Hypoxia Mesenchymal Stem Cells Accelerate Wound Closure Improvement by Controlling α-smooth Muscle actin Expression in the Full-thickness Animal Model

Nur Fitriani Hamra; Agung Putra; Arya Tjipta; Nur Dina Amalina; Taufiqurrachman Nasihun

doi:10.3889/oamjms.2021.5537

Authors

Nur Fitriani Hamra Postgraduate Student of Biomedical Sciences Department, Medical Faculty, Sultan Agung Islmaic University (UNISSULA), Semarang Central Java, Indonesia
Agung Putra Stem Cell and Cancer Research (SCCR) Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Central Java, Indonesia; Department of Posgraduate Biomedical Science, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Central Java, Indonesia; Department of Pathological Anatomy, Medical Faculty, Sultan Agung Islmaic University (UNISSULA), Semarang, Central Java, Indonesia http://orcid.org/0000-0003-4261-9437
Arya Tjipta Surgery Department, Medical Faculty, Universitas Sumatera Utara, Medan, North Sumatera, Indonesia
Nur Dina Amalina Stem Cell and Cancer Research (SCCR) Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semaran; Pharmacy Study Program, Chemistry Department, Faculty of Mathematics and Natrual Sciences, Universitas Negeri Semarang, Semarang, Central Java, Indonesiag, Central Java, Indonesia
Taufiqurrachman Nasihun Department of Biochemistry, Medical Faculty, Sultan Agung Islamic University (UNISSULA), Semarang, Central Java, Indonesia

DOI:

https://doi.org/10.3889/oamjms.2021.5537

Keywords:

HMSCs, α-SMA, Wound closure, full-thickness wound model

Abstract

BACKGROUND: The active myofibroblast producing extracellular matrix deposition regarding wound closure is characterized by alpha-smooth muscle actin (α-SMA) expression. However, the persistence of α-SMA expression due to prolonged inflammation may trigger scar formation. A new strategy to control α-SMA expression in line with wound closure improvement uses hypoxic mesenchymal stem cells (HMSCs) due to their ability to firmly control inflammation for early initiating cell proliferation, including the regulation of α-SMA expression associated with wound closure acceleration.

AIM: This study aimed to explore the role of HMSCs in accelerating the optimum wound closure percentages through controlling the α-SMA expression.

MATERIALS AND METHODS: Twenty-four full-thickness rats wound model were randomly divided into four groups: Sham (Sh), Control (C) by NaCl administration only, and two treatment groups by HMSCs at doses of 1.5×106 cells (T1) and HMSCs at doses of 3×106 cells (T2). HMSCs were incubated under hypoxic conditions. The α-SMA expression was analyzed under immunohistochemistry staining assay, and the wound closure percentage was analyzed by ImageJ software.

RESULTS: This study showed a significant increase in wound closure percentage in all treatment groups that gradually initiated on days 6 and 9 (p < 0.05). In line with the increase of wound closure percentages on day 9, there was also a significant decrease in α-SMA expression in all treatment groups (p < 0.05), indicating the optimum wound healing has preceded.

CONCLUSION: HMSCs have a robust ability to accelerated wound closure improvement to the optimum wound healing by controlling α-SMA expression depending on wound healing phases.

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References

Sorg H, Tilkorn DJ, Hager S, Hauser J, Mirastschijski U. Skin wound healing: An update on the current knowledge and concepts. Eur Surg Res. 2017;58(1-2):81-94. https://doi.org/10.1159/000454919 PMid:27974711 DOI: https://doi.org/10.1159/000454919

Pastar I, Stojadinovic O, Yin NC, Ramirez H, Nusbaum AG, Sawaya A, et al. Epithelialization in wound healing: A comprehensive review. Adv Wound Care (New Rochelle). 2014;3(7):445-64. https://doi.org/10.1089/wound.2013.0473 PMid:25032064 DOI: https://doi.org/10.1089/wound.2013.0473

Guo S, DiPietro LA. Factors affecting wound healing. J Dent Res. 2010;89(3):219-29. PMid:20139336 DOI: https://doi.org/10.1177/0022034509359125

Bainbridge P. Wound healing and the role of fibroblasts. J Wound Care. 2013;22(8):407-12. PMid:23924840 DOI: https://doi.org/10.12968/jowc.2013.22.8.407

Schreml S, Szeimies RM, Prantl L, Karrer S, Landthaler M, Babilas P. Oxygen in acute and chronic wound healing. Br J Dermatol. 2010;163(2):257-68. https://doi.org/10.1111/j.1365-2133.2010.09804.x PMid:20394633 DOI: https://doi.org/10.1111/j.1365-2133.2010.09804.x

