Effects Of Nordy On Contribution Of Endothelial Progenitor Cells To Neovascularization In Glioma

Both angiogenesis and vasculogenesis contribute to tumor neovascularization, which provides oxygen and nutrients for tumor cell survival and proliferation. Endothelial progenitor cells (EPCs) are important initiators of vasculogenesis in the process of tumor neovascularization. Accumulating evidences indicate that EPCs are actively recruited to sites of tumor where the cells differentiate in situ into mature ECs and integrate into new blood vessels. However, it is unclear how circulating EPCs contribute to the formation of tumor microvessels. EPCs can be mobilized from bone marrow into the blood stream and homed by a variety of angiogenic growth factors such as vascular endothelial growth factor (VEGF) and chemokines such as CXCL12 (stromal derived factor-1, SDF-1) and CXCL8 (interleukin-8, IL-8). Therefore, EPCs have been considerated as a promising target in anti-angiogenesis treatment strategy.Nordy is a chiral compound of a natural lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), synthesized in our laboratory. In previous study, we found that Nordy inhibited glioma cells proliferation, induced glioma cell apoptosis. Furthermore, it has been confirmed that Nordy inhibited angiogenesis in inflammatory diseases and cancers through its inhibitory effect on CXCR4-mediated production of CXCL8 and VEGF. It has also been found that Nordy could inhibit migration of glioma-derived microvascular endothelial cells to form tubule-like structures. However, the effect of Nordy on EPCs has not been identified.To investigate whether Nordy has influence on vasculogenic function of EPCs and their contribution to malignant glioma neovascularization, we isolated and cultured EPCs from human umbilical cord blood (HUCB) and examined their morphology and immunophenotype. Then, we explored the inhibitory effect of Nordy on the the response of EPCs to VEGF and CXCL8. Finally, we studied the influences of Nordy on EPCs contributions to malignant glioma neovascularization. The main results and conclusions are as follows:1. We isolated EPCs from HUCB. These EPCs expressed CD133, CD34, VEGFR-2 (KDR), CD31 and von Willebrand factor (vWF), and were capable of forming colonies. They could functionally uptake acetylated low-density lipoprotein (ac-LDL) and bind lectins. These EPCs were highly proliferative, migrated and formed capillary-like tubules in response to VEGF. Incubation of EPCs with 100μmol/L Nordy for 24 h initially inhibited the proliferative capacity of EPCs induced by VEGF (P < 0.05). Moreover,25～50μmol/L Nordy also exhibited inhibitory effect at 48～72 h. In addition, 25～100μmol/L Nordy impaired EPCs migratory and tubule-forming capability in vitro (P < 0.05).2. EPCs expressed high levels of CXCL8, CXCR1 and CXCR2 at mRNA and proteins, but reduced by treatment with 100μmol/L Nordy. Moreover, 100μmol/L Nordy impaired EPCs migratory capacity in vitro induced by CXCL8 (P < 0.05).3. When injected into mice bearing subcutaneously implanted human malignant glioma, EPCs specifically accumulated at the sites of tumors and differentiated into mature endothelial cells (ECs), which formed 18% of the tumor microvessel ECs. Nordy inhibited the growth of glioma xenografts, reduced tumor volume and microvessel density. Also, treatment with Nordy significantly inhibited production of VEGF and CXCL8 in tumor and attenuated incorporation of EPCs into tumor neovascularture. In addition, Nordy obviously influenced the morphology of tumor microvessles,that is, Nordy weakened the capability of EPCs-derived ECs to form typical lumina or strap-like structures in tumor, but EPCs-derived ECs just arranged by sing cell or clusters.In summary, the present results suggest that (1) EPCs constitute important components of tumor microvessel network and contribute to tumor neovascularization. (2) Nordy inhibited EPCs function induced by VEGF or CXCL8 in vitro and attenuated incorporation of EPCs into tumor neovascularture and also impacted tumor microvascular phenotype heterogeneity. This might be one of the mechanisms of the anti-cancer effection of Nordy.