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The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review
The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review Y. Mostmans, M. Cutolo, C. Giddelo, S. Decuman, K. Melsens, H. Declercq, E. Vandecasteele, F. De Keyser, O. Distler, J. Gutermuth, V. Smith PII: DOI: Reference:
S1568-9972(17)30148-9 doi:10.1016/j.autrev.2017.05.024 AUTREV 2028
To appear in:
Autoimmunity Reviews
Received date: Accepted date:
8 April 2017 13 April 2017
Please cite this article as: Mostmans Y, Cutolo M, Giddelo C, Decuman S, Melsens K, Declercq H, Vandecasteele E, De Keyser F, Distler O, Gutermuth J, Smith V, The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review, Autoimmunity Reviews (2017), doi:10.1016/j.autrev.2017.05.024
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ACCEPTED MANUSCRIPT The role of endothelial cells in the vasculopathy of systemic sclerosis: a systematic review SSc vasculopathy: the role of endothelial cells
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Y. Mostmans1, M. Cutolo2, C. Giddelo1, S. Decuman3, K. Melsens4, H. Declercq5, E. Vandecasteele6, F. De Keyser4, O. Distler7, J. Gutermuth1, V. Smith4
1. Vrije Universiteit Brussel (VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Department of
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Dermatology, Laarbeeklaan 101, 1090 Brussels, Belgium; Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium; and Department of Rheumatology, Ghent University, Ghent, Belgium Department of Basic Medical Sciences, Tissue Engineering and Biomaterials Group, Ghent University, Ghent, Belgium Department of Cardiology, Ghent University Hospital, Ghent, Belgium Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland
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Corresponding author:
Dr. Yora Mostmans* Department of Dermatology/Universitary Hospital Brussels (UZ Brussel) Vrije Universiteit Brussel (VUB) Laarbeeklaan 101 1090 Brussels, Belgium Tel. +32 2 477 63 55; Fax +32 2 477 63 57 Email: [email protected] *
Present address: Department of Immunology and Allergology (CIA) Centre Hospitalier Universitaire (CHU) Brugmann Université Libre de Bruxelles (ULB) Van Gehuchtenplein 4 1020 Brussels, Belgium Tel. +32 2 477 22 94; Fax +32 2 477 22 76 Email: [email protected] 1
ACCEPTED MANUSCRIPT Statement of author contribution:
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Yora Mostmans: substantial contributions to design of the study, acquisition of data and analysis and interpretation of data, drafting of the article, final approval of the version to be published.
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Maurizio Cutolo: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Christina Giddelo: acquisition of data and analysis and interpretation of data, critical revising of the article for important intellectual content, final approval of the version to be published.
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Saskia Decuman: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Karin Melsens: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Heidi De Clercq: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Els Vandecasteele: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Filip De Keyser: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Oliver Distler: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Jan Gutermuth: analysis and interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Vanessa Smith: substantial contributions to design of the study, acquisition of data and analysis and interpretation of data, drafting of the article, final approval of the version to be published. 2
ACCEPTED MANUSCRIPT Statement of conflict of interest:
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Yora Mostmans: no conflicts of interest to declare Maurizio Cutolo: no conflicts of interest to declare Christina Giddelo: no conflicts of interest to declare Saskia Decuman: no conflicts of interest to declare Karin Melsens: no conflicts of interest to declare Heidi De Clercq: no conflicts of interest to declare Els Vandecasteele: no conflicts of interest to declare Filip De Keyser: no conflicts of interest to declare Oliver Distler: has/had consultancy relationship and/or has received research funding from 4 D Science, Actelion, Active Biotec, Bayer, Biogen Idec, Boehringer Ingelheim Pharma, BMS, ChemomAb, EpiPharm, Ergonex, espeRare foundation, GSK,Roche-Genentech, Inventiva, Lilly, medac, MedImmune, Mitsubishi Tanabe, Pharmacyclics, Pfizer, Sanofi, Serodapharm and Sinoxa in the area of potential treatments of scleroderma and its complications. He has a patent on mir-29 for the treatment of systemic sclerosis licensed. Jan Gutermuth: no conflicts of interest to declare Vanessa Smith: no conflicts of interest to declare
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ACCEPTED MANUSCRIPT ABSTRACT
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Introduction: Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibroproliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs including the skin. In this study, all English speaking articles concerning the role of endothelial cells (ECs) in SSc vasculopathy and representing biomarkers are systematically reviewed and categorized according to endothelial cell (EC) (dys)function in SSc.
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Methods: A sensitive search on behalf of the EULAR study group on microcirculation in Rheumatic Diseases was developed in Pubmed, The Cochrane Library and Web of Science to identify articles on SSc vasculopathy and the role of ECs using the following Mesh terms: (systemic sclerosis OR scleroderma) AND pathogenesis AND (endothelial cells OR marker). All selected papers were read and discussed by two independent reviewers. The selection process was based on title, abstract and full text level. Additionally, both reviewers further searched the reference lists of the articles selected for reading on full text level for supplementary papers. These additional articles went through the same selection process.
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Results: In total 193 resulting articles were selected and the identified biomarkers were categorized according to description of EC (dys)function in SSc. The most representing and reliable biomarkers described by the selected articles were adhesion molecules for EC activation, anti-endothelial cell antibodies for EC apoptosis, vascular endothelial growth factor (VEGF), its receptor VEGFR-2 and endostatin for disturbed angiogenesis, endothelial progenitors cells for defective vasculogenesis, endothelin-1 for disturbed vascular tone control, Von Willebrand factor for coagulopathy and interleukin (IL)-33 for EC- immune system communication. Emerging, relatively new discovered biomarkers described in the selected articles, are VEGF165b, IL-17A and the adipocytokines. Finally, myofibroblasts involved in tissue fibrosis in SSc can derive from ECs or epithelial cells through a process known as endothelial-to-mesenchymal transition.
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Conclusion: This systematic review emphasizes the growing evidence that SSc is primarily a vascular disease where EC dysfunction is present and prominent in different aspects of cell survival (activation and apoptosis), angiogenesis and vasculogenesis and where disturbed interactions between ECs and various other cells contribute to SSc vasculopathy.
KEYWORDS biomarker, endothelial cells, systemic sclerosis, systematic review, vasculopathy, EULAR study group on microcirculation in Rheumatic Diseases 4
ACCEPTED MANUSCRIPT Abbreviations Ab: antibodies ACAs: anti-centromere antibodies
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ACE: angiotensin converting enzyme ADCC: antibody dependent cell mediated cytotoxicity
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ADMA: asymmetric dimethylarginine ADP: adenosine diphosphate
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AECA: anti-endothelial cell antibodies Ag: antigen
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AIF-1: allograft inflammatory factor 1 Ang I: angiotensin I
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Ang II: angiotensin II
Ang-2: angiopoietin 2
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Ang-1: angiopoietin 1
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Angptl3: angiopoietin-like protein 3 Anti-topo I: anti-topoisomerase ATIII: antithrombin III
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BALF: broncho-alveolar lavage fluid BCGF: B cell growth factor bFGF: basic fibroblast growth factor-2 BM: bone marrow BM-MSCs: bone marrow mesenchymal stem cells BMPRII: bone morphogenetic protein receptor II CACs: circulating angiogenic cells cAMP: cyclic adenosine monophosphate CCL: chemokine ligand CCN-1: cysteine-rich 61 matrix protein
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ACCEPTED MANUSCRIPT CD: cluster of differentiation CD44R: cluster of differentiation 44 receptor CEC: circulating endothelial cells
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CFU: colony-forming unit cGMP: cyclic guanosine monophosphate
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CIC: circulating immune complexes CKO: knock out
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CRP: C-reactive protein CTSB: cathepsin B
CTSV: cathepsin V
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CXCL: chemokine (C-X-C motif) ligand
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CTSL: cathepsin L
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CX3CR1: fractalkine receptor DAF: decay accelerating factor
DD: d-dimers
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dcSSc: diffuse cutaneous systemic sclerosis
DLCO: diffusing capacity for carbon monoxide
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DMVEC: dermal microvascular endothelial cells DRC3f: complement C3f-des-arginine DS: dermatansulphate DU: digital ulcers EC: endothelial cell ECA: endothelial cytotoxic activity ECs: endothelial cells EGFL7: epidermal growth factor-like protein 7 EMP: endothelial cell-derived microparticles ENA-78: epithelial-neutrophil activating peptide-78
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ACCEPTED MANUSCRIPT EndoMT: endothelial-to-mesenchymal transition eNOS: endothelial nitric oxide synthase EPC: endothelial progenitor cells
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ERK: extracellular signal-regulated kinase EScSG: the European Scleroderma Study Group
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ESR: erythrocyte sedimentation rate
EUSTAR: The European Scleroderma Trials and Research group
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F-AnxV(-): Annexin V non-binding
FASSc: fibrosing alveolitis associated with systemic sclerosis
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FEV: forced expiratory volume FKN: fractalkine
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FNG: fibrinogen
Fra-2: fos-related antigen-2
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FVC: forced vital capacity
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Fli1: friend leukemia integration factor 1
F2IP-M: tetranor-dicarboxylic acid metabolite of F2-isoprostanes F1+2: prothrombin fragments 1+2
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fVIII/vWF Ag: factor VIII/von Willebrand factor antigen Gal3: galectin 3
GLUT-1: glucose transporter 1 GPA: granulomatosis with polyangiitis GRO-α : growth-regulated oncogene-alpha GVHD: graft-versus-host disease HC: healthy controls HDEC: human dermal endothelial cells HMVEC: human microvascular endothelial cells HGF: hepatocyte growth factor
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ACCEPTED MANUSCRIPT HRC: hypertensive renal crisis HUVEC: human umbilical vein endothelial cells ICAM: intercellular adhesion molecule
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IF: immunofluorescence IFI16: gamma-interferon-inducible protein IFI-16
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IFN-γ: interferon gamma Ig: immunoglobulin
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IL: interleukin IP: interstitial pneumonia
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iNOS: inducible NO synthase JAM: junctional adhesion molecule
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KDR: kinase insert domain receptor
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KLK: human tissue kallikrein
LMP: leukocyte-derived microparticle
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Lp(a): lipoprotein (a)
MAC: membrane attack complex of complement MC: mast cells
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MCP: membrane cofactor protein
MDMP: monocyte-derived microparticle Mesh: medical subject heading MIF: macrophage migration inhibitory factor MMP: matrix metalloproteinases MMT: mesenchymal-to-mesenchymal transition MPs: microparticles mRNA: messenger ribonucleic acid MRSS: modified Rodnan skin thickness score MVD: microvessel density
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ACCEPTED MANUSCRIPT MVEC: microvascular endothelial cells Nb: number NEP: neutral endopeptidase
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NIH: national institute of health NO: nitric oxide
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NOS: nitric oxide synthase NOx: total nitrate and nitrite
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NVC: nail fold videocapillaroscopy OA: osteoarthritis
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OPG: osteoprotegerin OSM: oncostatin M
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PAP: pulmonary artery pressure
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PAI: plasminogen activator inhibitor
PCs: pericytes
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PDGF (-BB): platelet derived growth factor (-BB) PDMP: platelet-derived microparticle PEDF: pigment epithelium-derived factor
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PECAM: platelet endothelial cell adhesion molecule PH: pulmonary hypertension PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase PL: phospholipids PlGF: placental growth factor PRP: primary raynaud phenomenon PS: phosphatidylserine PSS: progressive systemic sclerosis PTX3: pentraxin 3 RA: rheumatoid arthritis
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ACCEPTED MANUSCRIPT RANTES: regulated on activation normally T cell expressed and secreted RAS: renin-angiotensin system RBP4: retinol binding protein-4
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RF: rheumatoid factor RhoA: GTPase Ras homolog gene family-member Ab
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RNAse: ribonuclease ROCKS: rho-associated creatine kinases
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ROS: reactive oxygen species RP: raynaud phenomenon
RVSP: right ventricular systolic pressure
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RT-PCR: reverse-transcriptase polymerase chain reaction
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sCD40L: soluble cluster of differentiation-40 ligand
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SCF: stem cell factor SDF-1: stromal cell-derived factor 1
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sENG: soluble endoglin sFKN: soluble fractalkine
sGP130: soluble glycoprotein 130
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sIL: soluble interleukin
sIL-2R: soluble IL-2 receptor sIL-6R: soluble IL-6 receptor siRNA: small interfering RNA SLE: systemic lupus erythematosus α-SMA: alpha smooth muscle actin SM22α: transgelin SNAIL-1: transcriptional repressor zinc finger protein SPARC: secreted protein, acidic and rich in cysteine SRC: scleroderma renal crisis
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ACCEPTED MANUSCRIPT SSc: systemic sclerosis sTie1: soluble tyrosine kinase 1 sTie2: soluble tyrosine kinase 2
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ST2: IL-1 receptor-related protein sVEGF: soluble vascular endothelial growth factor
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TAT: thrombin-antithrombin TBARS: thiobarbituric acid reactive substances
TGF-β: tumor growth factor beta
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TIMP: tissue inhibitor of metalloproteinases
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β-TG: beta-thromboglobulin
TLR: toll-like receptors
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TNF-α: tumor necrosis factor alpha
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TM: thrombomodulin
tPA: tissue type plasminogen activator
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TRAIL: tumor necrosis factor related apoptosis-inducing ligand TSLP: thymic stromal lymphopoietin TSP-1: thrombospondin 1
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TXAR: thromboxane A2 receptor
uPAR: urokinase-type plasminogen activator receptor U-II: urotensin-II VA: alveolar volume VC: vital capacity VCAM: vascular cell adhesion molecule VE-cadherin: vascular endothelial cadherin VEGF: vascular endothelial growth factor VEGFR: vascular endothelial growth factor receptor VSMC: vascular smooth muscle cells
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vWF: von willebrand factor
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ACCEPTED MANUSCRIPT The role of endothelial cells in the vasculopathy of systemic sclerosis: a systematic review 1. INTRODUCTION
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Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibroproliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs such as skin and lung (1). Dysregulation of endothelial cell (EC) function within the vascular wall plays an important role in vascular remodeling associated with the fibroproliferative vasculopathy observed in SSc. Endothelial cell injury is proposed as a crucial initiating event leading to vascular remodeling with intimal proliferation of arterioles and capillary breakdown and finally, blood vessel occlusion (2-4). Ongoing research continues to focus on the role of endothelial cells (ECs) in SSc vasculopathy and on identification of related biomarkers. This study, performed on behalf of the EULAR study group on microcirculation in Rheumatic Diseases, gives an overview on the large body of data of current research in this domain, obtained after systematically reviewing the literature.
