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Transcriptional Inhibition of Hypertrophic Scars by a Gene Silencer, Pyrrole–Imidazole Polyamide, Targeting the TGF-β1 Promoter  Hisayo Washio, Noboru.

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Presentation on theme: "Transcriptional Inhibition of Hypertrophic Scars by a Gene Silencer, Pyrrole–Imidazole Polyamide, Targeting the TGF-β1 Promoter  Hisayo Washio, Noboru."— Presentation transcript:

1 Transcriptional Inhibition of Hypertrophic Scars by a Gene Silencer, Pyrrole–Imidazole Polyamide, Targeting the TGF-β1 Promoter  Hisayo Washio, Noboru Fukuda, Hiroyuki Matsuda, Hiroki Nagase, Takayoshi Watanabe, Yoshiaki Matsumoto, Tadashi Terui  Journal of Investigative Dermatology  Volume 131, Issue 10, Pages (October 2011) DOI: /jid Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Changes in macroscopic and histopathological findings, and expression of growth factors and extracellular matrices in the incised skin. (a) Changes in macroscopic and histopathological findings of hypertrophied scar formation in rats at 14, 28, and 42 days after skin incisions. The hypertrophic scars were created dorsally by incisions into the skin with a scalpel. Four full-thickness, linear incisions, 1cm in length, down to and including the panniculus carnosus were made on the dorsal skin of rats. Skin samples were obtained from the scar lesions and 6-μm thick sections were stained with hematoxylin–eosin. Eight incisions were made per experiment (biopsy site=8). Representative data are shown in the figures because the changes were very similar in each experiment. Bar=5mm in macroscopic findings and 500μm in histopathological findings. (b) Changes in mRNA expression of growth factors (transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF)) and extracellular matrices (collagen type1 and fibronectin) in incisional wounds of skin. Total RNAs from the samples were extracted and reverse transcription PCR was performed. The values are expressed as the mean±SE (n=6). *P<0.05 versus day 0. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Distribution of FITC-labeled pyrrole–imidazole (PI) polyamide targeted to the rat transforming growth factor-β1 (TGF-β1) promoter in rat skin. Ten μg of FITC-labeled PI polyamide were injected subcutaneously into rat skin just after incision. The skin excised at each indicated time point was immediately embedded in OCT compound and frozen in liquid nitrogen. Nuclei were stained with 4′-6-diamidino-2-phenylindole (DAPI). Bar=500μm (original magnification × 400). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Effects of pyrrole–imidazole (PI) polyamide targeted to TGF-β1 promoter on the expression of transforming growth factor (TGF)-β1 mRNA and protein in hypertrophic scars on rat skin. The hypertrophic scar was created on the back by skin incisions with a scalpel. (a) Doses of 1 to 100μg of TGF-β1-targeted PI polyamide (Polyamide) were injected subcutaneously into wounds just after skin incisions. Total RNAs from scar lesion samples were extracted and reverse transcription PCR was performed. (b) One hundred μg of Polyamide or mismatch polyamide (Mismatch) were injected into wounds just after skin incisions and the resulting expression of TGF-β1 protein was determined by western blot analysis. The values are expressed as the mean±SE (n=6). *P<0.05 versus injection of saline (control). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effects of pyrrole-imidazole polyamide targeted to the transforming growth factor (TGF)-β1 promoter (Polyamide) on the fibrosis and the expression of growth factors and extracellular matrix mRNAs in skin scars. (a) Effects of Polyamide on development, histopathology, and stainings of TGF-β1 and vimentin in hypertrophic scars in rat skin. Hypertrophic scars were created on the back by skin incisions with a scalpel. Skin samples were obtained from scar lesions and 6-μm thick sections were stained with hematoxylin–eosin. Eight incisions were made per experiment (biopsy site=8). Representative data are shown in the figures because the changes were very similar in each experiment. (b) Effects of Polyamide on expression of growth factor (TGF-β1 and connective tissue growth factor (CTGF)) and extracellular matrix (collagen type1 and fibronectin) mRNAs in incisional wounds. The values are expressed as the mean±SE (n=6). *P<0.05 versus mismatch PI polyamide. Bar=5mm in macroscopic findings, 500μm in histopathological findings, and 50μm in histological findings. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effect of pyrrole–imidazole (PI) polyamide targeted to the transforming growth factor-β1 (TGF-β1) promoter on established hypertrophic scars on rat skin. Established hypertrophic scars were created on the dorsal skin by incision with a scalpel and left to develop for 42 days. TGF-β1-targeted PI polyamide (Polyamide) was subsequently injected at 3-day intervals during a series of either 3 or 10 groups of injections around the scars and the skin samples were obtained from the scar lesions. (a) Six-μm thick sections were stained with hematoxylin–eosin and used to evaluate TGF-β1 and vimentin immunohistochemically. (b) Effects of Polyamide on expression of TGF-β1 mRNA in incisional wounds of established hypertrophic scars. The values are expressed as the mean±SE (n=6). *P<0.05 versus injection of saline (control). Bar=5mm in macroscopic findings, 500μm in histopathological findings, and 50μm in histological findings. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Target sequence and structure of the synthetic pyrrole–imidazole (PI) polyamide that targets the rat transforming growth factor-β1 (TGF-β1) promoter. (a) The polyamide was designed to bind to the rat TGF-β1 promoter adjacent to the activator protein-1-binding site (TGAGTCA, -2,303 to -2,297). (b) Structure of the rat TGF-β1 specific polyamide (Polyamide) and mismatch polyamide (Mismatch). Mismatch was designed not to bind to the transcription-binding sites of the promoter. PI polyamides were synthesized by solid phase methods and were purified by high-performance liquid chromatography (0.1% AcOH/CH3CN 0–50% linear gradient, 0–40minutes, 254nm through a Chemcobond 5-ODS-H column, Chemco Scientific, Osaka, Japan). Im, imidazole; Py, pyrrole. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

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