Date: Monday, November 6, 2017
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology. Using human skin as an ex vivo organ model of fibrosis is an attractive tool to examine mechanisms underlying fibrosis and assess the potential effect of anti-fibrotic therapies. We have previously shown that a single injection of TGF-β induced dermal fibrosis in human skin ex vivo. In this study, we examined the effect of Bleomycin (BLM).
Methods: Human skin was stimulated with BLM using two different methods: injection and immersion. For the injection method, normal human skin was obtained from residual tissue following plastic surgery. Subcutaneous fat tissue was removed and skin tissue was cut into 1.5 cm x 1.5 cm sections. Skin tissues were injected intradermally with a total volume of 100 μl of 1×PBS: BLM (1 or 10 mU/ml) or 1× PBS as a vehicle control. Skin samples were cultured in an air-liquid interface with the epidermal side up. The culture medium was replaced after 72h. After 7 days, skin tissue corresponding to an area with 8-mm diameter centered around the injection site was harvested with a disposable 3 mm punch. For the immersion method, normal human skin was cut with a disposable 3 mm punch, and punches were cultured in medium containing BLM (1 or 10 mU/ml) or 1× PBS as a vehicle control. Skin tissues were harvested after 48 hours or 7 days post-treatment for real-time PCR and hydroxyproline assay, respectively.
Results: BLM significantly increased hydroxyproline levels and dermal thickness in a dose-dependent manner 7 days after injection. BLM had a similar effect on skin punches immersed in media for the same duration. The increase in hydroxyproline was paralleled by increased dermal thickness and Masson Trichrome staining. qRT-PCR analysis revealed that the expression levels of fibrosis-related genes, collagen 1A1, fibronectin, CTGF, and TGFb1, were significantly increased in BLM-treated skin compared with control skin.
Our findings show that BLM induces fibrosis in human skin and higher doses of BLM are more effective at inducing significant increases in collagen content, expression of fibrosis-associated genes, and dermal thickness. Moreover, immersing skin in medium containing BLM is more effective at inducing dermal fibrosis than intradermal BLM injections. BLM treatment using the immersion method may be an attractive tool for the functional analysis of skin fibrosis in human skin in organ culture, providing a tool with direct relevance to human fibrotic skin disease.
To cite this abstract in AMA style:Watanabe T, Mlakar L, Heywood J, Malaab M, Feghali-Bostwick CA. Human Skin in Organ Culture As an Ex Vivo Model for Assessing the Fibrotic Effects of Bleomycin [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/human-skin-in-organ-culture-as-an-ex-vivo-model-for-assessing-the-fibrotic-effects-of-bleomycin/. Accessed November 20, 2018.
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