MON-253 - TGFβ signaling in inguinal hernias: from mouse to human

Introduction: Over 1 in 4 men experience an inguinal hernia during their lifetime, with surgery their only available treatment option. The molecular basis of the hernias is unclear, staunching development of nonsurgical therapies. Recently, we found that humanized aromatase mice (Aromhum) with high aromatase expression and estradiol (E2) production in the lower abdominal muscle (LAM) develop LAM fibrosis and atrophy through fibroblast E2/ESR1 signaling, resulting in hernia development. Furthermore, single-cell (sc) RNA sequencing (scRNA-seq) of Aromhum LAM tissue revealed enrichment of the transforming growth factor beta (TGFβ) signaling pathway. However, it is unclear whether TGFβ signal transduction is elevated in hernia-affected tissue in both mouse and human or whether it is required for hernia development.

Methods: We first measured TGFβ ligands by multiplex ELISA and latent and active TGFβ by immunoblotting in LAM tissues of WT and Aromhum mice. We then defined TGFβ receptor (TGFBR2) expression and downstream SMAD2/3 phosphorylation in WT and Aromhum LAM. Next, primary LAM fibroblasts from Aromhum mice were treated with E2 with or without ESR1 antagonist (fulvestrant) to determine whether E2/ESR1 regulates expression of TGFβ ligands and receptors by qPCR. Additionally, Aromhum mice were treated with a TGFBR1 inhibitor, Galunisertib (50mg/kg, 3 times per week, intraperitoneal injection) to assess the in vivo role of TGFβ signaling in hernia development and LAM fibrosis/atrophy. Finally, we translated our findings to human by performing single-nucleus (sn) RNA-seq on paired human LAM tissues (i.e., the internal oblique muscle of the conjoint tendon) from the hernia-affected area and adjacent healthy controls (n=2) from inguinal hernia patients.

Results: TGFβ ligands 1, 2, and 3 by multiplex ELISA as well as latent and active TGFβ by immunoblotting were significantly increased in Aromhum LAM compared to WT LAM. TGFBR2 expression and SMAD2/3 phosphorylation were also elevated in Aromhum LAM compared to WT LAM. E2 treatment of primary Aromhum LAM fibroblasts upregulated expression of Tgfb2 and Tgfbr2, which is blocked by fulvestrant. Galunisertib treatment significantly reduced hernia size in Aromhum mice. Interestingly, preliminary snRNA-seq analysis of paired hernia-affected and healthy control LAM tissues from inguinal hernia patients revealed notably higher levels of a TGFβ ligand (TGFB1), TGFβ receptors (TGFBR1-3), activation modulators (ITGB3, THBS2), and latent binding proteins (LTBP1-4) in hernia-affected LAM.

Conclusion: TGFβ signal transduction is significantly elevated in Aromhum LAM and contributes to hernia development and possibly LAM fibrosis/atrophy. Preliminary data from snRNA-seq of human inguinal hernia patient LAM tissues demonstrate TGFβ pathway dysregulation, presenting a potential avenue for pharmacological therapeutic intervention.

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