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N. Loewen, J. Chen, J. Dudley, V. P. Sarthy, J. R. Mathura, Jr.; Angiogenic Network in Hypoxic Müller Cells Involves the Very Low Density Lipoprotein Receptor. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3770. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To study the genomic response of Müller cells and to identify novel angiogenic genes that are upregulated in hypoxia.
Rat Müller cells (rMC-1) were exposed to standard or hypoxic conditions. RNA was extracted from control and hypoxic samples and reverse transcribed to cDNA. Gene expression was analyzed with three independent microarrays. Expression levels of selected, upregulated genes were then quantitated with real-time PCR (RTPCR). Protein expression and subcellular localization were examined by immunohistochemistry. A network-based pathway analysis of expression was performed to investigate how those genes may contribute to angiogenesis.
19,571 of 28,000 known rat genes were expressed in Müller cells. 275 genes were upregulated by hypoxia from 2 to 14.9-fold (p < 0.05) whereas 234 genes were downregulated 2 to 4.6-fold (p < 0.05). Unexpected increases were seen in the expression of genes associated with in cell proliferation, differentiation and organogenesis besides predictable declines of cell function (cell cycle, energy metabolism, cell death). Very low density lipoprotein receptor (VLDLR) and tribbles 3 (TRIB3) were further analyzed because of recent implication in retinal neovascularization in the mouse and age related macular degeneration (VLDLR, use of statins) as well as in ocular mesodermal development and differentiation of the eye (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 RTPCR suggested 3.0-fold upregulation for VLDLR and 5.1-fold for TRIB3. Vascular endothelial growth factor (VEGF) was increased by 3.1-fold (p = 0.003, RTPCR 8.3-fold) and apelin (APLN), a novel factor of retinal angiogenesis, by 5.6-fold (p = 0.006, RTPCR 8.7-fold). A network with at least 15 directly interacting angiogenic genes was identified that used VLDLR as a key entry node. In the immunocytochemical study, VLDLR localized to the perinucleus, cytoplasm and cell membrane, while TRIB3 was found in nucleoli, the nucleus and cytoplasm. This pattern was the same in hypoxic and normoxic cells.
In Müller cells hypoxia triggers an angiogenic network response with VLDLR as an entry node. Müller cells further react to hypoxia by expressing genes such as TRIB3 that are responsible for proliferation, differentiation and organogenesis.
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