Abstract
Angiogenesis is very important for vascularized tissue engineering. In this study, we found that a two-dimensional co-culture of human bone marrow stromal cell (HBMSC) and human umbical vein endothelial cell (HUVEC) is able to stimulate the migration of co-cultured HUVEC and induce self-assembled network formation. During this process, expression of vascular endothelial growth factor (VEGF165) was upregulated in co-cultured HBMSC. Meanwhile, VEGF165-receptor2 (KDR) and urokinase-type plasminogen activator (uPA) were upregulated in co-cultured HUVEC. Functional studies show that neutralization of VEGF165 blocked the migration and the rearrangement of the cells and downregulated the expression of uPA and its receptor. Blocking of vascular endothelial-cadherin (VE-cad) did not affect the migration of co-cultured HUVEC but suppressed the self-assembled network formation. In conclusion, co-cultures upregulated the expression of VEGF165 in co-cultured HBMSC; VEGF165 then activated uPA in co-cultured HUVEC, which might be responsible for initiating the migration and the self-assembled network formation with the participation of VE-cad. All of these results indicated that only the direct contact of HBMSC and HUVEC and their respective dialogue are sufficient to stimulate secretion of soluble factors and to activate molecules that are critical for self-assembled network formation which show a great application potential for vascularization in tissue engineering.
Highlights
In vascularized tissue engineering, formation of blood vessel network is very important: oxygen and nutrient supply will be insufficient due to the lack of blood vessel network [1]; cell loss during the early post-implantational stage causes failure of bone engineering and subsequent bone repair [2]
We can see that there is no significant difference between the expression of vascular endothelial-cadherin (VE-cad) in human umbilical vein endothelial cell (HUVEC) and co-HUVEC during the development of self-assembled network (6 hours, 14 hours, and 18 hours), which were detected by Quantitative real time polymerase chain reaction (Q-PCR) and western blot (Fig. 2A, B)
Focusing on bone tissue engineering, our laboratory has been working on the cocultures of human bone marrow stromal cell (HBMSC) and HUVEC for about ten years to investigate the effects of HBMSC-HUVEC interactions on osteoblastic differentiation of HBMSC and angiogenesis of endothelial cells [22,23,24,25,26,27]
Summary
Formation of blood vessel network is very important: oxygen and nutrient supply will be insufficient due to the lack of blood vessel network [1]; cell loss during the early post-implantational stage causes failure of bone engineering and subsequent bone repair [2]. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) have been used for stimulating angiogenesis in many reports [3,4,5,6]. Cell-based approaches have been applied in order to improve tissue vascularization, among which endothelial cells (ECs) have attracted most of the attention [7,8,9,10,11,12]. For studying the angiogenesis process in vitro based on endothelial cells, numerous assay models have been applied. Among these models, three-dimensional (3D) assays culturing endothelial cells on a supportive matrix are most common [7,8,9,13,14,15,16]. Donovan et al demonstrated that the tube formation stimulated by Matrigel are not specific for endothelial cells: several non-endothelial cell types could be induced to form tube on Matrigel, indicating that the tube formation by endothelial cells on Matrigel may not represent true differentiation of this cell type [17]
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