Darby IA, Laverdet B, Bonté F, Desmoulière A. Fibroblasts and myofibroblasts in wound healing. Clin Cosmet Investig Dermatol. 2014;4(7):301-11. https://doi.org/10.2147/ccid.s50046 DOI: https://doi.org/10.2147/CCID.S50046

Landén NX, Li D, Ståhle M. Transition from inflammation to proliferation: A critical step during wound healing. Cell Mol Life Sci. 2016;73(20):3861-85. https://doi.org/10.1007/s00018-016-2268-0 PMid:27180275 DOI: https://doi.org/10.1007/s00018-016-2268-0

Faulknor RA, Olekson MA, Nativ NI, Ghodbane M, Gray AJ, Berthiaume F. Mesenchymal stromal cells reverse hypoxia-mediated suppression of α-smooth muscle actin expression in human dermal fibroblasts. Biochem Biophys Res Commun. 2015;458(1):8-13. https://doi.org/10.1016/j.bbrc.2015.01.013 PMid:25625213 DOI: https://doi.org/10.1016/j.bbrc.2015.01.013

Chen L, Xu Y, Zhao J, Zhang Z, Yang R, Xie J, et al. Conditioned medium from hypoxic bone marrow-derived mesenchymal stem cells enhances wound healing in mice. PLoS One. 2014;9(4):e96161. https://doi.org/10.1371/journal.pone.0096161 PMid:24781370 DOI: https://doi.org/10.1371/journal.pone.0096161

Yustianingsih V, Sumarawati T, Putra A. Hypoxia enhances self-renewal properties and markers of mesenchymal stem cells. Univ Med. 2019;38(3):164-71. https://doi.org/10.18051/univmed.2019.v38.164-171 DOI: https://doi.org/10.18051/UnivMed.2019.v38.164-171

Jun EK, Zhang Q, Yoon BS, Moon JH, Lee G, Park G, et al. Hypoxic conditioned medium from human amniotic fluid-derived

mesenchymal stem cells accelerates skin wound healing through TGF-β/SMAD2 and PI3K/AKT pathways. Int J Mol Sci. 2014;15(1):605-28. https://doi.org/10.3390/ijms15010605 PMid:24398984 DOI: https://doi.org/10.3390/ijms15010605

Trisnadi S, Muhar AM, Putra A, Kustiyah AR. Hypoxia-preconditioned mesenchymal stem cells attenuate peritoneal adhesion through TGF-β inhibition. Univ Med. 2020;39(2):97- 104. https://doi.org/10.18051/univmed.2020.v39.97-104 DOI: https://doi.org/10.18051/UnivMed.2020.v39.97-104

Muhar AM, Putra A, Warli SM, Munir D. Hypoxia-mesenchymal stem cells inhibit intra-peritoneal adhesions formation by upregulation of the il-10 expression. Open Access Maced J Med Sci. 2019;7(23):3937-43. https://doi.org/10.3889/oamjms.2019.713 PMid:32165932 DOI: https://doi.org/10.3889/oamjms.2019.713

Putra A, Pertiwi D, Milla MN, Indrayani UD, Jannah D, Sahariyani M, et al. Hypoxia-preconditioned MSCs have superior effect in ameliorating renal function on acute renal failure animal model. Open Access Maced J Med Sci. 2019;7(3):305-10. https://doi.org/10.3889/oamjms.2019.049 PMid:30833992 DOI: https://doi.org/10.3889/oamjms.2019.049

Han Y, Li X, Zhang Y, Han Y, Chang F, Ding J. Mesenchymal stem cells for regenerative medicine. Cells. 2019;8(8):886. https://doi.org/10.3390/cells8080886 PMid:31412678 DOI: https://doi.org/10.3390/cells8080886

Hu P, Yang Q, Wang Q, Shi C, Wang D, Armato U, et al. Mesenchymal stromal cells-exosomes: A promising cell-free therapeutic tool for wound healing and cutaneous regeneration. Burn Trauma. 2019;7:38. https://doi.org/10.1186/s41038-019-0178-8 PMid:31890717 DOI: https://doi.org/10.1186/s41038-019-0178-8

Lv FJ, Tuan RS, Cheung KM, Leung VY. Concise review: The surface markers and identity of human mesenchymal stem cells. Stem Cells. 2014;32(6):1408-19. https://doi.org/10.1002/stem.1681 PMid:24578244 DOI: https://doi.org/10.1002/stem.1681

Tomasek JJ, McRae J, Owens GK, Haaksma CJ. Regulation of alpha-smooth muscle actin expression in granulation tissue myofibroblasts is dependent on the intronic carg element and the transforming growth factor-beta1 control element. Am J Pathol. 2005;166(5):1343-51. https://doi.org/10.1016/s0002-9440(10)62353-x PMid:15855636 DOI: https://doi.org/10.1016/S0002-9440(10)62353-X

