Institut für Anatomie und Zellbiologie
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- Institut für Anatomie und Zellbiologie (277)
- Graduate School of Life Sciences (17)
- Institut für diagnostische und interventionelle Radiologie (Institut für Röntgendiagnostik) (11)
- Neurologische Klinik und Poliklinik (9)
- Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I) (8)
- Klinik und Poliklinik für Anästhesiologie (ab 2004) (7)
- Theodor-Boveri-Institut für Biowissenschaften (7)
- Institut für Pharmakologie und Toxikologie (6)
- Klinik und Poliklinik für Nuklearmedizin (5)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (4)
Sonstige beteiligte Institutionen
- Department of Biomedical Imaging, National Cerebral and Cardiovascular Research Center, Suita, Japan (2)
- Division of Medical Technology and Science, Department of Medical Physics and Engineering, Course of Health Science, Osaka University Graduate School of Medicine, Suita Japan (2)
- Institut for Molecular Biology and CMBI, Department of Genomics, Stem Cell Biology and Regenerative Medicine, Leopold-Franzens-University Innsbruck, Innsbruck, Austria (2)
- Johns Hopkins School of Medicine, The Russell H Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA (2)
- Naturalis Biodiversity Centre (2)
- Johns Hopkins School of Medicine (1)
- Johns Hopkins School of Medicine, Baltimore, MD, U.S. (1)
Da die Pathogenese der Multiplen Sklerose (MS) bis heute nicht vollständig geklärt ist, befassten wir uns mit der Rolle CEACAM1-exprimierender Immunzellen bei Patienten mit MS und untersuchten diese mittels durchflusszytometrischer Untersuchung. Bei CEACAM1 (Carcinoembryonic-antigen-related cell adhesion molecule) handelt es sich um ein Zelladhäsionsmolekül, das sowohl an inter- als auch intrazellulären Signalmechanismen modulatorisch beteiligt ist. Anhand unserer Ergebnisse scheint CEACAM1 keine zentrale Rolle in der Pathogenese der MS zu spielen. Es ließ sich jedoch eine signifikante Erhöhung CD56+dim NK-Zellen (natürliche Killerzellen) im peripheren Blut von Patienten mit schubförmig remittierender MS feststellen. Dies stützt die These, dass die „dim“-Subpopulation der NK-Zellen eine proinflammatorische Rolle in der Pathogenese der MS einnehmen könnte. Demnach sollte in Zukunft hinsichtlich der Entwicklung neuer Biomarker in der MS der Fokus auf NK-Zellen und Monozyten sowie deren Subpopulationen gerichtet werden.
Viele Organoide sind bisher nur stark vereinfachte Modelle der Originalgewebe, da sie nur aus dem Gewebsparenchym bestehen. Um neurale Organoide näher an das Originalgewebe zu bringen, ist ein wichtiger Schritt mesenchymale Anteile zu integrieren. In dieser Arbeit war die wichtige Fragenstellung, ob neurale Organoide sich mit mesodermalen Progenitorzellen zu einem gemeinsamen Gewebe vereinigen lassen.
Um die Generierung von neuro-mesenchymalen Organoiden zu erreichen, wurden geeignete Differenzierungsprotokolle zur Erzeugung neuroepithelialer und mesodermaler Aggregate aus humanen induzierten pluripotenten Stammzellen etabliert. Anschließend wurden die Sphäroide vereinigt und eingehend histologisch charakterisiert. Darüber hinaus wurde die Organoidentwicklung unter dem Einfluss von Hypoxie analysiert. Um die Organoide anschaulich mit der tatsächlichen Embryogenese vergleichen zu können, wurden Schnitte von Hühnerembryonen angefertigt. Die neuro-mesenchymalen Organoide wurden insgesamt 280 Tage kultiviert und an verschieden Zeitpunkten untersucht.
Die hier präsentierten Daten zeigen, dass die erzeugten neuro-mesenchymalen Organoide viele Aspekte der natürlichen Embryogenese in Zellkultur nachahmen können. So wurde die Ausbildung neuralrohrähnlicher Strukturen, die von einem perineuralen Gefäßplexus umgeben sind, gezeigt. Des Weiteren wurde eine Interaktion von Astrozyten/radiale Gliazellen mit dem entstehenden Gefäßnetz beobachtet. Schließlich zeigten sich das Einwandern von mikrogliaartigen Zellen aus dem mesenchymalen Organoidteil in das Nervengewebe.
Diese Arbeit bildet die Basis für die Generierung neuro-mesenchymaler Organoide als realistisches Modellsystem für die Entwicklung des Nervensystems. Solche Modellsysteme können für die Erforschung von Krankheiten, Toxizitätsstudien sowie Medikamententests verwendet werden.
Der epithelialen Präsenz des Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) in Prostatadrüsen wird eine tumorsupprimierende Funktion zugeschrieben. Maligne Veränderungen des Prostatadrüsenepithels bei einem PCa führen zu einer Abnahme der epithelialen CEACAM1-Expression, zu einem Verlust der Zellpolarität und zu einer erhöhten Zellproliferation (prostatische intraepitheliale Neoplasie (PIN)). Während des PIN-Stadiums exprimieren benachbarte Blut- und Lymphgefäße CEACAM1. CEACAM1 selbst wirkt pro-angiogen und stimuliert die Gefäßneubildung und auch die Neubildung von Lymphgefäßen, Lymphangiogenese. Seine Rolle in der Tumor-Lymphangiogenese und dadurch bedingten Metastasierung von Tumoren wurde bisher nicht ausreichend geklärt. Ziel dieser Arbeit war es, die Rolle von CEACAM1 bei der lymphogenen PCa-Metastasierung anhand von immunhistochemischen (IHC) Analysen am humanem PCa-Prostata- und Lymphknoten-(LN)-Gewebe, sowie im Mausmodell zu analysieren.
Laut den Immunfluoreszenzanalysen traten in den PIN-Arealen signifikant mehr CEACAM1-positive Blut- und Lymphgefäße auf, als in den darauffolgenden Tumorstadien. Weiter wurde eine CEACAM1-Expression in LN-Sinusgewebe bereits bei Niedrig-Risiko-Patienten (pN0) detektiert. Diese frühe CEACAM1-Expression trat auch in den LN im PCa-Mausmodell auf. Weiter wurde im LN-Gewebe von „Hoch-Risiko“-Patienten (pN1) eine luminale CEACAM1-Expression innerhalb der aus Tumorzellen bestehenden Drüsen beobachtet, die mit der CEACAM1-Expression in nativen Prostatadrüsen vergleichbar ist. Auch das angiogen-aktivierte Gefäßendothel von pN0- und pN1-LN war CEACAM1-positiv. Bei Hoch-Risiko-Patienten (pN1) nahmen die CEACAM1-positiven Blut- und Lymphgefäße im Tumorstroma mit zunehmender Dedifferenzierung des Gewebes ab. Die CEACAM1/PSA-Doppelimmun-fluoreszenzanalysen ergaben eine heterogene Expression der beiden Marker bei Intermediate-risk-Patienten und mit zunehmender Dedifferenzierung des Tumorgewebes einen epithelialen Verlust der CEACAM1-Expression in den PSA-positiven G3-Tumordrüsen. Das Fehlen von PSA in pN0-LN und die nachweisbare Expression von PSA in pN1-LN bestätigten PSA als geeigneten PCa-Zellmarker in LN. In pN1-LN ohne Drüsenbildung traten Zellansammlungen mit einer nach außen gerichteten CEACAM1-positiven Front und einem im Zentrum liegenden PSA-positiven Bereich auf. Diese Befunde belegen einen Zusammenhang zwischen der endothelialen CEACAM1-Expression im Sinus und der Mikrometastasierungswahrscheinlichkeit im pN0-LN-Gewebe von PCa-Patienten. Potentiell lässt sich daher im Niedrig-Risiko-PCa-Patientenkollektiv über eine CEACAM1-Bestimmung in LN das Risiko für eine Metastasierung frühzeitig erkennen.
Extracellular vesicle (EV)-mediated intercellular communication through exosomes, microvesicles (MVs) and apoptotic bodies has been shown to be implicated in various physiological as well as pathological processes such as the development and progression of atherosclerosis. While the cellular machinery controlling EV formation and composition has been studied extensively, little is known about the underlying morphological processes. This study focuses on a detailed ultrastructural analysis of the different steps of EV formation and release in Myocardial Endothelial (MyEnd) and Aortic Endothelial (AoEnd) cells cultured under serum starvation and inflammatory stimulation with TNF-α. Detailed morphological analyses were conducted applying and comparing different high- resolution light and electron microscopic methods. In this study, we could depict all steps of MV biogenesis named in literature. However, during the study of exosome biogenesis, we discovered a yet undescribed process: Instead of a direct fusion with the plasma membrane, multivesicular bodies were incorporated into a new distinct cellular compartment bound by fenestrated endothelium first. This may present a novel step in exosome biogenesis and warrants further study. Regarding the conditions of cell cultivation, we observed that the commonly used serum starvation causes MyEnd cells, but not AoEnd cells, to enter apoptosis after 48 hours. When preparing functional EV studies, we therefore recommend assessing the morphological condition of the serum-starved cells at different cultivation points first. When evaluating MV production, a statistical analysis showed that the more time AoEnd cells spent in cultivation under serum starvation, the higher the percentage of MV producing cells. However, additional TNF-α stimulation induced a significantly higher MV production than serum starvation alone. Lastly, our results show that TNF-α stimulation of AoEnd cells in vitro leads to the upregulation of CD44, an adhesion molecule critical in the early stages of atherosclerosis. CD44 was then depicted on the surface of generated MVs and exosomes. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells. This could be a novel mechanism by which MVs contribute to the development and progression of atherosclerotic disease and should be clarified by further studies.
LOX-catalyzed collagen stabilization is a proximal cause for intrinsic resistance to chemotherapy
(2018)
The potential of altering the tumor ECM to improve drug response remains fairly unexplored. To identify targets for modification of the ECM aiming to improve drug response and overcome resistance, we analyzed expression data sets from pre-treatment patient cohorts. Cross-evaluation identified a subset of chemoresistant tumors characterized by increased expression of collagens and collagen-stabilizing enzymes. We demonstrate that strong collagen expression and stabilization sets off a vicious circle of self-propagating hypoxia, malignant signaling, and aberrant angiogenesis that can be broken by an appropriate auxiliary intervention: Interfering with collagen stabilization by inhibition of lysyl oxidases significantly enhanced response to chemotherapy in various tumor models, even in metastatic disease. Inhibition of collagen stabilization by itself can reduce or enhance tumor growth depending on the tumor type. The mechanistical basis for this behavior is the dependence of the individual tumor on nutritional supply on one hand and on high tissue stiffness for FAK signaling on the other.
Fibroblast growth factor (FGF) signaling is involved in the pathogenesis of multiple sclerosis (MS). Data from neuropathology studies suggest that FGF signaling contributes to the failure of remyelination in MS. In MOG\(_{35–55}\)-induced EAE, oligodendrocyte-specific deletion of FGFR1 and FGFR2 resulted in a less severe disease course, reduced inflammation, myelin and axon degeneration and changed FGF/FGFR and BDNF/TrkB signaling. Since signaling cascades in oligodendrocytes could not be investigated in the EAE studies, we here aimed to characterize FGFR-dependent oligodendrocyte-specific signaling in vitro. FGFR inhibition was achieved by application of the multi-kinase-inhibitor dovitinib and the FGFR1/2/3-inhibitor AZD4547. Both substances are potent inhibitors of FGF signaling; they are effective in experimental tumor models and patients with malignancies. Effects of FGFR inhibition in oligodendrocytes were studied by immunofluorescence microscopy, protein and gene analyses. Application of the tyrosine kinase inhibitors reduced FGFR1, phosphorylated ERK and Akt expression, and it enhanced BDNF and TrkB expression. Furthermore, the myelin proteins CNPase and PLP were upregulated by FGFR inhibition. In summary, inhibition of FGFR signaling in oligodendrocytes can be achieved by application of tyrosine kinase inhibitors. Decreased phosphorylation of ERK and Akt is associated with an upregulation of BDNF/TrkB signaling, which may be responsible for the increased production of myelin proteins. Furthermore, these data suggest that application of FGFR inhibitors may have the potential to promote remyelination in the CNS.
Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disease of the central nervous system (CNS) affecting more than two million people worldwide. In MS, oligodendrocytes and myelin sheaths are destroyed by autoimmune-mediated inflammation, while remyelination is impaired. Recent investigations of post-mortem tissue suggest that Fibroblast growth factor (FGF) signaling may regulate inflammation and myelination in MS. FGF2 expression seems to correlate positively with macrophages/microglia and negatively with myelination; FGF1 was suggested to promote remyelination. In myelin oligodendrocyte glycoprotein (MOG)\(_{35–55}\)-induced experimental autoimmune encephalomyelitis (EAE), systemic deletion of FGF2 suggested that FGF2 may promote remyelination. Specific deletion of FGF receptors (FGFRs) in oligodendrocytes in this EAE model resulted in a decrease of lymphocyte and macrophage/microglia infiltration as well as myelin and axon degeneration. These effects were mediated by ERK/Akt phosphorylation, a brain-derived neurotrophic factor, and downregulation of inhibitors of remyelination. In the first part of this review, the most important pharmacotherapeutic principles for MS will be illustrated, and then we will review recent advances made on FGF signaling in MS. Thus, we will suggest application of FGFR inhibitors, which are currently used in Phase II and III cancer trials, as a therapeutic option to reduce inflammation and induce remyelination in EAE and eventually MS.
Cone-beam computed tomography is a powerful tool for 3D imaging of the appendicular skeleton, facilitating detailed visualization of bone microarchitecture. This study evaluated various combinations of acquisition and reconstruction parameters for the cone-beam CT mode of a twin robotic x-ray system in cadaveric wrist and elbow scans, aiming to define the best possible trade-off between image quality and radiation dose. Images were acquired with different combinations of tube voltage and tube current–time product, resulting in five scan protocols with varying volume CT dose indices: full-dose (FD; 17.4 mGy), low-dose (LD; 4.5 mGy), ultra-low-dose (ULD; 1.15 mGy), modulated low-dose (mLD; 0.6 mGy) and modulated ultra-low-dose (mULD; 0.29 mGy). Each set of projection data was reconstructed with three convolution kernels (very sharp [Ur77], sharp [Br69], intermediate [Br62]). Five radiologists subjectively assessed the image quality of cortical bone, cancellous bone and soft tissue using seven-point scales. Irrespective of the reconstruction kernel, overall image quality of every FD, LD and ULD scan was deemed suitable for diagnostic use in contrast to mLD (very sharp/sharp/intermediate: 60/55/70%) and mULD (0/3/5%). Superior depiction of cortical and cancellous bone was achieved in FD\(_{Ur77}\) and LD\(_{Ur77}\) examinations (p < 0.001) with LD\(_{Ur77}\) scans also providing favorable bone visualization compared to FD\(_{Br69}\) and FD\(_{Br62}\) (p < 0.001). Fleiss’ kappa was 0.618 (0.594–0.641; p < 0.001), indicating substantial interrater reliability. In this study, we demonstrate that considerable dose reduction can be realized while maintaining diagnostic image quality in upper extremity joint scans with the cone-beam CT mode of a twin robotic x-ray system. Application of sharper convolution kernels for image reconstruction facilitates superior display of bone microarchitecture.
Hintergrund - Die Multiple Sklerose (MS) ist bis heute eine nur teilweise verstandene Autoimmunerkrankung des zentralen Nervensystems (ZNS). Das Tiermodell der experimentellen autoimmunen Enzephalomyelitis (EAE) ermöglicht die Erforschung von Teilaspekten der Pathogenese der MS und kann zur Etablierung von Therapeutika herangezogen werden. Die MP4-abhängige EAE ermöglicht als Mausmodell die gezielte Erforschung der Rolle der B-Zelle als Akteur in der Pathogenese der MS. Diese Dissertation untersuchte den Effekt des S1P1-Rezeptor-Modulators FTY720 (Fingolimod) auf die Immunantwort der autoreaktiven B-Zellen in der Peripherie sowie im ZNS.
Methoden - MP4-immunisierte Mäuse erhielten 50 Tage nach dem EAE-Krankheitsbeginn oral appliziertes FTY720 über einen Zeitraum von 30 Tagen. Die Tiere wurden nach dem Auftreten der Krankheitssymptome täglich klinisch evaluiert. Die MP4-spezifische B-Zell-Immunantwort und die MP4-spezifische humorale Immunreaktion wurden mittels ELISPOT und ELISA ausgewertet. Die Verteilung der T- und B-Zell-Anteile im peripheren Blut der Mäuse sowie die Aufteilung der B-Zell-Subsets in der Milz wurden mittels Durchflusszytometrie quantifiziert. Mittels Immunhistochemie wurden die B- und T-Zell-Ansammlungen im ZNS der Mäuse hinsichtlich ihrer Entwicklung in tertiär lymphatische Organe (TLOs) untersucht.
Ergebnisse - In diesem Versuchsaufbau zeigte FTY720 keine signifikante Verbesserung des klinischen Krankheitsverlaufes der Tiere. Der Anteil von T-Zellen im peripheren Blut der Mäuse war unter der Therapie mit FTY720 signifikant reduziert, während die Anzahl an B-Zellen nicht-signifikant beeinflusst wurde. Bei der Untersuchung der B-Zell-Subtypen in der Milz fiel zunächst ein erhöhter Anteil an B220+-B-Zellen auf, während die Verteilung der weiteren Subsets nicht-signifikant verändert war. Unter der Therapie mit FTY720 zeigte sich keine Reduktion bereits etablierter B-Zell-Aggregate im ZNS, allerdings ist eine inhibierte Entwicklung in TLOs zu diskutieren.
Zusammenfassung - Diese Arbeit impliziert unterschiedliche Effekte von FTY720 auf die B-Zellen in einem B-Zell-abhängigen chronischen Mausmodell der MS.
Bei Patienten, die an einer speziellen Form der MS erkrankten, konnten Entzündungsinfiltrate in den Meningen nachgewiesen werden, die in ihrem Aufbau lymphoidem Gewebe ähnelten. Das Auftreten dieser Infiltrate war mit einem schwereren Krankheitsverlauf assoziiert. Das Mausmodell der B-Zell-abhängigen MP4-induzierten experimentellen autoimmunen Enzephalomyelitis (EAE) zeigt in den Kleinhirnen der Mäuse Infiltrate, die den Infiltraten beim Menschen ähneln.
Wir nutzten die MP4-induzierte EAE, um die Mechanismen der Erkrankungsentstehung und Progression besser zu verstehen. Ziel dieser Arbeit war es intakte und stabile Ribonukleinsäuren (RNA) aus den Infiltraten der Mäuse zu isolieren. Sowie Identifizierung von Gene, die während verschiedener Stadien der zerebralen Entzündungsreaktion hochreguliert waren.
Wir verglichen die Möglichkeiten der RNA-Isolation bei Paraffin-eingebettetem Gewebe und kryofixiertem Gewebe. Um die Vergleichbarkeit der Qualitäts- und Quantitätsanalyse zu gewährleisten, wurde für jede Probe eine RNA Integritätsnummer (RIN) ermittelt.
Wir führten eine Laser Capture Microdissection (LCM) und anschließende Gensequenzierung der zerebralen Infiltrate bei Mäusen durch, bei denen eine MP4-abhängige EAE ausgelöst wurde. Des Weiteren verglichen wir die Ergebnisse mit den Expressionsprofilen von sekundär lymphatischen Organen (SLOs).
Insgesamt konnten wir 43 Gene herausfiltern, die im Vergleich zu den Kontrollgruppen hochreguliert waren.
Die Entwicklung von ektopen lymphatischen Strukturen (ELS) im zentralen Nervensystem (ZNS) ist ein komplexer und bisher wenig verstandener Prozess. In dieser Studie beobachteten wir 43 Gene, die während der Entwicklung von B- Zell-Infiltraten in den Kleinhirnen von MP4-immunisierten Mäusen signifikant hochreguliert waren. Von diesen Genen wurden bereits 14 im Zusammenhang mit der MS erwähnt und sind zum Teil Gegenstand aktiver Forschung.
