Invest Ophthalmol Vis Sci. 2016 Nov 1;57(14):5984-5995. doi: 10.1167/iovs.16-19734.

Aberrant Collagen Composition of the Trabecular Meshwork Results in Reduced Aqueous Humor Drainage and Elevated IOP in MMP-9 Null Mice

Lies De Groef,1,2 Lien Andries,1 Anuja Siwakoti,3 Emiel Geeraerts,1 Ilse Bollaerts,1 Lut Noterdaeme,1 Isabelle Etienne,4 Anna-Pia Papageorgiou,5,6 Ingeborg Stalmans,2 Johan Billen,7 Judith A. West-Mays,3 and  Lieve Moons1

 

1Laboratory of Neural Circuit Development and Regeneration, Animal Physiology and Neurobiology Section, Department of Biology, KU Leuven, Leuven, Belgium

2Laboratory of Ophthalmology, Department of Neurosciences, KU Leuven, Leuven, Belgium

3Department of Pathology and Molecular Medicine, McMaster University Health Science Centre, Hamilton, Ontario, Canada

4ThromboGenics NV, Heverlee, Belgium

5Centre for Molecular and Vascular Biology (CMVB), Department of Cardiovascular Sciences, KU Leuven, Leuven, Belgium

6Cardiovascular Research Institute Maastricht (CARIM), Maastricht, The Netherlands

7Laboratory of Socioecology and Social Evolution, Ecology, Evolution and Biodiversity Conservation Section, Department of Biology, KU Leuven, Leuven, Belgium

Correspondence: Lieve Moons, Research Group Neural Circuit Development and Regeneration, Animal Physiology and Neurobiology Section, Department of Biology, KU Leuven, Naamsestraat 61 Box 2464, B-3000 Leuven, Belgium, lieve.moons@kuleuven.be

 

Abstract

Purpose – Homeostatic turnover of the trabecular meshwork extracellular matrix (ECM) is essential to regulate aqueous humor outflow and to maintain intraocular pressure homeostasis. In this study, we evaluated aqueous humor turnover, intraocular pressure, and trabecular meshwork organization in MMP-9 null mice.

Methods – Intraocular pressure and aqueous humor turnover were measured in MMP-9 null versus wild-type mice. Morphology of the anterior segment of the eye, with special attention to the structural organization of the trabecular meshwork, was investigated by means of optical coherence tomography, light microscopy, and transmission electron microscopy. Furthermore, using quantitative real-time polymerase chain reaction and immunostainings, we evaluated the ECM composition of the trabecular meshwork. Finally, the integrity and function of the retina and optic nerve were assessed, via optical coherence tomography, histologic techniques, and optomotor testing.

Results – MMP-9 null mice displayed early-onset ocular hypertension and reduced aqueous humor turnover. While transmission electron microscopic analysis did not reveal any abnormalities in the cellular organization of the trabecular meshwork, detailed investigation of collagen expression indicated that there is an aberrant trabecular meshwork ECM composition in MMP-9 null mice. Notably, at the age of 13 months, no glaucomatous neurodegeneration was seen in MMP-9 null mice.

Conclusions – Our observations corroborate MMP-9 as an important remodeler of the collagenous composition of the trabecular meshwork and provide evidence for a causal link between MMP-9 deficiency, trabecular meshwork ultrastructure, and ocular hypertension.

 

Supplement

1. Challenges for glaucoma treatment

In 2013, 64.3 million people suffered from glaucomatous optic neuropathies, making it the second most important cause of irreversible blindness. This number is predicted to rise to 76-80 million in 2020. By 2040, an increase in glaucoma prevalence of 74% (as compared to 2013) is foreseen, and 111.8 million people, aged 40 to 80 years, will suffer from glaucoma (Quigley and Broman 2006, Tham, Li et al. 2014). By then, Asia will contain the largest number of primary open angle glaucoma and primary angle-closure glaucoma patients, followed by Africa. The projected increase in the worldwide number of glaucoma patients largely results from the expected ageing of the world population, which affects some regions (e.g. Asia, Africa) more than others.

Glaucomatous neurodegeneration is thought to be inflicted by a combination of genetic predisposition and age-related and environmental stressors (Leske, Heijl et al. 2007, Calkins and Horner 2012). The most prevalent and important risk factor, as well as the sole target for clinical intervention, is ocular hypertension. First-line treatment consists of topical administration of intraocular pressure (IOP) lowering medications. In case these are ineffective or not well-tolerated, laser trabeculoplasty or filtration surgery (generally trabeculectomy) may be used to increase aqueous humor drainage. Although many patients benefit from IOP lowering therapies, these do not always succeed to stop the gradual worsening of visual function, and some patients continue to lose vision in spite of all current treatments (Heijl, Leske et al. 2002, Iwase, Suzuki et al. 2004). Moreover, glaucoma results in irreversible vision loss, and even when IOP is successfully lowered, disease progression is halted yet not reversed. This emphasizes the need for novel therapeutic strategies to stop and reverse glaucomatous neurodegeneration.

