Background Antiphospholipid syndrome is an autoimmune thrombophillia characterized by recurrent thromboembolism and or pregrancy morbidity in the presence of antiphospholipid antibodies (aPL). β2GPI which is the major autoantigen in the syndrome forms complexes with anti-B2GPI autoantibodies that activate platelets, monocytes and endothelial cells. Previous studies have shown that anti-β2GPI-β2GPI complexes activate TLR4 and TLR6 on endothelial cells leading to NFκB, MAPK activation and Tissue Factor and proinflammatory cytokine expression.1,2
Objectives To evaluate the whole transcriptome of endothelial cells that have been stimulated with aPL-B2GPI complexes.
Methods Human umbilical Vein Endothelial cells (HUVECs) were isolated from 2 APS patients and 4 Healthy control women upon delivery. Healthy donor HUVEC were stimulated with IgG isolated from APS patients with high aPL titers and healthy individuals in the presence of B2GPI. Consequently total mRNA was isolated, cDNA libraries were created and whole transcriptome sequencing (RNASeq) was performed. Gene expression data were validated in protein levels with immunohistochemistry in placenta tissues from APS patients and healthy individuals.
Results Whole transcriptome analysis of HUVECs stimulated with aPL-β2GPI complexes and IgG from healthy individuals revealed 680 differentially expressed genes, among which 377 were upregulated and 303 downregulated in the aPL stimulated endothelial cells. Characteristic examples of the upregulated genes are IL-6, IL-8, VCAM1, SELE and TGFB2 and TGFBR1. Bioinformatics analysis revealed that the upregulated genes belong mainly to the cytokine-cytokine receptor interaction (hsa053323), MAPK signaling pathway (hsa04010), TNF signaling pathway (hsa04668) and NOD-like receptor pathway (hsa04621). Characteristic examples of the downregulated genes include the CBX4, CBX8, BCOR and HDAC7 genes. Interestingly some of the proteins encoded by these genes play role in the epigenetic modification of DNA. Immunohistochemical staining on placenta biopsies from APS patients and healthy individuals for IL-6, IL-8, IL-18, NFkB, TF, TNF-a, E-SELECTIN, MAPK8, TGFB2 and TGFBR1 showed increased intensity in the signal of endothelial cells on APS specimens validating thus the RNASeq results in the tissues.
Conclusions RNASeq of endothelial cells treated with aPL and B2GPI reveals a thoroughly analysed proinflamatory and procoagulant phenotype. Moreover differential expression of DNA modifying proteins suggests the possible epigenetic regulation of gene expression on endothelial cells in APS syndrome. Ongoing experiments aim to analyze histone acetylation and methylation status of the promoters of the selected genes that were shown to be differentially expressed.
Pierangeli SS, Vega-Ostertag ME, Gonzalez EB. New targeted therapies for treatment of thrombosis in antiphospholipid syndrome. Expert reviews in molecular medicine. 2007;9(30):1–15.
Vega-Ostertag M, Casper K, Swerlick R, Ferrara D, Harris EN, Pierangeli SS. Involvement of p38 MAPK in the up-regulation of tissue factor on endothelial cells by antiphospholipid antibodies. Arthritis and rheumatism. 2005;52(5):1545–54.
Disclosure of Interest None declared
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