Purchase this article with an account.
Daniel Kampik, Mark D. Basche, Alexander J. Smith, Ulrich F. Luhmann, Prateek Buch, Daniel F. Larkin, Robin R. Ali; Non-integrating Lentiviral Gene Transfer Of E2F2 To Induce Corneal Endothelial Cell Replication. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6460.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Corneal endothelial cells (CEC) are considered non-replicative in humans but retain their proliferative capacity. A decrease in CEC density due to aging, injury or inherited disease causes stromal swelling and loss of transparency, requiring corneal transplantation. E2F2 is a transcription factor regulating the transition from G1 to the S phase of the cell cycle. We investigate whether lentiviral mediated gene transfer of E2F2 is able to induce mitosis and to increase CEC density.
We constructed a non-integrating HIV-based lentiviral vector delivering E2F2 under a CMV promoter (LNT-E2F2). A lentiviral vector delivering green fluorescent protein gene (LNT-GFP) served as control. Human corneal buttons with normal endothelium explanted during keratoplasty or whole donor corneas not suitable for transplantation were exposed to the virus for 2 hours and then maintained in ex vivo culture for one week. Confluent serum-starved ARPE19 cells served as an in vitro model for a contact-inhibited cell monolayer. Transgene overexpression upon viral exposure was verified by quantitative RT-PCR, Western blot and immunohistochemistry. 5-bromodeoxyuridine (BrdU) incorporation and Ki-67 immunostaining was used to detect G1 to S phase progression or cells in any active cell cycle phase, respectively.
In vitro, exposure to LNT-E2F2 resulted in a >200fold increase of E2F2 mRNA compared to uninfected or LNT-GFP infected controls. In human corneal ex vivo samples, exogenous E2F2 protein was detectable only in LNT-E2F2 infected samples whereas LNT-GFP or uninfected controls remained negative. Samples infected with LNT-E2F2 showed both increased BrdU and Ki67 nuclear staining. The increase in replicating cells was significant for cell culture monolayers, but not for corneal endothelial cells in situ. Increasing virus concentration up to 20 viral particles per cell had toxic effects and did not increase mitotic rate.
Although lentiviral E2F2 gene transfer induces mitosis in arrested cells in vitro, this alone does not seem to be a sufficient method to increase endothelial cell density in human donor corneas in ex vivo organ culture. Additional pathways are now being explored.
This PDF is available to Subscribers Only