We aimed to identify the plasma miRNA profile of antiphospholipid syndrome (APS) patients and to investigate the potential role of specific circulating miRNAs as non-invasive disease biomarkers. Ninety APS patients and 42 healthy donors were recruited. Profiling of miRNAs by PCR-array in plasma of APS patients identified a set of miRNAs differentially expressed and collectively involved in clinical features. Logistic regression and ROC analysis identified a signature of 10 miRNA ratios as biomarkers of disease. In addition, miRNA signature was related to fetal loss, atherosclerosis, and type of thrombosis, and correlated with parameters linked to inflammation, thrombosis, and autoimmunity. Hard clustering analysis differentiated 3 clusters representing different thrombotic risk profile groups. Significant differences between groups for several miRNA ratios were found. Moreover, miRNA signature remained stable over time, demonstrated by their analysis three months after the first sample collection. Parallel analysis in two additional cohorts of patients, including thrombosis without autoimmune disease, and systemic lupus erythematosus without antiphospholipid antibodies, each displayed specific miRNA profiles that were distinct from those of APS patients. In vitro, antiphospholipid antibodies of IgG isotype promoted deregulation in selected miRNAs and their potential atherothrombotic protein targets in monocytes and endothelial cells. Taken together, differentially expressed circulating miRNAs in APS patients, modulated at least partially by antiphospholipid antibodies of IgG isotype, might have the potential to serve as novel biomarkers of disease features and to typify patients’ atherothrombotic status, thus constituting a useful tool in the management of the disease.

Although IMTmean agreement is excellent under a strict protocol, limits of agreement might be too wide to consider carotid ultrasound a robust cardiovascular risk biomarker.

BackgroundThe course of antiphospholipid syndrome (APS) may rapidly progress from asymptomatic to severe manifestations. Thus, timely diagnosis is essential to improve accuracy of therapy. The role of circulating microRNAs (miRNAs) as potential biomarkers of disease has not yet been analysed in APS.ObjectivesTo investigate the contribution of circulating miRNAs to the pathogenesis of APS and their potential role as non-invasive biomarkers of the disease.MethodsNinety APS patients and 42 healthy donors (HD) were included in the study. Clinical and inflammatory parameters were analysed. As a complement to standard clinical follow-up, the ankle-brachial index (ABI) and carotid intima-media thickness (CIMT) were determined. miRNA expression profiling was performed in plasma by PCR-Array, and the Ingenuity Pathways analysis software (IPA) was used to identify specific miRNAs and target proteins associated to the pathogenesis of APS. RT-PCR and ELISA/Bioplex of selected genes and proteins were used to validate microarray data. The diagnostic value of specific miRNAs signatures (ratios) as disease biomarkers was evaluated by ROC curves analysis. To assess the specificity of these signatures in APS, the expression of the selected miRNAs was analysed in plasma from 23 thrombotic patients without associated autoimmune disease. Monocytes isolated from HD and endothelial cells (ECs) were treated in vitro with antiphospholipid antibodies (aPL-IgGs) purified from the serum of APS patients, and the changes promoted on the levels of selected miRNAs and their potential targets were analysed.ResultsThe PCR-Array identified 39 circulating miRNAs differentially expressed, including 19 up-regulated and 20 down-regulated in APS. IPA analysis recognized 11 miRNAs as potential modulators of target genes involved in the physiopathology of APS. Logistic Regression and ROC curve analyses identified a signature of 10 miRNA ratios as biomarkers for diagnosis of APS with great accuracy (AUC:0.81), along with 2 miRNA ratios as biomarkers for typifying the atherothrombotic status (pathologic CIMT) of APS (AUC:0.76). Patients with thrombosis but without associated autoimmune disease displayed a specific miRNA profile, distinct from that of APS patients. The miRNA signature was related to clinical features of APS such as the occurrence of foetal loss and the type of thrombosis suffered, and correlated with parameters linked to autoimmunity (aPL-IgG titers), inflammation, and thrombosis (ABI, ESR, TF, PAI-1, MCP-1, VEGF-A and VEGF-R1). In vitro treatment of monocytes and endothelial cells with aPL-IgG antibodies promoted a significant deregulation in the secreted levels of the selected miRNAs and atherothrombotic target proteins.ConclusionsmiRNA levels in the serum of APS patients -modulated by aPL-IgG antibodies- are potential novel biomarkers for diagnosis and typifying of their atherothrombotic status, thus constituting a useful tool in the prevention and management of the disease.AcknowledgementsSupported by CTS794 and ISCIII (FIS15/1333 and RI...