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A
B
C
D
Figure 7. Antiphospholipid antibodies modulate the expression of both the circulating miRNAs that integrate the signature in antiphospholipid syndrome (APS) and their putative protein targets. Human umbilical vein endothelial cells (HUVECs) were treated with antiphospholipid antibodies and secreted selected microRNAs (miRNAs) (A) and putative target protein (B) levels were determined in the supernatant. Monocytes were also treated with antiphospholipid antibodies and secreted selected miRNAs (C) and putative target proteins (D) levels were evaluated in the supernatant of culture. Differences were analyzed by Student t-test. Values are the means and Standard Error of Mean of 4 independent experiments performed in triplicate. P<0.05 was considered statistically significant. TF: tissue factor; PAI-1: plasminogen activator inhibitor-1; VEGF-A: vascular endothelial growth factor A; VEGF-R1: VEGF-Receptor-1; MCP-1: monocyte chemotactic protein.
Moreover, the main target of miR-34a is VEGF-A, a key inflammatory protein involved in numerous cardiovascu- lar and autoimmune pathologies, including APS.23,36 In the same way, miR-374 has been described as regulator of maintenance of vascular integrity.37 The remaining miRNAs members of the signature, including miR-296, miR-210, miR-206 and miRNA-15, have been found altered in severe pre-eclampsia, one of the leading causes of maternal mortality and neonatal morbidity world- wide.38-40 Thus, all the processes regulated by these miRNAs seem to orchestrate distinct aspects of APS pathogenesis.
To assess the specificity of the circulating miRNA signa- ture in APS we evaluated the miRNA profile in an addi- tional cohort of patients characterized by the presence of previous thrombotic events in the absence of an associat- ed autoimmune disease. The miRNAs analysis revealed a differential pattern of expression between these two cohorts. Those results substantiate previous studies that evidenced the presence of a distinct miRNA profile in monocytes and neutrophils of thrombotic non-autoim- mune patients compared to APS patients.9 This could reflect a differential mode of regulation and activity of miRNAs in thrombotic patients compared to APS patients, on which the role of autoantibodies might be crucial. Moreover, the analysis of a parallel autoimmune popula- tion (SLE patients) negative for aPL, also identified an miRNA signature distinct from that of APS, thus underly- ing the potential role of aPLs as regulators of thrombosis- related miRNAs in APS, and pointing to the presence of a specific miRNA profile relative to the pathogenesis of each disease.
Antiphospholipid syndrome patients recruited in this
study were mainly treated with anticoagulant and/or antiplatelet agents. All of them have been shown to influ- ence miRNAs expression, an epigenetic process that might help to delineate the mechanisms underlying their effects.9,41,42 Thus, we evaluated the potential effect of these treatments on the circulating miRNA expression profile. No significant differences were observed in our cohort of APS patients between those who received antiplatelet and those treated with anticoagulant agents, suggesting that the prothrombotic status induced by effects of aPLs, and the consequently deregulated miRNAs, were not differentially modulated among these drugs.
In order to understand the clinical relevance of the altered circulating miRNA signature, association and cor- relation studies were perfomed. Altered expression of var- ious miRNA ratios was associated with the presence of previous fetal losses. In line with these findings, several studies have shown that the misregulation of circulating placental miRNAs in maternal blood might lead to preg- nancy complications, thus acting as non-invasive diagnos- tic and prognostic biomarkers for pregnancy monitoring.42- 44 Association studies further established a significantly increased expression of 2 miRNA ratios in APS patients that had suffered arterial thrombosis in comparison with those who experienced venous thrombotic events. Interestingly, both miRNA ratios were integrated by the miR-20a, previously reported to be the main regulator of TF, whose expression levels have been found to be related to the development of arterial thrombosis in the setting of APS.8,45 Finally, we identified 2 miRNA ratios as clinical rel- evant biomarkers related to early atherosclerosis develop- ment in APS patients, which were integrated by the miR-
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