Data_Sheet_1_Differences in Endothelial Activation and Dysfunction Induced by Antiphospholipid Antibodies Among Groups of Patients With Thrombotic, Refractory, and Non-refractory Antiphospholipid Syndrome.DOCX

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by pregnancy morbidity or thrombosis and persistent antiphospholipid antibodies (aPL) that bind to the endothelium and induce endothelial activation, which is evidenced by the expression of adhesion molecules and the production of reactive oxygen species (ROS) and subsequent endothelial dysfunction marked by a decrease in the synthesis and release of nitric oxide (NO). These endothelial alterations are the key components for the development of severe pathological processes in APS. Patients with APS can be grouped according to the presence of other autoimmune diseases (secondary APS), thrombosis alone (thrombotic APS), pregnancy morbidity (obstetric APS), and refractoriness to conventional treatment regimens (refractory APS). Typically, patients with severe and refractory obstetric APS exhibit thrombosis and are classified as those having primary or secondary APS. The elucidation of the mechanisms underlying these alterations according to the different groups of patients with APS could help establish new therapies, particularly necessary for severe and refractory cases. Therefore, this study aimed to evaluate the differences in endothelial activation and dysfunction induced by aPL between patients with refractory obstetric APS and other APS clinical manifestations. Human umbilical vein endothelial cells (HUVECs) were stimulated with polyclonal immunoglobulin-G (IgG) from different groups of patients n = 21), including those with primary (VTI) and secondary thrombotic APS (VTII) and refractory primary (RI+), refractory secondary (RII+), and non-refractory primary (NR+) obstetric APS. All of them with thrombosis. The expression of adhesion molecules; the production of ROS, NO, vascular endothelial growth factor (VEGF), and endothelin-1; and the generation of microparticles were used to evaluate endothelial activation and dysfunction. VTI IgG induced the expression of adhesion molecules and the generation of microparticles and VEGF. RI+ IgG induced the expression of adhesion molecules and decreased NO production. RII+ IgG increased the production of microparticles, ROS, and endothelin-1 and reduced NO release. NR+ IgG increased the production of microparticles and endothelin-1 and decreased the production of VEGF and NO. These findings reveal differences in endothelial activation and dysfunction among groups of patients with APS, which should be considered in future studies to evaluate new therapies, especially in refractory cases.

抗磷脂综合征（Antiphospholipid Syndrome, APS）是一类以妊娠不良事件或血栓形成、持续存在的抗磷脂抗体（antiphospholipid antibodies, aPL）为特征的自身免疫性疾病。此类抗体可结合内皮细胞并诱导内皮激活，具体表现为黏附分子表达上调、活性氧（reactive oxygen species, ROS）生成增加，以及以一氧化氮（nitric oxide, NO）合成与释放减少为标志的后续内皮功能障碍。上述内皮细胞异常是APS发生严重病理进程的关键环节。

APS患者可根据合并其他自身免疫疾病（继发性APS）、单纯血栓形成（血栓性APS）、妊娠不良事件（产科APS），以及对常规治疗方案耐药（难治性APS）进行分型。通常，重症难治性产科APS患者会出现血栓形成，被归类为原发性或继发性APS。阐明不同APS患者亚群上述内皮异常的潜在机制，有助于开发新型治疗策略，对于重症难治性病例尤为关键。因此本研究旨在评估难治性产科APS患者与其他APS临床表型患者体内aPL诱导的内皮激活与功能障碍差异。

本研究共纳入21例不同APS患者亚群的多克隆免疫球蛋白G（Immunoglobulin G, IgG），用以刺激人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVECs），受试亚群包括原发性血栓性APS（VTI）、继发性血栓性APS（VTII）、难治性原发性产科APS（RI+）、难治性继发性产科APS（RII+）以及非难治性原发性产科APS（NR+），所有受试者均合并血栓形成。本研究通过检测黏附分子表达、ROS生成、NO、血管内皮生长因子（vascular endothelial growth factor, VEGF）、内皮素-1的产生，以及微粒生成情况，用以评估内皮激活与功能障碍程度。

实验结果显示：VTI IgG可诱导黏附分子表达及微粒、VEGF生成；RI+ IgG可诱导黏附分子表达并减少NO生成；RII+ IgG可增加微粒、ROS及内皮素-1的生成，并降低NO释放；NR+ IgG可增加微粒及内皮素-1的生成，并减少VEGF与NO的产生。本研究结果揭示了不同APS患者亚群在内皮激活与功能障碍方面的异质性，该差异应在未来评估新型治疗策略的研究中予以充分考量，尤其针对难治性APS病例。