Alex Ramos López ^1,2 Lina Youssef ^3,4 Patricia Molina ¹ Sergi Torramadé-Moix ¹ Julia Martinez-Sanchez ^1,2 Ana Belen Moreno-Castaño ¹ Miquel Blasco Pelicano ^5,6 Elena Guillén-Olmos ⁵ Blanca De Moner Rafel ^1,2,4 Marc Pino ¹ Marta Tortajada ³ Marta Camacho ³ Maria Borrell ³ Francesca Corvetto ³ Maria Jose Ramirez-Bajo ⁷ Pedro Ventura-Aguiar ^5,7 ELISENDA BANON-MANEUS ⁷ Jordi Rovira ⁷ Gines Escolar ¹ Enric Carreras ^2,4 Eduard Gratacos ^3,8 Maribel Diaz-Ricart ^1,2* Fatima Crispi ^3,9 Marta Palomo ^10,2

• ¹ Hemostasis and Erythropathology Laboratory, Hematopathology, IDIBAPS, Universitat de Barcelona, Barcelona, Spain., barcelona, Spain
• ² Barcelona Endothelium Team, Barcelona, Spain., barcelona, Spain
• ³ BCNatal Fetal Medicine Research Center, Sant Joan de Déu Hospital, Barcelona, Balearic Islands, Spain
• ⁴ Josep Carreras Leukaemia Research Institute (IJC), Badalona, Catalonia, Spain
• ⁵ Nephrology and Kidney Transplant Department, Center of Reference in Complex Glomerular Disease (CSUR), Hospital Clínic de Barcelona, IDIBAPS, Universitat de Barcelona, Barcelona, Spain., barcelona, Spain
• ⁶ Red de Investigación Cooperativa Orientada a Resultados en Salud RICORS2040, Madrid, Spain., barcelona, Spain
• ⁷ Laboratori Experimental de Nefrologia i Trasplantament (LENIT), Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain., barcelona, Spain
• ⁸ Centre for Biomedical Research on Rare Diseases (CIBER-ER), Madrid, Spain, barcelona, Spain
• ⁹ Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Madrid Community, Spain
• ¹⁰ Hematology External Quality Assessment Laboratory, Biomedical Diagnostic Center, Hospital Clinic of Barcelona, Barcelona, Spain., barcelona, Spain

Background: Preeclampsia (PE) is a pregnancy complication characterized by hypertension, proteinuria, endothelial dysfunction, and complement dysregulation. Placenta-derived extracellular vesicles (EVs), necessary in maternal-fetal communication, might contribute to PE pathogenesis. Moreover, neutrophil extracellular traps (NETs) play a pathogenic role in other complement-mediated pathologies and their contribution in PE remains unexplored.Material and Methods: EVs were isolated from PE (peEVs) and normotensive pregnant women sera. NETs were obtained incubating donor-pre-activated neutrophils with PE or control sera. Microvascular (HMEC) endothelial cells (ECs) were incubated with PE or control sera with or without (depleted sera) EVs or NETs, to assess changes in VCAM-1, ICAM-1, VE-cadherin, eNOS, VWF, ROS and C5b-9 deposits. Results were expressed as fold increase vs. control.Results: VWF, VCAM-1, and ROS expression was significantly higher in cells exposed to PE sera vs. control (12.3±8.1, 3.6±2.3 and 1.8 ±0.2, respectively, p<0.05), though significantly lower in cells exposed to depleted PE (dPE) sera (6.1±2.7, 0.7±0.6, and 1.2±0.1, respectively, vs control p<0.05). EC exposure to depleted control sera supplemented with peEVs (dC+peEVs) increased significantly VWF, VCAM-1, and ROS compared to non-supplemented sera (4.5±0.3, 2.8±2.0, and 1.4±0.2, respectively, p<0.05). ICAM-1, VE-cadherin and C5b-9 did not differ among groups.ECs incubated with PE-NETs increased VWF and VCAM-1 and decreased VE-Cadherin expression vs control (4±1.6, 5.9±1.2 and 0.5±0.1, respectively, p<0.05), and increased notably C5b-9 deposit (7.5±2.9, p<0.05). ICAM-1 and ROS did not differ.Conclusions: Both circulating extracellular vesicles and NETs from preeclampsia pregnant exhibit a deleterious effect on endothelial cells. Whereas extracellular vesicles trigger a prooxidant and proinflammatory state, NETs potentiate the activation of the complement system, as already described in preeclampsia.