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Roles of early events in the modifications undergone by bovine corneal endothelial cells during wound healing

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Abstract

A mechanical injury in bovine corneal endothelial (BCE) cells in culture induces: (1) a fast calcium wave (FCW); (2) slow increases in cytosolic sodium and calcium, critical for the healing process, and (3) a rise in the apoptotic rate with respect to quiescent cells. In order to investigate the nature of the stimuli that determine the ionic changes and apoptotic response, we performed here studies on a non-injury model of tissue restitution in BCE monolayers. For this, we employed cell cultures grown to confluence in the presence of a Parafilm strip. We observed that, previously to strip removal, most of the border cells had already developed the slow ionic modifications, while in the scratch wounds these changes gradually occur after several hours of healing. This finding suggests that, in BCE cells, the presence of a free edge is sufficient to trigger ionic modifications necessary for wound healing and to elicit an augmented apoptotic response. The apoptotic index of the migrating cells in the Parafilm model (PF) was determined to be approximately two-fold the one of scratch wounds, a result that, in agreement with our previous observations, we attributed to the absence of the FCW in the PF experiments. The findings of this work further contribute to the understanding of epithelial wound healing, a crucial adaptive, and homeostatic response.

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Data that support the findings of this study are available from the authors upon reasonable request.

References

  1. Horowitz JC, Thannickal VJ (2019) Mechanisms for the resolution of organ fibrosis. Physiology (Bethesda) 34:43–55. https://doi.org/10.1152/physiol.00033.2018

    Article  CAS  Google Scholar 

  2. Sommer K, Wiendl M, Muller TM, Heidbreder K, Voskens C, Neurath MF, Zundler S (2021) Intestinal mucosal wound healing and barrier integrity in IBD-crosstalk and trafficking of cellular players. Front Med (Lausanne) 8:643973. https://doi.org/10.3389/fmed.2021.643973

    Article  Google Scholar 

  3. Deyell M, Garris CS, Laughney AM (2021) Cancer metastasis as a non-healing wound. Br J Cancer 124:1491–1502. https://doi.org/10.1038/s41416-021-01309-w

    Article  Google Scholar 

  4. Ross MH, Pawlina W (2016) Histology: a text and atlas: with correlated cell and molecular biology. Lippincott Williams & Wilkins, Philadelphia

    Google Scholar 

  5. Silverthorn DU (2018) Human physiology: an integrated approach, 7th edn. Pearson, Upper Saddle River

    Google Scholar 

  6. Murrell M, Kamm R, Matsudaira P (2011) Tension, free space, and cell damage in a microfluidic wound healing assay. PLoS ONE 6:e24283. https://doi.org/10.1371/journal.pone.0024283

    Article  CAS  Google Scholar 

  7. Furuya K, Sokabe M, Grygorczyk R (2014) Real-time luminescence imaging of cellular ATP release. Methods 66:330–344. https://doi.org/10.1016/j.ymeth.2013.08.007

    Article  CAS  Google Scholar 

  8. Niethammer P (2016) The early wound signals. Curr Opin Genet Dev 40:17–22. https://doi.org/10.1016/j.gde.2016.05.001

    Article  CAS  Google Scholar 

  9. Matsubayashi Y, Ebisuya M, Honjoh S, Nishida E (2004) ERK activation propagates in epithelial cell sheets and regulates their migration during wound healing. Curr Biol 14:731–735. https://doi.org/10.1016/j.cub.2004.03.060

    Article  CAS  Google Scholar 

  10. Chifflet S, Hernandez JA, Grasso S (2005) A possible role for membrane depolarization in epithelial wound healing. Am J Physiol Cell Physiol 288:C1420–C1430. https://doi.org/10.1152/ajpcell.00259.2004

    Article  CAS  Google Scholar 

  11. Chifflet S, Justet C, Hernandez JA, Nin V, Escande C, Benech JC (2012) Early and late calcium waves during wound healing in corneal endothelial cells. Wound Repair Regen 20:28–37. https://doi.org/10.1111/j.1524-475X.2011.00749.x

