OC 16.2 - Antiphospholipid syndrome patients’ serum differentially activates venous and microcirculatory endothelium in vitro

Sunday, June 23, 2024

09:45 – 10:00 ICT

Room: 111 A-C

Presenting Author(s)

Massimo Radin, MD

Researcher
University of Turin
Turin, Piemonte, Italy

Co-Author(s)

DM

Daniele Mancardi

Researcher
University of Turin
Turin, Piemonte, Italy
FS

Francesco Shelton Agar

Researcher
University of Turin
Turin, Piemonte, Italy
CM

Clara Marinotti

Student
University of Turin
Turin, Piemonte, Italy
EA

Elisa Arrigo

PhD candidate
University of Turin
Turin, Piemonte, Italy
Irene Cecchi, MD,PhD

Researcher
University of Turin
Turin, Piemonte, Italy
Alice Barinotti, MSc

PhD-Student
University of Turin
Turin, Piemonte, Italy
SF

Silvia Grazietta Foddai

PhD-Student
University of Turin
Turin, Piemonte, Italy
DR

Dario Roccatello

Professor
University of Turin
Turin, Piemonte, Italy
SS

Savino Sciascia

Physician
University Center of Excellence on Nephrologic, Rheumatologic and Rare Diseases (ERK-Net, ERN-ReConnect and RITA-ERN Member) with Nephrology and Dialysis Unit and Center of Immuno-Rheumatology and Rare Diseases (CMID), Department of Clinical and Biological Sciences, ASL Città Di Torino and University of Turin, Turin, Italy
Turin, Piemonte, Italy

Background: Endothelial dysfunction plays a pivotal role in vascular events in Antiphospholipid syndrome (APS).

Aims: To evaluate the effect of APS patients serum on venous and microvascular endothelial cells.

Methods: HUVEC and HMEC-1 were treated with APS patients’ serum supplemented media. Commercial human serum was used as negative control. For migration assays, a 3-chamber transept was employed, in order to create gaps in the cell monolayer. Images were acquired at t0 and after six hours. 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 (Figure1).
When realizing tubulogenic assays, capillary-like structures were more abundant in APS treated HUVEC compared to control (increased master segments number/length and master junctions number). 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 (Figure2).
BAX expression was slightly reduced on HMEC-1, while BAX/BCL2 ratio was increased. BAX/BCL2 ratio was slightly diminished on HUVEC. An increased expression of Cyt C was observed in HUVEC, finding consistent with the overexpression of other intracellular proteins involved in cell death (TOM22 and VDAC). In HMEC-1 the same results were not observed, with the exception of an increased TOM22 expression.
When focusing on MMP-9, given its pro-angiogenic role and its involvement in ECM remodeling, the zimography assay showed an increased activity in both cells treated with APS serum, compared to controls, effect particularly pronounced in HUVEC cells.

Conclusion(s): Serum from APS patients significantly increased angiogenesis and tubule formation in venous endothelium, but not in microcirculation. An increase in MMP-9 activity and a notable shift in the expression of apoptosis-related proteins were observed.