Integrin binding peptides facilitate growth and interconnected vascular-like network formation of rat primary cortical vascular endothelial cells in vitro

Abstract

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, α_vβ₅ and α_vβ₈ 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.

Keywords: 3D culture; angiogenesis; brain microvascular endothelial cells; hydrogel; integrins; platelet endothelial cell adhesion molecule (PECAM-1); vascular endothelial growth factor (VEGF); vascularization.

Figure 1

Growth of primary rat BMECs in the integrin-binding array platform. BMECs after 7-day culture were identified with EC markers von Willibrand factor (VWF, red), PECAM-1 (green) and neucli marker DAPI (blue). Representative image showed the growth pattern of BMECs in wells of the selected integrin-binding peptide and the control well with standard collagen coating. Note that the majority of cells were ECs expressing VWF and PECAM1. Scale bar: 50 µm. BMECs: Brain microvascular endothelial cells; DAPI: 4′,6-diamidino-2-phenylindole; ECs: endothelial cells; PECAM-1: platelet endothelial cell adhesion molecule 1; VWF: von Willibrand factor.

Figure 2

Proliferation of primary rat BMECs in the integrin-binding array platform. Proliferation of BMECs in the integrin array platform was assessed by immunofluorecent labeling with EC marker VWF (red), thymidine aanlog BrdU (green) and DAPI (blue) after 7-day culture. Representative images showed the proliferation pattern of BMECs in wells of the selected integrin-binding peptide and the control well with standard collagen coating. Scale bar: 50 µm. BMECs: Brain microvascular endothelial cells; BrdU: 5-bromo-2’-dexoyuridine; DAPI: 4’,6-diamidino-2-phenylindole; PECAM-1: platelet endothelial cell adhesion molecule 1; VWF: von Willibrand factor.

Figure 3

Quantification analysis of the proliferation rate of the BMECs in the integrin-binding array platform. The number of VWF and BrdU double-labeled cells were counted against the total number of DAPI⁺ cells. Cells growing in integrin-binding peptides for α₅β₁ or α_vβ₅ had significantly higher ratio in comparison to cells growing in control wells. All experiments were performed in triplicate. BMECs: Brain microvascular endothelial cells; BrdU: 5-bromo-2′-dexoyuridine; DAPI: 4′,6-diamidino-2-phenylindole; VWF: von Willibrand factor. *P < 0.05, ***P < 0.001, ****P < 0.0001.

Figure 4

Growth of primary rat BMECs in three-dimensional culture in integrin binding peptide-conjugated hydrogels. Immunofluorescent staining with PECAM-1 (red), Phalloidin (green) and DAPI (blue) showed the growth pattern of primary rat BMECs in 3-D culture within selected integrin α5β1-binding peptide-conjugated hydrogels. Formation of interconnected tubular network structures with lumens was clearly seen at the x-y-z plane image (asterisks). Scale bar: 50 µm. BMECs: Brain microvascular endothelial cells; DAPI: 4′,6-diamidino-2-phenylindole; PECAM-1: platelet endothelial cell adhesion molecule 1.