In vitro and in vivo evaluation of antifibrotic properties of verteporfin as a composition of a collagen scaffold

Cover Page

Cite item

Full Text

Open Access Open Access
Restricted Access Access granted
Restricted Access Subscription Access

Abstract

Extensive skin damage requires specialized therapy that stimulates regeneration processes without scarring. In vivo and in vitro we examined the possibility of collagen gel application as a wound dressing and fibroblast attractant in combination with verteporfin as an antifibrotic agent. In vitro the effects of verteporfin on viability and myofibroblast markers expression were evaluated using fibroblasts isolated from human scar tissue. In vivo collagen gel and verteporfin (individually and in combination) were loaded into the wound to investigate the scarring signature during skin regeneration: deviations in skin layer thickness, collagen synthesis, and extracellular matrix fiber characteristics. The results indicate that verteporfin reduces the fibrotic phenotype by suppressing the expression of the contractile protein Sm22α without inducing cell death. However, the administration of verteporfin inside collagen gel interrupts its ability to direct wound healing in a scarless manner, which may be related to the incompatibility of the mechanisms by which collagen and verteporfin control regeneration.

Full Text

Restricted Access

About the authors

O. S. Rogovaya

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Author for correspondence.
Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

D. S. Abolin

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

O. L. Cherkashina

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

A. D. Smyslov

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

E. A. Vorotelyak

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

Е. P. Kalabusheva

Koltzov Institute of Developmental Biology of the Russian Academy of Sciences

Email: rogovaya26f@yandex.ru
Russian Federation, 119334, Moscow

References

  1. Cañedo-Dorantes, L., and Cañedo-Ayala, M. (2019) Skin acute wound healing: a comprehensive review, Int. J. Inflamm., 3706315, https://doi.org/10.1155/2019/3706315.
  2. Xue, M., and Jackson, C. J. (2015) Extracellular matrix reorganization during wound healing and its impact on abnormal scarring, Adv. Wound Care, 4, 119-136, https://doi.org/10.1089/wound.2013.0485.
  3. Karppinen, S.-M., Heljasvaara, R., Gullberg, D., Tasanen, K., and Pihlajaniemi, T. (2019) Toward understanding scarless skin wound healing and pathological scarring, F1000Res., 8, 787, https://doi.org/10.12688/f1000research.18293.1.
  4. Tomasek, J. J., Gabbiani, G., Hinz, B., Chaponnier, C., and Brown, R. A. (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling, Nat. Rev. Mol. Cell Biol., 3, 349-363, https://doi.org/10.1038/nrm809.
  5. Eudy, M., Eudy, C. L., and Roy, S. (2021) Apligraf as an alternative to skin grafting in the pediatric population, Cureus, 13, e16226, https://doi.org/10.7759/cureus.16226.
  6. Dibbs, R. P., Depani, M., and Thornton, J. F. (2022) Technical refinements with the use of biologic healing agents, Semin. Plast. Surg., 36, 008-016, https://doi.org/10.1055/s-0042-1742749.
  7. El Masry, M. S., Chaffee, S., Das Ghatak, P., Mathew-Steiner, S. S., Das, A., Higuita-Castro, N., Roy, S., Anani, R. A., and Sen, C. K. (2019) Stabilized collagen matrix dressing improves wound macrophage function and epithelialization, FASEB J., 33, 2144-2155, https://doi.org/10.1096/fj.201800352R.
  8. Parenteau-Bareil, R., Gauvin, R., and Berthod, F. (2010) Collagen-based biomaterials for tissue engineering applications, Materials, 3, 1863-1887, https://doi.org/10.3390/ma3031863.
  9. Mouw, J. K., Ou, G., and Weaver, V. M. (2014) Extracellular matrix assembly: a multiscale deconstruction, Nat. Rev. Mol. Cell Biol., 15, 771-785, https://doi.org/10.1038/nrm3902.