Sousa AM, Liu T, Guevara O, Stevens JA, Fanburg BL, Gaestel M, et al. Smooth muscle α-actin expression and myofibroblast differentiation by TGFβ are dependent upon MK2. J Cell Biochem. 2007;100(6):1581-92. https://doi.org/10.1002/ jcb.21154 PMid:17163490 DOI: https://doi.org/10.1002/jcb.21154

Chitturi RT, Balasubramaniam AM, Parameswar RA, Kesavan G, Haris KT, Mohideen K. The role of myofibroblasts in wound healing, contraction and its clinical implications in cleft palate repair. J Int Oral Health. 2015;7(3):75-80. PMid:25878485 DOI: https://doi.org/10.4103/0975-7406.163456

Tan J, Wu J. Current progress in understanding the molecular pathogenesis of burn scar contracture. Burn Trauma. 2017;5:14. https://doi.org/10.1186/s41038-017-0080-1 PMid:28546987 DOI: https://doi.org/10.1186/s41038-017-0080-1

Xue M, Jackson CJ. Extracellular matrix reorganization during wound healing and its impact on abnormal scarring. Adv Wound Care (New Rochelle). 2015;4(3):119-36. https://doi.org/10.1089/wound.2013.0485 PMid:25785236 DOI: https://doi.org/10.1089/wound.2013.0485

Nugraha A, Putra A. Tumor necrosis factor-α-activated mesenchymal stem cells accelerate wound healing through vascular endothelial growth factor regulation in rats. Univ Med. 2018;37(2):135. https://doi.org/10.18051/univmed.2018.v37.135-142 DOI: https://doi.org/10.18051/UnivMed.2018.v37.135-142

Lunardi LO, Martinelli CR, Lombardi T, Soares EG, Martinelli C. Modulation of MCP-1, TGF-β1, and α-SMA Expressions in granulation tissue of cutaneous wounds treated with local Vitamin B complex: An experimental study. Dermatopathology (Basel). 2014;1:98-107. https://doi.org/10.1159/000369163 PMid:27047929 DOI: https://doi.org/10.1159/000369163

Hinz B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol. 2007;127(3):526-37. PMid:17299435 DOI: https://doi.org/10.1038/sj.jid.5700613

Dong L, Hao H, Liu J, Ti D, Tong C, Hou Q, et al. A conditioned medium of umbilical cord mesenchymal stem cells overexpressing Wnt7a promotes wound repair and regeneration of hair follicles in mice. Stem Cells Int. 2017;2017:3738071. https://doi.org/10.1155/2017/3738071 PMid:28337222 DOI: https://doi.org/10.1155/2017/3738071

Shinde A V, Humeres C, Frangogiannis NG. The role of α-smooth muscle actin in fibroblast-mediated matrix contraction and remodeling. Biochim Biophys Acta Mol Basis Dis. 2017;1863(1):298-309. https://doi.org/10.1016/j.bbadis.2016.11.006 PMid:27825850 DOI: https://doi.org/10.1016/j.bbadis.2016.11.006

Li B, Wang JH. Fibroblasts and myofibroblasts in wound healing: Force generation and measurement. J Tissue Viability. 2011;20(4):108-20. https://doi.org/10.1016/j.jtv.2009.11.004 PMid:19995679 DOI: https://doi.org/10.1016/j.jtv.2009.11.004

Zhong ZF, Tan W, Tian K, Yu H, Qiang WA, Wang YT. Combined effects of furanodiene and doxorubicin on the migration and invasion of MDA-MB-231 breast cancer cells in vitro. Oncol Rep. 2017;37(4):2016-24. https://doi.org/10.3892/or.2017.5435 PMid:28184941 DOI: https://doi.org/10.3892/or.2017.5435

Gras C, Ratuszny D, Hadamitzky C, Zhang H, Blasczyk R, Figueiredo C. miR-145 contributes to hypertrophic scarring of the skin by inducing myofibroblast activity. Mol Med. 2015;21:296- 304. https://doi.org/10.2119/molmed.2014.00172 PMid:25876136 DOI: https://doi.org/10.2119/molmed.2014.00172

Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG. Hypertrophic scarring and keloids: Pathomechanisms and current and emerging treatment strategies. Mol Med. 2011;17(1-2):113-25. https://doi.org/10.2119/molmed.2009.00153 PMid:20927486 DOI: https://doi.org/10.2119/molmed.2009.00153

