Identifying cell type-specific mechanisms of inflammatory prostaglandin signaling in diabetic retinopathy

Abstract

Purpose : NSAID drugs inhibiting cyclooxygenase (COX) have shown some clinical efficacy slowing early-stage diabetic retinopathy (DR) progression driven by inflammation, but chronic NSAID use is associated with severe systemic side effects. Targeting individual prostanoids, the signaling lipids downstream of COX, offers a directed approach to circumvent side effects of NSAID use. Here we characterize prostanoids produced by retinal cells in conditions of systemic diabetes and evaluate prostanoid receptors as therapeutic targets for DR-relevant cytokine production and leukostasis.

Methods : Primary human Müller cells (hMC) and retinal microvascular endothelial cells (hRMEC) were treated with IL-1β modeling inflammation, palmitic acid (PA) for dyslipidemia, D-glucose for hyperglycemia, or controls for 24 hours, then media were harvested for prostanoid mass spectrometry. hMC were stimulated with PGE₂ ± antagonists to receptors EP1, EP2, EP3, or EP4, then media were collected for cytokine ELISA. hRMEC were stimulated with PGF_2α ± FP receptor antagonist AL8810. Cells were harvested for western blot or used in static adhesion assays, where monolayers were incubated with peripheral blood mononuclear cells (PBMCs) for 30 minutes, washed to remove nonadherent PBMCs, fixed in PFA and imaged.

Results : hMC produced PGE₂ in response to IL-1β and PA (p<0.001 each) but not glucose (p=0.97) vs vehicle. 1μM PGE₂ stimulated elevation of cytokines IL-6 (6.6-fold, p<0.001) and IL-8 (1.3-fold, p<0.001) in hMC. The EP2 antagonist PF-04418948 (1μM) but not other EP antagonists blocked PGE₂-induced expression of these proteins (p=0.013 vs PGE₂ for IL-6). hRMEC produced PGF_2α in response to IL-1β, PA, and glucose (p<0.001 each). 10μM PGF_2α elevated levels of adhesion proteins ICAM-1 (1.9-fold, p=0.018) and VCAM-1 (3.2-fold, p<0.001), and static adhesion of PBMCs to hRMEC increased 1.9-fold (p<0.001). PGF_2α-induced adhesion protein levels and static adhesion were each decreased to vehicle levels in hRMEC by 10μM AL8810 (p=0.042 vs PGF_2α in static adhesion).

Conclusions : Conditions of systemic diabetes lead to cell type-specific proinflammatory prostanoid elevation: PGE₂ in hMC and PGF_2α in hRMEC. hMC cytokine production is mediated by PGE₂-EP2 signaling whereas hRMEC leukostasis is via PGF_2α-FP signaling. These suggest specific targets for modulation with potential clinical impact in slowing DR progression at early stages.

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