult mammalian metabolism and circadian rhythm. Pax6 mice lack appropriate retinal structures for light perception and do not display normal daily rhythmic changes in energy metabolism. Despite β cell dysfunction and decreased insulin secretion, mutant mice have normal glucose tolerance. This is associated with reduced hepatic glucose production possibly due to altered circadian variation in expression of clock and metabolic genes, thereby evading hyperglycemia. Hence, our findings show that while the RED subdomain is important for β cell functional maturity, the Leca2 mutation impacts peripheral metabolism via loss of circadian rhythm, thus revealing pleiotropic effects of PAX6.
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http://dx.doi.org/10.1038/s42003-020-01337-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7599253PMC
October 2020

Effect of Dietary Sodium Modulation on Pig Adrenal Steroidogenesis and Transcriptome Profiles.

Hypertension 2020 12 19;76(6):1769-1777. Epub 2020 Oct 19.

From the Medizinische Klinik und Poliklinik IV, Klinikum der Universität München (T.V., I.-S.K., M.R., T.A.W.), Ludwig-Maximilians-Universität München, Munich, Germany.

Primary aldosteronism is a frequent form of endocrine hypertension caused by aldosterone overproduction from the adrenal cortex. Regulation of aldosterone biosynthesis has been studied in rodents despite differences in adrenal physiology with humans. We, therefore, investigated pig adrenal steroidogenesis, morphology, and transcriptome profiles of the zona glomerulosa (zG) and zona fasciculata in response to activation of the renin-angiotensin-aldosterone system by dietary sodium restriction. Six-week-old pigs were fed a low- or high-sodium diet for 14 days (3 pigs per group, 0.4 g sodium/kg feed versus 6.8 g sodium/kg). Plasma aldosterone concentrations displayed a 43-fold increase (=0.011) after 14 days of sodium restriction (day 14 versus day 0). Low dietary sodium caused a 2-fold increase in thickness of the zG (<0.001) and an almost 3-fold upregulation of (<0.05) compared with high dietary sodium. Strong immunostaining of the KCNJ5 (G protein-activated inward rectifier potassium channel 4), which is frequently mutated in primary aldosteronism, was demonstrated in the zG. mRNA sequencing transcriptome analysis identified significantly altered expression of genes modulated by the renin-angiotensin-aldosterone system in the zG (n=1172) and zona fasciculata (n=280). These genes included many with a known role in the regulation of aldosterone synthesis and adrenal function. The most highly enriched biological pathways in the zG were related to cholesterol biosynthesis, steroid metabolism, cell cycle, and potassium channels. This study provides mechanistic insights into the physiology and pathophysiology of aldosterone production in a species closely related to humans and shows the suitability of pigs as a translational animal model for human adrenal steroidogenesis.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.120.15998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011841PMC
December 2020

Impact of porcine cytomegalovirus on long-term orthotopic cardiac xenotransplant survival.

Sci Rep 2020 10 16;10(1):17531. Epub 2020 Oct 16.

Department of Anaesthesiology, University Hospital, Ludwig-Maximilians-Universität München, Munich, Germany.

Xenotransplantation using pig organs has achieved survival times up to 195 days in pig orthotopic heart transplantation into baboons. Here we demonstrate that in addition to an improved immunosuppressive regimen, non-ischaemic preservation with continuous perfusion and control of post-transplantation growth of the transplant, prevention of transmission of the porcine cytomegalovirus (PCMV) plays an important role in achieving long survival times. For the first time we demonstrate that PCMV transmission in orthotopic pig heart xenotransplantation was associated with a reduced survival time of the transplant and increased levels of IL-6 and TNFα were found in the transplanted baboon. Furthermore, high levels of tPA-PAI-1 complexes were found, suggesting a complete loss of the pro-fibrinolytic properties of the endothelial cells. These data show that PCMV has an important impact on transplant survival and call for elimination of PCMV from donor pigs.
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http://dx.doi.org/10.1038/s41598-020-73150-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7568528PMC
October 2020

Murine tissue factor disulfide mutation causes a bleeding phenotype with sex specific organ pathology and lethality.

Haematologica 2020 10 1;105(10):2484-2495. Epub 2020 Oct 1.

Department of Cardiology, University Heart Center, University Hospital, Zurich, Switzerland.

