Gene transfer of keratinocytes with a human factor XIII plasmid construct [Elektronische Ressource] / vorgelegt von Laura Gabriella Horváth
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Gene transfer of keratinocytes with a human factor XIII plasmid construct [Elektronische Ressource] / vorgelegt von Laura Gabriella Horváth


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Aus der Chirurgischen Universitätsklinik der Albert - Ludwigs - Universität Freiburg / Br. Abteilung Plastische und Handchirurgie Gene transfer of keratinocytes with a human Factor XIII - plasmid construct INAUGURAL – DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinschen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt 2002 von Laura Gabriella Horváth geboren in Gy őr 2 Dekan Professor Dr. J. Zentner 1. Gutachter Professor Dr. G.B. Stark 2. Gutachter Professor Dr. W. Vanscheidt Jahr der Promotion 2004 3TABLE OF CONTENTS I. INTRODUCTION 1 1.1. Normal wound healing 1.2. Transplantation of keratinocytes 3 1.3. Gene therapeutic approaches in wound healing 3 1.4. Factor XIII 5 1.4.1. History of blood coagulation factor XIII 5 1.4.2. Protein structure of FXIII 6 1.4.3. Role of FXIII in the blood coagulation 6 1.4.4. FXIII and wound healing 9 1.5. Aim of study 10 II. MATERIALS AND METHODS 11 2.1. Molecular cloning 11 2.1.1. Restriction 2.1.2. Ligation 12 2.2. Introduction of plasmid DNA into E. coli cells 13 2.3. Analyse of the DNA 13 2.3.1. Agarose gel electrophoresis 13 2.3.2. Recovery of DNA fragments 15 2.4. Isolation of plasmid DNA 15 2.4.1. Small scale purification of DNA 15 2.4.2. Large scale purification of DNA 16 2.5.



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Aus der Chirurgischen Universitätsklinik der Albert - Ludwigs - Universität Freiburg / Br. Abteilung Plastische und Handchirurgie
Gene transfer of keratinocytes with a human Factor XIII - plasmid construct
INAUGURAL  DISSERTATION zur Erlangung des Medizinischen Doktorgrades der Medizinschen Fakultät der Albert-Ludwigs-Universität Freiburg i. Br. Vorgelegt 2002 von Laura Gabriella Horváth geboren in Győr
Dekan1. Gutachter 2. Gutachter Jahr der Promotion
Professor Dr. J. Zentner Professor Dr. G.B. Stark Professor Dr. W. Vanscheidt 2004
TABLE OF CONTENTS I. INTRODUCTION 1 1.1. Normal wound healing 1 1.2. Transplantation of keratinocytes 3 1.3. Gene therapeutic approaches in wound healing 3 1.4. Factor XIII 5 1.4.1. History of blood coagulation factor XIII 5 1.4.2. Protein structure of FXIII 6 1.4.3. Role of FXIII in the blood coagulation 6 1.4.4. FXIII and wound healing 9 1.5. Aim of study 10 II. MATERIALS AND METHODS 11 2.1. Molecular cloning 11 2.1.1. Restriction 11 2.1.2. Ligation 12 2.2. Introduction of plasmid DNA into E. coli cells 13 2.3. Analyse of the DNA 13 2.3.1. Agarose gel electrophoresis 13 2.3.2. Recovery of DNA fragments 15 2.4. Isolation of plasmid DNA 15 2.4.1. Small scale purification of DNA 15 2.4.2. Large scale purification of DNA 16 2.5. Determination of concentration and purity of DNA 17 2.6. Protein-analyses 18 2.6.1. In vitro translation of the proteins 18 2.6.2. SDS polyacrilamide denaturing electrophoresis (SDS-PAGE) 19 Analysis of radioactive labeled gels by autoradiography 21 2.7. Keratinocyte culture 22 2.8. Transfection 23 2.9. Detection of FXIII protein 24 III. RESULTS 25 3.1. Molecular cloning 25 3.2. Screening of pCMX-pl1 recombinants for the successfully FXIII gene insertion 26 3.3. In vitro translation of FXIII protein 27 3.4. Transfection 28 IV. DISCUSSION 29 V. SUMMARY 36 VI. REFERENCES 37 VII. REAGENTS 45 VIII. LIST OF ABBREVIATIONS 46 AcknowledgmentsCurriculum vitae
I. INTRODUCTION I. INTRODUCTION Chronic, non-healing wounds still present a significant challenge for the clinician and cause considerable morbidity for the patient. New data have been collected and analyzed recently [74]. The latter report concludes that the prevalence of chronic leg ulcers, the most frequently occurring non-healing disorder, is 1-2% of the population at any given time. Taking into consideration that this affects a lot of people; it has not only medical, but also economical and social consequences. Therefore, the treatment of choice for chronic wound care should not only be up to the standards of modern patient care, but it is also desirable to be cost-effective. 