Single-Cell RNA Sequencing Reveals Novel Populations of Fibroblasts and Transcriptomic Changes Within Abdominal Skeletal Muscle in a Mouse Model of Aromatase-Induced Inguinal Hernia

Introduction: Inguinal hernia is a highly prevalent condition in men, of which the only currently available treatment is invasive surgical repair. An inguinal hernia often results from a protrusion of the intra-abdominal contents through a weakened region of the lower abdominal wall, but the etiology is unknown. One potential cause is aging-related steroid hormonal changes, which coincide with an increased incidence of hernia in aged men. Our group previously developed the first mouse model of inguinal hernia (Arom(Hum)) that is generated via the humanized expression of the enzyme aromatase, which converts androgens to estrogens. In the lower abdominal muscle (LAM), an aromatase-mediated increase in tissue estrogen causes fibroblast proliferation, fibrosis, and myocyte atrophy, resulting in hernias. However, the molecular mechanism of this phenotype remains unclear. In this study, we aimed to find genome-wide transcriptomic differences in Arom(Hum) compared to WT mice at a single-cell resolution. We hypothesized that in relation to WT mice, Arom(Hum) mice would have distinct fibroblast signatures that arise from the increased estrogen exposure to LAM tissue. Methods: LAM was harvested from 9-10-week-old male WT and Arom(Hum) mice (n=3 each) and digested into a single-cell suspension. Cells were processed via the 10X Genomics Chromium platform for single-cell RNA sequencing. The 6 samples combined yielded a total of ~63,000 cells. Data was analyzed using Cell Ranger v3, Seurat v3, Slingshot, and PROGENy R packages. Results: UMAP visualization of WT and Arom(Hum) LAM tissue revealed 22 cell clusters, which we grouped into 10 broad cell types through known marker gene expression. Arom(Hum) LAM contained a significantly higher proportion of fibroblasts than WT (44% vs. 27% of total analyzed cells), and Arom(Hum) fibroblasts expressed more pro-fibrotic genes, such as Timp1, Spon2, and Postn. In Arom(Hum) and WT combined, we found 6 clusters of fibroblast-like cells. Two of these clusters (clusters 2 and 3) were heavily represented by cells derived from Arom(Hum) mice (85-90% of cells in each cluster), which we termed “hernia-associated fibroblasts” (HAFs). Cluster 3 HAFs expressed high levels of Esr1 (gene encoding ERα), as well as estrogen-responsive genes such as Pgr and Greb1, and was enriched for estrogen, hypoxia, and TGFβ signaling pathways. Cluster 2 HAFs expressed genes associated with a pathological state, such as Lbp, Cthrc1, Mmp3, and Il33, and was enriched for the NF-κB and TNF-α signaling pathways. Conclusions: We found that LAM fibrosis in Arom(Hum) may result from the expansion of two distinct populations of HAFs - one is estrogen-responsive, and another is pathologic. Further in vitro / in vivo experiments are required to determine the relative contributions of these sub-populations of HAFs to fibrosis and inguinal hernias, leading to developing novel intervention strategies.