@phdthesis{Hondke2014,
  author    = {Hondke, Sylvia},
  title     = {Elucidation of WISP3 function in human mesenchymal stem cells and chondrocytes},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-109641},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2014},
  abstract  = {WISP3 is a member of the CCN family which comprises six members found in the 1990's: Cysteine-rich,angiogenic inducer 61 (CYR61, CCN1), Connective tissue growth factor (CTGF, CCN2), Nephroblastoma overexpressed (NOV, CNN3) and the Wnt1 inducible signalling pathway protein 1-3 (WISP1-3, CCN4-6).They are involved in the adhesion, migration, mitogenesis, chemotaxis, proliferation, cell survival, angiogenesis, tumorigenesis, and wound healing by the interaction with different integrins and heparan sulfate proteoglycans. Until now the only member correlated to the musculoskeletal autosomal disease Progressive Pseudorheumatoid Dysplasia (PPD) is WISP3. PPD is characterised by normal embryonic development followed by cartilage degradation over time starting around the age of three to eight years. Animal studies in mice exhibited no differences between knock out or overexpression compared to wild type litter mates, thus were not able to reproduce the symptoms observed in PPD patients. Studies in vitro and in vivo revealed a role for WISP3 in antagonising BMP, IGF and Wnt signalling pathways. Since most of the knowledge of WISP3 was gained in epithelial cells, cancer cells or chondrocyte cell lines, we investigated the roll of WISP3 in primary human mesenchymal stem cells (hMSCs) as well as primary chondrocytes. WISP3 knock down was efficiently established with three short hairpin RNAs in both cell types, displaying a change of morphology followed by a reduction in cell number. Simultaneous treatment with recombinant WISP3 was not enough to rescue the observed phenotype nor increase the endogenous expression of WISP3. We concluded that WISP3 acts as an essential survival factor, where the loss resulted in the passing of cell cycle control points followed by apoptosis. Nevertheless, Annexin V-Cy3 staining and detection of active caspases by Western blot and immunofluorescence staining detected no clear evidence for apoptosis. Furthermore, the gene expression of the death receptors TRAILR1 and TRAILR2,important for the extrinsic activation of apoptosis, remained unchanged during WISP3 mRNA reduction. Autophagy as cause of cell death was also excluded, given that the autophagy marker LC3 A/B demonstrated to be uncleaved in WISP3-deficient hMSCs. To reveal correlated signalling pathways to WISP3 a whole genome expression analyses of WISP3-deficient hMSCs compared to a control (scramble) was performed. Microarray analyses exhibited differentially regulated genes involved in cell cycle control, adhesion, cytoskeleton and cell death. Cell death observed by WISP3 knock down in hMSCs and chondrocytes might be explained by the induction of necroptosis through the BMP/TAK1/RIPK1 signalling axis. Loss of WISP3 allows BMP to bind its receptor activating the Smad 2/3/4 complex which in turn can activate TAK1 as previously demonstrated in epithelial cells. TAK1 is able to block caspase-dependent apoptosis thereby triggering the assembly of the necrosome resulting in cell death by necroptosis. Together with its role in cell cycle control and extracellular matrix adhesion, as demonstrated in human mammary epithelial cells, the data supports the role of WISP3 as tumor suppressor and survival factor in cells of the musculoskeletal system as well as epithelial cells.},
  subject      = {Knorpelzelle},
  language  = {en}
}
@phdthesis{Hafen2015,
  author    = {Hafen, Bettina},
  title     = {Physical contact between mesenchymal stem cells and endothelial precursors induces distinct signatures with relevance to tissue regeneration and engineering},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119417},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {The goal of the project VascuBone is to develop a tool box for bone regeneration, which on one hand fulfills basic requirements (e.g. biocompatibility, properties of the surface, strength of the biomaterials) and on the other hand is freely combinable with what is needed in the respective patient's situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA-approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging-based in vivo diagnostics, which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects. This project receives funding from the European Union's Seventh Framework Program (VascuBone 2010). The present study is embedded into this EU project. The intention of this study is to assess the changes of the global gene expression patterns of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) after direct cell-cell contact as well as the influence of conditioned medium gained from MSCs on EPCs and vice versa. EPCs play an important role in postnatal vasculogenesis. An intact blood vessel system is crucial for all tissues, including bone. Latest findings in the field of bone fracture healing and repair by the use of tissue engineering constructs seeded with MSCs raised the idea of combining MSCs and EPCs to enhance vascularization and therefore support survival of the newly built bone tissue. RNA samples from both experimental set ups were hybridized on Affymetrix GeneChips® HG-U133 Plus 2.0 and analyzed by microarray technology. Bioinformatic analysis was applied to the microarray data and verified by RT-PCR. This study gives detailed information on how EPCs and MSCs communicate with each other and therefore gives insights into the signaling pathways of the musculoskeletal system. These insights will be the base for further functional studies on protein level for the purpose of tissue regeneration. A better understanding of the cell communication of MSCs and EPCs and subsequently the targeting of relevant factors opens a variety of new opportunities, especially in the field of tissue engineering. The second part of the present work was to develop an ELISA (enzyme-linked immunosorbent assay) for a target protein from the lists of differentially expressed genes revealed by the