Hypoxia-induced expression of VEGF splice variants and protein in four retinal cell types

Abstract

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.

Keywords: hypoxia; splice variants; vascular endothelial growth factor.

Figure 1

Immunocytochemical staining of rat retinal cells: A) Müller cells with anti-CRALBP; B) astrocytes with anti-GFAP; C) retinal pigmented epithelium (unstained); D) microvascular endothelial cells with anti-VE-cadherin.

Figure 2

Muller cells were cultured in hypoxia or normoxia for 12 hours. The cells were lysed. Total RNA was isolated, reverse transcribed, and analyzed by a validated, splice variant specific, external calibration curve qRT-PCR method. The expression levels are reported as Vegf copy numbers/ng input RNA ± standard deviation.

Figure 3

Astrocytes were cultured in hypoxia or normoxia for 12 hours. The cells were lysed. Total RNA was isolated, reverse transcribed, and analyzed by a validated, splice variant specific, external calibration curve qRT-PCR method. The expression levels are reported as Vegf copy numbers/ng input RNA ± standard deviation.

Figure 4

Retinal pigment epithelial cells were cultured in hypoxia or normoxia for 12 hours. The cells were lysed. Total RNA was isolated, reverse transcribed, and analyzed by a validated, splice variant specific, external calibration curve qRT-PCR method. The expression levels are reported as Vegf copy numbers/ng input RNA ± standard deviation.

Figure 5

Retinal microvascular endothelial cells were cultured in hypoxia or normoxia for 12 hours. The cells were lysed. Total RNA was isolated, reverse transcribed, and analyzed by a validated, splice variant specific, external calibration curve qRT-PCR method. The expression levels are reported as Vegf copy numbers/ng input RNA ± standard deviation.

Figure 6

Müller cells, retinal microvascular endothelial cells, retinal pigmented epithelium cells and astrocytes were cultured in hypoxia or normoxia for 24 hours. Conditioned media were collected and assayed for total VEGF by ElISA. The VEGF levels were normaliszed to cellular protein.