Therapeutic potential of AAV-mediated MMP-3 secretion from corneal endothelium in treating glaucoma.

Intraocular pressure (IOP) is maintained as a result of the balance between production of aqueous humour (AH) by the ciliary processes and hydrodynamic resistance to its outflow through the conventional outflow pathway comprising the trabecular meshwork (TM) and Schlemm's canal (SC). Elevated IOP, which can be caused by increased resistance to AH outflow, is a major risk factor for open-angle glaucoma. Matrix metalloproteinases (MMPs) contribute to conventional aqueous outflow homeostasis in their capacity to remodel extracellular matrices, which has a direct impact on aqueous outflow resistance and IOP. We observed decreased MMP-3 activity in human glaucomatous AH compared to age-matched normotensive control AH. Treatment with glaucomatous AH resulted in significantly increased transendothelial resistance of SC endothelial and TM cell monolayers and reduced monolayer permeability when compared to control AH, or supplemented treatment with exogenous MMP-3.Intracameral inoculation of AAV-2/9 containing a CMV-driven MMP-3 gene (AAV-MMP-3) into wild type mice resulted in efficient transduction of corneal endothelium and an increase in aqueous concentration and activity of MMP-3. Most importantly, AAV-mediated expression of MMP-3 increased outflow facility and decreased IOP, and controlled expression using an inducible promoter activated by topical administration of doxycycline achieved the same effect. Ultrastructural analysis of MMP-3 treated matrices by transmission electron microscopy revealed remodelling and degradation of core extracellular matrix components. These results indicate that periodic induction, via use of an eye drop, of AAV-mediated secretion of MMP-3 into AH could have therapeutic potential for those cases of glaucoma that are sub-optimally responsive to conventional pressure-reducing medications.

Age-related changes in eye morphology and aqueous humor dynamics in DBA/2J mice using contrast-enhanced ocular MRI.

PURPOSE: Here, we are testing the hypothesis that dynamic contrast enhanced MRI (DCE-MRI) is a useful approach for non-invasively evaluating age-related changes in aqueous humor outflow and its contribution to elevated intraocular pressure in the DBA/2J model of pigmentary glaucoma. METHODS: A rodent-specific 7 T MRI was used to assess eye anatomy (anterior chamber (AC) and vitreous body (VB) morphology, eye size, lens size) and aqueous humor dynamics (via intravenous administration of Gd-DTPA and Gd-BOPTA contrast agents) in C57BL/6 and DBA/2J mice at 3 and 9 months of age. RESULTS: Gd-MRI was used to demonstrate an anterior solute pathway into the mouse AC. Topical latanoprost treatment in C57BL/6J mice reduced Gd-BOPTA accumulation in the AC. Age-related increases in AC area, AC depth and eye size were observed in DBA/2J mice compared to C57BL/6J mice. The rate of Gd-DTPA accumulation and peak Gd-DTPA intensity was lowest in 9-month old DBA/2J mice compared to 3-month old DBA/2J mice and C57BL/6J mice at both ages. Leakage of Gd-DTPA posteriorly into the VB was also observed in 9-month old DBA/2J mice. CONCLUSIONS: These studies support the idea that age-related changes in aqueous humor outflow contribute to elevated intraocular pressure (IOP) in the DBA/2J model of pigmentary glaucoma. Gd-MRI is a valuable tool for better understanding of mechanisms and dynamics of aqueous humor circulation in normal and glaucomatous mouse eyes or following topical administration of medicines to reduce IOP.

Manipulating ocular endothelial tight junctions: Applications in treatment of retinal disease pathology and ocular hypertension.

Protein levels of endothelial tight-junctions of the inner retinal microvasculature, together with those of Schlemm's canal, can be readily manipulated by RNA interference (RNAi), resulting in the paracellular clefts between such cells to be reversibly modulated. This facilitates access to the retina of systemically-deliverable low molecular weight, potentially therapeutic compounds, while also allowing potentially toxic material, for example, soluble Amyloid-ß1-40, to be removed from the retina into the peripheral circulation. The technique has also been shown to be highly effective in alleviation of pathological cerebral oedema and we speculate that it may therefore have similar utility in the oedematous retina. Additionally, by manipulating endothelial tight-junctions of Schlemm's canal, inflow of aqueous humour from the trabecular meshwork into the Canal can be radically enhanced, suggesting a novel avenue for control of intraocular pressure. Here, we review the technology underlying this approach together with specific examples of clinical targets that are, or could be, amenable to this novel form of genetic intervention.

Enhancement of Outflow Facility in the Murine Eye by Targeting Selected Tight-Junctions of Schlemm's Canal Endothelia.

The juxtacanalicular connective tissue of the trabecular meshwork together with inner wall endothelium of Schlemm's canal (SC) provide the bulk of resistance to aqueous outflow from the anterior chamber. Endothelial cells lining SC elaborate tight junctions (TJs), down-regulation of which may widen paracellular spaces between cells, allowing greater fluid outflow. We observed significant increase in paracellular permeability following siRNA-mediated suppression of TJ transcripts, claudin-11, zonula-occludens-1 (ZO-1) and tricellulin in human SC endothelial monolayers. In mice claudin-11 was not detected, but intracameral injection of siRNAs targeting ZO-1 and tricellulin increased outflow facility significantly. Structural qualitative and quantitative analysis of SC inner wall by transmission electron microscopy revealed significantly more open clefts between endothelial cells treated with targeting, as opposed to non-targeting siRNA. These data substantiate the concept that the continuity of SC endothelium is an important determinant of outflow resistance, and suggest that SC endothelial TJs represent a specific target for enhancement of aqueous movement through the conventional outflow system.

Autoregulated paracellular clearance of amyloid-ß across the blood-brain barrier.

The blood-brain barrier (BBB) is essential for maintaining brain homeostasis and protecting neural tissue from damaging blood-borne agents. The barrier is characterized by endothelial tight junctions that limit passive paracellular diffusion of polar solutes and macromolecules from blood to brain. Decreased brain clearance of the neurotoxic amyloid-ß (Aß) peptide is a central event in the pathogenesis of Alzheimer's disease (AD). Whereas transport of Aß across the BBB can occur via transcellular endothelial receptors, the paracellular movement of Aß has not been described. We show that soluble human Aß(1-40) monomers can diffuse across the paracellular pathway of the BBB in tandem with a decrease in the tight junction proteins claudin-5 and occludin in the cerebral vascular endothelium. In a murine model of AD (Tg2576), plasma Aß(1-40) levels were significantly increased, brain Aß(1-40) levels were decreased, and cognitive function was enhanced when both claudin-5 and occludin were suppressed. Furthermore, Aß can cause a transient down-regulation of claudin-5 and occludin, allowing for its own paracellular clearance across the BBB. Our results show, for the first time, the involvement of the paracellular pathway in autoregulated Aß movement across the BBB and identify both claudin-5 and occludin as potential therapeutic targets for AD. These findings also indicate that controlled modulation of tight junction components at the BBB can enhance the clearance of Aß from the brain.