FREE SHIPPING over $40
OVER 9000 FIVE-STAR REVIEWS
5% OFF ALL SUBSCRIPTIONS
100% HAPPINESS GUARANTEE
Analyze Diet
0
Home / Equine Research Bank / Stem Cells Int / Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic ...
Stem cells international2021; 2021; 8284690; doi: 10.1155/2021/8284690

Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells.

Abstract: Mesenchymal stromal cells (MSC) represent a promising therapeutic tool for tendon regeneration. Their tenogenic differentiation is crucial for tissue engineering approaches and may support their beneficial effects after cell transplantation . The transforming growth factor (TGF)-, signalling via intracellular Smad molecules, is a potent paracrine mediator of tenogenic induction. Moreover, scaffold topography or tendon matrix components induced tenogenesis via activation of the Rho/ROCK cascade, which, however, is also involved in pathological adaptations in extracellular matrix pathologies. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-3/Smad signalling in tenogenic differentiation in both human and equine MSC. Primary equine and human MSC isolated from adipose tissue were cultured as monolayers or on tendon-derived decellularized scaffolds to evaluate the influence of the ROCK inhibitor Y-27632 on TGF-3-induced tenogenic differentiation. The MSC were incubated with and without TGF-3 (10 ng/ml), Y-27632 (10 M), or both. On day 1 and day 3, the signalling pathway of TGF- and the actin cytoskeleton were visualized by Smad 2/3 and phalloidin staining, and gene expression of signalling molecules and tendon markers was assessed. ROCK inhibition was confirmed by disruption of the actin cytoskeleton. Activation of Smad 2/3 with nuclear translocation was evident upon TGF-3 stimulation. Interestingly, this effect was most pronounced with additional ROCK inhibition in both species ( < 0.05 in equine MSC). In line with that, the tendon marker scleraxis showed the strongest upregulation when TGF-3 and ROCK inhibition were combined ( < 0.05 in human MSC). The regulation pattern of tendon extracellular matrix components and the signalling molecules TGF-3 and Smad 8 showed differences between human and equine MSC. The obtained results showed that ROCK inhibition promotes the TGF-3/Smad 2/3 axis, with possible implications for future MSC priming regimes in tendon therapy.
Copyright © 2021 Michaela Melzer et al.
Get Full Text
Publication Date: 2021-10-08 PubMed ID: 34659420PubMed Central: PMC8519677DOI: 10.1155/2021/8284690Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedInCopy
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research looked at enhancing tendon regeneration by accelerating the transformation of Mesenchymal Stromal Cells (MSC) into tendon cells through manipulating Rho/ROCK and TGF-β3/Smad signaling. The study found that inhibiting Rho/ROCK signaling while stimulating TGF-β3/Smad signaling effectively transformed MSC into tendon cells, a finding that potentially improves MSC-based therapies for tendon injuries.

Study Rationale and Objective

  • The focus of the research is on two important biological processes for tendon regeneration — the process of MSC transforming into tendon cells (tenogenic differentiation) and the interplay between two signaling pathways, Rho/ROCK and TGF-β3/Smad, which regulate this transformation.
  • The goal is to optimize this process to make MSC-based tendon therapies more efficacious.

Methodology

  • MSC from human and equine were isolated and cultured under different conditions: no treatment, TGF-β3 treatment to stimulate tenogenic differentiation, Rho/ROCK inhibitor Y-27632 treatment to suppress Rho/ROCK signaling, and both TGF-β3 and Y-27632 treatments.
  • Both monolayer culture and culture on tendon-derived decellularized scaffolds were used to mimic realities of tissue construction and cell transplantation.
  • Gene expression of signaling molecules and tendon markers and the actin cytoskeleton were evaluated on days 1 and 3 to assess and compare the effectiveness of different treatments.

Findings

  • Rho/ROCK inhibition by Y-27632 was confirmed through the disruption of the actin cytoskeleton.
  • Activation of Smad 2/3 with nuclear translocation, a key process in TGF-β-mediated tenogenic differentiation, was observed when TGF-β3 was applied, and this effect was even more pronounced with additional ROCK inhibition.
  • The expression of scleraxis, a marker for tendon cells, increased the most when TGF-β3 and ROCK inhibition were combined, indicating the highest rate of tenogenic differentiation under this condition.
  • Differences were observed between human and equine MSC in the regulation pattern of tendon extracellular matrix components and the signaling molecules TGF-β3 and Smad 8.

