Antiangiogenic Cytokine TNFSF15 Inhibits Abnormal Vascular Vessel Growth And Alleviates Hemorrhage In Murine Hemangioblastoma Model

Objective:Surgical options are limited for hemangioblastoma (HB) occurring in critical (such as brainstem or base of skull) or multiple sites of the brain, and few effective medications are available. Strategies aiming at counter-balancing the abnormally high angiogenesis potential in the microenvironment of HB may be of therapeutic value.Methods:Immunohistochemical staining was used to detect the expression of VEGF/TNFSF15 and microglia/macrophages in human hemangioblastoma. bEnd.3 cells were stereotacticly injected into the basal ganglia of mice to establish hemangioblastoma model. HE staining to evaluate the vascular volume and hemorrhage volume. ELISA was used to detect the expression levels of VEGF/TNFSF15. Immunofluorescence staining was performed to analyze the inflammatory cells infiltration to hemangioblastoma. bEnd.3 by lentiviral transfection cells, causing the cells expressed high TNFSF15, by RT-PCR, Western blotting and ELISA, analysis of expression levels VEGF/TNFSF15. Cell proliferation assay and tube formation in vitro angiogenesis experiments to evaluate the ability of bEnd.3 cells.Results:Tumor necrosis factor superfamily-15 (TNFSF15) is an anti-angiogenic cytokine, TNFSF15 can inhibit the growh of endothelial cells (ECs), induce the apoptosis when ECs are in mtosis and prevent the transformation from endothelial projenitor cell to ECs. Immunohistochemistry analysis shows that TNFSF15 is highly expressed in human normal brain but diminished in HB, so increasing the expression of TNFSF15 may be an effective target to cure this kind of disease. Mouse endothelioma bEnd.3 cells produce large amounts of VEGF, a potent angiogenic and vascular permeability factor, but little TNFSF15.If implanted into wild type C57BL/6 mouse brain, these cells form vascular tumors that resemble human HB pathologically. When implanted into the brain of a transgenic mouse overexpressing TNFSF15, however, the cells form tumors that exhibit much smaller lesion volumes and greatly reduced hemorrhage, as well as decreased inflammatory cell infiltration, enhanced pericyte coverage, and decreased VEGF expression, compared with tumors formed in transgene-negative littermates. Also up-regulation the expression of TNFSF15 in bEnd.3 also could inhibit the growth of the vascular tumor and decrease the hemorrhage. Treatment of bEnd.3 cells with TNFSF15 either by gene transfer or with recombinant protein results in a marked reduction of VEGF expression and inhibition of cell growth. Death receptor 3 (DR3) is found to mediate TNFSF15 activity in the inhibition of VEGF expression in bEnd.3 cells. These findings indicate that down-regulation of VEGF expression by TNFSF15 leads to inhibition of brain vascular tumor growth and alleviation of tumor-associated hemorrhage.Conclusions:Compared to the normal vessel in human brain, there is high expression of VEGF and extreme low expression of TNFSF15 in human HB. This kind of abnormal expression of VEGF and TNFSF15 maybe induce or exacerbate the development of HBs. In the murine vascular model, TNFSF15 could inhibit the growth of the vascular and decrease the hemorrhage, decrease the inflammation and increase the pericyte recruitment to the vascular lesion border. Importantly, we found that TNFSF15 could prevent the expression of VEGF, the further explore showed that TNFSF15 inhibited the VEGF expression through DR3.