MON-718 Adrenal Cell-Derived Extracellular Vesicles Alter Gene Expression Of Markers Of Endothelial Function And Inflammation In Human Endothelial Cells

Abstract C.A. Carvajal: None. P. Carrion: None. M.P. Hernandez: None. J.A. Perez: None. A. Martinez: None. R. Pinilla: None. T. Uslar: None. R. Baudrand: None. A. Tapia-Castillo: None. C.E. Fardella: None. Small Extracellular vesicles (EVs) are nanosized vesicles (<200nm) released by most cell types and circulate in body fluids, regulating cell-cell communication by carrying lipids, proteins, RNA and miRNA out of cell. In this context, communication by EVs has been proposed be an active circuit in physiological and pathophysiological conditions, as primary aldosteronism (PA). Increased biosynthesis of aldosterone by adrenal cells can be accompanied by shedding EVs to blood stream that carry biological information that could affect the recipient cell status. Aim: To study the effect of adrenal-EVs in the gene expression of markers of endothelial function and inflammation in human endothelial cells. Methods: Human adrenocortical carcinoma cell line, HAC50, kindly donated, were incubated and plated in DMEM/F-12 medium, 10% cosmic calf serum and ITS. HAC50 were characterized by AngII response (100 nM), by testing gene expression (CYP11B2, HSD3B2, AT1R) and aldosterone biosynthesis. Losartan (10 uM), an AT1R antagonist, was used to block AngII response. HAC50 adrenal cells were cultured in T875 flasks to isolate HAC50-EVs, which were isolated by ultracentrifugation and characterized by nanotracking analysis, electron microscopy and w-blot markers according to MISEV2023. EVs (1000/cell) from AngII stimulated HAC50 (AngII-HAC50-EVs) were added to EaHy926 cells (human endothelial cell line). Relative gene expressions (fold changes, FC) of markers of inflammation (IL-1β, IL-6 and IL-8), and endothelial function (eNOS, ICAM1) were assessed by RT-qPCR. Statistical analysis was performed by Mann-Whitney or Kruskal-Wallis and Dunn post-hoc test using GraphPad Prism v10. Results: We observed a significative increase of relative gene expression of HSD3B2 (2.5 FC) and for CYP11B2 (32 FC) in HAC50 cells stimulated with 100 nM AngII vs vehicle (p<0.05). No differences were found in AT1R expression. Preincubation with 10 uM Losartan blunt the effect of AngII in CYP11B2 and HSD3B2 expression. An increase of aldosterone biosynthesis was also detected with 100 nM AngII (p<0.05). We observed a mean concentration of 4x109 vs 1x109 EVs/mL (p<0.05) and an EV mode size of 112 vs 110 nm (p NS), in AngII-HAC50-EVs vs control-HAC50-EVs. Further, incubation of endothelial cells with AngII-HAC50-EVs showed an increased of IL-6 (p<0.05) and decreased gene expression of NOS3 (p<0.05) compared with control-HAC50-EVs. Conclusion: HAC50 release more EVs to extracellular medium in presence of Ang-II. AngII-HAC50-EVs affect the expression of NOS3 and IL-6, suggesting changes in endothelial function and inflammatory properties of EAhy926. These results suggest that EVs could mediate communication between adrenal and endothelial cells, highlighting their potential role in PA pathophysiology. Acknowledgements: Grant ANID-FONDECYT 1212006 and 3200646; FONDEQUIP EQM150023; SOCHED & CETREN-UC. Monday, June 3, 2024