Title:Expression of Dominant Negative K6W-Ubiquitin in the Lens Epithelium via an Adenoviral Vector Delays Posterior Capsule Opacification in a Rabbit Model
Volume: 17
Issue: 2
Author(s): X. Bao, M. Hou, R. Peng, F. Luo and M. Wu*
Affiliation:
- State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060,China
Keywords:
Posterior capsule opacification (PCO), K6W-ubiquitin, ubiquitin-proteasome pathway (UPP), lens
epithelial cells (LECs), adenoviral vector
Abstract: Purpose: Ubiquitin is involved in cell proliferation and differentiation, and the
objective of this study is to investigate the potential of dominant negative Ubiquitin (Ub)
with a lysine to tryptophan mutation at the 6 position (K6W) through an adenoviral
expression vector in the prevention of posterior capsule opacification (PCO) in a rabbit
PCO model.
Methods: Recombinant dominant negative K6W-Ub adenovirus (RAd-K6W-Ub) with
green fluorescent protein (RAd-K6W-Ub/GFP) and RAd-GFP viruses (control) were
generated with QBI-HEK 293A cells. New Zealand rabbits receiving lens
phacoemulsification were given an intraoperative anterior chamber injection of the
viruses. The images of anterior segment photography taken by a slit lamp biomicroscopy
were analyzed by posterior capsule opacification manual software (POCOman) for PCO
grading. The intraocular pressure (IOP) was detected with a non-contact tonometer
(NCT). The expression of α-smooth muscle actin (α-SMA) was assessed by Western
blotting. Cell migration ability in cultured rabbit’s lens epithelial cells (LECs) was
evaluated by scratch healing assay.
Results: The expression of GFP and Ub in the lens epithelium was markedly upregulated
after 48 hours vector injection. Eyes injected with RAd-K6W-Ub showed a
significantly lower PCO degree compared with controls. Meanwhile, higher IOP and
corneal edema was observed in groups with a higher RAd-K6W-Ub virus dosage. The
expression of α-SMA was down-regulated in the RAd-K6W-Ub eyes as compared to
controls at the 15th day after injection. Cell migration was inhibited by RAd-K6W-Ub
infection.
Conclusions: RAd-K6W-Ub at an appropriate dosage could inhibit the proliferation of
LECs and the formation of PCO in rabbit models. However, a higher dosage of Rad-
K6W-Ub viral vector caused toxic effects to the surrounding tissues, such as corneal
edema and high IOP.
