Inhibition of Hsp90 Counteracts the Established Experimental Dermal Fibrosis Induced by Bleomycin

Abstract

Our previous study demonstrated that heat shock protein 90 (Hsp90) is overexpressed in the involved skin of patients with systemic sclerosis (SSc) and in experimental dermal fibrosis. Pharmacological inhibition of Hsp90 prevented the stimulatory effects of transforming growth factor-beta on collagen synthesis and the development of dermal fibrosis in three preclinical models of SSc. In the next step of the preclinical analysis, herein, we aimed to evaluate the efficacy of an Hsp90 inhibitor, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), in the treatment of established experimental dermal fibrosis induced by bleomycin. Treatment with 17-DMAG demonstrated potent antifibrotic and anti-inflammatory properties: it decreased dermal thickening, collagen content, myofibroblast count, expression of transforming growth factor beta receptors, and pSmad3-positive cell counts, as well as leukocyte infiltration and systemic levels of crucial cytokines/chemokines involved in the pathogenesis of SSc, compared to vehicle-treated mice. 17-DMAG effectively prevented further progression and may induce regression of established bleomycin-induced dermal fibrosis to an extent comparable to nintedanib. These findings provide further evidence of the vital role of Hsp90 in the pathophysiology of SSc and characterize it as a potential target for the treatment of fibrosis with translational implications due to the availability of several Hsp90 inhibitors in clinical trials for other indications.

S100A4-neutralizing monoclonal antibody 6B12 counteracts the established experimental skin fibrosis induced by bleomycin

“…To assess the effect of 6B12 on attenuating fibrosis, we chose the murine model of bleomycin-induced dermal fibrosis in a therapeutic design as previously described [ 19 , 20 ]. Briefly, to induce robust dermal fibrosis, bleomycin (Bleomedac, Medac GmbH, Wedel, Germany) was administered s.c. to 6-week-old male C57BL/6 mice (Velaz, s.r.o., Prague, Czech Republic) every other day for 6 weeks at a concentration of 0.5 mg/ml.…”

“…Assessments of dermal thickness and of the number of infiltrating leukocytes were performed on haematoxylin–eosin stained skin sections as previously described [ 20 , 21 ]. Infiltrating leukocytes were also identified by immunohistochemistry staining using rabbit anti-CD45 mAb (1:300, overnight at 4°C, Abcam, Berlin, Germany), co-stained with haematoxylin.…”

“…Infiltrating leukocytes were also identified by immunohistochemistry staining using rabbit anti-CD45 mAb (1:300, overnight at 4°C, Abcam, Berlin, Germany), co-stained with haematoxylin. The myofibroblast count was evaluated by immunohistochemistry staining and identification of alpha-smooth muscle actin (aSMA)-positive fibroblasts using mouse anti-aSMA mAb (1:1000, clone 1A4, Sigma‐Aldrich, St Louis, MO, USA), co-stained with haematoxylin as previously specified [ 20 ]. Further details on CD45 staining and quantification of myofibroblasts, inflammatory infiltrate and CD45-positive cells are available in Supplementary Material , available at Rheumatology online.…”

“…Further details on CD45 staining and quantification of myofibroblasts, inflammatory infiltrate and CD45-positive cells are available in Supplementary Material , available at Rheumatology online. Detection of pSmad3-positive cells was performed by immunofluorescence staining using rabbit anti-pSmad3 mAb (1:50, phospho S423 + S425, Abcam, Cambridge, UK) and co-staining of nuclei with 4,6-diamidino-2-phenylindole (DAPI, 1:1000, Invitrogen, Carlsbad, CA, USA) as described elsewhere [ 20 ]. The staining was visualized at 100- and 400-fold magnification using appropriate fluorescence filters on a BX53 microscope with a DP80 Colour lens camera using CellSens Standard software v.3.1‐Build 21199 (Olympus, Philadelphia, PA, USA) and assessed in a blinded manner, as previously depicted [ 20 ].…”

Frontiers of Targeted Therapy and Predictors of Treatment Response in Systemic Sclerosis

“…In their original article, Štorkánová et al present compelling evidence for a potential novel therapeutic target implicated in SSc-related skin fibrosis [ 6 ]. Using a modified murine model of bleomycin-induced dermal fibrosis, they demonstrate that the inhibition of Heat shock protein (Hsp)-90 by 17-dimethylaminoethylamino-17-demethoxy-geldanamycin (17-DMAG) prevents further progression and may induce regression of the established skin fibrosis with a comparable effect to that of nintedanib.…”