Tissue factor is highly expressed in sub-endothelial tissue. The extracellular allosteric disulfide bond Cys186-Cys209 of human tissue factor shows high evolutionary conservation and in vitro evidence suggests that it significantly contributes to tissue factor procoagulant activity. To investigate the role of this allosteric disulfide bond in vivo, we generated a C213G mutant tissue factor mouse by replacing Cys213 of the corresponding disulfide Cys190-Cys213 in murine tissue factor. A bleeding phenotype was prominent in homozygous C213G tissue factor mice. Pre-natal lethality of 1/3rd of homozygous offspring was observed between E9.5 and E14.5 associated with placental hemorrhages. After birth, homozygous mice suffered from bleedings in different organs and reduced survival. Homozygous C213G tissue factor male mice showed higher incidence of lung bleedings and lower survival rates than females. In both sexes, C213G mutation evoked a reduced protein expression (about 10-fold) and severely reduced pro-coagulant activity (about 1000-fold). Protein glycosylation was impaired and cell membrane exposure decreased in macrophages in vivo. Single housing of homozygous C213G tissue factor males reduced the occurrence of severe bleeding and significantly improved survival, suggesting that inter-male aggressiveness might significantly account for the sex differences. These experiments show that the tissue factor allosteric disulfide bond is of crucial importance for normal in vivo expression, post-translational processing and activity of murine tissue factor. Although C213G tissue factor mice do not display the severe embryonic lethality of tissue factor knock-out mice, their postnatal bleeding phenotype emphasizes the importance of fully functional tissue factor for hemostasis.
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http://dx.doi.org/10.3324/haematol.2019.218818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7556672PMC
October 2020

A decade of experience with genetically tailored pig models for diabetes and metabolic research.

Anim Reprod 2020 Aug 26;17(3):e20200064. Epub 2020 Aug 26.

Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, Munich, Germany.

The global prevalence of diabetes mellitus and other metabolic diseases is rapidly increasing. Animal models play pivotal roles in unravelling disease mechanisms and developing and testing therapeutic strategies. Rodents are the most widely used animal models but may have limitations in their resemblance to human disease mechanisms and phenotypes. Findings in rodent models are consequently often difficult to extrapolate to human clinical trials. To overcome this 'translational gap', we and other groups are developing porcine disease models. Pigs share many anatomical and physiological traits with humans and thus hold great promise as translational animal models. Importantly, the toolbox for genetic engineering of pigs is rapidly expanding. Human disease mechanisms and targets can therefore be reproduced in pigs on a molecular level, resulting in precise and predictive porcine (PPP) models. In this short review, we summarize our work on the development of genetically (pre)diabetic pig models and how they have been used to study disease mechanisms and test therapeutic strategies. This includes the generation of reporter pigs for studying beta-cell maturation and physiology. Furthermore, genetically engineered pigs are promising donors of pancreatic islets for xenotransplantation. In summary, genetically tailored pig models have become an important link in the chain of translational diabetes and metabolic research.
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http://dx.doi.org/10.1590/1984-3143-AR2020-0064DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534555PMC
August 2020

Progressive Proteome Changes in the Myocardium of a Pig Model for Duchenne Muscular Dystrophy.

iScience 2020 Sep 1;23(9):101516. Epub 2020 Sep 1.

Laboratory for Functional Genome Analysis, LAFUGA, Gene Center, LMU Munich, 81377 Munich, Germany. Electronic address:

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is characterized by progressive muscle weakness. Even though DMD manifests first in skeletal muscle, heart failure is a major cause of death in late-stage DMD. To get insights into DMD-associated cardiomyopathy, we performed a proteome analysis of myocardium from a genetically engineered porcine DMD model resembling clinical and pathological hallmarks of human DMD. To capture DMD progression, samples from 2-day- and 3-month-old animals were analyzed. Dystrophin was absent in all DMD samples, and components of the dystrophin-associated protein complex were decreased, suggesting destabilization of the cardiomyocyte plasma membrane and impaired cellular signaling. Furthermore, abundance alterations of proteins known to be associated with human cardiomyopathy were observed. Compared with data from skeletal muscle, we found clear evidence that DMD progression in myocardium is not only slower than in skeletal muscle but also involves different biological and biochemical pathways.
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http://dx.doi.org/10.1016/j.isci.2020.101516DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495112PMC
September 2020

The impact of transcription inhibition during in vitro maturation on the proteome of bovine oocytes†.

