Antiphospholipid antibodies induce proinflammatory and procoagulant pathways in endothelial cells

Abstract

Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thrombotic events and/or pregnancy morbidity in the presence of antiphospholipid antibodies detected either as anti-cardiolipin, anti-β2 Glycoprotein I (anti-β2GPI) or Lupus anticoagulant (LA). Endothelial deregulation characterizes the syndrome. To address gene expression changes accompanying the development of autoimmune phenotype in endothelial cells in the context of APS, we performed transcriptomics analysis in Human Umbilical Vein Endothelial Cells (HUVECs) stimulated with IgG from APS patients and β2GPI, followed by intersection of RNA-seq data with published microarray and ChIP-seq results (Chromatin Immunoprecipitation). Our strategy revealed that during HUVEC activation diverse signaling pathways such as TNF-α, TGF-β, MAPK38, and Hippo are triggered as indicated by Gene Ontology (GO) classification and pathway analysis. Finally, cell biology approaches performed side-by-side in naïve and stimulated cultured HUVECs, as well as, in placenta specimens derived from Healthy donors (HDs) and APS-patients verified the evolution of an APS-characteristic gene expression program in endothelial cells during the initial stages of the disease's development.

Keywords: Antiphospholipid syndrome (APS); Computational biology tools; Gene expression programs; Inflammatory and procoagulant phenotype; Transcriptional regulators; Transcriptomics analysis.

Fig. 1

Methodology, results and validation of RNA-seq in HUVECs treated with IgG and β2GPI isolated from APS patients A. Schematic representation of HUVECs' treatment and RNA sequencing. B. Transcriptomics and Bioinformatics analysis uncovered 395 genes that were activated during the first 6 h of treatment. Inflammatory and immune response genes as well as known APS-markers including genes that encode for transcription factors (e.g NF-κB, SMAD, YAP1), chemokines (e.g IL-8), growth factors (TGFβ-2), adhesion molecules (e.g E-Selectin, VCAM-1, ICAM-1), signal transduction molecules (e.g MAP2K3, MAP3K5) are upregulated. C. Verification of the results from transcriptomics analyses using gene-specific qPCR assays for selected genes (left panel). High correlation of the RNA-seq/qPCR results is detected (right panel).

Fig. 2

RNA-seq intersection with published data and gene ontology analysis. A. Snapshots from Genome Browser show that sequencing reads (signal) covering exon sequences of NFKB1, SELE, VCAM1, BMP2, IL7R are increased in treated samples compared to control samples whereas signals derived from ACTB exons, remain unaffected B. Direct intersection of previously published microarray results with our RNA-seq results revealed 31 common upregulated genes (2 B)·C-D KEGG pathway and Gene Ontology analysis (Biological Process) for the upregulated genes reveals that signal transduction pathways such as TNF-signaling, TGF-β signaling, MAPK38- signaling and Hippo pathway operate simultaneously upon HUVECs activation.

Fig. 3

Immunofluorescent antibody staining in naïve and treated HUVECs. AK. The mixture of APS total IgG and β2GPI induces a pronounced increase of the protein levels of the proinflammatory cytokines IL-6, IL-8 as well the transcription factor NF-κB1 and cell adhesion molecules Tissue Factor, ICAM-1, VCAM-1, Eselectin, P-selectin and TGFR1. There was no significant difference for the TGFR1 molecule (3 J). Visual analysis revealed that all the inflammatory mediators and adhesion molecules presented statistically significant difference between the untreated and treated endothelial cells (3 K).

Fig. 4

Upregulated transcription factors and their putative targets. A. (Upper panel) Classification of upregulated genes into families. (Bottom panel) Genes that encode for transcriptional regulators are activated in HUVECs upon treatment. B. Combined bioinformatics meta-analyses of published NF-κB-ChIP-seq results on HUVECs with our RNA-seq data reveals several NF-kB binding-events in 28 upregulated genes that includes known target genes of the above transcription factor C. Intersection of the RNA-seq results with the already published Smad1/5-ChIP-seq experiment in HUVECs identifies 14 direct target genes of Smad1/5 such as SMAD9, IPPK, CCDC68.

Fig. 5

Immunofluorescent antibody staining in placenta biopsies from APS patients and healthy women. A-K Placenta biopsies derived from APS patients as well as Healthy Donors show increased signal intensity for IL-6, IL-8, NF-κB1, ICAM1, VCAM-1, E-selectin, P-selectin, TGF-β2, and TGFR1 (5A-5D, 5F-5J). Slight difference in fluorescence intensity between HD and APS patient was observed for Tissue Factor (5E). Increased signal intensity was observed as well for the TNF-α molecule in the APS placenta biopsies (5 K).