  10. Hynes, R. O. (2014) Stretching the boundaries of extracellular matrix research, Nat. Rev. Mol. Cell Biol., 15, 761-763, https://doi.org/10.1038/nrm3908.
  11. Mathew-Steiner, S. S., Roy, S., and Sen, C. K. (2021) Collagen in wound healing, Bioengineering, 8, 63, https://doi.org/10.3390/bioengineering8050063.
  12. Zhang, Y., Wang, Y., Li, Y., Yang, Y., Jin, M., Lin, X., Zhuang, Z., Guo, K., Zhang, T., and Tan, W. (2023) Application of collagen-based hydrogel in skin wound healing, Gels, 9, 185, https://doi.org/10.3390/gels9030185.
  13. Potter, D. A., Veitch, D., and Johnston, G. A. (2019) Scarring and wound healing, Br. J. Hosp. Med. Lond. Engl., 80, C166-C171, https://doi.org/10.12968/hmed.2019.80.11.C166.
  14. Chen, K., Liu, Y., Liu, X., Guo, Y., Liu, J., Ding, J., Zhang, Z., Ni, X., and Chen, Y. (2023) Hyaluronic acid-modified and verteporfin-loaded polylactic acid nanogels promote scarless wound healing by accelerating wound re-epithelialization and controlling scar formation, J. Nanobiotechnol., 21, 241, https://doi.org/10.1186/s12951-023-02014-x.
  15. Wei, C., You, C., Zhou, L., Liu, H., Zhou, S., Wang, X., and Guo, R. (2023) Antimicrobial hydrogel microneedle loading verteporfin promotes skin regeneration by blocking mechanotransduction signaling, Chem. Eng. J., 472, 144866, https://doi.org/10.1016/j.cej.2023.144866.
  16. Zhang, C., Yang, D., Wang, T.-B., Nie, X., Chen, G., Wang, L.-H., You, Y.-Z., and Wang, Q. (2022) Biodegradable hydrogels with photodynamic antibacterial activity promote wound healing and mitigate scar formation, Biomater. Sci., 11, 288-297, https://doi.org/10.1039/D2BM01493A.
  17. Zhang, Y., Wang, S., Yang, Y., Zhao, S., You, J., Wang, J., Cai, J., Wang, H., Wang, J., Zhang, W., Yu, J., Han, C., Zhang, Y., and Gu, Z. (2023) Scarless wound healing programmed by core-shell microneedles, Nat. Commun., 14, 3431, https://doi.org/10.1038/s41467-023-39129-6.
  18. Mascharak, S., Talbott, H. E., Januszyk, M., Griffin, M., Chen, K., Davitt, M. F., Demeter, J., Henn, D., Bonham, C. A., Foster, D. S., Mooney, N., Cheng, R., Jackson, P. K., Wan, D. C., Gurtner, G. C., and Longaker, M. T. (2022) Multi-Omic analysis reveals divergent molecular events in scarring and regenerative wound healing, Cell Stem Cell, 29, 315-327.e6, https://doi.org/10.1016/j.stem.2021.12.011.
  19. Lee, M.-J., Byun, M. R., Furutani-Seiki, M., Hong, J.-H., and Jung, H.-S. (2014) YAP and TAZ regulate skin wound healing, J. Invest. Dermatol., 134, 518-525, https://doi.org/10.1038/jid.2013.339.
  20. Mascharak, S., desJardins-Park, H. E., Davitt, M. F., Griffin, M., Borrelli, M. R., Moore, A. L., Chen, K., Duoto, B., Chinta, M., Foster, D. S., Shen, A. H., Januszyk, M., Kwon, S. H., Wernig, G., Wan, D. C., Lorenz, H. P., Gurtner, G. C., and Longaker, M. T. (2021) Preventing engrailed-1 activation in fibroblasts yields wound regeneration without scarring, Science, 372, eaba2374, https://doi.org/10.1126/science.aba2374.
  21. International Guiding Principles for Biomedical Research Involving Animals (1985) In The Development of Science-based Guidelines for Laboratory Animal Care: Proceedings of the November 2003 International Workshop; National Academies Press (US), 2004.
  22. European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes, URL: https://www.ecolex.org/details/treaty/european-convention-for-the-protection-of-vertebrate-animals-used-for-experimental-and-other-scientific-purposes-tre-001042/ (accessed on November 26, 2023).
  23. Bredfeldt, J. S., Liu, Y., Pehlke, C. A., Conklin, M. W., Szulczewski, J. M., Inman, D. R., Keely, P. J., Nowak, R. D., Mackie, T. R., and Eliceiri, K. W. (2014) Computational segmentation of collagen fibers from second-harmonic generation images of breast cancer, J. Biomed. Opt., 19, 16007, https://doi.org/10.1117/1.JBO. 19.1.016007.