Biol Reprod 2020 10;103(5):1000-1011

Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, Germany.

Proper oocyte maturation is a prerequisite for successful reproduction and requires the resumption of meiosis to the metaphase II stage (MII). In bovine oocytes, nuclear maturation has been shown to occur in in vitro maturing cumulus-enclosed oocytes (COCs) in the absence of transcription, but their developmental capacity is reduced compared to transcriptionally competent COCs. To assess the impact of transcription during in vitro maturation of bovine COCs on the quantitative oocyte proteome, a holistic nano-LC-MS/MS analysis of germinal vesicle oocytes and MII oocytes matured with or without addition of the transcription inhibitor actinomycin D (ActD) was carried out. Analyzing eight biological replicates for each of the three groups, a total of 2018 proteins was identified. These could be clearly classified into proteins depending or not depending on transcription during oocyte maturation. Proteins whose abundance increased after maturation irrespective of transcription inhibition - and hence independent of transcription - were related to the cell cycle, reflecting the progression of meiosis, and to cellular component organization, which is crucial for cytoplasmic maturation. In contrast, transcription-dependent proteins were associated with cell-cell adhesion and translation. Since a high rate of protein synthesis in oocytes has been shown to correlate with their developmental competence, oocyte maturation in transcriptionally impaired COCs is apparently disturbed. Our experiments reveal that impaired transcription during in vitro maturation of COCs has a substantial effect on specific components of the oocyte proteome, and that transcription is required for specific classes of oocyte proteins predominantly involved in translation.
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http://dx.doi.org/10.1093/biolre/ioaa149DOI Listing
October 2020

Cold non-ischemic heart preservation with continuous perfusion prevents early graft failure in orthotopic pig-to-baboon xenotransplantation.

Xenotransplantation 2021 01 25;28(1):e12636. Epub 2020 Aug 25.

Department of Cardiac Surgery, University Hospital, LMU Munich, Munich, Germany.

Background: Successful preclinical transplantations of porcine hearts into baboon recipients are required before commencing clinical trials. Despite years of research, over half of the orthotopic cardiac xenografts were lost during the first 48 hours after transplantation, primarily caused by perioperative cardiac xenograft dysfunction (PCXD). To decrease the rate of PCXD, we adopted a preservation technique of cold non-ischemic perfusion for our ongoing pig-to-baboon cardiac xenotransplantation project.

Methods: Fourteen orthotopic cardiac xenotransplantation experiments were carried out with genetically modified juvenile pigs (GGTA1- KO/hCD46/hTBM) as donors and captive-bred baboons as recipients. Organ preservation was compared according to the two techniques applied: cold static ischemic cardioplegia (IC; n = 5) and cold non-ischemic continuous perfusion (CP; n = 9) with an oxygenated albumin-containing hyperoncotic cardioplegic solution containing nutrients, erythrocytes and hormones. Prior to surgery, we measured serum levels of preformed anti-non-Gal-antibodies. During surgery, hemodynamic parameters were monitored with transpulmonary thermodilution. Central venous blood gas analyses were taken at regular intervals to estimate oxygen extraction, as well as lactate production. After surgery, we measured troponine T and serum parameters of the recipient's kidney, liver and coagulation functions.

Results: In porcine grafts preserved with IC, we found significantly depressed systolic cardiac function after transplantation which did not recover despite increasing inotropic support. Postoperative oxygen extraction and lactate production were significantly increased. Troponin T, creatinine, aspartate aminotransferase levels were pathologically high, whereas prothrombin ratios were abnormally low. In three of five IC experiments, PCXD developed within 24 hours. By contrast, all nine hearts preserved with CP retained fully preserved systolic function, none showed any signs of PCXD. Oxygen extraction was within normal ranges; serum lactate as well as parameters of organ functions were only mildly elevated. Preformed anti-non-Gal-antibodies were similar in recipients receiving grafts from either IC or CP preservation.

Conclusions: While standard ischemic cardioplegia solutions have been used with great success in human allotransplantation over many years, our data indicate that they are insufficient for preservation of porcine hearts transplanted into baboons: Ischemic storage caused severe impairment of cardiac function and decreased tissue oxygen supply, leading to multi-organ failure in more than half of the xenotransplantation experiments. In contrast, cold non-ischemic heart preservation with continuous perfusion reliably prevented early graft failure. Consistent survival in the perioperative phase is a prerequisite for preclinical long-term results after cardiac xenotransplantation.
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http://dx.doi.org/10.1111/xen.12636DOI Listing
January 2021

Xeno-organ donor pigs with multiple genetic modifications - the more the better?

