Plastic Surgery Research Council

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Targeting Wnt Signaling to Reduce Capsular Fibrosis
Britta A. Kuehlmann, M.D., PhD, Clark Andrew Bonham, Geoffrey C. Gurtner, M.D., FACS.
Stanford University School of Medicine, Palo Alto, CA, USA.

PURPOSE: It is well known that Wnt signaling plays a central role in various forms of fibrosis and has been implicated in pulmonary, renal, liver and skin fibrosis. Multiple studies have unraveled that activation of the canonical Wnt pathway stimulates fibroblast and myofibroblast activation and thus increases excessive collagen deposition resulting in fibrosis. Genetic and pharmacological blockage of different Wnt markers in experimental settings improves fibrotic disease models with direct translational implications for future therapies.
METHODS: Capsular fibrosis was induced by inserting a silicone implant in a subcutaneous pocket in C57/BL6 mice and in Wnt11-knockout mice. Hematoxylin and eosin staining (H&E) was done to qualitatively asses the morphology, including the density of the capsules. Cells from the murine capsules were isolated and characterized by fluorescence-activated cell scanning (FACS), single cell quantitative polymerase chain reaction (qPCR) and single cell RNA sequencing to determine the cells responsible for the fibrotic response. For imaging purposes SEM, TEM and 3D confocal imaging of the capsules were performed.
RESULTS: FACS analysis and immunohistochemistry stains revealed that collagen depositing macrophages are responsible for the development of capsular fibrosis. qPCR revealed that the majority of the myeloid cells in the fibrotic capsule expressed macrophage genes and were found to significantly increase Col1a1 as well as Wnt11. Therefore, we looked at an intervention to reduce these macrophages by using Wnt11-knockout mice and subsequent induction of fibrosis in these mice. Using transcriptional analysis we found distinct markers to differentiate three macrophage subgroups (Group 1= Cd36, Group 2= Cd209, Group 3= Ccr2). FACS revealed that Cd36 and Ccr2 were significantly reduced in the capsule of Wnt11-knockout mice compared to Bl6 mice. Furthermore, Wnt11-knockout mice displayed significantly thinner capsules with a lower cellularity and loosely arranged fibers compared to the capsule of Bl6-mice after 3 months, confirmed by H&E stains. Our findings suggest that these cell subgroups are essential to capsule development through pro-fibrotic activity.
CONCLUSION: For the first time, we identify macrophage subgroups that are essential to capsule development through pro-fibrotic activity. Furthermore, we use a methodology to decrease capsular fibrosis in an animal model. These findings have promising therapeutic implications for the treatment of fibrosis around implants.


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