Abstract
Purpose :
Inflammation is an early pathogenic event in diabetic retinopathy (DR). Activation of the cannabinoid-2 (CB2) receptor by endocannabinoids (eCB) promotes anti-inflammatory effects, suggesting potential therapeutic relevance in DR. 5,6-epoxyeicosatrienoic acid ethanolamide (5,6-EET-EA) is an eCB thought to reduce inflammation via CB2 activation. We investigated the potential of 5,6-EET-EA to mitigate DR-relevant inflammation, including adhesion protein expression and vascular permeability, both in vitro and in vivo.
Methods :
Human Müller cells (HMC) were treated with 1ng/ml IL-1β +/- 0.5μM 5,6-EET-EA and proper control reagents for 2hrs, washed, and treated again with 5,6-EET-EA or vehicle only for 6hrs to generate conditioned media (CM). Human retinal microvascular endothelial cells (HRMEC) were treated with CM for 2hrs before lysis for qRT-PCR analysis of cell adhesion proteins E-selectin (SELE), VCAM, and ICAM. Transendothelial electrical resistance was measured in HRMEC treated with 1ng/ml TNFα +/- 0.5μM 5,6-EET-EA. In vivo, mice were intravitreally injected with 15ng/ml TNFα +/- 0.5μM 5,6-EET-EA. Retinal vascular permeability was measured by quantitative fluorescein angiography (qFA) 6hrs after treatment.
Results :
CM from IL-1β-treated HMC increased expression of SELE by 63-fold, VCAM by 90-fold, and ICAM by 3.8-fold in HRMEC (p<0.001). When 5,6-EET-EA was present during IL-1β treatment of HMC, the CM reduced these induction levels in HRMEC by 58% for SELE, 66% for VCAM, and 39% for ICAM (p<0.001). TNFα decreased HRMEC monolayer resistance by 15% 4hrs after treatment (p<0.001). 5,6-EET-EA restored 68% of the deficit after 12hrs of treatment (p<0.001) to levels comparable with controls (p=0.881). In vivo, TNFα caused a 4-fold increase in vessel leakage (p=0.009), whereas 5,6-EET-EA rescued TNFα-induced leakage to near-control levels (p=0.029 vs. TNFα, p=0.739 vs. control).
Conclusions :
Our results indicate that 5,6-EET-EA offers anti-inflammatory relief of DR-relevant damage at multiple endpoints. 5,6-EET-EA reduced expression of multiple adhesion proteins in HRMEC stimulated by DR-relevant inflammation, and it decreased vascular permeability in both HRMEC and an acute model of retinal inflammation in vivo. Administration of exogenous eCB and/or CB2 activation may serve as valuable anti-inflammatory therapies for early-stage DR treatment.
This is a 2021 ARVO Annual Meeting abstract.