An estrogen-sensitive fibroblast population drives abdominal muscle fibrosis in an inguinal hernia mouse model
Tanvi Potluri, Matthew J. Taylor, Jonah J. Stulberg, Richard L. Lieber, Hong Zhao, Serdar E. Bulun
Tanvi Potluri, Matthew J. Taylor, Jonah J. Stulberg, Richard L. Lieber, Hong Zhao, Serdar E. Bulun
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Research Article Endocrinology Muscle biology

An estrogen-sensitive fibroblast population drives abdominal muscle fibrosis in an inguinal hernia mouse model

Abstract

Greater than 25% of all men develop an inguinal hernia in their lifetime, and more than 20 million inguinal hernia repair surgeries are performed worldwide each year. The mechanisms causing abdominal muscle weakness, the formation of inguinal hernias, or their recurrence are largely unknown. We previously reported that excessively produced estrogen in the lower abdominal muscles (LAMs) triggers extensive LAM fibrosis, leading to hernia formation in a transgenic male mouse model expressing the human aromatase gene (Aromhum). To understand the cellular basis of estrogen-driven muscle fibrosis, we performed single-cell RNA sequencing on LAM tissue from Aromhum and wild-type littermates. We found a fibroblast-like cell group composed of 6 clusters, 2 of which were validated for their enrichment in Aromhum LAM tissue. One of the potentially novel hernia-associated fibroblast clusters in Aromhum was enriched for the estrogen receptor-α gene (Esr1hi). Esr1hi fibroblasts maximally expressed estrogen target genes and seemed to serve as the progenitors of another cluster expressing ECM-altering enzymes (Mmp3hi) and to upregulate expression of proinflammatory, profibrotic genes. The discovery of these 2 potentially novel and unique hernia-associated fibroblasts may lead to the development of novel treatments that can nonsurgically prevent or reverse inguinal hernias.

Authors

Tanvi Potluri, Matthew J. Taylor, Jonah J. Stulberg, Richard L. Lieber, Hong Zhao, Serdar E. Bulun

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Figure 1

Identification of scRNA-Seq cell clusters through gene marker expression analysis in WT and Aromhum mice.

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Identification of scRNA-Seq cell clusters through gene marker expression...
(A) Representative images of male WT and Aromhum mice prior to (top) and after dissection (bottom). Arrows indicate bulging from scrotal hernias (n = 5). (B) Lower abdominal muscle (LAM) tissue sections from WT and Aromhum mice stained with hematoxylin and eosin (H&E, left) depicting marked reduction in myofiber size in Aromhum mice and Masson’s trichrome staining (MTS, right) indicating increased fibrotic deposition in LAM tissues of Aromhum mice. Arrows point to atrophied muscle fibers. Scale bar: 100 μm (n = 5). (C) Experimental design. LAM was harvested from 9- to 10-week-old male mice (n = 3 per group). Single-cell suspension was achieved through enzymatic digestion, and scRNA-Seq was performed using 10x Genomics Chromium. (D) UMAP plot of WT cells alone and (E) Aromhum cells alone. When both groups were analyzed together, 22 cell clusters were found, and cells were grouped into 10 cell groupings based on canonical marker expression. (F) Dot plot representing the expression of known marker genes of individual cell types. Size of dots corresponds to frequency of expression within a cell group. Color of dots corresponds to average expression level within the cell group. (G) Compositional makeup of the total cell population from WT and Aromhum mice, represented as a percentage of total cells. Groups from D and E were used, with macrophages/monocytes and T cells combined as “immune cells,” and the lymphatic endothelial cells combined with the other endothelial cell clusters. Data were compared using 2-way ANOVA and corrected for multiple comparisons. *P < 0.05. (H) UMAP feature plots showing expression of fibro-adipogenic progenitor (FAP) marker Pdgfra in a majority of fibroblast-like cells from LAM of WT and Aromhum mice (n = 3). MuSCs, muscle satellite cell/stem cells; UMAP, uniform manifold approximation and projection.