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Dose-dependent pro- or anti-scar-preventing responses of freeze-dried amniotic membrane extract in relation to suppression of connective tissue growth factor and α-smooth muscle actin

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Abstract

Purpose

Dose-dependent pro- or anti-scar-preventing effects of amniotic membrane extract (AME) are unclear. This study investigated the dose-dependent effects of freeze-dried AME on connective tissue growth factor (CTGF) and α-smooth muscle action (α-SMA) (key determinants of excessive scarring) expression in an attempt to develop medication for scarless wound healing.

Methods

The inhibitory effect of freeze-dried AME on α-SMA was visualized through immunofluorescence confocal microscopy. The suppressive effect of 10 different concentrations of AME (0.0025–0.25 µg/mL) on CTGF and α-SMA was determined, and rates of collagen lattice contraction were measured.

Results

AME inhibited the trans-differentiation of fibroblasts to myofibroblasts, a hallmark of scar formation. AME downregulated CTGF and α-SMA in a dose-dependent manner; supplementation of 0.0175 µg/mL of AME in cell culture media downregulated CTGF (by 49%; p < 0.05) and α-SMA (by 33%; p < 0.05) relative to control values, the effect saturating with a subsequent increase in AME levels to 0.025 µg/mL. Collagen lattice contraction was maximally inhibited (75 ± 8%) upon treatment with 0.0175 µg/mL AME relative to the control (44 ± 6%).

Conclusion

AME downregulates α-SMA and inhibits the trans-differentiation of fibroblasts to myofibroblasts in a dose-dependent manner. Treatment of cells with high AME concentrations retained high α-SMA concentration levels. A delicate balance between the wound healing properties and pro-fibrotic abilities of AME should be considered for selecting an appropriate therapeutic dose.

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Change history

  • 08 May 2021

    The original version of this article has been revised: The second author's name has been corrected.

References

  • Blom IE, Goldschmeding R, Leask A (2002) Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy? Matrix Biol 21:473–482

    Article  CAS  Google Scholar 

  • Bose B (1979) Burn wound dressing with human amniotic membrane. Ann R Coll Surg Engl 61:444–447

    CAS  PubMed  PubMed Central  Google Scholar 

  • Brown G, Curtsinger L, White M, Mitchell RO, Pietsch J, Nordquist R, Fraunhofer A, Schultz GS (1988) Acceleration of tensile strength of incisions treated with EGF and TGF-beta. Ann Surg 208:788–794

    Article  CAS  Google Scholar 

  • Campaner AB, Ferreira LM, Gragnani A, Bruder JM, Cusick JL, Morgan JR (2006) Upregulation of TGF-β1 expression may be necessary but is not sufficient for excessive scarring. J Investig Dermatol 126:1168–1176

    Article  CAS  Google Scholar 

  • Chen Z, Zhang N, Chu HY, Yu Y, Zhang ZK, Zhang G, Zhang BT (2020) Connective tissue growth factor: from molecular understandings to drug discovery. Front Cell Dev Biol 29:93269. https://doi.org/10.3389/fcell.2020.593269

    Article  Google Scholar 

  • Cho Lee AR, Lim H, Lee J, Park KC (2005) Reversal of silver sulfadiazine impaired wound healing by epidermal growth factor. Biomaterials 26:4670–4676

    Article  Google Scholar 

  • Choi YS, Kim JY, Wee WR, Lee JH (1998) Effect of the application of human amniotic membrane on rabbit corneal wound healing after excimer laser photorefractive keratectomy. Cornea 17(4):389–395

    Article  CAS  Google Scholar 

  • Dudok D, Nagdee I, Cheung K, Liu H, Vedovelli L, Ghinelli E, Kenyon K, Parapuram S (2015) Effects of amniotic membrane extract on primary human corneal epithelial and limbal cells. Clin Exp Ophthalmol 43:443–448

    Article  Google Scholar 

  • Eisinger M, Sadan S, Silver IA, Flick RB (1988) Growth regulation of skin cells by epidermal cell-derived factors: implications for wound healing. Proc Natl Acad Sci USA 85:1937–1941

    Article  CAS  Google Scholar 

  • Fetterolf DE, Snyder RJ (2012) Scientific and clinical support for the use of dehydrated amniotic membrane in wound management. Wounds 24(10):299–307

    PubMed  Google Scholar 

  • Gajiwala K, Gajiwala AL (2004) Evaluation of lyophilized, gamma-irradiated amnion as a biological dressing. Cell Tissue Bank 5:73–80

    Article  Google Scholar 

  • Garrett Q, Khaw PT, Blalock TD, Schultz GS, Grotendorst GR et al (2004) Involvement of CTGF in TGF-1 stimulation of myofibroblast differentiation and collagen matrix contraction in the presence of mechanical stress. Investig Ophthalmol Vis Sci 45:1109–1116

    Article  Google Scholar 

  • Goldberg SR, Diegelmann RF (2010) Wound healing primer. Surg Clin N Am 90(6):1133–1146

    Article  Google Scholar 

  • Harris DC (1994) Quantitative chemical analysis, 4th edn. W.H. Freeman and Company, New York, pp 62–67

    Google Scholar 

  • Hao Y, Hui-Kang D, Hwang DG, Kim WS, Zhang F (2000) Identification of antiangiogenic and antiinflammatory proteins in human amniotic membrane. Cornea 19(3):348–352

    Article  CAS  Google Scholar 

  • He H, Li W, Tseng S, Zhang S, Chen SY, Day AJ, Tseng G (2009) Biochemical characterization and function of complexes formed by hyaluronan and the heavy chains of inter-α-inhibitor (HC∙HA) purified from extracts of human amniotic membrane. J Biol Chem 284(30):20136–20146

