Endothelial preconditioning method

Abstract

FIELD: biotechnology.SUBSTANCE: method for preconditioning endothelium. The method involves incubating EA.hy926 endothelial cells by placing the cell culture in a sealed incubation chamber and exposing it to a gas mixture containing argon and oxygen for 24 hours.EFFECT: gentle and effective activation of the lysosomal system in cells of endothelial origin.1 cl

Description

[0001] The invention relates to experimental medicine, specifically to methods of influencing a patient's body by mixing various gases. The invention can be used for the further development of medications and treatment methods for patients with pathological conditions characterized by endothelial dysfunction.

[0002] Technique level.

[0003] Currently, several technical solutions are known for correcting endothelial dysfunction and protecting cells.

[0004] In particular, a method for increasing the survival of human endothelial cells is known using the synthetic peptides UEHLERALNSS and UEQLERALNTS, which increase the survival of human endothelial cells (HUVEC) during oxidative stress modeling using H2O2. (RU 2751332) However, these peptides have a narrow specificity and require chemical synthesis and direct delivery to cells, which limits the clinical application of this method. In contrast, the claimed method utilizes an argon-oxygen mixture as an accessible and safe agent capable of activating natural mechanisms of lysosomal degradation and mitophagy, providing long-term endothelial protection.

[0005] Also known is the peptide β-aspartyl-alanine, which improves the functional activity of vascular endothelium in atherosclerosis, coronary heart disease, and cerebrovascular accidents, as well as a pharmaceutical composition based on it and a method for its use (RU 2769051). This technical solution is intended primarily for the correction of chronic vascular diseases. The use of an argon-oxygen mixture activates universal intracellular mechanisms – the lysosomal-autophagic system, which is especially significant in acute critical conditions (ischemia-reperfusion, sepsis, ARDS).

[0006] A method for correcting endothelial dysfunction using pharmacological preconditioning with nicorandil in an experimental model of gestosis is known (RU 2543359). However, this method has a narrow scope of application, and the therapeutic agent used is associated with safety and pharmacokinetic limitations. In contrast, an argon-oxygen mixture can be used in a wide range of critical conditions, acting immediately and without drug-induced complications.

[0007] A known method for preventing disruption of intercellular contacts between endothelial cells in endothelial dysfunction uses lithium chloride, which prevents the degradation of VE-cadherin and claudin proteins during the toxic effects of serum (RU 2741766). However, despite its proven effectiveness, LiCl exhibits pharmacological toxicity and limitations in its use. At the same time, the claimed method does not require the use of drugs: exposure to an argon-oxygen mixture activates natural defense processes, which not only maintains contacts but also increases mitochondrial potential and overall cellular resilience.

[0008] A known method for preconditioning against cell death involves using blood treated with ex vivo stressors (temperature, UV, oxidation), followed by the introduction of such blood to protect against apoptosis and necrosis (NZ 512849). This known method involves complex manipulations and subsequent risks. In contrast, the proposed method utilizes direct exposure of cells to a gas mixture, which is safer and simpler and allows for the activation of natural autophagic and mitophagic mechanisms.

[0009] In the scientific, medical and available patent literature studied by the authors of the claimed method, no information was found on methods of preconditioning the endothelium by incubating cells of endothelial origin in a gas mixture.

[0010] Disclosure of the essence of the invention.

[0011] Recent studies have shown that the lysosomal system of endothelial cells plays a key role in maintaining cellular homeostasis, regulating barrier function and angiogenesis. The endothelial lysosomal system is an important link integrating stress signals, metabolic disorders and inflammatory stimuli. Its activation through TFEB or pharmacological modulators can be considered as a promising therapeutic strategy for correcting endothelial dysfunction in critical conditions. [Johnson D, Colijn S, Richee J, Yano J, Burns M, Davis AE, Pham VN, Saric A, Jain A, Yin Y, Castranova D, Melani M, Fujita M, Grainger S, Bonifacino JS, Weinstein BM, Stratman AN. Angiogenesis is limited by LIC1-mediated lysosomal trafficking. Angiogenesis. 2024 Nov; 27(4):943-962. doi:10.1007 / s10456-024-09951-7. Epub 2024 Oct 2. PMID: 39356418; PMCID: PMC11653708.].

[0012] The essence of the claimed method for preconditioning endothelial dysfunction consists in stimulating the lysosomal activity of cells of endothelial origin Ea.Hy926 by incubating said cells in a gas mixture containing 79% argon and 21% oxygen for 24 hours.

[0013] To conduct the experiment, the authors of the proposed method selected cells of endothelial origin. They found that the basal level of lysosomal activity varies significantly between different cell types (cells of the neurovascular unit) and is lowest in endothelial cells. They also found that in response to incubation in an argon gas environment, lysosomal activity increases in endothelial cells, which, in turn, leads to an increase in the number of mitochondria with an increased transmembrane potential.