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2. METHODS 2.1 Search strategy
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A structured search on Pubmed, The Cochrane Library and Web of Science was performed without limitation on publication date to identify articles on SSc vasculopathy and the role of ECs. The Medical subject heading (Mesh) term for “systemic sclerosis” and its synonym “scleroderma” were used in combination with other groups of search terms: (systemic sclerosis OR scleroderma) AND pathogenesis AND (endothelial cells OR marker). The structured search was last updated on the 24th of November 2016. Details on search strategy are shown in Supplementary file 1. 2.2 Searching process
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All articles were screened by two independent reviewers (YM and CG). This selection process was based on title, abstract and full text level. All studies elaborating on the role of ECs in the pathogenesis of SSc vasculopathy were included in the systematic review. All study designs with the exception of reviews, editorials and case studies were included. Languages other than English were excluded. Titles and abstracts selected by either one of the reviewers were included for further screening. The final articles were withheld after reading and judgement of the full text. When different opinions existed among the two reviewers on full text level, consensus was reached. Additionally, both reviewers further searched the reference lists of the articles selected for reading on full text level for supplementary papers. These additional articles went through the same selection process performed by the same two reviewers. Supplementary file 2 depicts the flow of the searching process performed in this systematic review (5). The majority of articles were observational studies of which most were evaluated to be of fair quality using the quality assessment tool for observational studies adapted from the national institute of health (NIH) (supplementary file 3)(6). 3. RESULTS Of 2442 articles screened (on title, abstract and full text level) as potentially relevant to the topic of this study, 127 articles were included. From these articles, reviewers further searched the reference 13
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lists for more relevant articles not present in the database search, and identified 115 articles for additional screening. The flow of the searching and selection process is shown in supplementary file 2. Supplementary file 4 summarizes all 193 selected manuscripts on biomarkers reflecting EC dysfunction described in SSc vasculopathy. The majority of articles were observational studies, of which most were evaluated to be of fair quality according to the quality assessment tool for observational studies adapted from the national institute of health (NIH) (supplementary file 3)(6). The 193 selected articles were categorized according to description of EC (dys) function in SSc, as shown in Table 1 and Figure 1: EC-activation, EC-apoptosis, disturbed angiogenesis, endothelial-tomesenchymal transition (EndoMT), disturbed vasculogenesis, defective vascular tone control, coagulation cascade impairment, immune dysregulation, mast cell dysregulation, dysregulation of the vascular system through pericytes/ECs crosstalk, fibrosis and dysregulation of adipocytokines. Since many of these functional pathways interact, some markers were therefore categorized in more than one function group. 3.1 Markers of EC-activation
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In its early stages, SSc is characterized by EC injury and perivascular mononuclear cell infiltration with an accumulation of T and B lymphocytes and monocytes in the affected tissues (7). Interaction between these leukocytes and ECs is of major importance in the induction of EC permeability contributing to SSc vasculopathy. These interactions are highly dependent on the expression and function of cell adhesion molecules for the maintenance of transendothelial leukocyte migration. In total 38 articles concerning markers of EC-activation were selected (Table 1). The majority of selected articles covered adhesion molecules as biomarker of EC-activation: E-selectin (8-19), intercellular adhesion molecule (ICAM)(8, 9, 11, 12, 15-17, 20-24), junctional adhesion molecule (JAM)(25-27), platelet endothelial cell adhesion molecule (PECAM)(28), P-selectin (8, 9, 14, 15, 19, 29) and vascular cell adhesion molecule (VCAM)(9-12, 15, 17, 29-31). Nevertheless also other biomarkers in EC activation are described but less frequent: allograft inflammatory factor 1 (AIF-1)(32), asymetric dimethylarginine (ADMA)(33), fractalkine (FKN)(34, 35), fractalkine receptor (CX3CR1)(34), soluble endoglin (sENG)(31, 33, 36), Β1/β2/β4 integrins (16), von Willebrand factor (vWF) (37-44), counter adhesive proteins thrombospondin 1 (TSP-1) and secreted protein, acidic and rich in cysteine (SPARC)(13). The majority of discussed markers of EC activation are elevated in the sera of SSc or certain SSc subtypes (supplementary file 4). 3.2 EC-apoptosis Endothelial injury is regarded as a crucial initiating event leading to vascular remodeling with intimal proliferation of arterioles and capillary breakdown and blood vessel occlusion. EC apoptosis is recognized as an important component of the response to the injury. 29 articles investigating EC apoptosis and possible biomarkers were selected for this topic. Described biomarkers are: antiendothelial cell antibodies (AECA) (through antibody dependent cell mediated cytotoxicity (ADCC))(3, 37, 45-57), immunoglobulin (Ig) G anti-caspase-3 antibodies (Ab)(58), caspase-3 (59), gammainterferon-inducible protein (IFI)-16 Ab (60, 61), bone morphogenetic protein receptor II (BMPRII)(62), circulating endothelial cells (CEC)(31, 63), osteoprotegerin (OPG) and TNF-related apoptosis-inducing ligand (TRAIL)(64), membrane attack complex of complement (MAC)(65), collagen V (66), microparticles (MPs)(14, 67, 68) and Fos-related antigen-2 (Fra-2)(69). 14
ACCEPTED MANUSCRIPT Most markers of EC apoptosis were elevated in the sera of SSc patients (Table 2). 3.3 Disturbed angiogenesis
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A loss of vasculature in SSc results in tissue hypoxia, which normally promotes angiogenesis through the production of pro-angiogenic factors. Angiogenesis implicates endothelium sprouting from preexisting ECs and involves the proliferation and migration of mature ECs. However in SSc, angiogenesis is disturbed through expression of inefficient pro-angiogenic mediators, upregulation of powerful inhibitors of angiogenesis and by alteration of transcripts involved in signal transduction pathways (70). In total 56 articles on mechanisms of dysfunctional angiogenesis in SSc were selected (Table 1). Biomarkers described in these selected articles demonstrating this disturbance, shown in Table 2, are: vascular endothelial growth factor (VEGF)(28, 31, 33, 71-82), VEGF165b (83), vascular endothelial growth factor receptor (VEGFR) 1 (74, 78, 84, 85), VEGFR 2 (72, 74, 78, 85, 86), VEGFR 3 (78, 85), platelet-derived growth factor (PDGF)(77, 87), PDGF-BB (28), basic fibroblast growth factor-2 (bFGF, also known as FGF-2 or FGF-β)(73, 77, 79, 88), hepatocyte growth factor (HGF)(28, 77, 79), soluble tyrosine kinase 1 (sTie1)(89), sTie2 (31, 90), angiopoietin (Ang)-1 (Ang-1)(91), Ang-2 (28, 31, 91), angiopoietin-like protein 3 (Angptl3)(92), placental growth factor (PlGF) (31, 77, 93, 94), endostatin (31, 73, 75, 77, 81, 82, 95), tissue inhibitor of metalloproteinase 2 (TIMP-2)(75), growth-regulated oncogene-α (GRO-α)(96), glucose transporter 1 (GLUT-1)(72), pigment epithelium-derived factor (PEDF)(76), matrix metalloproteinases (MMP)-9 (77), MMP-12 (97-100) and pro-MMP-1 (77), urokinase-type plasminogen activator receptor (uPAR)(98, 99, 101), human tissue kallikreins (KLK) 1 (102, 103), KLK9 (102), KLK11 (102), KLK 12 (102), cathepsin B (CTSB)(104), cathepsin V (CTSV)(105), cathepsin L (CTSL)(106), friend leukemia integration factor 1 (Fli1)(107-109), galectin-3 (Gal3)(110), apelin (111), CCN1 (112) and chemokines epithelial-neutrophil activating peptide (ENA-78)(15), regulated on activation normally T-cell expressed and secreted (RANTES) (15), chemokine ligand (CXCL)1 (96), CXCL4 (113), CXCL5 (108), CXCL8 (15, 28-30, 96), CXCL9 (114), CXCL10 (114, 115), CXCL12 (116), CXCL16 (114, 117) and chemokine ligands (CCL)2 (19, 30), CCL13 (118), CCL23 (119).
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Most articles showed upregulation of proangiogenic and/or angiostatic biomarker(s) in SSc as shown in Table 2. 3.4 Endothelial-to-mesenchymal transition (EndoMT) In the pathogenesis of SSc, an accumulation of fibroblasts and myofibroblasts occurs and production of interstitial collagens and extracellular matrix components is excessive. Numerous studies have shown that myofibroblasts involved in tissue fibrosis can derive from ECs through a process known as EndoMT (120-127). It is a nonmalignant phenomenon of cellular transdifferentiation by which ECs undergo a phenotypical conversion where they lose vascular EC markers and gain mesenchymal cell markers (120-122). Seven articles concerning this topic were selected (127-133). Most included studies (127-131, 133) are in vitro studies (mostly using mouse models) suggestive of a role for EndoMT in SSc progressive fibrosis. 3.5 Disturbed vasculogenesis Vasculogenesis involves recruitment, mobilization and in situ differentiation of endothelial progenitor cells (EPC) from the bone marrow (BM). Recent data show that the morphology of SSc BM is normal but that the number of microvessels is severely reduced despite striking increase of VEGF 15
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(80). Furthermore the angiogenic potential of EC-like mesenchymal stromal cells was reduced after being stimulated with VEGF and stromal cell-derived factor-1 in vitro, suggesting that endothelial repair may be affected in SSc starting from the BM (134). EPC normally have the ability to develop into fully mature EC and contribute to neovascularization by targeting sites of endothelial injury (135). They represent a heterogeneous cell population originated from a single multipotent progenitor within the BM and consist of cells at different stages of maturation, ranging from early CD133+/VEGFR2+ to more mature CD34+/VEGFR2+ phenotypes (136). Impaired functioning of the EPC has been thought to be involved in the pathogenesis of SSc.
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In total 22 articles on the role of ECs in defective SSc vasculogenesis were selected. The most described biomarker in this selection are EPC (31, 63, 79, 80, 84, 88, 94, 116, 135, 137-147). Biomarkers other than EPC for this disturbed process, described in the selected articles, are: circulating angiogenic cells (CACs) (139), epidermal growth factor-like domain 7 (EGFL7) (148) and pentraxin 3 (PTX3) (88, 100, 146). 3.6 Defective vascular tone control and the nitric oxide (NO) paradox in SSc
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Endothelin-1 (ET-1) plays a prominent role in vascular tone regulation through its receptors ETA and ETB. ETA receptor predominates on vascular smooth muscle and mediates vasoconstriction, whereas the ETB receptor subtype, when present on vascular endothelium, mediates vasodilation through NO release (149-151). ET-1 is shown overexpressed in skin, lung tissue and plasma of SSc patients suggesting a role for this mediator in SSc vasculopathy (31, 38, 87, 152-158). In total 32 articles on the role of EC in vascular tone mediation were selected (Table 1). Suggested biomarkers, other than ET-1, of (direct or indirect) vascular tone dysregulation, described by the selected articles, are: urotensin-II (U-II)(159), angiotensin (Ang) I (160), Ang II (160), heptapeptide Ang (1-7)(160), angiotensin converting enzyme (ACE) (37-39, 160), 8-isoprostane (161-165), nitric oxide (NO)(11, 166-177) and inducible nitric oxide synthase (iNOS)(166). The latter is the result of ischemia caused by vascular obliteration known to occur in proliferative vasculopathy. As a result fibroblasts and ECs in skin and lung tissue produce reactive oxygen species (ROS), including NO, that selectively activates ECs or trigger fibroblasts to produce collagen (178). Synthesized ROS (i.e. superoxide anions, hydrogen peroxide, hydroxyl radicals and/or peroxynitrite) selectively activate ECs, leading to vascular inflammation (179, 180). 3.7 Coagulation cascade and complement system impairment The interaction between ECs and platelets plays an important role in vascular tone regulation in SSc. Damaged ECs release several molecular substances into the circulation that interfere with coagulation homeostasis (181). In a recent in vitro study, it is shown that activated platelets induce thymic stromal lymphopoietin (TSLP) production in human dermal microvascular ECs inducing profibrotic and EC-activating factors such as interleukin (IL)-13 (182). Abnormalities and impairment of the coagulation system, such as presence of circulating supranormal multimers of von Willebrand factor (vWF) or elevated levels of vWF, factor VIII antigen, fibrinogen (FNG), thrombomodulin (TM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) have been described in SSc in multiple studies (19, 37-44, 87, 156, 181, 183). However whether or not impairment of fibrinolysis is present is still under debate since both depressed fibrinolytic activity (41) and a normal plasma fibrinolytic profile (40) has been reported. In total 14 articles have been selected for this topic. 16
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The role of the complement system (CS) in the pathogenesis of SSc vasculopathy has not been exhaustively investigated. The CS classically functions as a recruiter of inflammatory cells and regulates coagulation cascade and angiogenesis by damaging EC integrity (184). On human ECs, a group of membrane-bound complement regulators, including membrane cofactor protein (MCP or CD46) and decay accelerating factor (DAF or CD55), participates in protection from activation of both alternative and classic CS. An impaired expression of these regulators is shown in SSc skin, suggesting that a defective endothelial protection might be mediated by reduced expression of the complement regulatory proteins. This could potentially lead to endothelium-bound membrane attack complex of complement (MAC or C5B-9) depositions which are known to cause EC apoptosis (65, 185). Furthermore comprehensive peptidomics analysis revealed the predominance of complement C3fdes-arginine (DRC3f), derived from C3b, in the sera of patients with SSc and the level of DRC3f was related to vascular involvement in SSc and to SSc disease activity (186).
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3.8 ECs and their immune signature on SSc vasculopathy
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Although articles on vascular abnormalities in SSc were reviewed and selected, also the (direct or indirect) interaction between ECs with the immune system can contribute to the emergence or maintenance of SSc vasculopathy. In total 27 articles suggesting and discussing this interaction were selected. The selected immunological biomarkers, mostly cytokines, important in this vascular ECimmune system communication, are: macrophage migration inhibitory factor (MIF)(187, 188), IL-1α (189-191) and IL-1β (189, 191), IL-1 receptor-related protein (ST2)(192, 193), IL-2 (189, 194-196), soluble IL-2 receptor (21, 22, 194, 195), B cell growth factor (BCGF)(194), IL-4 (189, 197), IL-6 (18, 29, 189, 198, 199), soluble IL-6 receptor (198), oncostatin M (OSM)(198), soluble glycoprotein 130 (sGP130)(198), interferon γ (IFN-γ)(189), tumor necrosis factor alpha (TNF-α)(29, 189), IL-8 (19, 29, 30, 200), IL-10 (197), IL-13 (30, 197, 201), IL-15 (202, 203), IL-17A (204) and IL-33 (30, 191-193, 205207). All cytokines described in the selected clinical studies are shown upregulated in SSc plasma or (late stage) SSc skin as shown in supplementary file 4 and Table 2.
