The vascular endothelium continually senses and responds to both biochemical and mechanical stimuli to regulate vascular function in health and disease. The purpose of this dissertation was to understand the molecular mechanisms by which endothelial cells (ECs) respond to sphingosine 1-phosphate (S1P) and fluid wall shear stress (WSS) to initiate angiogenesis. To accomplish this, a novel cell culture system was developed to study the combined effects of S1P and WSS on inducing EC invasion into three-dimensional (3-D) collagen matrices. EC invasion required the presence of S1P, with the effects of S1P being enhanced by WSS to an extent comparable with S1P combined with pro-angiogenic growth factor stimulation. The extent of EC invasion depended on the magnitude of WSS in a biphasic manner, with the greatest induction occurring at 5.3 dyn/cm2 WSS. Several proteins have been implicated in EC invasion, including calpain, Akt, vimentin, p21-activated kinase (PAK), and membrane type 1-matrix metalloproteinase (MT1-MMP). Interestingly, activations of calpain and MT1-MMP and phosphorylations of Akt, PAK, and vimentin coincided with, and were required for, S1P- and WSS- induced EC invasion. Further, inhibitors of calpain, MT1-MMP, Akt and PAK all attenuated invasion induced by WSS and S1P. Calpain inhibition reduced Akt phosphorylation, vimentin cleavage, and MT1-MMP membrane translocation, suggesting that calpain regulates MT1-MMP via Akt phosphorylation and vimentin remodeling. Akt inhibition also completely blocked MT1-MMP membrane translocation and decreased phosphorylation of PAK and vimentin. In summary, these results suggest a new molecular pathway by which the combination of S1P and WSS stimulates EC invasion through calpain, Akt, PAK and vimentin to regulate activation and membrane translocation of MT1-MMP in 3-D collagen matrices.