A comparison of inner and outer blood-retina barrier function under in vitro prostaglandin stimulation

Abstract

Purpose : In early diabetic retinopathy (DR), chronic retinal inflammation can disrupt the blood-retina barrier, leading to vessel leakage and fluid accumulation in the retina. The consequent retinal edema is the leading cause of vision loss in diabetics. Barrier integrity is essential to prevent DR progression to irreversible blindness. Our lab has shown that Müller glia produce prostaglandins PGE₂ and PGF_2α in response to palmitic acid and IL-1β—stimuli modeling dyslipidemia and inflammation in DR, respectively. However, effects of prostaglandins on blood-retina barrier function are, to date, unknown. We compared effects of PGE₂ and PGF_2α on inner and outer blood-retina barrier function in vitro using primary human retinal microvascular endothelial cells (hRMEC) and retinal pigment epithelial cells (hRPE).

Methods : hRMEC (passage P7; Cell Systems) and hRPE (P5; Lonza) were used. In electric cell-substrate impedance sensing (ECIS) assays, cells were grown to monolayers with resistances of 1200-1400W and stimulated with PGF_2α, PGE₂, agonists/antagonists, or controls for 24hrs. In transwell assays, cells formed monolayers on inserts for 3 days prior to adding PGE₂ and 70kD FITC-dextran, and fluorescence in the bottom well was measured after 24hrs.

Results : Barrier function was assessed by monolayer resistance in ECIS. In hRMEC and hRPE, PGF_2α up to 1μM did not affect barrier resistance. In hRPE, PGE₂ decreased resistance with an EC₅₀ of 9.19nM (R²=0.91), consistent with a proinflammatory role. However in hRMEC, PGE₂ increased resistance with an EC₅₀ of 644.3pM (R²=0.87). This was validated in transwell assays, where 100nM PGE₂ reduced dextran flux across hRMEC monolayers by 30.6% vs vehicle (p=0.01). The barrier-enhancing effects were similarly observed in ECIS with a selective agonist of the EP4 receptor at 1nM (p<0.01 vs vehicle, p=0.07 vs PGE₂), and an EP4 antagonist prevented this elevation by PGE₂ (p=0.02 vs PGE₂). Manipulation of other PGE₂ receptors yielded no change.

Conclusions : We show opposing responses of hRMEC of the inner blood-retina barrier and hRPE of the outer blood-retina barrier to PGE₂ that is elevated in early DR. Whereas PGE₂ stimulates permeability of hRPE monolayers, PGE₂ via receptor EP4 decreases hRMEC permeability. These opposite effects may indicate the importance of cell-specific anti-inflammatory therapeutic targeting for blood-retina barrier integrity in diabetes.

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