DOCSLIB.ORG

Impact of Liver-Derived Complement Component C5 on Atherosclerosis in Apoe-Deficient Mice

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Impact of Liver-Derived Complement Component C5 on Atherosclerosis in Apoe-Deficient Mice Impact of Liver-derived Complement Component C5 on Atherosclerosis in ApoE-deficient Mice Zhe Ma München 2019 Aus dem Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten Kliniker Ludwig-Maximilians-Universität München Direktor: Univ.-Prof. Dr. med. Christian Weber Impact of Liver-derived Complement Component C5 on Atherosclerosis in ApoE-deficient Mice Dissertation zum Erwerb des Doktorgrades der Naturwissenschaften an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München vorgelegt von Zhe Ma aus Henan, China 2019 Mit Genehmigung der Medizinischen Fakultät der Universität München Betreuerin: Univ.-Prof. Dr. rer. nat. Sabine Steffens Zweitgutachter: PD Dr. rer. nat. Andreas Herbst Dekan: Prof. Dr. med. dent. Reinhard Hickel Tag der mündlichen Prüfung: 23. 07. 2020 Dean’s Office Faculty of Medicine Affidavit Ma, Zhe Surname, first name Hermann-Lingg Str. 18 Street 80336, Munich Zip code, town Germany Country I hereby declare, that the submitted thesis entitled Impact of Liver-derived Complement Component C5 on Atherosclerosis in ApoE-deficient Mice is my own work. I have only used the sources indicated and have not made unauthorised use of services of a third party. Where the work of others has been quoted or reproduced, the source is always given. I further declare that the submitted thesis or parts thereof have not been presented as part of an examination degree to any other university. Munich, 07 04 2020 Zhe Ma Place, date Signature doctoral candidate Affidavit September 2018 Impact of Liver-derived Complement Component C5 on Atherosclerosis in ApoE-deficient Mice TABLE OF CONTENTS TABLE OF CONTENTS ABBREVIATIONS ······························································································································· IV 1. INTRODUCTION ····························································································································· 1 1.1 Atherosclerosis ··························································································································· 1 1.1.1 The healthy artery ··············································································································· 1 1.1.2 Progress in the field of atherogenesis ·················································································· 3 1.1.3 Immunocytes in atherosclerotic plaques ············································································· 3 1.1.4 Mouse models of atherosclerosis ························································································ 5 1.1.5 Atherosclerosis in ApoE-/- mice ··························································································· 6 1.2 Complement system and activation ···························································································· 6 1.2.1 Complement cascade ··········································································································· 6 1.2.2 Activation of the complement system ················································································· 6 1.3 Complement cascade and atherosclerosis ·················································································· 9 1.4 Complement-based therapies ··································································································· 11 1.4.1 Complement-system-associated diseases ·········································································· 11 1.4.2 Complement therapeutics ·································································································· 12 1.4.3 Liver-specific C5 small interfering RNA (siRNA) from Alnylam Pharmaceuticals, Inc. · 13 1.4.4 Complement component C5 and autoimmune diseases studies ········································ 13 1.5 Aims ········································································································································· 16 2. MATERIALS & METHODS············································································································ 17 2.1 Materials ··································································································································· 17 2.1.1 Equipment ························································································································· 17 2.1.2 Reagents and materials ······································································································ 18 2.1.3 Standard solutions and buffers ·························································································· 19 2.1.4 Antibodies for Immunofluorescence Microscopy ····························································· 20 2.2 Mouse strains and husbandry ··································································································· 23 2.3 Mouse models ·························································································································· 23 2.3.1 siRNA preparation and injection ······················································································· 