    Article  Google Scholar 

  12. Justet C, Evans F, Vasilskis E, Hernandez JA, Chifflet S (2013) ENaC contribution to epithelial wound healing is independent of the healing mode and of any increased expression in the channel. Cell Tissue Res 353:53–64. https://doi.org/10.1007/s00441-013-1635-5

    Article  CAS  Google Scholar 

  13. Block ER, Matela AR, SundarRaj N, Iszkula ER, Klarlund JK (2004) Wounding induces motility in sheets of corneal epithelial cells through loss of spatial constraints: role of heparin-binding epidermal growth factor-like growth factor signaling. J Biol Chem 279:24307–24312. https://doi.org/10.1074/jbc.M401058200

    Article  CAS  Google Scholar 

  14. Streichan SJ, Hoerner CR, Schneidt T, Holzer D, Hufnagel L (2014) Spatial constraints control cell proliferation in tissues. Proc Natl Acad Sci USA 111:5586–5591. https://doi.org/10.1073/pnas.1323016111

    Article  CAS  Google Scholar 

  15. Javaherian S, O’Donnell KA, McGuigan AP (2011) A fast and accessible methodology for micro-patterning cells on standard culture substrates using Parafilm inserts. PLoS ONE 6:e20909. https://doi.org/10.1371/journal.pone.0020909

    Article  CAS  Google Scholar 

  16. Luan S, Hao R, Wei Y, Chen D, Fan B, Dong F, Guo W, Wang J, Chen J (2017) A microfabricated 96-well wound-healing assay. Cytometry A 91:1192–1199. https://doi.org/10.1002/cyto.a.23286

    Article  CAS  Google Scholar 

  17. Justet C, Hernandez JA, Torriglia A, Chifflet S (2016) Fast calcium wave inhibits excessive apoptosis during epithelial wound healing. Cell Tissue Res 365:343–356. https://doi.org/10.1007/s00441-016-2388-8

    Article  CAS  Google Scholar 

  18. Gordon SR (2009) Cell migration along the basement membrane during wound repair. The corneal endothelium as a model system. In: Gefen A (ed) Bioengineering research of chronic wounds: a multidisciplinary study approach. Springer, Berlin, pp 43–84

    Chapter  Google Scholar 

  19. Eghrari AO, Riazuddin SA, Gottsch JD (2015) Overview of the cornea: structure, function, and development. Prog Mol Biol Transl Sci 134:7–23. https://doi.org/10.1016/bs.pmbts.2015.04.001

    Article  Google Scholar 

  20. Català P, Thuret G, Skottman H, Mehta JS, Parekh M, Ní Dhubhghaill S, Collin RWJ, Nuijts RMMA, Ferrari S, LaPointe VLS, Dickman MM (2022) Approaches for corneal endothelium regenerative medicine. Prog Retin Eye Res 87:100987. https://doi.org/10.1016/j.preteyeres.2021.100987

    Article  Google Scholar 

  21. Jurkunas UV, Bitar MS, Funaki T, Azizi B (2010) Evidence of oxidative stress in the pathogenesis of fuchs endothelial corneal dystrophy. Am J Pathol 177:2278–2289. https://doi.org/10.2353/ajpath.2010.100279

    Article  CAS  Google Scholar 

  22. Chifflet S, Hernandez JA, Grasso S, Cirillo A (2003) Nonspecific depolarization of the plasma membrane potential induces cytoskeletal modifications of bovine corneal endothelial cells in culture. Exp Cell Res 282:1–13

    Article  CAS  Google Scholar 

  23. Grasso S, Hernandez JA, Chifflet S (2007) Roles of wound geometry, wound size, and extracellular matrix in the healing response of bovine corneal endothelial cells in culture. Am J Physiol Cell Physiol 293:C1327–C1337. https://doi.org/10.1152/ajpcell.00001.2007

    Article  CAS  Google Scholar 

  24. Bruni E, Reichle A, Scimeca M, Bonanno E, Ghibelli L (2018) Lowering etoposide doses shifts cell demise from caspase-dependent to differentiation and caspase-3-independent apoptosis via DNA damage response, Inducing AML Culture Extinction. Front Pharmacol 9:1307. https://doi.org/10.3389/fphar.2018.01307

