Matrix viscoelasticity promotes pro-fibrotic macrophage and cardiac fibroblast signaling

Introduction: : Cardiac fibrosis is a chronic, immune-mediated condition regulated by the function of macrophages and cardiac fibroblasts. Macrophages are recruited to sites of injury upon where they help activate fibroblasts resulting in increased production of extracellular matrix proteins. Over time, fibrosis stiffens the injured myocardial wall and reduces tissue viscosity. While there has been much focus on elastic changes, it is not clear to what extent cellular crosstalk and viscous dissipation influence chronic remodeling and scar formation. Here, we utilize mechanically tunable culture systems to investigate the role of substrate mechanics in modulating macrophage and cardiac fibroblast extrinsic signaling and function.

Materials and

Methods: : Induced pluripotent stem cell derived cardiac fibroblasts (iCFs) and macrophages (iMACs) were generated using previously established methods [Whitehead et al., JMCC, 2022; Pouyanfard et al., Stem Cells, 2021] and cultured on 15kPa viscous as well as non-viscous RGD conjugated alginate hydrogels. iCFs were treated with the agonist TGFβ or were cultured against iMACs using an engineered coculture platform that enables the study of paracrine interactions between two cell populations cultured on mechanically tunable hydrogel surfaces. iCFs cultured alone were used as controls. Immunofluorescence was performed and mean fluorescence intensity of fibronectin quantified to evaluate resulting changes in iCF activation state.

Results, Conclusions, and Discussions:: When cultured on viscous surfaces fibronectin expression was enhanced in iCFs suggesting that viscous environments promote cell activation (Fig. 1A). Further increases in fibronectin were observed with addition of TGFβ, an agonist known to promote fibroblast activation (Fig. 1A). Moreover, given that cardiac fibroblasts and macrophages are known to coexist within the heart, co-culture experiments were also performed using an engineered device (Fig. 1B). Interestingly, biochemical cues produced by iMACs produced similar increases in iCF activation on viscous surfaces (Fig. 1C). Subsequent analyses will identify viscous dependent mechanotransduction pathways and molecular mechanisms involved in regulating macrophage-cardiac fibroblast paracrine interactions. These data suggest that viscous environments enhance cardiac fibroblast activation. In addition, through the use of a novel co-culture platform, viscous surfaces were also observed to promote pro-fibrotic paracrine interactions between macrophages and cardiac fibroblasts.

Acknowledgements and/or References (Optional):: This work was supported by the National Institutes of Health (R01AG045428 to A.J.E). Fellowship support was provided by the National Institutes of Health (1F32HL178303 to H.A.).