Exosomes from adipose-derived stem cells promote angiogenesis and reduce necrotic grade in hindlimb ischemia mouse models

Document Type : Original Article

Authors

1 Stem Cell Institute, University of Science Ho Chi Minh City, Viet Nam

2 Viet Nam National University, Ho Chi Minh City, Viet Nam

3 Laboratory of Stem Cell Research and Application, University of Science Ho Chi Minh City, Viet Nam

Abstract

Objective(s): Acute hindlimb ischemia is a peripheral arterial disease that severely affects the patient’s health. Injection of stem cells-derived exosomes that promote angiogenesis is a promising therapeutic strategy to increase perfusion and repair ischemic tissues. This study aimed to evaluate the efficacy of adipose stem cell-derived exosomes injection (ADSC-Exos) in treating acute mouse hindlimb ischemia.
Materials and Methods: ADSC-Exos were collected via ultracentrifugation. Exosome-specific markers were analyzed via flow cytometry. The morphology of exosomes was detected by TEM. A dose of 100 ug exosomes/100 ul PBS was locally injected into acute mice ischemic hindlimb. The treatment efficacy was evaluated based on the oxygen saturation level, limb function, new blood vessel formation, muscle structure recovery, and limb necrosis grade. 
Results: ADSC-exosomes expressed high positivity for markers CD9 (76.0%), CD63 (91.2%), and CD81 (99.6%), and have a cup shape. After being injected into the muscle, in the treatment group, many small and short blood vessels formed around the first ligation and grew down toward the second ligation. The SpO2 level, reperfusion, and recovery of the limb function are more positively improved in the treatment group. On day 28, the muscle’s histological structure in the treatment group is similar to normal tissue. Approximately 33.33% of the mice had grade I and II lesions and there were no grade III and IV observed in the treatment group. Meanwhile, in the placebo group, 60% had grade I to IV lesions. 
Conclusion: ADSC-Exos showed the ability to stimulate angiogenesis and significantly reduce the rate of limb necrosis.