Curr Opin Genet Dev 2020 10 30;64:60-65. Epub 2020 Jun 30.

Chair of Molecular Animal Breeding and Biotechnology, and Laboratory for Functional Genome Analysis (LAFUGA), Gene Center, LMU Munich, 81377 Munich, Germany; Center for Innovative Medical Models (CiMM), Department of Veterinary Sciences, LMU Munich, 85764 Oberschleißheim, Germany. Electronic address:

The number of donated human organs and tissues for patients with terminal organ failure falls far short of the need. Alternative sources, such as organs and tissues from animals, are therefore urgently required. During the past few years, major progress has been made in the development of genetically multi-modified donor pigs, and their organs have been shown to be safe and efficacious in life-supporting transplantation models into non-human primates, paving the way to clinical xenotransplantation studies. Here, we summarize recent developments in pig genome engineering and discuss efforts to develop the optimum donor pig for xenotransplantation. In addition, we speculate on how many genetic modifications may be required for initial xenotransplantation clinical trials.
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http://dx.doi.org/10.1016/j.gde.2020.05.034DOI Listing
October 2020

Physiological relevance of the neuronal isoform of inositol-1,4,5-trisphosphate 3-kinases in mice.

Neurosci Lett 2020 09 25;735:135206. Epub 2020 Jun 25.

Department of Biochemistry and Signal Transduction, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, D-20246, Hamburg, Germany. Electronic address:

Inositol-1,4,5-trisphosphate 3-kinase-A (ITPKA) is the neuronal isoform of ITPKs and exhibits both actin bundling and InsPkinase activity. In addition to neurons, ITPKA is ectopically expressed in tumor cells, where its oncogenic activity increases tumor cell malignancy. In order to analyze the physiological relevance of ITPKA, here we performed a broad phenotypic screening of itpka deficient mice. Our data show that among the neurobehavioral tests analyzed, itpka deficient mice reacted faster to a hotplate, prepulse inhibition was impaired and the accelerating rotarod test showed decreased latency of itpka deficient mice to fall. These data indicate that ITPKA is involved in the regulation of nociceptive pathways, sensorimotor gating and motor learning. Analysis of extracerebral functions in control and itpka deficient mice revealed significantly reduced glucose, lactate, and triglyceride plasma concentrations in itpka deficient mice. Based on this finding, expression of ITPKA was analyzed in extracerebral tissues and the highest level was found in the small intestine. However, functional studies on CaCo-2 control and ITPKA depleted cells showed that glucose, as well as triglyceride uptake, were not significantly different between the cell lines. Altogether, these data show that ITPKA exhibits distinct functions in the central nervous system and reveal an involvement of ITPKA in energy metabolism.
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http://dx.doi.org/10.1016/j.neulet.2020.135206DOI Listing
September 2020

Pig-to-non-human primate heart transplantation: The final step toward clinical xenotransplantation?

J Heart Lung Transplant 2020 08 15;39(8):751-757. Epub 2020 May 15.

Department of Cardiac Surgery, University Hospital, LMU Munich, Munich, Germany.

Background: The demand for donated human hearts far exceeds the number available. Xenotransplantation of genetically modified porcine organs provides an alternative. In 2000, an Advisory Board of the International Society for Heart and Lung Transplantation set the benchmark for commencing clinical cardiac xenotransplantation as consistent 60% survival of non-human primates after life-supporting porcine heart transplantations. Recently, we reported the stepwise optimization of pig-to-baboon orthotopic cardiac xenotransplantation finally resulting in consistent success, with 4 recipients surviving 90 (n = 2), 182, and 195 days. Here, we report on 4 additional recipients, supporting the efficacy of our procedure.

Results: The first 2 additional recipients succumbed to porcine cytomegalovirus (PCMV) infections on Days 15 and 27, respectively. In 2 further experiments, PCMV infections were successfully avoided, and 3-months survival was achieved. Throughout all the long-term experiments, heart, liver, and renal functions remained within normal ranges. Post-mortem cardiac diameters were slightly increased when compared with that at the time of transplantation but with no detrimental effect. There were no signs of thrombotic microangiopathy. The current regimen enabled the prolonged survival and function of orthotopic cardiac xenografts in altogether 6 of 8 baboons, of which 4 were now added. These results exceed the threshold set by the Advisory Board of the International Society for Heart and Lung Transplantation.

