Blockade of PDGF Receptors by Crenolanib Has Therapeutic Effect in Patient Fibroblasts and in Preclinical Models of Systemic Sclerosis  Katsunari Makino, Tomoko Makino, Lukasz Stawski, Julio C. Mantero, Robert Lafyatis, Robert Simms, Maria Trojanowska  Journal of Investigative Dermatology  Volume 137, Issue 8, Pages 1671-1681 (August 2017) DOI: 10.1016/j.jid.2017.03.032 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Crenolanib inhibits periostin, CCN2, and collagen protein levels in HC and SSc skin fibroblasts. (a) HC and SSc fibroblasts were pretreated for 1 hour with crenolanib (100 nmol/L), followed by 24 hours of treatment with TGF-β (2.5 ng/ml). (b) HC fibroblasts were transfected with siRNA targeting both PDGFRα and PDGFRβ for 24 hours, followed by 24 hours of treatment with TGF-β. (c) HC fibroblasts were stimulated with PDGFAA (10 ng/ml) or PDGFBB (10 ng/ml). (a, b, c) Cell lysates were subjected to immunoblotting. The values below each blot showed mean ± standard deviation of the relative protein levels by densitometry. Values are normalized relative to control. n = 3 per each group. ∗P versus control < 0.05; #P versus TGF-β < 0.05. HC, healthy control; PDGF, platelet-derived growth factor; PDGFR, platelet-derived growth factor receptor; siPDGFR, small interfering platelet-derived growth factor receptor; siRNA, small interfering RNA; SSc, systemic sclerosis; TGF, transforming growth factor. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions

Figure 2 Crenolanib inhibits proliferation of HC and SSc fibroblasts. (a, b) Proliferation of HC or SSc fibroblasts was measured by IncuCyte system (Essen BioScience, Ann Arbor, MI). Fibroblasts were cultured with 10% FBS or 1% FBS media containing PDGFAA, PDGFBB, crenolanib, or CCN2 as shown in the right panels. The line graphs represent mean (n = 3) confluence measurements (%). The bar graphs represent mean ± standard deviation (n = 3) of area under the curve (AUC). Values are normalized relative to control. ∗P < 0.05. (c) HC fibroblasts were pretreated for 1 hour with crenolanib (100 nmol/L), followed by 30 minutes of stimulation with PDGFAA (10 ng/ml) or PDGFBB (10 ng/ml). Cell lysates were subjected to immunoblotting. The values below each blot showed mean ± standard deviation of the relative protein levels. Values are normalized relative to control; n = 3 per each group. ∗P versus control < 0.05; #P versus PDGFAA or PDGFBB < 0.05. (d) HC and SSc fibroblasts were stimulated with PDGFAA (10 ng/ml) and/or CCN2 (25 ng/ml) for 30 minutes. Cell lysates were subjected to immunoblotting. The values below each blot showed mean ± standard deviation of the relative protein levels. Values are normalized relative to control. n = 3 per each group. ∗P versus PDGFAA < 0.05. Akt, protein kinase B; AUC, area under the curve; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; h, hour; HC, healthy control; n.s., not significant; PDGF, platelet-derived growth factor; SSc, systemic sclerosis. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Relationship between PDGFRα/β and CCN2 in HC and SSc skin. RNA of skin biopsy samples from HC subjects or SSc patients was analyzed by microarray. Modified Rodnan skin score is also added in microarray analysis. (a) Gene expressions between HC and SSc skin are shown in log2 scale. (b, c) Correlations were assessed by Pearson correlation coefficient. Correlation coefficient r and P values are shown in each panel. Gene expressions are shown in log2 scale. (b) PDGFRα versus CCN2. (c) PDGFRα versus periostin. (d) The intersection of each marker represents r and P values. Blue and red colors indicate high or low r-value. The circle size correlates with an absolute value of r. The number in each box shows P-value. No number in each box means that the P-value is less than 0.05. HC, healthy control; PDGFR, platelet-derived growth factor receptor; SSc, systemic sclerosis. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Crenolanib ameliorates skin fibrosis in Ang II-induced SSc mice model. (a) Skin fibrosis in C57BL/6J mice was induced by Ang II administered via subcutaneously installed osmotic pumps. Mice were treated daily for 2 weeks with 15 mg/kg crenolanib or vehicle. (b) Mice skin sections were stained with hematoxylin and eosin. Scale bar = 200 μm. (c) Dermal thickness is summarized as a graph. (d) Collagen contents were measured by hydroxyproline assay. Values are normalized relative to phosphate buffered saline control group. (e) Skin sections were stained with anti-αSMA antibody. Representative images are shown. The αSMA-positive cells were counted in all experimental mice in five random high-power fields per mouse using a light microscope. The mean score was used for analysis. Values are normalized relative to phosphate buffered saline control group. Each graph represents mean ± standard deviation of the indicated parameters. n = 5 per group. ∗P < 0.05. Ang II, angiotensin II; HE, hematoxylin and eosin; IP, intraperitoneal; PBS, phosphate buffered saline; SMA, smooth muscle actin; SSc, systemic sclerosis. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions

Figure 5 PDGFR-positive cells contribute to fibrosis in Ang II-induced SSc mice model. Skin fibrosis in PDGFRα-GFP mice was induced by Ang II administered via subcutaneously installed osmotic pumps. Skin was extracted 7 days after pump installation. Immunofluorescent staining was performed on cryosections using antibodies to (a) PDGFRβ, (b) procollagen, and (c) periostin and counterstained with DAPI. The green fluorescence represents the PDGFRα-positive cells. Arrows indicate the PDGFRα-positive cells stained with each antibody. Epidermis and hair follicle are stained nonspecifically by secondary antibody. Scale bar = 10 μm. Ang II, angiotensin II; GFP, green fluorescent protein; PBS, phosphate buffered saline; PDGFR, platelet-derived growth factor receptor; SSc, systemic sclerosis. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions

Figure 6 Crenolanib prevents Ang II-induced heart fibrosis. (a) Heart fibrosis in C57BL/6J mice was induced by Ang II pump. Mice were treated daily for 2 weeks with 15 mg/kg crenolanib or vehicle. Representative heart images of Picrosirius Red staining are shown. Scale bar = 500 μm. (b) Collagen content in the heart was quantified by measuring the ratio of red area to the total area in the Picrosirius Red staining. (c) Heart sections were stained with anti-αSMA antibody. (d) Heart fibrosis in PDGFRα-GFP mice was induced by Ang II pumps. The heart was extracted 14 days after pump installation. Immunofluorescent staining was performed on cryosections using antibodies to PDGFRβ, procollagen, and periostin and counterstained with DAPI. The green fluorescence represents the PDGFRα-positive cells. Scale bar = 10 μm. Ang II, angiotensin II; GFP, green fluorescent protein; PBS, phosphate buffered saline; PDGFR, platelet-derived growth factor receptor; SMA, smooth muscle actin. Journal of Investigative Dermatology 2017 137, 1671-1681DOI: (10.1016/j.jid.2017.03.032) Copyright © 2017 The Authors Terms and Conditions