23 2.3.2 Experimental protocol ······································································································· 23 2.4 Serum collection and body weighing ······················································································· 24 2.5 Blood and organ collection······································································································· 24 2.6 Assessment of atherosclerosis in mice ····················································································· 25 2.6.1 en-face preparation and Sudan IV staining ······································································· 25 2.6.2 Tissue sectioning ··············································································································· 26 2.6.3 Oil-Red-O Hematoxylin (ORO/HE) staining and atherosclerotic lesion quantification ··· 27 2.6.4 Immunofluorescence staining ··························································································· 27 2.6.5 Quantification of CD68+ macrophage and CD3e+ T cell infiltration in the plaque areas ·· 28 2.7 ELISA for C5 protein in the circulation ··················································································· 29 2.8 Human carotid artery tissues and atherosclerotic lesion analyses ············································ 30 2.9 Statistical analysis ···················································································································· 31 3. RESULTS ········································································································································ 32 I TABLE OF CONTENTS 3.1 mRNA Microarrays of Wt and ApoE-/- aortas of various ages reveal the presence of transcripts of complement cascade constituents in atherosclerotic plaques ····················································· 32 3.2 Liver-specific C5 siRNA reduces atherosclerosis burden in young ApoE-/- mice maintained on normal mouse chow ······················································································································· 37 3.2.1 Liver-specific C5 siRNA reduces serum C5 protein level in 32 weeks old ApoE-/- mice on a normal diet ······························································································································ 38 3.2.2 Liver-specific C5 siRNA does not change blood lipid levels, body weight, blood cell numbers, and leukocyte percentages in 32 weeks old ApoE-/- mice on a normal mouse chow ·· 39 3.2.3 Liver-specific C5 inhibition does not change blood leukocyte subsets in young ApoE-/- mice on normal mouse chow ····································································································· 44 3.2.4 Liver-specific C5 siRNA reduces atherosclerotic plaque burden in young ApoE-/- mice on a normal mouse chow ················································································································ 44 3.2.5 Liver-specific C5 siRNA inhibits macrophage/dendritic and T cell infiltration in atherosclerosis in young ApoE-/- mice on a normal chow ·························································· 46 3.2.6 Effect of C5 siRNA on complement protein activation and deposition in normal chow-fed 32 weeks old ApoE-/- mice ········································································································· 48 3.3 The role of liver-specific complement C5 in atherosclerosis in aged ApoE-/- mice maintained on a normal mouse chow ················································································································ 49 3.3.1 Liver-specific C5 siRNA reduced serum C5 protein level in aged ApoE-/- mice on a normal chow ······························································································································ 50 3.3.2 Liver-specific C5 siRNA does not change body weight and blood lipid level in aged ApoE-/- mice ······························································································································· 51 3.3.3 C5 inhibition does not change blood leukocytes subsets in aged ApoE-/- mice on normal chow ··········································································································································· 53 3.3.4
Load more

Recommended publications
  • Application Note
    APPLICATION NOTE Quantification of functional C5a using iLite® C5a Assay Ready Cells For research and professional use only. Not for use in diagnostic procedures. This application note contains a suggested protocol and performance data. Each individual laboratory must set up their own method and perform relevant validations. Background Complement component 5a (C5a) is a 74 amino acid small protein fragment of complement protein 5 (C5). C5 is cleaved to C5a and C5b by C5 convertase enzymes as a result of complement activation. Factors of the coagulation and fibrinolytic pathway are also able to cleave C5 to C5a. (1) C5a is a highly potent anaphylatoxin and chemoattracting peptide, with the ability to increase blood vessel permeability, stimulate cytokine release from myeloid cells, and expression of adhesion molecules on endothelial cells. (2) Main pro-inflammatory effector functions are induced by C5a binding to a seven-transmembrane G- protein-coupled receptor, C5aR1 (CD88). Rapidly after cleavage of C5 into C5a and C5b, C5a is metabolized by carboxypeptidases which removes the C-terminal arginine and forms C5a-desArg, a less potent ligand of C5aR1. C5a can also bind to a second receptor, C5aR2 (C5L2 or GPR77), however the biological effects associated with C5a binding C5aR2 are less well understood, both anti- inflammatory and pro-inflammatory effects have been described in literature. The C5a receptors are expressed in a wide range of cells and tissues, and the effect of C5a activation is dependent on the location of the C5a receptors. (3, 4) While the complement system is an important part of the innate immune defense against pathogens, research has shown that excessive complement activation including C5a-C5aR interaction plays a central role in several autoimmune and neurodegenerative disorders as well as in acute and chronic inflammatory conditions.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • A MASP-1 Által Indukált Proinflammatorikus Válasz, És Ezen Belül Az Adhéziós Tulajdonságok Vizsgálata Endotélsejtekben
    A MASP-1 által indukált proinflammatorikus válasz, és ezen belül az adhéziós tulajdonságok vizsgálata endotélsejtekben Doktori értekezés Schwaner Endre Semmelweis Egyetem Elméleti és Transzlációs Orvostudományok Doktori Iskola Témavezető: Dr. Cervenak László, Ph.D., tudományos főmunkatárs Hivatalos bírálók: Dr. Jeney Viktória, Ph.D., tudományos főmunkatárs Dr. Káldi Krisztina, Ph.D., egyetemi docens Szigorlati bizottság elnöke: Dr. Kellermayer Miklós, az MTA doktora, egyetemi tanár Szigorlati bizottság tagjai: Dr. Uzonyi Barbara, Ph.D., tudományos munkatárs Dr. Láng Orsolya, Ph.D., egyetemi docens Budapest 2019 TARTALOMJEGYZÉK 1. RÖVIDÍTÉSEK JEGYZÉKE ....................................................................................... 5 2. BEVEZETÉS ................................................................................................................ 8 2.1. A KOMPLEMENTRENDSZER ........................................................................... 9 2.1.1. A lektin út és a MBL-asszociált szerint proteázok ........................................ 11 2.2. A GYULLADÁS FOLYAMATA ....................................................................... 14 2.3. AZ ENDOTÉLSEJTEK ....................................................................................... 17 2.3.1. Az endotélsejtek funkciói .............................................................................. 18 2.3.1.1. Szelektív barrier képzés .......................................................................... 19 2.3.1.2. A vaszkuláris tónus szabályozása
    [Show full text]
  • The Complement System and Cardiovascular Disease
    The complement system and cardiovascular disease Citation for published version (APA): Hertle, E. (2016). The complement system and cardiovascular disease: the CODAM study. https://doi.org/10.26481/dis.20160401eh Document status and date: Published: 01/01/2016 DOI: 10.26481/dis.20160401eh Document Version: Publisher's PDF, also known as Version of record Please check the document version of this publication: • A submitted manuscript is the version of the article upon submission and before peer-review. There can be important differences between the submitted version and the official published version of record. People interested in the research are advised to contact the author for the final version of the publication, or visit the DOI to the publisher's website. • The final author version and the galley proof are versions of the publication after peer review. • The final published version features the final layout of the paper including the volume, issue and page numbers. Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal. If the publication is distributed under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license above, please follow below link for the End User Agreement: www.umlib.nl/taverne-license Take down policy If you believe that this document breaches copyright please contact us at: [email protected] providing details and we will investigate your claim.
    [Show full text]
  • Derivation of Ligands for the Complement C3a Receptor from the C-Terminus of C5a
    This is a repository copy of Derivation of ligands for the complement C3a receptor from the C-terminus of C5a. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/99926/ Version: Submitted Version Article: Halai, R., Bellows-Peterson, M.L., Branchett, W. et al. (8 more authors) (2014) Derivation of ligands for the complement C3a receptor from the C-terminus of C5a. European Journal of Pharmacology, 745. pp. 176-181. ISSN 0014-2999 https://doi.org/10.1016/j.ejphar.2014.10.041 Reuse This article is distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs (CC BY-NC-ND) licence. This licence only allows you to download this work and share it with others as long as you credit the authors, but you can’t change the article in any way or use it commercially. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Derivation of ligands for the complement C3a receptor from the C- terminus of C5a Reena Halaia,*, Meghan L Bellows-Petersonb,*, Will Branchettc, James Smadbeckb, Chris A Kieslichb, Daniel E Crokera, Matthew A Coopera, Dimitrios Morikisd, Trent M Woodruffe, Christodoulos A Floudasb,#, Peter N Monkc,# *equal contributing authors #equal corresponding authors ([email protected]; tel.
