Co-culture with fibroblasts in stiff 3D scaffolds increases CD54 and CD140a expression on macrophages

Vascular fibrosis is a major contributor to hypertension and cardiovascular disease and is caused by excessive extracellular matrix deposition and tissue stiffening. Macrophages and fibroblasts are key regulators of this process, yet most studies have relied on non-physiological two-dimensional (2D) in vitro culture systems, limiting their relevance. In this study, three-dimensional (3D) collagen-based scaffolds with tunable stiffnesses were developed to model the effect of fibrotic microenvironments on macrophage and fibroblast interactions. Bone-marrow derived macrophages (BMDMs) and mouse embryonic fibroblasts (MEFs) co-cultured in 3D maintained equivalent viability in scaffolds with a storage modulus of ~200 Pa and ~2000 Pa. Strikingly, during co-culture, macrophages, but not fibroblasts, exhibited a stiffness-dependent upregulation of CD54 (ICAM-1) and CD140a (PDGFRα), markers associated with inflammation and fibrosis. Confocal imaging revealed only occasional direct interaction between cell types, suggesting that the altered macrophage phenotype is driven by combined mechanical and soluble cues rather than increased physical interaction. Overall, these findings highlight the importance of dimensionality and stiffness in shaping macrophage-fibroblast crosstalk and provide a platform for dissecting mechanisms underlying vascular fibrosis.