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A. K. Dunnon, P. Saloupis, T. Pridgen, K. Wu, G. Malek, S. W. Cousins; Different Patterns of Neovascularization Are Determined by Different Pro-Angiogenic Stimuli. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3357.
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In human neovascular AMD, choroidal neovascularization (CNV) can manifest a range of severity in terms of lesion size, degree of fibrosis, hemorrhage and vessel maturation. Similarly, increased severity of experimental CNV can be induced in mice not only by local expression of pro-angiogenic factors but also by increased age, exposure to environmental toxins, inflammation and circulating marrow-derived mesenchymal progenitor cells. We desire to determine if similar factors control severity and maturation of neovascularization at extraocular sites. The traditional assay using subcutaneous injection of Matrigel fails to preserve key morphological features making it difficult to evaluate vessel severity and morphology. We developed a Matrigel-filled chamber device which is implanted subcutaneously to evaluate the impact of local expression of various pro-angiogenic factors on neovascularization size, morphology and hemorrhage.
Matrigel chambers were made by filling Tygon tubing with growth factor-depleted Matrigel supplemented with various combinations of pro-angiogenic, pro-fibrogenic and inflammatory growth factors. Chambers were implanted subcutaneously in young and old C57BL/6 mice. The neovascularization response was assessed at 8, 15 and 20 days post implantation. Mice were perfused intracardially. Chambers were excised, fixed, cyropreserved, and sectioned. Vascular morphology and size were determined by light and epifluorescence microscopy. Immunolabelling was performed to assess smooth muscle, endothelial cell and macrophage infiltration.
Chamber implants showed superior morphology compared to subcutaneous injection. Neovascularization grew along the surface of the implant. Chambers containing VEGF only showed modest growth of predominantly capillary type lesions with CD31>SMA positive cells. Chambers containing VEGF/FGF/PDGF developed well defined arborizing vascular fronds with mature feeder arterioles and smaller areas of ‘pure’ capillaries. Greater density of SMA positive cells was observed. Chambers containing a mixture of VEGF/PDGF/FGF plus inflammatory factors demonstrated the most severe lesions, with greater hemorrhage and inflammatory cells. Factor-depleted chambers developed minimal neovascularization.
Matrigel chamber implants provide excellent vascular morphology allowing assessment of neovascular severity and vessel maturation. Preliminary data suggest that inflammatory mediators combined with VEGF and PDGF promote the most severe lesions.
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