Angiotensin-(1-7) counteracts Angiotensin II-induced senescence in telomerase-immortalized human mesenchymal stem cells

Abstract

Objective: Angiotensin-(1-7) (Ang-(1-7)) exerts protective and anti-inflammatory effects, whereas angiotensin II (AngII) promotes pro-inflammatory and pro-oxidative processes. Cellular senescence is an irreversible cell cycle arrest that can be induced in vitro by a variety of cellular stresses, including AngII “stress-induced premature senescence (SIPS)”. This study aimed to evaluate the potential inhibitory role of Ang-(1-7) in AngII-induced senescence in telomerase-immortalized human mesenchymal stem cells (hMSCs-telo1). Materials and Methods: Senescence was induced using AngII at various time points and concentrations. Based on preliminary optimization experiments, cells were treated with AngII (10-⁶ M) for 48 hours, followed by Ang-(1-7) treatment (10-⁶ M) for an additional 24 hours. Following treatments, cellular senescence and functional responses were assessed using proliferation analysis based on population doubling levels (PDLs), senescence associated-beta-galactosidase (SA-β-gal) staining, and colony forming unit (CFU) assays. Results: No marked morphological differences were observed among the experimental groups. However, Ang-(1-7) treatment significantly reduced the proportion of SA-β-gal-positive cells in AngII-induced senescent hMSCs-telo1, showing a 1.65-fold decrease. In parallel, Ang-(1-7) partially restored cell proliferation capacity. Conclusion: Ang-(1-7) attenuates AngII-induced cellular senescence and improves proliferative potential in hMSCs-telo1 cells, suggesting a modulatory role in stress-induced senescence and potential relevance for stem cell-based therapies.

Keywords

• Angiotensin-(1-7)
• Angiotensin II
• Cellular senescence
• Telomerase-immortalized human mesenchymal stem cells

References

1. Miao Z, Jin J, Chen L, et al. Isolation of mesenchymal stem cells from human placenta: comparison with human bone marrow mesenchymal stem cells. Cell Biol Int 2006;30: 681-7. doi: 10.1016/j.cellbi.2006.03.009.
2. Tsai MS, Lee JL, Chang YJ, Hwang SM. Isolation of human multipotent mesenchymal stem cells from second-trimester amniotic fluid using a novel two-stage culture protocol. Hum Reprod 2004;19:1450-6. doi: 10.1093/humrep/deh279.
3. Al-Azab M, Safi M, Idiiatullina E, Al-Shaebi F, Zaky MY. Aging of mesenchymal stem cell: machinery, markers, and strategies of fighting. Cell Mol Biol Lett 2022;27:69. doi: 10.1186/s11658-022-00366-0.
4. Serakinci N, Fahrioglu U, Christensen R. Mesenchymal stem cells, cancer challenges and new directions. Eur J Cancer 2014; 50:1522-30. doi: 10.1016/j.ejca.2014.02.011.
5. Serakinci N, Christensen R, Graakjaer J, et al. Ectopically hTERT expressing adult human mesenchymal stem cells are less radiosensitive than their telomerase negative counterpart. Exp Cell Res 2007;313:1056-67. doi: 10.1016/j. yexcr.2007.01.002.
6. Foreman KE, Tang J. Molecular mechanisms of replicative senescence in endothelial cells. Exp Gerontol 2003;38:1251-7. doi: 10.1016/j.exger.2003.09.005
7. Minamino T, Komuro I. Vascular cell senescence: contribution to atherosclerosis. Circ Res 2007;100:15-26. doi: 10.1161/01. RES.000.025.6837.40544.4a.
8. Lian XJ, Gallouzi IE. Oxidative stress increases the number of stress granules in senescent cells and triggers a rapid decrease in p21waf1/cip1 Translation. J Biol Chem 2009;284:8877-87. doi: 10.1074/jbc.M806372200.

