Abstract
Purpose :
To evaluate the protective effects of vitamin D3 on blood retinal barrier (BRB) integrity and its role on the modulation of inflammatory pathways, by using an in vitro model of diabetic retinopathy (DR).
Methods :
Human retinal endothelial cells (HRECs) were exposed to high glucose (HG) challenge, with or without vitamin D3. MTT and LDH assays were carried out to evaluate cell toxicity. The inner BRB integrity was investigated through the measurement of trans endothelial electrical resistance (TEER) and the apical-to-basolateral permeability of sodium fluorescein (Na-F). Moreover, junction proteins such as zonula occludens-1 (ZO-1) and vascular endothelial-cadherin (VE-cadherin), were analyzed by immunocytochemistry and RT-PCR. The mRNA levels of intercellular adhesion molecule 1 (ICAM-1), interleukin-1β (IL-1β), toll-like receptor 4 (TLR-4) and vascular endothelial growth factor-A (VEGF-A) were assessed by RT-PCR. Additionally, ERK phosphorylation and nuclear translocation of p-NFκB were investigated by western blot and immunocytochemistry analyses, respectively. Matrigel assay was carried out on HRECs exposed to exogenous VEGF-A, to assess the anti-angiogenic activity of vitamin D3.
Results :
Vitamin D3 attenuated cell damage elicited by high glucose, maintaining cell viability, and reducing LDH release. Furthermore, vitamin D3 preserved BRB integrity as demonstrated by TEER, permeability assay and by the expression and localization of ZO-1 and VE-cadherin proteins. Vitamin D3 was able to significantly (p<0.05) attenuate the expression of ICAM-1, IL-1β, TLR-4 and VEGF-A, induced by HG exposure. In addition, HG challenge elicited a significant increase of phosphorylated ERK protein, as well as induced the nuclear translocation of p-NFκB, compared to control cells. On the contrary, vitamin D3 significantly (p<0.05) counteracted the activation of these pathways. Finally, vitamin D3 exerted an anti-angiogenic activity on HRECs cells treated with VEGF-A, as shown by Matrigel assay.
Conclusions :
These results provided new insights on the retinal protective role of vitamin D3, particularly regarding the early phase of DR, characterized by BRB breakdown.
Funding: MUR grant PRIN 2020FR7TCL; PIACERI 2020/ 22 Linea2 NanoRet.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.