Defining signalling pathways to characterise a novel anti-fibrotic agent

Lisa J Randall

doi:10.25907/00980

Hypertrophic scars (HS) represent a challenging clinical burden, characterized by excessive fibroblast proliferation, myofibroblast differentiation, and aberrant extracellular matrix (ECM) deposition following deep dermal injury. Existing treatments offer inconsistent efficacy, often leading to recurrence and adverse side effects, highlighting an urgent need for novel therapeutic strategies. This thesis investigates the anti-fibrotic potential of tomentosenol A, an acylphloroglucinol-meroterpenoid isolated from the propolis of the Australian stingless bee, Tetragonula carbonaria. My overarching objective was to elucidate the cellular and molecular mechanisms underlying the compounds anti-scarring activity in human dermal fibroblasts, focusing on known pro-fibrotic signalling pathways. Utilizing both normal adult human dermal fibroblasts (NAHDFs) and HS fibroblasts (HSFs) in in vitro studies, I first established that transforming growth factor-beta 1 (TGF-β1, 10 ng.mL⁻¹) robustly stimulates pro-scarring responses, including enhanced secreted cellular fibronectin (cFN) protein, increased smooth muscle alpha actin (α-SMA) incorporation into stress fibres, and elevated mRNA levels of profibrotic genes (FN1, ACTA2, COL1A1, COL3A1, CCN2). Tomentosenol A (6.25 µM) effectively inhibited all these TGF-β1-induced profibrotic responses in both NAHDFs and HSFs, suggesting the action of tomentosenol A as an anti-scarring agent. Further mechanistic investigations revealed that tomentosenol A modulates the canonical TGF-β signalling pathway by selectively inhibiting SMAD3 phosphorylation (IC₅₀ of 0.99 µM) in a dose-dependent manner, without affecting SMAD2 phosphorylation or total SMAD protein expression. This targeted inhibition of SMAD3, a key transcriptional mediator of fibrosis, underscores a precise mechanism of action avoiding the potentially detrimental systemic consequences associated with TGF-β receptor blockade. Beyond direct anti-fibrotic signalling, tomentosenol A demonstrated a multi-faceted impact on fibroblast fate. It significantly reduced NAHDF and HSF proliferation, as evidenced by real-time cell impedance measurements. Crucially, tomentosenol A induced caspase 3/7-dependent apoptotic signalling in unstimulated and TGF-β1-transformed NAHDFs, suggesting an ability to overcome pro-survival signals typically associated with scar-forming myofibroblasts. This pro-apoptotic effect was linked to an upregulation of pro-apoptotic genes (e.g., BCL2L11, PTEN, CDKN1A) and the induction of cell cycle arrest, indicated by decreased phosphorylation Histone H3. As a first step to compound optimisation, the anti-scarring activity of tomentosenol A was compared to several structurally related molecules, including the diastereoisomers S- and R-focifolidione. The study identified that the natural conformation of tomentosenol A is necessary for the potent anti-scarring effect observed in the in vitro setting Although S- and R- ficifolidione exhibited a capacity to inhibit myofibroblast differentiation independent of SMAD signalling, these analogues consistently lacked the comprehensive anti-fibrotic and pro-apoptotic profile characteristic of the parent compound, Tomentosenol A. Collectively, this research establishes compelling in vitro evidence for tomentosenol A as a highly promising anti-fibrotic agent. The demonstrated ability of tomentosenol A to selectively inhibit SMAD3 phosphorylation, suppress myofibroblast differentiation and aberrant pro-fibrotic mediator expression, and crucially, induce apoptosis and orchestrate cell cycle arrest, positions tomentosenol A as a valuable candidate compound for targeted therapeutic intervention in HS. These findings laid a foundation to chart a translational pathway towards further in vivo efficacy and comprehensive safety studies, with the goal of developing a much-needed clinical treatment for dermal fibrosis.

Defining signalling pathways to characterise a novel anti-fibrotic agent

Files and links (1)

Abstract

Details

Metrics