Differential Expression of IRBP by cultured rod and cone cells in presence of hypoxia and hyperglycemia

Abstract

Purpose : Interphotoreceptor-Retinoid-Binding-Protein (IRBP, RBP3) is a 124 kDa glycolipoprotein secreted by the rods and cones. IRBP traffics and protects visual cycle retinoid, and has significant free radical scavenging activity. Its expression is reduced in early stages of diabetes. Interestingly, increased levels of IRBP is protective against diabetic retinopathy (DR). We reported that that hypoxia increases VEGF expression by 661W cone photoreceptors in culture, suggesting a role of photoreceptors in proliferative DR (PDR) (Rodriguez et al, Arch Clin Exp. Ophthal 3:23,2021). Here, we compare hypoxia and VEGF on IRBP expression between rods and cones in high and low glucose.

Methods : Y79 (rod-like) cells were grown on laminate coated plates and then transferred to 24-well plates. Both Y79 and 661W (cone-like) cells were seeded to 5x10⁵ / well. Cells were treated with low (5.5 mM) and high (30 mM) glucose at 300 mM of CoCl₂ (to induce hypoxia) and 10 ng/mL of VEGF for 24 hrs. ELISA determined the IRBP concentration in the conditioned media.

Results : High and low glucose treatment did not change the level of IRBP expression by 661W cells (27.3 vs 30.8 ng/ml). Also, the addition of VEGF did not result in a change in IRBP levels. However, hypoxia resulted in an increase of IRBP expression in cells grown in both low and high glucose (to 119.6 ng/ml and 81.5 ng/ml). Furthermore, combined hypoxia and VEGF also resulted in an increase of IRBP level (to 123.67 ng/ml in high glucose and to 93.9 ng/ml in low glucose). Similarly high and low glucose did not change the level of IRBP in Y79 cells (31.6 vs 27.8 ng/ml). VEGF did not result in a change in IRBP level. However, hypoxia significantly reduced in IRBP levels in high glucose (to 2.02 ng/ml). Combined hypoxia and VEGF treatment also resulted in a significant reduction in IRBP level (to 11.2ng/ml).

Conclusions : Hypoxia results in a significant increase in IRBP expression in the conditioned media of 661W grown in low and high glucose. In contrast, hypoxia reduced IRBP levels in the conditioned media of Y79 grown in high glucose. The differential expression of IRBP under conditions of hypoxia and hyperglycemia may provide insight into the observed clinical measurements of vitreal IRBP in DR.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.