Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

HU Xuan ,  YI Yangyan , ZHU Yuanzheng , WANG Zhaohui , WU Shu , ZHANG Jing , WANG Jiangwen , NIE Jiaying

Department of Plastic Surgery, the Second Affiliated Hospital of Nanchang University, Nanchang Jiangxi, 330006, P.R.China;

Corresponding?author：

YI Yangyan, Email: yyy0218@126.com

Keywords：

Adipose-derived stem cells; exosomes; flap transplantation; angiogenesis; rat

DOI：

10.7507/1002-1892.201904023

Video：

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Objective To investigate the effect of adipose-derived stem cell derived exosomes (ADSC-Exos) on angiogenesis after skin flap transplantation in rats.Methods ADSCs were isolated and cultured by enzymatic digestion from voluntary donated adipose tissue of patients undergoing liposuction. The 3rd generation cells were observed under microscopy and identified by flow cytometry and oil red O staining at 14 days after induction of adipogenesis. After cells were identified as ADSCs, ADSC-Exos was extracted by density gradient centrifugation. And the morphology was observed by transmission electron microscopy, the surface marker proteins (CD63, TSG101) were detected by Western blot, and particle size distribution was measured by nanoparticle size tracking analyzer. Twenty male Sprague Dawley rats, weighing 250-300 g, were randomly divided into ADSC-Exos group and PBS group with 10 rats in each group. ADSC-Exos (ADSC-Exos group) and PBS (PBS group) were injected into the proximal, middle, and distal regions of the dorsal free flaps with an area of 9 cm×3 cm along the long axis in the two groups. The survival rate of the flap was measured on the 7th day, and then the flap tissue was harvested. The tissue morphology was observed by HE staining, and mean blood vessel density (MVD) was measured by CD31 immunohistochemical staining.Results ADSCs were identified by microscopy, flow cytometry, and adipogenic induction culture. ADSC-Exos was a round or elliptical membrane vesicle with clear edge and uniform size. It has high expression of CD63 and TSG101, and its size distribution was 30-200 nm, which was in accordance with the size range of Exos. The distal necrosis of the flaps in the ADSC-Exos group was milder than that in the PBS group. On the 7th day, the survival rate of the flaps in the ADSC-Exos group was 64.2%±11.5%, which was significantly higher than that in the PBS group (31.0%±6.6%; t=7.945, P=0.000); the skin appendages in the middle region of the flap in the ADSC-Exos group were more complete, the edema in the proximal region was lighter and the vasodilation was more extensive. MVD of the ADSC-Exos group was (103.3±27.0) /field, which was significantly higher than that of the PBS group [(45.3±16.2)/field; t=3.190, P=0.011].Conclusion ADSC-Exos can improve the blood supply of skin flaps by promoting the formation of neovascularization after skin flap transplantation, thereby improve the survival rate of skin flaps in rats.

Citation： HU Xuan, YI Yangyan, ZHU Yuanzheng, WANG Zhaohui, WU Shu, ZHANG Jing, WANG Jiangwen, NIE Jiaying. Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats. Chinese Journal of Reparative and Reconstructive Surgery, 2019, 33(12): 1560-1565. doi: 10.7507/1002-1892.201904023 Copy