1.1. Normal wound healing Skin completely covers the mammalian body and protects it from the outer environment. The outer layer of the skin is a multilayered, stratified squamous epithelium, the epidermis. Epidermal cell growth occurs among the cells of the basal layer. To maintain a constant thickness, cell growth in the lower epidermis balances the loss of cells from the superficial epidermis. Characteristic of all epithelial cells is the propensity to cover a free surface. When an injury occurs, the integrity of the epidermis is damaged. In order to cover the denuded surface, epithelial cells either move or grow over the wounded area. Although wounding stimulates both processes, the more important process in early wound closure is cell migration [23;59]. The migrating cells arise from the residual epithelium at the periphery of the wound or from the residual hair or sweat gland structures (i.e. skin appendages) at the wound base [23;49]. Within 1 or 2 days following epidermal injury, the epithelial cells behind the migrating front begin to proliferate, generating new populations of cells to cover the wound [13]. Wound healing is a well-organized response to tissue injury that involves several separate, but closely related phases, which include inflammation, proliferation and remodeling [66]. As a result of tissue injury, the coagulation cascade is activated in order to limit blood loss. The primary function of blood coagulation is hemostasis. Platelets become trapped in the clot, their aggregation and degranulation is essential for the hemostasis as well as for the subsequent normal inflammatory response. Platelets and damaged cells at the site of vascular injury release numerous biologically active mediators like platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-ß), which attract and activate fibroblasts, endothelial cells and macrophages [1;89].
I. INTRODUCTION As a part of the early inflammation reaction, infiltration by polymorphonuclear leukocytes (PMNs) ensues. The major function of PNM is to remove bacteria and debris from the wound, thereby to prevent clinically significant infection. Parallel to thrombocyte activation, a cascade of proteolytic reaction leads to activation of serin proteases that convert fibrinogen to fibrin. The activated form of factor XIII (FXIIIa) transforms soluble fibrin into its insoluble form, thus stabilizes fibrin and contributes to the formation of net-like structures. This event initiates the formation of a provisional wound matrix. Within 48-96 h post-injury monocytes enter the wound and differentiate into macrophages [61]. Macrophages just like neutrophils phagocytose and in addition they release cytokines like tumor-necrosis-factor-α, IL-1, bFGF and TGF-ß, which are responsible for both the production and the proliferation of the extracellular matrix (ECM) by fibroblasts. Furthermore they attract endothelial cells to migrate into the wound area and enhance neoangiogenesis, the formation of new blood vessels [93]. Fibroblasts are the main cells in the repair process that are responsible for production of the majority of structural proteins during tissue reconstruction. At 5 to 7 days following tissue injury, fibroblasts begin to synthesize collagen in order to provide strength and restoration of integrity encompassing the entire injury site. Collagens play an important role in all phases of wound healing. Immediately after injury, exposed collagen comes into contact with blood and promotes platelet aggregation and activation of chemotactic factors involved in the response to injury. Later, collagen becomes the foundation of the wound extracellular matrix and a significant component of the final scar. Remodeling is the last and longest phase of healing. The principal processes occurring during this phase are the dynamic remodeling of collagen, addition of elastin and the formation of the mature scar, whereby the tensile strength of the wound continues to increase. During this latter phase keratinocytes begin to migrate from the wound periphery to the center of the wound using the provisional wound matrix as a guideline. For their migration, localized proteolysis is necessary, which can be achieved by secretion of proteolytic enzymes into the extracellular matrix. The complexity of wound healing indicates that damaged tissue can only be repaired by a precise coordination of successive processes.
