2-D and 3-D Adipocyte Cell Culture
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2-D and 3-D Adipocyte Cell Culture Promising Tools for Basic Research and Approaches towards Clinical Therapies Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) der Fakultät Chemie und Pharmazie der Universität Regensburg vorgelegt von Anna Katharina Seitz aus Neumarkt i.d.OPf. im Juni 2010 Diese Doktorarbeit entstand in der Zeit von März 2006 bis Juni 2010 am Lehrstuhl Pharmazeutische Technologie der Universität Regensburg. Die Arbeit wurde von Prof. Dr. Achim Göpferich angeleitet. Promotionsgesuch eingereicht am: 21.06.2010 Mündliche Prüfung am: 30.07.2010 Prüfungsausschuss: Prof. Dr. S. Elz (Vorsitzender) Prof. Dr. A. Göpferich (Erstgutachter) Prof. Dr. T. Blunk (Zweitgutachter) Prof. Dr. E. Tamm (Drittprüfer) Meinen Eltern in Liebe und Dankbarkeit 2-D and 3-D Adipocyte Cell Culture Table of Contents 2-D and 3-D Adipocyte Cell Culture - Promising Tools for Basic Research and Approaches towards Clinical Therapies…………………………………………... 1 Chapter 1 Introduction……………………………………………….................... 3 1. Characteristics of adipose tissue………………………………… 4 1.1. Structure of adipose tissue………………………………………. 4 1.2. Functions of adipose tissue……………………………………… 4 1.3. Adipocyte differentiation………………………………………... 6 1.4. Adipocyte metabolism………………………………………….... 8 2. The adipocyte extracellular matrix…………………………….... 10 2.1. The extracellular matrix composition of adipose tissue………… 10 2.2. Extracellular matrix dynamics during adipogenesis……………. 14 2.3. Novel culture systems for the investigation of the extracellular matrix……………………………………………… 16 3. Adipocyte culture in reconstructive and plastic surgery………… 17 3.1. Injection lipolysis………………..………………………………. 17 3.2. Adipose tissue engineering………………………………………. 19 Chapter 2 Goals of the Thesis……………………………………………………. 41 Chapter 3 Human Adipose-derived Stem Cells as an in vitro Model for Human Adipogenesis - Evaluation of Culture Conditions………… 47 Chapter 4 In vitro Studies of the Lipolytic Effect of Therapeutic Substances Used for Injection Lipolysis…………………………………………... 71 Chapter 5 Effect of a Prolyl Hydroxylase Inhibitor on Adipogenesis in 2-D and 3-D Culture………………………………………………….......... 101 Chapter 6 Collagen Type XVI is Regulated during in vitro Adipogenesis of 3T3-L1……………………………………………………………….... 133 -v- 2-D and 3-D Adipocyte Cell Culture Chapter 7 Poly(ethylene glycol)-based Hydrogels for Adipose Tissue Engineering……………………………………………………. 165 Chapter 8 Summary and Conclusions………………………………………….… 187 Appendices …………………………………………………………………………. 195 I) List of Abbreviations…………………………………………...... 196 II) Curriculum Vitae…………………………………………............ 201 III) List of Publications………………………………………………. 203 IV) Eidesstattliche Erklärung………………………………………... 205 V) Acknowledgments………………………………………….......... 207 -vi- 2-D and 3-D Adipocyte Cell Culture Promising Tools for Basic Research and Approaches towards Clinical Therapies You must have a plan, if you don´t have a plan, you´ll become part of somebody else´s plan. (Terence McKenna) - 1 - Chapter 1 Introduction - 3 - Chapter 1 Introduction 1. Characteristics of adipose tissue 1.1. Structure of adipose tissue Adipose tissue represents a loose connective tissue mainly consisting of mature lipid-filled adipocytes and, a variety of other cell types such as stromal vascular cells including blood cells, endothelial cells, pericytes, adipose precursor cells, and fibroblasts as well as immune cells [1-3]. The mature adipocytes are closely arranged within the tissue resulting in a hexagonal configuration. Spaces between the cells are filled by a specific fiber network referred to as extracellular matrix (ECM) which is interspersed with capillaries. Not only due to its color, adipose tissue is classified into two subtypes, white adipose tissue (WAT) and brown adipose tissue (BAT), which differ in their abundance and function in humans [4]. BAT is principally found in neonates and only marginally in the adult body and is specialized for heat production by adaptive non-shivering thermogenesis [4-6]. The BAT is extremely well vascularized and the adipocytes are characterized by densely packed mitochondria as well as by the accumulation of triglycerides (TGs) in multiple small lipid droplets [4,7]. However, this work focuses only on WAT as it is the major object of the current adipose research. WAT is the predominant type of fat tissue in adult humans and is widely distributed in multiple depots in the body. The total fat mass is locally classified into subcutaneous (~ 80%) and internal (~ 20%) adipose compartments. The internal fat depots include visceral and nonvisceral fat as well as other small fat depots [4]. White fat acts as the major energy reservoir by storing TGs in periods of energy excess and releasing