The Antifibrotic Effect of α2AP Neutralization in Systemic Sclerosis Dermal Fibroblasts and Mouse Models of Systemic Sclerosis  Yosuke Kanno, En Shu, Hiroyuki Kanoh, Mariko Seishima  Journal of Investigative Dermatology  Volume 136, Issue 4, Pages 762-769 (April 2016) DOI: 10.1016/j.jid.2015.12.028 Copyright © 2015 The Authors Terms and Conditions

Figure 1 The profibrotic effect of α2AP in mice. (a) The level of α2AP in the serum from saline or bleomycin-administered mice was determined by ELISA (n = 4). (b) Representative skin sections from saline or α2AP-administered mice (hematoxylin and sirius red stain). Double head arrows indicate the dermal thickness. (c) The dermal thickness in the skin sections from mice (n = 6). (d) The collagen content in the skin from mice (n = 6). (e) The expression of each protein in the skin of mice was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). (f) Paraffin sections were stained with antibodies to α-SMA. The histogram shows quantitative representations of α-SMA (n = 6). The data represent the mean ± SEM. *P < 0.01. Scale bar = 200 μm. α2AP, α2-antiplasmin; α-SMA, α-smooth muscle actin; SEM, standard error of the mean. Journal of Investigative Dermatology 2016 136, 762-769DOI: (10.1016/j.jid.2015.12.028) Copyright © 2015 The Authors Terms and Conditions

Figure 2 The antifibrotic effect of α2AP neutralization in mouse models of SSc. (a) Representative skin sections from saline, bleomycin plus control IgG, or bleomycin plus α2AP-neutralizing antibodies-administered mice (hematoxylin and sirius red stain). Double head arrows indicate the dermal thickness. (b) The dermal thickness in the skin sections from mice (n = 6). (c) The collagen content in the skin from mice (n = 6). (d) The expression of each protein in the skin from mice was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). (e) Paraffin sections were stained with α-SMA antibodies. The histogram shows quantitative representations of α-SMA (n = 6). The data represent the mean ± SEM. *P < 0.01; **P < 0.05. Scale bar = 200 μm. α2AP, α2-antiplasmin; α-SMA, α-smooth muscle actin; SEM, standard error of the mean; SSc, systemic sclerosis. Journal of Investigative Dermatology 2016 136, 762-769DOI: (10.1016/j.jid.2015.12.028) Copyright © 2015 The Authors Terms and Conditions

Figure 3 The effect of α2AP neutralization in mouse models of SSc. (a) Representative lung sections from each group of mice (hematoxylin and sirius red stain). (b) Lung fibrosis score in the lung sections from each group of mice (n = 4). (c) The collagen content in the lung from each group of mice (n = 4). (d) The expression of type I collagen in the lung from mice was examined by a western blot analysis. The histogram shows quantitative representations of type I collagen (n = 3). (e) The level of anti-Scl-70 antibodies in the serum of mice was determined by ELISA (n = 4). The data represent the mean ± SEM. *P < 0.01; **P < 0.05; NS, not significant. Scale bar = 200 μm. α2AP, α2-antiplasmin; SEM, standard error of the mean; SSc, systemic sclerosis. Journal of Investigative Dermatology 2016 136, 762-769DOI: (10.1016/j.jid.2015.12.028) Copyright © 2015 The Authors Terms and Conditions

Figure 4 The effect of α2AP in human normal and SSc dermal fibroblasts. (a) The human normal dermal fibroblasts from N3 were stimulated by α2AP (4 nM) for 24 hours. The expression of each protein was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). (b) The expression of α2AP in human normal and SSc dermal fibroblasts was examined by a western blot analysis. The histogram shows quantitative representations of α2AP (normal fibroblasts, n = 4; SSc fibroblasts, n = 5). (c) The human dermal fibroblasts from SSc patient S4 were stimulated by control IgG (1 μg/ml) or α2AP-neutralizing antibodies (1 μg/ml) for 48 hours. The expression of each protein was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). The data represent the mean ± SEM. **P < 0.05. α2AP, α2-antiplasmin; SEM, standard error of the mean; SSc, systemic sclerosis. Journal of Investigative Dermatology 2016 136, 762-769DOI: (10.1016/j.jid.2015.12.028) Copyright © 2015 The Authors Terms and Conditions

Figure 5 The effect of α2AP receptor in SSc dermal fibroblasts. The human dermal fibroblasts from SSc patient S4 were transfected with control or ATGL siRNA. At 48 hours after transfection, the cells were used for experiments. The expression of each protein was examined by a western blot analysis. The histogram shows quantitative representations of each protein (n = 3). The data represent the mean ± SEM. *P < 0.01; **P < 0.05. α2AP, α2-antiplasmin; ATGL, adipose triglyceride lipase; SEM, standard error of the mean; siRNA, small interfering RNA; SSc, systemic sclerosis. Journal of Investigative Dermatology 2016 136, 762-769DOI: (10.1016/j.jid.2015.12.028) Copyright © 2015 The Authors Terms and Conditions