Keywords


1. Olinic DM, Stanek A, Tătaru DA, Homorodean C, Olinic M. Acute limb ischemia: An update on diagnosis and management. J Clin Med 2019; 8:1215.
2. Martyn Knowles CHT. Epidemiology of acute critical limb ischemia.  Critical Limb Ischemia: Springer, Cham.; 2017. p. 1-7.
3. Lawall H, Huppert P, Rümenapf G. S3-Leitlinie zur diagnostik, therapie und nachsorge der peripheren arteriellen verschlusskrankheit. Vasa 2016; 45:11-82.
4. Fluck F, Augustin AM, Bley T, Kickuth R. Current treatment options in acute limb ischemia. Rofo 2020; 192:319-326.
5. Baril DT, Patel VI, Judelson DR, Goodney PP, McPhee JT, Hevelone ND, et al. Outcomes of lower extremity bypass performed for acute limb ischemia. J Vasc Surg 2013; 58:949-956.
6. Sprengers R, Moll F, Verhaar M. Stem cell therapy in PAD. Eur J Vasc Endovasc Surg 2010; 39:S38-S43.
7. Norgren L, Hiatt WR, Dormandy JA, Nehler MR, Harris KA, Fowkes FG. Inter-society consensus for the management of peripheral arterial disease (TASC II). J Vasc Surg 2007; 45:S5-67.
8. Bian X, Ma K, Zhang C, Fu X. Therapeutic angiogenesis using stem cell-derived extracellular vesicles: an emerging approach for treatment of ischemic diseases. Stem Cell Res Ther 2019; 10:158.
9. Vu NB, Phi LT, Dao TT-T, Le HT-N, Pham PV. Adipose derived stem cell transplantation is better than bone marrow mesenchymal stem cell treating hindlimb ischemia in mice in treating hindlimb ischemia in mice. Biomed Res Ther 2016; 3:46.
10. Vu NB, Trinh VN-L, Phi LT, Phan NK, Pham PV. Human Menstrual Blood-Derived Stem Cell Transplantation for Acute Hind Limb Ischemia Treatment in Mouse Models. Regenerative Med 2015;1:205-215.
11. Zhang B, Adesanya TM, Zhang L, Xie N, Chen Z, Fu M, et al. Delivery of placenta-derived mesenchymal stem cells ameliorates ischemia induced limb injury by immunomodulation. Cell Physiol Biochem 2014; 2014:1998-2006.
12. Kocan B, Maziarz A, Tabarkiewicz J, Ochiya T, Banaś-Ząbczyk A. Trophic activity and phenotype of adipose tissue-derived mesenchymal stem cells as a background of their regenerative potential. Stem Cells Int 2017; 2017:1653254-1653254.
13. Beltrán-Camacho L, Rojas-Torres M, Durán-Ruiz MC. Current status of angiogenic cell therapy and related strategies applied in critical limb ischemia. Int J Mol Sci 2021; 22:2335.
14. Xia J, Minamino S, Kuwabara K, Arai S. Stem cell secretome as a new booster for regenerative medicine. Biosci Trends 2019; 13:299-307.
15. Van Der Pol E, Boing AN, Harrison P, Sturk A, Nieuwland R. Classification, functions, and clinical relevance of extracellular vesicles. Pharmacol Rev 2012; 64:676-705.
16. Record M, Carayon K, Poirot M, Silvente-Poirot S. Exosomes as new vesicular lipid transporters involved in cell-cell communication and various pathophysiologies. Biochim Biophys Acta 2014; 1841:108-120.
17. Lai RC, Yeo RWY, Lim SK. Mesenchymal stem cell exosomes. Semin Cell Dev Biol 2015; 40:82-88.
18. Pham P. Concise review: Extracellular vesicles from mesenchymal stem cells as cellular therapy. Biomed Res Ther 2017; 4:1562-1573.
19. Li X, Chen C, Wei L, Li Q, Niu X, Xu Y, et al. Exosomes derived from endothelial progenitor cells attenuate vascular repair and accelerate reendothelialization by enhancing endothelial function. Cytotherapy 2016; 18:253-262.
20. Gong M, Yu B, Wang J, Wang Y, Liu M, Paul C, et al. Mesenchymal stem cells release exosomes that transfer miRNAs to endothelial cells and promote angiogenesis. Oncotarget 2017; 8:45200-45212.
21. Liang X, Zhang L, Wang S, Han Q, Zhao RC. Exosomes secreted by mesenchymal stem cells promote endothelial cell angiogenesis by transferring miR-125a. J Cell Sci 2016; 129:2182-2189.
22. Du W, Zhang K, Zhang S, Wang R, Nie Y, Tao H, et al. Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer. Biomaterials 2017; 133:70-81.
23. Eirin A, Riester SM, Zhu X-Y, Tang H, Evans JM, O’Brien D, et al. MicroRNA and mRNA cargo of extracellular vesicles from porcine adipose tissue-derived mesenchymal stem cells. Gene 2014; 551:55-64.
24. Pu CM, Liu CW, Liang CJ, Yen YH, Chen SH, Jiang-Shieh YF, et al. Adipose-derived stem cells protect skin flaps against ischemia/reperfusion injury via IL-6 expression. J Invest Dermatol 2017; 137:1353-1362.