    [Show full text]
  • Complement System on the Attack in Autoimmunity
    Complement system on the attack in autoimmunity John P. Atkinson J Clin Invest. 2003;112(11):1639-1641. https://doi.org/10.1172/JCI20309. Commentary The antiphospholipid syndrome is characterized clinically by fetal loss and thrombosis and serologically by the presence of autoantibodies to lipid-binding proteins. In a model of this procoagulant condition in which these antibodies are injected into pregnant mice, fetal loss was prevented by blocking of complement activation. Specifically, interaction of complement component 5a (C5a) with its receptor is necessary for thrombosis of placental vasculature (see the related article beginning on page 1644). Inhibition of complement activation may have a therapeutic role in this disease. Find the latest version: https://jci.me/20309/pdf Complement system on the attack tion of the classical pathway is a time- honored concept, antibody can also in autoimmunity serve as a site that is protected from plasma and membrane inhibitors of John P. Atkinson complement activation. In particular, these regulators of complement activa- Washington University School of Medicine, St. Louis, Missouri, USA tion are designed to block amplifica- tion of the alternative pathway (AP) on The antiphospholipid syndrome is characterized clinically by fetal loss self tissue (6). To produce arthritis, anti- and thrombosis and serologically by the presence of autoantibodies to body binding at a site relatively defi- lipid-binding proteins. In a model of this procoagulant condition in cient in complement inhibitors allows which these antibodies are injected into pregnant mice, fetal loss was “firing” of the AP (5). Complement prevented by blocking of complement activation. Specifically, interac- component 5a (C5a), interacting with tion of complement component 5a (C5a) with its receptor is necessary its G-coupled receptor, is absolutely for thrombosis of placental vasculature (see the related article beginning required to recruit neutrophils and to on page 1644).
    [Show full text]
  • Exploring the Role of C5a-C5ar1 Signalling in Development Through Pluripotent Stem Cell Modelling
    Exploring the role of C5a-C5aR1 signalling in development through pluripotent stem cell modelling Owen Hawksworth BSc (Hons) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 Faculty of Medicine Abstract The complement system has been traditionally described as a powerful controller of innate immunity. With activation through the recognition of pathogenic surfaces, spontaneous hydrolysis, or extrinsic cleavage, a cleavage cascade is initiated culminating in the formation of the active complement fragments C3a, C3b, C5a, and C5b. These fragments direct immune cells to sites of inflammation, as well as tagging pathogens for destruction and directly lysing foreign cells. It is now clear however, that this complex family of proteins possesses a wide range of functions outside of immune regulation. From fertilisation and morphogenesis, to the control of foetal and adult stem cell populations, studies have described novel actions of complement factors. This thesis is focussed on the central complement receptor, C5aR1. Traditionally described as a potent activating and chemotactic receptor for immune cells, C5aR1 has been shown to function in a number of non- immune cell populations. Of note, our laboratory has previously described a role for C5aR1 in neural tube closure, with loss of C5aR1 signalling associated with increased neural tube defects under folate deficient conditions. However, a mechanistic role of C5aR1 at this stage of development was not described. In this thesis, pluripotent stem cells were utilised as an in vitro model of human development to interrogate the expression and function of C5aR1 at a number of developmental stages. Specifically, in pluripotent stem cells representative of the blastocyst inner cell mass, in neural rosettes representative of the developing ventricular zone, and in matured post-mitotic cortical neurons.
