The expression of growth factors and their receptors in retinal and endothelial cells cocultured in the rotating bioreactor.
Vascular endothelial growth factor (VEGF) has been strongly implicated in the development of choroidal neovascularization, which is seen in age-related macular degeneration. This study investigates whether retinal cells cultured individually or as cocultures with endothelial cells and maintained in the horizontally rotating bioreactor will express more VEGF and VEGF receptors. We measured the expression of VEGF isotypes and VEGF receptors for cells maintained in monolayer and horizontally rotating bioreactor culture at various times by using reverse transcription PCR and Western blot analysis. Retinal cells showed a twofold increase in VEGF-A mRNA expression after five days of culture in the bioreactor, compared with monolayer cultures (77 +/- 3 vs 42 +/- 2, P < .006). Further, we found that the expression of mRNA for VEGF-A growth factor was increased fivefold for retinal cells cocultured with endothelial cell (52 +/- 4 for one day vs 240 +/- 15, P < .001, cultured in the bioreactor for five days). Where the expression of VEGF receptors (FLK-1 and FLT-4) was low for monolayer retinal cultures, we found the expression of both VEGF receptors was higher after 5, 10, and 15 days of culture. Increased expression of these receptors was also found for cocultured retinal/endothelial cells. Further, we found that cultured retinal cells showed higher VEGF-C protein expression compared to monolayer cultures. Our protein analysis data showed that the expression of VEGF-A was increased by twofold (780 +/- 30 for one day vs 1520 +/- 36 for five days, P < .001) after five days in bioreactor cocultures. These findings suggest that retinal/endothelial cell coculture in the horizontally rotating bioreactor may be a very good model for investigating the role of growth factors in the neovascularization seen in human ocular disorders.
Kumar
Ravindra
R
Department of Microbiology, Biochemistry, and Immunology, Morehouse School of Medicine, 720 Westview Dr SW, Atlanta, GA 30310-1495, USA. rkumar@msm.edu
Harris-Hooker
Sandra
S
Sanford
Gary L
GL
D019149:Bioreactors; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000378:metabolism*; D006801:Humans; D009389:Neovascularization, Pathologic; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A; D040281:Vascular Endothelial Growth Factor Receptor-1