The sensitivity of multipotent mesenchymal stromal cells to short-term hypoxic stress in vitro depends on the efficiency of homotypic communication through gap junctions

Gap junctions (GJ) provide metabolic cooperation between cells through the direct exchange of cytoplasmic components. We analyzed the effect of short-term hypoxic stress on the efficiency of communication through the GJs in cultured multipotent mesenchymal stromal cells (MSCs) and characterized the sensitivity of MSCs to short-term hypoxic stress depending on the GJ function. Mitotically inactive MSCs were used in the experiments, in which the GJs were blocked with a specific inhibitor – carbenoxolone. The MSCs were continuously cultured at 20% O2. Further, MSCs with blocked and working GJs were subjected to hypoxic stress (0.1%, 24 hours). The efficiency of GJ communication was attenuated under hypoxic stress. The combined action of GJ inhibition and hypoxic stress was accompanied by an increase in ROS level as compared to the MSCs after hypoxic stress only. MSCs with blocked GJs were less sensitive to short-term hypoxic stress in comparison with MSCs integrated into the common network through working GJs. It was manifested in attenuation of hypoxia-induced angiogenic activity of MSCs. The angiogenic effects of conditioned medium from the MSCs with blocked GJs were almost twice less, which seems to be related to differences in the angiogenic mediators’ profiles: VEGF level decreased and FGF-2 level increased, while the monocyte chemoattractant protein 3 (MCP-3) level was unchanged. Thus, a decrease in the efficiency of direct MSCs- MSCs communication had a negative effect on mostly requested MSCs activity – the ability to induce angiogenesis. We conclude that blocking of GJ communication in MSCs is a negative event that impairs the coordination of MSCs’ response to the microenvironmental factors, in particular hypoxic stress, and reduces their functional plasticity.

1. Андреева Е.Р., Буравкова Л.Б. Паракринная активность мультипотентных мезенхимальных стромальных клеток и ее особенности в условиях гипоксии. Физиол. чел. 2013;39(3):104–113.

2. Murray I.R., Péault B. Q&A: mesenchymal stem cells—where do they come from and is it important? BMC Biol. 2015;13:99.

3. Caplan A.I. Mesenchymal stem cells: time to change the name! Stem Cells Transl. Med. 2017;6(6):1445–1451.

4. Tan L., Liu X., Dou H., Hou Y. Characteristics and regulation of mesenchymal stem cell plasticity by the microenvironment–specific factors involved in the regulation of MSC plasticity. Genes Dis. 2020;9(2):296–309.

5. Буравкова Л.Б., Андреева Е.Р., Григорьев А.И. Роль кислорода как физиологического фактора в проявлении функциональных свойств мультипотентных мезенхимальных стромальных клеток человека. Физиол. чел. 2012;38(4):121–130.

6. Buravkova L.B., Andreeva E.R., Gogvadze V., Zhivotovsky B. Mesenchymal stem cells and hypoxia: where are we? Mitochondrion. 2014;19(Part A):105–112.

7. Pulido-Escribano V., Torrecillas-Baena B., Camacho-Cardenosa M., Dorado G., Gálvez-Moreno M.Á., Casado-Díaz A. Role of hypoxia preconditioning in therapeutic potential of mesenchymal stem-cell-derived extracellular vesicles. World J. Stem Cells. 2022;14(7):453–472.

8. Antebi B., Rodriguez L.A., Walker K.P., Asher A.M., Kamucheka R.M., Alvarado L., Mohammadipoor A., Cancio L.C. Short-term physiological hypoxia potentiates the therapeutic function of mesenchymal stem cells. Stem Cell Res. Ther. 2018;9(1):265.

9. Ishiuchi N., Nakashima A., Doi S., Yoshida K., Maeda S., Kanai R., Yamada Y., Ike T., Doi T., Kato Y., Masaki T. Hypoxia-preconditioned mesenchymal stem cells prevent renal fibrosis and inflammation in ischemia reperfusion rats. Stem Cell Res Ther. 2020;11(1):130.

10. Udartseva O.O., Lobanova M.V., Andreeva E.R., Buravkov S.V., Ogneva I.V., Buravkova L.B. Acute hypoxic stress affects migration machinery of tissue O 2-adapted adipose stromal cells. Stem Cells Int. 2016;2016:7260562.

11. Ездакова М.И., Матвеева Д.К., Андреева Е.Р. Гомотипическая регуляция функциональной активности мультипотентных мезенхимных стромальных клеток: роль щелевых контактов. Цитология. 2022;64(6):523–533.

12. Dorshkind K., Green L., Godwin A., Fletcher W.H. Connexin-43-type gap junctions mediate communication between bone marrow stromal cells. Blood. 1993;82(1):38–45.

13. Chanson M., Derouette J.P., Roth I., Foglia B., Scerri I., Dudez T., Kwak B.R. Gap junctional communication in tissue inflammation and repair. Biochim. Biophys. Acta. 2005;1711(2):197–207.

14. Danon A., Zeevi-Levin N., Pinkovich D.Y., Michaeli T., Berkovich A., Flugelman M., Eldar Y.C., Rosen M.R., Binah O. Hypoxia causes connexin 43 internalization in neonatal rat ventricular myocytes. Gen. Physiol. Biophys. 2010;29(3):222–233.

