LAUSR

Background: Antiphospholipid syndrome (APS) is an autoimmune thrombophillia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies mainly anti-β2glycoprotein I (anti-β2GPI), which lead to monocyte and endothelial cell activation and subsequent tissue factor and proinflammatory cytokine expression such us IL-6 and IL-8 (1). Epigenetics describes changes in gene expression without alterations in the genomic sequence. Methylation of DNA at CpG islands by adding a methyl group to the nucleotide cytosine, is one of main epigenetic mechanisms. Objectives: To explore the possible differential methylation of IL8 and Tissue Factor (F3)gene promoters, which are critical for the pathophysiology of APS. Methods: Whole blood and serum were isolated from 27 APS patients and 25 healthy donors (HDs). Human umbilical vein endothelial cells (HUVECs) and peripheral blood monocytes were isolated from 3 HDs. Anti-β2GPI IgG was isolated and pooled from 8 APS patients.HUVECs and monocytes were stimulated with a mixture of IgG, β2GPI and CXCL4.Then mRNA was isolated and qPCR was performed for the assessment of IL8 and tissue factor (F3)gene expression.Whole blood DNA from APS patients and HDs,and DNA from the in vitro experiments was isolated and bisulphite treated. The methylation of 1CpG in the promoter of F3 gene and 1CpG in the promoter of IL8 gene was assessed with Methylation Specific PCR Results: DNA from APS patients had significantly lower methylation levels in the IL8 promoter compared to HDs (mean: APS 0,6715vs HD1,153, p < 0,0001). Patients with history of arterial thromboembolism had the lowest IL8 methylation levels compared to ones with history of venous thrombosis or pregnancy morbidity, p=0.0003.An inverse correlation was noted between the IL8 methylation levels and the time passed since the last clinical event. APS patients who suffered 3 or more clinical incidents had significantly lower methylation levels. APS DNA was significantly methylated in the F3 promoter compared to HD (APS 1,902vs HD1,335, p<0,0001). Negative association of F3 methylation was noted with anti-β2GPI, auto-antibody titers and APTT elongation. HD HUVECs and monocytes stimulated with anti-β2GPI IgG/β2GPI/CXCL4 obtained an activated and procoagulant phenotype. In HUVECs the triple mixture induced an 8fold increase for the SELE gene, 3fold for the IL8, 1.5fold for F3. At the same time 2-fold decreased F3 promoter methylation levels correlated inversely to F3 gene expression (r -0.7679, p=0.0008). No change was noted in the IL8 promoter methylation levels.Treated monocytes had a 40fold increase in the IL8 and 20 fold increase in the F3 gene levels, while methylation of IL-8 and F3 promoter regions was decreased by 2-fold. HUVECs stimulation resulted in the differential expression of genes involved in DNA epigenetic alterations. Significant difference was observed for MeCP2 (2fold), DNMT3B (2 fold) and HDAC9 genes (3fold), which correlate with theexpression of activation markers of SELE, F3 and IL8. Conclusion: APS patients expressed decreased relative IL8 methylation levels which associate with arterial clinical events and significantly with more recent events. The mixture of anti-β2GPI IgG-β2GPI-CXCL4 activate HD HUVECs and monocytes to a procoagulant phenotype and decrease methylation in the F3 and IL8 promoters. References: [1] Allen, K.L., et al., A novel pathway for human endothelial cell activation by antiphospholipid/anti-beta2 glycoprotein I antibodies. Blood, 2012. 119(3): p.884-93. Disclosure of Interests: None declared