Operative treatment of ruptured pubic symphysis by plating is often accompanied by complications. Trans-obturator cable fixation might be a more reliable technique; however, have not yet been tested for stabilization of ruptured pubic symphysis. This study compares symphyseal trans-obturator cable fixation versus plating through biomechanical testing and evaluates safety in a cadaver experiment. APC type II injuries were generated in synthetic pelvic models and subsequently separated into three different groups. The anterior pelvic ring was fixed using a four-hole steel plate in Group A, a stainless steel cable in Group B, and a titan band in Group C. Biomechanical testing was conducted by a single-leg-stance model using a material testing machine under physiological load levels. A cadaver study was carried out to analyze the trans-obturator surgical approach. Peak-to-peak displacement, total displacement, plastic deformation and stiffness revealed a tendency for higher stability for trans-obturator cable/band fixation but no statistical difference to plating was detected. The cadaver study revealed a safe zone for cable passage with sufficient distance to the obturator canal. Trans-obturator cable fixation has the potential to become an alternative for symphyseal fixation with less complications.
Salivary gland tumors are a rare tumor entity within malignant tumors of all tissues. The most common are malignant mucoepidermoid carcinoma, adenoid cystic carcinoma, and acinic cell carcinoma. Pleomorphic adenoma is the most recurrent form of benign salivary gland tumor. Due to their low incidence rates and complex histological patterns, they are difficult to diagnose accurately. Malignant tumors of the salivary glands are challenging in terms of differentiation because of their variability in histochemistry and translocations. Therefore, the primary goal of the study was to review the current literature to identify the recent developments in histochemical diagnostics and translocations for differentiating salivary gland tumors.
Salivary gland cancers are rare but aggressive tumors that have poor prognosis and lack effective cure. Of those, parotid tumors constitute the majority. Functioning as metabolic machinery contributing to cellular redox balance, peroxisomes have emerged as crucial players in tumorigenesis. Studies on murine and human cells have examined the role of peroxisomes in carcinogenesis with conflicting results. These studies either examined the consequences of altered peroxisomal proliferators or compared their expression in healthy and neoplastic tissues. None, however, examined such differences exclusively in human parotid tissue or extended comparison to peroxisomal proteins and their associated gene expressions. Therefore, we examined differences in peroxisomal dynamics in parotid tumors of different morphologies. Using immunofluorescence and quantitative PCR, we compared the expression levels of key peroxisomal enzymes and proliferators in healthy and neoplastic parotid tissue samples. Three parotid tumor subtypes were examined: pleomorphic adenoma, mucoepidermoid carcinoma and acinic cell carcinoma. We observed higher expression of peroxisomal matrix proteins in neoplastic samples with exceptional down regulation of certain enzymes; however, the degree of expression varied between tumor subtypes. Our findings confirm previous experimental results on other organ tissues and suggest peroxisomes as possible therapeutic targets or markers in all or certain subtypes of parotid neoplasms.
Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-β regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-β, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-β, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.
Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes.
SARS-CoV-2 infection can cause fatal inflammatory lung pathology, including thrombosis and increased pulmonary vascular permeability leading to edema and hemorrhage. In addition to the lung, cytokine storm-induced inflammatory cascade also affects other organs. SARS-CoV-2 infection-related vascular inflammation is characterized by endotheliopathy in the lung and other organs. Whether SARS-CoV-2 causes endotheliopathy by directly infecting endothelial cells is not known and is the focus of the present study. We observed 1) the co-localization of SARS-CoV-2 with the endothelial cell marker CD31 in the lungs of SARS-CoV-2-infected mice expressing hACE2 in the lung by intranasal delivery of adenovirus 5-hACE2 (Ad5-hACE2 mice) and non-human primates at both the protein and RNA levels, and 2) SARS-CoV-2 proteins in endothelial cells by immunogold labeling and electron microscopic analysis. We also detected the co-localization of SARS-CoV-2 with CD31 in autopsied lung tissue obtained from patients who died from severe COVID-19. Comparative analysis of RNA sequencing data of the lungs of infected Ad5-hACE2 and Ad5-empty (control) mice revealed upregulated KRAS signaling pathway, a well-known pathway for cellular activation and dysfunction. Further, we showed that SARS-CoV-2 directly infects mature mouse aortic endothelial cells (AoECs) that were activated by performing an aortic sprouting assay prior to exposure to SARS-CoV-2. This was demonstrated by co-localization of SARS-CoV-2 and CD34 by immunostaining and detection of viral particles in electron microscopic studies. Moreover, the activated AoECs became positive for ACE-2 but not quiescent AoECs. Together, our results indicate that in addition to pneumocytes, SARS-CoV-2 also directly infects mature vascular endothelial cells in vivo and ex vivo, which may contribute to cardiovascular complications in SARS-CoV-2 infection, including multipleorgan failure.
Ongoing research to fight cancer, one of the dominant diseases of the 21st century has led to big progress especially when it comes to understanding the tumor growth and metastasis. This includes the discovery of the molecular mechanisms of tumor vascularization, which is critically required for establishment of tumor metastasis.
Formation of new blood vessels is the first step in tumor vascularization. Therefore, understanding the molecular and cellular basis of tumor vascularization attracted a significant effort studying in biomedical research. The blood vessels for supplying tumor can be formed by sprouting from pre-existing vessels, a process called angiogenesis, or by vasculogenesis, that is de novo formation of blood vessels from not fully differentiated progenitor cell populations. Vasculogenic endothelial progenitor cells (EPCs) can either be activated from populations in the bone marrow reaching the pathological region via the circulation or they can be recruited from local reservoirs. Neovessel formation influences tumor progression, hence therapeutic response model systems of angiogenesis/vasculogenesis are necessary to study the underlying mechanisms. Although, initially the research in this area focused more on angiogenesis, it is now well understood that both angiogenesis and postnatal vasculogenesis contribute to neovessel formation in adult under both most pathological as well as physiological conditions. Studies in the last two decades demonstrate that in addition to the intimal layer of fully differentiated mature endothelial cells (ECs) and various smaller supplying vessels (vasa vasorum) that can serve as a source for new vessels by angiogenesis, especially the adventitia of large and medium size blood vessels harbors various vascular wall-resident stem and progenitor cells (VW-SPCs) populations that serve as a source for new vessels by postnatal vasculogenesis. However, little is known about the potential role of VW-SPCs in tumor vascularization.
To this end, the present work started first to establish a modified aortic ring assay (ARA) using mouse aorta in order to study the contribution of vascular adventitia-resident VW-SPCs to neovascularization in general and in presence of tumor cells. ARA is already established an ex vivo model for neovascularization allows to study the morphogenetic events of complex new vessel formation that includes all layers of mature blood vessels, a significant advantage over the assays that employ monolayer endothelial cell cultures. Moreover, in contrast to assays employing endothelial cells monocultures, both angiogenic and vasculogenic events take place during new vessel formation in ARA although the exact contribution of these two processes to new vessel formation cannot be easily distinguished in conventional ARA. Thus, in this study, a modified protocol for the ARA (mdARA) was established by either removing or keeping the aortic adventitia in place. The mdARA allows to distinguish the role of VW-SPCs from those of other aortic layers. The present data show that angiogenic sprouting from mature aortic endothelium was markedly delayed when the adventitial layer was removed. Furthermore, the network between the capillary-like sprouts was significantly reduced in absence of aortic adventitia. Moreover, the stabilization of new sprouts by assembling the NG2+ pericyte-like cells that enwrapped the endothelial sprouts from the outside was improved when the adventitial layer remained in place.
Next, mimicking the tumor-vessel adventitia-interaction, multicellular tumor spheroids (MCTS) and aortic rings (ARs) with or without adventitia of C57BL/6-Tg (UBC-GFP) mice were confronted within the collagen gel and cultured ex vivo. This 3D model enabled analysis of the mobilization, migration and capillary-like sprouts formation by VW-SPCs within tumor-vessel wall-interface in comparison to tumor-free side of the ARs. Interestingly, while MCTS preferred the uptake of single vascular adventitia-derived cells, neural spheroids were directly penetrated by capillary-like structures that were sprouted from the aortic adventitia. In summary, the model established in this work allows to study new vessel formation by both postnatal vasculogenesis and angiogenesis under same conditions. It can be applied in various mouse models including reporter mouse models, e.g. Cxcr1 CreER+/mTmG+/- mice, in which GFP-marked macrophages of the vessel wall were directly observed as they mobilized from their niche and migrated into collagen gel. Another benefit of the model is that it can be used for testing different factors such as small molecules, growth factors, cytokines, and drugs with both pro- and anti-angiogenic/vasculogenic effects.
Chronic obstructive pulmonary disease (COPD) is a major cause of morbidity and mortality worldwide and encompasses chronic bronchitis and emphysema. It has been shown that vascular wall remodeling and pulmonary hypertension (PH) can occur not only in patients with COPD but also in smokers with normal lung function, suggesting a causal role for vascular alterations in the development of emphysema. Mechanistically, abnormalities in the vasculature, such as inflammation, endothelial dysfunction, imbalances in cellular apoptosis/proliferation, and increased oxidative/nitrosative stress promote development of PH, cor pulmonale, and most probably pulmonary emphysema. Hypoxemia in the pulmonary chamber modulates the activation of key transcription factors and signaling cascades, which propagates inflammation and infiltration of neutrophils, resulting in vascular remodeling. Endothelial progenitor cells have angiogenesis capabilities, resulting in transdifferentiation of the smooth muscle cells via aberrant activation of several cytokines, growth factors, and chemokines. The vascular endothelium influences the balance between vaso-constriction and -dilation in the heart. Targeting key players affecting the vasculature might help in the development of new treatment strategies for both PH and COPD. The present review aims to summarize current knowledge about vascular alterations and production of reactive oxygen species in COPD. The present review emphasizes on the importance of the vasculature for the usually parenchyma-focused view of the pathobiology of COPD.