15. Wu X., Huang W., Luo G., Alain L.A. Hypoxia induces connexin 43 dysregulation by modulating matrix metalloproteinases via MAPK signaling. Mol. Cell. Biochem. 2013;384(1–2):155–162.

16. McNair A.J., Wilson K.S., Martin P.E., Welsh D.J., Dempsie Y. Connexin 43 plays a role in proliferation and migration of pulmonary arterial fibroblasts in response to hypoxia. Pulm. Circ. 2020;10(3):2045894020937134.

17. Glass B.J., Hu R.G., Phillips A.R., Becker D.L. The action of mimetic peptides on connexins protects fibroblasts from the negative effects of ischemia reperfusion. Biol. Open. 2015;4(11):1473–1480.

18. Ездакова М.И., Зорникова К.В., Буравков С.В., Андреева Е.Р. Функциональная активность непролиферирующих мезенхимных стромальных клеток, культивируемых в различной плотности. Клеточные технол. биол. мед. 2020(4):247–254.

19. Talbot J., Brion R., Lamora A., Mullard M., Morice S., Heymann D., Verrecchia F. Connexin43 intercellular communication drives the early differentiation of human bone marrow stromal cells into osteoblasts. J. Cell. Physiol. 2018;233(2):946–957.

20. Udartseva O.O., Zhidkova O.V., Ezdakova M.I., Ogneva I.V., Andreeva E.R., Buravkova L.B., Gollnick S.O. Low-dose photodynamic therapy promotes angiogenic potential and increases immunogenicity of human mesenchymal stromal cells. J. Photochem. Photobiol. B. 2019;199:111596.

21. Andreeva E., Andrianova I., Rylova J., Gornostaeva A., Bobyleva P., Buravkova L. Proinflammatory interleukins’ production by adipose tissue-derived mesenchymal stromal cells: the impact of cell culture conditions and cellto-cell interaction. Cell Biochem. Funct. 2015;33(6):386–393.

22. Wiesner M., Berberich O., Hoefner C., Blunk T., Bauer-Kreisel P. Gap junctional intercellular communication in adipose-derived stromal/stem cells is cell densitydependent and positively impacts adipogenic differentiation. J. Cell. Physiol. 2018;233(4):3315–3329.

23. Paquet J., Deschepper M., Moya A., LogeartAvramoglou D., Boisson-Vidal C., Petite H. Oxygen tension regulates human mesenchymal stem cell paracrine functions. Stem Cells Transl. Med. 2015;4(7):809–821.

24. Fuhrmann D.C., Brüne B. Mitochondrial composition and function under the control of hypoxia. Redox Biol. 2017;12:208–215.

25. Presley A.D., Fuller K.M., Arriaga E.A. MitoTracker Green labeling of mitochondrial proteins and their subsequent analysis by capillary electrophoresis with laser-induced fluorescence detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2003;793(1):141–150.

26. Cottet-Rousselle C., Ronot X., Leverve X., Mayol J.F. Cytometric assessment of mitochondria using fluorescent probes. Cytometry A. 2011;79(6):405–425.

27. Ездакова М.И., Матвеева Д.К., Буравков С.В., Андреева Е.Р. Роль щелевых контактов во взаимодействии эндотелиальных и стромальных клеток. Физиол. чел. 2021;47(3):124–136.

28. Zhu Y. Gap junction-dependent and-independent functions of Connexin43 in biology. Biology (Basel). 2022;11(2):283.

29. Zamorano M., Castillo R.L., Beltran J.F., Herrera L., Farias J.A., Antileo C., Aguilar-Gallardo C., Pessoa A., Calle Y., Farias J.G. Tackling ischemic reperfusion injury with the aid of stem cells and tissue engineering. Front. Physiol. 2021;12:705256.

30. Yang Y., Lee E.H., Yang Z. Hypoxia-conditioned mesenchymal stem cells in tissue regeneration application. Tissue Eng. Part B Rev. 2022;28(5):966–977.

31. Blebea J., Vu J.H., Assadnia S., McLaughlin P.J., Atnip R.G., Zagon I.S. Differential effects of vascular growth factors on arterial and venous angiogenesis. J. Vasc. Surg. 2002;35(3):532–538.

32. Yu J., Wu J., Bagchi I.C., Bagchi M.K., Sidell N., Taylor R.N. Disruption of gap junctions reduces biomarkers of decidualization and angiogenesis and increases inflammatory mediators in human endometrial stromal cell cultures. Mol. Cell. Endocrinol. 2011;344(1–2):25–34.

33. Suarez S., Ballmer-Hofer K. VEGF transiently disrupts gap junctional communication in endothelial cells. J. Cell Sci. 2001;114(Part 6):1229–1235.

34. Muto T., Tien T., Kim D., Sarthy V.P., Roy S. High glucose alters Cx43 expression and gap junction intercellular communication in retinal Müller cells: promotes Müller cell and pericyte apoptosis. Invest. Ophthalmol. Vis. Sci. 2014;55(7):4327–4337.

35. Duffy H.S., John G.R., Lee S.C., Brosnan C.F., Spray D.C. Reciprocal regulation of the junctional proteins claudin-1 and connexin43 by interleukin-1β in primary human fetal astrocytes. J. Neurosci. 2000;20(23):RC114.