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K. Engelmann, M. Valtink, D. Lindemann, R. H. W. Funk; HMW FGF-2 Mediates Cell Rescue After Retroviral Gene Transfer to Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1732.
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To evaluate retroviral vectors as a tool to transduce primary human corneal endothelial cells (pHCEC) and to assess a possible role of HMW FGF-2 in HCEC proliferation and survival. Such method may be applied to detect factors that are able to induce an increase in corneal endothelial cell density of insufficient donor corneas, hence increasing the number of available corneas for keratoplasty.
Retroviral vectors based on HIV-1 and MLV, both pseudotyped with either VSV-G env or FV env, were tested upon their infectivity in primary human corneal endothelial cells (pHCEC). HIV-1 based vectors which express either 18 kD FGF-2 or codon-optimized 22 kD, 22.5 kD, 24 kD or 34 kD isoforms of human FGF-2 were generated. Viruses were produced in 293T cells using a three plasmid system. Transduction efficiency was evaluated and optimized for pHCEC by FACS, and FGF-2 expression was assessed by western blotting. HT1080 epithelial cells and NIH 3T3 fibroblasts served as control cells. Toxic effect of virus infection under starvation or enriched nutrient conditions and cell survival mediated by FGF-2 overexpression were evaluated by means of resazurin conversion test.
HIV-1 based vectors appeared superior to MLV based vectors, and FV env appeared superior to VSV-G env, with gene transfer efficiencies >90 % in pHCEC (average titers 106-107). Transduction with FGF-2 resulted in overexpression of the respective isoform in all tested cell populations. It was also found that cultured pHCEC expressed the 18, 22.5 and 24 kD isoform constituently. Nuclear localization of the 22 - 34 kD isoforms was proven by western blotting after mild cell lysis (excluding nuclei) versus total cell lysis. Resazurin conversion test revealed a toxic effect of viral infection on pHCEC. FGF-2 overexpression especially of the higher molecular weight isoforms partially abolished this effect and was also found to promote cell survival under serum-free and starvation culture conditions.
Retroviral vectors can efficiently be used to transduce pHCEC. HMW FGF-2 is a potent survival factor in pHCEC.
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