Post-fabrication formation of a proper vasculature remains an unresolved challenge in bioprinting. Established strategies focus on the supply of the fabricated structure with nutrients and oxygen and either rely on the mere formation of a channel system using fugitive inks or additionally use mature endothelial cells and/or peri-endothelial cells such as smooth muscle cells for the formation of blood vessels in vitro. Functional vessels, however, exhibit a hierarchical organization and multilayered wall structure that is important for their function. Human induced pluripotent stem cell-derived mesodermal progenitor cells (hiMPCs) have been shown to possess the capacity to form blood vessels in vitro, but have so far not been assessed for their applicability in bioprinting processes. Here, we demonstrate that hiMPCs, after formulation into an alginate/collagen type I bioink and subsequent extrusion, retain their ability to give rise to the formation of complex vessels that display a hierarchical network in a process that mimics the embryonic steps of vessel formation during vasculogenesis. Histological evaluations at different time points of extrusion revealed the initial formation of spheres, followed by lumen formation and further structural maturation as evidenced by building a multilayered vessel wall and a vascular network. These findings are supported by immunostainings for endothelial and peri-endothelial cell markers as well as electron microscopic analyses at the ultrastructural level. Moreover, endothelial cells in capillary-like vessel structures deposited a basement membrane-like matrix at the basal side between the vessel wall and the alginate-collagen matrix. After transplantation of the printed constructs into the chicken chorioallantoic membrane (CAM) the printed vessels connected to the CAM blood vessels and get perfused in vivo. These results evidence the applicability and great potential of hiMPCs for the bioprinting of vascular structures mimicking the basic morphogenetic steps of de novo vessel formation during embryogenesis.
For protection from inhaled pathogens many strategies have evolved in the airways such as mucociliary clearance and cough. We have previously shown that protective respiratory reflexes to locally released bacterial bitter “taste” substances are most probably initiated by tracheal brush cells (BC). Our single‐cell RNA‐seq analysis of murine BC revealed high expression levels of cholinergic and bitter taste signaling transcripts (Tas2r108, Gnat3, Trpm5). We directly demonstrate the secretion of acetylcholine (ACh) from BC upon stimulation with the Tas2R agonist denatonium. Inhibition of the taste transduction cascade abolished the increase in [Ca\(^{2+}\)]\(_{i}\) in BC and subsequent ACh‐release. ACh‐release is regulated in an autocrine manner. While the muscarinic ACh‐receptors M3R and M1R are activating, M2R is inhibitory. Paracrine effects of ACh released in response to denatonium included increased [Ca\(^{2+}\)]\(_{i}\) in ciliated cells. Stimulation by denatonium or with Pseudomonas quinolone signaling molecules led to an increase in mucociliary clearance in explanted tracheae that was Trpm5‐ and M3R‐mediated. We show that ACh‐release from BC via the bitter taste cascade leads to immediate paracrine protective responses that can be boosted in an autocrine manner. This mechanism represents the initial step for the activation of innate immune responses against pathogens in the airways.
Ischemic insults to the heart and brain, i.e., myocardial and cerebral infarction, respectively, are amongst the leading causes of death worldwide. While there are therapeutic options to allow reperfusion of ischemic myocardial and brain tissue by reopening obstructed vessels, mitigating primary tissue damage, post-infarction inflammation and tissue remodeling can lead to secondary tissue damage. Similarly, ischemia in retinal tissue is the driving force in the progression of neovascular eye diseases such as diabetic retinopathy (DR) and age-related macular degeneration (AMD), which eventually lead to functional blindness, if left untreated. Intriguingly, the easily observable retinal blood vessels can be used as a window to the heart and brain to allow judgement of microvascular damages in diseases such as diabetes or hypertension. The complex neuronal and endocrine interactions between heart, retina and brain have also been appreciated in myocardial infarction, ischemic stroke, and retinal diseases. To describe the intimate relationship between the individual tissues, we use the terms heart-brain and brain-retina axis in this review and focus on the role of transforming growth factor β (TGFβ) and neurotrophins in regulation of these axes under physiologic and pathologic conditions. Moreover, we particularly discuss their roles in inflammation and repair following ischemic/neovascular insults. As there is evidence that TGFβ signaling has the potential to regulate expression of neurotrophins, it is tempting to speculate, and is discussed here, that cross-talk between TGFβ and neurotrophin signaling protects cells from harmful and/or damaging events in the heart, retina, and brain.
Fibroblast growth factors (FGFs) and their receptors (FGFRs) are involved in demyelinating pathologies including multiple sclerosis (MS). In our recent study, oligodendrocyte‐specific deletion of FGFR1 resulted in a milder disease course, less inflammation, reduced myelin and axon damage in EAE. The objective of this study was to elucidate the role of oligodendroglial FGFR2 in MOG\(_{35-55}\)‐induced EAE. Oligodendrocyte‐specific knockout of FGFR2 (Fgfr2\(^{ind-/-}\)) was achieved by application of tamoxifen; EAE was induced using the MOG\(_{35-55}\) peptide. EAE symptoms were monitored over 62 days. Spinal cord tissue was analysed by histology, immunohistochemistry and western blot. Fgfr2\(^{ind-/-}\) mice revealed a milder disease course, less myelin damage and enhanced axonal density. The number of oligodendrocytes was not affected in demyelinated areas. However, protein expression of FGFR2, FGF2 and FGF9 was downregulated in Fgfr2\(^{ind-/-}\) mice. FGF/FGFR dependent signalling proteins were differentially regulated; pAkt was upregulated and pERK was downregulated in Fgfr2\(^{ind-/-}\) mice. The number of CD3(+) T cells, Mac3(+) cells and B220(+) B cells was less in demyelinated lesions of Fgfr2\(^{ind-/-}\) mice. Furthermore, expression of IL‐1β, TNF‐α and CD200 was less in Fgfr2\(^{ind-/-}\) mice than controls. Fgfr2ind−/− mice showed an upregulation of PLP and downregulation of the remyelination inhibitors SEMA3A and TGF‐β expression. These data suggest that cell‐specific deletion of FGFR2 in oligodendrocytes has anti‐inflammatory and neuroprotective effects accompanied by changes in FGF/FGFR dependent signalling, inflammatory cytokines and expression of remyelination inhibitors. Thus, FGFRs in oligodendrocytes may represent potential targets for the treatment of inflammatory and demyelinating diseases including MS.
Although the bone marrow contains most hematopoietic activity during adulthood, hematopoietic stem and progenitor cells can be recovered from various extramedullary sites. Cells with hematopoietic progenitor properties have even been reported in the adult brain under steady‐state conditions, but their nature and localization remain insufficiently defined. Here, we describe a heterogeneous population of myeloid progenitors in the leptomeninges of adult C57BL/6 mice. This cell pool included common myeloid, granulocyte/macrophage, and megakaryocyte/erythrocyte progenitors. Accordingly, it gave rise to all major myelo‐erythroid lineages in clonogenic culture assays. Brain‐associated progenitors persisted after tissue perfusion and were partially inaccessible to intravenous antibodies, suggesting their localization behind continuous blood vessel endothelium such as the blood‐arachnoid barrier. Flt3\(^{Cre}\) lineage tracing and bone marrow transplantation showed that the precursors were derived from adult hematopoietic stem cells and were most likely continuously replaced via cell trafficking. Importantly, their occurrence was tied to the immunologic state of the central nervous system (CNS) and was diminished in the context of neuroinflammation and ischemic stroke. Our findings confirm the presence of myeloid progenitors at the meningeal border of the brain and lay the foundation to unravel their possible functions in CNS surveillance and local immune cell production.
Die Identifizierung endogener Stammzellen mit kardiogenem Potenzial und die Möglichkeit, deren Differenzierung zu steuern, würde einen Meilenstein in der kardioregenerativen Therapie darstellen. Innerhalb der Gefäßwand konnten unterschiedliche Stamm- und Vorläuferzellen identifiziert werden, die sog. Gefäßwand-residenten Stammzellen (VW-SCs). Zuletzt konnten aus CD34(+) VW-SCs, ohne genetische Manipulation, Kardiomyozyten generiert werden. Zusätzlich fungiert die Gefäßwand als Quelle inflammatorischer Zellen, die essenziell für die kardiogene Differenzierung der VW-SCs zu sein scheinen.
Ziel dieser Arbeit war es, das Verhalten von CD44(+) VW-SCs zu untersuchen, um herauszufinden, inwieweit dieser Stammzelltyp eine endogene Generierung von Kardiomyozyten unterstützen könnte. Dabei wurde mit infarzierten Mäuseherzen, dem Aortenringassay (ARA) und dem kardialen Angiogeneseassay (CAA) gearbeitet.
Sowohl in vivo in ischämischen Arealen infarzierter Mäuseherzen als auch ex vivo im CAA kam es zu einem signifikanten Anstieg von CD44(+) Zellen. Mittels Färbungen auf CD44 und Ki-67 konnte die Teilungsfähigkeit dieser Zellen demonstriert werden.
Ex vivo ließen sich aus CD44(+) Zellen F4/80(+) Makrophagen generieren. Die CD44(+) VW-SCs können sich dabei sowohl zu pro-inflammatorischen iNOS(+) M1- als auch zu anti-inflammatorischen IL-10(+) M2-Makrophagen differenzieren. Eine Modulation der kardialen Inflammation könnte einen entscheidenden Einfluss auf die Kardiomyogenese haben.
Unter VEGF-A kam es im CAA zu einer deutlichen Zunahme von CD44(+) Zellen. Unter Lenvatinib blieb das kardiale Sprouting gänzlich aus, die Anzahl der CD44(+) Zellen stagnierte und die VW-SCs verblieben in ihren physiologischen Nischen innerhalb der Gefäßwand.
Warum es nach einem MI kaum zu einer funktionellen Herzmuskelregeneration kommt, ist weiterhin unklar. Die therapeutische Beeinflussung koronaradventitieller CD44(+) VW-SCs und inflammatorischer Prozesse könnte dabei zukünftig eine wichtige therapeutische Option darstellen.
