@phdthesis{Bachmann2021, author = {Bachmann, Julia}, title = {Role of Adipose-Derived Stromal/Stem Cells in Cell-Assisted Lipotransfer - Characterization of their Secretory Capacity under Ischemia-Like Stress Conditions and Establishment of a 3D Adipose Tissue-ASC Co-Culture}, doi = {10.25972/OPUS-25178}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251786}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2021}, abstract = {The use of human adipose-derived mesenchymal stem cells (ASCs) for cell-based therapeutic approaches, in terms of repair and regeneration of various tissues and organs, offers an alternative therapeutic tool in the field of regenerative medicine. The ability of ASCs to differentiate along mesenchymal lineages is not the only property that makes these cells particularly attractive for therapeutic purposes. Their promising functions in promoting angiogenesis, reducing inflammation as well as in functional tissue restoration are largely related to the trophic effects of a broad panel of secreted cytokines and growth factors. However, in cell-based approaches, the cell-loaded construct often is exposed to an ischemic microenvironment characterized by severe oxidative and nutritional stress after transplantation due to the initial lack of vascular connection, resulting in reduced cell viability and altered cell behaviour. Therefore, the effective use of ASCs in regenerative medicine first requires a comprehensive characterization of the cells in terms of their viability, differentiation capacity and especially their secretory capabilities under ischemia-mimicking conditions in order to better understand their beneficial role. Accordingly, in the first part of this work, ASCs were investigated under different ischemic conditions, in which cells were exposed to both glucose and oxygen deprivation, with respect to viability and secretory function. Using mRNA gene expression analysis, significantly higher expression of selected angiogenic, anti-apoptotic and immunomodulatory factors (IL-6, VEGF, STC-1) could be demonstrated under harsh ischemic conditions. These results were reflected at the protein expression level by a significantly increased secretion of these factors. For stanniocalcin-1 (STC-1), a factor not yet described in ASCs, a particularly high expression with significant secreted amounts of the protein could be demonstrated under harsh ischemic conditions. Thus, the first part of this work, in addition to the characterization of the viability, provided first insights into the secretory response of ASCs under ischemic conditions. The response of ASCs to glucose deficiency in combination with severe hypoxia has been little explored to date. Thus, the focus of the second part of this work was on a more detailed investigation of the secretory response of ASCs under glucose and oxygen deprivation. For a more comprehensive analysis of the secretion profile, a cytokine antibody array was performed, which allowed the detection of a broad panel of secreted angiogenic factors (IL-8, ANG), matrix-regulating proteins (TIMP-1, TIMP-2), chemokines (MCP-1/CCL2, IP-10/CXCL 10) and other factors under ischemic conditions. To verify these results, selected factors were examined using ELISA. The analysis revealed that the secretion of individual factors (e.g., STC-1, VEGF) was significantly upregulated by the combination of glucose and oxygen deprivation compared to oxygen deprivation alone. In order to investigate the impact of the secretome of ischemic ASCs on cell types involved in tissue regeneration, the effect of conditioned medium of ischemia-challenged ASCs on both endothelial cells and fibroblasts was investigated in subsequent experiments. Significantly increased viability and tube formation of endothelial cells as well as activated migration of fibroblasts by the secreted factors of ischemic ASCs could be demonstrated. A direct correlation of these effects to STC-1, which was significantly upregulated under ischemic conditions and has been described as a regulator of key cellular functions, could not be verified. The particular secretory capacity of ASCs provides a valuable tool for cell-based therapies, such as cell-assisted lipotransfer (CAL), where by enriching fat grafts with isolated ASCs, a significantly improved survival rate of the transplanted construct is achieved with less resorption of the fat tissue as well as a reduction in adverse implications, such as fibrosis and cyst formation. In order to better understand the function of ASCs in CAL, an autologous transwell-based lipograft-ASC co-culture was established in the last part of this work, in which first investigations showed a markedly increased secretion of VEGF compared to lipografts without added ASCs. As the stability rate of the fat tissue and thus the success of CAL is presumably also dependent on the preparation of the tissue before transplantation, the conventional preparation method of fat tissue for vocal fold augmentation in laryngoplasty was additionally evaluated in vitro in a pilot experiment. By analyzing the viability and tissue structure of the clinically prepared injection material, a large number of dead cells and a clearly damaged tissue structure with necrotic areas could be demonstrated. In comparison, the preparation method of the fat tissue established in this work as small tissue fragments was able to provide a clearly intact, vital, and vascularized tissue structure. This type of adipose tissue preparation represents a promising alternative for clinical vocal fold augmentation. In conclusion, the results of this work contribute to a comprehensive characterization of ASCs under ischemic conditions, such as those prevalent at the transplantation site or in tissue regeneration. The results obtained, especially on the secretory capacity of ASCs, provide new insights into how ASCs mediate regenerative effects in an ischemic milieu and why their use for therapeutic purposes is highly attractive and promising.