  24. Liu, Y., Keikhosravi, A., Mehta, G. S., Drifka, C. R., and Eliceiri, K. W. (2017) Methods for quantifying fibrillar collagen alignment, Methods Mol. Biol., 1627, 429-451, https://doi.org/10.1007/978-1-4939-7113-8_28.
  25. Mussbacher, M., Salzmann, M., Brostjan, C., Hoesel, B., Schoergenhofer, C., Datler, H., Hohensinner, P., Basílio, J., Petzelbauer, P., Assinger, A., and Schmid, J. A. (2019) Cell type-specific roles of NF-κB linking inflammation and thrombosis, Front. Immunol., 10, 85, https://doi.org/10.3389/fimmu.2019.00085.
  26. Wnuk, D., Lasota, S., Paw, M., Madeja, Z., and Michalik, M. (2020) Asthma-derived fibroblast to myofibroblast transition is enhanced in comparison to fibroblasts derived from non-asthmatic patients in 3D in vitro culture due to Smad2/3 signalling, Acta Biochim. Pol., 67, 441-448, https://doi.org/10.18388/ abp.2020_5412.
  27. Shin, D., and Minn, K. W. (2004) The effect of myofibroblast on contracture of hypertrophic scar, Plast. Reconstr. Surg., 113, 633-640, https://doi.org/10.1097/01.PRS.0000101530.33096.5B.
  28. Wang, C., Zhu, X., Feng, W., Yu, Y., Jeong, K., Guo, W., Lu, Y., and Mills, G. B. (2016) Verteporfin inhibits YAP function through Up-regulating 14-3-3σ sequestering YAP in the cytoplasm, Am. J. Cancer Res., 6, 27-37.
  29. Shi-wen, X., Racanelli, M., Ali, A., Simon, A., Quesnel, K., Stratton, R. J., and Leask, A. (2021) Verteporfin inhibits the persistent fibrotic phenotype of lesional scleroderma dermal fibroblasts, J. Cell Commun. Signal., 15, 71-80, https://doi.org/10.1007/s12079-020-00596-x.
  30. El Ayadi, A., and Jay, J. W., Prasai, A. (2020) Current approaches targeting the wound healing phases to attenuate fibrosis and scarring, Int. J. Mol. Sci., 21, 1105, https://doi.org/10.3390/ijms21031105.
  31. Zlobina, K., Malekos, E., Chen, H., and Gomez, M. (2023) Robust classification of wound healing stages in both mice and humans for acute and burn wounds based on transcriptomic data, BMC Bioinformatics, 24, 166, https://doi.org/10.1186/s12859-023-05295-z.
  32. Galiano, R. D., Michaels, J., Dobryansky, M., Levine, J. P., and Gurtner, G. C. (2004) Quantitative and reproducible murine model of excisional wound healing, Wound Repair Regen., 12, 485-492, https://doi.org/10.1111/j.1067-1927.2004.12404.x.
  33. Ud-Din, S., Foden, P., Stocking, K., Mazhari, M., Al-Habba, S., Baguneid, M., McGeorge, D., and Bayat, A. (2019) Objective assessment of dermal fibrosis in cutaneous scarring, using optical coherence tomography, high-frequency ultrasound and immunohistomorphometry of human skin, Br. J. Dermatol., 181, 722-732, https://doi.org/10.1111/bjd.17739.
  34. Henn, D., Chen, K., Fehlmann, T., Trotsyuk, A. A., Sivaraj, D., Maan, Z. N., Bonham, C. A., Barrera, J. A., Mays, C. J., Greco, A. H., Moortgat Illouz, S. E., Lin, J. Q., Steele, S. R., Foster, D. S., Padmanabhan, J., Momeni, A., Nguyen, D., Wan, D. C., Kneser, U., Januszyk, M., Keller, A., Longaker, M. T., and Gurtner, G. C. (2021) Xenogeneic skin transplantation promotes angiogenesis and tissue regeneration through activated Trem2+ macrophages, Sci. Adv., 7, eabi4528, https://doi.org/10.1126/sciadv.abi4528.
  35. Brett, D. A (2008) Review of collagen and collagen-based wound dressings, Wounds Compend. Clin. Res. Pract., 20, 347-356.
  36. Ge, B., Wang, H., Li, J., Liu, H., Yin, Y., Zhang, N., and Qin, S. (2020) Comprehensive assessment of Nile tilapia skin (Oreochromis Niloticus) collagen hydrogels for wound dressings, Mar. Drugs, 18, 178, https://doi.org/10.3390/md18040178.
  37. Chermnykh, E. S., Kiseleva, E. V., Rogovaya, O. S., Rippa, A. L., Vasiliev, A. V., and Vorotelyak, E. A. (2018) Tissue-engineered biological dressing accelerates skin wound healing in mice via formation of provisional connective tissue, Histol. Histopathol., 33, 1189-1199, https://doi.org/10.14670/HH-18-006.

