Kuwar Ram, Wen Xuejun, Zhang Ning, Sun Dong
Department of Anatomy and Neurobiology, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA, USA.
Department of Chemical and Life Science Engineering, College of Engineering, Virginia Commonwealth University, Richmond, VA, USA.
Neural Regen Res. 2023 May;18(5):1052-1056. doi: 10.4103/1673-5374.355760.
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 αβ, αβ and αβ 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.
大脑中的新生血管形成和血管生成是正常脑发育以及损伤后修复/再生的重要生理过程。整合素是细胞表面黏附受体，在组织构建、分化和器官发生过程中介导细胞的重要功能，如存活、生长和发育。在本研究中，我们使用整合素结合阵列平台来鉴定促进大鼠原代脑微血管内皮细胞黏附、生长、发育和类血管网络形成的重要整合素类型及其结合肽。在出生后第7天从大鼠脑中分离出脑微血管内皮细胞。将细胞培养在定制设计的整合素阵列系统中，该系统包含与脊椎动物细胞中常见表达的16种整合素结合的短合成肽。培养7天后，对脑微血管内皮细胞进行免疫染色，使用包括血管性血友病因子和血小板内皮细胞黏附分子在内的内皮细胞标志物。在固定前48小时向培养物中加入5-溴-2'-脱氧尿苷以评估细胞增殖。在测试的16种整合素中，我们发现αβ、αβ和αβ极大地促进了培养内皮细胞的增殖。为了研究整合素结合肽在促进新生血管形成和血管生成中的作用，将上述三种整合素的结合肽固定在我们定制设计的水凝胶上，用于脑微血管内皮细胞的三维（3D）培养，并添加血管内皮生长因子。经过7天的3D培养后，将培养物固定并进行鬼笔环肽与血管性血友病因子或血小板内皮细胞黏附分子的双重标记，并在共聚焦显微镜下评估。在与整合素结合肽偶联的水凝胶中的3D培养中，脑微血管内皮细胞形成了具有清晰可辨管腔的相互连接的类血管网络，这让人联想到体内的脑微血管网络。通过新型整合素结合阵列系统，我们鉴定了脑微血管内皮细胞上介导细胞黏附和生长的特定整合素类型，随后使用与鉴定出的整合素特异性结合的结合肽对3D水凝胶培养系统进行功能化，导致脑微血管内皮细胞在3D培养中强劲生长并形成有管腔的类微血管网络。该技术可用于移植或脑损伤的体外和体内血管化，以促进神经损伤后脑组织的再生。
