P.033 BAG3 as a possible therapeutic target and biomarker in systemic sclerosis

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Introduction Skin fibrosis is the pathognomonic manifestation of SSc, characterized by excessive collagen deposition driven by activated myofibroblasts. BAG3 (Bcl2-associated athanogene 3) induces fibroblast-to-myofibroblast transition through regulation of autophagy and apoptotic pathways, potentially playing a central role in cutaneous SSc pathogenesis. This study investigated BAG3 expression patterns, receptor dynamics, and regulatory mechanisms in SSc skin-derived fibroblasts. Material and Methods BAG3 protein expression and its receptor IFITM2 were analyzed in dermal tissue biopsies from dcSSc patients compared to healthy controls using dual immunohistochemistry and co-localization immunofluorescence analysis with PDGFR-a fibroblast markers. Skin-derived fibroblasts were isolated from punch biopsies of 10 SSc patients and 7 healthy controls. BAG3 expression was quantified using Western blotting analysis of whole cell lysates and concentrated culture supernatants. IFITM2 surface expression was assessed by flow cytometry using specific monoclonal antibodies, measuring both percentage of positive cells and mean fluorescence intensity. TGF-b1-induced BAG3 regulation was examined in healthy control fibroblasts using dose-response studies (0,5ng/ml, 10ng/ml TGF-b1 for 48 hours), with BAG3 quantification by ELISA. Nintedanib’s modulatory effects on BAG3 release were evaluated using 50nM concentration with pre-treatment protocols before TGF-b1 stimulation. Results Immunohistochemistry analysis revealed significant bag3 overexpression in dcssc patient dermal tissue compared to hcs, with prominent co-localization in pdgfr-a-positive dermal fibroblasts throughout the dermis and particularly concentrated in areas of dense collagen deposition (figure 1A). IFITM2 receptor demonstrated corresponding spatial overexpression patterns in SSc dermal tissue (figure 1B). Western blotting confirmed significantly elevated BAG3 protein levels in SSc skin fibroblasts versus healthy controls in both whole cell lysates and culture supernatants, indicating both increased intracellular expression and enhanced secretion (figure 1C, F). Flow cytometric analysis revealed upregulation of IFITM2 receptor in SSc fibroblasts (70%vs 40% in controls, p=0.0016), with significantly increased mean fluorescence intensity(p=0.007), demonstrating enhanced receptor density (figure 1D). TGF-b1 dose-dependently induced BAG3 protein secretion in healthy fibroblasts (75 pg/ml at 10ng/ml vs 40 pg/ml at 5ng/ml TGF-b1, p<0.01), establishing TGF-b1 as a key regulatory stimulus.Notably, nintedanib pre-treatment completely suppressed TGF-b1-induced BAG3 expression to near-baseline levels, suggesting its anti-fibrotic action through BAG3 pathway inhibition (figure 1E). Conclusions SSc skin fibroblasts exhibit constitutive BAG3 overexpression coupled with enhanced IFITM2 receptor sensitivity. TGF-b1-driven BAG3 production represents a critical pathogenic mechanism targeted by nintedanib through pathway suppression. These findings identify BAG3 as a fundamental driver of cutaneous fibrosis.

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