}, subject = {Adipose}, language = {en} } @phdthesis{Kraehnke2019, author = {Kr{\"a}hnke, Martin}, title = {Chondrogenic differentiation of bone marrow-derived stromal cells in pellet culture and silk scaffolds for cartilage engineering - Effects of different growth factors and hypoxic conditions}, doi = {10.25972/OPUS-19299}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192999}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2019}, abstract = {Articular cartilage lesions that occur upon intensive sport, trauma or degenerative disease represent a severe therapeutic problem. At present, osteoarthritis is the most common joint disease worldwide, affecting around 10\% of men and 18\% of women over 60 years of age (302). The poor self-regeneration capacity of cartilage and the lack of efficient therapeutic treatment options to regenerate durable articular cartilage tissue, provide the rationale for the development of new treatment options based on cartilage tissue engineering approaches (281). The integrated use of cells, biomaterials and growth factors to guide tissue development has the potential to provide functional substitutes of lost or damaged tissues (2,3). For the regeneration of cartilage, the availability of mesenchymal stromal cells (MSCs) or their recruitment into the defect site is fundamental (281). Due to their high proliferation capacity, the possibility to differentiate into chondrocytes and their potential to attract other progenitor cells into the defect site, bone marrow-derived mesenchymal stromal cells (BMSCs) are still regarded as an attractive cell source for cartilage tissue engineering (80). However, in order to successfully engineer cartilage tissue, a better understanding of basic principles of developmental processes and microenvironmental cues that guide chondrogenesis is required.}, subject = {Hypoxie}, language = {en} } @phdthesis{Chaudhari2013, author = {Chaudhari, Sweena M.}, title = {Role of Hypoxia-Inducible Factor (HIF) 1α in Dendritic Cells in Immune Regulation of Atherosclerosis}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-91853}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2013}, abstract = {Atherosclerosis is the underlying cause of cardiovascular diseases and a major threat to human health worldwide. It involves not only accumulation of lipids in the vessel wall but a chronic inflammatory response mediated by highly specific cellular and molecular responses. Macrophages and dendritic cells (DCs) play an essential role in taking up modified lipids and presenting them to T and B lymphocytes, which promote the immune response. Enhanced activation, migration and accumulation of inflammatory cells at the local site leads to formation of atherosclerotic plaques. Atherosclerotic plaques become hypoxic due to reduced oxygen diffusion and high metabolic demand of accumulated cells. The various immune cells experience hypoxic conditions locally and inflammatory stimuli systemically, thus up-regulating Hypoxia-inducible factor 1α. Though the role of HIF1α in macrophages and lymphocytes has been elucidated, its role in DCs still remains controversial, especially with respect to atherosclerosis. In this project work, the role of HIF1α in DCs was investigated by using a cell specific knockout mouse model where HIF1α was deleted in CD11c+ cells. Aortic root sections from atherosclerotic mice showed presence of hypoxia and up-regulation of HIF1α which co-localized with CD11c+ cells. Atherosclerotic splenic DCs also displayed enhanced expression of HIF1α, proving non-hypoxic stimulation of HIF1α due to systemic inflammation. Conditional knockout (CKO) mice lacking HIF1α in CD11c+ cells, under baseline conditions did not show changes in immune responses suggesting effects of HIF1α only under inflammatory conditions. When these mice were crossed to the Ldlr-/- line and placed on 8 weeks of high fat diet, they developed enhanced plaques with higher T-cell infiltration as compared to the wild-type (WT) controls. The plaques were of a complex phenotype, defined by increased percent of smooth muscle cells (SMCs) and necrotic core area and reduced percent of macrophages and DCs. The mice also displayed enhanced T-cell activation and a Th1 bias in the periphery. The CKO DCs themselves exhibited increased expression of IL 12 and a higher capacity to proliferate and polarize naive T cells to the Th1 phenotype in vitro. The DCs also showed decreased expression of STAT3, in line with the inhibitory effects of STAT3 on DC activation seen in previous studies. When STAT3 was overexpressed in DCs in vitro, IL 12 was down-regulated, but its expression increased significantly on STAT3 inhibition using a mutant vector. In addition, when STAT3 was overexpressed in DCs in vivo using a Cre regulated lentiviral system, the mice showed decreased plaque formation compared to controls. Interestingly, the effects of STAT3 modulation were similar in WT and CKO mice, intending that STAT3 lies downstream of HIF1α. Finally, using a chromatin immunoprecipitation assay (ChIP), it was confirmed that HIF1α binds to hypoxia responsive elements (HREs) in the Stat3 gene promoter thus regulating its expression. When DCs lack HIF1α, STAT3 expression is not stimulated and hence IL 12 production by DCs is uninhibited. This excessive IL 12 can activate naive T cells and polarize them to the Th1 phenotype, thereby enhancing atherosclerotic plaque progression. This project thus concludes that HIF1α restrains DC activation via STAT3 generation and prevents excessive production of IL 12 that helps to keep inflammation and atherosclerosis under check.