Activated Endothelium Changes The Activity Of Multipotent Mesenchymal Stromal Cells During Physiological Hypoxia Or Short Hypoxic Stress In Vitro

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Abstract

Multipotent mesenchymal stromal cells (MSCs) are used for supplemental therapy of ischemic and inflammatory diseases. After systemic administration, transmigration of MSCs to the target tissue is accompanied by interaction with activated endothelial cells (ECs) at the site of injury. In this study, we investigated the influence of TNF-α-activated ECs on the functions of MSCs under different levels of hypoxia. For this purpose, MSCs and TNF-α activated ECs were cocultured in a direct cell-to-cell setting for a short period of time. MSCs retained their stromal phenotype and multilineage differentiation potential after interaction with activated ECs. At the same time, changes in molecules involved in MSC-cell and MSC-extracellular matrix interaction were detected. The paracrine activity of MSCs and activated ECs after interaction was demonstrated by both upregulated transcription and increased levels of pleiotropic IL-6 and IL-8. Proteases/antiproteases profiles were also altered after interaction. These data suggest that short-term interaction of MSCs with activated ECs may play an important role in tissue repair and remodeling processes. In particular, it may promote the migratory phenotype of MSCs. In comparison to physiological hypoxia – 5% O2, acute hypoxic stress (0.1% O2, 24 h) attenuated the stimulatory effects of ECs on MSCs.

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About the authors

O. V. Zhidkova

Institute of Biomedical Problems, Russian Academy of Sciences

Author for correspondence.
Email: flain-fish@yandex.ru
Russian Federation, Moscow

E. R. Andreeva

Institute of Biomedical Problems, Russian Academy of Sciences

Email: andreeva1564@gmail.com
Russian Federation, Moscow

L. B. Buravkova

Institute of Biomedical Problems, Russian Academy of Sciences

Email: buravkova@imbp.ru
Russian Federation, Moscow

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2. Fig. 1. Experimental scheme for evaluating the effects of interaction between mesenchymal stromal and endothelial cells.

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3. Fig. 2. Characteristics of cultured MSCs with different O2 content. (a) – representative micrography of cultured MSCs at 5% O2, scale segment of 100 microns; (b) – characteristics of viability of MSCs using fluorescent dyes annexin/propidium iodide (Ann/PI); representative histograms are presented (lower left quadrant – population living cells, upper left quadrant – cells in a state of early apoptosis, upper right quadrant – cells in a state of late apoptosis, lower right quadrant – cells in a state of necrosis); (c) – production of ROS; (d) – expression of differentiation regulatory genes in MSCs, the multiplicity of changes in gene expression compared to the monoculture of MSCs at 5% O2 is presented; (e) – production of IL-6, IL-8 in MSCs. The graphs show the median, interquartile range, maximum and minimum values, n ≥ 4, * – p < 0.05 compared with the monoculture of MSC cultivated at 5% O2.

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4. Fig. 3. Effect of TNF-α activation and O2 deprivation on EC at different O2 levels. (a) – intact and TNF-α activated EC after short–term cultivation at 5% O2, light microscopy, representative micrographs of EC at 5% O2, scale segment of 100 microns; (b) - viability of EC Representative histograms are presented (the lower left quadrant is a population of living cells, the upper left quadrant is cells in a state of early apoptosis, the upper right quadrant is cells in a state of late apoptosis, the lower right quadrant is cells in a state of necrosis); (c) – expression of adhesion molecules on EC at different O2 levels, representative histograms at 5% O2. ECs – intact EC, ECs TNF-α – activated EC, control cells stained with isotypic IgG1 antibodies.

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5. Fig. 4. Morphology and transcription profile of MSCs after interaction with EC activated TNF-α. (a) – type of co-culture after 24 hours at 5 and 0.1% O2. Representative images obtained by combining microphotographs taken in phase contrast and fluorescence mode are presented, endothelial cells are stained with fluorescent dye PKH27. The scale segment is 100 microns. The arrows indicate MSC; (b), (c) – expression of MSC differentiation genes after interaction with EC activated at a reduced O2 content. The graphs show the multiplicity of changes in gene expression in MSCs after co-cultivation compared with the monoculture of MSCs (dotted line) under the same conditions. The data are presented as median, interquartile range, maximum and minimum values, n ≥ 3. * – p < 0.05 compared with the monoculture of MSC cultivated at 5% O2. MSC+EC TNF-α –MSC after 24 hours of co-cultivation with activated TNF-α EC.

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6. Fig. 5. Expression of MSCs intercellular interaction molecules after co-cultivation with TNF-α activated EC. The graphs show the proportion of positively colored MSCs before and after interaction with activated EC (median, minimum and maximum values). In the same graphs, the size of the bubbles corresponds to the value of the median fluorescence intensity of the stained cells, n ≥ 3. * – p < 0.05 compared with the monoculture of MSC under the same cultivation conditions. 1 – MSK 5% O2, 2 – MSK after co–cultivation with EC 5% O2, 3 – MSK 0.1% O2, 4 - MSK after co-cultivation with EC 0.1% O2.

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7. Fig. 6. Changes in the paracrine activity of EC and MSC after interaction with a reduced O2 content. (a) – concentration of IL-6 in the medium from mono- and co–cultures of MSC and activated EC; (b) - concentration of IL-8 in the medium from mono- and co-cultures of MSC and activated EC; (c) – changes in the expression of IL6 and IL8 genes in MSCs; (d) – a change in the expression of IL6 and IL8 genes in the EC. The graphs show the multiplicity of differences in the expression of genes of interest in MSCs (c) and EC (d) after interaction compared with a monoculture of cells (dotted line) under the same cultivation conditions. The data are presented as median, interquartile range, maximum and minimum values, n = 3, * – p < 0.05 compared with the MSC monoculture under the same cultivation conditions. ** – p < 0.05 compared to the EC monoculture under the same cultivation conditions. MSC+EC TNF-α – MSC after interaction with activated EC. EC TNF-α+MSC – activated ECS after interaction with MSC.

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8. Fig. 7. Paracrine effect of EC on the mobility of MSCs. (a) – non-directional migration of MSCs in the "wound" model in an air-conditioned environment from activated ECS. Representative micrographs immediately after the application of the "wound" (0 h) and a day later (24 h); (b) – the area of the wound closure. On the graph, the closing area is represented in % relative to the original area of the wound; (c) – directed migration of MSCs through the transwell membrane in an air-conditioned environment from activated ECS. Representative micrographs taken after 24 hours of incubation of cells in the wells of tablets; (d) – the graph shows the proportion of cells that migrated in 24 hours (the ratio of cells that migrated in the conditioned environment from EC, compared with the number of cells that migrated in the growth medium). The data are presented as median, interquartile range, maximum and minimum values, n = 3. * – p < 0.05 compared with the number of cells migrated in the growth medium (Cntrl). CM 5% O2 – conditioned environment from activated EC,

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9. Fig. 8. Determination of the level of proteases in MSC lysates before and after 24 hours of co-cultivation with EC activated TNF-α. (a) – representative photographs of membranes; (b), (c) – the layout and names of detecting antibodies to various proteases.

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10. Fig. 9. Effects of hypoxic stress on the interaction of activated EC and MSCs.

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