bc54f0c9fbdd7f9d241065416714917d 319567.pdf 0aea028700698ebb2abef83857c6282002a023bc 319567.pdf 5fd1ed35dd0a6e14e1320b58ff0ce609e6b12821e7b8c4c19de0e80a7f8f09a4 319567.pdf Title: Small-Extracellular-Vesicle-Derived miRNA Profile Identifies miR-483-3p and miR-326 as Regulators in the Pathogenesis of Antiphospholipid Syndrome (APS) Subject: Primary antiphospholipid syndrome (PAPS) is a systemic autoimmune disease associated with recurrent thrombosis and/or obstetric morbidity with persistent antiphospholipid antibodies (aPL). Although these antibodies drive endothelial injury and thrombophilia, the underlying molecular mechanism is still unclear. Small extracellular vesicles (sEVs) contain miRNAs, key players in intercellular communication. To date, the effects of miRNA-derived sEVs in PAPS are not well understood. We characterised the quantity, cellular origin and miRNA profile of sEVs isolated from thrombotic APS patients (PAPS, n = 50), aPL-carrier patients (aPL, n = 30) and healthy donors (HD,n = 30). We found higher circulating sEVs mainly of activated platelet origin in PAPS and aPL patients compared to HD, that were highly engulfed by HUVECs and monocyte. Through miRNA-sequencing analysis, we identified miR-483-3p to be differentially upregulated in sEVs from patients with PAPS and aPL, and miR-326 to be downregulated only in PAPS sEVs. In vitro studies showed that miR-483-3p overexpression in endothelial cells induced an upregulation of the PI3K-AKT pathway that led to endothelial proliferation/dysfunction. MiR-326 downregulation induced NOTCH pathway activation in monocytes with the upregulation of NFKB1, tissue factor and cytokine production. These results provide evidence that miRNA-derived sEVs contribute to APS pathogenesis by producing endothelial cell proliferation, monocyte activation and adhesion/procoagulant factors. Keywords: small extracellular vesicle; microRNA profiling; antiphospholipid syndrome; pathogenesis Author: Cristina Solé, Maria Royo, Sebastian Sandoval, Teresa Moliné and Josefina Cortés-Hernández Creator: LaTeX with hyperref Producer: pdfTeX-1.40.21 CreationDate: Wed Jul 19 10:10:47 2023 CEST ModDate: Wed Jul 19 11:02:39 2023 CEST Custom Metadata: no Metadata Stream: no Tagged: no UserProperties: no Suspects: no Form: none JavaScript: no Pages: 19 Encrypted: no Page size: 595.276 x 841.89 pts (A4) Page rot: 0 File size: 5747325 bytes Optimized: no PDF version: 1.7 name type encoding emb sub uni object ID ------------------------------------ ----------------- ---------------- --- --- --- --------- OEZHVW+URWPalladioL-Bold Type 1 Custom yes yes no 10 0 MXGVMY+URWPalladioL-Roma Type 1 Custom yes yes no 15 0 FHQCFO+URWPalladioL-Ital Type 1 Custom yes yes no 19 0 HCRKXL+VnURWPalladioL Type 1 Custom yes yes no 23 0 KESBIZ+EURM10 Type 1 Builtin yes yes no 28 0 GIFXFT+VnURWPalladioL-Bold 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Destination: section.2 Children: Item: Title: Clinical Characteristics Destination: subsection.2.1 Item: Title: Small Extracellular Vesicles (sEVs) in Patients with PAPS and aPL Destination: subsection.2.2 Item: Title: sEVs from aPL and PAPS Patients Produce Endothelial Dysfunction Destination: subsection.2.3 Item: Title: PAPS sEVs Produced Monocyte Activation Destination: subsection.2.4 Item: Title: Differential Expression Profiles of sEV-Derived miRNA between PAPS and aPL Patients Destination: subsection.2.5 Item: Title: sEV-Derived miRNAs Produce HUVEC Cell Activation via PTEN Pathway and Monocyte Activation via NOTCH1 Destination: subsection.2.6 Item: Title: Discussion Destination: section.3 Item: Title: Materials and Methods Destination: section.4 Children: Item: Title: Patients Destination: subsection.4.1 Item: Title: Sample Collection Destination: subsection.4.2 Item: Title: Anti-Phospholipid Antibody Detection Destination: subsection.4.3 Item: Title: Small RNA-Sequencing of sEV-miRNAs Destination: subsection.4.4 Item: Title: Cell Cultures Destination: subsection.4.5 Item: Title: RNA Extraction and RT-qPCR Gene Expression Destination: subsection.4.6 Item: Title: Apoptosis and Proliferation Assay Destination: subsection.4.7 Item: Title: Luciferase Assay Destination: subsection.4.8 Item: Title: Immunofluorescence on Primary Cells Destination: subsection.4.9 Item: Title: Measurement of Cytokines Levels by Enzyme-Linked Immunosorbent Assay (ELISA) Destination: subsection.4.10 Item: Title: Statistical Analysis Destination: subsection.4.11 Item: Title: References Destination: section.A. Info: Title: Small-Extracellular-Vesicle-Derived miRNA Profile Identifies miR-483-3p and miR-326 as Regulators in the Pathogenesis of Antiphospholipid Syndrome (APS) Author: Cristina Solé, Maria Royo, Sebastian Sandoval, Teresa Moliné and Josefina Cortés-Hernández Subject: Primary antiphospholipid syndrome (PAPS) is a systemic autoimmune disease associated with recurrent thrombosis and/or obstetric morbidity with persistent antiphospholipid antibodies (aPL). Although these antibodies drive endothelial injury and thrombophilia, the underlying molecular mechanism is still unclear. Small extracellular vesicles (sEVs) contain miRNAs, key players in intercellular communication. To date, the effects of miRNA-derived sEVs in PAPS are not well understood. We characterised the quantity, cellular origin and miRNA profile of sEVs isolated from thrombotic APS patients (PAPS, n = 50), aPL-carrier patients (aPL, n = 30) and healthy donors (HD,n = 30). We found higher circulating sEVs mainly of activated platelet origin in PAPS and aPL patients compared to HD, that were highly engulfed by HUVECs and monocyte. Through miRNA-sequencing analysis, we identified miR-483-3p to be differentially upregulated in sEVs from patients with PAPS and aPL, and miR-326 to be downregulated only in PAPS sEVs. In vitro studies showed that miR-483-3p overexpression in endothelial cells induced an upregulation of the PI3K-AKT pathway that led to endothelial proliferation/dysfunction. MiR-326 downregulation induced NOTCH pathway activation in monocytes with the upregulation of NFKB1, tissue factor and cytokine production. These results provide evidence that miRNA-derived sEVs contribute to APS pathogenesis by producing endothelial cell proliferation, monocyte activation and adhesion/procoagulant factors. 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