Effects of dopamine on BDNF / TrkB mediated signaling and plasticity on cortico-striatal synapses
(2021)
Progressive loss of voluntary movement control is the central symptom of Parkinson's disease (PD). Even today, we are not yet able to cure PD. This is mainly due to a lack of understanding the mechanisms of movement control, network activity and plasticity in motor circuits, in particular between the cerebral cortex and the striatum. Brain-derived neurotrophic factor (BDNF) has emerged as one of the most important factors for the development and survival of neurons, as well as for synaptic plasticity. It is thus an important target for the development of new therapeutic strategies against neurodegenerative diseases. Together with its receptor, the Tropomyosin receptor kinase B (TrkB), it is critically involved in development and function of the striatum. Nevertheless, little is known about the localization of BDNF within presynaptic terminals in the striatum, as well as the types of neurons that produce BDNF in the cerebral cortex. Furthermore, the influence of midbrain derived dopamine on the control of BDNF / TrkB interaction in striatal medium spiny neurons (MSNs) remains elusive so far. Dopamine, however, appears to play an important role, as its absence leads to drastic changes in striatal synaptic plasticity. This suggests that dopamine could regulate synaptic activity in the striatum via modulation of BDNF / TrkB function. To answer these questions, we have developed a sensitive and reliable protocol for the immunohistochemical detection of endogenous BDNF. We find that the majority of striatal BDNF is provided by glutamatergic, cortex derived afferents and not dopaminergic inputs from the midbrain. In fact, we found BDNF in cell bodies of neurons in layers II-III and V of the primary and secondary motor cortex as well as layer V of the somatosensory cortex. These are the brain areas that send dense projections to the dorsolateral striatum for control of voluntary movement. Furthermore, we could show that these projection neurons significantly downregulate the expression of BDNF during the juvenile development of mice between 3 and 12 weeks.
In parallel, we found a modulatory effect of dopamine on the translocation of TrkB to the cell surface in postsynaptic striatal Medium Spiny Neurons (MSNs). In MSNs of the direct pathway (dMSNs), which express dopamine receptor 1 (DRD1), we observed the formation of TrkB aggregates in the 6-hydroxydopamine (6-OHDA) model of PD. This suggests that DRD1 activity controls TrkB surface expression in these neurons. In contrast, we found that DRD2 activation has opposite effects in MSNs of the indirect pathway (iMSNs). Activation of DRD2 promotes a rapid decrease in TrkB surface expression which was reversible and depended on cAMP. In parallel, stimulation of DRD2 led to induction of phospho-TrkB (pTrkB). This effect was significantly slower than the effect on TrkB surface expression and indicates that TrkB is transactivated by DRD2. Together, our data provide evidence that dopamine triggers dual modes of plasticity on striatal MSNs by acting on TrkB surface expression in DRD1 and DRD2 expressing MSNs. This surface expression of the receptor is crucial for the binding of BDNF, which is released from corticostriatal afferents. This leads to the induction of TrkB-mediated downstream signal transduction cascades and long-term potentiation (LTP). Therefore, the dopamine-mediated translocation of TrkB could be a mediator that modulates the balance between dopaminergic and glutamatergic signaling to allow synaptic plasticity in a spatiotemporal manner. This information and the fact that TrkB is segregated to persistent aggregates in PD could help to improve our understanding of voluntary movement control and to develop new therapeutic strategies beyond those focusing on dopaminergic supply.
Biofabrication, including printing technologies, has emerged as a powerful approach to the design of disease models, such as in cancer research. In breast cancer, adipose tissue has been acknowledged as an important part of the tumor microenvironment favoring tumor progression. Therefore, in this study, a 3D-printed breast cancer model for facilitating investigations into cancer cell-adipocyte interaction was developed. First, we focused on the printability of human adipose-derived stromal cell (ASC) spheroids in an extrusion-based bioprinting setup and the adipogenic differentiation within printed spheroids into adipose microtissues. The printing process was optimized in terms of spheroid viability and homogeneous spheroid distribution in a hyaluronic acid-based bioink. Adipogenic differentiation after printing was demonstrated by lipid accumulation, expression of adipogenic marker genes, and an adipogenic ECM profile. Subsequently, a breast cancer cell (MDA-MB-231) compartment was printed onto the adipose tissue constructs. After nine days of co-culture, we observed a cancer cell-induced reduction of the lipid content and a remodeling of the ECM within the adipose tissues, with increased fibronectin, collagen I and collagen VI expression. Together, our data demonstrate that 3D-printed breast cancer-adipose tissue models can recapitulate important aspects of the complex cell–cell and cell–matrix interplay within the tumor-stroma microenvironment
Megakaryocytes (MKs) release platelets into the lumen of bone marrow (BM) sinusoids while remaining to reside within the BM. The morphogenetic events of this complex process are still not fully understood. We combined confocal laser scanning microscopy with transmission and serial block-face scanning electron microscopy followed by 3D-reconstruction on mouse BM tissue sections. These analyses revealed that MKs in close vicinity to BM sinusoid (BMS) wall first induce the lateral retraction of CXCL12-abundant reticular (CAR) cells (CAR), followed by basal lamina (BL) degradation enabling direct MK-sinusoidal endothelial cells (SECs) interaction. Subsequently, an endothelial engulfment starts that contains a large MK protrusion. Then, MK protrusions penetrate the SEC, transmigrate into the BMS lumen and form proplatelets that are in direct contact to the SEC surface. Furthermore, such processes are induced on several sites, as observed by 3D reconstructions. Our data demonstrate that MKs in interaction with CAR-cells actively induce BMS wall alterations, including CAR-cell retraction, BL degradation, and SEC engulfment containing a large MK protrusion. This results in SEC penetration enabling the migration of MK protrusion into the BMS lumen where proplatelets that are adherent to the luminal SEC surface are formed and contribute to platelet release into the blood circulation.
Contribution of adventitia-derived stem and progenitor cells to new vessel formation in tumors
(2021)
Blocking tumor vascularization has not yet come to fruition to the extent it was hoped for, as angiogenesis inhibitors have shown only partial success in the clinic. We hypothesized that under- appreciated vascular wall-resident stem and progenitor cells (VW-SPCs) might be involved in tumor vascularization and influence effectiveness of anti-angiogenic therapy. Indeed, in patient samples, we observed that vascular adventitia-resident CD34\(^+\) VW-SPCs are recruited to tumors in situ from co-opted vessels. To elucidate this in detail, we established an ex vivo model using concomitant embedding of multi-cellular tumor spheroids (MCTS) and mouse aortic rings (ARs) into collagen
gels, similar to the so-called aortic ring assay (ARA). Moreover, ARA was modified by removing the ARs’ adventitia that harbors VW-SPCs. Thus, this model enabled distinguishing the contribution of VW-SPCs from that of mature endothelial cells (ECs) to new vessel formation. Our results show that the formation of capillary-like sprouts is considerably delayed, and their number and network formation were significantly reduced by removing the adventitia. Substituting iPSC-derived neural spheroids for MCTS resulted in distinct sprouting patterns that were also strongly influenced by the
presence or absence of VW-SPCs, also underlying the involvement of these cells in non-pathological vascularization. Our data suggest that more comprehensive approaches are needed in order to block all of the mechanisms contributing to tumor vascularization.
Die Arten des Schweizer Ranunculus-auricomus-Komplexes sind nur zu einem Teil bekannt. Zur vollständigeren Erfassung des Komplexes wurden Exkursionen in die südwestliche und östliche Schweiz unternommen. Es wurden sieben neue Arten entdeckt, die hier beschrieben und abgebildet sind. Ihre Taxonomie und Gefährdung wird diskutiert. R. chalarocarpus W. Koch ex Dunkel ist bereits bei Koch provisorisch erwähnt, R. clavicornis Dunkel wird nun gültig beschrieben. Beide Arten sind aufgrund ihres Vorkommens in Auwäldern und feuchten Laubwäldern stark gefährdet, R. clavicornis sogar fast ausgestorben. Der neu beschriebene R. thurgoviae kommt im Osten der Schweiz vor (Kanton Thurgau). Die bislang bekannte Verbreitung von R. allobrogorum Dunkel, R. crenulatus Dunkel, R. genevensis Dunkel und R. lineatus ist fast vollständig auf den Kanton Genf beschränkt. Die Arten des Ranunculus auricomus-Komplexes sind ein sensibler Indikator für Veränderungen der Vegetation und Umwelt und sollten diesbezüglich deutlich mehr Gewicht bekommen.
Bis zum Jahr 2100 prognostiziert der Weltklimarat (IPCC 2021) einen Anstieg des Meeresspiegels von bis zu 63-101 cm gegenüber heutigen Wasserständen. Im Rahmen des Generalplans Küstenschutz Schleswig-Holstein(GKSH) soll als Klimafolgeanpassung eine Erhöhung und Profiländerung der meisten Nordseedeiche und Elbedeiche erfolgen (zusammen 363,3 km mit einer Vegetationsfläche von 3.500 ha). Diese Maßnahmen werden mit einem vollständigen Verlust der alten Deichvegetation einhergehen und zur Freisetzung von großen Mengen an CO₂ aus dem Bodenkohlenstoff führen. Die Seedeiche der Nordseeküste (262 km) zählen zu den artenreichen, semi-natürlichen und von Schafen beweideten Grasländern (Fläche von 2600 ha) in Schleswig-Holstein mit bis zu 18 Gras- und 64 zweikeim-blättrigen Blütenpflanzen und an die Vegetation gebundene 800-1000 Arten von Invertebraten (darunter 200 Käferarten). Auf die Außenböschung dringen Pflanzen der Salzwiesengesellschaften vor. Die steileren, wärmeexponierten (überwiegend nach Osten und Süden ausgerichtet) und durch Vertritt lückigen Innenböschungen der Seedeiche sind wertvolle Refugien wärmeliebender, konkurrenzschwacher Arten von Magerstandorten und Trittgesellschaften wie die folgenden mediterran-subatlantischen Arten: Knotenklettenkerbel (Torilis nodosa), Zwergklee/Armblütiger Klee (Trifolium micranthum) und Vogelfußklee (Trifolium ornithopodioides). Für die Erhaltung beider Kleearten (die aktuelle Verbreitung wird dokumentiert) besitzt Schleswig-Holstein eine nationale und nordwest-europäisch-kontinentale Verantwortlichkeit. Folgende Maßnahmen zum Schutz der reichhaltigen Deichvegetation und Teilen seiner Invertebratenfauna bei der Deichverstärkung im Rahmen des GKSH werden vorgeschlagen: 1. Abheben der Grasnarbe mit Wurzelraum und zeitnahe Wiederverlegung der alten Grasnarbe (Soden) auf das neue Deichprofil; das ist auch wichtig zum Erhalt des Bodenkohlenstoffs (Klimaschutz). 2. Einsaat von neuen Deichprofilen mit Saatgut von artenreichen Deichabschnitten. 3. Aufnahme substanzieller Forschungsprogramme/Forschungsförderung zur Ökologie der Seedeiche. Weiterhin sollte auf den Einsatz von Herbiziden auf Deichen zur Bekämpfung von Disteln verzichtet werden.
