Vascular Endothelial Growth Factor Is Involved in Protective Effects of Mesenchymal Stem Cells on Cultured Neurons in Hypoxic Condition.

INTRODUCTION: Transplantation of autologous bone marrow mesenchymal stem cells (MSCs) is increasingly being considered in cell-based therapeutic strategies for traumatic brains. Pluripotent MSCs can differentiate into neural cells, but few reports completely address the therapeutic effect of transplanting the cells into damaged brains in vivo. The early remarkable functional effect of MSC-transplantation cannot be readily attributed to tissue regeneration from MSCs. Hypoxia is the major characteristic of ischemic microenvironment. Low oxygen tension is thought to be an integral component of the MSC native bone marrow microenvironment. Thus, we hypothesized that paracrine actions exerted by the cells through the release of soluble factors might be partly important mechanisms of tissue repair and functional improvement after injection of MSCs. In the current study, we demonstrate that hypoxia induces expression of vascular endothelial growth factor (VEGF) in rat bone marrow MSCs, and VEGF markedly inhibits apoptosis of neonatal rat neurons induced by hypoxia in vitro.

METHODS: Adult Sprague-Dawley rat bone marrow MSCs and neonatal rat neurons were purified and cultured. MSCs were characterized by high expression of CD44, the lack of CD45 and CD11b molecules, their typical spindle-shaped morphology, together with their ability to differentiate into osteogenic, chondrogenic, and adipogenic cells, and neurons by expression of neuron specific enolase and neuronal nuclei marker NeuN. In the experiments, MSCs were co-cultured with or without neurons at the density of 2×10⁵ cells/ml (ratio of 1:1) in DMEM/F12 containing 15% FBS. After 24 hours, cells were cultured with hypoxic DMEM/F12 containing 5% FBS in hypoxia condition (93% N₂ + 2% O₂ +5% CO₂), and were treated with or without 0.2 nM VEGF and/or 100 nM SiRNA specific for VEGF mRNA. 72 hours after hypoxic culture, cells were harvested for analysis of cell apoptosis by flow cytometry. Level of the VEGF protein in medium was detected by ELISA.

RESULTS: The apoptotic ratio of normoxic cultured neurons was (11.46±2.68)% at 72 hours. The apoptotic ratio of hypoxic cultured neurons increased to (38.95±4.23)% (P<0.01). The apoptotic ratio of neurons co-cultured with MSCs in hypoxic condition decreased to (17.52±3.19)% (P<0.01). VEGF protein in the co-cultured media was (178.15±11.93) pg/ml, which were undetectable in media of normoxic or hypoxic cultured neurons. However, specific SiRNA enhanced the apoptotic ratio of hypoxic-induced neurons co-cultured with MSCs (30.81±4.09)%, and inhibited expression of VEGF protein in MSCs. Moreover, 0.2 nM VEGF reversed the role of specific SiRNA, of which the apoptotic ratio was (16.83±3.16)% (P<0.01).

CONCLUSION: These results showed that hypoxia induced expression of VEGF in bone marrow MSCs, and co-culture with MSCs improved the viability of neurons in hypoxia condition in vitro. We conclude that VEGF is involved in protective effects of Mesenchymal stem cells on cultured neurons in hypoxic condition. These evidences support paracrine hypothesis for mesenchymal stem cell-mediated acute neuroprotective effect and functional improvement, and the paracrine action is one of the multiple mechanisms of MSCs-based therapy for traumatic brains.