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3.9 EC interaction with other cells
Macrophages: They exist of diverse phenotypes, from "classically activated" M1 to "alternatively activated" M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic. M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant for stabilizing pericytes, and also promote anastomosis of sprouting ECs in vitro whereas M2c macrophages secrete the highest levels of MMP-9, a protease involved in vascular remodeling (208). A crosstalk between ECs and macrophages has been shown in cardiac microvascular inflammation (209). Furthermore ET-1 seems to induce the M2 phenotype in cultured human macrophages (210). Therefore similar intercellular interaction is suggested but not yet deeply investigated in SSc (211). Mast cells (MC): It is known for ECs to interact with mast cells through the production of Stem Cell Factor (SCF; c-kit ligand) to influence mast cell proliferation and differentiation (212). MC are increased in number in the lesional skin of SSc patients and are a valid source of TGF-β when degranulation occurs (213). Additionally SCF is shown to be strongly expressed on ECs in the SSc skin (214).
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4. DISCUSSION
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Pericytes (PCs): Among other cell types, ECs secrete PDGF-BB which recruits and induces proliferation of PCs progenitors (215). PDGF-BB serum levels are shown elevated in SSc patients (28). There is increasing evidence that PCs exhibit a phenotype similar to that of BM mesenchymal stem cells (BM-MSCs)(215). In a recent in vitro study SSc BM-MSCs are cocultured with healthy controls (HC) MVEC and cell reprogramming towards a pro-angiogenic behavior is observed (216). Fibroblasts: There is considerable evidence that EC products can modulate fibroblast properties in vivo. It is shown that SSc fibroblasts are particularly sensitive to EC-induced phenotypic modulation through IL-1 and bFGF and that damaging the EC vascular monolayer alters fibroblast responses (217). Furthermore it is shown that EC apoptosis plays a very important role in activating fibrogenic pathways through mediators that induce resistance to apoptosis in fibroblasts. These mediators in turn increase proliferation and inhibit apoptosis of EC and vascular smooth muscle cells (VSMC)(218, 219). A more recent study shows that SSc MVEC recruit and activate dermal fibroblasts by induction of a CCN2/TGF-β-dependent mesenchymal-to-mesenchymal transition this way clarifying the possible vascular connection of SSc-associated fibrosis (220). Furthermore evidence is provided that fibroblasts from SSc patients over-express MMP-12, which cleaves uPAR of normal MVEC, thus contributing to the failure of SSc-ECs to induce an efficient angiogenic program (99). Adipocytes: Recent studies have been focusing on the role of adipocytokines in the pathogenesis of SSc. Described markers that emphasize this emerging new interaction are: resistin (221), leptin (28, 221), adiponectin (221), chemerin (109) and retinol binding protein4 (RBP4)(222). Four articles have been selected concerning this interaction. Serum levels of possible biomarkers, shown in supplementary file 4, were mostly normal although correlations with the presence of digital ulcers (DU) were shown for elevated resistin and chemerin levels.
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In this systematic review on the role of EC in SSc vasculopathy, 193 selected articles present one or more aspects of EC dysfunction (Figure 1). Multiple significantly elevated activation markers, but mostly adhesion molecules, in the sera of SSc patients underline the significant involvement of EC activation in SSc pathogenesis. Dysregulation of apoptosis and proliferation within the vascular wall plays an important role in vascular remodeling associated with fibroproliferative vasculopathy observed in SSc. Apoptosis of ECs is recognized as an important component of the response to the injury phenomenon. Multiple selected studies have shown that AECA might be key players responsible for mediating this EC injury through ADCC. It is suggested that this ADCC is induced via the Fas pathway (46, 48). Though an earlier study not only failed to show Fas receptor binding by apoptosis-inducing AECA, it also showed that purified IgG AECA from SSc patients can induce apoptosis in human umbilical vein EC (HUVEC) through a membrane phospholipid reassembly with anionic phospholipid exposure and phosphatidylserine reaching the surface from its intracellular position (47). Additionally the apoptotic phenotype of endothelial progenitor cells (EPC) was investigated for possible mechanisms inducing apoptosis. Bone marrow studies showed significant titers of AECA, correlating with the number of apoptotic progenitors and in vitro assays on normal progenitors confirmed these results through induction of apoptosis by addition of AECA+purified IgG (55). Furthermore up-regulation of adhesion 18
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molecule expression (E-selectin, ICAM-1 and VCAM-1) and increased leukocyte adhesion (U937 cells) following AECA binding has also been shown to be an AECA binding effect (56). Because of these contradictory underlying mechanisms (activation versus apoptosis), more studies are needed to define the pathogenic role of AECA and its various subtypes before considering it as a useful biomarker.
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MPs are released from a variety of cells during activation and apoptosis via an exocytic budding process. Although long considered as inert debris, they are now appreciated as important mediators of cellular cross-talk regulating inflammation, coagulation, vascular functions, apoptosis and cell proliferation (223). Increased (67) as well as decreased (14) numbers of this EC apoptosis biomarker have been described in SSc. This contrast could be explained by a discrepancy in definition of examined MPs (according to size, Annexin V binding/non-binding MPs, CD144+/CD146+ EMP), isolation procedures and dissimilar use of fluorochromes and cell-specific, monoclonal antibodies. This implicates the need of a clear cut consensus on the exact characterization of MPs.
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Furthermore, it is shown in SSc that ECs lose their potential to participate in a normal angiogenesis process in response to EC injury. Mainly biomarkers suggestive for dysfunctional angiogenesis in relation to overexpression of pro-angiogenic mediators are described. The most suggestive ones in the selected articles are VEGF, VEGFR-2 and CXCL8. Despite elevation of VEGF in plasma and skin, adaptive angiogenesis is absent and a progressive loss of capillaries is observed (71). This paradox is further supported by the knowledge that short-time upregulation of VEGF is a strong inducer of angiogenesis, while chronic and uncontrolled overexpression of VEGF, which occurs in SSc, leads to the formation of irregularly shaped sac-like vessels with reduced blood flow in the newly formed vessels (74). However, most studies could not make the distinction between proangiogenic and antiangiogenic VEGF splice variants, which have been uncovered only recently. Indeed, although VEGF had conventionally been considered to be a family of proangiogenic mediators, the VEGF premRNA is shown to be differentially spliced in its terminal exon to form two distinct subfamilies of proteins, the so-called proangiogenic VEGFxxx isoforms and antiangiogenic VEGFxxxb isoforms (mainly VEGF165 and VEGF165b)(224). In a recent study, an increase in VEGF in SSc patients appears to be the result of a significant increase in the anti-angiogenic VEGF165b isoform instead of VEGF165. Furthermore in this study elevated circulating levels of VEGF165b in SSc patients are shown associated with early disease. Additionally by using blocking antibodies, severe impairment of in vitro angiogenesis is ameliorated in their study. Therefore this switch from pro-angiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia and needs further investigation (83). The most often described upregulated angiogenesis inhibitor in the selected articles is endostatin (31, 73, 75, 77, 81, 82, 95). Furthermore also alteration of transcripts involved in signal transduction pathways, i.e. the Ang/Tie2 signalling pathway (90) and the mechanism of uPAR truncation (98, 99, 101), is described. Also downregulation of the critical proangiogenic transcript Fli1 is mentioned in several articles (107-109) to play a part in the disturbed angiogenesis known in SSc vasculopathy. In seven selected articles, the role of EndoMT in SSc is also emphasized (127-133). In vitro studies using MVEC obtained from SSc patients confirmed a role for ET-1 and TGFβ as enhancers for EndoMT (127, 133). Nevertheless the mechanisms behind this transition are still not yet fully understood.
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ACCEPTED MANUSCRIPT While EndoMT is a known center stage in the fibrotic processes of many diseases (120-126), its role in the pathogenesis of SSc vasculopathy still needs clarification.
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The most often described biomarker of disturbed vasculogenesis are EPC. Nevertheless whether or not they can be used as biomarker in practice is still under debate since both decreased (79, 94, 137, 141, 143, 145) and increased (31, 63, 84, 138, 140, 142, 144, 147) levels of circulating EPC have been described in the selected literature. These contradictory results, displayed in supplementary file 5, could be due to the use of different protocols to quantify EPC, the use of different combinations of surface markers and differences in subsets of patients, used medications and disease durations between the studies. EPC can be defined by colony-forming unit (CFU) assays that use culture-based methods and by flow cytometry where expression of cell-surface antigens including CD34, CD133 and VEGFR2 is assayed. However, protocols for isolating and counting EPC are not yet standardized therefore causing discrepancy in results between studies. Recently the European League Against Rheumatism Scleroderma Trials and Research group (EUSTAR) proposed recommendations for the identification and measurement of EPC (225). Ever since these recommendations were published, mostly decreasing levels of EPC have been reported.
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In the dysregulation of endothelial dependent control of arteriolar/venular tone, NO is the most investigated biomarker in SSc vasculopathy. Nevertheless the exact status of NO production as biomarker in SSc (vasculopathy) is still controversial since both increased (11, 166-173, 175) and decreased (172, 174, 175) total nitrate (metabolites) levels in SSc patients have been reported (Supplementary file 6). Decreased NO levels could be explained by the rapid reaction of NO and superoxide anions to generate the reactive intermediate peroxynitrite (11). ET-1 is the second most described elevated biomarker in dysfunctional vascular tone control known to occur in SSc vasculopathy. Furthermore, multiple selected articles also emphasized the role of the reninangiotensin system (RAS) and its products in the integrated regulation of vascular tone.
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Additionally, EC also interact with the coagulation system by upregulating multiple markers of coagulation (most importantly vWF), this way contributing to the prothrombotic state of SSc patients (41). However whether or not impairment of fibrinolysis is present, is still under debate since both depressed fibrinolytic activity (41) and a normal plasma fibrinolytic profile (40) has been reported. The importance of the interaction of EC with the complement system in SSc vasculopathy has not been deeply investigated. Only three articles were selected concerning this topic. Although the immune system plays its own particular part in SSc pathogenesis, its interaction with EC and therefore its role in SSc vasculopathy, is not negligible. IL-33, one of the most described cytokines in the selected articles is abnormally expressed in early stage SSc skin (193) and is elevated in the sera of early SSc patients (30, 205, 206). Furthermore it is shown that the IL-33/ST2 axis increases proliferation, migration and morphologic differentiation of human ECs with increased endothelial permeability, consistently with increased angiogenesis in vivo (193). These findings support the hypothesis that IL-33 might mediate very early pathogenic events of SSc through recruitment and stimulation of ST2-expressing cells (immune cells and fibroblast/myofibroblast), this way modulating inflammatory and fibrotic processes in SSc. IL-2 is known to increase binding of natural killer cells to the endothelium (226). Needleman et al. show significantly higher IL-2 levels in SSc patients compared to HC using a CTLL bioassay (189). However when the same authors use an ELISA to determine both levels, no statistical significance between both groups was shown. Their 20
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ELISA findings suggest similar IL-2 levels in SSc patients and HC but increased IL-2 biologic activity in SSc patient sera. The latter is supported by the hypothesis of Kahaleh and LeRoy (196) that an IL-2 inhibitor is present in sera from HC but reduced in SSc sera. Famularo et al (194) also found an increase in mean IL-2 serum levels in SSc patients compared to controls. In contrast, Degiannis et al (195) found comparable levels of IL-2 in plasma from SSc patients and controls. Both authors use an ELISA detection method for IL-2 quantification. More recently IL-17A is investigated and authors observe that this cytokine induces the expression of adhesion molecules and chemokines in HUVEC. Furthermore it mediates vascular inflammation through ERK phosphorylation (204) clarifying the link between IL-17 producing T cells and endothelial injury in SSc vasculopathy.
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Finally the selected articles also provide limited evidence of EC interaction with other inflammatory (MC), vascular (PCs) or adipose cells. The latter is a relatively new discovery, which involves a new type of cytokines, the adipocytokines, this way expanding the spectrum of SSc vasculopathy. Although recent data suggest a role for macrophages in angiogenesis (208-211), there is currently no scientific evidence (in the form of (randomized) control trials, observational and/or in vitro studies) proving that EC-macrophage interaction contributes to SSc vasculopathy.
5. CONCLUSION
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Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by proliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs including the skin. This systematic review focuses on the role of ECs in SSc vasculopathy. The most representing and reliable biomarkers described by the selected articles were adhesion molecules (ICAM1, VCAM1, E-selectin, P-selectin) for EC activation, AECA for EC apoptosis, VEGF, its receptor VEGFR-2 and endostatin for disturbed angiogenesis, circulating EPC for defective vasculogenesis, ET-1 for disturbed vascular tone control, vWF for coagulopathy and IL-33 for EC- immune system communication. Emerging, relatively new discovered biomarkers described in the selected articles are VEGF165b, IL-17A and the adipocytokines. Although the etiology of SSc remains unclear, this systematic review summarizes the growing evidence that SSc is primarily a vascular disease where EC dysfunction is present and prominent in different aspects of cell survival (activation and apoptosis), angiogenesis and vasculogenesis and where disturbed interactions between ECs and various other cells contribute to SSc vasculopathy.
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Figure 1. Endothelial cell dysfunction in SSc vasculopathy: a multifunctional approach
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Figure legend: this figure summarizes the different dysfunctional aspects of EC in SSc vasculopathy.
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Table 1. Number of selected articles according to EC (dys)function in SSc vasculopathy
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Table legend: data are given as number of articles selected in the systematic review, categorized according to EC (dys)function. Some articles (n=33) covered more than one topic and were therefore categorized in more than one function group. This way the grand total exceeds 193 articles.
Table 2. Regulation of EC apoptosis markers described by the selected articles
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Table legend: data are given as number of selected articles describing significant upregulation, significant downregulation or normal regulation (i.e. no significant difference in regulation between controls and SSc population) of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc or certain SSc subtypes. Selected basic science articles (in vitro experiments, animal studies) involving the process of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc are not included in this table.
