The stem and progenitor cells and the functional activity of liver from age-different Wistar rats

The liver has a big potential for self-healing, but the activity of regeneration decreases with age. Changes are occurring, including in the functional activity of various liver cell populations, the study of the characteristics of which can become the basis for the development of new therapeutic approaches to the liver diseases treatment at older people. The aim of this research was to study the level of stem and progenitor cells and the functional activity of the healthy liver from age- different rats. Material and methods. Experiments were carried out on Wistar rats aged 6 and 12 months. Ultrasound and histological examination of the liver from rats was used to assess morphological changes. The lipid profile of blood serum was evaluated by biochemical methods. Cytometric methods were used to study the surface and intracellular antigens of stem and progenitor cells isolated from the bone marrow, arterial blood and liver of rats. Results and discussion. In 12-month-old male Wistar rats, compared with 6-month-old rats, excessive formation of extracellular matrix components, disruption of tissue architecture, development of portal hypertension, as well as an increase in the concentration of cholesterol, triglycerides, high- and low-density lipoproteins were revealed. We identified age- related differences in the content of hematopoietic and mesenchymal stem cells, epithelial cells (CD45–CD326+) in the bone marrow, blood and liver of rats. In the liver parenchyma, the populations of hepatocyte precursors (CD45– CD326+CD133+), oval cells (CD45–CD326+CD133+CD90+). At the same time, the level of all cell populations in the liver parenchyma of rats expressing the intracellular marker Sox9 was higher in one-year-old animals compared to younger ones, regardless of the cell phenotype. Conclusions. In the liver of 12-month-old rats, compared to 6-month-old rats, the number of cells expressing Sox9, lymphocytes with an inflammatory phenotype increases, the number of stem cells and various populations of epithelial and endothelial cells decreases, which leads to a decrease in the regenerative capacity of the liver, disruption of the tissue architecture of the organ and changes in lipid metabolism. These changes largely determine the increased susceptibility with age to the development of chronic liver diseases.

1. Fraser H.C., Kuan V., Johnen R., Zwierzyna M., Hingorani A.D., Beyer A., Partridge L. Biological mechanisms of aging predict age-related disease cooccurrence in patients. Aging Cell. 2022;21(4):e13524. doi: 10.1111/acel.13524

2. Radonjić T., Dukić M., Jovanović I., Zdravković M., Mandić O., Popadić V., Popović M., Nikolić N., Klašnja S., Divac A., Todorović Z., Branković M. Aging of liver in its different diseases. Int. J. Mol. Sci. 2022;23(21):13085. doi: 10.3390/ijms232113085

3. Moon C.M., Kim S.K., Heo S.H., Shin S.S. Hemodynamic changes in the portal vein with age: evaluation using four-dimensional flow MRI. Sci. Rep. 2023;13(1):7397. doi: 10.1038/s41598-023-34522-z

4. Helmersson-Karlqvist J., Ridefelt P., Lind L., Larsson A. Reference values for 34 frequently used laboratory tests in 80-year-old men and women. Maturitas. 2016;92:97–101. doi: 10.1016/j.maturitas.2016.07.015

5. Lasry A., Ben-Neriah Y. Senescence-associated inflammatory responses: aging and cancer perspectives. Trends Immunol. 2015;36(4):217–228. doi: 10.1016/j.it.2015.02.009

6. Zhao Y., Yang Y., Li Q., Li J. Understanding the unique microenvironment in the aging liver. Front. Med. (Lausanne). 2022;9:842024. doi: 10.3389/fmed.2022.842024

7. Antwi M.B., Dumitriu G., Simón-Santamaria J., Romano J.S., Li R., Smedsrød B., Vik A., Eskild W., Sørensen K.K. Liver sinusoidal endothelial cells show reduced scavenger function and downregulation of Fc gamma receptor IIb, yet maintain a preserved fenestration in the Glmpgt/gt mouse model of slowly progressing liver fibrosis. PLoS One. 2023;18(11):e0293526. doi: 10.1371/journal.pone.0293526

8. Wang W., Xu K., Shang M., Li X., Tong X., Liu Z., Zhou L., Zheng S. The biological mechanism and emerging therapeutic interventions of liver aging. Int. J. Biol. Sci. 2024;20(1):280–295. doi: 10.7150/ijbs.87679

9. Tanimizu N., Ichinohe N., Ishii M., Kino J., Mizuguchi T., Hirata K., Mitaka T. Liver progenitors isolated from adult healthy mouse liver efficiently differentiate to functional hepatocytes in vitro and repopulate liver tissue. Stem Cells. 2016;34(12):2889–2901. doi: 10.1002/stem.2457/

10. Yamazaki T., Enosawa S., Kasahara M., Fukuda A., Sakamoto S., Shigeta T., Nakazawa A., Tokiwa T. Isolation of hepatic progenitor cells from human liver with cirrhosis secondary to biliary atresia using EpCAM or Thy-1 markers. Cell. Med. 2012;15(1– 3):121–126. doi: 10.3727/215517912X639441

11. Fernández A., Mazuecos L., Pintado C., Rubio B., López V., de Solís A.J., Rodríguez M., Andrés A., Gallardo N. Effects of moderate chronic food restriction on the development of postprandial dyslipidemia with ageing. Nutrients. 2019;11(8):1865. doi: 10.3390/nu11081865

12. Guillén J., Robinson S., Decelle T., Exner C., van Vlissingen M.F. Approaches to animal research project evaluation in Europe after implementation of Directive 2010/63/EU. Lab. Anim. (NY). 2015;44(1):23–31. doi: 10.1038/laban.604

