Matrix stiffness modulates tip cell formation through the p-PXN-Rac1-YAP signaling axis.

Endothelial tip cell outgrowth of blood-vessel sprouts marks the initiation of angiogenesis which is critical in physiological and pathophysiological procedures. However, how mechanical characteristics of extracellular matrix (ECM) modulates tip cell formation has been largely neglected. In this study, we found enhanced CD31 expression in the stiffening outer layer of hepatocellular carcinoma than in surrounding soft tissues. Stiffened matrix promoted sprouting from endothelial cell (EC) spheroids and upregulated expressions of tip cell-enriched genes in vitro. Moreover, tip cells showed increased cellular stiffness, more actin cytoskeleton organization and enhanced YAP nuclear transfer than stalk and phalanx ECs. We further uncovered that substrate stiffness regulates FAK and Paxillin phosphorylation in focal adhesion of ECs promoting Rac1 transition from inactive to active state. YAP is subsequently activated and translocated into nucleus, leading to increased tip cell specification. p-Paxillin can also loosen the intercellular connection which also facilitates tip cell specification. Collectively our present study shows that matrix stiffness modulates tip cell formation through p-PXN-Rac1-YAP signaling axis, shedding light on the role of mechanotransduction in tip cell formation. This is of special significance in biomaterial design and treatment of some pathological situations.