Abstract
Purpose :
Vitamin D is a hormone with known protective roles in endothelial cell (EC) function. The majority of vitamin D action is mediated through the vitamin D receptor (VDR). VDR is a nuclear receptor whose engagement by vitamin D impacts the expression of many genes with important roles in regulation of angiogenesis and inflammation. We previously showed calcitriol, the active form of vitamin D, is a potent inhibitor of retinal neovascularization in vivo and retinal EC capillary morphogenesis in vitro. However, the detailed mechanisms involved remained unknown.
Methods :
Using retinal EC prepared from wild-type (Vdr+/+) and Vdr-deficient (Vdr−/−) mice, we determined the expression of VDR by qPCR and Western blot analysis. The cell morphology was examined by phase microscopy. The expression and localization of cell adhesion and junctional molecules were confirmed by Western blot and immunofluorescence analysis, respectively. Cell migration was assessed by scratch wound assay and confirmed by transmigration assay. Cell adhesion to various matrix proteins were assessed using various extracellular matrix (ECM) proteins. The expression of various ECM proteins was confirmed by Western blot analysis of cell lysates and conditioned medium. The expression of various inflammatory mediators and genes involved in iron homeostasis was assessed by qPCR and Western blot analysis.
Results :
We showed that retinal EC express VDR and its expression is induced by calcitriol. The lack of VDR expression had a significant impact on endothelial cell-cell and cell-matrix interactions. Vdr−/− retinal EC proliferated at a slower rate and were more adherent and less migratory. They also exhibited increased expression levels of inflammatory markers driven in part by sustained activation of STAT1 and NF-κB pathways and were more sensitive to oxidative challenge. These changes were attributed, in part, to down-regulation of endothelial nitric oxide synthetase, enhanced BMP6 and hepcidin expression, and increased intracellular iron levels.
Conclusions :
Our results indicate that VDR expression plays a fundamental role in maintaining the proper angiogenic and inflammatory state of retinal EC.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.