13. Lessa A.S., Paredes B.D., Dias J.V., Carvalho A.B., Quintanilha L.F., Takiya C.M., Tura B.R., Rezende G.F., Campos de Carvalho A.C., Resende C.M., Goldenberg R.C. Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats. BMC Vet. Res. 2010;6:6. doi: 10.1186/1746-6148-6-6

14. Cardiff R.D., Miller C.H., Munn R.J. Manual hematoxylin and eosin staining of mouse tissue sections. Cold Spring Harb. Protoc. 2014;2014(6):655– 658. doi: 10.1101/pdb.prot073411

15. Pan E.S., Pakhomova A.V., Ermakova N.N., Afanas’ev S.A., Rebrova T.Y., Zhukova M.A., Sandrikina L.A., Putrova O.D., Kogai L.V., Pershina O.V., Dygai A.M., Skurikhin E.G. Age-related features of ketanserin effects in experimental liver cirrhosis. Bull. Exp. Biol. Med. 2022;174(2):205–209. doi: 10.1007/s10517-023-05674-9

16. Gholami Bahnemiri M., Mirabedini S., Mohammadi P., Barmaki H., Qaffaripour Z., Rezapour M., Alijanpour M. Determination of serum alkaline phosphatase reference in healthy children aged 1-18 years. Caspian J. Intern. Med. 2022;13(4):749–756. doi: 10.22088/cjim.13.4.749

17. Maeso-Díaz R., Ortega-Ribera M., Lafoz E., Lozano J.J., Baiges A., Francés R., Albillos A., Peralta C., García-Pagán J.C., Bosch J., Cogger V.C., Gra-cia-Sancho J. Aging influences hepatic microvascular biology and liver fibrosis in advanced chronic liver disease. Aging Dis. 2019;10(4):684–698. doi: 10.14336/AD.2019.0127

18. Pinto C., Ninfole E., Benedetti A., Maroni L., Marzioni M. Aging-related molecular pathways in chronic cholestatic conditions. Front. Med. (Lausanne). 2020;6:332. doi: 10.3389/fmed.2019.00332

19. Skurikhin E.G., Afanas’ev S.A., Zhukova M.A., Rebrova T.Y., Muslimova E.F., Pan E.S., Ermakova N.N., Pershina O.V., Pakhomova A.V., Putrova O.D., … Dygai A.M. Age-related features of the viscosity of plasma and mitochondrial membranes of hepatocytes in liver cirrhosis. Bull. Exp. Biol. Med. 2021;171(6):707– 712. doi: 10.1007/s10517-021-05300-6

20. Asumda F.Z., Chase P.B. Age-related changes in rat bone-marrow mesenchymal stem cell plasticity. BMC Cell Biology. 2011;12:44. doi: 10.1186/1471-2121-12-44

21. Kaur S., Sehgal R., Shastr S.M., McCaughan G., McGuire H.M., Fazekas St. de Groth B., Sarin S., Trehanpati N., Seth D. Circulating endothelial progenitor cells present an inflammatory phenotype and function in patients with alcoholic liver cirrhosis. Front. Physiol. 2018;9:556. doi: 10.3389/fphys.2018.00556

22. Lazarevic I., Soldati S., Mapunda J.A., Rudolph H., Rosito M., de Oliveira A.C., Enzmann G., Nishihara H., Ishikawa H., Tenenbaum T., Schroten H., Engelhardt B. The choroid plexus acts as an immune cell reservoir and brain entry site in experimental autoimmune encephalomyelitis. Fluids Barriers CNS. 2023;20(1):39. doi: 10.1186/s12987-023-00441-4

23. Qian L., Zhang H., Gu Y., Li D., He S., Wang H., Cheng Y., Yang W., Yu H., Zhao X., … Zhang Y. Reduced production of laminin by hepatic stellate cells contributes to impairment in oval cell response to liver injury in aged mice. Aging (Albany NY). 2018;10(12):3713–3735. doi: 10.18632/aging.101665

24. Athwal V.S., Pritchett J., Martin K., Llewellyn J., Scott J., Harvey E., Zaitoun A.M., Mullan A.F., Zeef L.A.H., Friedman S.L., … Piper Hanley K. SOX9 regulated matrix proteins are increased in patients serum and correlate with severity of liver fibrosis. Sci. Rep. 2019;9(1):11547. doi: 10.1038/s41598-019-47715-2

25. Ming Z., Vining B., Bagheri-Fam S., Harley V. SOX9 in organogenesis: shared and unique transcriptional functions. Cell. Mol. Life Sci. 2022;79(10):522. doi: 10.1007/s00018-022-04543-4

26. Marcos R., Lopes C., Malhão F., Correia-Gomes C., Fonseca S., Lima M., Gebhardt R., Rocha E. Stereological assessment of sexual dimorphism in the rat liver reveals differences in hepatocytes and Kupffer cells but not hepatic stellate cells. J. Anat. 2016;228(6):996–1005. doi: 10.1111/joa.12448

27. DeLeve L.D. Liver sinusoidal endothelial cells in hepatic fibrosis. Hepatology. 2015;61(5):1740–1746. doi: 10.1002/hep.27376

28. Maeso-Díaz R., Ortega-Ribera M., Fernández-Iglesias A., Hide D., Muñoz L., Hessheimer A.J., Vila S., Francés R., Fondevila C., Albillos A., … Gracia-Sancho J. Effects of aging on liver microcirculatory function and sinusoidal phenotype. Aging Cell. 2018;17(6):e12829. doi: 10.1111/acel.12829