Insulin-Like Growth Factor 1 Induced Expression of Vascular Endothelial Growth Factor, Which Is Dependent on MAP Kinase and Phosphatidylinositol 3-Kinase Signaling in Human Bone Marrow Stromal Cells.

INTRODUCTION: Administration of bone marrow stromal cells after traumatic brain injury provides functional benefit. Its protective mechanisms include neurogenesis and angiogenesis. Different traumatic brain injuries have shown that vascular endothelial growth factor (VEGF) induced neuroprotection, neurogenesis, and angiogenesis. Insulin-like growth factor 1 (IGF-1) is a 7.5-kDa peptide with structural homology to proinsulin. In the adult organism, the liver is the major source of IGF-1. Although IGF-1 is very low in the normal adult rat brain, a number of studies have shown that IGF-1 is strongly induced in the CNS, and exerted its mitogenic and trophic effects on a variety of cell-types after different traumatic brain injuries, suggesting its repair roles after brain damage. We tested the hypothesis that IGF-1 induces expression of VEGF in human bone marrow stromal cells, and its signaling pathway.

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 washed twice in PBS, then cultured in serum-free DMEM/F12. After overnight incubation, cells were exposed to IGF-1 (100 ng/ml) in the presence of no kinase inhibitor or a 1 h pretreatment with 50 μM PD98059, or 200 nM wortmannin. Cells were harvested after 2 h for analysis of active phosphorylated kinases (Akt and MAPK) 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 by Western blot.

RESULTS: 100 ng/ml IGF-1 increased significantly both VEGF mRNA and VEGF protein, which were very low level in control cells. 24 hours after treatment, ratio of RT-PCR product between VEGF and β-actin reach to (38.93±6.73)% from (18.61±4.25)% of control cells (P<0.01). VEGF protein increased to (123.45±20.86)pg/ml from (46.97±8.91)pg/ml (P<0.01). Moreover, IGF-1 induced active phosphorylated kinases (Akt and MAPK). And PD98059 or wortmannin inhibited effects of IGF-1.

CONCLUSION: IGF-1 enhanced expression of VEGF mRNA and VEGF protein in human bone marrow stromal cells, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling. These results suggest that VEGF be involved in therapeutic effects of bone marrow stromal cells in ischemic disorders, including stroke.