[0014] An in vitro study examining the effects of cell incubation in an argon-containing gas environment revealed a significant increase in the red fluorescence of the mitochondrial probe JC-1 in endothelial cells. This indicates an increase in the population of healthy mitochondria with a higher transmembrane potential after cell incubation in an argon-containing gas environment.

[0015] The method of endothelial preconditioning involves placing a culture of Ea.Hy926 endothelial cells in a sealed incubation chamber and supplying them with a gas mixture containing 79% argon and 21% oxygen for 24 hours. The choice of the gas mixture composition (Ar 79% / O221%) was due to the need to isolate the effect of argon while maintaining a physiological proportion of oxygen comparable to a standard cultivation atmosphere. The exposure duration of 24 hours is based on data on the kinetics of inert gas transport: for a 1.0 cm deep medium layer, the saturation duration is about 16 hours for argon and about 30 hours for xenon, while for a 0.5 cm thick layer - 5 and 8 hours, respectively [Katz, I., Palgen, M., Murdock, J., Martin, A. R., Farjot, G., & Caillibotte, G. (2016). Gas transport during in vitro and in vivo preclinical testing of inert gas therapies. Medical gas research, 6(1), 14-19. https: / / doi.org / 10.4103 / 2045-9912.179342].Thus, a 24-hour exposure ensures that a steady-state concentration of argon is achieved in the medium, which allows for the correct interpretation of the observed cellular effects.

[0016] As a result of the incubation procedure, a more than 2-fold increase in lysosome activity and an increase in the number of highly energetic mitochondria were observed in the cells compared to control conditions, indicating metabolic activation of the cells.

[0017] The claimed method allows for gentle and effective activation of the lysosomal system in cells of endothelial origin.

[0018] The claimed method can be used for the further development of drugs and methods for correcting pathological conditions in which endothelial dysfunction is observed: tissue ischemia-reperfusion syndrome in stroke and myocardial infarction, systemic inflammatory response syndrome after extensive surgery, combined trauma, severe sepsis and septic shock, pulmonary edema and acute respiratory distress syndrome, burn disease, and others.

[0019] Implementation of the invention.

[0020] Continuous cultures of EA.hy926 vascular endothelial cells were thawed from stock in a water bath at 37°C, then resuspended in a complete nutrient medium (CGM) of the following composition: for vascular endothelial culture, the CGM composition was DMEM with a glucose content of 1 g / l + P12 (1: 1) with 10% fetal bovine serum (FBS), 1% L-glutamine. Cultivation conditions were CO2 incubator, 5% CO2, 37°C. Upon reaching a monolayer, cells were subcultured using a 0.05% trypsin solution with EDTA.

[0021] During the experiment, the resulting cell culture was divided into 4 equal portions and placed in culture dishes at a density of 200 thousand cells / dish:

[0022] 1. control followed by JC-1 staining,

[0023] 2. control followed by LysoTracker Green staining,

[0024] 3. cells after 24 hours in an argon gas environment followed by JC-1 staining,

[0025] 4. Cells after 24 hours in an argon gas environment, followed by staining with LysoTracker Green.

[0026] After 24 hours, the standard PPS in all 4 culture dishes was replaced with Dulbecco's Modified Eagle Medium (DMEM) without sodium bicarbonate with Fetal Bovine Serum (FBS).

[0027] Then, the two dishes were transferred to a desiccator connected to a cylinder containing a gas mixture consisting of 21% oxygen and 79% argon. The cells were left in the desiccator for 24 hours. The control dishes were kept in a thermostat for 24 hours at a standard atmospheric gas composition.

[0028] After 24 hours, the cells were stained with the vital dyes JC-1 and LysoTracker Green. For this purpose, working solutions of 10 μg / ml LumiTracker® Mito JC-1 (Lumiprobe, Russia) and 1 μM LysoTracker Green (Invitrogen, CJIIA) were prepared in a medium without sodium bicarbonate. The dye solutions were added to control cells and cells incubated in an argon gas environment, and the cells were incubated for 30 minutes for JC-1 and 40 minutes for LysoTracker Green in a thermostat. The dyes were then washed once with a warm medium without sodium bicarbonate and imaging was performed on an LSM900 confocal microscope (Zeiss, Germany). The average fluorescence intensity of the dyes was then calculated using ImageJ.

[0029] As a result of calculating the average fluorescence intensity of dyes using the ImageJ program, a significant change in the mitochondrial transmembrane potential in endothelial cells after incubation in a gas environment with argon was revealed. In Ea.Hy926 endothelial cells, a tendency towards an increase in the number of highly energetic mitochondria was revealed after incubation in a gas environment with argon: the Control group 1.28 (1.15; 1.32), the Argon group: 1.46 (1.24; 1.77).

[0030] Thus, it has been established that exposure to an argon-oxygen mixture leads to a more than twofold increase in lysosomal activity, which, in turn, is accompanied by an increase in the number of mitochondria with an increased transmembrane potential in cells.

[0031] The endothelial preconditioning method has high practical significance, since clinicians can use it in their work to correct endothelial dysfunction in critically ill patients.