Sphingosine-1-Phosphate

Camille L. Duran, Roland Kaunas, Kayla J. Bayless

Angiogenesis is the process of new blood vessel growth from pre-existing structures. During sprout initiation, endothelial cells (ECs) are activated by pro-angiogenic factors to degrade the basement membrane, migrate into the surrounding matrix, and form structures that anastomose to connect neighboring vessels. Sphingosine 1-phosphate (S1P) is a biologically active lysosphingolipid that is secreted by platelets and promotes angiogenesis under normal and pathological conditions by acting on ECs. In addition to biochemical factors, the endothelium is continuously subjected to mechanical forces in the form of wall shear stress (WSS) from fluid forces. Here, we describe an in vitro, three-dimensional (3D) endothelial sprouting assay that is significantly enhanced by S1P, WSS, angiogenic growth factors (GFs), and fibronectin. This assay is assembled by seeding primary human endothelial cells onto 3D collagen matrices containing S1P and other pro-angiogenic factors. Once attached, physiological levels of WSS are applied to induce robust sprouting responses. This approach promotes the initiation of angiogenic sprouts stimulated by S1P, and allows the study of 3D sprouting of primary human endothelial cells induced in response to these physiological factors.