TGF-β1 处理后的 DPSCs 通过 VEGF-Ang-Tie2 信号通路调节三维共培养的 HUVECs 和 DPSCs 的血管生成发芽。

BACKGROUND: Maintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-β1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-β1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels. METHODS: We utilized TGF-β1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling. RESULTS: Western blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-β1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs. CONCLUSIONS: This study revealed that TGF-β1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

背景：维持血管的稳定性和成熟度对于血管发挥其生理功能至关重要。平滑肌细胞（SMCs）、周细胞和间充质干细胞（MSCs）参与了新形成血管的成熟过程。本研究旨在探讨转化生长因子β 1（TGF-β1）处理是否能增强牙髓干细胞（DPSCs）的周细胞样特性，以及经过 7 天 TGF-β1 处理的 DPSCs（T-DPSCs）如何稳定新形成的血管。

方法：我们利用 TGF-β1 处理 DPSCs 1、3、5 和 7 天。采用 Western blot 和免疫荧光法分析 SMC 标志物的表达。通过功能收缩试验评估 T-DPSCs 的收缩性。通过 MTT、划痕愈合和 Transwell 迁移试验检测 T-DPSC 条件培养基（T-DPSC-CM）对人脐静脉内皮细胞（HUVEC）增殖和迁移的影响。最重要的是，我们使用体外 3D 共培养球体发芽试验来研究血管内皮生长因子（VEGF）-血管生成素（Ang）-Tie2 信号对 HUVEC 和 T-DPSC 共培养球体中血管生成发芽的调节作用。使用 Ang 血管生成素 2（Ang2）和 VEGF 处理共培养球体，以探讨它们在血管生成发芽中的作用。使用 Tie2 和 VEGFR2 的抑制剂阻断 Ang1/Tie2 和 VEGF/VEGFR2 信号。

结果：Western blot 和免疫荧光结果表明，TGF-β1 处理后 SMC 特异性标志物（α-SMA 和 SM22α）的表达明显增加。T-DPSCs 的收缩性大于 DPSCs。T-DPSC-CM 抑制 HUVEC 迁移。体外发芽试验表明，T-DPSCs 包围 HUVECs，类似于周细胞样细胞。与与 DPSCs 共培养相比，与 T-DPSCs 共培养时观察到 HUVEC 发芽的数量较少。VEGF 和 Ang2 共同刺激显著增强了 HUVEC 和 T-DPSC 共培养球体的发芽，而 VEGF 或 Ang2 单独对 HUVEC 的发芽没有显著影响。阻断 Tie2 信号逆转了 T-DPSCs 对发芽的抑制作用，而阻断 VEGFR 信号则增强了 T-DPSCs 对发芽的抑制作用。

结论：本研究表明，TGF-β1 可诱导 DPSC 分化为功能性周细胞样细胞。T-DPSCs 通过 Ang1/Tie2 和 VEGF/VEGFR2 信号维持血管稳定性。