I. INTRODUCTION 1.2. Transplantation of keratinocytes Rheinwald and Green were the first to establish a human keratinocyte culture that could be propagated for several hundred generations [83]. Later, advances in the cell culture technique rendered the in vitro cultivation of multilayer keratinocytes, the cultured epidermal cell sheets (CEA), which have become a recognized method for the coverage of wounds [21;41]. Recently a modification of keratinocyte autografts has been executed. The new technique includes transplantation of human keratinocytes suspended as single cells in fibrin glue (KFGS, Keratinocytes Fibrin Glue Suspension). This method has been successfully applied for the treatment of chronic wounds or burns [53;88]. Fibrin glue is a biological substance, containing mainly fibrinogen, thrombin and blood clotting factor XIII, which mimics the end of the coagulation cascade. Because of its physiological property it has been extensively used in surgical field for the fixation of tissue,as well as for a delivery of growth factors [32;92]. Both clinical and experimental results show that the application of KFGS leads to complete epithelization of experimental wounds at least equivalence to the treatment with sheet grafts. Moreover, earlier availability, easier handling, better epithelization quality and cost reduction were achieved compared to standard epidermal sheet grafts. The KFGS technique combines the effect of fibrin glue to serve as a matrix, physiologically involved in the early stage of wound healing with the mechanical adhesion of cell suspensions to wound tissue. Furthermore, due to the transplantation of a non contact-inhibited single cell suspension in an appropriate biological carrier, the grafted cells can attach, proliferate, and differentiate in vivo [47]. 1.3. Gene therapeutic approaches in wound healing Recent advances in molecular biology have opened new perspectives in the therapy of wound healing disturbances. This new field, known as gene therapy, is defined as the genetic modification of cells for therapeutic purpose[30]. The accessibility of the skin and the proliferative capacity of cultured epidermal cells make keratinocytes, the main cells of the epidermis, ideal candidates for genetic manipulation and gene therapy. The goal of gene therapy is to transfer the DNA of interest into cells, thereby allowing the DNA to be synthesized and the protein, coded by this DNA, expressed in these cells. Following this aim, the genetic information coded for this protein (DNA fragment) has to be integrated into a DNA-vector (plasmid vector) through covalent bindings. The recombinant plasmid that is
I. INTRODUCTION formed, is a circular, extrachromosomally localized element, which can be make produced by bacteria. In order to achieve an efficient transcription, the DNA is inserted next to a strong promotor in the vector. DNA can be transferred into cells by a variety of techniques, which can be broadly divide into two categories; the non-viral and the viral methods. Both methods have advantages and disadvantages. However, non-viral gene transfer efficiencies are lower than those obtained by viral methods, they represent an easy method and can be applied repeatedly. Furthermore,in vitro retroviral gene transfer is laborious and has its limitations, especially regarding its clinical applicability.used non-viral gene transfer method is the liposomalA widely transfection, called lipofection. This technique relies upon the anionic properties of DNA, the cationic lipids and the negatively charged cell surface. The liposomes are mixed with the DNA of interest resulting in a