    [Show full text]
  • Dimerization of Complement C5a Receptors in Inflammatory Responses
    Dimerization of complement C5a receptors in inflammatory responses By: AHMED ALI ABD A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Medicine, Dentistry & Health The Medical School Department of Department of Infection, Immunity & Cardiovascular Disease August/2017 Abstract Inflammation is a complex pathophysiologic process that occurs in response to tissue injury induced though various stimuli. It involves cellular and humoral responses in which various cell types and inflammatory mediators are engaged. The inflammatory response must be tightly controlled, otherwise it results in chronic inflammation and perhaps continuous tissue damage. During the activation of the complement cascade, several small fragments, known as anaphylatoxins, are released. One of these anaphylatoxins is produced from the complement protein C5 and is known as C5a. C5a is a multifunctional polypeptide that is involved in cellular immune responses. The receptors for C5a, C5a1 and C5a2, are among the large family known as G protein-coupled receptors. Unlike C5a1, C5a2 is incapable of signalling through G proteins but can induce β- arrestin translocation and recruitment. C5a2 function is still enigmatic and it has been suggested as a decoy receptor for its ligands, as a signalling receptor or as a signalling modifier of C5a1, possibly through the formation of heterodimers. In the current study, we aimed to study possible interactions between the C5a1 and C5a2 receptors. To achieve these goals, heterologous expression of different C5a receptors was studied in clearly defined settings using transfected RBL cells. The possibility of direct physical interaction between the two receptors was explored using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) using tagged C5a1 and C5a2 receptors.
    [Show full text]
  • Contribution of the Anaphylatoxin Receptors, C3ar and C5ar, to the Pathogenesis of Pulmonary Fibrosis † Hongmei Gu,*,1 Amanda J
    THE JOURNAL • RESEARCH • www.fasebj.org Contribution of the anaphylatoxin receptors, C3aR and C5aR, to the pathogenesis of pulmonary fibrosis † Hongmei Gu,*,1 Amanda J. Fisher,*,1 Elizabeth A. Mickler,* Frank Duerson III,* Oscar W. Cummings, ‡ ‡ Marc Peters-Golden, Homer L. Twigg III,*,§ Trent M. Woodruff,{ David S. Wilkes,*,§ and Ragini Vittal ,2 † *Division of Pulmonary and Critical Care Medicine, Department of Medicine, Department of Pathology, and §Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana, USA; {School of Biomedical Sciences, The University of Queensland, ‡ Brisbane, Queensland, Australia; and Division of Pulmonary and Critical Care Medicine, University of Michigan, Ann Arbor, Michigan, USA ABSTRACT: Complement activation, an integral arm of innate immunity, may be the critical link to the pathogenesis of idiopathic pulmonary fibrosis (IPF). Whereas we have previously reported elevated anaphylatoxins—complement component 3a (C3a) and complement component 5a (C5a)—in IPF, which interact with TGF-b and augment epithelial injury in vitro, their role in IPF pathogenesis remains unclear. The objective of the current study is to determine the mechanistic role of the binding of C3a/C5a to their respective receptors (C3aR and C5aR) in the progression of lung fibrosis. In normal primary human fetal lung fibroblasts, C3a and C5a induces mesenchymal activation, matrix synthesis, and the expression of their respective receptors. We investigated the role of C3aR and C5aR in lung fibrosis by using bleomycin-injured mice with fibrotic lungs, elevated local C3a and C5a, and overexpression of their receptors via pharmacologic and RNA interference interventions. Histopathologic examination revealed an arrest in disease progression and attenuated lung collagen deposition (Masson’s trichrome, hydroxyproline, collagen type I a 1 chain, and collagen type I a 2 chain).
