Jun Drives A Fibrogenic Cutaneous Wound Healing Response Through Differential Action On Distinct Fibroblast Subpopulations
Mimi R. Borrelli, MD, Alexandra L. Moore, MD, Shamik Maschara, Bryan A. Duoto, Heather E. desJardens-Park, Ronak A. Patel, Jan Sokol, Derrick C. Wan, Gerlinde Wernig, Michael T. Longaker.
Stanford, Palo Alto, CA, USA.
PURPOSE: Skin fibrosis and scarring can result in devastating disfigurement and permanent functional loss. Currently, there are no treatment modalities able to prevent or reverse this fibrotic process, and scars and their consequences result in an enormous medico-economic burden. Thus, it is of paramount importance to better understand the key pathogenic mechanisms driving this pathological fibrotic process. We recently described a mouse model in which overexpression of JUN, an AP-1 transcription factor, can be induced to produce global tissue fibrosis. Here, we explored the effects of JUN overexpression in skin scarring and fibrosis and the ability of our mouse to model hypertrophic scarring and excessive skin fibrosis in response to wounding.Methods: Stented excisional dorsal wounds were created in transgenic JUN (c-JuntetO R26-M2rtTA) and Rosa (Rosa26-rtTA) control mice. Doxycycline (2mg/ml) was used to induce JUN overexpression in the wound beds on the day of surgery (POD 0) and every other day until the wounds healed (postoperative day 14, POD 14). On alternate days throughout the healing response, wounds were harvested and analyzed histologically for thickness and collagen deposition, and by fluorescent activated cell sorting (FACS) to compare the relative percentages of fibroblast subpopulations (Fig. 1A). To study the role of JUN in human scars, dermal fibroblasts were isolated from hypertrophic scars (HTS) and healthy control skin (NS) and transduced to knock out JUN using a CRISPR-Cas 9 method. Proliferation and apoptosis were compared in the knock-out (KO) and non-KO human dermal fibroblasts (Fig. 1B).Results: The wounds of JUN mice healed at a significantly accelerated rate between POD5 and POD14 (*p<0.05) (Fig. 1C). Compared to the wounds of Rosa control mice, the scars of JUN mice on POD14 were significantly thicker, and although collagen content was not different, it was more disordered on Hematoxylin and Eosin staining, and brighter and more branched upon computational assessment of Picrosirius stained wounds (*p<0.05) (Fig. 1D). JUN overexpression resulted in a significant expansion of reticular fibroblasts at the expense of lipofibroblasts, evident on POD 7 (*p<0.05) (Fig. 1E). Translating these results to human scars; JUN CRISPR-Cas 9 deletion increased apoptosis and decreased proliferation of primary cultures of HTS and NS fibroblasts (Fig. 1F).Conclusion: JUN overexpression increases the fibrotic cutaneous wound healing response by significantly expanding reticular dermal fibroblasts at the expense of the dermal lipofibroblasts. In addition, assessment of HTS and NS fibroblasts isolated from human skin indicate that JUN also mediates the fibrotic response in human disease by inhibiting apoptosis and driving proliferation of the dermal fibroblast subsets. Thus, our novel inducible JUN mouse model can be used to explore the mechanisms driving HTS and other pathological skin fibrosis and facilitate targeted identification of new treatments.
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