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In Vivo Fluorescence Imaging of E-Selectin: Quantitative Detection of Endothelial Activation in Arthritis

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In Vivo Fluorescence Imaging of E-Selectin: Quantitative Detection of Endothelial Activation in Arthritis In Vivo Fluorescence Imaging of E-Selectin: Quantitative Detection of Endothelial Activation in Arthritis A thesis submitted for the degree of PhD by Luke Gompels August 2010 Kennedy Institute of Rheumatology Faculty of Medicine Imperial College SUPERVISORS Dr Ewa Paleolog Prof. Dorian Haskard 1 Abstract Rheumatoid arthritis (RA) is a chronic progressive systemic inflammatory disease, characterized by synovial inflammation and localized destruction of cartilage and bone. Heterogeneity in the clinical presentation of RA and uncertainty about which patients will respond to treatment makes diagnosis and management challenging. Fluorescent imaging in the near infrared (NIR) spectrum significantly decreases tissue autofluorescence offering unique potential to detect specific molecular targets in vivo. E-selectin or endothelial adhesion molecule-1 (ELAM-1), a 115kDa glycoprotein induced on endothelial cells in response to pro-inflammatory cytokines involved in RA, such as interleukin (IL)-1 and tumour necrosis factor (TNF. E-selectin has been well validated as a potential biomarker of disease activity. My study aimed to investigate whether E-selectin targeted optical imaging in vivo could be developed as a sensitive, specific and quantifiable preclinical molecular imaging technique, and also whether this approach could be used to delineate the molecular effects of novel therapies. I utilised anti-E-selectin antibody labelled with NIR fluorophore in a mouse model of paw swelling induced by intra-plantar injection of TNFand in acute collagen-induced arthritis (CIA) in DBA/1 mice, a widely used model of RA. E-selectin generated signal, localised to points of maximal clinical inflammation in the inflamed mouse paw in both models with significant differences to control antibody. Binding of anti-E-selectin antibody was also demonstrated by immunohistochemistry in both models. The ability of E-selectin targeted imaging to detect sub-clinical endothelial activation was also investigated, demonstrating that E- selectin may be an excellent way of determining subclinical vascular activation in CIA. Finally the effect of novel targeted therapy – RB200 which blocks epidermal growth factor (EGF) signalling was investigated. This demonstrated that E-selectin targeted signal could be absolutely abrogated to a level seen in unimmunised healthy control animals, following combination treatment with RB200 and the TNF inhibitor etanercept. E-selectin targeted optical imaging is a viable in vivo imaging technique that can also be applied to quantify disease and investigate the effects of novel molecular therapies. It holds significant promise as a molecular imaging technique for future translation into the clinic for patients with rheumatoid arthritis and other inflammatory diseases. 2 Table of Contents Abstract ................................................................................................................... 2 List of Figures ............................................................................................................ 8 List of Tables ............................................................................................................ 11 List of Appendices ................................................................................................... 11 List of Abbreviations ................................................................................................ 12 Statement of Originality ........................................................................................... 14 Acknowledgements .................................................................................................. 15 CHAPTER 1 ........................................................................................... 16 1 Introduction ....................................................................................................17 1.1 Rheumatoid Arthritis ..............................................................................17 1.1.1 RA: Chronic Inflammation of The Synovium ......................................... 20 1.1.1.1 Histopathological changes to synovial architecture in RA ............... 20 1.1.1.2 Cellular and soluble mediators of joint damage in RA ..................... 21 1.1.2 Targeted Therapeutic Approaches for the Treatment of RA ................... 22 1.2 The Vascular Endothelium: An Interface for the Interaction between Cytokines and Cell Surface Receptors....................................................24 1.2.1 The Adhesion Cascade: A Complex Multistep Process .......................... 24 1.2.2 E-Selectin: A Pivotal Endothelial Cell Adhesion Molecule for Leukocytes .............................................................................................. 27 1.2.2.1 E-selectin expression during the inflammatory response ................. 28 1.2.2.2 E-selectin imaging in animal models of inflammatory disease ........ 29 1.2.2.3 E-selectin expression in RA .............................................................. 30 1.2.3 Angiogenesis is Central to Promoting and Maintaining RA ................... 31 1.2.4 Increased Vessel Permeability in RA: Cause or Consequence? .............. 37 1.3 In Vivo Molecular Imaging Approaches in Arthritis ..............................40 1.3.1 E-Selectin: An Excellent Target for Molecular Imaging in RA .............. 42 1.3.2 In Vivo Fluorescence Imaging And Optical Imaging ............................. 43 1.3.2.1 Principles of fluorescent imaging ..................................................... 43 1.3.2.2 Potential molecular approaches for in vivo fluorescence imaging ... 48 1.3.2.2.1 Targeted approaches for dye deposition ....................................... 48 1.3.2.2.2 Protease activity probes ................................................................ 49 1.3.2.2.3 Bioluminescence .......................................................................... 50 1.3.3 Fluorescent Imaging in Arthritis ............................................................. 51 3 1.3.3.1.1 Invasive In Vivo Optical Imaging ................................................. 51 1.3.3.1.2 Non- targeted imaging in animal models of arthritis ................... 51 1.3.3.1.3 Targeting of specific molecular changes in arthritis .................... 52 1.3.3.2 Activity-Based Probes in Arthritis .................................................... 53 1.4 Animal Models of RA and Inflammation ...............................................55 1.4.1 Collagen Induced Arthritis: A Well Validated Pre Clinical Model of RA ................................................................................................................. 55 1.4.2 Acute Models of Inflammation ............................................................... 57 1.5 Hypothesis and Objectives ......................................................................59 CHAPTER 2 ........................................................................................... 60 2 Materials and Methods ..................................................................................61 2.1 Chemical Components ............................................................................61 2.2 Cell Lines ................................................................................................61 2.3 Fluorescence Reflectance Imaging Device .............................................61 2.4 Production of Anti-E-Selectin and Anti-DNP Isotype Control Antibodies ................................................................................................................62 2.4.1 Cell Culture for Anti-E-Selectin and Anti-DNP Antibodies ................... 62 2.4.2 Affinity Purification of Cultured Antibodies .......................................... 63 2.4.3 Measurement of Antibody Concentration Following Production and Purification to Ensure Accurate Quantification for Labelling and Imaging ................................................................................................................. 64 2.4.4 Determination of Antibody Purity by SDS PAGE Prior to Labelling ..... 65 2.4.5 Fluorescent Labelling of Anti E-Selectin and Control Antibodies.......... 65 2.4.5.1 Techniques of fluorescent labelling .................................................. 66 2.4.5.2 Measurement of the fluorescent labelling reaction ........................... 67 2.4.5.2.1 Quantification of fluorophore labelling ........................................ 67 2.4.5.2.2 Procedure for determining the dye:protein ratio .......................... 68 2.4.6 Measurement of The Immunoreactivity of DNP and E-Selectin Antibodies Pre and Post Labelling by ELISA ......................................... 70 2.4.7 Endothelial PY4.1 Cell Culture ............................................................... 71 2.4.7.1 Measurement of reactivity for anti-E-selectin and anti-DNP antibodies to endothelial cells before and after stimulation with TNF ................................................................................................ 72 2.5 Animal Models of Inflammation for In Vivo Imaging Experiments .......73 2.5.1 Laboratory Animal Care and Regulatory Approval ................................ 73 4 2.5.2 Collagen Induced Arthritis ...................................................................... 74 2.5.2.1 Purification of type II collagen ......................................................... 74 2.5.2.2 Induction of arthritis ......................................................................... 75 2.5.2.3 Evaluation
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