Abstract
Background:
Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine.
Methods:
Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and MPG∆NLS) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software.
Conclusion:
CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p < 0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF siRNA/MPG∆NLS complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of 128 ± 22 nm (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean 90 ± 20 nm, n = 563).
Conclusion:
Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.
Similar content being viewed by others
References
Block L, Gosain A, King TW. Emerging therapies for scar prevention. Adv Wound Care (New Rochelle). 2015;4:607–14.
Brown BC, Mckenna SP, Siddhi K, McGrouthe DA, Bayat A. The hidden cost of skin scars: quality of life after skin scarring. J Plast Reconstr Aesthet Surg. 2008;61:1049–58.
Young VL, Hutchison J. Insights into patient and clinician concerns about scar appearance: semi quantitative structured surveys. Plast Reconstr Surg. 2009;124:256–65.
Cross KJ, Mustoe TA. Growth factors in wound healing. Surg Clin North Am. 2003;83:531–45.
Reish RG, Eriksson E. Scars: a review of emerging and currently available therapies. Plast Reconstr Surg. 2008;122:1068–78.
Campaner AB, Ferreira LM, Gragnani A, Bruder JM, Cusick JL, Morgan JR. Upregulation of TGF-beta1 expression may be necessary but is not sufficient for excessive scarring. J Invest Dermatol. 2006;126:1168–76.
Blom IE, Goldschmeding R, Leask A. Gene regulation of connective tissue growth factor: new targets for antifibrotic therapy. Matrix Biol. 2002;21:473–82.
Chujo S, Shirasaki F, Kawara S, Inagaki Y, Kinbara T, Inaoki M, et al. Connective tissue growth factor causes persistent proalpha2(I) collagen gene expression induced by transforming growth factor-beta in a mouse fibrosis model. J Cell Physiol. 2005;203:447–56.
Frazier K, Williams S, Kothapalli D, Klapper H, Grotendors GR. Stimulation of fibroblast cell growth, matrix production and granulation tissue formation by CTGF. J Invest Dermatol. 1996;107:404–11.
Sonnylal S, Shi-Wen X, Leoni P, Naff K, Van Pelt CS, Nakamura H, et al. Selective expression of CTGF in fibroblasts in vivo promotes systemic tissue fibrosis. Arthritis Rheum 2010;62:1523–32.
Leask A, Holmes A, Abraham DJ. Connective tissue growth factor: a new and important player in the pathogenesis of fibrosis. Curr Rheumatol Rep. 2002;4:136–42.
Li G, Xie Q, Shi Y, Li D, Zhang M, Jiang S, et al. Inhibition of CTGF by siRNA prevents liver fibrosis in rats. J Gene Med. 2006;8:889–900.
Nishio N, Ito S, Suzuki H, Isobe K. Antibodies to wounded tissue enhance cutaneous wound healing. Immunology. 2009;128:369–80.
Sisco M, Kryger ZB, O’Shaughnessy KD, Kim PS, Schultz GS, Ding XZ, et al. Antisense inhibition of connective tissue growth factor (CTGF/CCN2) mRNA limits hypertrophic scarring without affecting wound healing in vivo. Wound Repair Regen. 2008;16:661–73.
Daniels JT, Schultz GS, Blalock TD, Garrett Q, Grotendorst GR, Dean NM, et al. Mediation of transforming growth factor-beta(1)-stimulated matrix contraction by fibroblasts: a role for connective tissue growth factor in contractile scarring. Am J Pathol. 2003;163:2043–52.
Whitehead KA, Langer R, Anderson DG. Knocking down barriers: advances in siRNA delivery. Nat Rev Drug Discov. 2009;8:129–38.
Eguchi A, Dowdy SF. siRNA delivery using peptide transduction domains. Trends Pharmacol Sci. 2009;30:341–5.
Wang JF, Olson ME, Ma L, Brigstock DR, Hart DA. Connective tissue growth factor siRNA modulates mRNA levels for a subset of molecules in normal and TGF-beta 1-stimulated porcine skin fibroblasts. Wound Repair Regen. 2004;12:205–16.
Berthod F, Germain L, Li H, Xu W, Damour O, Auger FA. Collagen fibril network and elastic system remodeling in a reconstructed skin transplanted on nude mice. Matrix Biol. 2001;20:463–73.
White JF, Werkmeister JA, Darby IA, Bisucci T, Birk DE, Ramshaw JA. Collagen fibril formation in a wound healing model. J Struct Biol. 2002;137:23–30.
Moon H, Yong H, Lee AR. Optimum scratch assay condition to evaluate connective tissue growth factor for anti-scar therapy. Arch Pharm Res. 2012;35:383–8.
Kim SC, Cho Lee A. Preparation of reproducible and responsive scar model and histology analysis. J Pharm Investig. 2010;40:45–9.
Reid RR, Mogford JE, Butt R, deGiorgio-Miller A, Mustoe TA. Inhibition of procollagen C-proteinase reduces scar hyperthrophy in a rabbit model of cutaneous scarring. Wound Repair Regen. 2006;14:138–41.
Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr. Design, synthesis and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry. 1997;36:3008–17.
Crombez L, Aldrian-Herrada G, Konate K, Nguyen QN, McMaster GK, Brasseur R, Heitz F, et al. A new potent secondary amphipathic cell-penetrating peptide for siRNA delivery into mammalian cells. Mol Ther. 2009;17:95–103.
Mok H, Park TG. Self-crosslinked and reducible fusogenic peptides for intracellular delivery of siRNA. Biopolymers. 2007;89:881–8.
Lee SH, Kim SH, Park TG. Intracellular siRNA delivery system using polyelectrolyte complex micelles prepared from VEGF siRNA-PEG conjugate and cationic fusogenic peptide. Biochem Biophys Res Commun. 2007;357:511–6.
Acknowledgements
This work was supported by the research grant from 2017 Duksung Women’s University. We thank to Ms. Youli Lee for her excellent technical assistance in quantitative analysis of collagen morphology. We thank to Prof. KC Park at Dermatology Department, School of Medicine, Seoul National University for his valuable discussion and donation of primary human fibroblasts.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflicts of interest
The authors have no financial conflicts of interest.
Ethical statement
The animal study was performed after receiving approval of the Institutional Animal Care and Use Committee in Duksung Women’s University (IACUC no. 2018-003-009).
Rights and permissions
About this article
Cite this article
Cho Lee, AR., Woo, I. Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements. Tissue Eng Regen Med 15, 711–719 (2018). https://doi.org/10.1007/s13770-018-0166-2
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13770-018-0166-2