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K.J. Bayless, E.A. Mendoza; Studying Angiogenic Responses in Three–Dimensional Type II Collagen Matrices in vitro . Invest. Ophthalmol. Vis. Sci. 2006;47(13):5337.
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Identifying the mechanisms through which angiogenesis occurs in type II collagen matrices is critical to design effective therapies that stimulate or inhibit retinal neovascularization.
Primary human endothelial cells were seeded on the upper surface of three–dimensional type II collagen matrices. Experiments were conducted under serum–free conditions in the presence of molecules that are known to stimulate angiogenesis, including vascular endothelial growth factor, fibroblast growth factor and sphingosine–1–phosphate, a platelet–derived lipid.
Marked endothelial cell invasion of three–dimensional type II collagen matrices required a combination of angiogenic growth factors and sphingosine–1–phosphate. Blockade of the alpha 2 beta 1 integrin resulted in nearly complete abolishment of invasion, indicating that the alpha 2 beta 1 integrin was involved in mediating the response.Gelatin zymogram analyses of conditioned media from invading cultures indicated that matrix metalloproteinase–2 activation correlated with the invasion response. The addition of hydroxamate–based inhibitors of matrix metalloproteinases GM6001 and TAPI–2 dose–dependently blocked invasion into collagen type II matrices. Addition of tissue inhibitor of metalloproteinase–1 (TIMP–1) resulted in invasion that was similar to control treatment, suggesting a membrane–associated metalloproteinase is responsible for invasion in collagen type II. This was confirmed by the addition of TIMP–2 and TIMP–3, which inhibited invasion significantly. Ongoing studies are aimed at identifying the proteinases that are responsible for invasion in type II collagen matrices as well as incorporating hyaluronic acid into the model to more closely mimic the chemical composition of the vitreous.
These data aid our understanding of basic mechanisms in endothelial cells that control neovascularization responses, namely that integrins and membrane–associated metalloproteinases mediate the process.
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