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Z.–M. Bian, S.G. Elner, V.M. Elner; Synergistically Induced VEGF Expression by Thrombin and TGF–ß2 in Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4906.
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The aim of the present study was to investigate the interplay between thrombin and other pro–angiogenic factors and the signal transduction pathways leading to VEGF expression in human RPE cells.
Human RPE cells were stimulated with thrombin alone or in the presence of TNF–α, monocytes, or TGF–ß2. The conditioned media and stimulated cells were used to determine VEGF protein production and mRNA levels by ELISA, Western blotting, immunocytochemistry, and RT–PCR. Specific inhibitors were employed to delineate the signal pathways mediating VEGF expression.
Thrombin induced time– and dose–dependent increases in VEGF mRNA synthesis and protein production. About 80% of thrombin–induced VEGF secretion was abrogated by inhibitors of MAPK/ERK kinase (MEK) (U0126), p38 (SB202190), c–Jun NH2–terminal kinase (JNK) (Sp100126), protein tyrosine kinase (PTK) (genistein), phosphatidylinositol 3–kinase (PI3K) (Ly294002), protein kinase C (PKC) (Ro31–8220), nuclear factor–ΚB (NF–ΚB) [caffeic acid phenethyl ester (CAPE)], and reactive oxygen species (ROS) [N–acetyl–Cysteine (Nac) and diphenyleneiodonium (DPI)]. The induced VEGF expression, however, was resistant to AG490, an inhibitor of Jak2. While additive VEGF expression was observed in cells stimulated by thrombin plus TNF–α or monocytes, induction of VEGF by TGF–ß2 plus thrombin was synergistic. Thus, RPE VEGF production, co–stimulated by TGF–ß2 plus thrombin, averaged threefold more than the sum of VEGF induced by each agent used separately. Moreover, the calcium chelator, BAPTA effectively blocked VEGF secretion induced by thrombin (65%) or by thrombin plus TGF–ß2 (20%), but not that induced by TGF–ß2 alone.
Thrombin plus TNF–α or monocytes additively and thrombin plus TGF–ß2 synergistically stimulate VEGF expression. Multiple signal pathways are involved in the stimulation of VEGF expression in human RPE cells.
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