9. Erusalimsky JD. Vascular endothelial senescence: from mechanisms to pathophysiology. J Appl Physiol (1985) 2009;106:326-32. doi: 10.1152/japplphysiol.91353.2008.
10. Touyz RM, Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res 2002;35:1001-15. doi: 10.1590/ s0100-879x2002000900001.
11. Benter IF, Diz DI, Ferrario CM. Cardiovascular actions of angiotensin(1-7). Peptides 1993;14:679-84. doi: 10.1016/0196- 9781(93)90097-z.
12. Benter IF, Ferrario CM, Morris M, Diz DI. Antihypertensive actions of angiotensin-(1-7) in spontaneously hypertensive rats. Am J Physiol 1995;269(1 Pt 2):H313-9. doi: 10.1152/ ajpheart.1995.269.1.H313.
13. Benter IF, Yousif MH, Dhaunsi GS, Kaur J, Chappell MC, Diz DI. Angiotensin-(1-7) prevents activation of NADPH oxidase and renal vascular dysfunction in diabetic hypertensive rats. Am J Nephrol 2008;28:25-33. doi: 10.1159/000108758.
14. Al-Maghrebi M, Benter IF, Diz DI. Endogenous angiotensin-(1-7) reduces cardiac ischemia-induced dysfunction in diabetic hypertensive rats. Pharmacol Res 2009. 59: 263-8. doi: 10.1016/j.phrs.2008.12.008.
15. Dikalov SI, Nazarewicz RR. Angiotensin II-induced production of mitochondrial reactive oxygen species: potential mechanisms and relevance for cardiovascular disease. Antioxid Redox Signal 2013;19:1085-94. doi: 10.1089/ ars.2012.4604.
16. Chappell MC. Emerging evidence for a functional angiotensin-converting enzyme 2-angiotensin-(1- 7)-MAS receptor axis: more than regulation of blood pressure? Hypertension 2007;50:596-9. doi: 10.1161/ HYPERTENSIONAHA.106.076216.
17. Ferrario CM. Angiotensin-converting enzyme 2 and angiotensin-(1-7): an evolving story in cardiovascular regulation. Hypertension 2006;47:515-21. doi: 10.1161/01. 10.1161/01.HYP.0000196268.08909.fb.
18. El-Hashim AZ, Khajah MA, Babyson RS, et al. Ang-(1-7)/ MAS1 receptor axis inhibits allergic airway inflammation via blockade of Src-mediated EGFR transactivation in a murine model of asthma. PLoS One 2019;14:e0224163. doi: 10.1371/ journal.pone.0224163.
19. El-Hashim AZ, Renno WM, Raghupathy R, Abduo HT, Akhtar S, Benter IF. Angiotensin-(1-7) inhibits allergic inflammation, via the MAS1 receptor, through suppression of ERK1/2 – and NF-κB-dependent pathways. Br J Pharmacol 2012;166: 1964- 76. doi: 10.1111/j.1476-5381.2012.01905.x.
20. Renno WM, Al-Banaw AG, George P, Abu-Ghefreh AA, Akhtar S, Benter IF. Angiotensin-(1-7) via the mas receptor alleviates the diabetes-induced decrease in GFAP and GAP-43 immunoreactivity with concomitant reduction in the COX-2 in hippocampal formation: an immunohistochemical study. Cell Mol Neurobiol 2012; 32:1323-36. doi: 10.1007/s10571- 012-9858-7.
21. Liu Y, Jiao R, Ma ZG, et al. Sanguinarine inhibits angiotensin IIinduced apoptosis in H9c2 cardiac cells via restoring reactive oxygen species-mediated decreases in the mitochondrial membrane potential. Mol Med Rep 2015;12:3400-8. doi: 10.3892/mmr.2015.3841.
22. Zhao L, Li AQ, Zhou TF, Zhang MQ, Qin XM. Exendin-4 alleviates angiotensin II-induced senescence in vascular smooth muscle cells by inhibiting Rac1 activation via a cAMP/PKA-dependent pathway. Am J Physiol Cell Physiol 2014;307:C1130-41. doi: 10.1152/ajpcell.00151.2014.
23. Kim HJ, Ham SA, Kim MY, et al. PPARδ coordinates angiotensin II-induced senescence in vascular smooth muscle cells through PTEN-mediated inhibition of superoxide generation. J Biol Chem 2011;286:44585-93. doi: 10.1074/jbc. M111.222562.
24. Xiao X, Zhang C, Ma X, et al. Angiotensin-(1-7) counteracts angiotensin II-induced dysfunction in cerebral endothelial cells via modulating Nox2/ROS and PI3K/NO pathways. Exp Cell Res 2015;336:58-65. doi: 10.1016/j.yexcr.2015.06.010
25. Murugan D, Lau YS, Lau CW, Mustafa MR, Huang Y. Angiotensin 1-7 Protects against Angiotensin II-Induced Endoplasmic Reticulum Stress and Endothelial Dysfunction via Mas Receptor. PLoS One 2015;10:e0145413. doi: 10.1371/ journal.pone.0145413.
26. Durand MJ, Zinkevich NS, Riedel M, et al. Vascular Actions of Angiotensin 1-7 in the Human Microcirculation: Novel Role for Telomerase. Arterioscler Thromb Vasc Biol 2016;36:1254- 62. doi: 10.1161/ATVBAHA.116.307518.
