Extracellular vesicles-encapsulated microRNA-29b-3p from bone marrow‐derived mesenchymal stem cells promotes fracture healing via modulation of the PTEN/PI3K/AKT axis

• DOI: 10.1016/j.yexcr.2022.113026
• Publication Date: 2022-01-10T15:56:09Z
• Authors: Jizhou Yang; Jian Gao; Feng Gao; Yi Zhao; Bowen Deng; Xiaohong Mu; Lin Xu
• Keywords: Fracture Healing; Male; 0301 basic medicine; PTEN Phosphohydrolase; Mesenchymal Stem Cells; Cell Line; Mice, Inbred C57BL; Disease Models, Animal; Extracellular Vesicles; Mice; MicroRNAs; Phosphatidylinositol 3-Kinases; 03 medical and health sciences; Bone Density; Bone Marrow; Human Umbilical Vein Endothelial Cells; Animals; Humans; Proto-Oncogene Proteins c-akt; Signal Transduction

Bone marrow-derived mesenchymal stem cells (BM-MSCs) are well-established as vital regulators of fracture healing, whereas angiogenesis is one of the critical processes during the course of bone healing. Accordingly, the current study sought to determine the functions of microRNA (miR)-29b-3p from BM-MSCs-derived extracellular vesicles (EVs) on the angiogenesis of fracture healing via the PTEN/PI3K/AKT axis. Firstly, BM-MSCs-EVs were extracted and identified. The lentiviral protocol was adopted to construct miR-29b-3pKD-BMSCs or miR-negative control-BMSCs, which were then co-cultured with human umbilical vein endothelial cells (HUVECs) in vitro to determine the roles of EVs-encapsulated miR-29b-3p on the proliferation, migration, and angiogenesis of HUVECs in vitro with the help of a CCK-8 assay, scratch test, and tube formation assay. Subsequent database prediction, luciferase activity assay, RT-qPCR, and Western blot assay findings identified the downstream target gene of miR-29b-3p, PTEN, and a signaling pathway, PI3K/AKT. Furthermore, the application of si-PTEN attenuated the effects induced by miR-29b-3pKD-EVs. Finally, a mouse model of femoral fracture was established with a locally instilled injection of equal volumes of BM-MSCs-EVs and miR-29b-3pKD-BM-MSCs-EVs. Notably, the mice treated with BMSC-EVs presented with enhanced neovascularization at the fracture site, in addition to increased bone volume (BV), BV/tissue volume, and mean bone mineral density; whereas miR-29b-3pKD-BMSCs-EVs-treated mice exhibited decreased vessel density with poor fracture healing capacity. Collectively, our findings elicited that BM-MSCs-EVs carrying miR-29b-3p were endocytosed by HUVECs, which consequently suppressed the PTEN expression and activated the PI3K/AKT pathway, thereby promoting HUVEC proliferation, migration, and angiogenesis, and ultimately facilitating fracture healing.