Supplementary files

Supplementary Files
Action
1. JATS XML
Download
2. Fig. 1. Assessment of death and proliferation of different types of fibroblasts (norm and scar) under the influence of VP. a is the proportion of living cells after VP treatment. b is the proportion of dying cells after treatment with VP. b is the number of proliferating cells. The data on the graphs is presented as an average with a spread in the form of a standard deviation. Statistically significant differences: ** p ≤ 0.01 and **** p ≤ 0.0001

Download (147KB)
Indexing metadata
3. Fig. 2. Analysis of the cell cycle of normal (norm) and scarred (scar) fibroblasts after exposure to VP. The data on the graphs are presented as an average with a spread in the form of a standard deviation

Download (118KB)
Indexing metadata
4. Fig. 3. The effect of VP exposure on the contracting abilities and phenotype of different types of fibroblasts (norm and scar) in collagen gel. a – Dynamics of collagen gel contraction. b – Immunofluorescence detection of Sm22a (green staining) and YAP1 (red staining). The inserts show large-scale cells with cytoplasmic localization of YAP1. The cores are painted with DAPI. Confocal microscopy. The scale segment is 100 microns

Download (197KB)
Indexing metadata
5. Fig. 4. Quantitative PCR analysis of fibrotic markers expressed by fibroblasts of different types (norm and scar) in collagen gel on day 5 after exposure to VP. The graphs are presented as an average with a spread in the form of a standard deviation. The results were normalized to the level of GAPDH expression. Statistically significant difference between the scar and the norm: * p < 0.05; ** p < 0.01; *** p < 0.001. Statistically significant difference from the group "Norm + VP": # p < 0.05; ## p < 0.01

Download (130KB)
Indexing metadata
6. Fig. 5. Analysis of the dynamics of wound healing based on morphometric parameters. a – The appearance of the wound in different groups on the 0, 7, 14 and 22 postoperative days; b – the dynamics of wound closure during the 22 days of the experiment; c – the rate of wound closure in the first week. *** p < 0.001 relative to the control group; ### p < 0.001 relative to the "VP" group. g – Measurement of the thickness of the epidermis in the wound area. d – Measurement of the thickness of the dermis in the wound area, * p < 0.05. The data on the graphs are presented as an average with a spread in the form of a standard deviation

Download (257KB)
Indexing metadata
7. 6. Immunofluorescence detection of wound healing markers: collagens I + III, P-cadherin, E-cadherin, YAP on slices of skin wounds of mice on the 22nd postoperative day in different groups. Fluorescence microscopy. The scale segment is 100 microns

Download (217KB)
Indexing metadata
8. Fig. 7. Digital analysis of collagen fibers on histological preparations of skin wounds of mice on the 22nd postoperative day. a – Visualization of the stages of analysis of the structure of collagen fibers on histological preparations using heat maps constructed using CT-FIRE and CurveAlign algorithms. b – Analysis of the main components based on CT-FIRE and CurveAlign data for the "Control" and "VP" groups. b – The directions of the original coordinate axes in the space of the main components. g – Analysis of the main components based on CT-FIRE and CurveAlign data for the "Gel" and "Gel + VP" groups. d – The directions of the original coordinate axes in the space of the main components. e – Analysis of the main components based on CT-FIRE and CurveAlign data for all experimental groups. g – Directions of the initial coordinate axes in the space of the main components

Download (439KB)
Indexing metadata

Copyright (c) 2024 Russian Academy of Sciences