Supplementary file 1. Details on search strategy using the Prisma 2009 checklist Supplementary file 2. Flow diagram of search process according to PRISMA 2009 guidelines
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Supplementary file 3. Quality assessment of the selected articles Supplementary file 4. Summary of all 193 selected articles
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Supplementary file 5. Discrepancy in selected literature concerning levels of circulating EPC in SSc patients
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Supplementary file 6. Discrepancy in literature concerning levels of NO (metabolites)
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[187] Selvi E, Tripodi SA, Catenaccio M, Lorenzini S, Chindamo D, Manganelli S, et al. Expression of macrophage migration inhibitory factor in diffuse systemic sclerosis. Ann Rheum Dis 2003; 62(5): 460-4. [188] Corallo C, Paulesu L, Cutolo M, Ietta F, Carotenuto C, Mannelli C, et al. Serum levels, tissue expression and cellular secretion of macrophage migration inhibitory factor in limited and diffuse systemic sclerosis. Clin Exp Rheumatol 2015; 33(4 Suppl 91): S98-105. [189] Needleman BW, Wigley FM, Stair RW. Interleukin-1, interleukin-2, interleukin-4, interleukin6, tumor necrosis factor alpha, and interferon-gamma levels in sera from patients with scleroderma. Arthritis Rheum 1992; 35(1): 67-72. [190] Maekawa T, Jinnin M, Ohtsuki M, Ihn H. Serum levels of interleukin-1α in patients with systemic sclerosis. J Dermatol 2013; 40(2): 98-101. [191] Zhang YJ, Zhang Q, Yang GJ, Tao JH, Wu GC, Huang XL, et al. Elevated serum levels of interleukin-1β and interleukin-33 in patients with systemic sclerosis in Chinese population. Z Rheumatol 2016. [192] Manetti M, Ibba-Manneschi L, Liakouli V, Guiducci S, Milia AF, Benelli G, et al. The IL1-like cytokine IL33 and its receptor ST2 are abnormally expressed in the affected skin and visceral organs of patients with systemic sclerosis. Ann Rheum Dis 2010; 69(3): 598-605. [193] Choi YS, Choi HJ, Min JK, Pyun BJ, Maeng YS, Park H, et al. Interleukin-33 induces angiogenesis and vascular permeability through ST2/TRAF6-mediated endothelial nitric oxide production. Blood 2009; 114(14): 3117-26. [194] Famularo G, Procopio A, Giacomelli R, Danese C, Sacchetti S, Perego MA, et al. Soluble interleukin-2 receptor, interleukin-2 and interleukin-4 in sera and supernatants from patients with progressive systemic sclerosis. Clin Exp Immunol 1990; 81(3): 368-72. [195] Degiannis D, Seibold JR, Czarnecki M, Raskova J, Raska K. Soluble interleukin-2 receptors in patients with systemic sclerosis. Clinical and laboratory correlations. Arthritis Rheum 1990; 33(3): 375-80. [196] Kahaleh MB, LeRoy EC. Interleukin-2 in scleroderma: correlation of serum level with extent of skin involvement and disease duration. Ann Intern Med 1989; 110(6): 446-50. [197] Hasegawa M, Fujimoto M, Kikuchi K, Takehara K. Elevated serum levels of interleukin 4 (IL-4), IL-10, and IL-13 in patients with systemic sclerosis. J Rheumatol 1997; 24(2): 328-32. [198] Hasegawa M, Sato S, Fujimoto M, Ihn H, Kikuchi K, Takehara K. Serum levels of interleukin 6 (IL-6), oncostatin M, soluble IL-6 receptor, and soluble gp130 in patients with systemic sclerosis. J Rheumatol 1998; 25(2): 308-13. [199] Khan K, Xu S, Nihtyanova S, Derrett-Smith E, Abraham D, Denton CP, et al. Clinical and pathological significance of interleukin 6 overexpression in systemic sclerosis. Ann Rheum Dis 2012; 71(7): 1235-42. [200] Sakamoto N, Kakugawa T, Hara A, Nakashima S, Yura H, Harada T, et al. Association of elevated α-defensin levels with interstitial pneumonia in patients with systemic sclerosis. Respir Res 2015; 16: 148. [201] Riccieri V, Rinaldi T, Spadaro A, Scrivo R, Ceccarelli F, Franco MD, et al. Interleukin-13 in systemic sclerosis: relationship to nailfold capillaroscopy abnormalities. Clin Rheumatol 2003; 22(2): 102-6. [202] Suzuki J, Morimoto S, Amano H, Tokano Y, Takasaki Y, Hashimoto H. Serum levels of interleukin 15 in patients with rheumatic diseases. J Rheumatol 2001; 28(11): 2389-91. [203] Wuttge DM, Eriksson P, Sirsjö A, Hansson GK, Stemme S. Expression of interleukin-15 in mouse and human atherosclerotic lesions. Am J Pathol 2001; 159(2): 417-23. [204] Xing X, Yang J, Yang X, Wei Y, Zhu L, Gao D, et al. IL-17A induces endothelial inflammation in systemic sclerosis via the ERK signaling pathway. PLoS One 2013; 8(12): e85032. [205] Manetti M, Guiducci S, Ceccarelli C, Romano E, Bellando-Randone S, Conforti ML, et al. Increased circulating levels of interleukin 33 in systemic sclerosis correlate with early disease stage and microvascular involvement. Ann Rheum Dis 2011; 70(10): 1876-8. 33
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[206] Terras S, Opitz E, Moritz RK, Höxtermann S, Gambichler T, Kreuter A. Increased serum IL-33 levels may indicate vascular involvement in systemic sclerosis. Ann Rheum Dis 2013; 72(1): 144-5. [207] Yanaba K, Yoshizaki A, Asano Y, Kadono T, Sato S. Serum IL-33 levels are raised in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis. Clin Rheumatol 2011; 30(6): 825-30. [208] Spiller KL, Anfang RR, Spiller KJ, Ng J, Nakazawa KR, Daulton JW, et al. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials 2014; 35(15): 4477-88. [209] Pabois A, Pagie S, Gérard N, Laboisse C, Pattier S, Hulin P, et al. Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation. Biochem Pharmacol 2016; 104: 95-107. [210] Soldano S, Pizzorni C, Paolino S, Trombetta AC, Montagna P, Brizzolara R, et al. Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages. PLoS One 2016; 11(11): e0166433. [211] Manetti M. Deciphering the alternatively activated (M2) phenotype of macrophages in scleroderma. Exp Dermatol 2015; 24(8): 576-8. [212] Coleman JW, Holliday MR, Kimber I, Zsebo KM, Galli SJ. Regulation of mouse peritoneal mast cell secretory function by stem cell factor, IL-3 or IL-4. J Immunol 1993; 150(2): 556-62. [213] Hügle T, Hogan V, White KE, van Laar JM. Mast cells are a source of transforming growth factor β in systemic sclerosis. Arthritis Rheum 2011; 63(3): 795-9. [214] Yamamoto T, Katayama I, Nishioka K. Expression of stem cell factor in the lesional skin of systemic sclerosis. Dermatology 1998; 197(2): 109-14. [215] da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells 2008; 26(9): 2287-99. [216] Cipriani P, Marrelli A, Benedetto PD, Liakouli V, Carubbi F, Ruscitti P, et al. Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine. Angiogenesis 2013; 16(3): 595-607. [217] Denton CP, Xu S, Black CM, Pearson JD. Scleroderma fibroblasts show increased responsiveness to endothelial cell-derived IL-1 and bFGF. J Invest Dermatol 1997; 108(3): 269-74. [218] Raymond MA, Vigneault N, Luyckx V, Hébert MJ. Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells. Biochem Biophys Res Commun 2002; 291(2): 2619. [219] Laplante P, Raymond MA, Gagnon G, Vigneault N, Sasseville AM, Langelier Y, et al. Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis. J Immunol 2005; 174(9): 5740-9. [220] Serratì S, Chillà A, Laurenzana A, Margheri F, Giannoni E, Magnelli L, et al. Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor β-dependent mesenchymal-to-mesenchymal transition. Arthritis Rheum 2013; 65(1): 258-69. [221] Olewicz-Gawlik A, Danczak-Pazdrowska A, Kuznar-Kaminska B, Batura-Gabryel H, Katulska K, Wojciech S, et al. Circulating adipokines and organ involvement in patients with systemic sclerosis. Acta Reumatol Port 2015; 40(2): 156-62. [222] Toyama T, Asano Y, Takahashi T, Aozasa N, Akamata K, Noda S, et al. Clinical significance of serum retinol binding protein-4 levels in patients with systemic sclerosis. J Eur Acad Dermatol Venereol 2013; 27(3): 337-44. [223] Distler JH, Jüngel A, Huber LC, Seemayer CA, Reich CF, Gay RE, et al. The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles. Proc Natl Acad Sci U S A 2005; 102(8): 2892-7. [224] Qiu Y, Hoareau-Aveilla C, Oltean S, Harper SJ, Bates DO. The anti-angiogenic isoforms of VEGF in health and disease. Biochem Soc Trans 2009; 37(Pt 6): 1207-13. [225] Distler JH, Allanore Y, Avouac J, Giacomelli R, Guiducci S, Moritz F, et al. EULAR Scleroderma Trials and Research group statement and recommendations on endothelial precursor cells. Ann Rheum Dis 2009; 68(2): 163-8. 34
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[226] Aronson FR, Libby P, Brandon EP, Janicka MW, Mier JW. IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury. J Immunol 1988; 141(1): 158-63.
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Activation Apoptosis
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Fibrosis
Defective vascular tone control
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Coagulopathy
Dysfunctional EC
Disturbed angiogenesis
Defective vasculogenesis
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MC dysregulation
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Dysregulation of adipocytokines
Dysregulation of vascular system through pericyte/EC interaction
Immune dysregulation EndoMT
Figure 1. Endothelial cell dysfunction in SSc vasculopathy: a multifunctional approach Figure 1 legend: this figure summarizes the different dysfunctional aspects of EC in SSc vasculopathy. Abbreviations used in figure 1: EC: endothelial cell; EndoMT: endothelial-mesenchymal transition; MC: mast cell; SSc: systemic sclerosis
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ACCEPTED MANUSCRIPT Table 1. Number of selected articles according to EC (dys)function in SSc vasculopathy TOTAL
EC activation
38
EC apoptosis
29
Disturbed angiogenesis
56
EndoMT
7
Defective vasculogenesis
22
Defective vascular tone control
32
IP SC R
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Coagulation cascade and complement impairment
17
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Immune dysregulation
27
Mast cell dysregulation
1
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Dysregulation of the vascular system through pericytes/ECs crosstalk Fibrosis
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EC function
2
5 4
GRAND TOTAL
240
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Dysregulation of adipocytokines
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Table legend: data are given as number of articles selected in the systematic review, categorized according to EC (dys)function. Some articles (n=33) covered more than one topic and were therefore categorized in more than one function group. This way the grand total exceeds 193 articles. Abbreviations used in table: EC: endothelial cell, EndoMT: endothelial-mesenchymal transition, EPC: endothelial progenitor cells
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ACCEPTED MANUSCRIPT Table 2. Regulation of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication described by the selected articles Norma Upregulat Downregula lly TOT ion tion regulat AL ed
Biomarkers
4
0
1
5
0
0
1
1
0
0
1
2
0
0
2
1
1
0
2
1
0
0
1
0
0
1
1
0
0
1
1
1
1
0
2
1
0
0
1
0
1
0
1
1
0
0
1
1
0
0
1
Collagen V
1
0
0
1
VEGF
9
1
1
11
VEGF165bVEGF165bVEGF165bVEGF165bVEGF 165bVEGF165b
1
0
0
1
VEGFR-1
0
1
0
1
VEGFR-2
1
0
0
1
VEGFR-3
0
0
1
1
PDGF-BB
1
0
0
1
PDGF
1
0
1
2
FGF-2 Markers of disturbed Serum βFGF angiogenesi HGF s
2
0
0
2
1
0
1
2
3
0
0
3
sTie1
0
0
1
1
sTie2
1
0
1
2
Ang-1
0
1
0
1
Ang-2
3
0
0
3
Angptl3
1
0
0
1
PlGF
4
0
0
4
PEDF
0
0
1
1
Endostatin
6
0
1
7
IP
AECA
T
Examin ed SSc tissue
1
SC R
IgG anti-caspase-3 Ab Caspase-3 IFI16 Ab Serum CEC Markers of EC apoptosis
NU
OPG TRAIL MAC (C5b9)
MA
MPs IFI16 Ab BMPRII MAC (C5b9)
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Lung
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Fra-2
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Skin
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0
1
1
GRO-α
1
0
0
1
MMP-12
1
0
0
1
MMP-9
1
0
0
1
Pro-MMP-1
1
0
0
1
CTSB
1
0
0
1
1
0
1
1
0
0
1
1
0
0
1
0
0
1
1
1
0
0
1
1
0
0
1
1
0
0
1
0
1
0
1
4
0
0
4
1
0
0
1
2
0
0
2
2
0
0
2
3
0
0
3
1
0
0
1
1
0
0
1
VEGF
3
0
0
3
VEGF165bVEGF165bVEGF165bVEGF165b
1
0
0
1
VEGFR-1
2
0
1
3
VEGFR-2
5
0
0
5
VEGFR-3
2
0
0
2
GLUT-1
1
0
0
1
Tie1
0
1
0
1
MMP-12
3
0
0
3
uPAR
1
0
0
1
KLK1
0
1
1
2
KLK9
0
1
0
1
KLK11
0
1
0
1
KLK12
0
1
0
1
CTSB
1
0
0
1
CTSV
0
1
0
1
CTSL
1
0
0
1
CCN-1
0
1
0
1
IP 0
SC R
CTSV CTSL Galectin 3 CCN-1
NU
Apelin CXCL1 CXCL4
MA
CXCL5 CXCL8 CXCL9
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CXCL10
CCL13
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CCL23
TE
CXCL16 CCL2
Skin
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TIMP2
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ACCEPTED MANUSCRIPT 1
0
0
1
CXCL5
0
1
0
1
CXCL8
1
0
0
1
CXCL9
1
0
0
1
CXCL10
1
0
0
1
CXCL16
1
0
0
1
1
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
2
0
0
2
3
0
1
4
2
0
0
2
1
0
0
1
2
0
0
2
3
0
0
3
1
0
0
1
0
0
1
1
1
0
1
2
IL-1α
1
0
2
3
TNF-α
0
0
1
1
IFNγ
0
0
1
1
IL-8
2
1
0
3
IL-10
1
0
0
1
IL-13
3
0
0
3
IL-15
2
0
0
2
IL-17A
1
0
0
1
IL-33
6
0
0
6
MIF
2
0
0
2
ST2
1
0
0
1
IL-6
2
0
0
2
IL-8
1
0
0
1
IL-17A
1
0
0
1
IL-33
1*
0*
0*
1
IP 0
SC R
CXCR3 CXCR6 CXCL12 CCL13
NU
CCL23 BM
VEGF MIF
MA
IL-2 sIL-2R BCGF
D
IL-4
OSM
CE P
AC
Serum Markers of disturbed EC-immune system communica tion
IL-1β
TE
IL-6 sIL-6R
Skin
T
CXCL4
* In early SSc, IL-33 protein was downregulated or absent in EC, while IL-33 mRNA was normally expressed or even upregulated. In late SSc, IL-33 was constitutively found in most EC.
