Three-dimensional multilayers of smooth muscle cells as a new experimental model for vascular elastic fiber formation studies

•We fabricated rat aortic SMCs into 3DCMs by employing fibronectin-gelatin nanofilms.•The 3DCMs cultured in the optimized conditions exhibited layered elastic fibers.•The elastic fiber formation in 3DCMs can be pharmacologically manipulated.•Macrophage infiltration decreases elastic fiber formation in 3DCMs.•3DCMs may offer a new experimental tool for the studies of vascular elastic fibers.

ObjectiveElastic fiber formation is disrupted with age and by health conditions including aneurysms and atherosclerosis. Despite considerable progress in the understanding of elastogenesis using the planar culture system and genetically modified animals, it remains difficult to restore elastic fibers in diseased vessels. To further study the molecular mechanisms, in vitro three-dimensional vascular constructs need to be established. We previously fabricated vascular smooth muscle cells (SMCs) into three-dimensional cellular multilayers (3DCMs) using a hierarchical cell manipulation technique, in which cells were coated with fibronectin-gelatin nanofilms to provide adhesive nano-scaffolds. Since fibronectin is known to assemble and activate elastic fiber-related molecules, we further optimized culture conditions.Methods and resultsElastica stain, immunofluorescence, and electron microscopic analysis demonstrated that 3DCMs, which consisted of seven layers of neonatal rat aortic SMCs cultured in 1% fetal bovine serum (FBS) in Dulbecco's modified Eagle's medium, exhibited layered elastic fibers within seven days of being in a static culture condition. In contrast, the application of adult SMCs, 10% FBS, ε-poly(lysine) as an alternative adhesive for fibronectin, or four-layered SMCs, failed to generate layered elastic fiber formation. Radioimmunoassay using [3H]valine further confirmed the greater amount of cross-linked elastic fibers in 3DCMs than in monolayered SMCs. Layered elastic fiber formation in 3DCMs was inhibited by the lysyl oxidase inhibitor β-aminopropionitrile, or prostaglandin E2. Furthermore, infiltration of THP-1-derived macrophages decreased the surrounding elastic fiber formation in 3DCMs.Conclusion3DCMs may offer a new experimental vascular model to explore pharmacological therapeutic strategies for disordered elastic fiber homeostasis.