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S. V. Klein, Y. Liang, E. Abari, K. Schmidt, I. Semkova, K. L. Meyer, W. J. Brunken, N. Kociok, M. Koch, A. M. Joussen; Influence of Netrin-4 and Its Receptors on the Neovascularization in the Mouse Retina. Invest. Ophthalmol. Vis. Sci. 2010;51(13):48.
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Vascular diseases of the retina are the most common reasons for blindness. The pathological proliferation of vessels is mostly accompanied by degeneration of the neuronal tissue. Netrins are path finding proteins, known to play a role in establishment of the nervous system and in normal and pathological neoangiogenesis. Especially macrophages are involved in the development of neovascularisations and neutrophil granulocytes play a main role in the regulation of VEGF expression in endothelial cells. The aim of this study was to examine if and how frequently netrin-4 and its receptors are expressed on those cells.
Red blood cells were lysed from C57/Bl6 whole blood and leukocytes were marked with cell specific antibodies. To determine receptors, cells were additionally stained with antibodies against netrin receptors and analysed using flow cytometry. Leukocytes were separated to obtain homogeneous populations of neutrophil granulocytes and monocytes. Activated peritoneal macrophages were obtained 3 days after intraperitoneal injection of 0.1ml thioglycolate.Total RNA was isolated from these cells and Real Time RT-PCR was carried out, verified and quantified.
FACS Analysis showed that leukocytes were positive for netrin-4 and its six receptors DCC, Neogenin and Unc5H1 to 4. The corresponding Real Time RT-PCR confirmed these results and showed that the expression profiles of the tested netrin receptors differ between the cell types.
The combination of Real Time RT-PCR and flow cytometry allows analysing the receptors on RNA and protein level. The fact that netrin-4 and its receptors are expressed in the majority of leukocytes suggests its importance as path finding molecule. Furthermore the different expression profiles of the netrin-4 receptors on the particular cell types indicate the importance of netrin-4’s functions.
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