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Table legend: data are given as number of selected articles describing significant upregulation, significant downregulation or normal regulation (i.e. no significant difference in regulation between controls and SSc population) of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc or certain SSc subtypes. Selected basic science articles (in vitro experiments, animal studies) involving the process of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc are not included in this table. Abbreviations used in table: Ab: antibodies; AECA: anti-endothelial cell antibodies; Ang-: Angiopoietin-; Angptl3: Angiopoietin-like protein 3; BCGF: B cell growth factor; BM: bone marrow; BMPRII: bone morphogenetic protein receptor II; CCL: chemokine ligand; CCN-1: cysteine-rich 61 matrix protein; CEC: circulating endothelial cells; CTSB: cathepsin B; CTSL: cathepsin L; CTSV: cathepsin V; CXCL: chemokine (C-X-C motif) ligand; EC: endothelial cell; FGF: fibroblast growth factor; Fli1: Friend leukemia integration factor 1; Fra-2: Fos-related antigen-2; GLUT-1: glucose transporter 1; GRO-α: Growth-Regulated Oncogene-α; HGF: hepatocyte growth factor; IFI16: Gamma-interferon-inducible protein IFI-16; IFNγ: interferon gamma; IL: interleukin-; KLK: human tissue kallikreins; MAC: membrane attack complex of complement; MIF: macrophage migration inhibitory factor; MMP: matrix metalloproteinases; MPs: microparticles; mRNA: messenger ribonucleic acid; OPG: osteoprotegerin; OSM: oncostatin M; PDGF: platelet-derived growth factor; PEDF: pigment epithelium-derived factor; PlGF: placental growth factor; sIL-2R: soluble IL-2 receptor; sIL-6R: sIL-6 receptor; SSc: systemic sclerosis; ST2: IL-1 receptor-related protein; Tie: soluble tyrosine kinase; TIMP2: tissue inhibitor of metalloproteinase 2; TNF-α: tumor necrosis factor alpha; TRAIL: TNF-related apoptosisinducing ligand; uPAR: urokinase-type plasminogen activator receptor; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor
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S1568-9972(17)30148-9 doi:10.1016/j.autrev.2017.05.024 AUTREV 2028
To appear in:
Autoimmunity Reviews
Received date: Accepted date:
8 April 2017 13 April 2017
Please cite this article as: Mostmans Y, Cutolo M, Giddelo C, Decuman S, Melsens K, Declercq H, Vandecasteele E, De Keyser F, Distler O, Gutermuth J, Smith V, The role of endothelial cells in the vasculopathy of systemic sclerosis: A systematic review, Autoimmunity Reviews (2017), doi:10.1016/j.autrev.2017.05.024
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ACCEPTED MANUSCRIPT The role of endothelial cells in the vasculopathy of systemic sclerosis: a systematic review SSc vasculopathy: the role of endothelial cells
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Y. Mostmans1, M. Cutolo2, C. Giddelo1, S. Decuman3, K. Melsens4, H. Declercq5, E. Vandecasteele6, F. De Keyser4, O. Distler7, J. Gutermuth1, V. Smith4
1. Vrije Universiteit Brussel (VUB), Universitair Ziekenhuis Brussel (UZ Brussel), Department of
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Dermatology, Laarbeeklaan 101, 1090 Brussels, Belgium; Research Laboratory and Academic Unit of Clinical Rheumatology, Department of Internal Medicine, University of Genova, Genova, Italy Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, Ghent, Belgium; and Department of Rheumatology, Ghent University, Ghent, Belgium Department of Basic Medical Sciences, Tissue Engineering and Biomaterials Group, Ghent University, Ghent, Belgium Department of Cardiology, Ghent University Hospital, Ghent, Belgium Department of Rheumatology, University Hospital Zurich, Zurich, Switzerland
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Corresponding author:
Dr. Yora Mostmans* Department of Dermatology/Universitary Hospital Brussels (UZ Brussel) Vrije Universiteit Brussel (VUB) Laarbeeklaan 101 1090 Brussels, Belgium Tel. +32 2 477 63 55; Fax +32 2 477 63 57 Email: [email protected] *
Present address: Department of Immunology and Allergology (CIA) Centre Hospitalier Universitaire (CHU) Brugmann Université Libre de Bruxelles (ULB) Van Gehuchtenplein 4 1020 Brussels, Belgium Tel. +32 2 477 22 94; Fax +32 2 477 22 76 Email: [email protected] 1
ACCEPTED MANUSCRIPT Statement of author contribution:
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Yora Mostmans: substantial contributions to design of the study, acquisition of data and analysis and interpretation of data, drafting of the article, final approval of the version to be published.
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Maurizio Cutolo: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Christina Giddelo: acquisition of data and analysis and interpretation of data, critical revising of the article for important intellectual content, final approval of the version to be published.
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Saskia Decuman: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Karin Melsens: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Heidi De Clercq: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published.
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Els Vandecasteele: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Filip De Keyser: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Oliver Distler: interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Jan Gutermuth: analysis and interpretation of data, critical revising of the article for important intellectual content and final approval of the version to be published. Vanessa Smith: substantial contributions to design of the study, acquisition of data and analysis and interpretation of data, drafting of the article, final approval of the version to be published. 2
ACCEPTED MANUSCRIPT Statement of conflict of interest:
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Yora Mostmans: no conflicts of interest to declare Maurizio Cutolo: no conflicts of interest to declare Christina Giddelo: no conflicts of interest to declare Saskia Decuman: no conflicts of interest to declare Karin Melsens: no conflicts of interest to declare Heidi De Clercq: no conflicts of interest to declare Els Vandecasteele: no conflicts of interest to declare Filip De Keyser: no conflicts of interest to declare Oliver Distler: has/had consultancy relationship and/or has received research funding from 4 D Science, Actelion, Active Biotec, Bayer, Biogen Idec, Boehringer Ingelheim Pharma, BMS, ChemomAb, EpiPharm, Ergonex, espeRare foundation, GSK,Roche-Genentech, Inventiva, Lilly, medac, MedImmune, Mitsubishi Tanabe, Pharmacyclics, Pfizer, Sanofi, Serodapharm and Sinoxa in the area of potential treatments of scleroderma and its complications. He has a patent on mir-29 for the treatment of systemic sclerosis licensed. Jan Gutermuth: no conflicts of interest to declare Vanessa Smith: no conflicts of interest to declare
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ACCEPTED MANUSCRIPT ABSTRACT
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Introduction: Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibroproliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs including the skin. In this study, all English speaking articles concerning the role of endothelial cells (ECs) in SSc vasculopathy and representing biomarkers are systematically reviewed and categorized according to endothelial cell (EC) (dys)function in SSc.
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Methods: A sensitive search on behalf of the EULAR study group on microcirculation in Rheumatic Diseases was developed in Pubmed, The Cochrane Library and Web of Science to identify articles on SSc vasculopathy and the role of ECs using the following Mesh terms: (systemic sclerosis OR scleroderma) AND pathogenesis AND (endothelial cells OR marker). All selected papers were read and discussed by two independent reviewers. The selection process was based on title, abstract and full text level. Additionally, both reviewers further searched the reference lists of the articles selected for reading on full text level for supplementary papers. These additional articles went through the same selection process.
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Results: In total 193 resulting articles were selected and the identified biomarkers were categorized according to description of EC (dys)function in SSc. The most representing and reliable biomarkers described by the selected articles were adhesion molecules for EC activation, anti-endothelial cell antibodies for EC apoptosis, vascular endothelial growth factor (VEGF), its receptor VEGFR-2 and endostatin for disturbed angiogenesis, endothelial progenitors cells for defective vasculogenesis, endothelin-1 for disturbed vascular tone control, Von Willebrand factor for coagulopathy and interleukin (IL)-33 for EC- immune system communication. Emerging, relatively new discovered biomarkers described in the selected articles, are VEGF165b, IL-17A and the adipocytokines. Finally, myofibroblasts involved in tissue fibrosis in SSc can derive from ECs or epithelial cells through a process known as endothelial-to-mesenchymal transition.
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Conclusion: This systematic review emphasizes the growing evidence that SSc is primarily a vascular disease where EC dysfunction is present and prominent in different aspects of cell survival (activation and apoptosis), angiogenesis and vasculogenesis and where disturbed interactions between ECs and various other cells contribute to SSc vasculopathy.
KEYWORDS biomarker, endothelial cells, systemic sclerosis, systematic review, vasculopathy, EULAR study group on microcirculation in Rheumatic Diseases 4
ACCEPTED MANUSCRIPT Abbreviations Ab: antibodies ACAs: anti-centromere antibodies
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ACE: angiotensin converting enzyme ADCC: antibody dependent cell mediated cytotoxicity
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ADMA: asymmetric dimethylarginine ADP: adenosine diphosphate
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AECA: anti-endothelial cell antibodies Ag: antigen
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AIF-1: allograft inflammatory factor 1 Ang I: angiotensin I
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Ang II: angiotensin II
Ang-2: angiopoietin 2
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Ang-1: angiopoietin 1
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Angptl3: angiopoietin-like protein 3 Anti-topo I: anti-topoisomerase ATIII: antithrombin III
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BALF: broncho-alveolar lavage fluid BCGF: B cell growth factor bFGF: basic fibroblast growth factor-2 BM: bone marrow BM-MSCs: bone marrow mesenchymal stem cells BMPRII: bone morphogenetic protein receptor II CACs: circulating angiogenic cells cAMP: cyclic adenosine monophosphate CCL: chemokine ligand CCN-1: cysteine-rich 61 matrix protein
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ACCEPTED MANUSCRIPT CD: cluster of differentiation CD44R: cluster of differentiation 44 receptor CEC: circulating endothelial cells
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CFU: colony-forming unit cGMP: cyclic guanosine monophosphate
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CIC: circulating immune complexes CKO: knock out
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CRP: C-reactive protein CTSB: cathepsin B
CTSV: cathepsin V
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CXCL: chemokine (C-X-C motif) ligand
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CTSL: cathepsin L
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CX3CR1: fractalkine receptor DAF: decay accelerating factor
DD: d-dimers
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dcSSc: diffuse cutaneous systemic sclerosis
DLCO: diffusing capacity for carbon monoxide
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DMVEC: dermal microvascular endothelial cells DRC3f: complement C3f-des-arginine DS: dermatansulphate DU: digital ulcers EC: endothelial cell ECA: endothelial cytotoxic activity ECs: endothelial cells EGFL7: epidermal growth factor-like protein 7 EMP: endothelial cell-derived microparticles ENA-78: epithelial-neutrophil activating peptide-78
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ACCEPTED MANUSCRIPT EndoMT: endothelial-to-mesenchymal transition eNOS: endothelial nitric oxide synthase EPC: endothelial progenitor cells
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ERK: extracellular signal-regulated kinase EScSG: the European Scleroderma Study Group
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ESR: erythrocyte sedimentation rate
EUSTAR: The European Scleroderma Trials and Research group
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F-AnxV(-): Annexin V non-binding
FASSc: fibrosing alveolitis associated with systemic sclerosis
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FEV: forced expiratory volume FKN: fractalkine
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FNG: fibrinogen
Fra-2: fos-related antigen-2
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FVC: forced vital capacity
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Fli1: friend leukemia integration factor 1
F2IP-M: tetranor-dicarboxylic acid metabolite of F2-isoprostanes F1+2: prothrombin fragments 1+2
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fVIII/vWF Ag: factor VIII/von Willebrand factor antigen Gal3: galectin 3
GLUT-1: glucose transporter 1 GPA: granulomatosis with polyangiitis GRO-α : growth-regulated oncogene-alpha GVHD: graft-versus-host disease HC: healthy controls HDEC: human dermal endothelial cells HMVEC: human microvascular endothelial cells HGF: hepatocyte growth factor
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ACCEPTED MANUSCRIPT HRC: hypertensive renal crisis HUVEC: human umbilical vein endothelial cells ICAM: intercellular adhesion molecule
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IF: immunofluorescence IFI16: gamma-interferon-inducible protein IFI-16
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IFN-γ: interferon gamma Ig: immunoglobulin
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IL: interleukin IP: interstitial pneumonia
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iNOS: inducible NO synthase JAM: junctional adhesion molecule
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KDR: kinase insert domain receptor
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KLK: human tissue kallikrein
LMP: leukocyte-derived microparticle
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Lp(a): lipoprotein (a)
MAC: membrane attack complex of complement MC: mast cells
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MCP: membrane cofactor protein
MDMP: monocyte-derived microparticle Mesh: medical subject heading MIF: macrophage migration inhibitory factor MMP: matrix metalloproteinases MMT: mesenchymal-to-mesenchymal transition MPs: microparticles mRNA: messenger ribonucleic acid MRSS: modified Rodnan skin thickness score MVD: microvessel density
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ACCEPTED MANUSCRIPT MVEC: microvascular endothelial cells Nb: number NEP: neutral endopeptidase
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NIH: national institute of health NO: nitric oxide
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NOS: nitric oxide synthase NOx: total nitrate and nitrite
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NVC: nail fold videocapillaroscopy OA: osteoarthritis
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OPG: osteoprotegerin OSM: oncostatin M
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PAP: pulmonary artery pressure
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PAI: plasminogen activator inhibitor
PCs: pericytes
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PDGF (-BB): platelet derived growth factor (-BB) PDMP: platelet-derived microparticle PEDF: pigment epithelium-derived factor
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PECAM: platelet endothelial cell adhesion molecule PH: pulmonary hypertension PI3K: phosphatidylinositol-4,5-bisphosphate 3-kinase PL: phospholipids PlGF: placental growth factor PRP: primary raynaud phenomenon PS: phosphatidylserine PSS: progressive systemic sclerosis PTX3: pentraxin 3 RA: rheumatoid arthritis
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ACCEPTED MANUSCRIPT RANTES: regulated on activation normally T cell expressed and secreted RAS: renin-angiotensin system RBP4: retinol binding protein-4
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RF: rheumatoid factor RhoA: GTPase Ras homolog gene family-member Ab
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RNAse: ribonuclease ROCKS: rho-associated creatine kinases
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ROS: reactive oxygen species RP: raynaud phenomenon
RVSP: right ventricular systolic pressure
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RT-PCR: reverse-transcriptase polymerase chain reaction
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sCD40L: soluble cluster of differentiation-40 ligand
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SCF: stem cell factor SDF-1: stromal cell-derived factor 1
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sENG: soluble endoglin sFKN: soluble fractalkine
sGP130: soluble glycoprotein 130
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sIL: soluble interleukin
sIL-2R: soluble IL-2 receptor sIL-6R: soluble IL-6 receptor siRNA: small interfering RNA SLE: systemic lupus erythematosus α-SMA: alpha smooth muscle actin SM22α: transgelin SNAIL-1: transcriptional repressor zinc finger protein SPARC: secreted protein, acidic and rich in cysteine SRC: scleroderma renal crisis
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ACCEPTED MANUSCRIPT SSc: systemic sclerosis sTie1: soluble tyrosine kinase 1 sTie2: soluble tyrosine kinase 2
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ST2: IL-1 receptor-related protein sVEGF: soluble vascular endothelial growth factor
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TAT: thrombin-antithrombin TBARS: thiobarbituric acid reactive substances
TGF-β: tumor growth factor beta
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TIMP: tissue inhibitor of metalloproteinases
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β-TG: beta-thromboglobulin
TLR: toll-like receptors
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TNF-α: tumor necrosis factor alpha
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TM: thrombomodulin
tPA: tissue type plasminogen activator
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TRAIL: tumor necrosis factor related apoptosis-inducing ligand TSLP: thymic stromal lymphopoietin TSP-1: thrombospondin 1
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TXAR: thromboxane A2 receptor
uPAR: urokinase-type plasminogen activator receptor U-II: urotensin-II VA: alveolar volume VC: vital capacity VCAM: vascular cell adhesion molecule VE-cadherin: vascular endothelial cadherin VEGF: vascular endothelial growth factor VEGFR: vascular endothelial growth factor receptor VSMC: vascular smooth muscle cells
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vWF: von willebrand factor
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ACCEPTED MANUSCRIPT The role of endothelial cells in the vasculopathy of systemic sclerosis: a systematic review 1. INTRODUCTION
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Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by fibroproliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs such as skin and lung (1). Dysregulation of endothelial cell (EC) function within the vascular wall plays an important role in vascular remodeling associated with the fibroproliferative vasculopathy observed in SSc. Endothelial cell injury is proposed as a crucial initiating event leading to vascular remodeling with intimal proliferation of arterioles and capillary breakdown and finally, blood vessel occlusion (2-4). Ongoing research continues to focus on the role of endothelial cells (ECs) in SSc vasculopathy and on identification of related biomarkers. This study, performed on behalf of the EULAR study group on microcirculation in Rheumatic Diseases, gives an overview on the large body of data of current research in this domain, obtained after systematically reviewing the literature.
