Stromal-Derived Factor and Vascular Endothelial Growth Factor Synergistically Induce Mesenchymal Stem Cells To Differentiate into Endothelial Cells in Hypoxia Condition, which Incorporated Tumor Vasculogenesis and Angiogenesis.

INTRODUCTION: High level of vascular endothelial growth factor (VEGF) and stromal-derived factor (SDF-1) are often expressed in many carcinoma tissues that are accompanied by intense neovascularization. VEGF is a key regulator of blood vessel formation during both vasculogenesis and angiogenesis. Mesenchymal stem cells (MSCs) in peripheral blood that are released from the bone marrow can be strongly attracted by SDF-1. Low oxygen tension is thought to be an integral component of the tumor microenvironment. To examine tumor angiogenesis in the tumor microenvironment, we studied that SDF-1 and VEGF both can induce MSCs to differentiate into endothelial cells (ECs) in hypoxia condition.

METHODS: Adult Sprague-Dawley rat bone marrow MSCs were purified and maintained in DMEM/F12 containing 10% FBS, and 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. Subconfluent MSCs were used, were maintained with DMEM/F12 containing 10% FBS in hypoxia condition (93% N₂ + 2% O₂ +5% CO₂), and were treated with or without VEGF and/or SDF-1. During induction, cell morphology, the vascular endothelial markers VE-cadherin and von Willebrand factor (vWF), and mesenchymal cell marker CD44 were observed. After induction, functions of cells were examined.

RESULTS: MSCs treated with both VEGF and SDF-1 for 7 days in hypoxic condition changed morphologically and adopted a caudate appearance with rod-shaped microtubulated structures resembling Weibel-Palade bodies, and formed mature endothelial cell tubules in Matrigel(R) cultures after 15 days. Almost every cell expressed VE-cadherin and vWF [(98.32±1.39)%], but CD44 expression was markedly down-regulated. Functional characteristics of vWF release upon histamine stimulation in response to hypoxia were indistinguishable between the MSC-derived endothelial-like cells and cultured mature ECs. The best condition for the differentiation was the combination of 0.3 nM VEGF and 100 ng/ml SDF-1 at a culture duration of 10 to 15 days. However, the positive ratio of both VE-cadherin and vWF in cells induced by different concentration of SDF-1 alone or VEGF alone was lower [(8.35±2.89)% and (27.33±3.26)%, respectively]. The positive ratio of both VE-cadherin and vWF in cells induced by the best combination of 0.3 nM VEGF and 100 ng/ml SDF-1 in normoxic culture condition was (34.91±3.47)%. Finally, in xenogenic transplantation studies using a SCID mouse model, non-small cell lung cancer cells were injected subcutaneously with the MSC-derived endothelial-like cells induced by the best condition. As a result, co-transplantation of the cells promoted the formation of blood vessels. Immunohistochemistry and FISH analysis showed that the MSC-derived cells incorporated tumor vasculogenesis and angiogenesis.

CONCLUSION: Stromal-derived factor and vascular endothelial growth factor synergistically induce bone marrow-derived mesenchymal stem cells to differentiate into endothelial cells in hypoxia condition, which incorporated tumor vasculogenesis and angiogenesis. These results suggest that targeting MSCs-derived angiogenesis might be a novel efficient antiangiogenesis strategy to treat cancer.