Conclusions: The results of our current and previous experimental cardiac xenotransplantations together fulfill for the first time the pre-clinical efficacy suggestions. PCMV-positive donor animals must be avoided.
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http://dx.doi.org/10.1016/j.healun.2020.05.004DOI Listing
August 2020

Proteome profile of neutrophils from a transgenic diabetic pig model shows distinct changes.

J Proteomics 2020 07 27;224:103843. Epub 2020 May 27.

Department of Veterinary Sciences, LMU, Munich, Germany. Electronic address:

INS transgenic pigs develop a stable diabetic phenotype early after birth and therefore allow studying the influence of hyperglycemia on primary immune cells in an early stage of diabetes mellitus in vivo. Since immune response is altered in diabetes mellitus, with deviant neutrophil function discussed as one of the possible causes in humans and mouse models, we investigated these immune cells in INS transgenic pigs and wild type controls at protein level. A total of 2371 proteins were quantified by label-free LC-MS/MS. Subsequent differential proteome analysis of transgenic animals and controls revealed clear differences in protein abundances, indicating a deviant behavior of granulocytes in the diabetic state. Interestingly, abundance of myosin regulatory light chain 9 (MLC-2C) was increased 5-fold in cells of diabetic pigs. MLC-2C directly affects cell contractility by regulating myosin ATPase activity, can act as transcription factor and was also associated with inflammation. It might contribute to impaired neutrophil cell adhesion, migration and phagocytosis. Our study provides novel insights into proteome changes in neutrophils from a large animal model for permanent neonatal diabetes mellitus and points to dysregulation of neutrophil function even in an early stage of this disease. Data are available via ProteomeXchange with identifier PXD017274. SIGNIFICANCE: Our studies provide novel basic information about the neutrophil proteome of pigs and contribute to a better understanding of molecular mechanisms involved in altered immune cell function in an early stage diabetes. We demonstrate proteins that are dysregulated in neutrophils from a transgenic diabetic pig and have not been described in this context so far. The data presented here are highly relevant for veterinary medicine and have translational quality for diabetes in humans.
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http://dx.doi.org/10.1016/j.jprot.2020.103843DOI Listing
July 2020

Integration of nano- and biotechnology for beta-cell and islet transplantation in type-1 diabetes treatment.

Cell Prolif 2020 May 27;53(5):e12785. Epub 2020 Apr 27.

Biotalentum Ltd, Hungary, Godollo, Hungary.

Regenerative medicine using human or porcine β-cells or islets has an excellent potential to become a clinically relevant method for the treatment of type-1 diabetes. High-resolution imaging of the function and faith of transplanted porcine pancreatic islets and human stem cell-derived beta cells in large animals and patients for testing advanced therapy medicinal products (ATMPs) is a currently unmet need for pre-clinical/clinical trials. The iNanoBIT EU H2020 project is developing novel highly sensitive nanotechnology-based imaging approaches allowing for monitoring of survival, engraftment, proliferation, function and whole-body distribution of the cellular transplants in a porcine diabetes model with excellent translational potential to humans. We develop and validate the application of single-photon emission computed tomography (SPECT) and optoacoustic imaging technologies in a transgenic insulin-deficient pig model to observe transplanted porcine xeno-islets and in vitro differentiated human beta cells. We are progressing in generating new transgenic reporter pigs and human-induced pluripotent cell (iPSC) lines for optoacoustic imaging and testing them in transplantable bioartificial islet devices. Novel multifunctional nanoparticles have been generated and are being tested for nuclear imaging of islets and beta cells using a new, high-resolution SPECT imaging device. Overall, the combined multidisciplinary expertise of the project partners allows progress towards creating much needed technological toolboxes for the xenotransplantation and ATMP field, and thus reinforces the European healthcare supply chain for regenerative medicinal products.
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http://dx.doi.org/10.1111/cpr.12785DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260069PMC
May 2020

Functional changes of the liver in the absence of growth hormone (GH) action - Proteomic and metabolomic insights from a GH receptor deficient pig model.

Mol Metab 2020 06 18;36:100978. Epub 2020 Mar 18.

