May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
rAd-p21WAF1/Cip1 Gene Therapy to Prevent Glaucoma Surgery Failure: Effects on the Proliferative and Remodeling Phases of the Wound Healing Cascade
Author Affiliations & Notes
  • B.A. Faha
    Pharmacology, Canji, Inc., San Diego, CA, United States
  • R. Nickells
    Ophthal. & Visual Sci., Univ. of Wisc., Madison, WI, United States
  • I. Atencio
    Ophthal. & Visual Sci., Univ. of Wisc., Madison, WI, United States
  • Footnotes
    Commercial Relationships  B.A. Faha, Canji, Inc. E; R. Nickells, Canji, Inc. C; I. Atencio, Canji, Inc. E.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1219. doi:
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      B.A. Faha, R. Nickells, I. Atencio; rAd-p21WAF1/Cip1 Gene Therapy to Prevent Glaucoma Surgery Failure: Effects on the Proliferative and Remodeling Phases of the Wound Healing Cascade . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1219.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Failure of glaucoma surgery is due to the proliferation of conjunctival ocular fibroblasts and subsequent scarring. We have shown that adjunctive treatment with rAd-p21, a recombinant adenovirus encoding p21WAF1/Cip1, inhibited wound healing in rabbit and monkey models of filtration surgery (Perkins et al., 2002; ARVO abstract #1940, 2002). The purpose of this study was to characterize further the effects of rAd-p21on conjunctival fibroblasts 1) in vitro and 2) by in situ analyses of the surgical site in rabbits. Methods: Primary human Tenon’s fibroblasts were grown to confluence and treated with rAd-p21. Culture supernatants and cell lysates were analyzed for pro-collagen type I C-peptide (PIP) by ELISA. Rabbits underwent filtration surgery with intraoperative administration via a Weck-Cel sponge soaked with saline, MMC (0.4 mg/ml), or rAd-p21 (7 x 1010 particles). Animals were sacrificed on days 1, 3, 7, and 14 after surgery (n = 3/group/timept.). Twenty-four hours prior to sacrifice animals received 50 mg/kg BrdU intraperitoneally. The surgical site was analyzed by immunohistochemistry (IHC) for p21, Mib-1 (Ki-67), BrdU, and smooth muscle actin on serial sections. A quantitative assessment of BrdU incorporation was performed by counting the # positive conjunctival fibroblasts compared to the total # fibroblasts. Results: Treatment of Tenon’s fibroblasts with rAd-p21 resulted in an inhibition of PIP production and secretion compared to control adenovirus, which is consistent with previous studies showing reduced collagen deposition in vivo (Perkins et al., 2002). In rabbits, IHC showed that rAd-p21 treatment reduced BrdU and Mib-1 staining in p21 positive areas adjacent to the sclerectomy site. Peak BrdU incorporation occurred 3 days post surgery in saline treated eyes, with ~30% of fibroblasts staining positive, which declined to ~3% by day 7 with no incorporation on day 14. Treatment with rAd-p21 inhibited BrdU incorporation on day 3 by ~95%, with only 1.3% cells positive. Treatment with MMC significantly inhibited BrdU incorporation, however, severe tissue effects were evident. Qualitatively, reduced SMA staining on day 7 was observed in rAd-p21 treated eyes compared to control. Conclusion: These results demonstrate the p21-specific inhibition of 1) fibroproliferation in situ following filtration surgery and 2) inhibition of pro-collagen type I C-peptide production, a collagen precursor molecule. These data suggest that rAd-p21 inhibits wound healing by preventing cell division and subsequent ECM deposition.

Keywords: gene transfer/gene therapy • wound healing • adenovirus 
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