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Doctoral Thesis Doctoral thesis submitted in partial fulfilment of the requirements for the academic degree “Doctor of Philosophy (PhD)” Integrin alpha V and alpha 5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells. Innsbruck, December 2016 Department of Plastic, Reconstructive and Aesthetic Surgery Dissertation committee, support and supervision: Prof. Dr. med. Gerhard Pierer, Prof. Dr. Günther Lepperdinger, Dr. Christian Ploner, PhD submitted by: Dr. med. univ. Evi M. Morandi 1 This work was written and submitted by Dr. med. univ. Evi M. Morandi Andreas Hofer Str. 26 6020 Innsbruck, Austria Supervisor: Dr. Christian Ploner, PhD The original paper containing this work has been accepted for publication in the open access online journal Scientific Reports on 09.06.2016 and was published on 01.07.2016: ITGAV and ITGA5 diversely regulate proliferation and adipogenic differentiation of human adipose derived stem cells. Morandi EM, Verstappen R, Zwierzina ME, Geley S, Pierer G, Ploner C. Sci Rep. 2016 Jul 1;6:28889. Innsbruck, December 2016 2 To my husband. 3 Table of contents 1. Introduction .........................................................................................................................6 1.1. Integrins, the niche and their physiologic significance in tissue engineering .........................6 1.2. Integrin signaling and relevant intracellular pathways ..........................................................7 1.3. Integrins and disease ............................................................................................................9 1.4. Integrins and differentation ................................................................................................ 11 1.5. RDG motif recognizing integrins and their substrates ......................................................... 12 2. Materials and Methods ......................................................................................................... 14 2.1 Isolation and cell culture of human ASC ............................................................................ 14 2.2. Isolation of lipid droplet containing adipocytes by isopycnic centrifugation. ...................... 15 2.3. Immunohistochemistry ...................................................................................................... 16 2.4. Proliferation assay ............................................................................................................. 16 2.5. Adhesion assay .................................................................................................................. 16 2.6. Spreading assay ................................................................................................................. 17 2.7. ASC trilineage differentiation ............................................................................................ 17 2.8. Plasmid Construction and lentiviral transduction ................................................................ 18 2.9. RNA isolation and quantitative RT-PCR ............................................................................ 19 2.10. Protein isolation from in-vivo differentiated adipocytes ..................................................... 22 2.11. Immunoblotting ................................................................................................................. 22 2.12. Flow cytometry ................................................................................................................. 23 2.13. Pathscan Multi-Target Sandwich ELISA ............................................................................ 23 2.14. Statistics ............................................................................................................................ 24 3. Results .............................................................................................................................. 25 3.1. Integrin expression patterns of differentiated adipocytes and ASC ..................................... 25 3.2. RGD-receptors ITGA5 and ITGAV are repressed during adipogenesis............................... 30 3.3. Loss of ITGAV moderately induced cell death and reduced cell proliferation ..................... 32 3.4. Loss of ITGAV impacts on cell adhesion and spreading ..................................................... 35 3.5. Intracellular signaling pathways are differentially regulated by ITGAV and ITGA5 ........... 38 3.6. Loss of ITGAV mediated upregulation of p21 ................................................................... 41 3.7. Loss of ITGAV promotes adipogenic differentiation of ASC ............................................. 42 3.8. Intracellular signaling mediated by ITGAV and ITGA5 in adipogenesis............................. 45 3.9. Pharmacological inhibition of ITGAV/B3 and ITGAV/B5 by cilengitide mimics loss of ITGAV in ASC ............................................................................................................................. 47 4 4. Discussion ......................................................................................................................... 51 5. Clinical and translational significance of this work............................................................. 59 5.1. Joining the ranks of plastic surgeons in basic research – how to connect basic research to plastic surgery ............................................................................................................................... 59 5.2. Big story short - A brief history and the fundamentals of tissue engineering ....................... 61 5.3. Lipografting: an excellent example of translational application of scientific knowledge in clinical plastic surgery................................................................................................................... 65 6. Personal acknowledgements .............................................................................................. 67 7. Funding statement ............................................................................................................. 68 8. Conflicts of interest ........................................................................................................... 68 9. Personal contribution ......................................................................................................... 68 10. Abstract ............................................................................................................................. 69 11. References ......................................................................................................................... 70 12. List of abbreviations .......................................................................................................... 86 13. Curriculum vitae ................................................................................................................ 90 5 1. Introduction 1.1. Integrins, the niche and their physiologic significance in tissue engineering In regenerative medicine, exerting influence on cell viability and differentiation is of great interest in regenerative medicine, as reconstructing complex soft tissue defects still remains a major clinical challenge. Tissue engineering techniques, extracellular matrix (ECM) scaffolds and the application of multipotent adipose derived stem cells (ASC) (1) are largely investigated attempts in preclinical and translational research. Knowledge about possible external influence on ASC physiology as well as clinical experience in this field is still limited. Although mesenchymal stem cells (MSC) and their developmental potential are well characterized, the use of this knowledge for the efficient generation and manipulation of MSC-derived cells for tissue engineering in medical applications remain unclear. An upcoming body of literature describes multiple effects of the extracellular matrix (ECM) on MSC and ASC physiology, including proliferation and differentiation. Figure 1: simplified scheme: cell niche, cellular interactions and specific functions in cell physiology that are influenced by biochemical, molecular and mechanical cues from the niche and cell cell interactions. 6 The ECM mediates these effects by specific molecular composition and mechanical properties (2-4). Characterizing interactions between cells and the ECM is therefore crucial for in-vitro expansion and differentiation of MSC as well as ASC as a multitude of functional, spatial and mechanical cues impact on cells and tissues, each of them being molecularly translated to finally end up in cellular response and herein answering the three major questions about cell fate: where is the cell, what is its purpose and what is the exact role in the integrity of the specific tissue? Answering these questions is not only crucial to cell fate but is also considered the key to success in tissue engineering. The surrounding three-dimensional environment of cells is referred to as the niche. The niche itself is composed of paracrine messenger substances, proteins that compose the ECM, other cells and the intrinsic factors influencing the physiology of the cells, thus of all molecular and mechanical cues with impact on stem cell fate. Moreover, also oxygen content, reactive oxygen species and growth factor gradients as well as cell crosstalk and cell-ECM interactions contribute to the niche. However, only little is known about its maintenance, regulation and homeostasis. On the other hand,
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