Abstract
Purpose :
Increased retinal inflammation has been shown to coincide with diabetic retinopathy (DR) progression, and inflammatory cytokines are experimentally linked to several hallmark DR features, including leukostasis. Cannabinoid receptor 2 (CB2) agonism has been shown to decrease leukocyte recruitment and adhesion in diseases such as arthritis and atherosclerosis, suggesting CB2 agonism could have similar therapeutic potential for leukostasis in DR.
Methods :
Human retinal microvascular endothelial cells (hRMEC) were treated with TNFα (0.1ng/mL) in the presence or absence of CB2 agonists HU-308 or CB65 (1 μM). ICC: 30min treatment, cultures where fixed, stained for NFκB, and imaged. qRT-PCR: 4hr treatment, total RNA was isolated and adhesion molecule expression was assayed. Western blot: 8hr treatment, total protein was isolated and adhesion molecule protein levels were assayed. Static Adhesion: 8hr treatment, PBMC were added to treated monolayers for 30mins, washed to remove nonadherent leukocytes, fixed, imaged, and PBMC adhesion was quantified per mm2 retinal area. In vivo: C57BL/6 mice were given daily intraperitoneal (IP) injections of vehicle or HU-308 (5mg/kg) for 4wks, starting at 6wks post STZ-injections, or mice were given an intravitreal (IVIT) injection of vehicle or HU-308 the day before leukostasis readings. Leukostasis was assayed 10wks post STZ injections.
Results :
HU-308 and CB65 significantly reduced TNFα-induced expression of leukocyte adhesion molecules ICAM-1 (24%, 40%), VCAM-1 (55%, 44%) and E-selectin (41%, 27%), respectively, in hRMEC (n=4, p<0.0001). HU-308 and CB65 significantly reduced TNFα-induced protein levels of ICAM-1 (30%, 29%) and VCAM-1 (30%, 28%), respectively, in hRMEC (n=4, p<0.0066). HU-308 and CB65 reduced TNFα-induced NFκB translocation by 29% and 27%, respectively, (n=4, p<0.0001). HU-308 and CB65 reduced TNFα-induced leukocyte adhesion to hRMEC monolayers, each by 44% (n=4, p<0.0298). In vivo, IP and IVIT HU-308 administration significantly decreased STZ-induced leukostasis, each by 30% (n=4-7, p<0.024).
Conclusions :
Consistent with our hypothesis, CB2 agonism decreased adhesion molecule expression, NFκB translocation, leukocyte adhesion to endothelial monolayers, and leukostasis in STZ-treated mice. CB2 agonism may therefore have therapeutic potential for addressing leukostasis, a hallmark feature characteristic of DR.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.