Stiffness of the aligned fibers affects structural and functional integrity of the oriented endothelial cells

Promoting healthy endothelialization of the tissue-engineered vascular grafts is of great importance in preventing the occurrence of undesired post-implantation complications including neointimal hyperplasia, late thrombosis, and neoatherosclerosis. Previous researches have demonstrated the crucial role of scaffold topography or stiffness in modulating the behavior of the monolayer endothelial cells (ECs). However, effects of the stiffness of scaffolds with anisotropic topography on ECs within vivo like oriented morphology has received little attention. In this study, aligned fibrous substrates (AFSs) with tunable stiffness (14.68-2141.72 MPa), similar to the range of stiffness of the healthy and diseased subendothelial matrix, were used to investigate the effects of fiber stiffness on ECs' attachment, orientation, proliferation, function, remodeling and dysfunction. The results demonstrate that stiffness of the AFSs, capable of providing topographical cues, is a crucial endothelium-protective microenvironmental factor by maintaining stable and quiescent endothelium with in vivo like orientation and strong cell-cell junctions. Stiffer AFSs exacerbated the disruption of endothelium integrity, the occurrence of endothelial-to-mesenchymal transition (EndMT), and the inflammation-induced activation in the endothelial monolayer. This study provides new insights into the understanding on how the stiffness of biomimicking anisotropic substrate regulates the structural and functional integrity of the in vivo like endothelial monolayer, and offers essential designing parameters in engineering biomimicking small-diameter vascular grafts for the regeneration of viable blood vessels. STATEMENT OF SIGNIFICANCE: In vascular tissue engineering, promoting endothelialization on scaffold surface has been considered as a paramount strategy to reduce post-implantation complications. Electrospun aligned fibers have been known to provide contact guidance effect in directing endothelial cells' oriented growth, however, whether the formed EC monolayer in 'correct' orientation shape is of 'correct' function hasn't been explored yet. Given the recognized important role of substrate stiffness in endothelial function, AFSs across physiologically relevant range of moduli (14.68-2141.72 MPa) while maintaining consistent surface chemistry and topographical features were employed to investigate the fiber stiffness effects on ECs function in anisotropic morphology. This study will provide more insightful perspectives in the physiologically remodeling progression of vascular endothelium and design of vascular scaffolds.