Synergism of Matrix Stiffness and Vascular Endothelial Growth Factor on Mesenchymal Stem Cells for Vascular Endothelial Regeneration

Abstract

Mesenchymal stem cells (MSCs) hold tremendous potential for vascular tissue regeneration. Research has demonstrated that individual factors in the cell microenvironment such as matrix elasticity and growth factors regulate MSC differentiation to vascular lineage. However, it is not well understood how matrix elasticity and growth factors combine to direct the MSC fate. This study examines the combined effects of matrix elasticity and vascular endothelial growth factor (VEGF) on both MSC differentiation into endothelial lineage and MSC paracrine signaling. MSCs were seeded in soft nanofibrous matrices with or without VEGF, and in Petri dishes with or without VEGF. Only MSCs seeded in three-dimensional soft matrices with VEGF showed significant increases in the expression of endothelial markers (vWF, eNOS, Flt-1, and Flk-1), while eliminating the expression of smooth muscle marker (SM-α-actin). MSCs cultured in VEGF alone on two-dimensional dishes showed increased expression of both early-stage endothelial and smooth muscle markers, indicating immature vascular differentiation. Furthermore, MSCs cultured in soft matrices with VEGF showed faster upregulation of endothelial markers compared with MSCs cultured in VEGF alone. Paracrine signaling studies found that endothelial cells cultured in the conditioned media from MSCs differentiated in the soft matrix and VEGF condition exhibited increased migration and formation of capillary-like structures. These results demonstrate that VEGF and soft matrix elasticity act synergistically to guide MSC differentiation into mature endothelial phenotype while enhancing paracrine signaling. Therefore, it is critical to control both mechanical and biochemical factors to safely regenerate vascular tissues with MSCs.

Three-dimensional, soft neotissue arrays as high throughput platforms for the interrogation of engineered tissue environments

“…For this work, we chose a soft, fibrous hydrogel platform prepared from an electrospinning method as previously described [8, 15]. Recent studies have highlighted the importance of 3-dimensional, fibrous matrices to optimize stem cell niche environments [24, 25], as well as their candidacy as platforms for MSC differentiation into vascular lineages [8, 15]. We find electrospinning, followed by UV polymerization to represent a highly versatile technique whereby stiffness and microstructure can be reproduced with high fidelity.…”

“…Several reports have observed the differentiation of MSCs into vascular lineages in vitro [36, 37]. The majority of literature regarding stem cell differentiation into vascular lineages involves precise soluble factor regiment [36,], application of shear [38], matrix rigidity [8, 15], composition of the ECM [39–41], or multiple factors [15, 42]. For example, it has been shown that the administration of vascular endothelial growth factor (VEGF) [36] or combination with shear stress [43] or matrix elasticity [15] instructs MSC differentiation into vascular lineages.…”

“…Several reports have demonstrated ECM protein microarrays [10], soluble factor screening [11], biomaterial chemistry screening [12, 13], and multiple signal integration arrays (i.e. elasticity and chemical factor) with encouraging results [14, 15]. However, despite the versatility afforded by current microarray technologies, the incorporation of multiple signals within engineered microarrays remain limited.…”

Novel biomaterials to study neural stem cell mechanobiology and improve cell-replacement therapies

“…A major next step will be to understand how these two classes of inputs are integrated to direct NSC behavior under physiological conditions, as well as how they can go awry in disease or injury. Encouragingly, there have already been studies that have reported the synergistic effect of tuned matrix stiffness and biochemical stimulation in MSCs [76, 77]. However, current culture platforms preclude high-throughput study of the additional parameter space.…”

Development of Mural Cells: From In Vivo Understanding to In Vitro Recapitulation

“…One study implicated the potential role of low stiffness (2 kPa) in directing MSC toward an EC versus an SMC fate, although it is unclear whether it is the nanofibrous architecture compared with polystyrene culture dishes or the signaling from lower stiffness of the nanofibers that might be directing EC fate [187]. Although there is little direct evidence to date that stiffness guides mural fate, it is well known that stiffness can modulate a progenitor's shape, cytoskeletal organization, and consequently its differentiation (reviewed in depth by Reilly et al [188]).…”