Implications

  • The results suggest that combined Rho/ROCK inhibition and TGF-β3/Smad stimulation could enhance tenogenic differentiation in MSC, which could optimize MSC-based therapies for tendon injuries.
  • The observed species differences in response to treatments warrant further investigation to reach more widely applicable conclusions.

Cite This Article

APA
Melzer M, Schubert S, Müller SF, Geyer J, Hagen A, Niebert S, Burk J. (2021). Rho/ROCK Inhibition Promotes TGF-β3-Induced Tenogenic Differentiation in Mesenchymal Stromal Cells. Stem Cells Int, 2021, 8284690. https://doi.org/10.1155/2021/8284690

Publication

Stem cells international
ISSN: 1687-966X
NlmUniqueID: 101535822
Country: United States
Language: English
Volume: 2021
Pages: 8284690
PII: 8284690

Researcher Affiliations

Melzer, Michaela
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Schubert, Susanna
  • Saxon Incubator for Clinical Translation, University of Leipzig, 04103 Leipzig, Germany.
  • Institute of Human Genetics, University of Leipzig Hospitals and Clinics, 04103 Leipzig, Germany.
Müller, Simon Franz
  • Biomedical Research Center Seltersberg (BFS), Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Geyer, Joachim
  • Biomedical Research Center Seltersberg (BFS), Institute of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Hagen, Alina
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Niebert, Sabine
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.
Burk, Janina
  • Equine Clinic (Surgery, Orthopedics), Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, 35392 Giessen, Germany.

