Mast cells contribute to choroidal neovascularization in ex vivo model of AMD.

Abstract

Purpose : Mast cells are granulocytes that play a role in immune function through degranulation; however, their role in angiogenesis remains elusive. Neovascular AMD involves the growth of permeable microvessels from the choroid. Mast cells are elevated in number in all forms of AMD and contain many proangiogenic factors that may contribute to the neovascularization seen in AMD. This study determines the effect of mast cell activation and stabilization using an ex vivo model of microvascular angiogenesis.

Methods : The CSA is a microvascular sprouting assay that models the neovascular form of AMD. Using ex vivo RPE/Bruch’s Membrane/Choroid/Scleral tissue from 3-month-old C57BL/6J mice cultured for up to 10 days, we assessed the effects activating or stabilizing mast cells on vascular sprouting. The tissues were stimulated with Ketotifen Fumarate (2.0 µg/mL), a mast cell stabilizer, and/or 48/80 (6.25 µg/mL), a mast cell activator/degranulator every two days starting on Day 2. HBSS was used a vehicle control. Sprouting was imaged every two days and quantified through standardized SWIFT-Choroid macro based on ImageJ software. Total vascular sprouting was measured in pixels (6600 pixels = 1 mm²) and compared using statistical tests. Western blots were used to assess the levels of TGF-β released into supernatants after treatments.

Results : Choroidal sprouting area was significant increased after treatment of mast cell degranulator, 48/80, compared to vehicle control (p<0.05, N=8). The difference in sprouting was detected two days after the first treatment and was maintained until the end of the experiment. Western blots revealed that 48/80 treatment led to increased TGF-β levels in supernatant (p<0.05, N=3). Sprouting was drastically reduced when a mast cell stabilizer was added (p< 0.0005, N = 4) and this trend was maintained until the end of the experiment.

Conclusions : Earlier studies have demonstrated that there is an elevated number of degranulated mast cells present in the choroid of AMD patients. Our data reveal that degranulation of mast cells significant increases the sprouting in the mouse choroid. Furthermore, stabilizing mast cells dramatically reduced the neovascular sprouting response. Mast cells may serve as a possible therapeutic target along with VEGF for reducing neovascularization seen in wet AMD. Further studies are needed to investigate which mast cell component(s) drives this neovascular response.

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