    Article  CAS  Google Scholar 

  25. Yang CF, Zhong YJ, Ma Z, Li L, Shi L, Chen L, Li C, Wu D, Chen Q, Li YW (2018) NOX4/ROS mediate ethanolinduced apoptosis via MAPK signal pathway in L02 cells. Int J Mol Med 41:2306–2316. https://doi.org/10.3892/ijmm.2018.3390

    Article  CAS  Google Scholar 

  26. Altairac S, Zeggai S, Perani P, Courtois Y, Torriglia A (2003) Apoptosis induced by Na+/H+ antiport inhibition activates the LEI/L-DNase II pathway. Cell Death Differ 10:548–557. https://doi.org/10.1038/sj.cdd.4401195

    Article  CAS  Google Scholar 

  27. Kanno S, Fukuda Y (1994) Fibronectin and tenascin in rat tracheal wound healing and their relation to cell proliferation. Pathol Int 44:96–106. https://doi.org/10.1111/j.1440-1827.1994.tb01693.x

    Article  CAS  Google Scholar 

  28. Galluzzi L et al (2018) Molecular mechanisms of cell death: recommendations of the nomenclature committee on cell death 2018. Cell Death Differ 25:486–541. https://doi.org/10.1038/s41418-017-0012-4

    Article  Google Scholar 

  29. Justet C, Chifflet S, Hernandez JA (2019) Calcium oscillatory behavior and its possible role during wound healing in bovine corneal endothelial cells in culture. Biomed Res Int 2019:8647121. https://doi.org/10.1155/2019/8647121

    Article  CAS  Google Scholar 

  30. Shabir S, Southgate J (2008) Calcium signalling in wound-responsive normal human urothelial cell monolayers. Cell Calcium 44:453–464. https://doi.org/10.1016/j.ceca.2008.02.008

    Article  CAS  Google Scholar 

  31. Handly LN, Wollman R (2017) Wound-induced Ca(2+) wave propagates through a simple release and diffusion mechanism. Mol Biol Cell 28:1457–1466. https://doi.org/10.1091/mbc.E16-10-0695

    Article  CAS  Google Scholar 

  32. Poujade M, Grasland-Mongrain E, Hertzog A, Jouanneau J, Chavrier P, Ladoux B, Buguin A, Silberzan P (2007) Collective migration of an epithelial monolayer in response to a model wound. Proc Natl Acad Sci USA 104:15988–15993. https://doi.org/10.1073/pnas.0705062104

    Article  Google Scholar 

  33. Charras G, Yap AS (2018) Tensile forces and mechanotransduction at cell-cell junctions. Curr Biol 28:R445–R457. https://doi.org/10.1016/j.cub.2018.02.003

    Article  CAS  Google Scholar 

  34. Nikolic DL, Boettiger AN, Bar-Sagi D, Carbeck JD, Shvartsman SY (2006) Role of boundary conditions in an experimental model of epithelial wound healing. Am J Physiol Cell Physiol 291:C68-75. https://doi.org/10.1152/ajpcell.00411.2005

    Article  CAS  Google Scholar 

  35. Cordeiro JV, Jacinto A (2013) The role of transcription-independent damage signals in the initiation of epithelial wound healing. Nat Rev Mol Cell Biol 14:249–262

    Article  CAS  Google Scholar 

  36. Li F, Huang Q, Chen J, Peng Y, Roop DR, Bedford JS, Li CY (2010) Apoptotic cells activate the “phoenix rising” pathway to promote wound healing and tissue regeneration. Sci Signal 3:ra13. https://doi.org/10.1126/scisignal.2000634

    Article  CAS  Google Scholar 

  37. Tan JQ, Zhang HH, Lei ZJ, Ren P, Deng C, Li XY, Chen SZ (2013) The roles of autophagy and apoptosis in burn wound progression in rats. Burns 39:1551–1556. https://doi.org/10.1016/j.burns.2013.04.018

    Article  Google Scholar 

  38. Johnson A, Francis M, DiPietro LA (2014) Differential apoptosis in mucosal and dermal wound healing. Adv Wound Care (New Rochelle) 3:751–761. https://doi.org/10.1089/wound.2012.0418