Laboratory for Functional Genome Analysis, (LAFUGA), Gene Center, LMU Munich, 81377 Munich, Germany; Institute of Molecular Animal Breeding and Biotechnology, Gene Center and Department of Veterinary Sciences, LMU Munich, 81377 Munich, Germany; German Center for Diabetes Research (DZD), 85764 Neuherberg, Germany. Electronic address:

Objective: The liver is a central target organ of growth hormone (GH), which stimulates the synthesis of insulin-like growth factor 1 (IGF1) and affects multiple biochemical pathways. A systematic multi-omics analysis of GH effects in the liver has not been performed. GH receptor (GHR) deficiency is a unique model for studying the consequences of lacking GH action. In this study, we used molecular profiling techniques to capture a broad spectrum of these effects in the liver of a clinically relevant large animal model for Laron syndrome.

Methods: We performed holistic proteome and targeted metabolome analyses of liver samples from 6-month-old GHR-deficient (GHR-KO) pigs and GHR-expressing controls (four males, four females per group).

Results: GHR deficiency resulted in an increased abundance of enzymes involved in amino acid degradation, in the urea cycle, and in the tricarboxylic acid cycle. A decreased ratio of long-chain acylcarnitines to free carnitine suggested reduced activity of carnitine palmitoyltransferase 1A and thus reduced mitochondrial import of fatty acids for beta-oxidation. Increased levels of short-chain acylcarnitines in the liver and in the circulation of GHR-KO pigs may result from impaired beta-oxidation of short-chain fatty acids or from increased degradation of specific amino acids. The concentration of mono-unsaturated glycerophosphocholines was significantly increased in the liver of GHR-KO pigs without morphological signs of steatosis, although the abundances of several proteins functionally linked to non-alcoholic fatty liver disease (fetuin B, retinol binding protein 4, several mitochondrial proteins) were increased. Moreover, GHR-deficient liver samples revealed distinct changes in the methionine and glutathione metabolic pathways, in particular, a significantly increased level of glycine N-methyltransferase and increased levels of total and free glutathione. Several proteins revealed a sex-related abundance difference in the control group but not in the GHR-KO group.

Conclusions: Our integrated proteomics/targeted metabolomics study of GHR-deficient and control liver samples from a clinically relevant large animal model identified a spectrum of biological pathways that are significantly altered in the absence of GH action. Moreover, new insights into the role of GH in the sex-related specification of liver functions were provided.
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http://dx.doi.org/10.1016/j.molmet.2020.100978DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184181PMC
June 2020

Gene Regulatory and Expression Differences between Mouse and Pig Limb Buds Provide Insights into the Evolutionary Emergence of Artiodactyl Traits.

Cell Rep 2020 04;31(1):107490

Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide-Junta de Andalucía, 41013 Seville, Spain. Electronic address:

Digit loss/reductions are evolutionary adaptations in cursorial mammals such as pigs. To gain mechanistic insight into these processes, we performed a comparative molecular analysis of limb development in mouse and pig embryos, which revealed a loss of anterior-posterior polarity during distal progression of pig limb bud development. These alterations in pig limb buds are paralleled by changes in the mesenchymal response to Sonic hedgehog (SHH) signaling, which is altered upstream of the reduction and loss of Fgf8 expression in the ectoderm that overlaps the reduced and vestigial digit rudiments of the pig handplate, respectively. Furthermore, genome-wide open chromatin profiling using equivalent developmental stages of mouse and pig limb buds reveals the functional divergence of about one-third of the regulatory genome. This study uncovers widespread alterations in the regulatory landscapes of genes essential for limb development that likely contributed to the morphological diversion of artiodactyl limbs from the pentadactyl archetype of tetrapods.
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http://dx.doi.org/10.1016/j.celrep.2020.03.054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7166081PMC
April 2020

Large Animal Models of Diabetes.

Methods Mol Biol 2020 ;2128:115-134

Department of Visceral, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, Dresden, Germany.

Safe and reliable large animal diabetes models are a key prerequisite for advanced preclinical studies on diabetes. Chemical induction is the standard model of diabetes in rodents but is often critiqued in higher animals due to reduced efficacy, relevant side effects, and inadequate mortality rate. In this chapter, we aim to describe both pharmacological and surgical approaches for reproducible and safe diabetes models in minipigs and primates. In addition, genetically modified pig models for diabetes research are described.
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http://dx.doi.org/10.1007/978-1-0716-0385-7_9DOI Listing
March 2021

A comprehensive and comparative phenotypic analysis of the collaborative founder strains identifies new and known phenotypes.