    [Show full text]
  • (CXCL‑13) and Complement Component C5a in Different Stages of ANCA Associated Vasculitis
    www.nature.com/scientificreports OPEN The signifcance of metalloproteinase 3 (MMP‑3), chemokine CXC ligand 13 (CXCL‑13) and complement component C5a in diferent stages of ANCA associated vasculitis Aleksandra Rymarz *, Magdalena Mosakowska & Stanisław Niemczyk The aim of the study was to evaluate the signifcance of metalloproteinase 3 (MMP‑3), chemokine CXC ligand 13 (CXCL‑13) and complement component 5a (C5a) in diferent stages of ANCA associated vasculitis (AAV). 89 adults were included into the study. 28 patients with active AAV (Birmingham Vasculitis Activity Score, BVAS > 3) formed the Active Group. 24 individuals who were in remission after 6 months of induction therapy formed the Short R Group, while 34 patients with longitudinal remission formed the Long R Group. 28 patients without autoimmune diseases similar in terms of age, gender and stage of kidney disease formed the Control Group. Receiver operating characteristic curve analysis (ROC) was used to evaluate MMP‑3, CXCL‑13 and C5a as markers of the diferent phases of vasculitis. In ROC analysis, MMP‑3, CXCL‑13 and C5a presented a good ability in distinguishing active vasculitis (Active Group) from the Control Group (AUC > 0.8), whereas only CXCL‑13 displayed potential ability in distinguishing active vasculitis (Active Group) from long term remission (Long R Group, AUC = 0.683). MMP‑3 signifcantly and positively correlated with serum creatinine concentration (r = 0.51, p = 0.011; r = 0.44, p = 0.009; r = −0.66, p < 0.001) and negatively with eGFR (r = −0.5, p = 0.012; r = −0.35, p = 0.039; r = −0.63, p < 0.001) in the Short R, Long R and Control Groups.
    [Show full text]
  • Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology)
    Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience I Abstract Macrophages (MΦ) are important to host defence and inflammation but plasticity in their gene expression profiles contributes to their functional heterogeneity. C5a is one of the most potent proinflammatory agents generated upon complement activation and binds to a specific receptor C5aR. Overproduction of C5a can contribute to numerous immune and inflammatory conditions. The objective of this thesis was to focus on human macrophage populations differentiated from primary human monocytes, and examine effects of C5a, C5aR agonists and antagonists, and TLR4- C5aR crosstalk on inflammatory profiles of human monocytes and macrophages. Chapter One summarizes roles for C5a activation on human macrophages, briefly surveying C5a, C5aR and some important analogues of C5a that had been developed to study roles for C5a in vivo. This chapter discusses new insights into C5aR-related biological functions and diseases, macrophage polarization and the usefulness of mouse models for human biology in relation to how closely human and mouse macrophages reproduce functional responses. Chapter Two addresses macrophage polarization. Macrophage heterogeneity was previously achieved by differentiating monocytes with either GM-CSF or M-CSF (generating GM- MΦ or M-MΦ), but was mostly studied on murine rather than human cells. This chapter describes a comparison of gene expression in populations of human macrophages (GM-MΦ versus M-MΦ), both in the basal state as well as in response to stimulation by LPS, using a combination of real-time PCR and cDNA microarray analysis, cellular migration, and functional responses.
    [Show full text]
  • Contribution of Complement Anaphylatoxin Receptors to Melanoma Growth: Potential Therapeutic Targets
    Contribution of complement anaphylatoxin receptors to melanoma growth: potential therapeutic targets Jamileh Alham Nabizadeh BBiot. Honours A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2015 Australian Institute for Bioengineering and Nanotechnology ABSTRACT The incidence of melanoma is increasing world-wide, and Australia has the highest rate of melanoma in the world. Hence there is an urgent need for safe and effective treatments. The aim of this thesis was to elucidate our understanding of the role that the complement system, in particular the complement anaphylatoxins C3a and C5a, plays in tumour development and growth. Given the mounting evidence for a role for the C5a receptor (C5aR1) in the growth of other tumour types, Chapter 3 explored the role of C5aR signalling in a murine model of melanoma. Immunostaining revealed that B16 murine melanoma cells expressed both receptors for C5a (C5aR1 and C5aR2). Although C5aR1 was capable of ERK activation in response to recombinant mouse C5a, there was no effect on cell proliferation or migration. Melanoma growth was significantly retarded in C5aR1-deficient (C5aR1-/-) mice, and the growth of established melanomas was inhibited by treatment of wild-type mice with a C5aR1 antagonist (C5aR1A), thus suggesting the potential of C5aR1 as a therapeutic target for melanoma. In accord with the premise that C5aR2 acts as a modulator of C5aR1, tumour growth was enhanced in mice lacking C5aR2. Given that the growth of C5aR-expressing tumour cells was retarded in C5aR1-/- mice, we hypothesised that the protective effect of C5aR1 inhibition could be due to changes in the immune response.
    [Show full text]