1.	Lin B, Lin Y, Lin D, et al. Effects of Bezafibrate on the survival of random skin flaps in rats. J Reconstr Microsurg, 2016, 32(5): 395-401.
2.	Yang M, Sheng L, Li H, et al. Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model. Microsurgery, 2010, 30(4): 275-281.
3.	Rao RR, Stegemann JP. Cell-based approaches to the engineering of vascularized bone tissue. Cytotherapy, 2013, 15(11): 1309-1322.
4.	Choudhery MS, Khan M, Mahmood R, et al. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int, 2012, 36(8): 747-753.
5.	Zhongrun Q, Qi S, Xi Y, et al. The role of extracellular vesicles: an epigenetic view of the cancer microenvironment. Biomed Res Int, 2015, 2015: 649161.
6.	Zhang Y, Hu YW, Zheng L, et al. Characteristics and roles of exosomes in cardiovascular disease. DNA Cell Biol, 2017, 36(3): 202-211.
7.	Sahoo S, Losordo DW. Exosomes and cardiac repair after myocardial infarction. Circ Res, 2014, 114(2): 333-344.
8.	Zhang H, Xiang M, Meng D, et al. Inhibition of myocardial ischemia/reperfusion injury by exosomes secreted from mesenchymal stem cells. Stem Cells Int, 2016, 2016: 4328362.
9.	Farzamfar S, Hasanpour A, Nazeri N, et al. Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury. J Cell Physiol, 2019, 234(8): 12290-12300.
10.	張靜, 易陽艷, 陽水發, 等. 脂肪干細胞來源外泌體對人臍靜脈血管內皮細胞增殖、遷移及管樣分化的影響. 中國修復重建外科雜志, 2018, 32(10): 1351-1357.
11.	Mcfarlane RM, Deyoung G, Henry RA, et al. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg, 1965, 35(2): 177-182.
12.	Zhou KL, Zhang YH, Lin DS, et al. Effects of calcitriol on random skin flap survival in rats. Sci Rep, 2016, 6: 18945.
13.	Cai L, Cao B, Lin D. Effects of traditional Chinese medicine huangqi injection (radix astragali) on random skin flap survival in rats. J Reconstr Microsurg, 2015, 31(8): 565-570.
14.	Bai Y, Han YD, Yan XL, et al. Adipose mesenchymal stem cell-derived exosomes stimulated by hydrogen peroxide enhanced skin flap recovery in ischemia-reperfusion injury. Biochem Biophys Res Commun, 2018, 500(2): 310-317.
15.	Annex, Brian H. Therapeutic angiogenesis for critical limb ischaemia. Nature Reviews Cardiology, 2013, 10(7): 387-396.
16.	Pu CM, Liu CW, Liang CJ, et al. Adipose-derived stem cells protect skin flaps against ischemia/reperfusion injury via IL-6 expression. J Invest Dermatol, 2017, 137(6): 1353-1362.
17.	Du W, Zhang K, Zhang S, et al. Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer. Biomaterials, 2017, 133: 70-81.
18.	Merino-González C, Zu?iga FA, Carlos E, et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: potencial clinical application. Front Physiol, 2016, 7: 24.
19.	Hessvik NP, Llorente A. Current knowledge on exosome biogenesis and release. Cell Mol Life Sci, 2018, 75(2): 193-208.
20.	Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles, 2013, 2(1): 19861.
21.	Kishore R, Khan M. More than tiny sacks: stem cell exosomes as cell-free modality for cardiac repair. Circ Res, 2016, 118(2): 330-343.
22.	Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med, 2015, 13: 49.
23.	Han YD, Bai Y, Yan XL, et al. Co-transplantation of exosomes derived from hypoxia-preconditioned adipose mesenchymal stem cells promotes neovascularization and graft survival in fat grafting. Biochem Biophys Res Commun, 2018, 497(1): 305-312.
24.	Arslan F, Lai RC, Smeets MB, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res, 2013, 10(3): 301-312.
25.	Schlaudraff KU, Bezzola T, Montandon D, et al. Mixed arterio-venous insufficiency in random skin flaps in the rat: Is the application of medicinal leeches beneficial? J Surg Res, 2008, 150(1): 85-91.
26.	李俊杰, 高自勉, 高偉陽, 等. 皮瓣延遲術對大鼠三血管體穿支皮瓣成活的影響及其機制. 中華燒傷雜志, 2014, 30(4): 337-343.
27.	Guo YX, Ji X, Liu JB, et al. Effects of exosomes on pre-metastatic niche formation in tumors. Mol Cancer, 2019, 18: 39.
28.	Kang T, Jones T M, Naddell C, et al. Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31. Stem Cells Translational Medicine, 2016, 5(4): 440-450.
29.	Todorova D, Simoncini S, Lacroix R, et al. Extracellular vesicles in angiogenesis. Circ Res, 2017, 120(10): 1658-1673.