Mamm Genome 2020 02 14;31(1-2):30-48. Epub 2020 Feb 14.

German Mouse Clinic, Institute of Experimental Genetics, Helmholtz Zentrum München, German Research Center for Environmental Health, Ingolstädter Landstrasse 1, 85764, Neuherberg, Germany.

The collaborative cross (CC) is a large panel of mouse-inbred lines derived from eight founder strains (NOD/ShiLtJ, NZO/HILtJ, A/J, C57BL/6J, 129S1/SvImJ, CAST/EiJ, PWK/PhJ, and WSB/EiJ). Here, we performed a comprehensive and comparative phenotyping screening to identify phenotypic differences and similarities between the eight founder strains. In total, more than 300 parameters including allergy, behavior, cardiovascular, clinical blood chemistry, dysmorphology, bone and cartilage, energy metabolism, eye and vision, immunology, lung function, neurology, nociception, and pathology were analyzed; in most traits from sixteen females and sixteen males. We identified over 270 parameters that were significantly different between strains. This study highlights the value of the founder and CC strains for phenotype-genotype associations of many genetic traits that are highly relevant to human diseases. All data described here are publicly available from the mouse phenome database for analyses and downloads.
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http://dx.doi.org/10.1007/s00335-020-09827-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060152PMC
February 2020

Cellular and Molecular Probing of Intact Human Organs.

Cell 2020 02 13;180(4):796-812.e19. Epub 2020 Feb 13.

Insititute for Tissue Engineering and Regenerative Medicine (iTERM), Helmholtz Zentrum München, 85764 Neuherberg, Germany; Institute for Stroke and Dementia Research (ISD), University Hospital, Ludwig Maximilian University of Munich (LMU), 81377 Munich, Germany; Munich Cluster for Systems Neurology (SyNergy), 81377 Munich, Germany. Electronic address:

Optical tissue transparency permits scalable cellular and molecular investigation of complex tissues in 3D. Adult human organs are particularly challenging to render transparent because of the accumulation of dense and sturdy molecules in decades-aged tissues. To overcome these challenges, we developed SHANEL, a method based on a new tissue permeabilization approach to clear and label stiff human organs. We used SHANEL to render the intact adult human brain and kidney transparent and perform 3D histology with antibodies and dyes in centimeters-depth. Thereby, we revealed structural details of the intact human eye, human thyroid, human kidney, and transgenic pig pancreas at the cellular resolution. Furthermore, we developed a deep learning pipeline to analyze millions of cells in cleared human brain tissues within hours with standard lab computers. Overall, SHANEL is a robust and unbiased technology to chart the cellular and molecular architecture of large intact mammalian organs.
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http://dx.doi.org/10.1016/j.cell.2020.01.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557154PMC
February 2020

Genetically encoded Ca -sensor reveals details of porcine endothelial cell activation upon contact with human serum.

Xenotransplantation 2020 09 14;27(5):e12585. Epub 2020 Feb 14.

Chair for Molecular Animal Breeding and Biotechnology, LMU Munich, Munich, Germany.

The activation of the endothelial surface in xenografts is still a poorly understood process and the consequences are unpredictable. The role of Ca -messaging during the activation of endothelial cells is well recognized and routinely measured by synthetic Ca -sensitive fluorophors. However, these compounds require fresh loading immediately before each experiment and in particular when grown in state-of-the-art 3D cell culture systems, endothelial cells are difficult to access with such sensors. Therefore, we developed transgenic pigs expressing a Ca -sensitive protein and examined its principal characteristics. Primary transgenic endothelial cells stimulated by ATP showed a definite and short influx of Ca into the cytosol, whereas exposure to human serum resulted in a more intense and sustained response. Surprisingly, not all endothelial cells reacted identically to a stimulus, rather activation took place in adjacent cells in a timely decelerated way and with distinct intensities. This effect was again more pronounced when cells were stimulated with human serum. Finally, we show clear evidence that antibody binding alone significantly activated endothelial cells, whereas antibody depletion dramatically reduced the stimulatory potential of serum. Transgenic porcine endothelial cells expressing a Ca -sensor represent an interesting tool to dissect factors inducing activation of porcine endothelial cells after exposure to human blood or serum.
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http://dx.doi.org/10.1111/xen.12585DOI Listing
September 2020