Molecular analysis of cell growth regulating factor in retinal, capillary endothelial cells

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Conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB) and retinal pericyte cell line (TR-rPCT) were established from transgenic rats harboring the temperature-sensitive simian virus 40 large T-antigen gene. The paracrine interaction between TR-iBRB and TR-rPCT cells has been investigated to elucidate the cell growth regulating factor in pericyte. TR-iBRB cells were seeded on the insert of Transwell chamber and TR-rPCT cell were seeded under the insert (contact co-culture model) and on the well chamber (non-contact co-culture model). The conditioned medium for TR-rPCT cells was also used to observe the cell growth of TR-iBRB cells. The growth of TR-iBRB cells was significantly slowed down with increasing concentration of TR-rPCT conditioned medium. In contact co-culture model, the growth of TR-iBRB cells was arrested 4 days after the beginning of the co-culture. These results suggest that some factor released from pericyte cells is involved in regulating the cell growth in retinal capillary endothelial cells.The [^<14>C] L-cystine uptake by TR-iBRB cells appeared to be mediated through a saturable Na^+-independent process with a Michaelis-Menten constant of 9.2 μM, suggesting system xc, which consists of XCT and 4F2hc mRNA, mediated L-cystine uptake. After 100 μM diethyl maleate treatment, which is a model agent for oxidative stress, the XCT mRNA level and L-cystine uptake activity in TR-iBRB cells were enhanced in a time-dependent manner. Concomitantly, the glutathione concentration in TR-iBRB cells was increased. In contrast, the 4F2hc mRNA level was unchanged up to 24 hours and was induced for more than 24 hours by DEM pretreatment. These findings suggest L-cystine transport is activated by induction of system xc under the oxidative stress conditions at the inner blood-retinal barrier.