Identification of Anti-Beta2 Glycoprotein I Auto-antibody Regulated Gene Targets in the Primary Antiphospholipid Syndrome Using Gene Microarray Analysis
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AuthorsHamid, Colleen G.
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AbstractAnti-Beta2-Glycoprotein I antibodies (anti-2GPI) are strongly associated with thrombosis in patients with primary antiphospholipid syndrome (PAPS). Anti-2GPI activate endothelial cells (EC) resulting in a pro-thrombotic and pro-inflammatory phenotype. In order to characterise EC gene regulation in response to anti-2GPI, early global gene expression was assessed in human umbilical vein endothelial cells (HUVEC) in response to affinity purified anti-2GPI. Sera were collected from patients with PAPS and IgG was purified using HiTrap Protein G Sepharose columns. Polyclonal anti-2GPI were prepared by passing patient IgG through NHS activated sepharose coupled to human 2GPI. Anti-2GPI preparations were characterized by confirming their 2GPI co-factor dependence, binding to 2GPI 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-2GPI (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, IL1 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-2GPI-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.
PublisherUniversity of Wolverhampton
DescriptionA thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosophy