Zhang Rongxin, Yang Hui, Li Min, Yao Qizhi, Chen Changyi
Molecular Surgeon Research Center, Division of Vascular and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
Exp Hematol. 2005 Dec;33(12):1554-63. doi: 10.1016/j.exphem.2005.08.011.
In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The objective of this study was to develop a new strategy that utilizes cytokines and lipopolysaccharide (LPS) to accelerate endothelial-like cell differentiation from peripheral blood CD14(+) monocytes.
Peripheral blood CD14(+) monocytes were purified with immunobeads and cultured with an angiogenic growth factor-rich growth medium (EGM-2) with or without initial treatment of LPS in combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days (the day 4 cultures). The cells were then continuously cultured in EGM-2 medium for an additional 4 or 10 days (the day 8 or day 14 cultures). Cell markers were determined by flow cytometry analysis and immunofluorescence staining. Cytokine/chemokine profile was studied by Bio-Plex immunoassay.
In the group of initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2, the majority of suspended CD14(+) monocytes were attached and changed their morphology to endothelial-like cells, which expressed high levels of endothelial cell markers CD31, von Willebrand factor, and vascular endothelial growth factor receptor-1 as well as two major endothelial tight junction proteins zonula occludens -1 and occludin in the day 8 cultures. Endothelial nitric oxide synthase expression was substantially increased. Endothelial-like cells were also able to uptake acetylated low-density lipoprotein and bind to Ulex europeus lectin. In addition, endothelial-like cells showed a unique cytokine/chemokine profile with substantial increases of macrophage inflammatory protein-1beta, IL-6, granulocyte colony-stimulating factor, and IL-8.
Initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2 is an effective strategy for acceleration of endothelial-like cell differentiation from peripheral blood CD14(+) monocytes in vitro.
内皮细胞的体外分化在血管组织工程和多种疾病的细胞治疗中具有潜在应用价值。本研究的目的是开发一种新策略，利用细胞因子和脂多糖（LPS）来加速外周血CD14(+)单核细胞向内皮样细胞的分化。
用免疫磁珠纯化外周血CD14(+)单核细胞，并在富含血管生成生长因子的生长培养基（EGM-2）中培养，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）和白细胞介素-4（IL-4）联合使用LPS进行初始处理或不进行初始处理的情况下培养4天（第4天培养物）。然后将细胞在EGM-2培养基中继续培养另外4天或10天（第8天或第14天培养物）。通过流式细胞术分析和免疫荧光染色确定细胞标志物。通过生物芯片免疫测定研究细胞因子/趋化因子谱。
在LPS与GM-CSF、IL-4和EGM-2联合进行初始处理的组中，大多数悬浮的CD14(+)单核细胞附着并将其形态改变为内皮样细胞，这些细胞在第8天培养物中表达高水平的内皮细胞标志物CD31、血管性血友病因子和血管内皮生长因子受体-1以及两种主要的内皮紧密连接蛋白闭合蛋白-1和闭合蛋白。内皮型一氧化氮合酶表达显著增加。内皮样细胞还能够摄取乙酰化低密度脂蛋白并与欧洲荆豆凝集素结合。此外，内皮样细胞显示出独特的细胞因子/趋化因子谱，巨噬细胞炎性蛋白-1β、IL-6、粒细胞集落刺激因子和IL-8大幅增加。
LPS与GM-CSF、IL-4和EGM-2联合进行初始处理是体外加速外周血CD14(+)单核细胞向内皮样细胞分化的有效策略。
