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SiRNA Knockdown of Tissue Inhibitor of Metalloproteinase-1 in Keloid Fibroblasts Leads to Degradation of Collagen Type I  Masayo Aoki, Koichi Miyake,

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Presentation on theme: "SiRNA Knockdown of Tissue Inhibitor of Metalloproteinase-1 in Keloid Fibroblasts Leads to Degradation of Collagen Type I  Masayo Aoki, Koichi Miyake,"— Presentation transcript:

1 siRNA Knockdown of Tissue Inhibitor of Metalloproteinase-1 in Keloid Fibroblasts Leads to Degradation of Collagen Type I  Masayo Aoki, Koichi Miyake, Rei Ogawa, Teruyuki Dohi, Satoshi Akaishi, Hiko Hyakusoku, Takashi Shimada  Journal of Investigative Dermatology  Volume 134, Issue 3, Pages (March 2014) DOI: /jid Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Knockdown of tissue inhibitor of metalloproteinase (TIMP)-1 and -2 messenger RNA and protein expression using a lentiviral vector encoding siRNA in keloid–derived fibroblasts (KFs). (a) Quantitative reverse transcriptase-PCR (qRT-PCR) analysis of TIMP-1/-2 mRNA levels in KFs 72 h after transduction with 3 transduction units per cell of small interfering RNAs targeting TIMP (siTIMP)-1 (A–C), siTIMP-2 (A–C), or a non-targeting control vector (LV-TH). All values were normalized to the level of an endogenous control (glyceraldehyde 3-phosphate dehydrogenase mRNA) in untreated control samples. Bars are means±SE of five independent experiments (*P< vs. untreated control samples, n=5). (b) Western blot showing TIMP-1/-2 protein expression in KFs 72 h after transduction. (c) Western blot showing TIMP-1/-2 protein expression in normal fibroblasts 72 h after transduction. (d) Transduction efficiency of siTIMP-1 B, siTIMP-2 A, or a non-targeting control vector (LV-TH) in KFs; dot plots are representative (n=18). GFP, green fluorescent protein; MW, molecular weight. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Interstitial collagenase (matrix metalloproteinase (MMP)-1) messenger RNA expression and activity in samples of the culture medium conditioned by keloid–derived fibroblasts (KFs) transduced with lentiviral vector encoding tissue inhibitor of metalloproteinase (TIMP)-1 or -2 siRNA. (a) Quantitative reverse transcriptase-PCR (qRT-PCR) analysis of MMP-1 mRNA levels in KFs 72 h after transduction with 3 transduction units per cell of siTIMP-1, siTIMP-2, or a non-targeting control vector (LV-TH). All values are normalized to the level of glyceraldehyde 3-phosphate dehydrogenase mRNA in untreated control samples. Bars are means±SE of six independent experiments (n=6). (b) Levels of MMP-1/TIMP-1 and (c) MMP-1/TIMP-2 complexes detected by ELISAs in conditioned medium 3–5 days after transduction. Bars are means±SE of four independent experiments (*P<0.05 vs. LV-TH, n=4). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Gelatinase A (matrix metalloproteinase (MMP)-2) messenger RNA expression and activity in the culture medium conditioned by keloid–derived fibroblasts (KFs) transduced with lentiviral vector encoding tissue inhibitor of metalloproteinase (TIMP)-1 or -2 siRNA. (a) Quantitative reverse transcriptase-PCR (qRT-PCR) analysis of MMP-2 mRNA levels in KFs 72 h after transduction with 3 transduction units per cell of siTIMP-1, siTIMP-2, or a non-targeting control vector (LV-TH). All values are normalized to the level of endogenous glyceraldehyde 3-phosphate dehydrogenase mRNA in untreated control samples. Bars show means±SE of four independent experiments (n=4). (b) Gelatin zymography showing MMP-2 activity in conditioned medium 3–5 days after transduction. Two representative gels are pictured. The graph shows active-MMP-2/pro-MMP-2 density ratios determined by densitometry. The bars are means±SD of four independent experiments (*P<0.01 vs. LV-TH, n=4). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Promotion of collagen type I degradation in culture medium conditioned by keloid–derived fibroblasts (KFs) transduced with lentiviral vector encoding tissue inhibitor of metalloproteinase (TIMP)-1 siRNA. (a) Quantitative reverse transcriptase-PCR (qRT-PCR) analyses of collagen (COL)1A2 and COL3A1 messenger RNA levels in KFs after transduction. All values are normalized to the level of glyceraldehyde 3-phosphate dehydrogenase mRNA in untreated control samples. Bars show means±SE (*P<0.01, n=4). (b) Collagen type I concentrations of conditioned medium determined by ELISA after transduction. Bars are means±SD (*P<0.05, n=3). (c) Western blot showing collagen type I in medium conditioned by normal fibroblast (NF)/KF transductants. (d) Collagen type I degradation evaluated on the basis of quantification using western blot band densitometry of 95 kDa (full-length) and 30 kDa (degraded) collagen type I in the medium conditioned by KFs. Bars are means±SD (*P<0.05, n=3). MW, molecular weight. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 In vitro anti-proliferative, pro-apoptotic effects and G2/M phase cell cycle arrest induced by tissue inhibitor of metalloproteinase (TIMP)-1/-2 siRNA in keloid–derived fibroblasts (KFs). (a) A methyl thiazol tetrazolium (MTT) viability assay of normal fibroblasts (NFs)/KFs 3 days after transduction with 3 transduction units per cell of siTIMP-1, siTIMP-2, or a non-targeting control vector (LV-TH). All data are normalized to untreated control samples. Bars are means±SD of four independent experiments (*P<0.05 vs. LV-TH, n=4). Annexin V-APC apoptosis analysis of NFs/KFs 5 days after transduction; (b) dot plots are representative of KFs. (c) Bars are means±SD of four independent experiments (*P<0.05 vs. LV-TH, n=4). (d) PI cell cycle analysis of KFs 5 days after transduction. Histograms are representative. (e) Bars are means±SD of four independent experiments (*P<0.05 vs. LV-TH, n=4). Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 siTIMP-1 (small interfering RNAs targeting tissue inhibitor of metalloproteinase-1)-induced reductions in the complexity of collagen bundles in ex vivo cultures of keloid tissues. Immunostaining of keloid tissues transduced with (a) LV-TH, (b) siTIMP-1, or (c) siTIMP-2 shows expression of TIMP-1 or TIMP-2 in cells expressing (d) LV-TH, (e) siTIMP-1, or (f) siTIMP-2. For each condition in (a–c) and (d–f), images were obtained at identical sensitivities. Hematoxylin and eosin (H&E) staining of keloid tissues transduced with (g) LV-TH, (h) siTIMP-1, or (i) siTIMP-2. The asterisk indicates the area in which the complexity of the collagen bundles was diminished, and the collagen bundles ran parallel to epidermis. (j–l) Higher magnification of the dermis in keloid tissues transduced with (j) LV-TH, (k) siTIMP-1, or (l) siTIMP-2. Bar=100 μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

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