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Abstract
Background
Vascular endothelial growth factor (VEGF) plays an essential role in promoting angiogenesis during tumor development. In addition, VEGF can mediate the inflammatory response in tumors. VEGF increases the level of neutrophil migration by upregulating interleukin-8 (IL-8) in endothelial cells in vitro. However, it is unclear if VEGF can mediate IL-8 production in vivo.
Methods
To address this issue, this study examined the effect of VEGF on IL-8 production in vivo using an adenovirus transduction and mouse ear assay.
Results
Adenovirus-encoded VEGF (VEGF-Ad) increased the level of IL-8 production in endothelial cells in vitro compared to the control-adenovirus (CTL-Ad). The mouse ear assay showed that VEGF-Ad increased the level of IL-8 production in the endothelium. Immunohistochemistry showed that the IL-8 proteins were expressed in the vasculature within a human glioblastoma, which is known to strongly express VEGF.
References
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Fig. 1.
IL-8 production by VEGF-Ad infection in HUVECs. HUVECs were grown subconfluently in 6-well plates and in-fected with 10 MOI of VEGF-Ad or CTL-Ad. After incubation of 3 days, cells were lysed and VEGF expression was examined by western blot analysis (A). Alternatively, super-natants were harvested and IL-8 proteins were measured by using ELISA (B).
Fig. 2.
IL-8 production by VEGF-Ad infection in mouse ears. Mouse ears were infected with 100 MOI of VEGF-Ad or CTL-Ad. After 7 days, mice were sacrificed and ears were excised to be frozen. The ears were cut into 5 mm and fixed with cold methanol. Immunohistochemistry was performed as described under “methods”. vWF, von Willebrand factor.
Fig. 3.
IL-8 and vWF colocalization in the VEGF-Ad infected mouse ears. Mouse ears were infected with 100 MOI of VEGF-Ad or CTL-Ad. After 7 days, mice were sacrificed and ears were excised to be frozen. The ears were cut into 5 μm and fixed with cold methanol. Colocalization of IL-8 and vWF was observed by using confocal microscopy. vWF, von Willebrand factor.
Fig. 4.
VEGF and IL-8 expression in human glioblastoma. The tissues were deparaffinized and fixed with 10% formaldehyde, then treated with 0.1% trypsin for 30 min to improve antigen accessibility. After blocking nonspecific binding to secondary antibodies by treatment with blocking solution (Dako Inc.) for 1 h, the tissues were incubated overnight at 4oC with primary antibodies against VEGF or IL-8 polyclonal antibodies. The tissues were washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Vector Laboratories) for 1 h. After washing, tissues were stained using the Vectastain ABC kit.
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