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Title: Identification of anti-beta₂ glycoprotein I auto-antibody regulated gene targets in the primary antiphospholipid syndrome using gene microarray analysis
Author: Hamid, Colleen G.
ISNI:       0000 0001 3528 1461
Awarding Body: University of Wolverhampton
Current Institution: University of Wolverhampton
Date of Award: 2007
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Anti-Beta2-Glycoprotein I antibodies (anti-b2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-b2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-b2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-b2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-b2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human b2GPI. Anti-b2GPI preparations were characterized by confirming their b2GPI co-factor dependence, binding to b2GPI and ability to induce leukocyte adhesion molecule expression and IL-8 production in vitro. Two microarray experiments tested differential global gene expression in 6 individual HUVEC donors in response to 5 different PAPS polyclonal anti-b2GPI (50 mg/ml) compared to 5 normal control IgG (50 mg/ml) after 4 hours incubation . Total HUVEC RNA was extracted and cRNA was prepared and hybridised to Affymetrix HG-133A (Exp.1) and HG-133A_2 (Exp.2) gene chips. Data were analyzed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. Significant change in gene expression was defined as greater than two fold increase or decrease in expression (p<0.05). Novel genes not previously associated with PAPS were induced including chemokines CCL20, CXCL3, CX3CL1, CXCL5, CXCL2 and CXCL1, the receptors Tenascin C, OLR1, IL-18 receptor 1 and growth factors, CSF2, CSF3, IL-6, IL1b and FGF18. Downregulated genes were transcription factors/signaling molecules including ID2. Microarray results were confirmed for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C) using quantitative real-time RT-PCR analysis. This study revealed a complex anti-b2GPI-regulated gene expression profile in HUVEC in vitro. The novel chemokines and pro-inflammatory cytokines identified in this study may contribute to the vasculopathy associated with PAPS.
Supervisor: Frampton, G. ; Murphy, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Anti-ß2 Glycoprotein I antibodies ; Endothelial cells ; Thrombosis ; Primary antiphospholipid syndrome ; Antiphospholipid antibodies