engleski

Elevated NaCl concentrations in cell culture media increases oxidative stress of Human Aortic Endothelial Cells

Introduction: As one of the main risk factors for development of cardiovascular diseases, high blood pressure is closely related to salt intake. The plasma sodium concentration in population averages between 134 and 148 mmol/l. Present study aimed to assess the effects of elevated NaCl concentration on oxidative stress in human aortic endothelial cells (HAEC). Methods: HAEC (Innoprot ; Barcelona, Spain) were grown at 37℃, 5% CO2 in Endothelial Cell Basal Medium (Medium 200) supplemented with low serum growth supplement (LSGS). The osmolality of Control medium was 270 mosmol/kg (133 mmol/l sodium). High NaCl medium was prepared by adding NaCl to the total osmolality of 320 mosmol/kg (158 mmol/l) and 350 mosmol/kg (173 mmol/l). During experiment, Control medium was replaced by the high NaCl medium. The experiments were performed on cells at about 80% confluence, at cells’ passage P5. Intracellular Reactive Oxygen Species (ROS) production: The samples of the HAEC seeded in T-25 flasks were collected after 72-hour exposure to 270 mosmol/kg, 320 mosmol/kg and 350 mosmol/kg NaCl and prepared for staining protocol. The levels of hydrogen peroxide (H2O2) and peroxynitrite (ONOO-) were determined using Dichlorofluorescein diacetate (DCF-DA). The cells were resuspended in 100 µL of 1 PBS and incubated with DCF-DA (10 µM final concentration) for 30 min at +4°C. Following the incubation period, samples were resuspended in 350 µL of 1 PBS and analysed. Following the initial readings, 50 μL of 1 mM phorbol 12-myristate 13-acetate (PMA) was added to each sample to stimulate ROS production. After 15- minute incubationthe samples were read on FACS Canto II flow cytometer (BD Bioscience ; 488 excitation laser and 530/30 BP analysis filter). Data analysis and visualization were performed using Flow Logic software (Inivai Technologies, Mentone, Australia). Results: The median fluorescence intensity of DCF- DA (marker of intracellular production of H2O2 and ONOO-) was significantly increased at 350 mosmol/kg (173 mmol/l) NaCl compared to Control. Conclusion: Increased NaCl concentration significantly increases endothelial cellular oxidative stress. Keywords: HAEC cells, NaCl, oxidative stress, Dichlorofluorescein diacetate (DCF-DA), high salt Acknowledgements: This study was supported under the Faculty of Medicine Osijek Institutional grant IP1-MEFOS-2019 (PI I. Drenjančević), IP1-MEFOS- 2020 (PI I. Drenjančević), IP3-MEFOS- 2021 project (PI I. Drenjančević), IP1-MEFOS- 2022 project (PI I. Drenjančević) and by European Structural and Investment Funds through a grant to the Croatian National Science Center of Excellence for Personalized Health Care, Josip Juraj Strossmayer University of Osijek # KK.01.1.1.01.0010. Thank to Cedars - Sinai Medical Center’s International Research and Innovation in Medicine Program, the Association for Regional Cooperation in the Fields of Health, Science and Technology (RECOOP HST Association) for their support of our organization as participating Cedars – Sinai Medical Center - RECOOP Research Centers (CRRC).

HAEC cells, NaCl, oxidative stress, Dichlorofluorescein diacetate (DCF-DA), high salt

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