}, subject = {Dendritische Zelle}, language = {en} } @article{ShannonHein2013, author = {Shannon, Graver and Hein, Melanie}, title = {Tumor cell response to bevacizumab single agent therapy in vitro}, series = {Cancer Cell International}, journal = {Cancer Cell International}, doi = {10.1186/1475-2867-13-94}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97185}, year = {2013}, abstract = {Background Angiogenesis represents a highly multi-factorial and multi-cellular complex (patho-) physiologic event involving endothelial cells, tumor cells in malignant conditions, as well as bone marrow derived cells and stromal cells. One main driver is vascular endothelial growth factor (VEGFA), which is known to interact with endothelial cells as a survival and mitogenic signal. The role of VEGFA on tumor cells and /or tumor stromal cell interaction is less clear. Condition specific (e.g. hypoxia) or tumor specific expression of VEGFA, VEGF receptors and co-receptors on tumor cells has been reported, in addition to the expression on the endothelium. This suggests a potential paracrine/autocrine loop that could affect changes specific to tumor cells. Methods We used the monoclonal antibody against VEGFA, bevacizumab, in various in vitro experiments using cell lines derived from different tumor entities (non small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer (BC) and renal cell carcinoma (RCC)) in order to determine if potential VEGFA signaling could be blocked in tumor cells. The experiments were done under hypoxia, a major inducer of VEGFA and angiogenesis, in an attempt to mimic the physiological tumor condition. Known VEGFA induced endothelial biological responses such as proliferation, migration, survival and gene expression changes were evaluated. Results Our study was able to demonstrate expression of VEGF receptors on tumor cells as well as hypoxia regulated angiogenic gene expression. In addition, there was a cell line specific effect in tumor cells by VEGFA blockade with bevacizumab in terms of proliferation; however overall, there was a limited measurable consequence of bevacizumab therapy detected by migration and survival. Conclusion The present study showed in a variety of in vitro experiments with several tumor cell lines from different tumor origins, that by blocking VEGFA with bevacizumab, there was a limited autocrine or cell-autonomous function of VEGFA signaling in tumor cells, when evaluating VEGFA induced downstream outputs known in endothelial cells.}, language = {en} } @phdthesis{Klinkenberg2011, author = {Klinkenberg, J{\"o}rn}, title = {Physiological Role of Fatty Acid Desaturation in Agrobacterium-induced Arabidopsis Crown Galls}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75262}, school = {Universit{\"a}t W{\"u}rzburg}, year = {2011}, abstract = {Crown gall development is accompanied by hypoxia, drought and oxidative stress. These abiotic stress factors are known to have an impact on fatty acid (FA) desaturation. Thus, an alteration in the lipid profile of plant tumors was expected. A comprehensive lipid analysis of Arabidopsis thaliana crown galls induced by Agrobacterium tumefaciens showed an increase in the degree of FA desaturation. The poly unsaturated fatty acid (PUFA) linolenic acid (18:3) of endoplasmic reticulum (ER) derived phospholipids was especially affected. The increased levels of desaturated FAs were reflected by a strong induction of two genes encoding desaturases, FAD3 and SAD6. In contrast to FAD3, which encodes the ER membrane bound fatty acid desaturase enzyme that synthesizes 18:3 PUFAs in the ER, the function of SAD6 is unknown. The ability of SAD6 to complement the extreme dwarf growth phenotype of the ssi2-2 mutant allele suggests that SAD6 is a functional stearoyl-acyl-carrier-protein delta-9 desaturase (SAD) which catalyzes the first step in FA desaturation and forms stearic acid (18:1). Overexpression of the SAD6 gene in Arabidopsis (SAD6-OE) to a similar degree as in tumors resulted in a light-dependent chlorosis phenotype and caused a similar shift in the lipid profile towards unsaturated phospholipids. Posttranscriptional down-regulation of SAD6 overexpression by RNA reverted the chlorosis phenotype and the changes in the lipid profile, showing that SAD6 overexpression forms the unsaturated FA profile and the phenotype in SAD6-OE. The subcellular localization of the SAD6 protein in chloroplasts, which is obligatory for SAD function was demonstrated. SSI2, which encodes the major contributor to the 18:1 FA levels in Arabidopsis is down-regulated in crown galls pointing to a replacement of SSI2 function by SAD6 in the tumor. SAD6 transcripts were almost undetectable in Arabidopsis under normal growth condition, whereas under hypoxia the gene was strongly activated. In the tumor hypoxia most likely caused the very high transcription of SAD6. Hypoxia is known to limit FA desaturation and it is associated with an elevated reactive oxygen species (ROS) production which is detrimental for unsaturated FAs. Thus, up-regulation of SAD6 in the crown gall, most likely serves as an adaptive mechanism to activate desaturation under low oxygen concentrations and to maintain the levels of unsaturated FA under oxidative stress. The ER localized FAD3 most likely is responsible for the rise in 18:3 of the phospholipid class to cope with drought stress in crown galls. This hypothesis was supported by the loss of function mutant, fad3-2, which developed significantly smaller tumors as the wild type under low relative humidity.Taken together, this study suggests that the induction of SAD6 and FAD3 shapes the tumor lipid profile by increasing the levels of unsaturated FAs. Unsaturated fatty acids prepare the crown gall to cope with ongoing hypoxia, drought and oxidative stress during growth and development.}, subject = {Agrobacterium tumefaciens}, language = {en} }