NORMALIZING ABERRANT VASCULATURE CHARACTERISTICS GENERATED BY ELEVATED EXTRACELLULAR MATRIX STIFFNESS

Tumor blood vasculature tends to be heterogeneously distributed, densely branched, tortuous, malformed, and hyperpermeable relative to that found within physiological tissues. Furthermore, tumors are known to stiffen as they progress, a phenomenon that has largely been attributed to heightened extracellular matrix protein cross-linking. As cells are capable of sensing and responding to the stiffness of their surrounding matrix via mechanisms that include changes in the phosphorylation of focal adhesion proteins, like FAK and Src, we hypothesized that there is a relationship between abnormal tumor vasculature characteristics and tumor stiffening. By grafting glycated collagen constructs to the chorioallantoic membranes of chicken embryos cultured ex ovo, and with two- and three-dimensional human umbilical vein endothelial cell culture systems, we show that increasing the stiffness of a tissue significantly enhances the extent of angiogenic vascularization it experiences, and that neovessels or monolayers formed within stiff tissues or on stiff substrates are significantly more permeable than their compliant counterparts. Furthermore, we demonstrated that membrane type-one matrix metalloproteinase activity inhibition can nullify the effect that matrix stiffening has on vascularization ex ovo, and that inhibiting the phosphorylation of FAK tyrosine residue 397 (Y397) can alleviate stiffness-induced hyperpermeability in vitro and ex ovo. Using western blotting techniques, we show that Src Y418 and VE-cadherin Y685 phosphorylation significantly increase with increasing matrix stiffness, and that FAK inhibition prevents the latter from occurring. Together, these data indicate that matrix stiffening promotes the phosphorylation of Src, and that Src interacts with activated FAK to phosphorylate tyrosine residues on the cytoplasmic tail of VE-cadherin, which regulate endothelial barrier junction integrity via the mediation of catenin attachment. We conclude by suggesting co-treatments to enhance the efficacy of existing chemotherapeutics, and by conceptualizing future projects that expand upon the work presented here