1,25(OH)₂D₃ Attenuates Retinal Angiogenesis by Inhibiting Proangiogenic Activity of Pericytes

Abstract

Purpose : We showed that the active form of vitamin D (calcitriol; 1,25(OH)₂D₃) is a potent inhibitor of retinal neovascularization. However, the underlying molecular and cellular mechanisms involved remained poorly understood. We previously showed that 1,25(OH)₂D₃ minimally affected retinal endothelial cell (EC) proliferation and migration in culture. Perivascular supporting cells including pericytes (PC) play important roles during angiogenesis, vascular maturation, and stabilization of blood vessels. How 1,25(OH)₂D₃ affects retinal PC proliferation and migration, and whether these effects are mediated through vitamin D receptor (Vdr), were unknown.

Methods : Retinal PC from wild type (Vdr+/+) and Vdr-deficient (Vdr-/-) mice were prepared as described by us. The impact of 1,25(OH)₂D₃ on proliferation, migration, adhesion, and Vdr expression of PC were determined and compared with those from Vdr-/- cells. Vdr expression was confirmed by Western blotting and qPCR analysis of cells incubated with solvent control or 1,25(OH)₂D₃. Vascular endothelial growth factor (VEGF) levels were determined by ELISA. The impact of VEGF and PDGF on migration of PC was determined by transwell assay. The role of VEGF in inhibition of PC migration was confirmed using VEGF antagonists, sFlt-1 and VEGF-R2 inhibitor.

Results : Retinal PC expressed significantly higher Vdr levels compared with retinal EC. 1,25(OH)₂D₃ significantly decreased PC proliferation and migration resulting in a G₀/G₁ cell cycle arrest. In contrast, 1,25(OH)₂D₃ did not inhibit the proliferation of Vdr-/- PC, but it did inhibit their migration. PC adhesion to various extracellular matrix (ECM) proteins and ECM production were also affected by incubation with 1,25(OH)₂D₃. Vdr-/- PC were more adherent compared with Vdr+/+ cells. Mechanistically, incubation of Vdr+/+ PC with 1,25(OH)₂D₃ resulted in increased expression of VEGF and attenuation of signaling through VEGF-R2 and PDGF-Rβ. Incubation with soluble VEGF-R1 (sFlt-1) partially reversed the effect of VEGF on Vdr+/+ PC migration. In addition, incubation of Vdr+/+ PC with VEGF or inhibition of VEGF-R2 increased Vdr expression.

Conclusions : Retinal PC are the major source of Vdr expression. Our results suggest an important role for retinal PC as a target for vitamin D and Vdr action, and attenuation of angiogenesis through a VEGF-mediated inhibition of PDGG-Rβ and VEGF-R2 autocrine signaling.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.