Loss of the interleukin-6 receptor leads to differential regulation of VEGF family members in Müller glial cells under hypoxic conditions in vitro

Abstract

Purpose : Interleukin-6 (IL-6) is a pleiotropic cytokine that plays a pivotal role in retinal inflammation in diabetic retinopathy (DR), as prolonged retinal inflammation contributes to tissue ischemia and neovascularization stimulated by vascular endothelial growth factor (VEGF). IL-6 has two major signaling mechanisms – cis-signaling through a membrane-bound IL-6 receptor (IL-6R) and trans-signaling through a soluble receptor – both of which occur in Müller glial cells (MGCs). MGCs are the major source of VEGF in the mature retina, and the contribution of IL-6 signaling to regulation of VEGF family members and receptors is not well-defined. The purpose of this study was to investigate the contribution of IL-6 signaling to hypoxia-induced expression of VEGFA, VEGFB, VEGFR1, and VEGFR2 in MGCs.

Methods : MGCs were isolated from wildtype (WT) C57BL/6J and MGC Il6ra^-/- (KO) mouse pups at P7 and cultured in vitro. WT and KO cells were exposed to 3% O₂ in a hypoxia chamber for 24 hours. WT and KO MGCs were also treated with IL-6 (50 ng/mL) or IL-6 + sIL-6R (150 ng/mL) to induce cis- and trans-signaling, respectively. Expression levels of VEGFA, VEGFB, VEGFR1, and VEGFR2 were measured using RT-PCR and western blot analysis.

Results : At baseline, KO MGCs expressed increased levels of Vegfa (2.90-fold, p=0.009), Vegfb (2.03-fold, p=0.009), and Vegfr2 (7.32-fold, p=0.001) relative to WT MGCs. Increased expression of Vegfa in KO MGCs was sustained in hypoxic conditions. Furthermore, hypoxia increased expression of Vegfr1 (3.09-fold, p=0.002) in WT MGCs but not in KO MGCs. Conversely, hypoxia induced a significant decrease in KO expression levels of Vegfb (0.25-fold, p<0.0001) and Vegfr2 (0.06-fold, p<0.0001), while in WT MGCs these genes were unaffected. Similarly, induction of IL-6 cis- and trans-signaling in WT MGCs increased the expression levels of Vegfa (1.42-fold, p=0.046, and 2.6-fold, p<0.0001 respectively) relative to untreated group. Conversely, trans-signaling induction reduced expression levels of Vegfb (0.72-fold, p=0.0006), while cis-signaling had no effect on its levels.

Conclusions : Loss of IL-6 receptor in MGCs differentially alters expression of VEGFA, VEGFB, and VEGF receptors in vitro under hypoxic conditions, suggesting a role for IL-6 signaling in regulation of the VEGF signaling pathway in MGCs that warrants further investigation.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.