Department of Cardiology, The Key Lab of Cardiovascular Disease of Wenzhou, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang, People's Republic of China.
Department of Pediatrics, The Second School of Medicine, Wenzhou Medical University, Wenzhou, Zhejiang, People's Republic of China.
Drug Des Devel Ther. 2020 Mar 16;14:1157-1167. doi: 10.2147/DDDT.S222244. eCollection 2020.
Multipotent mesenchymal stromal cells (MSCs) have recently been reported to promote vasculogenesis by differentiating into endothelial cells and releasing numerous cytokines and paracrine factors. However, due to low cell activity, their potential for clinical application is not very satisfactory. This study aimed to explore the effects and mechanisms of 1,25-dihydroxyvitamin D (1,25(OH)D) on the vasculogenesis of MSCs.
MSCs were isolated from the femurs and tibias of rats and characterized by flow cytometry. After treatment with different concentrations of 1,25(OH)D (0 µM, 0.1 µM and 1 µM), the proliferation of MSCs was analyzed by Cell Counting Kit-8 (CCK-8), and the migratory capability was measured by Transwell assays and cell scratch tests. Capillary-like structure formation was observed by using Matrigel. Western blotting was used to detect the expression of FLK-1 and vWF to investigate the differentiation of MSCs into endothelial cells. Western blotting and gelatin zymography were used to detect the expression and activities of VEGF, MMP-2 and MMP-9 secreted by MSCs under the influence of 1,25(OH)D Finally, the VDR antagonist pyridoxal-5-phosphate (P5P) and the PI3K/AKT pathway inhibitor LY294002 were utilized to test the phosphorylation levels of key kinases in the PI3K/AKT pathway by Western blotting and the formation of capillary-like structures in Matrigel.
The proliferation and migratory capability of MSCs and the ability of MSCs to form a tube-like structure in Matrigel were enhanced after treatment with 1,25(OH)D. Moreover, MSCs treated with 1,25(OH)D showed high expression of vWF and Flk-1. There was a significant increase in the expression of VEGF, MMP-2 and MMP-9 secreted by MSCs treated with 1,25(OH)D as well as in the activity of MMP-2 and MMP-9. The phosphorylation level of AKT increased with time after 1,25(OH)D treatment, while LY294002 weakened AKT phosphorylation. In addition, the ability to form capillary-like structures was reduced when the VDR and PI3K/AKT pathways were blocked.
This study confirmed that 1,25(OH)D treatment can strengthen the ability of MSCs to promote vasculogenesis in vitro, and the mechanism may be related to the activation of the PI3K/AKT pathway.
多能间充质干细胞（MSCs）最近被报道通过分化为内皮细胞和释放大量细胞因子和旁分泌因子来促进血管生成。然而，由于细胞活性低，其临床应用潜力并不十分令人满意。本研究旨在探讨 1,25-二羟维生素 D（1,25(OH)D）对 MSCs 血管生成的作用及其机制。
从大鼠股骨和胫骨中分离 MSCs，并通过流式细胞术进行鉴定。用不同浓度的 1,25(OH)D（0µM、0.1µM 和 1µM）处理 MSCs 后，通过 Cell Counting Kit-8（CCK-8）分析 MSCs 的增殖情况，通过 Transwell 测定和细胞划痕试验检测 MSCs 的迁移能力。用 Matrigel 观察毛细血管样结构的形成。用 Western blot 检测 FLK-1 和 vWF 的表达，以研究 MSCs 向内皮细胞的分化。用 Western blot 和明胶酶谱法检测受 1,25(OH)D 影响的 MSCs 分泌的 VEGF、MMP-2 和 MMP-9 的表达和活性。最后，用 VDR 拮抗剂吡哆醛-5-磷酸（P5P）和 PI3K/AKT 通路抑制剂 LY294002 通过 Western blot 检测 PI3K/AKT 通路中关键激酶的磷酸化水平，并在 Matrigel 中检测毛细血管样结构的形成。
用 1,25(OH)D 处理后，MSCs 的增殖和迁移能力以及在 Matrigel 中形成管状结构的能力增强。此外，用 1,25(OH)D 处理的 MSCs 中 vWF 和 Flk-1 的表达水平较高。用 1,25(OH)D 处理的 MSCs 分泌的 VEGF、MMP-2 和 MMP-9 的表达以及 MMP-2 和 MMP-9 的活性均显著增加。用 1,25(OH)D 处理后，AKT 的磷酸化水平随时间增加，而 LY294002 减弱 AKT 的磷酸化。此外，当阻断 VDR 和 PI3K/AKT 通路时，形成毛细血管样结构的能力降低。
本研究证实，1,25(OH)D 处理可增强 MSCs 体外促进血管生成的能力，其机制可能与激活 PI3K/AKT 通路有关。
