Evaluation of Adenovirus-Mediated Down-Regulation of Connective Tissue Growth Factor on Postoperative Wound Healing After Experimental Glaucoma Surgery

ABSTRACT

Purpose: This study was aimed to determine whether adenovirus-mediated down-regulation of connective tissue growth factor (CTGF) can modulate postoperative scarring in a rabbit receiving simplified glaucoma surgery.

Methods: In vitro studies were performed using a replication-deficient recombinant adenovirus that transcribes a small interfering RNA (siRNA) specific to the CTGF gene under the control of the modified CMV promoter. Primary tenon cells from a New Zealand White rabbit were transduced with 10–100 plaque-forming units (pfu) per cell of the viral vector. Seventy-two hours later, CTGF expression was analyzed by Western blot analysis. In vivo studies were conducted using 10 New Zealand White rabbits, which underwent simplified glaucoma surgery and received a postoperative subconjunctival injection of 5 µl suspension of adenovirus carrying shRNA for CTGF (2 × 10¹¹ pfu/ml) in the right eye, and the same amount of null virus in the left eye. Eyes were enucleated 5 d after the surgery, and immunohistochemical and histological examinations of the surgical outcome were performed.

Results: Western blot analysis showed that CTGF was depleted to less than 10% of its original level in cells transduced with the adenovirus expressing CTGF-specific siRNA. This demonstrates RNA interference (RNAi)-mediated CTGF inactivation in vitro. Immunohistochemical analysis also showed that CTGF was significantly depleted in eyes transduced with the adenovirus expressing CTGF siRNA. This demonstrates RNAi-mediated CTGF inactivation in vivo. In addition, less scar tissue was observed on histological evaluation in the transduced eyes, demonstrating that inhibition of CTGF expression can modulate the wound healing process after surgery.

Conclusions: Down-regulation of CTGF is effective in inhibiting postoperative scarring in vivo. This suggests that RNAi with CTGF siRNA may potentially pave the road for a novel therapeutic strategy to improve glaucoma surgery results.

KEYWORDS:

• Adenovirus
• connective tissue growth factor
• fibrosis
• gene therapy
• glaucoma
• glaucoma surgery
• wound healing

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (NRF-20120R1A1A2004976).