Hypoxia-Inducible Factor 1 Mediated Expression of Hypoxia-Induced Vascular Endothelial Growth Factor in Human Bone Marrow Stromal Cells, Which Is Involved in MAP Kinase and Phosphatidylinositol 3-Kinase Signaling.

INTRODUCTION: Recent studies indicate that transplantation of bone marrow stromal cells after rat traumatic brain injury provided functional recovery. Hypoxia is the major characteristic of ischemic microenvironment. Vascular endothelial growth factor (VEGF) has been demonstrated in vivo and in vitro to be the principal mediator of hypoxia-induced angiogenesis. Recent studies indicate that VEGF implicated in neuroprotection. We tested the hypothesis that hypoxia induces expression of vascular endothelial growth factor (VEGF) in human bone marrow stromal cells (hBMSC) by hypoxia-inducible factor 1 (HIF-1).

METHODS: Human bone marrow adherent cells were cultured, and were passaged in DMEM/F12 containing 10% FBS. The fifth passage cells were identified as stromal cells. Subconfluent cells were used, and were cultured with hypoxic DMEM/F12 in hypoxia condition (94% N₂ + 1% O₂ +5% CO₂), and were treated with 50 μM PD98059, or 200 nM wortmannin, or SiRNA specific for HIF-1mRNA. Cells were harvested after 6 h for analysis of active phosphorylated kinases (Akt and MAPK) and the HIF-1-alpha protein or after 24 h for analysis of VEGF protein and mRNA. VEGF mRNA was detected by semi-quantitative RT-PCR, and VEGF protein by ELISA, and kinases and the HIF-1-alpha protein by Western blot.

RESULTS: Hypoxia increased significantly level of both VEGF mRNA and VEGF protein in hBMSC s, which were low level in normoxic cultured cells. 24 hours after treatment, ratio of RT-PCR product between VEGF and β-actin reach to (46.25±7.54)% from (19.61±4.57)% of control cells (P<0.01). VEGF protein increased to (142.77±22.33) pg/ml from (45.85±9.69) pg/ml (P<0.01). In addition, hypoxia induced active phosphorylated kinases (Akt and MAPK), and enhanced level of the HIF-1-alpha protein. Moreover, PD98059, or wortmannin, or SiRNA specific for the HIF-1-alpha mRNA inhibited induction of the VEGF gene by hypoxia.

CONCLUSION: The expression of VEGF mRNA and VEGF increased in human bone marrow stromal cells during hypoxia condition, which is involved in MAP kinase and phosphatidylinositol 3-kinase signaling. These results support that these cells play an important role in therapy of ischemic/hypoxic injury such as cerebral ischemia and myocardial ischemia. The hMSC capacity to increase expression of VEGF may be the key to the benefit provided by transplanted hMSCs in the ischemic injuries.