1. Lin B, Lin Y, Lin D, et al. Effects of Bezafibrate on the survival of random skin flaps in rats. J Reconstr Microsurg, 2016, 32(5): 395-401.
2. Yang M, Sheng L, Li H, et al. Improvement of the skin flap survival with the bone marrow-derived mononuclear cells transplantation in a rat model. Microsurgery, 2010, 30(4): 275-281.
3. Rao RR, Stegemann JP. Cell-based approaches to the engineering of vascularized bone tissue. Cytotherapy, 2013, 15(11): 1309-1322.
4. Choudhery MS, Khan M, Mahmood R, et al. Bone marrow derived mesenchymal stem cells from aged mice have reduced wound healing, angiogenesis, proliferation and anti-apoptosis capabilities. Cell Biol Int, 2012, 36(8): 747-753.
5. Zhongrun Q, Qi S, Xi Y, et al. The role of extracellular vesicles: an epigenetic view of the cancer microenvironment. Biomed Res Int, 2015, 2015: 649161.
6. Zhang Y, Hu YW, Zheng L, et al. Characteristics and roles of exosomes in cardiovascular disease. DNA Cell Biol, 2017, 36(3): 202-211.
7. Sahoo S, Losordo DW. Exosomes and cardiac repair after myocardial infarction. Circ Res, 2014, 114(2): 333-344.
8. Zhang H, Xiang M, Meng D, et al. Inhibition of myocardial ischemia/reperfusion injury by exosomes secreted from mesenchymal stem cells. Stem Cells Int, 2016, 2016: 4328362.
9. Farzamfar S, Hasanpour A, Nazeri N, et al. Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury. J Cell Physiol, 2019, 234(8): 12290-12300.
10. 張靜, 易陽艷, 陽水發, 等. 脂肪干細胞來源外泌體對人臍靜脈血管內皮細胞增殖、遷移及管樣分化的影響. 中國修復重建外科雜志, 2018, 32(10): 1351-1357.
11. Mcfarlane RM, Deyoung G, Henry RA, et al. The design of a pedicle flap in the rat to study necrosis and its prevention. Plast Reconstr Surg, 1965, 35(2): 177-182.
12. Zhou KL, Zhang YH, Lin DS, et al. Effects of calcitriol on random skin flap survival in rats. Sci Rep, 2016, 6: 18945.
13. Cai L, Cao B, Lin D. Effects of traditional Chinese medicine huangqi injection (radix astragali) on random skin flap survival in rats. J Reconstr Microsurg, 2015, 31(8): 565-570.
14. Bai Y, Han YD, Yan XL, et al. Adipose mesenchymal stem cell-derived exosomes stimulated by hydrogen peroxide enhanced skin flap recovery in ischemia-reperfusion injury. Biochem Biophys Res Commun, 2018, 500(2): 310-317.
15. Annex, Brian H. Therapeutic angiogenesis for critical limb ischaemia. Nature Reviews Cardiology, 2013, 10(7): 387-396.
16. Pu CM, Liu CW, Liang CJ, et al. Adipose-derived stem cells protect skin flaps against ischemia/reperfusion injury via IL-6 expression. J Invest Dermatol, 2017, 137(6): 1353-1362.
17. Du W, Zhang K, Zhang S, et al. Enhanced proangiogenic potential of mesenchymal stem cell-derived exosomes stimulated by a nitric oxide releasing polymer. Biomaterials, 2017, 133: 70-81.
18. Merino-González C, Zu?iga FA, Carlos E, et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: potencial clinical application. Front Physiol, 2016, 7: 24.
19. Hessvik NP, Llorente A. Current knowledge on exosome biogenesis and release. Cell Mol Life Sci, 2018, 75(2): 193-208.
20. Webber J, Clayton A. How pure are your vesicles? J Extracell Vesicles, 2013, 2(1): 19861.
21. Kishore R, Khan M. More than tiny sacks: stem cell exosomes as cell-free modality for cardiac repair. Circ Res, 2016, 118(2): 330-343.
22. Zhang J, Guan J, Niu X, et al. Exosomes released from human induced pluripotent stem cells-derived MSCs facilitate cutaneous wound healing by promoting collagen synthesis and angiogenesis. J Transl Med, 2015, 13: 49.
23. Han YD, Bai Y, Yan XL, et al. Co-transplantation of exosomes derived from hypoxia-preconditioned adipose mesenchymal stem cells promotes neovascularization and graft survival in fat grafting. Biochem Biophys Res Commun, 2018, 497(1): 305-312.
24. Arslan F, Lai RC, Smeets MB, et al. Mesenchymal stem cell-derived exosomes increase ATP levels, decrease oxidative stress and activate PI3K/Akt pathway to enhance myocardial viability and prevent adverse remodeling after myocardial ischemia/reperfusion injury. Stem Cell Res, 2013, 10(3): 301-312.
25. Schlaudraff KU, Bezzola T, Montandon D, et al. Mixed arterio-venous insufficiency in random skin flaps in the rat: Is the application of medicinal leeches beneficial? J Surg Res, 2008, 150(1): 85-91.
26. 李俊杰, 高自勉, 高偉陽, 等. 皮瓣延遲術對大鼠三血管體穿支皮瓣成活的影響及其機制. 中華燒傷雜志, 2014, 30(4): 337-343.
27. Guo YX, Ji X, Liu JB, et al. Effects of exosomes on pre-metastatic niche formation in tumors. Mol Cancer, 2019, 18: 39.
28. Kang T, Jones T M, Naddell C, et al. Adipose-Derived Stem Cells Induce Angiogenesis via Microvesicle Transport of miRNA-31. Stem Cells Translational Medicine, 2016, 5(4): 440-450.
29. Todorova D, Simoncini S, Lacroix R, et al. Extracellular vesicles in angiogenesis. Circ Res, 2017, 120(10): 1658-1673.

Chinese Journal of Reparative and Reconstructive Surgery

Effect of adipose-derived stem cell derived exosomes on angiogenesis after skin flap transplantation in rats

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