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J.R. Mathura, V.J. Dudley, V.P. Sarthy; Hypoxia does not alter VEGF mRNA levels in rat Müller cell lines. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1899.
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Purpose: It has been shown previously by other investigators that freshly isolated Müller cells increase their production of vascular endothelial growth factor under hypoxic conditions in vitro. In an attempt to identify a model to test the role of various factors in retinal ischemia, we investigated the effects of hypoxia on VEGF mRNA production in two rat Müller cell lines. Methods:Two well–characterized rat Müller cell lines, rMC–1 and E 6/7, were used in these experiments. Total RNA was isolated from Müller cells exposed to either control or hypoxic conditions. RT–PCR was performed on RNA isolated from control and hypoxic samples using primers for rat VEGF and GAPDH, the internal control. The resultant PCR products were run on agarose gels and the gels were analyzed and quantified using optical density measurement of the VEGF band relative to GAPDH band. Results: Bands of appropriate size for VEGF (186 bp) and GAPDH (703 bp) were found by PCR. DNA Sequencing of the VEGF band established that the PCR product was homologous to the published sequence for rat VEGF. The VEGF bands in both cell lines were strong in normal and hypoxic conditions. Optical density analysis of the bands in three separate experiments showed no significant increase in VEGF expression in Müller cells grown in hypoxia. Conclusions:VEGF mRNA is highly expressed in the rMC–1 and E 6/7 rat Müller cell lines and is not significantly changed after exposure to hypoxia. This indicates that the rat Müller cell lines have lost a normal regulatory mechanism/s that controls VEGF expression.
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