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D. J. Paull, J. S. Ellis, A. Khalili, A. Kato, S. Brocchini, P. T. Khaw; The Role of Human Tenon’s Fibroblasts in Angiogenesis Using a 3D Co-Culture. Invest. Ophthalmol. Vis. Sci. 2009;50(13):916.
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Matrix Metalloproteinases (MMPs) play a key role in angiogenesis. Following glaucoma filtration surgery (GFS), Human Tenon’s Fibroblasts (HTFs) are crucial for the remodelling of the extracellular matrix through the upregulation of MMPs. Recent studies have suggested that HTFs express receptors for vascular endothelial growth factor (VEGF). However the exact mechanism between MMPs and VEGF in wound healing following GFS has yet to be fully understood. Here we describe an improved in vitro 3D co-culture model of angiogenesis using HTFs and endothelial cells (HUVECS). Furthermore we have investigated the relationship between VEGF and MMPs using an established model of collagen contraction.
The 3D model was established by coating low passage HUVECs onto beads before embedding into a fibrin gel. HTFs were then seeded either on top or inside of the gel to investigate their effect on sprouting and vessel formation. The gel was maintained in cultured medium designed to enhance angiogenesis. The effect of VEGF HTFs mediated gel contraction in vitro was also investigated. RT-PCR was further used to investigate changes in mRNA expression of MMPs in HTFs following the addition of VEGF.
The in vitro contraction model showed that VEGF supplemented growth media significantly increased the rate of gel contraction in vitro (P<0.0001). Preliminary PCR results suggest that VEGF modulates the expression of the metalloproteinases (including MMP-2 and -9). Through seeding the fibroblasts in the 3D co-culture the amount of early sprouting and subsequent vessel network formation was increased, with lumens clearly formed after 4 days.
We have improved an in vitro co-culture model of angiogenesis, establishing it as a co-culture model for ocular research. The role that these fibroblasts play on encouraging lumen formation and networking must be further investigated, but the role of VEGF on metalloproteinases suggests this may play a significant role.
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