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Jeremy Lavine, Harris Perlman; Beta-adrenergic receptor antagonism reduces choroidal sprouting angiogenesis ex vivo. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3270. doi: https://doi.org/.
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We previously demonstrated that beta-adrenergic receptor (beta-AR) blockade inhibits laser-induced choroidal neovascularization (CNV) by 50-80%. Additionally, we showed that beta2-AR signaling increases vascular endothelial growth factor (VEGF) expression in primary mouse microglia, choroidal endothelial cells (EC), retinal pericytes, and retinal pigment epithelium (RPE). However, cell lines are an artificial model that lack cell-cell interactions, 3-dimensionality, and are immortalized. Therefore, we tested the ability of beta-AR antagonism to inhibit angiogenesis during the choroidal sprouting assay: a microvascular angiogenesis model that includes choroidal EC, pericytes, RPE, and choroidal macrophages.
Male C57BL/6J mice were euthanized at 10-12 weeks of age, and eyes were enucleated. Peripheral RPE-choroid-scleral complex were dissected into 1x1 mm explants and plated in Matrigel. Choroidal explants were cultured for 6 days with the pan beta-AR antagonist propranolol or PBS control. Photographs were taken on Day 3 – Day 6 using the Nikon Ti2 widefield microscope. Area of growth was calculated using Nikon Elements, excluding the choroid explant area. Data were analyzed using two-way ANOVA and Dunnett’s multiple comparisons test.
We found that angiogenesis is first detectable on Day 3. We performed a dose-response curve using 0.05 microM, 0.5 microM, and 5 microM propranolol from Day 4 – 6, and found that propranolol had no effect upon angiogenesis growth area compared to control (N=10 per group). Our positive control, sunitinib, is a receptor tyrosine kinase inhibitor, which strongly inhibits VEGF receptor 2 (VEGFR2), reduced angiogenesis growth area by 40% on Day 6 (N=6, p<0.0001). Next, we investigated the initial phase of the choroidal spouting assay by adding propranolol and sunitinib on Day 0. Propranolol 0.5 microM (N=6, p=0.007) and 5 microM (N=7, p=0.01) reduced angiogenesis growth area by 36% and 34% respectively. Sunitinib decreased angiogenesis growth area by 68% (N=6, p<0.0001).
Propranolol decreases choroidal sprouting angiogenesis during the initial phase of the assay. This suggests that early angiogenesis is driven by different factors than during later stages. Sunitinib, an inhibitor of VEGFR2 and other receptor tyrosine kinases, is effective at all stages of the assay.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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