Unit:
Faculty of MedicineLibrary of the School of Health Sciences
Dissertation committee:
Αθανάσιος Τζιούφας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Παναγιώτης Βλαχογιαννόπουλος, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μενέλαος Μανουσάκης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Μιχαήλ Βουλγαρέλης, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Νικόλαος Σύψας, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Ευσταθία Καψογεώργου, Αναπ. Καθηγήτρια, Ιατρική Σχολή, ΕΚΠΑ
Αθανάσιος Πρωτογέρου, Καθηγητής, Ιατρική Σχολή, ΕΚΠΑ
Original Title:
Η παθογένεια του ενδοθηλίου στο αντιφωσφολιπιδικό σύνδρομο
Translated title:
The pathogenesis of endothelium in antiphospholipid syndrome
Summary:
Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies, mainly anti-β2glycoprotein I (anti-β2GPI). The autoantibodies lead to monocyte and endothelial cell activation and subsequent secretion of tissue factor (F3) and proinflammatory cytokines, like interleukins 6 (IL6) and 8 (IL8). The etiology of the syndrome remains largely unknown, with the contribution of environmental, genetic and epigenetic factors considered significant. The evaluation of the endothelial pathogenesis in antiphospholipid syndrome was the objective of this study and is divided in three main sections.
In the first section we sought to identify 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 healthy women and stimulated with IgG isolated from APS patients in the presence of B2GPI. Consequently, total mRNA was isolated, cDNA libraries were created and whole transcriptome sequencing (RNA-seq) was performed. Gene expression data were validated in protein levels with immunofluorescent assays in treated cells and placenta biopsies from APS patients and healthy individuals.
Results: Whole transcriptome analysis of HUVECs stimulated with APS total IgG andβ2GPI complexes and IgG from healthy individuals revealed 906 differentially expressed genes, among which 395 were upregulated and 511 downregulated in the aPL stimulated endothelial cells. Characteristic examples of the upregulated genes are IL-6, IL-8, VCAM-1, E-selectin and TGF-β2 and TGFR1. Bioinformatics analysis revealed that the upregulated genes belong mainly to the TNF-α signaling, TGF-β signaling, MAPK38-signaling and Hippo pathways. Several transcription factors in APS treated HUVECs are upregulated and NF-κB and SMAD chip-seq intersection with RNA-seq reveals target genes such as IL-6, IL-8, E-Selectin and Tissue factor. Immunofluorescent assays on treated HUVECs and placenta biopsies from APS patients for the above molecules further validate the RNASeq results.
Conclusions: RNA-seq of APS treated HUVECs reveals a thoroughly analysed proinflamatory and procoagulant phenotype.
In the second section we aimed to identify epigenetic changes and factors potentially implicated in the pathophysiology of APS. To this end, we compared DNA methylation levels of the IL8 and F3 genes between healthy donors (HDs) and APS patients, using whole blood as a source.
Results: Methylation was significantly reduced in the IL8 promoter and significantly increased in the F3 gene body in APS patients compared to HDs and correlated with specific clinical parameters. In an ex vivo model partially mimicking APS, stimulation of monocytes with a mixture of β2GPI, anti-β2GPI and CXCL4 also induces DNA methylation changes in the above genes, along with increase of their expression. Stimulation of human umbilical vein endothelial cells (HUVECs) with the same mixture also results in transcriptional upregulation of epigenetic factors, including MΕCP2, DNMT3, TET1, HDAC9 and ARID5B.
Conclusions: The above data support that epigenetic alterations could be implicated in the pathophysiology of APS and prompt further investigation of their potential diagnostic or therapeutic utility.
In the thord section, given the fact that anti-β2GPI antibodies recognize complexes of β2GPI dimers with CXCL4 chemokine and activate platelets and Thrombospondin 1 (TSP-1) is secreted by platelets andexhibitsprothrombotic and proinflammatoryproperties. Therefore, we investigated its implication in APS.
Methods: Plasma from APSpatients (n=100), Systemic Lupus Erythematosus (SLE) (n=27) and healthy donors (HD) (n=50) was analyzed for TSP-1, IL-1β, IL-17A and free active TGF-β1 by ELISA. Human Umbilical Vein Endothelial Cells (HUVECs) and HD monocytes were treated with total HD-IgG or anti-β2GPI, β2GPI and CXCL4and CD4+T-cells were stimulated by monocyte supernatants.TSP-1, IL-1β, IL-17A TGF-β1 levels were quantified by ELISA and Real-Time PCR.
Results: Higher plasma levels of TSP-1 and TGF-β1, which positively correlated each other, were observed in APS but not HDs or SLE patients. Patients with arterial thrombotic eventsor those undergoing a clinical event had the highest TSP-1 levels. These patients also haddetectable IL-1β, IL-17Ain their plasma. HD-derived monocytes and HUVECs stimulated with anti-β2GPI-IgG-β2GPI-CXCL4 secreted the highestTSP-1 and IL-1β levels. Supernatants from anti-β2GPI-β2GPI-CXCL4 treated monocytes induced IL-17A expression from CD4+ T-cells. Transcript levels followed a similar pattern.
Conclusions: These findings supporta possible implication of TSP-1 in APS pathology. In vitro cell treatments along with patient plasma measurements in APS patients suggest that high TSP-1 levels could mark a prothrombotic state and an underlying inflammatory process.
Main subject category:
Health Sciences
Keywords:
Antiphospholipid, Endothelium, Thrombospondin, Methylytaion, Epegenetic, IL-8, Τissue factor, Τranscriptome sequencing
Number of references:
337