non-covalently bound liposome-DNA complex. For lipofection, the liposome-DNA complex is added to the target cells. It adsorbs to the cell membrane and delivers the DNA into the cytoplasm. The cell will then express the protein, coded by this DNA. Application of gene therapy to wound healing can take the following general approaches: to create a plasmid construct containing the gene of interest, to carry out transfection in vitro or in vivo to isolate human keratinocytes from the skin and cultivate them in vitro by transferring the gene of interest throughto create genetically engineered keratinocytes transfection check the overexpressing of the protein, coded by gene, in the supernatantto to re-implant these cells to the wound area, for the purpose of covering the skin defect and expressing the proteins at the site of injury Transplantation of genetically modified, autologous keratinocytes, which express growth factor genes could be a new gene-therapeutic approach in the therapy of chronic wounds.Several groups reported on the successful in vitro gene transfer of keratinocytes with different gene transfer methods and different growth factors [29;31;51;97;98]. Some of them showed stimulated proliferation of epithelial cells in vivo after transplantation of genetically modified keratinocytes [29].Skin substitutes, which used genetically modified allogeneic cells, could be applied to wounds to act as a temporary dressing that sustained the local synthesis and
I. INTRODUCTION secretion of growth factors for wound healing [73]. It may also be used as an in vivo Drug-Delivery-System for other therapeutic proteins in the treatment of chronic wounds. 1.4. Factor XIII 1.4.1. History of blood coagulation factor XIII The story of blood coagulation factor XIII (FXIII) began in 1923, when Barkan et al. [3] observed the insolubility of fibrin clots in weak bases when formed in the presence of Ca++. In 1944 Robbins et al. [84] repeated and extended this experiment with purified fibrinogen and concluded that Ca++not enough to keep the clot insoluble in weak acids and bases. Heitself is presumed the existence of an additional serum factor. Two Hungarian researcher, Laki and Lóránd are considered to be the discoverer of FXIII. They were the first to realize that this thermolabile and non-dialysable serum factor was required to make the fibrin clot insoluble in concentrated urea solution [58;62;63]. They called this protein fibrin-stabilizing factor (FSF). As an acknowledgment of their contribution, the fibrin-stabilizing factor had also been called for some time, Laki  Lórand or L-L factor. The factor was purified, and the enzymatic nature of fibrin-stabilizing factor was first revealed and explored by Loewy et al [60]. Based on the knowledge of the enzyme nature of FSF provided by Loewys group, Buluk et al [12] first proposed a reaction sequence for fibrin stabilization that included the activation of FSF by thrombin in the presence of calcium ions. In 1961, the first congenital hemorrhagic diathesis was diagnosed in Switzerland that was due to deficiency in fibrin stabilizing factor [25]. Compared to other, more common hemorrhagic diathesis like hemophilia, wound healing disorders and aborts could be observed besides enhanced bleeding tendency. Soon after this discovery, in 1963, the International Committee on Blood Clotting Factors acknowledged fibrin-stabilizing factor as a clotting factor and termed it factor XIII. In 1986 and 1990 the next important step in the FXIII research was taken: it succeeded to elucidate the complete cDNA sequence of factor XIII A [42;48;90] and the B subunit of factor XIII [43].
