ANTIPHOSPHOLIPID SYNDROME PATIENTS’ SERUM DIFFERENTIALLY ACTIVATES VENOUS AND MICROCIRCULATORY ENDOTHELIUM IN VITRO

Background: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the presence circulating anti-phospholipids antibodies (aPL) and several clinical manifestations, with recurrent thrombosis being the most common. The involvement of endothelial (dys)function has been demonstrated to play a pivotal role in vascular events, that can arise at any level of the vascular bed. Objectives: We aimed to evaluate the effect of serum from APS patients on endothelial cells behavior in terms of migration, proliferation and intracellular response and how aPL are related to specific function of microvascular cells compared to venous cells. Methods: Human Umbilical Vein Endothelial Cells (HUVEC) and Human Microvascular Endothelial Cells (HMEC-1, ATCC CRL-3243) were cultured in EndoGRO-MV Complete Culture Media Kit (Millipore), with Pen/Strep (1%v/v) and incubated at 37ºC/5% CO2. For treatment, the media was supplemented with APS patients’ serum. Human serum (HC, H4522, Sigma-Aldrich) was used as negative control. For migration assays, cells were seeded on 12 wells plates endowed with a 3-chamber transept and incubated O/N. The next day the transept was removed to create gaps in the cell monolayer. Microscopy images were acquired at time 0 and after six hours. In vitro tubular structures formation was investigated through Corning®Matrigel®matrix cell culture. Western blot analysis was realized to evaluate VDAC, TOM22, CYT C, BAX and BCL2L10 expression. Zymography was employed to quantify MMP-9 activity. Results: APS serum induced a significant higher migration in HUVEC compared to HMEC-1, which exhibited no differences compared to the control group (Figure 1). When realizing tubulogenic assays, capillary-like structures were more abundant in APS treated HUVEC compared to control, with an increased number and length of master segments, as well as an increased number of master junctions. No significant difference was observed in the number of isolated segments. This pattern was reversed in HMEC-1, which showed a possibly reduced formation of capillary-like structures, matching the poor results in terms of migration. (Figure 2) When focusing on apoptotic markers, we observed a slight BAX expression reduction on HMEC-1 cells following treatment and, although not significant, an increased BAX/BCL2 ratio, which may be sign of increased apoptosis. On the other side, BAX/BCL2 ratio was slightly diminished on treated HUVEC. Moreover, an increased expression of Cyt C was observed in HUVEC compared to control serum, and results were consistent with the overexpression of other intracellular proteins involved in cell death such as TOM22 and VDAC. In HMEC-1, however, the same results were not observed, with the exception of an increased TOM22 expression. We then focused on MMP-9, given its pro-angiogenic role and its involvement in ECM remodeling. The zimography assay showed an increased MMP-9 activity in cells treated with APS serum, compared to controls, effect that was particularly pronounced in HUVEC cells, despite being elevated in both cell lines. Conclusion: Serum from APS patients significantly increased angiogenesis and tubule formation in venous endothelium, but not in microcirculation. Additionally, an increase in MMP-9 activity and a notable shift in the expression of apoptosis-related proteins were observed. These findings might contribute to our understanding of pathogenesis of the syndrome. Further exploration of the underlying mechanisms are needed to in identify novel therapeutic strategies for treating vascular events induced by aPL. Figure 1. A) Representative images of HUVEC during treatment. Pictures were acquired at the beginning of the treatment (T0) and after 6 hours (T6). B) Graphical representation of HUVEC and HMEC-1 cells migration response to treatment. Figure 2. A) Tubulogenic assay result image showing tubules formation in HUVEC. B) Graphical representation of the relative quantification of master junctions, master segments, total master segments length and isolated segments produced by HUVEC and HMEC-1 cells following APS and control serum treatment. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared. DOI: 10.1136/annrheumdis-2024-eular.2606 Keywords: Autoantibodies, Rare/orphan diseases, Interdisciplinary research Citation: , volume 83, supplement 1, year 2024, page 540Session: Antiphospholipid syndrome (Poster View)

Autoantibodies, Rare/orphan diseases, Interdisciplinary research