Conflict of Interest Statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

References

This article includes 42 references
  1. Clayton RA, Court-Brown CM. The epidemiology of musculoskeletal tendinous and ligamentous injuries.. Injury 2008 Dec;39(12):1338-44.
    doi: 10.1016/j.injury.2008.06.021pubmed: 19036362google scholar: lookup
  2. Lemme NJ, Li NY, DeFroda SF, Kleiner J, Owens BD. Epidemiology of Achilles Tendon Ruptures in the United States: Athletic and Nonathletic Injuries From 2012 to 2016.. Orthop J Sports Med 2018 Nov;6(11):2325967118808238.
    pmc: PMC6259075pubmed: 30505872doi: 10.1177/2325967118808238google scholar: lookup
  3. Kasashima Y, Takahashi T, Smith RK, Goodship AE, Kuwano A, Ueno T, Hirano S. Prevalence of superficial digital flexor tendonitis and suspensory desmitis in Japanese Thoroughbred flat racehorses in 1999.. Equine Vet J 2004 May;36(4):346-50.
    doi: 10.2746/0425164044890580pubmed: 15163043google scholar: lookup
  4. Trump M. A retrospective study of the prevalence of injuries to the suspensory ligament, digital flexor tendons and associated structures in a nonracehorse referral-hospital population . Hobokken, NJ: University of Zurich; 2014.
    doi: 10.5167/uzh-109107google scholar: lookup
  5. Stanley RL, Fleck RA, Becker DL, Goodship AE, Ralphs JR, Patterson-Kane JC. Gap junction protein expression and cellularity: comparison of immature and adult equine digital tendons.. J Anat 2007 Sep;211(3):325-34.
    doi: 10.1111/j.1469-7580.2007.00781.xpmc: PMC2375813pubmed: 17848160google scholar: lookup
  6. Leong NL, Kator JL, Clemens TL, James A, Enamoto-Iwamoto M, Jiang J. Tendon and Ligament Healing and Current Approaches to Tendon and Ligament Regeneration.. J Orthop Res 2020 Jan;38(1):7-12.
    doi: 10.1002/jor.24475pmc: PMC7307866pubmed: 31529731google scholar: lookup
  7. Pacini S, Spinabella S, Trombi L, Fazzi R, Galimberti S, Dini F, Carlucci F, Petrini M. Suspension of bone marrow-derived undifferentiated mesenchymal stromal cells for repair of superficial digital flexor tendon in race horses.. Tissue Eng 2007 Dec;13(12):2949-55.
    doi: 10.1089/ten.2007.0108pubmed: 17919069google scholar: lookup
  8. Godwin EE, Young NJ, Dudhia J, Beamish IC, Smith RK. Implantation of bone marrow-derived mesenchymal stem cells demonstrates improved outcome in horses with overstrain injury of the superficial digital flexor tendon.. Equine Vet J 2012 Jan;44(1):25-32.
    pubmed: 21615465doi: 10.1111/j.2042-3306.2011.00363.xgoogle scholar: lookup
  9. Smith RK, Werling NJ, Dakin SG, Alam R, Goodship AE, Dudhia J. Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy.. PLoS One 2013;8(9):e75697.
    doi: 10.1371/journal.pone.0075697pmc: PMC3783421pubmed: 24086616google scholar: lookup
  10. Manning CN, Martel C, Sakiyama-Elbert SE, Silva MJ, Shah S, Gelberman RH, Thomopoulos S. Adipose-derived mesenchymal stromal cells modulate tendon fibroblast responses to macrophage-induced inflammation in vitro.. Stem Cell Res Ther 2015 Apr 16;6(1):74.
    doi: 10.1186/s13287-015-0059-4pmc: PMC4416344pubmed: 25889287google scholar: lookup
  11. Shen H, Kormpakis I, Havlioglu N, Linderman SW, Sakiyama-Elbert SE, Erickson IE, Zarembinski T, Silva MJ, Gelberman RH, Thomopoulos S. The effect of mesenchymal stromal cell sheets on the inflammatory stage of flexor tendon healing.. Stem Cell Res Ther 2016 Sep 27;7(1):144.
    doi: 10.1186/s13287-016-0406-0pmc: PMC5039894pubmed: 27677963google scholar: lookup
  12. Barsby T, Guest D. Transforming growth factor beta3 promotes tendon differentiation of equine embryo-derived stem cells.. Tissue Eng Part A 2013 Oct;19(19-20):2156-65.
    pubmed: 23611525doi: 10.1089/ten.tea.2012.0372google scholar: lookup
  13. Maeda T, Sakabe T, Sunaga A, Sakai K, Rivera AL, Keene DR, Sasaki T, Stavnezer E, Iannotti J, Schweitzer R, Ilic D, Baskaran H, Sakai T. Conversion of mechanical force into TGF-β-mediated biochemical signals.. Curr Biol 2011 Jun 7;21(11):933-41.
    doi: 10.1016/j.cub.2011.04.007pmc: PMC3118584pubmed: 21600772google scholar: lookup
  14. Zhang YJ, Chen X, Li G, Chan KM, Heng BC, Yin Z, Ouyang HW. Concise Review: Stem Cell Fate Guided By Bioactive Molecules for Tendon Regeneration.. Stem Cells Transl Med 2018 May;7(5):404-414.
