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J. P. Eastlack, J. He, H. E. P. Bazan; The Induction of an Angiogenic Response by Platelet-Activating Factor (PAF) in Corneal Myofibroblasts. Invest. Ophthalmol. Vis. Sci. 2008;49(13):1476. doi: https://doi.org/.
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Although the exact mechanisms underlying corneal neovascularization remain unclear, cytokines and growth factors play an important role in the development of corneal neovascularization. Vascular endothelial growth factor (VEGF) and PAF are two major angiogenic cytokines, whereas thrombospodin (TSP) is anti-angiogenic. Earlier studies in our lab have shown that PAF is a potent inducer of corneal neovascularization (IOVS. 2004; 45:2915). Recently, we have found that myofibroblasts express VEGF, TSP-1 and the PAF receptor. The purpose of the current study is to investigate the effect of PAF on VEGF and TSP-1 expression in corneal myofibroblasts.
Myofibroblasts were obtained from rabbit corneal keratocytes and identified with anti-α-SMA antibody. Cells were treated with PAF (100 nM) for 24 hrs. Expression of VEGF and TSP-1 was examined by immunofluorescence and immunoblotting. To study the effect of myofibroblasts on vessel formation in vitro, Vybrant® CM-DiI labeled human umbilical vein endothelial cells (HUVECs) were cultured on myofibroblasts in a thin layer of collagen gel. CD31 was used as the cell marker of HUVEC. Images were recorded by fluorescence microscope, Nikon Eclipse TE200, equipped with a Nikon digital camera DXM1200 using the MetaVue imaging software.
VEGF and TSP-1 were not detectable in keratocytes, but positively stained in myofibroblasts. PAF induced a significant increase in the expression of VEGF and decrease in the expression of TSP-1. HUVECs co-cultured with corneal myofibroblasts formed a typical structure of vessel-like tubes within 1 week. Addition of anti-VEGF antibody to the medium completely prevented the formation of vessel-like tubes. Addition of PAF to the medium increased vessel-like tube formation by HUVECs co-cultured with myofibroblasts.
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