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2. METHODS 2.1 Search strategy
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A structured search on Pubmed, The Cochrane Library and Web of Science was performed without limitation on publication date to identify articles on SSc vasculopathy and the role of ECs. The Medical subject heading (Mesh) term for “systemic sclerosis” and its synonym “scleroderma” were used in combination with other groups of search terms: (systemic sclerosis OR scleroderma) AND pathogenesis AND (endothelial cells OR marker). The structured search was last updated on the 24th of November 2016. Details on search strategy are shown in Supplementary file 1. 2.2 Searching process
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All articles were screened by two independent reviewers (YM and CG). This selection process was based on title, abstract and full text level. All studies elaborating on the role of ECs in the pathogenesis of SSc vasculopathy were included in the systematic review. All study designs with the exception of reviews, editorials and case studies were included. Languages other than English were excluded. Titles and abstracts selected by either one of the reviewers were included for further screening. The final articles were withheld after reading and judgement of the full text. When different opinions existed among the two reviewers on full text level, consensus was reached. Additionally, both reviewers further searched the reference lists of the articles selected for reading on full text level for supplementary papers. These additional articles went through the same selection process performed by the same two reviewers. Supplementary file 2 depicts the flow of the searching process performed in this systematic review (5). The majority of articles were observational studies of which most were evaluated to be of fair quality using the quality assessment tool for observational studies adapted from the national institute of health (NIH) (supplementary file 3)(6). 3. RESULTS Of 2442 articles screened (on title, abstract and full text level) as potentially relevant to the topic of this study, 127 articles were included. From these articles, reviewers further searched the reference 13
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lists for more relevant articles not present in the database search, and identified 115 articles for additional screening. The flow of the searching and selection process is shown in supplementary file 2. Supplementary file 4 summarizes all 193 selected manuscripts on biomarkers reflecting EC dysfunction described in SSc vasculopathy. The majority of articles were observational studies, of which most were evaluated to be of fair quality according to the quality assessment tool for observational studies adapted from the national institute of health (NIH) (supplementary file 3)(6). The 193 selected articles were categorized according to description of EC (dys) function in SSc, as shown in Table 1 and Figure 1: EC-activation, EC-apoptosis, disturbed angiogenesis, endothelial-tomesenchymal transition (EndoMT), disturbed vasculogenesis, defective vascular tone control, coagulation cascade impairment, immune dysregulation, mast cell dysregulation, dysregulation of the vascular system through pericytes/ECs crosstalk, fibrosis and dysregulation of adipocytokines. Since many of these functional pathways interact, some markers were therefore categorized in more than one function group. 3.1 Markers of EC-activation
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In its early stages, SSc is characterized by EC injury and perivascular mononuclear cell infiltration with an accumulation of T and B lymphocytes and monocytes in the affected tissues (7). Interaction between these leukocytes and ECs is of major importance in the induction of EC permeability contributing to SSc vasculopathy. These interactions are highly dependent on the expression and function of cell adhesion molecules for the maintenance of transendothelial leukocyte migration. In total 38 articles concerning markers of EC-activation were selected (Table 1). The majority of selected articles covered adhesion molecules as biomarker of EC-activation: E-selectin (8-19), intercellular adhesion molecule (ICAM)(8, 9, 11, 12, 15-17, 20-24), junctional adhesion molecule (JAM)(25-27), platelet endothelial cell adhesion molecule (PECAM)(28), P-selectin (8, 9, 14, 15, 19, 29) and vascular cell adhesion molecule (VCAM)(9-12, 15, 17, 29-31). Nevertheless also other biomarkers in EC activation are described but less frequent: allograft inflammatory factor 1 (AIF-1)(32), asymetric dimethylarginine (ADMA)(33), fractalkine (FKN)(34, 35), fractalkine receptor (CX3CR1)(34), soluble endoglin (sENG)(31, 33, 36), Β1/β2/β4 integrins (16), von Willebrand factor (vWF) (37-44), counter adhesive proteins thrombospondin 1 (TSP-1) and secreted protein, acidic and rich in cysteine (SPARC)(13). The majority of discussed markers of EC activation are elevated in the sera of SSc or certain SSc subtypes (supplementary file 4). 3.2 EC-apoptosis Endothelial injury is regarded as a crucial initiating event leading to vascular remodeling with intimal proliferation of arterioles and capillary breakdown and blood vessel occlusion. EC apoptosis is recognized as an important component of the response to the injury. 29 articles investigating EC apoptosis and possible biomarkers were selected for this topic. Described biomarkers are: antiendothelial cell antibodies (AECA) (through antibody dependent cell mediated cytotoxicity (ADCC))(3, 37, 45-57), immunoglobulin (Ig) G anti-caspase-3 antibodies (Ab)(58), caspase-3 (59), gammainterferon-inducible protein (IFI)-16 Ab (60, 61), bone morphogenetic protein receptor II (BMPRII)(62), circulating endothelial cells (CEC)(31, 63), osteoprotegerin (OPG) and TNF-related apoptosis-inducing ligand (TRAIL)(64), membrane attack complex of complement (MAC)(65), collagen V (66), microparticles (MPs)(14, 67, 68) and Fos-related antigen-2 (Fra-2)(69). 14
ACCEPTED MANUSCRIPT Most markers of EC apoptosis were elevated in the sera of SSc patients (Table 2). 3.3 Disturbed angiogenesis
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A loss of vasculature in SSc results in tissue hypoxia, which normally promotes angiogenesis through the production of pro-angiogenic factors. Angiogenesis implicates endothelium sprouting from preexisting ECs and involves the proliferation and migration of mature ECs. However in SSc, angiogenesis is disturbed through expression of inefficient pro-angiogenic mediators, upregulation of powerful inhibitors of angiogenesis and by alteration of transcripts involved in signal transduction pathways (70). In total 56 articles on mechanisms of dysfunctional angiogenesis in SSc were selected (Table 1). Biomarkers described in these selected articles demonstrating this disturbance, shown in Table 2, are: vascular endothelial growth factor (VEGF)(28, 31, 33, 71-82), VEGF165b (83), vascular endothelial growth factor receptor (VEGFR) 1 (74, 78, 84, 85), VEGFR 2 (72, 74, 78, 85, 86), VEGFR 3 (78, 85), platelet-derived growth factor (PDGF)(77, 87), PDGF-BB (28), basic fibroblast growth factor-2 (bFGF, also known as FGF-2 or FGF-β)(73, 77, 79, 88), hepatocyte growth factor (HGF)(28, 77, 79), soluble tyrosine kinase 1 (sTie1)(89), sTie2 (31, 90), angiopoietin (Ang)-1 (Ang-1)(91), Ang-2 (28, 31, 91), angiopoietin-like protein 3 (Angptl3)(92), placental growth factor (PlGF) (31, 77, 93, 94), endostatin (31, 73, 75, 77, 81, 82, 95), tissue inhibitor of metalloproteinase 2 (TIMP-2)(75), growth-regulated oncogene-α (GRO-α)(96), glucose transporter 1 (GLUT-1)(72), pigment epithelium-derived factor (PEDF)(76), matrix metalloproteinases (MMP)-9 (77), MMP-12 (97-100) and pro-MMP-1 (77), urokinase-type plasminogen activator receptor (uPAR)(98, 99, 101), human tissue kallikreins (KLK) 1 (102, 103), KLK9 (102), KLK11 (102), KLK 12 (102), cathepsin B (CTSB)(104), cathepsin V (CTSV)(105), cathepsin L (CTSL)(106), friend leukemia integration factor 1 (Fli1)(107-109), galectin-3 (Gal3)(110), apelin (111), CCN1 (112) and chemokines epithelial-neutrophil activating peptide (ENA-78)(15), regulated on activation normally T-cell expressed and secreted (RANTES) (15), chemokine ligand (CXCL)1 (96), CXCL4 (113), CXCL5 (108), CXCL8 (15, 28-30, 96), CXCL9 (114), CXCL10 (114, 115), CXCL12 (116), CXCL16 (114, 117) and chemokine ligands (CCL)2 (19, 30), CCL13 (118), CCL23 (119).
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Most articles showed upregulation of proangiogenic and/or angiostatic biomarker(s) in SSc as shown in Table 2. 3.4 Endothelial-to-mesenchymal transition (EndoMT) In the pathogenesis of SSc, an accumulation of fibroblasts and myofibroblasts occurs and production of interstitial collagens and extracellular matrix components is excessive. Numerous studies have shown that myofibroblasts involved in tissue fibrosis can derive from ECs through a process known as EndoMT (120-127). It is a nonmalignant phenomenon of cellular transdifferentiation by which ECs undergo a phenotypical conversion where they lose vascular EC markers and gain mesenchymal cell markers (120-122). Seven articles concerning this topic were selected (127-133). Most included studies (127-131, 133) are in vitro studies (mostly using mouse models) suggestive of a role for EndoMT in SSc progressive fibrosis. 3.5 Disturbed vasculogenesis Vasculogenesis involves recruitment, mobilization and in situ differentiation of endothelial progenitor cells (EPC) from the bone marrow (BM). Recent data show that the morphology of SSc BM is normal but that the number of microvessels is severely reduced despite striking increase of VEGF 15
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(80). Furthermore the angiogenic potential of EC-like mesenchymal stromal cells was reduced after being stimulated with VEGF and stromal cell-derived factor-1 in vitro, suggesting that endothelial repair may be affected in SSc starting from the BM (134). EPC normally have the ability to develop into fully mature EC and contribute to neovascularization by targeting sites of endothelial injury (135). They represent a heterogeneous cell population originated from a single multipotent progenitor within the BM and consist of cells at different stages of maturation, ranging from early CD133+/VEGFR2+ to more mature CD34+/VEGFR2+ phenotypes (136). Impaired functioning of the EPC has been thought to be involved in the pathogenesis of SSc.
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In total 22 articles on the role of ECs in defective SSc vasculogenesis were selected. The most described biomarker in this selection are EPC (31, 63, 79, 80, 84, 88, 94, 116, 135, 137-147). Biomarkers other than EPC for this disturbed process, described in the selected articles, are: circulating angiogenic cells (CACs) (139), epidermal growth factor-like domain 7 (EGFL7) (148) and pentraxin 3 (PTX3) (88, 100, 146). 3.6 Defective vascular tone control and the nitric oxide (NO) paradox in SSc
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Endothelin-1 (ET-1) plays a prominent role in vascular tone regulation through its receptors ETA and ETB. ETA receptor predominates on vascular smooth muscle and mediates vasoconstriction, whereas the ETB receptor subtype, when present on vascular endothelium, mediates vasodilation through NO release (149-151). ET-1 is shown overexpressed in skin, lung tissue and plasma of SSc patients suggesting a role for this mediator in SSc vasculopathy (31, 38, 87, 152-158). In total 32 articles on the role of EC in vascular tone mediation were selected (Table 1). Suggested biomarkers, other than ET-1, of (direct or indirect) vascular tone dysregulation, described by the selected articles, are: urotensin-II (U-II)(159), angiotensin (Ang) I (160), Ang II (160), heptapeptide Ang (1-7)(160), angiotensin converting enzyme (ACE) (37-39, 160), 8-isoprostane (161-165), nitric oxide (NO)(11, 166-177) and inducible nitric oxide synthase (iNOS)(166). The latter is the result of ischemia caused by vascular obliteration known to occur in proliferative vasculopathy. As a result fibroblasts and ECs in skin and lung tissue produce reactive oxygen species (ROS), including NO, that selectively activates ECs or trigger fibroblasts to produce collagen (178). Synthesized ROS (i.e. superoxide anions, hydrogen peroxide, hydroxyl radicals and/or peroxynitrite) selectively activate ECs, leading to vascular inflammation (179, 180). 3.7 Coagulation cascade and complement system impairment The interaction between ECs and platelets plays an important role in vascular tone regulation in SSc. Damaged ECs release several molecular substances into the circulation that interfere with coagulation homeostasis (181). In a recent in vitro study, it is shown that activated platelets induce thymic stromal lymphopoietin (TSLP) production in human dermal microvascular ECs inducing profibrotic and EC-activating factors such as interleukin (IL)-13 (182). Abnormalities and impairment of the coagulation system, such as presence of circulating supranormal multimers of von Willebrand factor (vWF) or elevated levels of vWF, factor VIII antigen, fibrinogen (FNG), thrombomodulin (TM), tissue plasminogen activator (t-PA), plasminogen activator inhibitor 1 (PAI-1) have been described in SSc in multiple studies (19, 37-44, 87, 156, 181, 183). However whether or not impairment of fibrinolysis is present is still under debate since both depressed fibrinolytic activity (41) and a normal plasma fibrinolytic profile (40) has been reported. In total 14 articles have been selected for this topic. 16
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The role of the complement system (CS) in the pathogenesis of SSc vasculopathy has not been exhaustively investigated. The CS classically functions as a recruiter of inflammatory cells and regulates coagulation cascade and angiogenesis by damaging EC integrity (184). On human ECs, a group of membrane-bound complement regulators, including membrane cofactor protein (MCP or CD46) and decay accelerating factor (DAF or CD55), participates in protection from activation of both alternative and classic CS. An impaired expression of these regulators is shown in SSc skin, suggesting that a defective endothelial protection might be mediated by reduced expression of the complement regulatory proteins. This could potentially lead to endothelium-bound membrane attack complex of complement (MAC or C5B-9) depositions which are known to cause EC apoptosis (65, 185). Furthermore comprehensive peptidomics analysis revealed the predominance of complement C3fdes-arginine (DRC3f), derived from C3b, in the sera of patients with SSc and the level of DRC3f was related to vascular involvement in SSc and to SSc disease activity (186).
