Plastic Surgery Research Council
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Presenter: Philip Lofti, MD
Co-Authors: Low YC; Soares M; Ham MJ; Wilson SC; Lalezarzadeh F; Ojo CO; Sartor RA; Levine JP; Saadeh PB; Ceradini DJ
NYU School of Medicine

Introduction: PUMA is central in governing intracellular ROS production and contributes to several pathologic processes. Here, we hypothesize that PUMA is a critical determinant of radiation-induced ROS production and contributes significantly to chronic radiation fibrosis of the skin. Further, we postulate that therapeutic silencing of PUMA expression will effectively lower ROS overproduction and prevent cutaneous radiation fibrosis in a preclinical animal model.

Methods: 3T3 fibroblasts were transfected with PUMA or nonsense siRNA and irradiated (15 Gy). Protein and RNA expression profiles were generated. Dorsal skin of C57 mice was treated with topical PUMA or nonsense siRNA and irradiated (40 Gy). Skin was analyzed after 1 and 4 weeks post irradiation for ROS endproducts (8-OHDG ELISA) and pro-fibrosis gene expression. Dermal and vessel-wall thickness was quantified histologically. Tensiometry was used to quantify skin stiffness.

Results: Irradiation induced a 5.6-fold increase in PUMA expression compared to nonirradiated cells at day 3, an effect which was reduced 72% with siPUMA treatment (5.6-fold vs. 1.6-fold increase, p<.001). PUMA silencing also reduced TGF≤-beta (6-fold vs. 2-fold increase, p<.001) and MnSOD upregulation (10-fold vs. 2.5-fold increase, p<.001). Notably SMAD3 expression was unchanged. This correlated with a reduction of ROS endproducts in vivo (9.1 ng/ml control vs. 0.9 ng/ml treated, p<.001). Epidermal thickness was significantly decreased in irradiated vs. treated skin (1606?m vs. 361?m, p<.001). Vessel wall thickness was significantly improved as well (18.9 ?m control vs. 9.3 ?m treated, p<.001). PUMA silenced skin showed a 58% reduction in stiffness compared to the irradiated control (0.25 N/mm control vs. 0.10 N/mm treated p<.001).

Conclusion: PUMA is a critical mediator of ROS overproduction, TGF≤ beta expression, and ensuing skin fibrosis following radiation injury. Therapeutic inhibition of PUMA blocks the cycle of ROS overproduction and prevents cutaneous fibrosis. Topical PUMA inhibition is a locally effective translatable strategy to prevent clinical radiation induced fibrosis.

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