free fatty acids during energy deprivation. The growth of WAT results from increased adipocyte number (hyperplasia) and, respectively, adipocyte size (hypertrophy) [2]. Due to the accumulation of lipids, adipocytes reach sizes ranging from 20 µm to 200 µm in diameter [4]. The mature unilocular adipocyte contains a large single droplet surrounded by a thin rim of cytoplasm, and a nucleus located peripherally which gives the cell a so called signet-ring form [8]. However, developing adipocytes temporarily contain multiple lipid droplets, before these finally coalesce into a single large droplet. Multilocular appearance is also present at periods of nutrient deprivation due to mobilization of TGs. 1.2. Functions of adipose tissue Until the 1980s, the most important functions of fat seemed to be heat insulation, mechanical cushion, and of course, a source of energy. Adipose tissue stores energy in times of nutritional excess in the form of TGs and release free fatty acids and glycerol when energy is required - 4 - Chapter 1 Introduction [9,10]. However, during the last two decades, particularly through the identification of leptin in 1994 [11], it became increasingly clear that the fat tissue also has active roles in physiological processes by mediating endocrine, paracrine, and autocrine signals. Aside from the primary function as energy reservoir, the WAT is also described as a highly active metabolic and endocrine organ secreting a large number of protein factors and signals termed as adipokines [5,12,13]. Adipokines and other adipocyte-secreted factors include leptin, adiponectin, resistin, visfatin, apelin, and inflammatory cytokines like tumor necrosis factor alpha (TNF-α), monocyte chemoattractant protein-1 (MCP-1), and plasminogen activator inhibitor protein (PAI) as well as growth factors, enzymes, complement factors, and matrix proteins [4,6]. These factors act as mediators for multiple physiological processes, i.e., energy homeostasis, insulin sensitivity, lipid metabolism, inflammatory and immunological responses, angiogenesis, blood pressure, and reproductive function [14,15]. A large body of evidence indicates that many factors secreted by adipocytes are dysregulated in obesity contributing to hyperglycemia, hyperlipidemia, chronic inflammation, and insulin resistance [9,16-18]. Thus, WAT dysfunction is involved in the pathogenesis of type 2 diabetes and other metabolic diseases, such as atherosclerosis. Particularly leptin, a hormone that is primarily synthesized and secreted by mature adipocytes is elevated in human obesity and in animal models of obesity. Different roles of leptin are suggested including regulation of body fat mass, modulation of insulin action in liver, production of steroids in ovary, and direct effects on adrenocortical steroidogenesis. Another adipokine, the cytokine TNF-α, is upregulated in obesity which contributes to insulin resistance by the inhibition of the insulin receptor signalling through the insulin receptor substrate 1 (IRS-1) [19,20]. The two vascular function-related proteins angiotensinogen and plasminogen activator inhibitor type I (PAI-1) are responsible for mediating vasoregulatory functions of adipose tissue such as blood supply and fatty acid efflux from fat [21]. Angiotensin II, the cleavage product of angiotensinogen, increases lipogenesis in adipocytes and, thus, angiotensin II may play a role in the control of adiposity through regulation of lipid synthesis and storage in adipocytes [22]. Higher levels of PAI-1, a fibrinolytic protein, have been reported in obesity. Therefore, PAI-1 is assumed to be involved in the development of cardiovascular diseases associated with obesity [23,24]. Taken together, multiple studies from literature have clearly shown that adipose tissue can be regarded as endocrine organ which is involved in various physiological processes in addition to its main role in regulation of energy balance. - 5 - Chapter 1 Introduction 1.3. Adipocyte differentiation The adipose lineage arises from a pluripotent stem cell population of mesodermal origin which has the capacity to differentiate into various cell types such as adipocytes, chondrocytes, osteoblasts, and myocytes [9]. These multipotential precursor cells reside in the stromal vascular compartment of adipose tissue and undergo a commitment step, in which cells become restricted to the adipocyte lineage. Figure 1 illustrates an overview of the different stages of adipocyte development [9]. Figure 1: Overview of the adipocyte development (from Gregoire et al. [9]). Abbreviations: C/EBP=CCAAT/enhancer binding protein, PPAR=peroxisome proliferator-activated receptor, pref-1=preadipocyte factor 1, ECM=extracellular matrix. Adipocyte differentiation is induced on the molecular level by a coordinated transcriptional