Substrate stiffness engineered to replicate disease conditions influence senescence and fibrotic responses in primary lung fibroblasts

Abstract

In fibrosis remodelling of ECM leads to changes in composition and stiffness. Such changes can have a major impact on cell functions including proliferation, secretory profile and differentiation. Several studies have reported that fibrosis is characterised by increased senescence and accumulating evidence suggests that changes to the ECM including altered composition and increased stiffness may contribute to premature cellular senescence. This study investigated if increased stiffness could modulate markers of senescence and/or fibrosis in primary human lung fibroblasts. Using hydrogels representing stiffnesses that fall within healthy and fibrotic ranges, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to 7 days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiffer (±15 kPa) hydrogels showed higher Yes-associated protein-1 (YAP) nuclear translocation compared to soft hydrogels. When looking at senescence-associated proteins we also found higher secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) but no change in transforming growth factor-β1 (TGF-β1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiffer matrices. With respect to genes associated with fibrosis, fibroblasts on stiffer hydrogels compared to soft had higher expression of smooth muscle alpha (α)-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after 7 days. Our results show that exposure of lung fibroblasts to fibrotic stiffness activates genes and secreted factors that are part of fibrotic responses and part of the Senescence-associated secretory phenotype (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing progression of a fibrotic response.

Serglycin secreted by late-stage nucleus pulposus cells is a biomarker of intervertebral disc degeneration

“…Pro-NPCs: progenitor NPCs, marked with ACAN , SOX9 , UBE2C, and TOP2A ; Fibro- NPCs: fibrotic NPCs, marked with ACAN , SOX9 , FBLN1 34 , 35 ; IR-NPCs: integral regulatory NPCs, marked with ACAN , SOX9 , CH3L2 36 ; Met-NPCs: metabolic NPCs, marked with ACAN , SOX9 , DKK1 37 ; Adh-NPCs: adhesive NPCs, marked with ACAN , SOX9, and MSMO1 36 ; and SR-NPCs: stress-responsive NPCs, marked with ACAN , SOX9, and CP .…”

Metabolic pathways in immune senescence and inflammaging: Novel therapeutic strategy for chronic inflammatory lung diseases. An EAACI position paper from the Task Force for Immunopharmacology

“…205 Enhanced matrix stiffness seen in disease may also affect fibroblast senescence and the release of functional SASP. 206 Elevated 27-hydroxycholesterol (27-OHC) levels seen in COPD airways enhance fibroblast senescence via the prostaglandin E2 (PGE2) pathway. 207 Activators of the PGE2 receptor (EP4) reversed senescence of alveolar organoids.…”

Current possibilities and future opportunities provided by three-dimensional lung ECM-derived hydrogels

“…Alternatively, natural ECM components have also been used to generate single component hydrogels that readily support cell attachment, but are more limited in the possibilities for tuning their biomechanical properties. Examples of such hydrogels include collagen type I, fibrin, gelatin (methacrylate) and hyaluronan ( Bourke et al, 2011 ; Tjin et al, 2017 ; Sun et al, 2020 ; Martinez-Garcia et al, 2021b ; Hui et al, 2021 ; Blokland et al, 2022 ; Loebel et al, 2022 ; Martinez-Garcia et al, 2022 ). Such hydrogels provide the 3D environment for cells, modelling the dimensionality and possibly the biomechanical mimicry of the in vivo situation, but they are not reflective of the complexity of the ECM components within the tissue microenvironment.…”