    doi: 10.1002/sctm.17-0206pmc: PMC5905226pubmed: 29573225google scholar: lookup
  15. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P. TGF-beta receptor-mediated signalling through Smad2, Smad3 and Smad4.. EMBO J 1997 Sep 1;16(17):5353-62.
    doi: 10.1093/emboj/16.17.5353pmc: PMC1170167pubmed: 9311995google scholar: lookup
  16. Burch ML, Zheng W, Little PJ. Smad linker region phosphorylation in the regulation of extracellular matrix synthesis.. Cell Mol Life Sci 2011 Jan;68(1):97-107.
    pubmed: 20820849doi: 10.1007/s00018-010-0514-4google scholar: lookup
  17. Kishore V, Bullock W, Sun X, Van Dyke WS, Akkus O. Tenogenic differentiation of human MSCs induced by the topography of electrochemically aligned collagen threads.. Biomaterials 2012 Mar;33(7):2137-44.
    pmc: PMC3279298pubmed: 22177622doi: 10.1016/j.biomaterials.2011.11.066google scholar: lookup
  18. Yang G, Rothrauff BB, Lin H, Gottardi R, Alexander PG, Tuan RS. Enhancement of tenogenic differentiation of human adipose stem cells by tendon-derived extracellular matrix.. Biomaterials 2013 Dec;34(37):9295-306.
    doi: 10.1016/j.biomaterials.2013.08.054pmc: PMC3951997pubmed: 24044998google scholar: lookup
  19. Burk J, Plenge A, Brehm W, Heller S, Pfeiffer B, Kasper C. Induction of Tenogenic Differentiation Mediated by Extracellular Tendon Matrix and Short-Term Cyclic Stretching.. Stem Cells Int 2016;2016:7342379.
    pmc: PMC5007347pubmed: 27630718doi: 10.1155/2016/7342379google scholar: lookup
  20. Xu B, Song G, Ju Y, Li X, Song Y, Watanabe S. RhoA/ROCK, cytoskeletal dynamics, and focal adhesion kinase are required for mechanical stretch-induced tenogenic differentiation of human mesenchymal stem cells.. J Cell Physiol 2012 Jun;227(6):2722-9.
    doi: 10.1002/jcp.23016pubmed: 21898412google scholar: lookup
  21. Maharam E, Yaport M, Villanueva NL, Akinyibi T, Laudier D, He Z, Leong DJ, Sun HB. Rho/Rock signal transduction pathway is required for MSC tenogenic differentiation.. Bone Res 2015;3:15015.
    doi: 10.1038/boneres.2015.15pmc: PMC4605238pubmed: 26509098google scholar: lookup
  22. Roth SP, Schubert S, Scheibe P, Groß C, Brehm W, Burk J. Growth Factor-Mediated Tenogenic Induction of Multipotent Mesenchymal Stromal Cells Is Altered by the Microenvironment of Tendon Matrix.. Cell Transplant 2018 Oct;27(10):1434-1450.
    doi: 10.1177/0963689718792203pmc: PMC6180728pubmed: 30251565google scholar: lookup
  23. Knipe RS, Tager AM, Liao JK. The Rho kinases: critical mediators of multiple profibrotic processes and rational targets for new therapies for pulmonary fibrosis.. Pharmacol Rev 2015;67(1):103-17.
    pmc: PMC4279074pubmed: 25395505doi: 10.1124/pr.114.009381google scholar: lookup
  24. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement.. Cytotherapy 2006;8(4):315-7.
    doi: 10.1080/14653240600855905pubmed: 16923606google scholar: lookup
  25. Schubert S, Brehm W, Hillmann A, Burk J. Serum-free human MSC medium supports consistency in human but not in equine adipose-derived multipotent mesenchymal stromal cell culture.. Cytometry A 2018 Jan;93(1):60-72.
    pubmed: 28926198doi: 10.1002/cyto.a.23240google scholar: lookup
  26. Hagen A, Lehmann H, Aurich S, Bauer N, Melzer M, Moellerberndt J, Patané V, Schnabel CL, Burk J. Scalable Production of Equine Platelet Lysate for Multipotent Mesenchymal Stromal Cell Culture.. Front Bioeng Biotechnol 2020;8:613621.
    pmc: PMC7859354pubmed: 33553119doi: 10.3389/fbioe.2020.613621google scholar: lookup
  27. Burk J, Erbe I, Berner D, Kacza J, Kasper C, Pfeiffer B, Winter K, Brehm W. Freeze-thaw cycles enhance decellularization of large tendons.. Tissue Eng Part C Methods 2014 Apr;20(4):276-84.
    doi: 10.1089/ten.tec.2012.0760pmc: PMC3968887pubmed: 23879725google scholar: lookup
  28. Brandt L, Schubert S, Scheibe P, Brehm W, Franzen J, Gross C, Burk J. Tenogenic Properties of Mesenchymal Progenitor Cells Are Compromised in an Inflammatory Environment.. Int J Mol Sci 2018 Aug 28;19(9).
    pmc: PMC6163784pubmed: 30154348doi: 10.3390/ijms19092549google scholar: lookup
