摘要Neovascularization and angiogenesis in the brain are important physiological processes for normal brain development and repair/regeneration following insults.Integrins are cell surface adhesion receptors mediating important function of cells such as survival,growth and development during tissue organization,differentiation and organogenesis.In this study,we used an integrin-binding array platform to identify the important types of integrins and their binding peptides that facilitate adhesion,growth,development,and vascular-like network formation of rat primary brain microvascular endothelial cells.Brain microvascular endothelial cells were isolated from rat brain on post-natal day 7.Cells were cultured in a custom-designed integrin array system containing short synthetic peptides binding to 16 types of integrins commonly expressed on cells in vertebrates.After 7 days of culture,the brain microvascular endothelial cells were processed for immunostaining with markers for endothelial cells including von Willibrand factor and platelet endothelial cell adhesion molecule.5-Bromo-2'-dexoyuridine was added to the culture at 48 hours prior to fixation to assess cell proliferation.Among 16 integrins tested,we found that α5β1,αvβ5 and αvβ8 greatly promoted proliferation of endothelial cells in culture.To investigate the effect of integrin-binding peptides in promoting neovascularization and angiogenesis,the binding peptides to the above three types of integrins were immobilized to our custom-designed hydrogel in three-dimensional(3D)culture of brain microvascular endothelial cells with the addition of vascular endothelial growth factor.Following a 7-day 3D culture,the culture was fixed and processed for double labeling of phalloidin with von Willibrand factor or platelet endothelial cell adhesion molecule and assessed under confocal microscopy.In the 3D culture in hydrogels conjugated with the integrin-binding peptide,brain microvascular endothelial cells formed interconnected vascular-like network with clearly discernable lumens,which is reminiscent of brain microvascular network in vivo.With the novel integrin-binding array system,we identified the specific types of integrins on brain microvascular endothelial cells that mediate cell adhesion and growth followed by functionalizing a 3D hydrogel culture system using the binding peptides that specifically bind to the identified integrins,leading to robust growth and lumenized microvascular-like network formation of brain microvascular endothelial cells in 3D culture.This technology can be used for in vitro and in vivo vascularization of transplants or brain lesions to promote brain tissue regeneration following neurological insults.