3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.
Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44—a marker of acinar cells—diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.
Die Rolle von Connexinen und Gap Junction-vermittelter Kommunikation in pluripotenten Stammzellen sowie der frühen Embryonalentwicklung sind bis heute nicht vollständig aufgeklärt. Mutationen in humanen Connexinen verursachen eine Vielzahl von Krankheiten. Connexin-defiziente iPS Zellen stellen eine gute Basis für die Erforschung der Rolle von Connexinen während der Embryonalentwicklung und bei der Krankheitsentstehung dar.
Das Ziel der vorliegenden Arbeit war es, das CRISPR/Cas9-System in pluripotenten Stammzellen erfolgreich anzuwenden und ein Protokoll zur Erstellung verschiedener Cx43-Defektmutanten zu entwerfen. Nach der Etablierung der CRSIPR/Cas9-Methode in HEK293T-Zellen konnte in der vorliegenden Arbeit darüber hinaus erfolgreich eine Cx43-Defizienz in FSiPS-Zellen erzeugt werden. Weiterhin wurden mehrere Cx43-Mutanten geschaffen und initial auf Pluripotenzmarker und ihr Differenzierungspotential untersucht.
Diese Arbeit bildet die Basis für weitere Untersuchungen des Cx43 in iPS-Zellklonen und davon abgeleiteten Zelltypen sowie artifiziellen 3D-Gewebekulturen. Darüber hinaus bildet sie die Grundlage für die Bildung weiterer Connexin-Defektmutanten sowie von iPS-Zellen mit krankheitsrelevanten Mutationen.
Energy demand of neurons in brain that is covered by glucose supply from the blood is ensured by glucose transporters incapillaries and brain cells. In brain, the facilitative diffusion glucose transporters GLUT1-6 and GLUT8, and the Na+-D-glucosecotransporters SGLT1 are expressed. The glucose transporters mediate uptake of D-glucose across the blood-brain barrier anddelivery of D-glucose to astrocytes and neurons. They are critically involved in regulatory adaptations to varying energy demandsin response to differing neuronal activities and glucose supply. In this review, a comprehensive overview about verified andproposed roles of cerebral glucose transporters during health and diseases is presented. Our current knowledge is mainly based onexperiments performed in rodents. First, the functional properties of human glucose transporters expressed in brain and theircerebral locations are described. Thereafter, proposed physiological functions of GLUT1, GLUT2, GLUT3, GLUT4, andSGLT1 for energy supply to neurons, glucose sensing, central regulation of glucohomeostasis, and feeding behavior are compiled, and their roles in learning and memory formation are discussed. In addition, diseases are described in which functionalchanges of cerebral glucose transporters are relevant. These are GLUT1 deficiency syndrome (GLUT1-SD), diabetes mellitus, Alzheimer’s disease (AD), stroke, and traumatic brain injury (TBI). GLUT1-SD is caused by defect mutations in GLUT1. Diabetes and AD are associated with changed expression of glucose transporters in brain, and transporter-related energy defi-ciency of neurons may contribute to pathogenesis of AD. Stroke and TBI are associated with changes of glucose transporter expression that influence clinical outcome
Voltage-gated calcium channels (VGCCs) are widely distributed within the central nervous system (CNS) and presumed to play an important role in the pathophysiology of a broad spectrum of CNS disorders including Alzheimer’s and Parkinson’s disease as well as multiple sclerosis. Several calcium channel blockers have been in clinical practice for many years so that their toxicity and side effects are well studied. However, these drugs are primarily used for the treatment of cardiovascular diseases and most if not all effects on brain functions are secondary to peripheral effects on blood pressure and circulation. While the use of calcium channel antagonists for the treatment of CNS diseases therefore still heavily depends on the development of novel strategies to specifically target different channels and channel subunits, this review is meant to provide an impulse to further emphasize the importance of future research towards this goal.
Absorption of monosaccharides is mainly mediated by Na\(^+\)-d-glucose cotransporter SGLT1 and the facititative transporters GLUT2 and GLUT5. SGLT1 and GLUT2 are relevant for absorption of d-glucose and d-galactose while GLUT5 is relevant for d-fructose absorption. SGLT1 and GLUT5 are constantly localized in the brush border membrane (BBM) of enterocytes, whereas GLUT2 is localized in the basolateral membrane (BLM) or the BBM plus BLM at low and high luminal d-glucose concentrations, respectively. At high luminal d-glucose, the abundance SGLT1 in the BBM is increased. Hence, d-glucose absorption at low luminal glucose is mediated via SGLT1 in the BBM and GLUT2 in the BLM whereas high-capacity d-glucose absorption at high luminal glucose is mediated by SGLT1 plus GLUT2 in the BBM and GLUT2 in the BLM. The review describes functions and regulations of SGLT1, GLUT2, and GLUT5 in the small intestine including diurnal variations and carbohydrate-dependent regulations. Also, the roles of SGLT1 and GLUT2 for secretion of enterohormones are discussed. Furthermore, diseases are described that are caused by malfunctions of small intestinal monosaccharide transporters, such as glucose-galactose malabsorption, Fanconi syndrome, and fructose intolerance. Moreover, it is reported how diabetes, small intestinal inflammation, parental nutrition, bariatric surgery, and metformin treatment affect expression of monosaccharide transporters in the small intestine. Finally, food components that decrease d-glucose absorption and drugs in development that inhibit or downregulate SGLT1 in the small intestine are compiled. Models for regulations and combined functions of glucose transporters, and for interplay between d-fructose transport and metabolism, are discussed.
The size of the synaptic subcomponents falls below the limits of visible light microscopy. Despite new developments in advanced microscopy techniques, the resolution of transmission electron microscopy (TEM) remains unsurpassed. The requirements of tissue preservation are very high, and human post mortem material often does not offer adequate quality. However, new reprogramming techniques that generate human neurons in vitro provide samples that can easily fulfill these requirements. The objective of this study was to identify the culture technique with the best ultrastructural preservation in combination with the best embedding and contrasting technique for visualizing neuronal elements. Two induced neural stem cell lines derived from healthy control subjects underwent differentiation either adherent on glass coverslips, embedded in a droplet of highly concentrated Matrigel, or as a compact neurosphere. Afterward, they were fixed using a combination of glutaraldehyde (GA) and paraformaldehyde (PFA) followed by three approaches (standard stain, Ruthenium red stain, high contrast en-bloc stain) using different combinations of membrane enhancing and contrasting steps before ultrathin sectioning and imaging by TEM. The compact free-floating neurospheres exhibited the best ultrastructural preservation. High-contrast en-bloc stain offered particularly sharp staining of membrane structures and the highest quality visualization of neuronal structures. In conclusion, compact neurospheres growing under free-floating conditions in combination with a high contrast en-bloc staining protocol, offer the optimal preservation and contrast with a particular focus on visualizing membrane structures as required for analyzing synaptic structures.
In enterocytes, protein RS1 (RSC1A1) mediates an increase of glucose absorption after ingestion of glucose-rich food via upregulation of Na+-D-glucose cotransporter SGLT1 in the brush-border membrane (BBM). Whereas RS1 decelerates the exocytotic pathway of vesicles containing SGLT1 at low glucose levels between meals, RS1-mediated deceleration is relieved after ingestion of glucose-rich food. Regulation of SGLT1 is mediated by RS1 domain RS1-Reg, in which Gln-Ser-Pro (QSP) is effective. In contrast to QSP and RS1-Reg, Gln-Glu-Pro (QEP) and RS1-Reg with a serine to glutamate exchange in the QSP motif downregulate the abundance of SGLT1 in the BBM at high intracellular glucose concentrations by about 50%. We investigated whether oral application of QEP improves diabetes in db/db mice and affects the induction of diabetes in New Zealand obese (NZO) mice under glucolipotoxic conditions. After 6-day administration of drinking water containing 5 mM QEP to db/db mice, fasting glucose was decreased, increase of blood glucose in the oral glucose tolerance test was blunted, and insulin sensitivity was increased. When QEP was added for several days to a high fat/high carbohydrate diet that induced diabetes in NZO mice, the increase of random plasma glucose was prevented, accompanied by lower plasma insulin levels. QEP is considered a lead compound for development of new antidiabetic drugs with more rapid cellular uptake. In contrast to SGLT1 inhibitors, QEP-based drugs may be applied in combination with insulin for the treatment of type 1 and type 2 diabetes, decreasing the required insulin amount, and thereby may reduce the risk of hypoglycemia.
Background
Elbow imaging is challenging with conventional multidetector computed tomography (MDCT), while cone-beam CT (CBCT) provides superior options. We compared intra-individually CBCT versus MDCT image quality in cadaveric elbows.
Methods
A twin robotic x-ray system with new CBCT mode and a high-resolution clinical MDCT were compared in 16 cadaveric elbows. Both systems were operated with a dedicated low-dose (LD) protocol (equivalent volume CT dose index [CTDI\(_{vol(16 cm)}\)] = 3.3 mGy) and a regular clinical scan dose (RD) protocol (CTDI\(_{vol(16 cm)}\) = 13.8 mGy). Image quality was evaluated by two radiologists (R1 and R2) on a seven-point Likert scale, and estimation of signal intensity in cancellous bone was conducted. Wilcoxon signed-rank tests and intraclass correlation coefficient (ICC) statistics were used.
Results
The CBCT prototype provided superior subjective image quality compared to MDCT scans (for RD, p ≤ 0.004; for LD, p ≤ 0.001). Image quality was rated very good or excellent in 100% of the cases by both readers for RD CBCT, 100% (R1) and 93.8% (R2) for LD CBCT, 62.6% and 43.8% for RD MDCT, and 0.0% and 0.0% for LD MDCT. Single-measure ICC was 0.95 (95% confidence interval 0.91–0.97; p < 0.001). Software-based assessment supported subjective findings with less “undecided” pixels in CBCT than dose-equivalent MDCT (p < 0.001). No significant difference was found between LD CBCT and RD MDCT.
