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An Improved Protocol for the Matrigel Duplex Assay: A Method to Measure Retinal Angiogenesis
Kathleen C Brown1, Reagan S Light1, Kushal J Modi1
1Department of Biomedical Sciences, Joan C. Edwards School of Medicine, Marshall University, Huntington, WV, 25755 USA.
Bio-Protocol
|December 14, 2023
Summary
The Matrigel Duplex Assay models retinal neovascular diseases like diabetic retinopathy and age-related macular degeneration. This assay effectively measures the anti-angiogenic effects of drugs, such as PP2, on retinal microvascular endothelial cells.
Area of Science:
- Ophthalmology
- Cell Biology
- Biomedical Engineering
Background:
- Neovascular diseases of the retina, including diabetic retinopathy (DR) and age-related macular degeneration (AMD), cause vision loss due to abnormal blood vessel growth.
- Conventional angiogenesis assays do not accurately mimic the specific angiogenic processes occurring in the retina.
- Retinal angiogenesis involves new blood vessel growth into non-perfused areas, contributing to vision impairment.
Purpose of the Study:
- To introduce and validate the Matrigel Duplex Assay (MDA) as a model for retinal angiogenesis.
- To assess the utility of the MDA in measuring the anti-angiogenic activity of compounds.
- To investigate the effect of the Src kinase inhibitor PP2 on human retinal microvascular endothelial cells (HRMECs) using the MDA.
Main Methods:
- The Matrigel Duplex Assay involves seeding HRMECs in a phenol red-containing Matrigel layer, followed by overlaying a phenol red-free Matrigel layer.
- HRMECs migrate from the primary to the secondary Matrigel layer over 24 hours, mimicking angiogenic sprouting.
- Angiogenic sprouts are visualized using phase contrast microscopy and quantified with ImageJ software.
Main Results:
- The MDA successfully models the migration of angiogenic capillaries relevant to retinal neovascular diseases.
- The study demonstrated the ability of the MDA to measure the angiogenesis-inhibitory activity of the Src kinase inhibitor PP2 in HRMECs.
- The results indicate that PP2 has anti-angiogenic properties in the context of retinal angiogenesis.
Conclusions:
- The Matrigel Duplex Assay is a valuable tool for studying retinal angiogenesis and screening anti-angiogenic drugs.
- The MDA provides a more representative model of retinal pathological angiogenesis compared to conventional assays.
- This assay can be applied to investigate growth factor activity and signaling pathways in retinal angiogenesis.