Kuntardjo N, Dharmana E, Chodidjah C, Nasihun TR, Putra A. TNF-α-activated MSC-CM topical gel effective in increasing PDGF level, fibroblast density, and wound healing process compared to subcutaneous injection combination. Maj Kedokt Bandung. 2019;51(1):1-6. https://doi.org/10.15395/mkb.v51n1.1479 DOI: https://doi.org/10.15395/mkb.v51n1.1479

El Kahi CG, Atiyeh BS, Hussein IA, Dibo SA, Jurjus A, et al. Modulation of wound contracture α-smooth muscle actin and multispecific vitronectin receptor integrin αvβ3 in the rabbit’s experimental model. Int Wound J. 2009;6(3):214-24. https://doi.org/10.1111/j.1742-481x.2009.00597.x DOI: https://doi.org/10.1111/j.1742-481X.2009.00597.x

Yoon D, Yoon D, Sim H, Hwang I, Lee J, Chun W. Accelerated wound healing by fibroblasts differentiated from human embryonic stem cell-derived mesenchymal stem cells in a pressure ulcer animal model. Stem Cells Int. 2018;2018:4789568. https://doi. org/10.1155/2018/4789568 PMid:30693037 DOI: https://doi.org/10.1155/2018/4789568

El Ayadi A, Jay JW, Prasai A. Current approaches targeting the wound healing phases to attenuate fibrosis and scarring. Int J Mol Sci. 2020;21(3):1105. https://doi.org/10.3390/ijms21031105 PMid:32046094 DOI: https://doi.org/10.3390/ijms21031105

Yoshida M, Arai T, Hoshino S, Inoue K, Yano Y, Yanagita M, et al. IL-10 inhibits transforming growth factor-ß-induction of Type I collagen mRNA expression via both JNK and p38 pathways in human lung fibroblasts. EXCLI J. 2005;4:49-60.

Steen EH, Wang X, Balaji S, Butte MJ, Bollyky PL, Keswani SG. The role of the anti-inflammatory cytokine interleukin-10 in tissue fibrosis. Adv Wound Care (New Rochelle). 2020;9(4):184-98. https://doi.org/10.1089/wound.2019.1032 PMid:32117582 DOI: https://doi.org/10.1089/wound.2019.1032

Deng G, Li K, Chen S, Chen P, Zheng H, Yu BI, et al. Interleukin 10 promotes proliferation and migration, and inhibits tendon differentiation via the JAK/Stat3 pathway in tendon derived stem cells in vitro. Mol Med Rep. 2018;18(6):5044-52. https://doi.org/10.3892/mmr.2018.9547 PMid:30320384 DOI: https://doi.org/10.3892/mmr.2018.9547

Ren Z, Hou Y, Ma S, Tao Y, Li J, Cao H, et al. Effects of CCN3 on fibroblast proliferation, apoptosis and extracellular matrix production. Int J Mol Med. 2014;33(6):1607-12. https://doi.org/10.3892/ijmm.2014.1735 PMid:24715059 DOI: https://doi.org/10.3892/ijmm.2014.1735

Hinz B, Phan SH, Thannickal VJ, Prunotto M, Desmoulière A, Varga J, et al. Recent developments in myofibroblast biology: Paradigms for connective tissue remodeling. Am J Pathol. 2012;180(4):1340-55. https://doi.org/10.1016/j.ajpath.2012.02.004 PMid:22387320 DOI: https://doi.org/10.1016/j.ajpath.2012.02.004

Faulknor RA, Olekson MA, Ekwueme EC, Krzyszczyk P, Freeman JW, Berthiaume F. Hypoxia impairs mesenchymal stromal cell-induced macrophage M1 to M2. Technology (Singap World Sci). 2017;5(2):81-6. https://doi.org/10.1142/s2339547817500042 PMid:29552603 DOI: https://doi.org/10.1142/S2339547817500042

Serra MB, Barroso WA, Neves N, Silva N, Carlos A, Borges R, et al. From inflammation to current and alternative therapies involved in wound healing. Int J Inflam. 2017;2017:3406215. PMid:28811953 DOI: https://doi.org/10.1155/2017/3406215

Talele NP, Fradette J, Davies JE, Kapus A, Hinz B. Expression of α-smooth muscle actin determines the fate of mesenchymal stromal cells. Stem Cell Rep. 2015;4(6):1016-30. https://doi.org/10.1016/j.stemcr.2015.05.004 PMid:26028530 DOI: https://doi.org/10.1016/j.stemcr.2015.05.004

Hinz B, Dugina V, Ballestrem C, Wehrle-haller B, Chaponnier C. Alpha-smooth muscle actin is crucial for focal adhesion maturation in myofibroblasts. Mol Biol Cell. 2003;14(6):2508-19. https://doi.org/10.1091/mbc.e02-11-0729 PMid:12808047 DOI: https://doi.org/10.1091/mbc.e02-11-0729