  29. Hillmann A, Paebst F, Brehm W, Piehler D, Schubert S, Tárnok A, Burk J. A novel direct co-culture assay analyzed by multicolor flow cytometry reveals context- and cell type-specific immunomodulatory effects of equine mesenchymal stromal cells.. PLoS One 2019;14(6):e0218949.
    doi: 10.1371/journal.pone.0218949pmc: PMC6597077pubmed: 31247035google scholar: lookup
  30. Noronha NC, Mizukami A, Caliári-Oliveira C, Cominal JG, Rocha JLM, Covas DT, Swiech K, Malmegrim KCR. Correction to: Priming approaches to improve the efficacy of mesenchymal stromal cell-based therapies.. Stem Cell Res Ther 2019 May 17;10(1):132.
    doi: 10.1186/s13287-019-1259-0pmc: PMC6525348pubmed: 31101067google scholar: lookup
  31. Huveneers S, Danen EH. Adhesion signaling - crosstalk between integrins, Src and Rho.. J Cell Sci 2009 Apr 15;122(Pt 8):1059-69.
    pubmed: 19339545doi: 10.1242/jcs.039446google scholar: lookup
  32. Wang D, Pun CCM, Huang S, Tang TCM, Ho KKW, Rothrauff BB, Yung PSH, Blocki AM, Ker EDF, Tuan RS. Tendon-derived extracellular matrix induces mesenchymal stem cell tenogenesis via an integrin/transforming growth factor-β crosstalk-mediated mechanism.. FASEB J 2020 Jun;34(6):8172-8186.
    doi: 10.1096/fj.201902377RRpubmed: 32301551google scholar: lookup
  33. Vermeulen S, Roumans N, Honig F, Carlier A, Hebels DGAJ, Eren AD, Dijke PT, Vasilevich A, de Boer J. Mechanotransduction is a context-dependent activator of TGF-β signaling in mesenchymal stem cells.. Biomaterials 2020 Nov;259:120331.
    doi: 10.1016/j.biomaterials.2020.120331pubmed: 32836056google scholar: lookup
  34. Xu T, Wu M, Feng J, Lin X, Gu Z. RhoA/Rho kinase signaling regulates transforming growth factor-β1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway.. Int J Mol Med 2012 Nov;30(5):1119-25.
    pubmed: 22922645doi: 10.3892/ijmm.2012.1107google scholar: lookup
  35. Ji H, Tang H, Lin H, Mao J, Gao L, Liu J, Wu T. Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.. Biomed Rep 2014 Nov;2(6):787-792.
    doi: 10.3892/br.2014.323pmc: PMC4179758pubmed: 25279146google scholar: lookup
  36. Roth SP, Glauche SM, Plenge A, Erbe I, Heller S, Burk J. Automated freeze-thaw cycles for decellularization of tendon tissue - a pilot study.. BMC Biotechnol 2017 Feb 14;17(1):13.
    doi: 10.1186/s12896-017-0329-6pmc: PMC5307874pubmed: 28193263google scholar: lookup
  37. Roth SP, Brehm W, Groß C, Scheibe P, Schubert S, Burk J. Transforming Growth Factor Beta 3-Loaded Decellularized Equine Tendon Matrix for Orthopedic Tissue Engineering.. Int J Mol Sci 2019 Nov 3;20(21).
    doi: 10.3390/ijms20215474pmc: PMC6862173pubmed: 31684150google scholar: lookup
  38. Rodríguez-Vita J, Sánchez-Galán E, Santamaría B, Sánchez-López E, Rodrigues-Díez R, Blanco-Colio LM, Egido J, Ortiz A, Ruiz-Ortega M. Essential role of TGF-beta/Smad pathway on statin dependent vascular smooth muscle cell regulation.. PLoS One 2008;3(12):e3959.
    doi: 10.1371/journal.pone.0003959pmc: PMC2597201pubmed: 19088845google scholar: lookup
  39. Rys JP, DuFort CC, Monteiro DA, Baird MA, Oses-Prieto JA, Chand S, Burlingame AL, Davidson MW, Alliston TN. Discrete spatial organization of TGFβ receptors couples receptor multimerization and signaling to cellular tension.. Elife 2015 Dec 10;4:e09300.
    doi: 10.7554/eLife.09300pmc: PMC4728123pubmed: 26652004google scholar: lookup
  40. Kamaraju AK, Roberts AB. Role of Rho/ROCK and p38 MAP kinase pathways in transforming growth factor-beta-mediated Smad-dependent growth inhibition of human breast carcinoma cells in vivo.. J Biol Chem 2005 Jan 14;280(2):1024-36.
    pubmed: 15520018doi: 10.1074/jbc.m403960200google scholar: lookup
  41. Munger JS, Sheppard D. Cross talk among TGF-β signaling pathways, integrins, and the extracellular matrix.. Cold Spring Harb Perspect Biol 2011 Nov 1;3(11):a005017.
    pmc: PMC3220354pubmed: 21900405doi: 10.1101/cshperspect.a005017google scholar: lookup
  42. Hinz B. The extracellular matrix and transforming growth factor-β1: Tale of a strained relationship.. Matrix Biol 2015 Sep;47:54-65.
    pubmed: 25960420doi: 10.1016/j.matbio.2015.05.006google scholar: lookup

Citations

This article has been cited 12 times.
Subscribe to our newsletter
Join 99,383 horse owners like you who receive our email updates on horse health and nutrition.