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3.8 ECs and their immune signature on SSc vasculopathy
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Although articles on vascular abnormalities in SSc were reviewed and selected, also the (direct or indirect) interaction between ECs with the immune system can contribute to the emergence or maintenance of SSc vasculopathy. In total 27 articles suggesting and discussing this interaction were selected. The selected immunological biomarkers, mostly cytokines, important in this vascular ECimmune system communication, are: macrophage migration inhibitory factor (MIF)(187, 188), IL-1α (189-191) and IL-1β (189, 191), IL-1 receptor-related protein (ST2)(192, 193), IL-2 (189, 194-196), soluble IL-2 receptor (21, 22, 194, 195), B cell growth factor (BCGF)(194), IL-4 (189, 197), IL-6 (18, 29, 189, 198, 199), soluble IL-6 receptor (198), oncostatin M (OSM)(198), soluble glycoprotein 130 (sGP130)(198), interferon γ (IFN-γ)(189), tumor necrosis factor alpha (TNF-α)(29, 189), IL-8 (19, 29, 30, 200), IL-10 (197), IL-13 (30, 197, 201), IL-15 (202, 203), IL-17A (204) and IL-33 (30, 191-193, 205207). All cytokines described in the selected clinical studies are shown upregulated in SSc plasma or (late stage) SSc skin as shown in supplementary file 4 and Table 2.
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3.9 EC interaction with other cells
Macrophages: They exist of diverse phenotypes, from "classically activated" M1 to "alternatively activated" M2 macrophages. M2 macrophages, including the subsets M2a and M2c, are considered to promote angiogenesis and tissue regeneration, while M1 macrophages are considered to be anti-angiogenic. M2a macrophages secrete the highest levels of PDGF-BB, a chemoattractant for stabilizing pericytes, and also promote anastomosis of sprouting ECs in vitro whereas M2c macrophages secrete the highest levels of MMP-9, a protease involved in vascular remodeling (208). A crosstalk between ECs and macrophages has been shown in cardiac microvascular inflammation (209). Furthermore ET-1 seems to induce the M2 phenotype in cultured human macrophages (210). Therefore similar intercellular interaction is suggested but not yet deeply investigated in SSc (211). Mast cells (MC): It is known for ECs to interact with mast cells through the production of Stem Cell Factor (SCF; c-kit ligand) to influence mast cell proliferation and differentiation (212). MC are increased in number in the lesional skin of SSc patients and are a valid source of TGF-β when degranulation occurs (213). Additionally SCF is shown to be strongly expressed on ECs in the SSc skin (214).
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4. DISCUSSION
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Pericytes (PCs): Among other cell types, ECs secrete PDGF-BB which recruits and induces proliferation of PCs progenitors (215). PDGF-BB serum levels are shown elevated in SSc patients (28). There is increasing evidence that PCs exhibit a phenotype similar to that of BM mesenchymal stem cells (BM-MSCs)(215). In a recent in vitro study SSc BM-MSCs are cocultured with healthy controls (HC) MVEC and cell reprogramming towards a pro-angiogenic behavior is observed (216). Fibroblasts: There is considerable evidence that EC products can modulate fibroblast properties in vivo. It is shown that SSc fibroblasts are particularly sensitive to EC-induced phenotypic modulation through IL-1 and bFGF and that damaging the EC vascular monolayer alters fibroblast responses (217). Furthermore it is shown that EC apoptosis plays a very important role in activating fibrogenic pathways through mediators that induce resistance to apoptosis in fibroblasts. These mediators in turn increase proliferation and inhibit apoptosis of EC and vascular smooth muscle cells (VSMC)(218, 219). A more recent study shows that SSc MVEC recruit and activate dermal fibroblasts by induction of a CCN2/TGF-β-dependent mesenchymal-to-mesenchymal transition this way clarifying the possible vascular connection of SSc-associated fibrosis (220). Furthermore evidence is provided that fibroblasts from SSc patients over-express MMP-12, which cleaves uPAR of normal MVEC, thus contributing to the failure of SSc-ECs to induce an efficient angiogenic program (99). Adipocytes: Recent studies have been focusing on the role of adipocytokines in the pathogenesis of SSc. Described markers that emphasize this emerging new interaction are: resistin (221), leptin (28, 221), adiponectin (221), chemerin (109) and retinol binding protein4 (RBP4)(222). Four articles have been selected concerning this interaction. Serum levels of possible biomarkers, shown in supplementary file 4, were mostly normal although correlations with the presence of digital ulcers (DU) were shown for elevated resistin and chemerin levels.
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In this systematic review on the role of EC in SSc vasculopathy, 193 selected articles present one or more aspects of EC dysfunction (Figure 1). Multiple significantly elevated activation markers, but mostly adhesion molecules, in the sera of SSc patients underline the significant involvement of EC activation in SSc pathogenesis. Dysregulation of apoptosis and proliferation within the vascular wall plays an important role in vascular remodeling associated with fibroproliferative vasculopathy observed in SSc. Apoptosis of ECs is recognized as an important component of the response to the injury phenomenon. Multiple selected studies have shown that AECA might be key players responsible for mediating this EC injury through ADCC. It is suggested that this ADCC is induced via the Fas pathway (46, 48). Though an earlier study not only failed to show Fas receptor binding by apoptosis-inducing AECA, it also showed that purified IgG AECA from SSc patients can induce apoptosis in human umbilical vein EC (HUVEC) through a membrane phospholipid reassembly with anionic phospholipid exposure and phosphatidylserine reaching the surface from its intracellular position (47). Additionally the apoptotic phenotype of endothelial progenitor cells (EPC) was investigated for possible mechanisms inducing apoptosis. Bone marrow studies showed significant titers of AECA, correlating with the number of apoptotic progenitors and in vitro assays on normal progenitors confirmed these results through induction of apoptosis by addition of AECA+purified IgG (55). Furthermore up-regulation of adhesion 18
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molecule expression (E-selectin, ICAM-1 and VCAM-1) and increased leukocyte adhesion (U937 cells) following AECA binding has also been shown to be an AECA binding effect (56). Because of these contradictory underlying mechanisms (activation versus apoptosis), more studies are needed to define the pathogenic role of AECA and its various subtypes before considering it as a useful biomarker.
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MPs are released from a variety of cells during activation and apoptosis via an exocytic budding process. Although long considered as inert debris, they are now appreciated as important mediators of cellular cross-talk regulating inflammation, coagulation, vascular functions, apoptosis and cell proliferation (223). Increased (67) as well as decreased (14) numbers of this EC apoptosis biomarker have been described in SSc. This contrast could be explained by a discrepancy in definition of examined MPs (according to size, Annexin V binding/non-binding MPs, CD144+/CD146+ EMP), isolation procedures and dissimilar use of fluorochromes and cell-specific, monoclonal antibodies. This implicates the need of a clear cut consensus on the exact characterization of MPs.
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Furthermore, it is shown in SSc that ECs lose their potential to participate in a normal angiogenesis process in response to EC injury. Mainly biomarkers suggestive for dysfunctional angiogenesis in relation to overexpression of pro-angiogenic mediators are described. The most suggestive ones in the selected articles are VEGF, VEGFR-2 and CXCL8. Despite elevation of VEGF in plasma and skin, adaptive angiogenesis is absent and a progressive loss of capillaries is observed (71). This paradox is further supported by the knowledge that short-time upregulation of VEGF is a strong inducer of angiogenesis, while chronic and uncontrolled overexpression of VEGF, which occurs in SSc, leads to the formation of irregularly shaped sac-like vessels with reduced blood flow in the newly formed vessels (74). However, most studies could not make the distinction between proangiogenic and antiangiogenic VEGF splice variants, which have been uncovered only recently. Indeed, although VEGF had conventionally been considered to be a family of proangiogenic mediators, the VEGF premRNA is shown to be differentially spliced in its terminal exon to form two distinct subfamilies of proteins, the so-called proangiogenic VEGFxxx isoforms and antiangiogenic VEGFxxxb isoforms (mainly VEGF165 and VEGF165b)(224). In a recent study, an increase in VEGF in SSc patients appears to be the result of a significant increase in the anti-angiogenic VEGF165b isoform instead of VEGF165. Furthermore in this study elevated circulating levels of VEGF165b in SSc patients are shown associated with early disease. Additionally by using blocking antibodies, severe impairment of in vitro angiogenesis is ameliorated in their study. Therefore this switch from pro-angiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia and needs further investigation (83). The most often described upregulated angiogenesis inhibitor in the selected articles is endostatin (31, 73, 75, 77, 81, 82, 95). Furthermore also alteration of transcripts involved in signal transduction pathways, i.e. the Ang/Tie2 signalling pathway (90) and the mechanism of uPAR truncation (98, 99, 101), is described. Also downregulation of the critical proangiogenic transcript Fli1 is mentioned in several articles (107-109) to play a part in the disturbed angiogenesis known in SSc vasculopathy. In seven selected articles, the role of EndoMT in SSc is also emphasized (127-133). In vitro studies using MVEC obtained from SSc patients confirmed a role for ET-1 and TGFβ as enhancers for EndoMT (127, 133). Nevertheless the mechanisms behind this transition are still not yet fully understood.
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ACCEPTED MANUSCRIPT While EndoMT is a known center stage in the fibrotic processes of many diseases (120-126), its role in the pathogenesis of SSc vasculopathy still needs clarification.
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The most often described biomarker of disturbed vasculogenesis are EPC. Nevertheless whether or not they can be used as biomarker in practice is still under debate since both decreased (79, 94, 137, 141, 143, 145) and increased (31, 63, 84, 138, 140, 142, 144, 147) levels of circulating EPC have been described in the selected literature. These contradictory results, displayed in supplementary file 5, could be due to the use of different protocols to quantify EPC, the use of different combinations of surface markers and differences in subsets of patients, used medications and disease durations between the studies. EPC can be defined by colony-forming unit (CFU) assays that use culture-based methods and by flow cytometry where expression of cell-surface antigens including CD34, CD133 and VEGFR2 is assayed. However, protocols for isolating and counting EPC are not yet standardized therefore causing discrepancy in results between studies. Recently the European League Against Rheumatism Scleroderma Trials and Research group (EUSTAR) proposed recommendations for the identification and measurement of EPC (225). Ever since these recommendations were published, mostly decreasing levels of EPC have been reported.
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In the dysregulation of endothelial dependent control of arteriolar/venular tone, NO is the most investigated biomarker in SSc vasculopathy. Nevertheless the exact status of NO production as biomarker in SSc (vasculopathy) is still controversial since both increased (11, 166-173, 175) and decreased (172, 174, 175) total nitrate (metabolites) levels in SSc patients have been reported (Supplementary file 6). Decreased NO levels could be explained by the rapid reaction of NO and superoxide anions to generate the reactive intermediate peroxynitrite (11). ET-1 is the second most described elevated biomarker in dysfunctional vascular tone control known to occur in SSc vasculopathy. Furthermore, multiple selected articles also emphasized the role of the reninangiotensin system (RAS) and its products in the integrated regulation of vascular tone.
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Additionally, EC also interact with the coagulation system by upregulating multiple markers of coagulation (most importantly vWF), this way contributing to the prothrombotic state of SSc patients (41). However whether or not impairment of fibrinolysis is present, is still under debate since both depressed fibrinolytic activity (41) and a normal plasma fibrinolytic profile (40) has been reported. The importance of the interaction of EC with the complement system in SSc vasculopathy has not been deeply investigated. Only three articles were selected concerning this topic. Although the immune system plays its own particular part in SSc pathogenesis, its interaction with EC and therefore its role in SSc vasculopathy, is not negligible. IL-33, one of the most described cytokines in the selected articles is abnormally expressed in early stage SSc skin (193) and is elevated in the sera of early SSc patients (30, 205, 206). Furthermore it is shown that the IL-33/ST2 axis increases proliferation, migration and morphologic differentiation of human ECs with increased endothelial permeability, consistently with increased angiogenesis in vivo (193). These findings support the hypothesis that IL-33 might mediate very early pathogenic events of SSc through recruitment and stimulation of ST2-expressing cells (immune cells and fibroblast/myofibroblast), this way modulating inflammatory and fibrotic processes in SSc. IL-2 is known to increase binding of natural killer cells to the endothelium (226). Needleman et al. show significantly higher IL-2 levels in SSc patients compared to HC using a CTLL bioassay (189). However when the same authors use an ELISA to determine both levels, no statistical significance between both groups was shown. Their 20
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ELISA findings suggest similar IL-2 levels in SSc patients and HC but increased IL-2 biologic activity in SSc patient sera. The latter is supported by the hypothesis of Kahaleh and LeRoy (196) that an IL-2 inhibitor is present in sera from HC but reduced in SSc sera. Famularo et al (194) also found an increase in mean IL-2 serum levels in SSc patients compared to controls. In contrast, Degiannis et al (195) found comparable levels of IL-2 in plasma from SSc patients and controls. Both authors use an ELISA detection method for IL-2 quantification. More recently IL-17A is investigated and authors observe that this cytokine induces the expression of adhesion molecules and chemokines in HUVEC. Furthermore it mediates vascular inflammation through ERK phosphorylation (204) clarifying the link between IL-17 producing T cells and endothelial injury in SSc vasculopathy.
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Finally the selected articles also provide limited evidence of EC interaction with other inflammatory (MC), vascular (PCs) or adipose cells. The latter is a relatively new discovery, which involves a new type of cytokines, the adipocytokines, this way expanding the spectrum of SSc vasculopathy. Although recent data suggest a role for macrophages in angiogenesis (208-211), there is currently no scientific evidence (in the form of (randomized) control trials, observational and/or in vitro studies) proving that EC-macrophage interaction contributes to SSc vasculopathy.
5. CONCLUSION
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Systemic sclerosis (SSc) is an autoimmune connective tissue disorder characterized by proliferative vasculopathy, immunological abnormalities and progressive fibrosis of multiple organs including the skin. This systematic review focuses on the role of ECs in SSc vasculopathy. The most representing and reliable biomarkers described by the selected articles were adhesion molecules (ICAM1, VCAM1, E-selectin, P-selectin) for EC activation, AECA for EC apoptosis, VEGF, its receptor VEGFR-2 and endostatin for disturbed angiogenesis, circulating EPC for defective vasculogenesis, ET-1 for disturbed vascular tone control, vWF for coagulopathy and IL-33 for EC- immune system communication. Emerging, relatively new discovered biomarkers described in the selected articles are VEGF165b, IL-17A and the adipocytokines. Although the etiology of SSc remains unclear, this systematic review summarizes the growing evidence that SSc is primarily a vascular disease where EC dysfunction is present and prominent in different aspects of cell survival (activation and apoptosis), angiogenesis and vasculogenesis and where disturbed interactions between ECs and various other cells contribute to SSc vasculopathy.