Conclusions
In cadaveric elbow studies, the tested cone-beam CT prototype delivered superior image quality compared to high-end multidetector CT and showed a potential for considerable dose reduction.
Background: Dendritic cells (DCs) rendered suppressive by treatment with mitomycin C and loaded with the autoantigen myelin basic protein demonstrated earlier their ability to prevent experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). This provides an approach for prophylactic vaccination against autoimmune diseases. For clinical application such DCs are difficult to generate and autoantigens hold the risk of exacerbating the disease.
Methods: We replaced DCs by peripheral mononuclear cells and myelin autoantigens by glatiramer acetate (Copaxone ®), a drug approved for the treatment of MS. Spleen cells were loaded with Copaxone®, incubated with mitomycin C (MICCop) and injected into mice after the first bout of relapsing-remitting EAE. Immunosuppression mediated by MICCop was investigated in vivo by daily assessment of clinical signs of paralysis and in in vitro restimulation assays of peripheral immune cells. Cytokine profiling was performed by enzyme-linked immunosorbent assay (ELISA). Migration of MICCop cells after injection was examined by biodistribution analysis of 111Indium-labelled MICCop. The number and inhibitory activity of CD4+CD25+FoxP3+ regulatory T cells were analysed by histology, flow cytometry and in vitro mixed lymphocyte cultures. In order to assess the specificity of MICCop-induced suppression, treated EAE mice were challenged with the control protein ovalbumin. Humoral and cellular immune responses were then determined by ELISA and in vitro antigen restimulation assay.
Results: MICCop cells were able to inhibit the harmful autoreactive T-cell response and prevented mice from further relapses without affecting general immune responses. Administered MICCop migrated to various organs leading to an increased infiltration of the spleen and the central nervous system with CD4+CD25+FoxP3+ cells displaying a suppressive cytokine profile and inhibiting T-cell responses.
Conclusion: We describe a clinically applicable cell therapeutic approach for controlling relapses in autoimmune encephalomyelitis by specifically silencing the deleterious autoimmune response.
Die Multiple Sklerose (MS) ist eine chronisch-entzündliche Autoimmunerkrankung des zentralen Nervensystems (ZNS) und stellt die häufigste Ursache frühzeitiger Behinderung junger Erwachsener dar. Kennzeichnend sind multifokale ZNS-Läsionen, die durch Inflammation, Demyelinisierung und Axonschäden geprägt sind und zu multiplen neurologischen Defiziten führen. Derzeit ist es mithilfe der verlaufsmodifizierenden Therapie möglich, die Immunantwort abzuschwächen und damit die Krankheitsprogression zu verzögern. Geheilt werden kann die Erkrankung jedoch bislang nicht. Dabei ist nicht hinreichend geklärt, ob die neuen Therapieoptionen über die Immunmodulation/-suppression hinaus einen anhaltenden Schutz vor der langfristigen Neurodegeneration bieten.
Basierend auf den vielversprechenden Ergebnissen klinischer Studien zur Therapie der schubförmig-remittierenden MS mit dem Anti-CD52-Antikörper Alemtuzumab, der zu einer Depletion CD52-exprimierender Immunzellen führt, wurden diesbezüglich Analysen in MS-Tiermodellen durchgeführt. Da die Untersuchung der zugrunde liegenden Patho- und Effektormechanismen am Menschen kaum möglich ist, ist die MS-Forschung für ein tiefergehendes Verständnis auf Tiermodelle angewiesen. Die experimentelle autoimmune Enzephalomyelitis (EAE) ist hierbei das am weitesten verbreitete Modell der MS, wofür vor allem der C57BL/6 (B6) -Mausstamm verwendet wird, da auf diesem Hintergrund die meisten genmodifizierten Mäuse gezüchtet werden. Jene tierexperimentellen Studien, in denen ein muriner Anti-CD52-Antikörper im frühen Krankheitsstadium der EAE (Auftreten erster paralytischer Symptome) verabreicht wurde, erbrachten den Hinweis einer neuroprotektiven und scheinbar regenerativen Wirkung des Antikörpers.
Über einen neuroprotektiven Effekt von Alemtuzumab im schwer behandelbaren chronisch-progredienten Stadium der MS ist jedoch wenig bekannt. Die vorliegende Arbeit ist die erste detaillierte Untersuchung zum Einfluss des murinen Anti-CD52-Antikörpers auf die Demyelinisierung, den Axonschaden und die Hirnatrophie in der MP4-induzierten EAE der B6-Maus im chronischen Verlauf der Erkrankung (ab stabilem Plateau der klinischen Symptomatik). MP4 ist ein Myelinfusionsprotein aus MBP (Myelin-Basisches-Protein) und PLP (Proteolipidprotein), welches in B6-Mäusen durch aktive Immunisierung eine EAE induziert, die chronisch verläuft und als eines von wenigen Modellen neben der T-Zell-Abhängigkeit die an Bedeutung zunehmende B-Zell-Komponente der MS darstellt. Histopathologisch finden sich in der chronischen MP4-induzierten EAE eine ausgeprägte Rückenmarks- und Kleinhirnschädigung, die vor allem im Kleinhirn durch eine B-Zell-Aggregation charakterisiert ist.
Nachdem die MP4-immunisierten Mäuse im chronischen Stadium der EAE an fünf aufeinanderfolgenden Tagen mit 10 mg/kg Körpergewicht murinem Anti-CD52-spezifischem IgG2a-Isotypantikörper bzw. murinem unspezifischem IgG2a-Isotyp-Kontroll-Antikörper behandelt worden waren, wurde die Lymphozytendepletion im peripheren Blut durchflusszytometrisch ermittelt und deren Einfluss auf MP4-spezifische Antikörper anhand eines indirekten Enzyme-linked Immunosorbent Assays (ELISAs) untersucht. Als Marker für Axonschäden wurde im Serum vorhandenes phosphoryliertes Neurofilament-Heavy (pNF-H) mithilfe eines indirekten Sandwich-ELISAs quantitativ bestimmt. Rückenmark und Kleinhirn wurden ultrastrukturell auf Veränderungen der Myelinisierung (mittels g-Ratio: Axondurchmesser geteilt durch Gesamtdurchmesser der Nervenfaser) und auf Axonpathologien (verringerter Abstand benachbarter Neurofilamente, axolytische Axone, axonaler Verlust) untersucht. Die Hirnatrophie wurde MRT-basiert gemessen und der klinische Verlauf täglich evaluiert.
Durch die Anti-CD52-Antikörperbehandlung wurde die T- und B-Zellzahl zwar drastisch vermindert, die MP4-spezifische Antikörperproduktion blieb davon jedoch unbeeinträchtigt. Ein günstiger Effekt auf die De- und Remyelinisierung war nicht festzustellen. Das Hirnvolumen und die klinische Präsentation der Mäuse blieben ebenfalls unverändert. Während kein Unterschied der pNF-H-Konzentration zu erkennen war, konnte ultrastrukturell jedoch ein geringerer Axonschaden nachgewiesen werden.
Insgesamt legen diese Ergebnisse nahe, dass der Anti-CD52-Antikörper im chronischen Verlauf der EAE/MS wenig Einfluss auf die neurodegenerativen Prozesse nimmt und die Regeneration nicht fördern kann. Die Ursache liegt vermutlich in der Undurchlässigkeit der Bluthirnschranke für Antikörper sowie dem limitierten Verständnis der Antikörperwirkung im ZNS. Die vorliegende Studie regt somit zur Etablierung von ZNS-wirksamen Antikörpern an und unterstreicht die Bedeutung der Entwicklung von selektiveren neuroprotektiven und remyelinisierungsfördernden Behandlungsansätzen, die eine wertvolle Ergänzung zur verlaufsmodifizierenden Therapie darstellen könnten.
Pemphigus vulgaris (PV) ist eine blasenbildende Autoimmunerkrankung, die durch Autoantikörper gegen Dsg1 und Dsg3 gekennzeichnet ist. Der genaue Pathomechanismus, der zu einem PV-IgG vermittelten Verlust der interzellulären Adhäsion führt, ist noch unklar. Die Dsg3-Depletion und die Modulation von Signalkaskaden stellen hierbei kennzeichnende Merkmale der Erkrankung dar. Mit den Ergebnissen der vorliegenden Arbeit ist eine bessere Einordnung der Dsg3-Depletion in den pathogenetischen Kontext von Pemphigus vulgaris möglich. Die Experimente zeigen, dass die Dsg3-Depletion von Differenzierungsprozessen abhängig ist und mit einem Adhäsionsverlust einhergehen kann. Die Hemmung der PKC verhindert hierbei sowohl die PV-IgG vermittelten Effekte in der Zellkultur als auch die Blasenbildung im Mausmodell in vivo und in humaner Haut ex vivo. Des Weiteren liefert die Arbeit neue Erkenntnisse, welche für die suprabasale Lokalisation der Blasenbildung bedeutsam sein könnten.
Rekrutierung von Stromazellen aus gefäßwandresidenten Vorläuferzellen während der Tumorgenese
(2021)
Tumore bestehen nicht nur aus malignen Zellen, sondern ebenfalls aus einer Vielzahl an nicht tumorigenen Zellen, die den Tumor auf vielfältige Weise unterstützen und den Tumor vor therapeutischen Maßnahmen schützen. Die Frage der Herkunft dieser Zellen insbesondere in einem nicht vaskularisierten Tumor ist daher auch für die Entwicklung zukünftiger Therapeutika relevant. In dieser Arbeit wurde eine Methode etabliert, die im dreidimensionalen Raum die Untersuchung des Einflusses von Tumorzellen auf die vaskuläre Adventitia am Model der Mausaorta ermöglicht. Dazu erfolgte die Einbettung von Alginatbeads aus verschiedenen Tumorzelllinien in eine gemeinsame Kollagenmatrix mit murinen Aortenringen. Während des zehntägigem Versuchszeitraums wurde die Aussprossung von Zellen aus den Aortenringen beobachtet und quantifiziert. Es wurde festgestellt, dass die Auswanderung während des Versuchszeitraums zunimmt und dass die Konfrontation mit der Zytokinmischung der Tumorzellen zu einer stärkeren Aussprossung führt, als die Stimulation mit VEGF oder keine Stimulation. Eine gerichtete Auswanderung der Zellen in Richtung der Tumorbeads konnte nicht nachgewiesen bzw. bestätigt werden. Kapilläre Aussprossungen waren nur in geringem Ausmaß zu beobachten. Bei Charakterisierung der ausgewanderten Zellen mittels immunhistochemischer Färbungen waren keine F4/80-positiven und nur einzelne CD34-positive Zellen zu finden. CD31-positive Endothelzellen stellten die Mehrheit der ausgewanderten Zellen bei Tumorzellkonfrontation. Perizyten, die mit dem Marker NG2 gefärbt wurden, stellten eine Mehrheit der migrierten Zellen bei allen Bedingungen.
