Restorative Dental Sciences, Endodontics, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.
Int Endod J. 2023 Sep;56(9):1092-1107. doi: 10.1111/iej.13943. Epub 2023 Jun 20.
Prevascularization is vital to accelerate functional blood circulation establishment in transplanted engineered tissue constructs. Mesenchymal stem cells (MSCs) or mural cells could promote the survival of implanted endothelial cells (ECs) and enhance the stabilization of newly formed blood vessels. However, the dynamic cell-cell interactions between MSCs, mural cells and ECs in the angiogenic processes remain unclear. This study aimed to explore the interactions of human umbilical vein ECs (HUVECs) and dental pulp stem cells (DPSCs) in an in vitro cell coculture model.
Human umbilical vascular ECs and DPSCs were directly cocultured or indirectly cocultured with transwell inserts in endothelial basal media-2 (EBM-2) supplemented with 5% FBS for 6 days. Expression of SMC-specific markers in DPSCs monoculture and HUVEC+DPSC cocultures was assessed by western blot and immunofluorescence. Activin A and transforming growth factor-beta 1 (TGF-β1) in conditioned media (CM) of HUVECs monoculture (E-CM), DPSCs monoculture (D-CM) and HUVEC+DPSC cocultures (E+D-CM) were analysed by enzyme-linked immunosorbent assay. TGF-β RI kinase inhibitor VI, SB431542, was used to block TGF-β1/ALK5 signalling in DPSCs.
The expression of SMC-specific markers, α-SMA, SM22α and Calponin, were markedly increased in HUVEC+DPSC direct cocultures compared to that in DPSCs monoculture, while no differences were demonstrated between HUVEC+DPSC indirect cocultures and DPSCs monoculture. E+D-CM significantly upregulated the expression of SMC-specific markers in DPSCs compared to E-CM and D-CM. Activin A and TGF-β1 were considerably higher in E+D-CM than that in D-CM, with upregulated Smad2 phosphorylation in HUVEC+DPSC cocultures. Treatment with activin A did not change the expression of SMC-specific markers in DPSCs, while treatment with TGF-β1 significantly enhanced these markers' expression in DPSCs. In addition, blocking TGF-β1/ALK5 signalling inhibited the expression of α-SMA, SM22α and Calponin in DPSCs.
TGF-β1 was responsible for DPSC differentiation into SMCs in HUVEC+DPSC cocultures, and TGF-β1/ALK5 signalling pathway played a vital role in this process.
血管预形成对于加速移植工程组织构建中的功能性血液循环建立至关重要。间充质干细胞（MSCs）或壁细胞可以促进植入的内皮细胞（ECs）的存活，并增强新形成的血管的稳定性。然而，在血管生成过程中，MSCs、壁细胞和 ECs 之间的动态细胞-细胞相互作用仍不清楚。本研究旨在探索人脐静脉内皮细胞（HUVECs）和牙髓干细胞（DPSCs）在体外细胞共培养模型中的相互作用。
将人脐血管内皮细胞和牙髓干细胞直接共培养或通过 Transwell 插入物间接共培养于内皮基础培养基-2（EBM-2）中，添加 5%胎牛血清培养 6 天。通过 Western blot 和免疫荧光法评估 DPSCs 单核培养物和 HUVEC+DPSC 共培养物中 SMC 特异性标志物的表达。通过酶联免疫吸附试验分析 HUVEC 单核培养物（E-CM）、DPSCs 单核培养物（D-CM）和 HUVEC+DPSC 共培养物（E+D-CM）条件培养基中激活素 A 和转化生长因子-β1（TGF-β1）的含量。使用 TGF-βRI 激酶抑制剂 VI（SB431542）阻断 DPSCs 中的 TGF-β1/ALK5 信号通路。
与 DPSCs 单核培养物相比，HUVEC+DPSC 直接共培养物中 SMC 特异性标志物 α-SMA、SM22α 和 Calponin 的表达明显增加，而 HUVEC+DPSC 间接共培养物与 DPSCs 单核培养物之间无差异。与 E-CM 和 D-CM 相比，E+D-CM 显著上调了 DPSCs 中 SMC 特异性标志物的表达。E+D-CM 中的激活素 A 和 TGF-β1 明显高于 D-CM，HUVEC+DPSC 共培养物中 Smad2 磷酸化水平升高。激活素 A 处理并未改变 DPSCs 中 SMC 特异性标志物的表达，而 TGF-β1 处理则显著增强了 DPSCs 中这些标志物的表达。此外，阻断 TGF-β1/ALK5 信号通路抑制了 DPSCs 中 α-SMA、SM22α 和 Calponin 的表达。
TGF-β1 负责 HUVEC+DPSC 共培养物中 DPSCs 向 SMC 的分化，TGF-β1/ALK5 信号通路在这一过程中发挥了重要作用。
