Hypoxia-induced expression of VEGF splice variants and protein in four retinal cell types.
The purpose of this study was to investigate the hypoxia-induced Vegf120, Vegf164 and Vegf188 mRNA expression profiles in rat Müller cells (MC), astrocytes, retinal pigmented epithelial cells (RPE) and retinal microvascular endothelial cells (RMEC) and correlate these findings to VEGF secreted protein. Cultured cells were exposed to normoxia or hypoxia. Total RNA was isolated from cell lysates and Vegf splice variant mRNA copy numbers were assayed by a validated qRT-PCR external calibration curve method. mRNA copy numbers were normalized to input total RNA. Conditioned medium was collected from cells and assayed for total VEGF protein by ELISA. Hypoxia increased total Vegf mRNA and secreted protein in all the retinal cell types, with the highest levels observed in MC and astrocytes ranking second. Total Vegf mRNA levels in hypoxic RPE and RMEC were comparable; however, the greatest hypoxic induction of each Vegf splice variant mRNA was observed in RMEC. RPE and RMEC ranked 3rd and 4th respectively, in terms of secreted total VEGF protein in hypoxia. The Vegf120, Vegf164 and Vegf188 mRNA splice variants were all increased in hypoxic cells compared to normoxic controls. In normoxia, the relative Vegf splice variant mRNA levels ranked from highest to lowest for each cell type were Vegf164 > Vegf120 > Vegf188. Hypoxic induction did not alter this ranking, although it did favor an increased stoichiometry of Vegf164 mRNA over the other two splice variants. MC and astrocytes are likely to be the major sources of total Vegf, Vegf164 splice variant mRNAs, and VEGF protein in retinal hypoxia.
Department of Ophthalmology and Visual Sciences, Vanderbilt University School of Medicine, 8009 Medical Center East, 1215 21st Avenue South, Nashville, TN 37232-8808, USA.
William M
WM
Watkins
Gary W
GW
McCollum
Sara R
SR
Savage
Megan E
ME
Capozzi
John S
JS
Penn
David G
DG
Morrison
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D018507:Gene Expression Regulation, Developmental; D000860:Hypoxia / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D020033:Protein Isoforms; D012333:RNA, Messenger / Q000235:genetics*; D051381:Rats; D020318:Rats, Long-Evans; D060888:Real-Time Polymerase Chain Reaction; D012160:Retina / Q000378:metabolism* / Q000473:pathology; D012164:Retinal Diseases / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics*
D020033:Protein Isoforms; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A
hypoxia; splice variants; vascular endothelial growth factor