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Impact of elevation of NaCl concentrations in cell culture media on Human Aortic Endothelial Cells

Introduction: Cardiovascular diseases (CVDs) are a leading health problem worldwide. High blood pressure is one of the main risk factors for their development and blood pressure levels are closely associated with salt intake. The plasma sodium concentration in population generally varies between 134 and 148 mmol/l. The aim of this study was to assess the effects of elevation of NaCl concentrations in cell culture media on oxidative stress and survival of human aortic endothelial cells (HAEC). Methods: HAEC cells (from Innoprot ; Barcelona, Spain) were grown at 37°C, 5% CO2 in Endothelial Cell Basal Medium (Medium 200) supplemented with low serum growth supplement (LSGS). The osmolality of this medium (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), 350 mosmol/kg (173 mmol/l) and 380 mosmol/kg (188 mmol/l). To elevate NaCl, control medium was replaced by the high NaCl medium. The experiments were performed on cells at about 80% confluence. For all experiments, cells were used at passage P5. MTT test: The cells were seeded in 96 well plate. After 24 h, 48h and 72h 10 µL of MTT stock solution was added into each well and the plate was placed in an incubator for 4 h to produce formazan crystals. After incubation, 100 µL of MTT solvent was added to dissolve created formazan crystals and microplate was read at 595nm. Intracellular Reactive Oxygen Species (ROS) Production by Human Aortic Endothelial Cells (HAEC): Cells were seeded in T-25 flasks. The samples were collected after 72-hour exposure to two concentrations (320 mosmol/kg and 350 mosmol/kg) of NaCl. The cells were washed in 1 phosphate-buffered saline (PBS) and prepared for staining protocol. Dihydroethidium (DHE) was used to determine the level of O2∙− in HAECs. The cells were resuspended in 100 µL of 1 PBS and incubated with DHE (10 µM final concentration) for 30 min at +4 °C. Following the incubation period, samples were rinsed and resuspended in 350 µL of 1 PBS and analysed. After initial readings, 50 μL of 1 mM phorbol 12-myristate 13-acetate (PMA) was added to each sample to stimulate ROS production. After a 15-minute incubation, the samples were read again on the cytometer. FACS Canto II flow cytometer (BD Bioscience ; 488 excitation laser and 530/30 BP analysis filter) was used for the assessment of intracellular ROS production. Data analysis and visualization were performed using Flow Logic software (Inivai Technologies, Mentone, Australia). Results: The cell viability was significantly reduced after 24h at 350 mosmol/kg and 380 mosmol/kg concentrations of NaCl, while after 48h and 72h viability was reduced at 320 mosmol/kg, 350 mosmol/kg and 380 mosmol/kg concentrations. The level of O2∙− was significantly increased in both experimental groups. Conclusion: Increased NaCl concentration significantly reduces cell viability and increases cellular oxidative stress. Financial support: 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) 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.

Human Aortic Endothelial Cells, NaCl, oxidative stress

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