Anti- 2GPI-antibody-induced endothelial cell gene expression profiling reveals induction of novel pro-inflammatory genes potentially involved in primary antiphospholipid syndrome

Abstract

Objective: To determine the effects of primary antiphospholipid syndrome (PAPS)-derived anti-b 2 GPI antibodies on gene expression in human umbilical vein endothelial cells (HUVEC) by gene profiling using microarrays. Methods: Anti-b 2 GPI antibodies purified from sera of patients with PAPS or control IgG isolated from normal subjects were incubated with HUVEC for 4 h before isolation of RNA and processing for hybridisation to Affymetrix Human Genome U133A-2.0 arrays. Data were analysed using a combination of the MAS 5.0 (Affymetrix) and GeneSpring (Agilent) software programmes. For selected genes microarray data were confirmed by real-time PCR analysis or at the protein level by ELISA. Results: A total of 101 genes were found to be upregulated and 14 genes were downregulated twofold or more in response to anti-b 2 GPI antibodies. A number of novel genes not previously associated with APS 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. The majority of downregulated genes were transcription factors/signalling molecules including ID2. Quantitative real-time RT-PCR analysis confirmed the microarray results for selected genes (CSF3, CX3CL1, FGF18, ID2, SOD2, Tenascin C). Conclusions: This study reveals a complex gene expression response in HUVEC to anti-b 2 GPI antibodies with multiple chemokines, pro-inflammatory cytokines, pro-thrombotic and pro-adhesive genes regulated by these antibodies in vitro. Some of these newly identified anti-b 2 GPI antibody-regulated genes could contribute to the vasculopathy associated with this disease.

High Avidity Anti-β2-Glycoprotein i Antibodies Activate Human Coronary Artery Endothelial Cells and Trigger Peripheral Blood Mononuclear Cell Migration

“…Several past reports have addressed the activation of human umbilical vein endothelial cells (HUVEC) by a~2GPI (15)(16)(17) and their functional impact which mainly focused on monocyte adhesion (18,19). However, to our current knowledge, none has considered the avidity of the antibodies in relation to any of their cellular effects.…”

“…These reports describe the effects of a~2GPI autoantibodies isolated from patients with APS or monoclonal aPL subpopulations and were conducted mainly on HUVEC. A study by Hamid et al using gene expression microarrays in HUVEC, reported that human polyclonal IgG a~2GPI derived from APS patients induced changes in 115 genes involving apoptosis, coagulation, metabolism, transcription factors/signaling, cytokines/chemokines, adhesion and other molecules, including MCP-1, GRan, IL-8 and IL-6 (17). Several studies observed upregulation ofIL-6 at the protein level by a~2GPI (15,23,24) IgG fractions from patients with APS and~2GPI independent human monoclonal aPL elevated MCP-1 as well as IL-8 production at both mRNA and protein levels in a time-dependent manner in HUVEC (26).…”

Changes in regulation of human monocyte proteins in response to IgG from patients with antiphospholipid syndrome

“…11 Furthermore, TF was not identified among 101 genes found to be induced in a genomics analysis of anti-b 2 GPI stimulated endothelial cells which are another major source of TF. 26 We hypothesize that this discrepancy between TF gene activation, protein expression and activity may be explained by aPL leading to a cellular re-distribution of intracellular TF to the cell surface as has been shown in monocytes with a high response to LPS. 35 Ultimately, further experiments are required to address this issue.…”

“…Induction of an ankyrin repeat and BTB (POZ) domain containing gene was identified in an earlier genomics analysis of aPL-induced endothelial cell gene expression. 26 Plasminogen activator inhibitor type-1 (PAI-1) is the main inhibitor of urokinase, PAI-2 is predominantly intra-cellular and has a number of anti-inflammatory, anti-proliferative and antiapoptotic functions. 27 Therefore, reduced levels of PAI-2 may allow unopposed action of PAI-1 as found in APS 28 to promote thrombogenesis.…”

Blocking the Glucocorticoid Receptor with RU-486 Does Not Prevent Glucocorticoid Control of Autoimmune Mouse Hearing Loss

“…Hearing loss has been correlated with circulating autoantibodies against endothelial cells and their membrane phospholipids [18], such as cardiolipin and β2-glycoprotein 1. These autoantibodies have been shown to activate the inflammatory response of endothelial cells [25,26], which includes breaking tight junctions to facilitate intercellular movement of immune cells [27,28]. Such autoantibody impact on tight junctions of the blood labyrinth barrier is one proposed mechanism for hearing loss by disrupting the sensitive ion homeostatic functions within the stria vascularis.…”