Blood vessel maturation in a 3-dimensional spheroidal coculture model: direct contact with smooth muscle cells regulates endothelial cell quiescence and abrogates VEGF responsiveness

Paracrine interactions between endothelial cells (EC) and mural cells act a s critical regulators of vessel wall assembly, vessel maturation and define a plasticity window for vascular remodeling. The present study was aimed a t studying blood vessel maturation processes in a novel 3-dimensional spher oidal coculture system of EC and smooth muscle cells (SMC). Coculture spher oids differentiate spontaneously in a calcium-dependent manner to organize into a core of SMC and a surface layer of EC, thus mimicking the physiologi cal assembly of blood vessels with surface lining EC and underlying mural c ells. Coculture of EC with SMC induces a mature, quiescent EC phenotype as evidenced by 1) a significant increase in the number of junctional complexe s of the EC surface layer, 2) a down-regulation of PDGF-B expression by coc ultured EC, and 3) an increased resistance of EC to undergo apoptosis. Furt hermore, EC cocultured with SMC become refractory to stimulation with VEGF (lack of CD34 expression on VEGF stimulation; inability to form capillary-l ike sprouts in a VEGF-dependent manner in a S-dimensional in gel angiogenes is assay). In contrast, costimulation with VEGF and Ang-2 induced sprouting angiogenesis originating from coculture spheroids consistent with a model of Ang-2-mediated vessel destabilization resulting in VEGF responsiveness. Ang-2 on its own was able to stimulate endothelial cells in the absence of Ang-l producing SMC, inducing lateral sheet migration as well as in gel spr outing angiogenesis. Taken together, the data establish the spheroidal EC/S MC system as a powerful cell. culture model to study paracrine interactions in the vessel wall and provide functional evidence for smooth muscle cell- mediated quiescence effects on endothelial cells.