I. INTRODUCTION 1.4.2. Protein structure of FXIII FXIII is a protein containing 732 amino acids, which has an important role in the regulation of maintaining balance between two opposing processes: the coagulation and the fibrinolysis. It is distributed intracellular in platelets, megakaryocytes and macrophages, extracellular it is found in plasma [17]. The plasma protein consists of a heterotetramer (A2B2) of a molecular weight of 300.000-350.000. It contains two copies of each the A and B subunits that are hold together by non-covalent bindings. The A subunit contains the active sites, whereas the B unit is non-catalytic, seems to fulfill the role of a carrier. Furthermore, it protects and stabilize the A chains in the plasma against degradation. FXIII molecules from the blood platelets, megakaryocytes and macrophages are homodimers (A2)composed of two identical A subunits with the molecular weight of about 80.000. The A catalytic subunits are identical both in the plasma and intracellular. The B units are synthesized in the liver [101], the primary site for the synthesis of the A chains, cells of bone marrow origin were identified [75], the production of A chains by hepatocytes is underlying. Recent observation pointed out the importance of a common coding polymorphism in the A-subunit gene of FXIII (FXIIIVal34Leu). This point mutation occurs very close to the thrombin activation site and results in a ValineLeucin change. It suggest to be protective against myocardial infarction [56], venous thrombosis [15] and brain infarction [28]. However, this mutation promotes formation of intracerebral hemorrhage [14]. 1.4.3. Role of FXIII in the blood coagulation In the cascade of events leading to the final clot e.g. at the end of both the intrinsic and the extrinsic coagulation pathway is the activation of thrombin from its precursor prothrombin. Thrombin fulfills two main roles: to activate blood coagulation factor XIII and to build fibrin from its previous form fibrinogen by proteolytic action. The activation of FXIII is a multistep process, which requires the presence of calcium and4 fibrinogen besides thrombin. At the beginning of the activation by thrombin a 37 aminoacid-containing-peptide will be released from the NH2terminal of the molecule. Immediately after the cleavage calcium ions induce changes in the conformation of the molecule and subsequently the tetramer molecule becomes dissociated. As a consequence of the dissociation, FXIII changes into its enzymatically active form (FXIIIa-also called activated fibrinstabilizing factor, fibrinoligase or plasma transglutaminase). FXIIIa contains a cystein
I. INTRODUCTION in the active center [68] and acts as a transglutaminase. It catalyses covalent cross-linking reactions between proteins by forming of peptide like bonds by a transamidation reaction between aχ-Carboxyl-group of glutamine and anε-Amino-group of lysine. Clotting factor XIII stabilizes the aggregates of fibrin formed during blood clotting. The early fibrin, deriving from fibrinogen, is soluble with little stability. Activated FXIII catalyses the formation of strong covalent bonds among the fibrin fibers, through which fibrin aggregates get their stable, insoluble form (Figure 1.). Moreover, FXIII possesses a high degree of substrate specificity for numerous, physiologically important matrixproteins like fibronectin, collagen, von Willebrand-factor, actin andα2antiplasmin, which will be cross-liked to fibrin (Table 1.). SubstratetCross-linkedFunctiono fibrin fibrin stabilization of the clot 2-antiplasminfibrintchlortormebsiisntancetofibrinolysesbyinhibitionoffibronectin fibrin formation of provisional matrix for cell migration collagen fibrin formation of provisional matrix for cell migration von Willebrand factor fibrin attachment of clot to the vessel wall von Willebrand factor collagen attachment of clot to the vessel wall fibronectin collagen attachment of clot to the vessel wall Factor V actin attachment and spreading of endothelial cells Table 1.: Factor XIIIa-mediated cross-linking reactions This latter is the most potent inhibitor of plasmin and protects clot against a quick dissolution. During tissue injury basic structures of the extracellular matrix like collagen and fibrinogen are released. Cross-linking of these proteins to fibrin is not only for the adhesion of clot to the vessel wall of great importance, but it enhances the reorganization of both cellular and extracellular matrices [80].
I. INTRODUCTION 8 The fibrin-rich clot, composed of cross-linked fibrin, collagen, fibronectin andα2-antiplasmin provides a provisional matrix for cell, primary for fibroblast, migration and attachment. Besides the normal coagulation, plasma FXIII is also consequently involved in tissue repair and wound healing. Role of FXIII in the blood coa ulation cascade
intrinsic pathway extrinsic pathway
Meaning of the arrows: activation
Fibrin ( soluble )
Fibrin ( insoluble )
Fibrin degradation products
FXIII inactive
+ Ca+
XIII active
Figure 1.: FXIII and the coagulation cascade. FXIII is circulating in an inactive form and will be activated by thrombin in the presence of calcium and fibrinogen. Activated FXIII catalyses the formation of insoluble fibrin aggregates. Moreover, it bindsα2 to cross-liked fibrin, antiplasmin through which it inhibits the fibrinolytic action of plasmin.