Beta-2 Adrenergic Receptor Expression in Monocyte-Derived Macrophages Mediates the Inhibition of Choroidal Neovascularization

Abstract

Purpose : Anti-VEGF therapy is widely used to treat patients with neovascular age-related macular degeneration (nAMD), but the substantial treatment burden has spurred the exploration of alternative options. Beta-blockers, initially recognized for their anti-angiogenic properties in the treatment of infantile hemangiomas, have demonstrated efficacy in diminishing laser-induced choroidal neovascularization (CNV) size in mice and topical timolol-dorzolamide reduces anti-VEGF injection burden in treatment-resistant patients compared to placebo. Using retinal cell cultures, beta2-adrenergic receptor (AR) agonism increases VEGF and interleukin-6 (IL-6) expression, two pro-angiogenic factors. Further, Ccr2^-/- mice, which are deficient in classical monocytes and classical monocyte-derived macrophages (MDMs), do not demonstrate CNV lesion reduction with beta-blocker treatment. Based upon these data, we hypothesized that classical MDMs increase CNV area through beta2-AR (Adrb2) driven VEGF and IL-6 expression.

Methods : We generated conditional macrophage-specific Adrb2 knockouts by crossing Adrb2^flox and Cx3cr1^CreER mice. All experiments were performed on female Adrb2^fl/fl :: Cx3cr1^CreER/+ or Adrb2^fl/fl controls. All mice were given two intraperitoneal tamoxifen (100 mg/kg) injections at either 6 or 10 weeks of age to knockout Adrb2 in tissue resident macrophages or all macrophages, respectively. Mice underwent laser induced-CNV at 10 weeks of age with daily saline injections to create stress and increase endogenous (nor)epinephrine levels. Eyes were collected on Day 14 day post-laser for immunofluorescence imaging of choroidal wholemounts.

Results : Adrb2^fl/fl :: Cx3cr1^CreER/+ mice injected with tamoxifen at 10 weeks demonstrated a 1.4-fold decrease (p<0.05, N=14 mice per group) in CNV size compared to Adrb2^fl/fl controls. Alternatively, Adrb2^fl/fl :: Cx3cr1^CreER/+ mice that underwent 6-week tamoxifen injections showed no change in CNV size (p=0.59, N=11-14 mice per group).

Conclusions : Both 6-week and 10-week tamoxifen-treated mice have Adrb2 deletion in tissue resident macrophages, while only the 10-week tamoxifen-treated mice have Adrb2 deletion in MDMs. These data suggest that beta2-AR expression in MDMs is necessary for laser-induced CNV. Future studies will investigate macrophage heterogeneity by multi-parameter flow cytometry and IL-6 expression.

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