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ACCEPTED MANUSCRIPT APPENDICES
Figure 1. Endothelial cell dysfunction in SSc vasculopathy: a multifunctional approach
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Figure legend: this figure summarizes the different dysfunctional aspects of EC in SSc vasculopathy.
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Table 1. Number of selected articles according to EC (dys)function in SSc vasculopathy
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Table legend: data are given as number of articles selected in the systematic review, categorized according to EC (dys)function. Some articles (n=33) covered more than one topic and were therefore categorized in more than one function group. This way the grand total exceeds 193 articles.
Table 2. Regulation of EC apoptosis markers described by the selected articles
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Table legend: data are given as number of selected articles describing significant upregulation, significant downregulation or normal regulation (i.e. no significant difference in regulation between controls and SSc population) of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc or certain SSc subtypes. Selected basic science articles (in vitro experiments, animal studies) involving the process of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc are not included in this table.
Supplementary file 1. Details on search strategy using the Prisma 2009 checklist Supplementary file 2. Flow diagram of search process according to PRISMA 2009 guidelines
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Supplementary file 3. Quality assessment of the selected articles Supplementary file 4. Summary of all 193 selected articles
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Supplementary file 5. Discrepancy in selected literature concerning levels of circulating EPC in SSc patients
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Supplementary file 6. Discrepancy in literature concerning levels of NO (metabolites)
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[206] Terras S, Opitz E, Moritz RK, Höxtermann S, Gambichler T, Kreuter A. Increased serum IL-33 levels may indicate vascular involvement in systemic sclerosis. Ann Rheum Dis 2013; 72(1): 144-5. [207] Yanaba K, Yoshizaki A, Asano Y, Kadono T, Sato S. Serum IL-33 levels are raised in patients with systemic sclerosis: association with extent of skin sclerosis and severity of pulmonary fibrosis. Clin Rheumatol 2011; 30(6): 825-30. [208] Spiller KL, Anfang RR, Spiller KJ, Ng J, Nakazawa KR, Daulton JW, et al. The role of macrophage phenotype in vascularization of tissue engineering scaffolds. Biomaterials 2014; 35(15): 4477-88. [209] Pabois A, Pagie S, Gérard N, Laboisse C, Pattier S, Hulin P, et al. Notch signaling mediates crosstalk between endothelial cells and macrophages via Dll4 and IL6 in cardiac microvascular inflammation. Biochem Pharmacol 2016; 104: 95-107. [210] Soldano S, Pizzorni C, Paolino S, Trombetta AC, Montagna P, Brizzolara R, et al. Alternatively Activated (M2) Macrophage Phenotype Is Inducible by Endothelin-1 in Cultured Human Macrophages. PLoS One 2016; 11(11): e0166433. [211] Manetti M. Deciphering the alternatively activated (M2) phenotype of macrophages in scleroderma. Exp Dermatol 2015; 24(8): 576-8. [212] Coleman JW, Holliday MR, Kimber I, Zsebo KM, Galli SJ. Regulation of mouse peritoneal mast cell secretory function by stem cell factor, IL-3 or IL-4. J Immunol 1993; 150(2): 556-62. [213] Hügle T, Hogan V, White KE, van Laar JM. Mast cells are a source of transforming growth factor β in systemic sclerosis. Arthritis Rheum 2011; 63(3): 795-9. [214] Yamamoto T, Katayama I, Nishioka K. Expression of stem cell factor in the lesional skin of systemic sclerosis. Dermatology 1998; 197(2): 109-14. [215] da Silva Meirelles L, Caplan AI, Nardi NB. In search of the in vivo identity of mesenchymal stem cells. Stem Cells 2008; 26(9): 2287-99. [216] Cipriani P, Marrelli A, Benedetto PD, Liakouli V, Carubbi F, Ruscitti P, et al. Scleroderma Mesenchymal Stem Cells display a different phenotype from healthy controls; implications for regenerative medicine. Angiogenesis 2013; 16(3): 595-607. [217] Denton CP, Xu S, Black CM, Pearson JD. Scleroderma fibroblasts show increased responsiveness to endothelial cell-derived IL-1 and bFGF. J Invest Dermatol 1997; 108(3): 269-74. [218] Raymond MA, Vigneault N, Luyckx V, Hébert MJ. Paracrine repercussions of preconditioning on angiogenesis and apoptosis of endothelial cells. Biochem Biophys Res Commun 2002; 291(2): 2619. [219] Laplante P, Raymond MA, Gagnon G, Vigneault N, Sasseville AM, Langelier Y, et al. Novel fibrogenic pathways are activated in response to endothelial apoptosis: implications in the pathophysiology of systemic sclerosis. J Immunol 2005; 174(9): 5740-9. [220] Serratì S, Chillà A, Laurenzana A, Margheri F, Giannoni E, Magnelli L, et al. Systemic sclerosis endothelial cells recruit and activate dermal fibroblasts by induction of a connective tissue growth factor (CCN2)/transforming growth factor β-dependent mesenchymal-to-mesenchymal transition. Arthritis Rheum 2013; 65(1): 258-69. [221] Olewicz-Gawlik A, Danczak-Pazdrowska A, Kuznar-Kaminska B, Batura-Gabryel H, Katulska K, Wojciech S, et al. Circulating adipokines and organ involvement in patients with systemic sclerosis. Acta Reumatol Port 2015; 40(2): 156-62. [222] Toyama T, Asano Y, Takahashi T, Aozasa N, Akamata K, Noda S, et al. Clinical significance of serum retinol binding protein-4 levels in patients with systemic sclerosis. J Eur Acad Dermatol Venereol 2013; 27(3): 337-44. [223] Distler JH, Jüngel A, Huber LC, Seemayer CA, Reich CF, Gay RE, et al. The induction of matrix metalloproteinase and cytokine expression in synovial fibroblasts stimulated with immune cell microparticles. Proc Natl Acad Sci U S A 2005; 102(8): 2892-7. [224] Qiu Y, Hoareau-Aveilla C, Oltean S, Harper SJ, Bates DO. The anti-angiogenic isoforms of VEGF in health and disease. Biochem Soc Trans 2009; 37(Pt 6): 1207-13. [225] Distler JH, Allanore Y, Avouac J, Giacomelli R, Guiducci S, Moritz F, et al. EULAR Scleroderma Trials and Research group statement and recommendations on endothelial precursor cells. Ann Rheum Dis 2009; 68(2): 163-8. 34
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[226] Aronson FR, Libby P, Brandon EP, Janicka MW, Mier JW. IL-2 rapidly induces natural killer cell adhesion to human endothelial cells. A potential mechanism for endothelial injury. J Immunol 1988; 141(1): 158-63.
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Activation Apoptosis
SC R
IP
Fibrosis
Defective vascular tone control
MA
NU
Coagulopathy
Dysfunctional EC
Disturbed angiogenesis
Defective vasculogenesis
AC
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MC dysregulation
TE
D
Dysregulation of adipocytokines
Dysregulation of vascular system through pericyte/EC interaction
Immune dysregulation EndoMT
Figure 1. Endothelial cell dysfunction in SSc vasculopathy: a multifunctional approach Figure 1 legend: this figure summarizes the different dysfunctional aspects of EC in SSc vasculopathy. Abbreviations used in figure 1: EC: endothelial cell; EndoMT: endothelial-mesenchymal transition; MC: mast cell; SSc: systemic sclerosis
36
ACCEPTED MANUSCRIPT Table 1. Number of selected articles according to EC (dys)function in SSc vasculopathy TOTAL
EC activation
38
EC apoptosis
29
Disturbed angiogenesis
56
EndoMT
7
Defective vasculogenesis
22
Defective vascular tone control
32
IP SC R
NU
Coagulation cascade and complement impairment
17
MA
Immune dysregulation
27
Mast cell dysregulation
1
TE
D
Dysregulation of the vascular system through pericytes/ECs crosstalk Fibrosis
T
EC function
2
5 4
GRAND TOTAL
240
CE P
Dysregulation of adipocytokines
AC
Table legend: data are given as number of articles selected in the systematic review, categorized according to EC (dys)function. Some articles (n=33) covered more than one topic and were therefore categorized in more than one function group. This way the grand total exceeds 193 articles. Abbreviations used in table: EC: endothelial cell, EndoMT: endothelial-mesenchymal transition, EPC: endothelial progenitor cells
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ACCEPTED MANUSCRIPT Table 2. Regulation of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication described by the selected articles Norma Upregulat Downregula lly TOT ion tion regulat AL ed
Biomarkers
4
0
1
5
0
0
1
1
0
0
1
2
0
0
2
1
1
0
2
1
0
0
1
0
0
1
1
0
0
1
1
1
1
0
2
1
0
0
1
0
1
0
1
1
0
0
1
1
0
0
1
Collagen V
1
0
0
1
VEGF
9
1
1
11
VEGF165bVEGF165bVEGF165bVEGF165bVEGF 165bVEGF165b
1
0
0
1
VEGFR-1
0
1
0
1
VEGFR-2
1
0
0
1
VEGFR-3
0
0
1
1
PDGF-BB
1
0
0
1
PDGF
1
0
1
2
FGF-2 Markers of disturbed Serum βFGF angiogenesi HGF s
2
0
0
2
1
0
1
2
3
0
0
3
sTie1
0
0
1
1
sTie2
1
0
1
2
Ang-1
0
1
0
1
Ang-2
3
0
0
3
Angptl3
1
0
0
1
PlGF
4
0
0
4
PEDF
0
0
1
1
Endostatin
6
0
1
7
IP
AECA
T
Examin ed SSc tissue
1
SC R
IgG anti-caspase-3 Ab Caspase-3 IFI16 Ab Serum CEC Markers of EC apoptosis
NU
OPG TRAIL MAC (C5b9)
MA
MPs IFI16 Ab BMPRII MAC (C5b9)
AC
CE P
Lung
TE
Fra-2
D
Skin
38
ACCEPTED MANUSCRIPT 0
0
1
1
GRO-α
1
0
0
1
MMP-12
1
0
0
1
MMP-9
1
0
0
1
Pro-MMP-1
1
0
0
1
CTSB
1
0
0
1
1
0
1
1
0
0
1
1
0
0
1
0
0
1
1
1
0
0
1
1
0
0
1
1
0
0
1
0
1
0
1
4
0
0
4
1
0
0
1
2
0
0
2
2
0
0
2
3
0
0
3
1
0
0
1
1
0
0
1
VEGF
3
0
0
3
VEGF165bVEGF165bVEGF165bVEGF165b
1
0
0
1
VEGFR-1
2
0
1
3
VEGFR-2
5
0
0
5
VEGFR-3
2
0
0
2
GLUT-1
1
0
0
1
Tie1
0
1
0
1
MMP-12
3
0
0
3
uPAR
1
0
0
1
KLK1
0
1
1
2
KLK9
0
1
0
1
KLK11
0
1
0
1
KLK12
0
1
0
1
CTSB
1
0
0
1
CTSV
0
1
0
1
CTSL
1
0
0
1
CCN-1
0
1
0
1
IP 0
SC R
CTSV CTSL Galectin 3 CCN-1
NU
Apelin CXCL1 CXCL4
MA
CXCL5 CXCL8 CXCL9
D
CXCL10
CCL13
AC
CE P
CCL23
TE
CXCL16 CCL2
Skin
T
TIMP2
39
ACCEPTED MANUSCRIPT 1
0
0
1
CXCL5
0
1
0
1
CXCL8
1
0
0
1
CXCL9
1
0
0
1
CXCL10
1
0
0
1
CXCL16
1
0
0
1
1
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
2
0
0
2
3
0
1
4
2
0
0
2
1
0
0
1
2
0
0
2
3
0
0
3
1
0
0
1
0
0
1
1
1
0
1
2
IL-1α
1
0
2
3
TNF-α
0
0
1
1
IFNγ
0
0
1
1
IL-8
2
1
0
3
IL-10
1
0
0
1
IL-13
3
0
0
3
IL-15
2
0
0
2
IL-17A
1
0
0
1
IL-33
6
0
0
6
MIF
2
0
0
2
ST2
1
0
0
1
IL-6
2
0
0
2
IL-8
1
0
0
1
IL-17A
1
0
0
1
IL-33
1*
0*
0*
1
IP 0
SC R
CXCR3 CXCR6 CXCL12 CCL13
NU
CCL23 BM
VEGF MIF
MA
IL-2 sIL-2R BCGF
D
IL-4
OSM
CE P
AC
Serum Markers of disturbed EC-immune system communica tion
IL-1β
TE
IL-6 sIL-6R
Skin
T
CXCL4
* In early SSc, IL-33 protein was downregulated or absent in EC, while IL-33 mRNA was normally expressed or even upregulated. In late SSc, IL-33 was constitutively found in most EC.
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Table legend: data are given as number of selected articles describing significant upregulation, significant downregulation or normal regulation (i.e. no significant difference in regulation between controls and SSc population) of markers of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc or certain SSc subtypes. Selected basic science articles (in vitro experiments, animal studies) involving the process of EC apoptosis, disturbed angiogenesis and EC-immune system communication in SSc are not included in this table. Abbreviations used in table: Ab: antibodies; AECA: anti-endothelial cell antibodies; Ang-: Angiopoietin-; Angptl3: Angiopoietin-like protein 3; BCGF: B cell growth factor; BM: bone marrow; BMPRII: bone morphogenetic protein receptor II; CCL: chemokine ligand; CCN-1: cysteine-rich 61 matrix protein; CEC: circulating endothelial cells; CTSB: cathepsin B; CTSL: cathepsin L; CTSV: cathepsin V; CXCL: chemokine (C-X-C motif) ligand; EC: endothelial cell; FGF: fibroblast growth factor; Fli1: Friend leukemia integration factor 1; Fra-2: Fos-related antigen-2; GLUT-1: glucose transporter 1; GRO-α: Growth-Regulated Oncogene-α; HGF: hepatocyte growth factor; IFI16: Gamma-interferon-inducible protein IFI-16; IFNγ: interferon gamma; IL: interleukin-; KLK: human tissue kallikreins; MAC: membrane attack complex of complement; MIF: macrophage migration inhibitory factor; MMP: matrix metalloproteinases; MPs: microparticles; mRNA: messenger ribonucleic acid; OPG: osteoprotegerin; OSM: oncostatin M; PDGF: platelet-derived growth factor; PEDF: pigment epithelium-derived factor; PlGF: placental growth factor; sIL-2R: soluble IL-2 receptor; sIL-6R: sIL-6 receptor; SSc: systemic sclerosis; ST2: IL-1 receptor-related protein; Tie: soluble tyrosine kinase; TIMP2: tissue inhibitor of metalloproteinase 2; TNF-α: tumor necrosis factor alpha; TRAIL: TNF-related apoptosisinducing ligand; uPAR: urokinase-type plasminogen activator receptor; VEGF: vascular endothelial growth factor; VEGFR: vascular endothelial growth factor receptor
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