Related Experiment Video
Updated: Dec 27, 2025
An Ex Vivo Choroid Sprouting Assay of Ocular Microvascular Angiogenesis
Published on: August 6, 2020
Free fatty acid receptor 4 activation protects against choroidal neovascularization in mice
Yohei Tomita1, Bertan Cakir1, Chi-Hsiu Liu1
1Department of Ophthalmology, Boston Children's Hospital, Harvard Medical School, 300 Longwood Ave, Boston, MA, 02115, USA.
Abstract:
To examine whether free fatty acid receptor 4 (FFAR4) activation can protect against choroidal neovascularization (CNV), which is a common cause of blindness, and to elucidate the mechanism underlying the inhibition, we used the mouse model of laser-induced CNV to mimic angiogenic aspects of age-related macular degeneration (AMD). Laser-induced CNV was compared between groups treated with an FFAR4 agonist or vehicle, and between FFAR4 wild-type (Ffar4+/+) and knock out (Ffar4-/-) mice on a C57BL/6J/6N background. The ex vivo choroid-sprouting assay, including primary retinal pigment epithelium (RPE) and choroid, without retina was used to investigate whether FFAR4 affects choroidal angiogenesis. Western blotting for pNF-ĸB/NF-ĸB and qRT-PCR for Il-6, Il-1β, Tnf-α, Vegf, and Nf-ĸb were used to examine the influence of FFAR4 on inflammation, known to influence CNV. RPE isolated from Ffar4+/+ and Ffar4-/- mice were used to assess RPE contribution to inflammation. The FFAR4 agonist suppressed laser-induced CNV in C57BL/6J mice, and CNV increased in Ffar4-/- compared to Ffar4+/+ mice. We showed that the FFAR4 agonist acted through the FFAR4 receptor. The FFAR4 agonist suppressed mRNA expression of inflammation markers (Il-6, Il-1β) via the NF-ĸB pathway in the retina, choroid, RPE complex. The FFAR4 agonist suppressed neovascularization in the choroid-sprouting ex vivo assay and FFAR4 deficiency exacerbated sprouting. Inflammation markers were increased in primary RPE cells of Ffar4-/- mice compared with Ffar4+/+ RPE. In this mouse model, the FFAR4 agonist suppressed CNV, suggesting FFAR4 to be a new molecular target to reduce pathological angiogenesis in CNV.
Insights
Free fatty acid receptor 4 (FFAR4) activation protects against choroidal neovascularization (CNV) by reducing inflammation. FFAR4 deficiency worsens CNV, highlighting FFAR4 as a therapeutic target for blinding diseases like AMD.
Area of Science:
- Ophthalmology
- Molecular Biology
- Immunology
Background:
- Choroidal neovascularization (CNV) is a major cause of vision loss, often linked to age-related macular degeneration (AMD).
- The role of free fatty acid receptor 4 (FFAR4) in regulating pathological angiogenesis and inflammation in the eye remains largely unexplored.
Purpose of the Study:
- To investigate the protective effects of FFAR4 activation against CNV.
- To elucidate the underlying molecular mechanisms, focusing on inflammation and angiogenesis.
- To assess FFAR4's role in retinal pigment epithelium (RPE) mediated inflammation.
Main Methods:
- Laser-induced CNV mouse model to mimic AMD.
- Pharmacological activation of FFAR4 using an agonist and genetic deletion (knockout) of FFAR4.
- Ex vivo choroid-sprouting assay to assess angiogenesis.
- Western blotting and qRT-PCR to analyze inflammatory markers (e.g., IL-6, IL-1β, TNF-α, NF-κB) and VEGF.
- Analysis of primary RPE cells from FFAR4 wild-type and knockout mice.
Main Results:
- FFAR4 agonist treatment significantly suppressed laser-induced CNV.
- CNV development was exacerbated in FFAR4 knockout mice compared to wild-type controls.
- FFAR4 activation reduced the mRNA expression of key inflammation markers (IL-6, IL-1β) via the NF-κB pathway.
- FFAR4 deficiency led to increased inflammation in primary RPE cells.
- The FFAR4 agonist inhibited neovascularization in the ex vivo choroid-sprouting assay.
Conclusions:
- FFAR4 activation demonstrates a protective effect against CNV in a mouse model.
- FFAR4 plays a crucial role in regulating ocular inflammation and angiogenesis relevant to CNV.
- FFAR4 represents a promising novel molecular target for therapeutic interventions aimed at reducing pathological angiogenesis in CNV and potentially AMD.
