Expression of multiple angiogenic cytokines in cultured normal human prostate epithelial cells: Predominance of vascular endothelial growth factor

Abstract

The cytokines that regulate angiogenesis in normal and malignant prostate tissue are not well studied. Using an RT-PCR-based screen, we observed that cultured, low-passage normal human prostate epithelial cells (PrECs) express a variety of cytokines which have been shown to have angiogenic and/or endothelial cell-activating properties in various systems. These include vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), transforming growth factor-alpha (TGF-alpha), transforming growth factor-beta (TGF-beta), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Expression of VEGF, bFGF, GM-CSF, G-CSF, TGF-alpha and TNF-alpha in these cells was confirmed by immunohistochemistry. Culture medium conditioned by normal human PrECs for periods of up to 96 hr were found to contain VEGF, GM-CSF, G-CSF, IL-8, TGF-beta1 and TGF-beta2 but not TNF-alpha or bFGF, as determined by ELISA. Of these, VEGF was by far the most prominently expressed angiogenic cytokine (approx. 2,500 pg/ml conditioned medium at 96 hr vs. 30 to 100 pg/ml conditioned medium for the other cytokines). PrEC-conditioned medium induced an approximately 2-fold stimulation of [3H]-thymidine incorporation in cultured human umbilical cord endothelial cells (HUVECs) deprived of the endothelial growth factors VEGF and bFGF; this stimulation was abolished by neutralizing antibodies directed against VEGF but not bFGF, IL-8, GM-CSF or TNF-alpha. VEGF expression by PrECs was not markedly altered by administration or deprivation of other angiogenic cytokines for which these cells have receptors, suggesting that there is not a hierarchy of cytokines controlling its expression; however, retinoic acid, a component of PrEC growth medium, was found to modestly suppress VEGF at physiological concentrations (0.1 ng/ml). These data suggest that normal PrECs express a variety of angiogenic cytokines, most prominently VEGF, to recruit a supporting vasculature, even in culture. Our data also suggest that the ability of malignant PrECs to stimulate angiogenesis may be intrinsic and does not need to be acquired during oncogenesis.