    Article  Google Scholar 

  39. Takada H, Furuya K, Sokabe M (2014) Mechanosensitive ATP release from hemichannels and Ca2+ influx through TRPC6 accelerate wound closure in keratinocytes. J Cell Sci 127:4159–4171. https://doi.org/10.1242/jcs.147314

    Article  CAS  Google Scholar 

  40. Huang Z, Xie N, Illes P, Di Virgilio F, Ulrich H, Semyanov A, Verkhratsky A, Sperlagh B, Yu S-G, Huang C, Tang Y (2021) From purines to purinergic signalling: molecular functions and human diseases. Signal Transduct Target Ther 6:162. https://doi.org/10.1038/s41392-021-00553-z

    Article  CAS  Google Scholar 

  41. Morachevskaya EA, Sudarikova AV (2021) Actin dynamics as critical ion channel regulator: ENaC and Piezo in focus. Am J Physiol Cell Physiol 320:C696–C702. https://doi.org/10.1152/ajpcell.00368.2020

    Article  CAS  Google Scholar 

  42. Jain R, Watson U, Vasudevan L, Saini DK (2018) ERK activation pathways downstream of GPCRs. Int Rev Cell Mol Biol 338:79–109. https://doi.org/10.1016/bs.ircmb.2018.02.003

    Article  CAS  Google Scholar 

  43. van der Vliet A (2008) NADPH oxidases in lung biology and pathology: host defense enzymes, and more. Free Radical Biol Med 44:938–955. https://doi.org/10.1016/j.freeradbiomed.2007.11.016

    Article  CAS  Google Scholar 

  44. Clerkin JS, Naughton R, Quiney C, Cotter TG (2008) Mechanisms of ROS modulated cell survival during carcinogenesis. Cancer Lett 266:30–36. https://doi.org/10.1016/j.canlet.2008.02.029

    Article  CAS  Google Scholar 

  45. Ray PD, Huang B-W, Tsuji Y (2012) Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 24:981–990. https://doi.org/10.1016/j.cellsig.2012.01.008

    Article  CAS  Google Scholar 

  46. Moreno-Caceres J, Mainez J, Mayoral R, Martin-Sanz P, Egea G, Fabregat I (2016) Caveolin-1-dependent activation of the metalloprotease TACE/ADAM17 by TGF-beta in hepatocytes requires activation of Src and the NADPH oxidase NOX1. FEBS J 283:1300–1310. https://doi.org/10.1111/febs.13669

    Article  CAS  Google Scholar 

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Acknowledgements

We thank Drs. Lucía Canclini and Hugo Marcel Rodriguez for expert advice on statistical analysis. We also thank Frigorífico Las Piedras and Frigorífico Lorsinal S.A., for supplying us with fresh bovine eyes.

Funding

This study was supported by grants from the Comisión Sectorial de Investigación Científica (CSIC), Universidad de la República, Uruguay (Proyecto Grupos I + D 2014 to S. Chifflet and Magister and Doctoral Fellowships to C. Justet); and Programa de Desarrollo de las Ciencias Básicas (PEDECIBA), Uruguay (to S. Chifflet and J. A. Hernandez).

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Authors and Affiliations

  1. Departamento de Bioquímica, Facultad de Medicina, Universidad de la República, Gral. Flores 2125, 11800, Montevideo, Uruguay

    Cristian Justet & Silvia Chifflet

  2. Sección Biofísica y Biología de Sistemas, Facultad de Ciencias, Universidad de la República, Iguá s/n esq. Mataojo, 11400, Montevideo, Uruguay

    Julio A. Hernández

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  1. Cristian Justet
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  2. Julio A. Hernández
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  3. Silvia Chifflet
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Contributions

CJ and SC designed research; CJ performed research; CJ, SC and JAH analyzed data; CJ took responsibility for statistical analysis. CJ, SC and JAH wrote the paper.

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Correspondence to Silvia Chifflet.

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Justet, C., Hernández, J.A. & Chifflet, S. Roles of early events in the modifications undergone by bovine corneal endothelial cells during wound healing. Mol Cell Biochem 478, 89–102 (2023). https://doi.org/10.1007/s11010-022-04495-0

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