Extracellular Stiffness Modulates the Expression of Functional Proteins and Growth Factors in Endothelial Cells

Abstract

Angiogenesis, the formation of blood vessels from pre-existing ones, is of vital importance during the early stages of bone healing. Extracellular stiffness plays an important role in regulating endothelial cell behavior and angiogenesis, but how this mechanical cue affects proliferation kinetics, gene regulation, and the expression of proteins implicated in angiogenesis and bone regeneration remains unclear. Using collagen-coated polyacrylamide (PAAm) hydrogels, human umbilical vein endothelial cells (HUVECs) are exposed to an environment that mimics the elastic properties of collagenous bone, and cellular proliferation and gene and protein expressions are assessed. The proliferation and gene expression of HUVECs are not differentially affected by culture on 3 or 30 kPa PAAm hydrogels, henceforth referred to as low and high stiffness gels, respectively. Although the proliferation and gene transcript levels remain unchanged, significant differences are found in the expressions of functional proteins and growth factors implicated both in the angiogenic and osteogenic processes. The down-regulation of the vascular endothelial growth factor receptor-2 protein with concomitant over-expression of caveolin-1, wingless-type 2, bone morphogenic protein 2, and basic fibroblast growth factor on the high stiffness PAAm hydrogel suggests that rigidity has a pro-angiogenic effect with inherent benefits for bone regeneration.

Regulating osteogenesis and adipogenesis in adipose‐derived stem cells by controlling underlying substrate stiffness

“…The increased levels of ASC proteins and mRNAs observed on stiff matrices suggest that there is a potential relationship between the Rho/ROCK and Wnt/β‐catenin pathways. Mechano‐chemical stimuli generated as a result of culture on stiff matrices leads to ASCs spreading and an increase in cell tension, which is known to initiate RhoA and ROCK and trigger the activation of β‐catenin (Samuel et al, ; Santos et al, ). In this study, the Wnt/β‐catenin pathway had a negative effect on adipogenic differentiation whereas other studies have shown that the epidermal Wnt/β‐catenin pathway is a potent initiator of adipogenesis.…”

Vascularization and Angiogenesis in Tissue Engineering: Beyond Creating Static Networks

“…Several studies have shown that vascular organization is also dependent on the mechanical properties of the matrix in which the endothelial cells reside. A study by Santos et al demonstrates that HUVECs grown on collagen-coated polyacrylamide hydrogels of low (3 kPa) and high (30 kPa) stiffness display similar proliferation and gene transcription levels, but show a lower expression of the functional VEGF receptor-2 protein on the stiffer substrate [65], which means that the extent to which endothelial cells are responsive to angiogenetic processes depends on the mechanical environment. In a different study, Mason et al used a system where the stiffness of a collagen hydrogel can be changed without significant changes to the collagen density and architecture.…”

Angiogenesis in Tissue Engineering: As Nature Intended?

“…Increased stiffness of polyacrylamide (PA) scaffolds from 1 to 10 kPa has been shown to boost the endothelial cell response to VEGF by increasing VEGFR-2 internalisation (LaValley et al, 2017). Similarly, increased stiffness of collagen coated PA gels in a range from 3 to 3,000 Pa affected the expression of functional proteins and GF in HUVECs, but did not affect their proliferation and gene expression (Santos et al, 2015). Differentiation of endothelial progenitor cells was proportional to the stiffness of the PDMS scaffolds (Xue et al, 2017).…”