Poster CC7 - Matrix viscoelasticity mediates macrophage phenotype and regulates cardiac fibroblasts matrix production

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 in our lab [1] and elsewhere [2]. iMACs were stimulated with media (control), interferon-γ/liposaccharides (IFNγ/LPS), or interleukin-4 and interleukin 13 (IL4/IL13) to adopt a naïve, inflammatory, or healing activation state, respectively. iCFs and iMACs were cocultured together prior to immunostaining for cell-derived fibronectin (FN-EDA). In addition, iCFs were treated with transforming growth factor-β (TGFβ), angiotensin II (ANG II), or low molecular weight hyaluronic acid (LMW HA) prior to cell lysis. Western blot analysis was subsequently performed for cellular fibronectin (FN-EDA) and Actin. Material stiffness as well as stress relaxation was measured in RGD conjugated alginate hydrogels using parallel plate rheometry.

Results, Conclusions, and Discussions: When cocultured with naïve and healing activated iMACs, iCFs expressed significantly more FN-EDA when compared to fibroblast alone (none) and–counter intuitively– inflammatory coculture conditions (IFNγ/LPS, Fig. 1A). Cytokine analysis showed that iMACs produce more immune-mediated agonists, e.g., TGFβ, and their single addition to iCF culture (or Angiotensin II and low molecular weight hyaluronic acid (LMW HA)) was sufficient to induce dose-dependent increased FN-EDA expression (Fig. 1B). To assess viscoelastic regulation of iCF matrix production, RGD conjugated alginate hydrogels were fabricated with soft/stiff elastic and fast/slow stress relaxation properties (Fig. 1C). Subsequent analyses will show that iMAC and iCF co-culture have enhanced effects on viscoelastic hydrogels vs. their control counterparts. These data demonstrate that healing macrophages interactions with cardiac fibroblasts can modulate fibroblast activation and enhance fibronectin expression.

Acknowledgements (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 (T32HL007444 to H.A.).