27. Matsusaka T, Ichikawa I. Biological functions of angiotensin and its receptors. Annu Rev Physiol 1997;59:395- 412. doi: 10.1146/annurev.physiol.59.1.395.
28. Ruiz-Ortega M, Lorenzo O, Suzuki Y, Rupérez M, Egido J. Proinflammatory actions of angiotensins. Curr Opin Nephrol Hypertens 2001;10:321-9. doi: 10.1097/00041552-200105000- 00005.
29. Zhuo JL. Intracrine renin and angiotensin II: a novel role in cardiovascular and renal cellular regulation. J Hypertens 2006;24:1017-20. doi: 10.1097/01.hjh.0000226188.90815.56.
30. Dhaunsi GS, Yousif MH, Akhtar S, Chappell MC, Diz DI, Benter IF. Angiotensin-(1-7) prevents diabetes-induced attenuation in PPAR-gamma and catalase activities. Eur J Pharmacol 2010;638:108-14. doi: 10.1016/j.ejphar.2010.04.030.
31. Moon JY, Tanimoto M, Gohda T, et al. Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-A(y)/ Ta mice. Am J Physiol Renal Physiol 2011;300:F1271-82. doi: 10.1152/ajprenal.00065.2010.
32. Jin HR, Kim WJ, Song JS, et al. Functional and morphologic characterizations of the diabetic mouse corpus cavernosum: comparison of a multiple low-dose and a single high-dose streptozotocin protocols. J Sex Med 2009;6:3289-304. doi: 10.1111/j.1743-6109.2009.01464.x.
33. Kilarkaje N, Yousif MH, El-Hashim AZ, Makki B, Akhtar S, Benter IF. Role of angiotensin II and angiotensin-(1-7) in diabetes-induced oxidative DNA damage in the corpus cavernosum. Fertil Steril 2013;100:226-33. doi: 10.1016/j. fertnstert.2013.02.046.
34. Joshi S, Chittimalli K, Jahan J, Vasam G, Jarajapu YP. ACE2/ ACE imbalance and impaired vasoreparative functions of stem/progenitor cells in aging. Geroscience 2021; 43:1423-36. doi: 10.1007/s11357-020-00306-w.
35. Valero-Breton M, Tacchi F, Abrigo J, Simon F, Cabrera D, Cabello-Verrugio C. Angiotensin-(1-7) improves skeletal muscle regeneration. Eur J Transl Myol 2023; 33:12037. doi: 10.4081/ejtm.2023.12037.
36. Deng Y, Wang SY, Wang QG, et al. AVE 0991 suppresses astrocyte-mediated neuroinflammation of Alzheimer’s disease by enhancing autophagy. J Inflamm Res 2023;16:391-406. doi: 10.2147/JIR.S392599. PMID: 36755969
37. Liu M, Shi P, Sumners C. Direct anti-inflammatory effects of angiotensin-(1-7) on microglia. J Neurochem 2016;136:163- 71. doi: 10.1111/jnc.13386.
38. Woo KM, Guo Y, Mehrabian Z, Walther T, Miller NR, Bernstein SL. Angiotensin-(1-7) provides potent long-term neurorepair/ neuroregeneration in a rodent white matter stroke model: Nonarteritic ischemic optic neuropathy (rNAION). Cells 2025;14:. doi: 10.3390/cells14040289.
39. Han Z, Wang K, Ding S, Zhang M. Cross-talk of inflammation and cellular senescence: a new insight into the occurrence and progression of osteoarthritis. Bone Res 2024;12: 69. doi: 10.1038/s41413-024-00375-z.
40. Ahmed S, Passos JF, Birket MJ, et al. Telomerase does not counteract telomere shortening but protects mitochondrial function under oxidative stress. J Cell Sci 2008; 121(Pt 7):1046-53. doi: 10.1242/jcs.019372.
41. Santos JH, Meyer JN, Van Houten B. Mitochondrial localization of telomerase as a determinant for hydrogen peroxide-induced mitochondrial DNA damage and apoptosis. Hum Mol Genet 2006;15:1757-68. doi: 10.1093/hmg/ddl098.
42. Minamino T, Mitsialis SA, Kourembanas S. Hypoxia extends the life span of vascular smooth muscle cells through telomerase activation. Mol Cell Biol 2001;21:3336-42. doi: 10.1128/MCB.21.10.3336-3342.2001.
43. Sharma NK, Reyes A, Green P, et al. Human telomerase acts as a hTR-independent reverse transcriptase in mitochondria. Nucleic Acids Res 2012;40:712-25. doi: 10.1093/nar/gkr758.
44. Sampaio WO, Souza dos Santos RA, Faria-Silva R, da Mata Machado LT, Schiffrin EL, Touyz RM. Angiotensin-(1-7) through receptor Mas mediates endothelial nitric oxide synthase activation via Akt-dependent pathways. Hypertension 2007;49:185-92. doi: 10.1161/01.HYP.0000251865.35728.2f.
45. Romero A, Dongil P, Valencia I, et al. Pharmacological blockade of NLRP3 inflammasome/IL-1β-Positive loop mitigates endothelial cell senescence and dysfunction. Aging Dis 2022;13:284-97. doi: 10.14336/AD.2021.0617.
46. Valencia S, Shamoon L, Romero A, De la Cuesta F, Sánchez- Ferrer CF, Peiró C. Angiotensin-(1-7), a protective peptide against vascular aging. Peptides 2022;152: 170775. doi: 10.1016/j.peptides.2022.170775.