Abstract
Purpose: :
To identify novel angiogenic genes in Müller cells that are upregulated in hypoxia and involved in retinal neovascularization.
Methods: :
rMC–1 Müller cell were exposed to either standard or hypoxic conditions. Gene expression was analyzed with a microarray. RNA was extracted from control and hypoxic samples and reverse transcribed to cDNA. Two candidate genes, VLDLR and TRIB3, were PCR–amplified and subcloned. Semi–quantitative PCR was employed to verify expression levels and to compare them to a house keeping gene (16S). Expression of VLDLR and TRIB3 in cell cultures was confirmed with immunohistochemistry.
Results: :
In hypoxic rMC–1 cells 95 genes were upregulated 8 to 2 fold and 77 genes were downregulated 0.2 to 0.5 fold. Two candidate genes, very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization in the mouse (VLDLR) and in early differentiation and proliferation (TRIB3), respectively. Gene chip analysis demonstrated 3.1 fold upregulation of VLDLR (p 0.001) and 2.9 fold upregulation of TRIB3 (p 0.025), while semi–quantitative PCR suggested 100–300 (VLDLR) and 3–10 fold higher levels (TRIB3). Immunohistochemistry detected expression of both proteins in rat Müller cells.
Conclusions: :
VLDLR and TRIB3 are upregulated by hypoxia in rat Müller cells and warrant further investigation whether they contribute to the central role of Müller cells in retinal neovascularization.
Keywords: Muller cells • hypoxia • retinal neovascularization