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C.G. Wong, D. Rabin, A. Yeh; Can Bioengineered Sustained-Release VEGF/bFGF Wafers Elicit Apoptosis in Cultured Human Adult RPE Cells? . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1707.
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Purpose: To evaluate if sustained-release VEGF and bFGF that are incorporated into a symmetrically round non-biodegradable Hydron matrix can elicit apoptosis in cultured adult human RPE cells. Methods: Both human recombinant VEGF and bFGF (PeproTech) are incorporated into a 95% EtOH/Hydron solution and subsequently engineered as round wafers (1 ug to 4 ug per growth factor) directly onto the surface matrix of the plastic tissue culture wells. Such VEGF/bFGF-containing Hydron implants have been shown to produce a florid irreversible retinal neovascular response and hemorrhage in pigmented rabbits. After both VEGF/bFGF-containing wafers and blank wafers were engineered directly onto the tissue culture wells and subsequently air-dried under sterile conditions, adult human D507 RPE cells were plated directly onto the tissue culture wells with complete DMEM containing 10% FBS. The cells subsequently were monitored at 1,2,4, and 7 days. Blank Hydron wafers that lacked growth factors were used as negative controls. Cells were stained with PAS and H & E at day 7. Results: Cells grew to confluency around blank wafers but did not grow directly onto the blank Hydron wafers. In contrast, cells reached total confluency and also grew on the VEGF/bFGF wafers. Interestingly, by day 7 the area between the VEGF-bFGF-containing wafers and the plastic tissue culture plate was devoid of cells, thereby forming a "halo." No such halo-like area was noted in the wells with blank Hydron wafers. Conclusions: Preliminary observations indicate that cell death had occurred in the area between the RPE cells on the tissue culture plate and the RPE cells on the VEGF/bFGF-containing wafers. Both TUNEL and annexin V staining will ascertain if apoptosis rather than necrosis occurred at the cellular interface between the VEGF/bFGF-containing wafers and the tissue culture well. Additional experiments will determine what mechanistic role sustained levels of multiple cytokines in the presence of either normal or environmentally injured RPE could play in the formation of drusen, which is a putative marker of AMD, and what role apoptosis of the RPE could play in the extremely early phase of AMD.
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