Die in dieser Arbeit etablierte Methode des Aortenring-Bead-Konfrontationsassays ermöglicht es, in Echtzeit den Einfluss von Tumorzellen auf die Gefäßwand im dreidimensionalen Raum zu beobachten. Der Aortenring-Bead-Konfrontationsassay bietet eine Vielzahl an Variationsmöglichkeiten und stellt daher eine vielversprechende Möglichkeit dar, die Lücke zwischen zweidimensionalen in vitro-Experimenten und kostenintensiven in vivo-Versuchen zu schließen.
Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state.
A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated beta-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27\(^{KIP1}\) (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27\(^{KIP1}\) gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene.
Bei der Autoimmunerkrankung Pemphigus vulgaris führen Antikörper zur charakteristischen suprabasalen Akantholyse und Blasenbildung der Epidermis, indem sie an spezifische Antigene, Dsg3 (Desmoglein 3) und Dsg1 (Desmoglein 1), auf der Zelloberfläche der Keratinozyten binden. Die Art und Weise, wie die multiplen zellulären Pathomechanismen zusammenwirken und das potenziell tödliche Krankheitsbild hervorrufen, ist jedoch bislang noch weitgehend unklar. In der vorliegenden Arbeit wurden entscheidende, durch die Autoantikörper hervorgerufene, pathologische intrazelluläre Prozesse genauer untersucht und deren Stellenwert beleuchtet.
Induced pluripotent stem cells (iPSCs) have been recognised as a virtually unlimited source of stem cells that can be generated in a patient-specific manner. Due to these cells’ potential to give rise to all differentiated cell types of the human body, they have been widely used to derive differentiated cells for drug screening and disease modelling purposes. iPSCs also garner much interest as they can potentially serve as a source for cell replacement therapy. Towards the realisation of these biomedical applications, this thesis aims to address challenges that are associated with scale-up, safety and biofabrication.
Firstly, the manufacture of a high number of human iPSCs (hiPSCs) will require standardised procedures for scale-up and the development of a flexible bioprocessing method, since standard adherent hiPSC culture exhibits limited scalability and is labour-intensive. While the quantity of cells that are required for cell therapy depends largely on the tissue and defect that these replacing cells are meant to correct, an estimate of 1 × 10^9 has been suggested to be sufficient for several indications, including myocardial infarction and islet replacement for diabetes. Here, the development of an integrated, microcarrier-free workflow to transition standard adherent hiPSC culture (6-well plates) to scalable stirred suspension culture in bioreactors (1 L working volume, 2.4 L maximum working volume) is presented. The two-phase bioprocess lasts 14 days and generates hiPSC aggregates measuring 198 ± 58 μm in diameter on the harvesting day, yielding close to 2 × 10^9 cells. hiPSCs can be maintained in stirred suspension for at least 7 weeks with weekly passaging, while exhibiting pluripotency-associated markers TRA-1-60, TRA-1-81, SSEA-4, OCT4, and SOX2. These cells retain their ability to differentiate into cells of all the three germ layers in vitro, exemplified by cells positive for AFP, SMA, or TUBB3. Additionally, they maintain a stable karyotype and continue to respond to specification cues, demonstrated by directed differentiation into beating cardiomyocyte-like cells. Therefore, the aim of manufacturing high hiPSC quantities was met using a state-of-the-art scalable suspension bioreactor platform.
Secondly, multipotent stem cells such as induced neural stem cells (iNSCs) may represent a safer source of renewable cells compared to pluripotent stem cells. However, pre-conditioning of stem cells prior to transplantation is a delicate issue to ensure not only proper function in the host but also safety. Here, iNSCs which are normally maintained in the presence of factors such as hLIF, CHIR99021, and SB431542 were cultured in basal medium for distinct periods of time. This wash-out procedure results in lower proliferation while maintaining key neural stem cell marker PAX6, suggesting a transient pre-differentiated state. Such pre-treatment may aid transplantation studies to suppress tumourigenesis through transplanted cells, an approach that is being evaluated using a mouse model of experimental focal demyelination and autoimmune encephalomyelitis.
Thirdly, biomedical applications of stem cells can benefit from recent advancements in biofabrication, where cells can be arranged in customisable topographical layouts. Employing a 3DDiscovery bioprinter, a bioink consisting of hiPSCs in gelatin-alginate was extruded into disc-shaped moulds or printed in a cross-hatch infill pattern and cross-linked with calcium ions. In both discs and printed patterns, hiPSCs recovered from these bioprints showed viability of around 70% even after 4 days of culture when loaded into gelatin-alginate solution in aggregate form. They maintained pluripotency-associated markers TRA-1-60 and SSEA-4 and continued to proliferate after re-plating. As further proof-of-principle, printed hiPSC 3D constructs were subjected to targeted neuronal differentiation, developing typical neurite outgrowth and resulting in a widespread network of cells throughout and within the topology of the printed matrix. Staining against TUBB3 confirmed neuronal identity of the differentiated cellular progeny. In conclusion, these data demonstrate that hiPSCs not only survive the 3D-printing process but were able to differentiate along the printed topology in cellular networks.
Neurogene Entzündung ist charakterisiert durch Vasodilatation, Plasmaextravasation und Leukozytenmigration.
Im Zuge dieser Dissertationsarbeit konnte ein in vivo Versuchsmodell zur Quantifizierung neurogener Entzündungsreaktionen in den Atemwegen etabliert werden. Der bakterielle Bitterstoff Cycloheximid ist in der Lage, eine Erhöhung der Plasmaextravasation und Migration neutrophiler Granulozyten zu bewirken. Somit kann Cycloheximid nicht nur protektive Schutzreflexe auslösen, sondern führt auch lokal zu einer neurogenen Entzündungsreaktion. Das carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) ist an der Regulierung der endothelialen Barrierefunktion beteiligt. Die Versuche zeigen bei CC1-/--Mäusen eine Verminderung der basalen Permeabilität in trachealen postkapillären Venolen. Nach Stimulation mit Cycloheximid zeigen CC1-/--Mäuse im Vergleich mit WT-Mäusen eine verminderte Plasmaextravasation in bronchialen postkapillären Venolen. Auch die Permeabilität des Endothels für neutrophile Granulozyten scheint durch CEACAM1-Defizienz in trachealen und bronchialen Venolen herabgesetzt zu werden. Die Anwesenheit des CEACAM1-Moleküls verursacht offenbar eine verminderte Stabilität der endothelialen Barriere in postkapillären Venolen der Atemwege. Diese Ergebnisse zeigen eine gegenteilige Funktion von CEACAM1 in postkapillären Venolen der Atemwege im Vergleich mit großen, herznahen Blutgefäßen. Des Weiteren scheint sich die Rolle von CEACAM1 in der Entstehung von akuten und chronischen Entzündungsreaktionen zu unterscheiden. Das in dieser Arbeit etablierte Versuchsmodell stellt eine Möglichkeit dar, neurogene Entzündungsreaktionen als Reaktion auf verschiedene gustatorische Stimulanzien zu testen und zu quantifizieren.
Die vorliegende Arbeit hatte zum Ziel, neuroendokrine Zellen in den Atemwegen bei Mäusen zu untersuchen, welche Kontakt zu sensorischen Nervenfasern ausbilden. In vorangegangenen Versuchen konnte bereits die Menge des ausgeschütteten CGRPs nach Stimulation mit Bitterstoffen bestimmt werden. Die Methode zur Messung der Freisetzung von CGRP aus verschiedenen Organen wurde von Prof. Reeh und seiner Arbeitsgruppe etabliert. Ziel der vorliegenden Arbeit war es, zu untersuchen, woher das ausgeschüttete CGRP kommt und ob die Stimulation von Bürstenzellen mit Bitterstoffen zur Ausschüttung von CGRP aus den neuroendokrinen Zellen führt. Anhand der elektronenmikroskopischen Auswertung und der dreidimensionalen Rekonstruktion konnte gezeigt werden, dass es Kontakt zwischen den neuroendokrinen Zellen im Epithel der Trachea und sensorischen Nervenfasern gibt. Die immunhistochemischen Versuche zeigten, dass es nach Stimulation mit Denatonium höchstwahrscheinlich zur Ausschüttung von CGRP durch die intraepithelialen Fasern gekommen ist. Diese Annahme spiegelt sich in der veränderten Morphologie sowie der geringeren Quantität der intraepithelialen Fasern nach Stimulation mit Denatonium deutlich wider. Dass es weder bei der Anzahl der neuroendokrinen Zellen, noch bei der Erscheinung und Anzahl der extraepithelialen Fasern nach Denatoniumstimulation zu einer Veränderung gekommen ist, unterstützt diese Annahme ebenfalls. Im Hinblick auf die durchgeführten Versuche mit den TRPM5-gendefizienten Mäusen zeigte sich, dass die Stimulation mit Denatonium keine Auswirkungen auf die Anzahl der neuroendokrinen Zellen hatte. Dieses Ergebnis unterstützt die Erkenntnisse der vorangegangenen Untersuchungen, welche gezeigt haben, dass das CGRP nicht von den neuroendokrinen Zellen ausgeschüttet wurde. Des Weiteren lässt das Ergebnis darauf schließen, dass die Ausschüttung von CGRP nicht abhängig von der Anwesenheit von Bürstenzellen ist. Insgesamt zeigen die Untersuchungen, dass es nach Stimulation mit Bittersubstanzen zu einer CGRP-Ausschüttung durch die intraepithelialen Fasern gekommen ist. Interessant wäre es weiterhin zu klären, welche Effekte diese Ausschüttung bewirkt und welche Bedeutung der Freisetzung von Substanz P in diesem Zusammenhang zukommt.