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Chi Zhang, ALEXANDRO DAVID GUERRERO, FREDDI HUAN TRAN; Wnt pathway inhibitors regulate the tube formation of human retinal microvascular endothelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):112. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Ocular neovascularization in the retina is a major cause of irreversible visual loss. The mechanisms through which retinal neovascularization occurs are still being uncovered, one such mechanism is thought to be driven by Wnt signaling given its capacity to govern the expression of the pro angiogenic factor vascular endothelial growth factor (VEGF). Activated Wnt signaling stabilizes cytoplasmic β-catenin by disrupting the Axin complex which then attenuates β-catenin phosphorylation. Non-phosphorylated β-catenin associates with TCF/LEF transcriptional promoter to activate Wnt target genes, such as VEGF. Wnt mediated VEGF is critical for the tube formation of human retinal endothelial cells and therefore their capacity to contribute to neovascularization. In our lab we have novel small molecule inhibitors targeting Wnt and thus we wanted to examine whether their effect upon primary human retinal microvascular endothelial cells (HRMEC) in vitro.
The 3T3 luciferase based reporter cell with stable expression of firefly luciferase under the control of TCF/LEF promoters was utilized to confirm the inhibitory profile of Wnt inhibitors and activation profile of Wnt3a soluble protein. Then HRMEC cells were seeded in matrigels and treated with Wnt3a, Wnt3a together with Wnt inhibitors, or Vehicle. Tube formation HRMEC was examined via fluorescent microscopy. These assays were performed separately at least 2 times and there were greater than 3 technical repeats within each experiment.
Application of Wnt3a to the 3T3 reporter cells lead to increased luciferase readings. In contrast to this Wnt inhibitors attenuated Wnt3a mediated Wnt signaling activation. HRMEC cells receiving Wnt3a in the presence of Wnt inhibitors exhibited lessened tube formation and this was also consistent with decreased levels of total tube length, branching points and loops.
Application of small molecule Wnt inhibitors hampered the ability of HRMEC cells to form tubes in vitro. These data suggest that the tube formation of HRMEC cells is highly dependent on Wnt signaling. Therefore, this may serve as an in vitro tool to assess Wnt mediated neovascularization mechanisms in vitro.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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