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H. Yang, Z. Xu, P.M. Iuvone, J.L. Arbiser, S.E. Crawford, V.M. Stellmach, H.E. Grossniklaus; Vascular Endothelial Growth Factor (VEGF) and Pigment Epithelium Derived Factor (PEDF) in Micrometastatic Ocular Melanoma in a Murine Model . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3410.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have shown that melanoma cells express VEGF and PEDF, angiostatin alters the melanoma VEGF/PEDF ratio in vitro, and low dose angiostatin decreases hepatic micrometastatic melanoma in a mouse model. To further define these processes, we evaluated the relationship of VEGF and PEDF on in vitro proliferation and analyzed VEGF and PEDF mRNA–expression in micrometastatic uveal melanoma cells. Methods:B16LS9 mouse and Mel 290 human melanoma cell lines were transfected to overexpress VEGF and PEDF with pB.VEGF or human PEDF vectors. Expression of VEGF and PEDF was confirmed by Western blot. Mouse VEGF or PEDF siRNA duplexes were transfected into B16LS9 via Oligofectamine to block VEGF or PEDF. Cell proliferation was measured with the CyQuant cell proliferation assay. 5x105 cells/2.5µl aliquots of tissue culture B16LS9 melanoma cells previously treated with or without 0.1 µg/ml murine angiostatin for 12 hours were used in our model. Frozen sections from the liver were stained with H&E under RNase–free conditions. Melanoma cells were isolated by laser capture microdissection and the total RNA was extracted and amplified. RT–PCR was performed to detect the levels of VEGF, PEDF, and ß–actin mRNA expression. Results: B16LS9 cells overexpressing VEGF showed increased proliferation and underexpressing VEGF showed decreased proliferation. Mel290 cells with overexpression of PEDF showed decreased proliferation and B16LS9 cells with underexpression of PEDF showed increased proliferation. Micrometastatic uveal melanoma cells express VEGF and PEDF mRNA. Ratios of VEGF and PEDF mRNA to ß–actin mRNA were determined. Both study groups (with/without pre–angiostatin treatment) had similar levels of VEGF mRNA (VEGF/ß–actin: 0.05). PEDF mRNA–expression was higher in the micrometastatic uveal melanoma cells derived from B16LS9 cells pre–treated with angiostatin prior to inoculation compared to the control cells(PEDF/ß–actin: 2.16 vs. 0.30). Conclusions: VEGF and PEDF have an inverse relationship regarding in vitro proliferation of B16LS9 melanoma. Low dose angiostatin affects the expression of PEDF in micrometastatic B16LS9 melanoma cells resulting in a decreased VEGF/PEDF ratio.
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