    Article  CAS  Google Scholar 

  • Hemmann S, Graf J, Roderfeld M, Roeb E (2007) Expression of MMPs and TIMPs in liver fibrosis—a systematic review with special emphasis on anti-fibrotic strategies. J Hepatol 46:955–975

    Article  CAS  Google Scholar 

  • Imazumi R, Akasaka Y, Inomata N, Okada E, Ito K, Ishikawa Y, Maruma Y (2009) Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions. Histopathology 54:722–730

    Article  Google Scholar 

  • Kang M, Choi S, Cho Lee AR (2013) Effect of freeze-dried bovine amniotic membrane extract on full thickness wound healing. Arch Pharmacal Res 36:472–481

    Article  CAS  Google Scholar 

  • Kim JS, Kim JC, Na BK, Jeong JM, Song CY (2000) Amniotic membrane patching promotes healing and inhibits proteinase activity on wound healing following acute corneal alkali burn. Exp Eye Res 709:329–337

    Article  Google Scholar 

  • Koizumi NJ, Inatomi TJ, Sotozono CJ (2000) Growth factor mRNA and protein in preserved human amniotic membrane. Curr Eye Res 20:173–177

    Article  CAS  Google Scholar 

  • Koob T, Lim J, Massee M, Zabek N, Rennert R, Gurtner G, Li W (2014) Angiogenic properties of dehydrated human amnion/chorion allografts: therapeutic potential for soft tissue repair and regeneration. Vasc Cell 6:10

    Article  Google Scholar 

  • Lee J, Cho Lee AR, Lee JH (2012) Method for extracting component of Amnion membrane by solubilization of amnion membranes and extract thereof. Patent No. 10-1187423, Korea

  • Li W, He H, Chen YT, Hayashida Y, Tseng S (2008) Reversal of myofibroblasts by amniotic membrane stromal extract. J Cell Physiol 215(3):657–664

    Article  CAS  Google Scholar 

  • Liang L, Li W, Ling S, Sheha H, Qiu W, Li C, Liu Z (2009) Amniotic membrane extraction solution for ocular chemical burns. Clin Exp Ophthalmol 37:855–863

    Article  Google Scholar 

  • Mahbod M, Shahhoseini S, Khabazkhoob M, Beheshtnehad A, Bakhshandeh G, Atyabi F, Hashemi H (2014) Amniotic membrane extract preparation: what is the best method? J Ophthalmic Vis Res 9(3):314–319

    PubMed  PubMed Central  Google Scholar 

  • Moon H, Yong H, Cho Lee AR (2012) Optimum scratch assay condition to evaluate connective tissue growth factor expression for anti-scar therapy. Arch Pharmacal Res 35:383–388

    Article  CAS  Google Scholar 

  • Nakamura T, Yoshitami M, Rigby H, Fullwood NJ, Ito W, Inatomi T, Sotosomo C, Nakamura T, Shimiz Y, Kinoshita S (2004) Sterilized, freeze-dried amniotic membrane: a useful substrate for ocular surface reconstruction. Investig Ophthalmol Vis Sci 45:93–99

    Article  Google Scholar 

  • Piscatelli SJ, Michaels B, Gregory P, Jennings RW, Longaker MT, Harrison MR, Siebert JW (1994) Fetal fibroblast contraction of collagen matrices in vitro: the effects of epidermal growth factor and transforming growth factor-b. Ann Plast Surg 33:38–45

    Article  CAS  Google Scholar 

  • Rahman I, Said DG, Maharajan VS, Dua HS (2009) Amniotic membrane in ophthalmology: indications and limitations. In: Cambridge ophthalmology symposium, pp 1954–1961

  • Riau AK, Beuerman RW, Lim LS, Mehta JS (2010) Preservation, sterilization and de-epithelialization of human amniotic membrane for use in ocular surface reconstruction. Biomaterials 31:216–225

    Article  CAS  Google Scholar 

  • Röck T, Bartz-Schmidt KU, Landenberger J, Bramkamp M, Röck D (2018) Amniotic membrane transplantation in reconstructive and regenerative ophthalmology. Ann Transplant 23:160–165

    Article  Google Scholar 

  • Russo A, Bonci P, Bonci P (2012) The effects of different preservation processes on the total protein and growth factor content in a new biological product developed from human amniotic membrane. Cell Tissue Bank 13:353–361

    Article  CAS  Google Scholar 

  • Stevens LJ, Page-McCaw A (2012) A secreted MMP is required for reepithelialization during wound healing. Mol Biol Cell 23:1068–1079

    Article  CAS  Google Scholar 

  • Tseng SCG (2001) Amniotic membrane transplantation for ocular surface reconstruction. Biosci Rep 21:481–489

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the Duksung Women’s University Research Grants 2018. We thank Professor Kyung Chan Park, Department of Dermatology, School of Medicine, Seoul National University for providing human primary dermal fibroblasts and his scientific inputs.

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Authors and Affiliations

  1. College of Pharmacy, Duksung Women’s University, Ssangmun-dong 419, Dobong-ku, Seoul, 132-714, Korea

    Mirin Kang & Ae-Ri Cho Lee

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  1. Mirin Kang
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  2. Ae-Ri Cho Lee
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Correspondence to Ae-Ri Cho Lee.

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All authors (M. Kang and A.-R. Cho Lee) declare no conflict of interest with any person or any organization.

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The original version of this article has been revised: The second author’s name has been corrected.

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Kang, M., Cho Lee, AR. Dose-dependent pro- or anti-scar-preventing responses of freeze-dried amniotic membrane extract in relation to suppression of connective tissue growth factor and α-smooth muscle actin. J. Pharm. Investig. 51, 327–336 (2021). https://doi.org/10.1007/s40005-021-00518-y

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