Abstract
Purpose: :
Retinal pigment epithelial cells (RPEs) secrete vascular endothelial growth factor (VEGF) in a polarized fashion (apical side < basal side). Although the previous studies showed various factors to affect secretion of VEGF by RPEs, most of them used non-polarized RPEs, which might not exactly reflect an in vivo condition. This study was conducted to investigate factors affecting secretion of VEGF from polarized RPEs.
Methods: :
Porcine polarized RPE was established in Boyden chamber by our previous report (Sonoda S, et al. Nat Protoc, 2009). Polarized or non-polarized RPEs were treated with tumor necrosis factor alpha (TNF-α) or thrombin in various conditions. VEGF concentration of upper chamber or lower chamber was measured by enzyme-linked immunosorbent assay.
Results: :
In polarized RPEs, thrombin (1-20 U/ml) increased the amount of VEGF (both upper chamber and lower chamber together) 1.63-3.42 times of control in a dose or a time dependent manner. The amount of VEGF in lower chamber was 2.5 times greater than that of upper chamber. The similar results were obtained in non-polarized RPEs. While, TNF-α increased the amount of VEGF in non-polarized RPEs (1.15 times of controls); however, it decreased that of VEGF in polarized RPEs both in upper chamber and lower chamber.
Conclusions: :
Although thrombin enhanced VEGF secretion in polarized RPEs similarly non-polarized RPEs, TNF-a affected it differently dependent upon its polarization status. In a physiological condition, polarized RPEs would work not only as a mechanical barrier but also as a functional barrier against insults such as TNF-α. Polarized RPE might be more suitable than for non-polarized RPEs for analyzing the actual functions.
Keywords: retinal pigment epithelium • cytokines/chemokines • vascular endothelial growth factor