25. Hu G-w, Li Q, Niu X, Hu B, Liu J, Zhou S-m, et al. Exosomes secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells attenuate limb ischemia by promoting angiogenesis in mice. Stem Cell Res Ther 2015; 6:10-10.
26. Komaki M, Numata Y, Morioka C, Honda I, Tooi M, Yokoyama N, et al. Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis. Stem Cell Res Ther 2017; 8:219.
27. Zhang B, Wu X, Zhang X, Sun Y, Yan Y, Shi H, et al. Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4/β-catenin pathway. Stem Cells Transl Med 2015; 4:513-522.
28. Goto T, Fukuyama N, Aki A, Kanabuchi K, Kimura K, Taira H, et al. Search for appropriate experimental methods to create stable hind-limb ischemia in mouse. Tokai J Exp Clin Med 2006; 31:128-132.
29. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006; 8:315-317.
30. Stipp CS, Kolesnikova TV, Hemler ME. Functional domains in tetraspanin proteins. Trends Biochem Sci 2003; 28:106-112.
31. Xiong M, Zhang Q, Hu W, Zhao C, Lv W, Yi Y, et al. Exosomes from adipose-derived stem cells: The emerging roles and applications in tissue regeneration of plastic and cosmetic surgery. Front Cell Dev Biol 2020; 8:574223.
32. Jin J, Shi Y, Gong J, Zhao L, Li Y, He Q, et al. Exosome secreted from adipose-derived stem cells attenuates diabetic nephropathy by promoting autophagy flux and inhibiting apoptosis in podocyte. Stem Cell Res Ther 2019; 10:95.
33. Huang B, Lu J, Ding C, Zou Q, Wang W, Li H. Exosomes derived from human adipose mesenchymal stem cells improve ovary function of premature ovarian insufficiency by targeting SMAD. Stem Cell Res Ther 2018; 9:216-216.
34. Jankovičová J, Sečová P, Michalková K, Antalíková J. Tetraspanins, more than markers of extracellular vesicles in reproduction. Int J Mol Sci 2020; 21:7568.
35. Mitchell R, Mellows B, Sheard J, Antonioli M, Kretz O, Chambers D, et al. Secretome of adipose-derived mesenchymal stem cells promotes skeletal muscle regeneration through synergistic action of extracellular vesicle cargo and soluble proteins. Stem Cell Res Ther 2019; 10:116.
36. Barranco I, Padilla L, Parrilla I, Álvarez-Barrientos A, Pérez-Patiño C, Peña FJ, et al. Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles. Sci Rep 2019; 9:11584.
37. Aref Z, de Vries MR, Quax PHA. Variations in Surgical procedures for inducing hind limb ischemia in mice and the impact of these variations on neovascularization assessment. Int J Mol Sci 2019; 20:3704.
38. Faber JE, Chilian WM, Deindl E, van Royen N, Simons M. A brief etymology of the collateral circulation. Arterioscler Thromb Vasc Biol 2014; 34:1854-1859.
39. Simon F, Oberhuber A, Floros N, Busch A, Wagenhäuser MU, Schelzig H, et al. Acute limb ischemia-much more than just a lack of oxygen. Int J Mol Sci 2018; 19:374.
40. Ni J, Li H, Zhou Y, Gu B, Xu Y, Fu Q, et al. Therapeutic potential of human adipose-derived stem cell exosomes in stress urinary incontinence - an in vitro and in vivo study. Cell Physiol Biochem 2018; 48:1710-1722.
41. Murphy MB, Moncivais K, Caplan AI. Mesenchymal stem cells: Environmentally responsive therapeutics for regenerative medicine. Exp Mol Med 2013; 45:e54-e54.
42. Sawada K, Takedachi M, Yamamoto S, Morimoto C, Ozasa M, Iwayama T, et al. Trophic factors from adipose tissue-derived multi-lineage progenitor cells promote cytodifferentiation of periodontal ligament cells. Biochem Biophys Res Commun 2015; 464:299-305.
43. Procházka V, Jurčíková J, Laššák O, Vítková K, Pavliska L, Porubová L, et al. Therapeutic potential of adipose-derived therapeutic factor concentrate for treating critical limb ischemia. Cell Transplant 2016; 25:1623-1633.
44. Komaki M, Numata Y, Morioka C, Honda I, Tooi M, Yokoyama N, et al. Exosomes of human placenta-derived mesenchymal stem cells stimulate angiogenesis. Stem Cell Res Ther 2017; 8:219.
45. Yang Y, Cai Y, Zhang Y, Liu J, Xu Z. Exosomes secreted by adipose-derived stem cells contribute to angiogenesis of brain microvascular endothelial cells following oxygen-glucose deprivation in vitro through microRNA-181b/TRPM7 axis. J Mol Neurosci 2018; 65:74-83.
46. Shao L, Zhang Y, Lan B, Wang J, Zhang Z, Zhang L, et al. MiRNA-sequence indicates that mesenchymal stem cells and exosomes have similar mechanism to enhance cardiac repair. Biomed Res Int 2017; 2017:4150705.