What is the clinical evidence from this study?

Study design: Other. Population: Fibrosis. Intervention: MEK inhibitors and TGFβ receptor inhibitors vs. Control medium. Primary outcome: ECM remodeling between fibroblast clusters.

What does this research mean for the field?

After initial fibroblast activation by TGFβ, non-canonical MEK-ERK signaling drives fibrotic extracellular matrix remodeling, and high concentrations of TGFβ receptor inhibitors can paradoxically stimulate this profibrotic activity. Novelty: ClaimNovelty.NOVEL_FINDING. Consensus alignment: ConsensusAlignment.NEUTRAL.

April 24, 2025Open Access

Novel high throughput 3D ECM remodeling assay identifies MEK as key driver of fibrotic fibroblast activity

Key Result

Non-canonical activation of MEK-ERK signaling drives fibrotic ECM remodeling, and high concentrations of TGFβ receptor inhibitors paradoxically stimulate this MEK-mediated profibrotic activity.

Structured PICO

Does inhibition of specific signaling pathways (e.g., MEK, ROCK, LOX) reduce TGFβ-induced 3D ECM remodeling in primary human fibroblasts?

Population

Primary human dermal and lung fibroblasts (normal and TGFβ-activated) embedded in 3D collagen matrix.

Intervention

Anti-fibrotic compounds (including ROCK inhibitors, LIMK inhibitors, LOX inhibitors, TGFβ receptor inhibitors, and MEK inhibitors) and profibrotic stimuli (TGFβ, S1P).

Comparator

Control medium or untreated normal fibroblasts.

Outcome

3D fibroblast-mediated ECM remodeling (quantified by reflection microscopy) and cell viability (cytotoxicity).surrogate

A novel 3D high-throughput assay reveals that non-canonical MEK-ERK signaling drives fibrotic ECM remodeling and that high doses of TGFβ receptor inhibitors paradoxically stimulate this profibrotic activity.

Limitations

The model does not recapitulate the complex interplay between immune cells and fibroblasts that drives in vivo fibrosis.

Abstract

In fibrotic tissues, activated fibroblasts remodel the collagen-rich extracellular matrix (ECM). Intervening with this process represents a candidate therapeutic strategy to attenuate disease progression. Models that generate quantitative data on 3D fibroblast-mediated ECM remodeling with the reproducibility and throughput needed for drug testing are lacking. Here, we develop a model that fits this purpose and produces combined quantitative information on drug efficacy and cytotoxicity. We use microinjection robotics to design patterns of fibrillar collagen-embedded fibroblast clusters and apply automated microscopy and image analysis to quantify ECM remodeling between-, and cell viability within clusters of TGFβ-activated primary human skin or lung fibroblasts. We apply this assay to compound screening and reveal actionable targets to suppress fibrotic ECM remodeling. Strikingly, we find that after an initial phase of fibroblast activation by TGFβ, canonical TGFβ signaling is dispensable and, instead, non-canonical activation of MEK-ERK signaling drives ECM remodeling. Moreover, we reveal that higher concentrations of two TGFβ receptor inhibitors while blocking canonical TGFβ signaling, in fact stimulate this MEK-mediated profibrotic ECM remodeling activity.

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