Development of a three-dimensional experimental vascular model with smooth muscle cell-derived cross-linked elastic fiber assembly

Abstract

Elastic lamellae are stratified extracellular structures essential for maintaining the integrity of large vessels. While numerous studies have elucidated the roles of individual molecules in elastic fiber formation, the mechanisms governing three-dimensional (3D) elastic fiber assembly in blood vessels remain incompletely understood. Advancing comprehensive understanding of these mechanisms requires overcoming the limitation of genetically modified animal models and conventional planar culture systems. Here, we present a 3D experimental vascular model (3D-VM) consisting of rat embryonic aortic smooth muscle cells (SMCs) that recapitulates multilayered SMCs and SMC-derived cross-linked elastic lamellae, constructed by a layer-by-layer technique utilizing fibronectin and gelatin. Electron microscopy confirmed the presence of stratified elastic lamellae, and mass spectrometry detected abundant desmosines and isodesmosines. The model exhibited an average burst pressure of 0.178 ± 0.042 MPa and withstood arterial pressures for at least 5 months after implantation in the adult rat aorta. Transcriptomic analysis revealed a gene expression profile in the 3D-VM that closely resembled that of native rat aortic tissues rather than planar-cultured SMCs. Gene ontology and pathway enrichment analyses identified significant positive correlations with genes associated with vascular development and extracellular matrix organization. Several elastic fiber-related genes were highly expressed at mRNA and protein levels in the 3D-VM compared with the adult aorta. Furthermore, fibulin-4 is a well-recognized elastic fiber component, and the 3D-VM generated with fibulin-4-deficient SMCs failed to form elastic fibers, highlighting the model's utility. These results suggest that the 3D-VM provides a platform for investigating the molecular mechanisms underlying 3D elastic fiber formation. STATEMENT OF SIGNIFICANCE: Elastic fibers confer tissues with distensibility and elastic recoil, allowing tissues to withstand repeated mechanical stress throughout life, particularly in dynamic organs such as arteries. Understanding the molecular mechanisms that govern elastic fiber formation is essential for developing therapeutic strategies for progressive diseases associated with elastic fiber dysfunction. To overcome the limitations of genetically modified animal models and conventional planar culture systems, which primarily elucidate the roles of individual molecules, we successfully established a three-dimensional vascular model composed of multilayered smooth muscle cells (SMCs) and SMC-derived cross-linked functional elastic lamellae. This model enables spatiotemporal analysis of elastic fiber formation and provides a platform for investigating the precise mechanisms that coordinate the interplay among multiple molecules.

Keywords: Blood vessel; Elastic fiber; Experimental model; Extracellular matrix.