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Yanhong Wei, Junsong Gong, Rajesh Thimmulappa, Shyam Biswal, Elia J Duh; Direct regulation of endothelial cells by Nrf2 during retinal vascular development. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5369.
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Nrf2 is a transcription factor well-known for regulating the stress response in multiple pathologic settings. We have previously demonstrated that Nrf2 has a cytoprotective role in the retina in ischemia-reperfusion injury and diabetic retinopathy. However, its role in vascular development is less appreciated, including the direct role of Nrf2 in endothelial cells. The objective of this study is to gain insights into Nrf2 function in endothelial cells during vascular development.
For in vivo studies, retinal flatmounts were prepared at P5 and blood vessels were visualized by PECAM-1 staining. Cell-specific knockouts of Nrf2 were used to dissect the specific role of Nrf2 in endothelial cells. Complementary ex vivo studies of vascularization were performed using the aortic ring assay. Finally, cultured human retinal endothelial cells (HREC) were used for in vitro studies.
Endothelial cell-specific deletion of Nrf2 resulted in reduction of vascular density as well as endothelial cell (EC) sprouting, similar to global Nrf2 knockout. Deletion of endothelial Nrf2 led to increased hypoxic area and VEGF expression in retina. Aortic ring segments from endothelial cell-specific Nrf2 knockout mice exhibited a marked reduction in vascularization compared to wild-type mice. Knockdown of Nrf2 in HREC resulted in impairment of multiple angiogenic capabilities, including proliferation, migration, and tube formation. Blockade of either Dll4 or Notch signaling restored the vascular phenotype in the retina and in Nrf2 knockouts.
These studies indicate that Nrf2 plays a direct role in regulating endothelial cells during retinal vascular development.
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