Novel therapies for diseases of the ocular surface

Author: Yazad Irani

Irani, Yazad, 2015 Novel therapies for diseases of the ocular surface, Flinders University, School of Medicine

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Ocular surface disease and corneal neovascularization can lead to visual impairment. The principal aims of the work described in this thesis were to develop novel treatments for such conditions, and to assess their efficacy in rat models of disease.

Ocular surface disease results from loss of or damage to, corneal epithelial stem cells or their niche. Oral mucosal epithelial cells were harvested from male inbred Sprague-Dawley rats and cultured on porous silicon membrane scaffolds, coated with factors to replicate the stem cell niche. The cells were characterised, and found to express epithelial cell markers CK3 and CK19, as well as the putative stem cell markers p63 and ABCG2. Ocular surface disease was induced in female inbred Sprague-Dawley rats by n-heptanol debridement. Porous silicon scaffolds loaded with oral mucosal cells were implanted subconjunctivally. Cell samples were taken from the ocular surface and the presence of transplanted cells was detected by PCR for the male sry gene. Despite successful transfer of cells to the ocular surface, transplanted cells were not detected on the central cornea after 8 weeks, indicating daughter cells had not migrated over the ocular surface.

A novel composite of porous silicon and polycaprolactone was then developed as an improved dual purpose ophthalmic implant, to deliver drugs and cells to the eye. The composite biomaterial exhibited some characteristics, including flexibility and increased stability, which made it more suitable than porous silicon membrane for ocular implantation. The loading and subsequent release of a small molecule (fluorescein diacetate) and biologic growth factors (epithelial growth factor, insulin and transferrin) in active form were demonstrated. Further, the composite supported attachment and growth of mammalian cells, and displayed acceptable biocompatibility when implanted in the eye of live rats. The material may be of use as an artificial stem cell niche for the transfer of epithelial stem cells to the cornea.

The normal cornea is avascular. Corneal neovascularization results from an imbalance in pro- and anti-angiogenic factors. Anti-vascular endothelial growth factor A (VEGF-A) therapy has shown promise in reducing progression of neovascularization in humans but has little effect on established vessels. The related molecule VEGF-B is a survival factor for endothelial cells. A recombinant antibody fragment reactive with human, rat and mouse VEGF-B was engineered from an established hybridoma. Corneal neovascularization was induced in Sprague-Dawley rats by silver nitrate cautery and the anti-VEGF-B antibody fragment or a control fragment was administered as eye drops or by subconjunctival injection. Treatment extended for 14 days either immediately, to determine the effect on growing vessels, or for 14 days after cautery to treat established vessels, at which time rats were killed, perfused with haematoxylin, and the corneal flatmounts imaged to quantify vascularization. Topical treatment neither prevented growth of vessels nor caused regression of established vessels. However, subconjunctival administration of the anti-VEGF-B antibody fragment significantly reduced the neovascular area of established vessels. Anti-VEGF-B biologics may thus hold promise for the regression of established corneal vessels.

Keywords: Ocular surface disease, Corneal neovascularization, VEGF-B, Stem Cells, Limbal stem cell deficiency

Subject: Ophthalmology thesis

Thesis type: Doctor of Philosophy
Completed: 2015
School: School of Medicine
Supervisor: Prof. Keryn Williams