 

2. Matrix metalloproteinases in the trabecular meshwork

Although glaucoma presents as a neurodegenerative disorder affecting RGC axons in the optic nerve and their somata in the retina, the elicitors of this optic neuropathy are often located outside the neuroretina. Disturbances in aqueous humor outflow, leading to ocular hypertension, are considered the major risk factor for the development of glaucoma.

The IOP is the result of the continuous production of aqueous humor by the ciliary body into the posterior chamber, and the continuous aqueous humor outflow from the anterior chamber. Microscopic examination reveals that the trabecular meshwork is a filter-like tissue, composed of fenestrated beams through which aqueous humor flows to exit the anterior chamber. Importantly, the trabecular meshwork extracellular matrix (ECM) is highly dynamic, well-hydrated and composed of many bio-active molecules that influence outflow resistance, as such it provides a conduit for aqueous humor and determines the outflow resistance and thereby IOP. In fact, the abundant ECM in the trabecular meshwork is generally considered to be the source of aqueous humor outflow resistance (Flammer, Orgül et al. 2002, Wierzbowska, Robaszkiewicz et al. 2010, Calkins and Horner 2012).

Multiple surveys in glaucoma patients, as well as in vitro studies in anterior segment explant or cell cultures, reported changes in the expression and activity of several matrix metalloproteinases (MMPs) in the aqueous humor and trabecular meshwork, in response to elevated IOP. Indeed, MMPs are well-known for their ability to cleave and remodel the ECM and thereby act as crucial regulators of trabecular meshwork ultrastucture and aqueous humor outflow resistance (De Groef, Van Hove et al. 2013). Their role is believed to be part of a complex equilibrium of ECM biosynthesis versus proteolysis in the trabecular meshwork, and changes in aqueous humor outflow resistance (i.e., IOP) might be the result of qualitative alterations in ECM composition, rather than quantitative differences (De Groef, Van Hove et al. 2013). Notably, although beyond the scope of this study, MMPs can also modulate cell-cell and cell-ECM communication, (in)activation of growth factors, cytokines and cell adhesion molecules, and influence on various signaling cascades regulating mobility, proliferation, survival, etc.

 

3. MMPs as targets for IOP lowering glaucoma therapies

Current IOP lowering medications aim to either reduce aqueous humor production or increase the drainage of excess aqueous humor. The latter is achieved mostly through increased uveoscleral outflow, and to a lesser extent by enhancing drainage via the trabecular meshwork. In other words, these medications do not adequately target the trabecular meshwork, which represents the largest surface area for drainage, and as a result they do not succeed to reduce IOP by more than 25% (Beidoe and Mousa 2012).

In this study, we revealed the importance of MMP-9 during IOP homeostasis and identified MMP-9 as a potential target for novel IOP lowering therapies focusing at the trabecular meshwork. Restoration of MMP-9 activity in the trabecular meshwork of hypertensive eyes could re-establish optimal ECM composition and aqueous outflow resistance, thereby counteracting IOP elevations. Analogous to what has been shown for MMP-1, controlled overexpression of MMP-9 in the trabecular meshwork via gene therapy, might prove an efficient, long-lasting approach to restore the endogenous balance in trabecular ECM turnover. Indeed, MMP-1 viral vector-mediated gene therapy in sheep with ocular hypertension is a convincing proof-of-principle of the high potential of MMP gene therapy to restore self-regulatory ECM turnover in the trabecular meshwork (Gerometta, Spiga et al. 2010, Spiga and Borras 2010). Moreover, the success of viral vector-mediated gene therapy in patients with, e.g., Leber’s hereditary optic neuropathy, Leber’s congenital amaurosis, and Usher syndrome (Bainbridge, Mehat et al. 2015, Lopes and Williams 2015, Feuer, Schiffman et al. 2016), as well as the dozens of clinical trials using viral vector-mediated gene therapy to treat ocular diseases that are currently ongoing (JournalofGeneMedicine 2017), indicate that this approach could be taken to the clinic within a reasonable time frame. Alternatively, as evidence points out that ocular hypertension could be treated by increasing MMP activity, MMP balance in the trabecular meshwork could also be modulated via MMP activating drugs, e.g. tissue plasminogen activator (tPA) and plasmin, which both have already been approved for other indications in the eye (Tripathi and Tripathi 2005, Tsui, Pan et al. 2012, Gerometta, Kumar et al. 2013, Candia, Gerometta et al. 2014).

 

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