PROJECT SUMMARY Angiogenesis refers to the process by which new blood vessels grow from pre-existing ones. Excessive angiogenesis is a hallmark of many cancers and contributes to cancer progression. During tumor growth, the angiogenic switch is deregulated resulting in an abnormal vasculature characterized by hyperpermeable, tortuous, and immature blood vessels. Consequences of the irregular tumor vasculature include poor tumor perfusion resulting in hypoxia and hindrance to therapeutic drug delivery. While it is well established that chemical cues are heavily involved in regulating tumor angiogenesis, the influence of mechanical cues are becoming more apparent. Notably, the extracellular matrix is significantly stiffer than normal tissue in many cancers, and elevated matrix stiffness has been shown to promote angiogenesis and disrupt barrier integrity. Prior work in our lab has shown both in vitro and in vivo that increased matrix crosslinking (resulting in a stiffer matrix) increases endothelial sprouting and causes more permeable blood vessels to form. However, the mechanisms governing the effects of matrix stiffness on endothelial cell function are still poorly understood. Uncovering the mechanisms driving stiffness-mediated effects on endothelial cells is critical for developing novel approaches to normalize tumor vasculature and promote normal vessel growth. Interestingly, our preliminary data obtained through RNA-sequencing indicate that endothelial biglycan expression is regulated by matrix stiffness in vitro and in vivo. Previously, biglycan has been identified as a tumor endothelial cell marker and biglycan overexpression is correlated to poor prognosis in several cancers. Notably, biglycan can promote angiogenesis as an autocrine agent. While it is established that biglycan is upregulated in disease and contributes to pathological endothelial signaling and behavior, our data indicates that ECM stiffness may drive these effects. Given these findings, we will use tailored biomaterials, transgenic mice, and state of the art biochemical assays to investigate the hypothesis that matrix stiffness increases cellular contractility to drive elevated endothelial biglycan expression and promote aberrant angiogenesis and hyperpermeability. In aim 1, the molecular mechanism governing matrix stiffness driven endothelial biglycan expression will be determined. In aim 2, the effects of matrix stiffness-mediated endothelial biglycan expression in promoting increased angiogenesis and a hyperpermeable phenotype will be investigated. This work will provide critical insight into how the mechanical properties of tumors regulate the structure and integrity of vasculature and uncover potential therapeutic targets for vasculature normalization with a focus on endothelial biglycan regulation.