CD36 Antagonism Minimizes Skin Scarring By Inhibiting JUN-dependent Fibrotic Pathways Within Fibrogenic Fibroblast Subpopulations
Mimi R. Borrelli, MBBS, MSc, Julia Garcia, PhD, Michelle Griffin, MD, PhD, Nestor M. Deleon Diaz, Alexandra L. Moore, MD, Shamik Mascharak, MS, Bryan Duoto, MS, Heather desJardins-Park, Sandeep Adem, MS, Tristan Lerbs, PhD, Marc Gastou, PhD, Lui Cui, PhD, Michael Januszyk, MD, PhD, Hermann P. Lorenz, MD, Derrick C. Wan, MD, Geoffrey C. Gurtner, MD, Howard Chang, MD, PhD, Gerlinde Wernig, MD, Michael T. Longaker, MD, MA.
Stanford, menlo park, CA, USA.
PURPOSE: Skin fibrosis is the end result of injury in human skin. In the US alone >100 million new scars are formed every year, and with no therapy able to prevent or reverse skin fibrosis the medico-economic burden is enormous. Excessive fibrosis, as seen in hypertrophic scarring (HTS), can lead to devastating disfigurement and permanent functional loss. Incomplete understanding of the key pathogenic mechanisms driving pathological skin fibrosis has significantly hindered development of effective treatment strategies. We recently identified JUN, the AP-1 transcription factor, as a key driver of global tissue fibrosis. Here we investigate the role of JUN in skin scarring. METHODS: Primary cultures of human dermal fibroblasts (HDF) were derived from HTS and unwounded skin. JUN expression was deleted using CRISPR/Cas9, and the downstream genetic/epigenetic consequences were assessed by RNA-sequencing (RNAseq), the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATACseq), and gene ontology pathway analysis to identify the genes mediating JUN-dependent fibrosis. HDF were treated with salvianolic acid (SAB), a CD36 antagonist, to determine the in vitro effects on HDF proliferation, apoptosis, and production of collagen and TGFβ. The in vivo effects of CD36 antagonism were explored using a novel inducible mouse model of HTS. Dorsal excisional stented wounds were created in JUN (c-JuntetO R26M2rtTA) mice, and either SAB (1mg/ml) or PBS (control) was administred via intraparietal injection every 24h for 14 days. Wounds were compared macroscopically for scar morphology and re-epithelialization rates. On day-14, wounds were harvested and compared for histological fibrosis as well as for the composition fibroblast subpopulations and immune cells by flow cytometry (Fig.1A). PCRs were conducted on genes upregulated in JUN fibroblasts to investigate the mechanism by which CD36 antagonism lead to decreased scarring. RESULTS: JUN deletion significantly altered HDF gene expression (Fig.1Bi-ii) and chromatin accessibility (Fig.1Ci), affecting genes involved in key fibroproliferative pathways (e.g. PI3K/AKT/mTOR, PPARγ, ECM) (Fig.1Biii). Targeted exploration of the 100 candidate genes with closing epigenetic landscapes and reduced gene expression following JUN knock-out (KO) in HTS-HDF (Fig.1Cii) implicated CD36 as a surface mediating JUN-dependent fibrosis and proliferative pathways (Fig.1Ciii). Immunofluorescence showed close association between JUN and CD36 proteins in HDF (Fig.1D). In vitro CD36 antagonism using SAB decreased fibroblast proliferation (Fig.1Ei), increased apoptosis, and decreased production of collagen and TGFβ (Fig.1Eii-iii, 1F). In vivo, SAB treatment did not alter wound closure rate (Fig.1Gi-ii), but significantly prevented the development of HTS; SAB-treated wounds were less raised, less pigmented, had thinner dermal layers, and more ordered collagen fiber networks (Fig.1H). At a cellular level, SAB-treated JUN wounds had fewer immune cells and fewer profibrotic reticular fibroblasts. JUN lipofibroblasts exhibited increased expression of genes in the PPARγ pathway (PLIN1, FABP4, FABP5) linking CD36 signaling to adipocyte differentiation.
CONCLUSIONS: JUN drives pathological skin fibrosis following wounding, and CD36 antagonism with SAB can be used to inhibit JUN-dependent fibrotic pathways within key fibroblast subpopulations and decrease pathological scarring.
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