microarray analysis. This project was in cooperation with Immundiagnostik AG, Bensheim, Germany. The development of the ELISA aimed to have an in vitro diagnostic tool to monitor e.g. the quality of cell seeded tissue engineering constructs. The target protein chosen from the lists was klotho. Klotho seemed to be a very promising candidate since it is described in the literature as anti-aging protein. Furthermore, studies with klotho knock-out mice showed that these animals suffered from several age-related diseases e.g. osteoporosis and atherosclerosis. As a co-receptor for FGF23, klotho plays an important role in bone metabolism. The present study will be the first one to show that klotho is up-regulated in EPCs after direct cell-cell contact with MSCs. The development of an assay with a high sensitivity on one hand and the capacity to differentiate between secreted and shedded klotho on the other hand will allow further functional studies of this protein and offers a new opportunity in medical diagnostics especially in the field of metabolic bone disease.},
  subject      = {Vorl{\"a}uferzelle},
  language  = {en}
}
@phdthesis{Simann2015,
  author    = {Simann, Meike},
  title     = {Aufkl{\"a}rung der Effekte von Fibroblasten-Wachstumsfaktor 1 und 2 auf die Adipogenese und Osteogenese von prim{\"a}ren humanen Knochenmark-Stroma-Zellen},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119322},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2015},
  abstract  = {Regulating and reverting the adipo-osteogenic lineage decision of trabecular human bone marrow stromal cells (hBMSCs) represents a promising approach for osteoporosis therapy and prevention. Fibroblast growth factor 1 (FGF1) and its subfamily member FGF2 were scored as lead candidates to exercise control over lineage switching processes (conversion) in favor of osteogenesis previously. However, their impact on differentiation events is controversially discussed in literature. Hence, the present study aimed to investigate the effects of these FGFs on the adipogenic and osteogenic differentiation and conversion of primary hBMSCs. Moreover, involved downstream signaling mechanisms should be elucidated and, finally, the results should be evaluated with regard to the possible therapeutic approach. This study clearly revealed that culture in the presence of FGF1 strongly prevented the adipogenic differentiation of hBMSCs as well as the adipogenic conversion of pre-differentiated osteoblastic cells. Lipid droplet formation was completely inhibited by a concentration of 25 ng/µL. Meanwhile, the expression of genetic markers for adipogenic initiation, peroxisome proliferator-activated receptor gamma 2 (PPARg2) and CCAAT/enhancer binding protein alpha (C/EBPa), as well as subsequent adipocyte maturation, fatty acid binding protein 4 (FABP4) and lipoprotein lipase (LPL), were significantly downregulated. Yet, the genetic markers of osteogenic commitment and differentiation were not upregulated during adipogenic differentiation and conversion under FGF supplementation, not supporting an event of osteogenic lineage switching. Moreover, when examining the effects on the osteogenic differentiation of hBMSCs and the osteogenic conversion of pre-differentiated adipocytic cells, culture in the presence of FGF1 markedly decreased extracellular matrix (ECM) mineralization. Additionally, the gene expression of the osteogenic marker alkaline phosphatase (ALP) was significantly reduced and ALP enzyme activity was decreased. Furthermore, genetic markers of osteogenic commitment, like the master regulator runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 4 (BMP4), as well as markers of osteogenic differentiation and ECM formation, like collagen 1 A1 (COL1A1) and integrin-binding sialoprotein (IBSP), were downregulated. In contrast, genes known to inhibit ECM mineralization, like ANKH inorganic pyrophosphate transport regulator (ANKH) and osteopontin (OPN), were upregulated. ANKH inhibition revealed that its transcriptional elevation was not crucial for the reduced matrix mineralization, perhaps due to decreased expression of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) that likely annulled ANKH upregulation. Like FGF1, also the culture in the presence of FGF2 displayed a marked anti-adipogenic and anti-osteogenic effect. The FGF receptor 1 (FGFR1) was found to be crucial for mediating the described FGF effects in adipogenic and osteogenic differentiation and conversion. Yet, adipogenic conversion displayed a lower involvement of the FGFR1. For adipogenic differentiation and osteogenic differentiation/conversion, downstream signal transduction involved the extracellular signal-regulated kinases 1 and 2 (ERK1/2) and the mitogen-activated protein kinase (MAPK)/ERK kinases 1 and 2 (MEK1/2), probably via the phosphorylation of FGFR docking protein FGFR substrate 2a (FRS2a) and its effector Ras/MAPK. The c-Jun N-terminal kinase (JNK), p38-MAPK, and protein kinase C (PKC) were not crucial for the signal transduction, yet were in part responsible for the rate of adipogenic and/or osteogenic differentiation itself, in line with current literature. Taken together, to the best of our knowledge, our study was the first to describe the strong impact of FGF1 and FGF2 on both the adipogenic and osteogenic differentiation and conversion processes of primary hBMSCs in parallel. It clearly revealed that although both FGFs were not able to promote the differentiation and lineage switching towards the osteogenic fate, they strongly prevented adipogenic differentiation and lineage switching, which seem to be elevated during osteoporosis. Our findings indicate that FGF1 and FGF2 entrapped hBMSCs in a pre-committed state. In conclusion, these agents could be applied to potently prevent unwanted adipogenesis in vitro. Moreover, our results might aid in unraveling a pharmacological control point to eliminate the increased adipogenic differentiation and conversion as potential cause of adipose tissue accumulation and decreased osteoblastogenesis in bone marrow during aging and especially in osteoporosis.},
  subject      = {Mesenchymzelle},
  language  = {en}
}