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Marchien Geesje Dallinga, Bahar Arik-Yetin, Richelle Kayser, Ilse Vogels, Ingeborg Klaassen, Cornelis van Noorden, Reinier Schlingemann, Ocular angiogenesis group; CD34 marks angiogenic tip cells in human microvascular endothelial cell cultures.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2306. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Current therapies for proliferative retinopathies target vascular endothelial growth factor (VEGF). Due to unsatisfactory results thus far, novel complementary approaches for anti-angiogenesis are needed. Angiogenic sprouts are led by specialized tip cells. With their distinct phenotype and proteins expression, tip cells are potential targets for novel anti-angiogenic therapies.
Recently, our group discovered that tip cells exist in cultures of Human Umbilical Vein Endothelial Cells (HUVECs), and that they can be identified by staining for the protein CD34. To confirm our results in a different cell type that better resembles endothelial cells that perform angiogenesis in vivo, we studied cultures of human microvascular endothelial cells (hMVECs). We used qPCR analysis to study the expression of known tip cell genes. To study the number of tip cells, we used FACS analysis.
We found that, like HUVECs, hMVEC cultures possess a subset of CD34+ cells with filopodia. qPCR analysis of these CD34+ cells revealed an upregulation of 9 out of 11 known tip cells genes. Furthermore, when we studied the regulation of these CD34+ cells, we found that addition of Vascular Endothelial Growth Factor (VEGF) has a stimulating effect on the number of CD34+ cells. A coating of Delta-like Ligand 4 (Dll4) reduced the number of CD34+ cells. These results resemble the in vivo regulation of tip cells. Using FACS sorting of CD34+ and CD34- cells, we also show that the CD34+ cells are actively generated within a culture of CD34- cells.
In conclusion: we used hMVECs to confirm that there is a subset of CD34+ tip cells in endothelial cell cultures that expresses tip cell genes and other in vivo characteristics, amongst which tip cell morphology. This will allow us to cut down on costly, time-consuming in vivo experiments and study tip cells in vitro in an efficient and more detailed fashion.
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