Matrix Metalloproteinases and Glaucoma Treatment.

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that degrade extracellular matrix (ECM) components such as collagen and have important roles in multiple biological processes, including development and tissue remodeling, both in health and disease. The activity of MMPs is influenced by the expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). In the eye, MMP-mediated ECM turnover in the juxtacanalicular region of the trabecular meshwork (TM) reduces outflow resistance in the conventional outflow pathway and helps maintain intraocular pressure (IOP) homeostasis. An imbalance in the MMP/TIMP ratio may be involved in the elevated IOP often associated with glaucoma. The prostaglandin analog/prostamide (PGA) class of topical ocular hypotensive medications used in glaucoma treatment reduces IOP by increasing outflow through both conventional and unconventional (uveoscleral) outflow pathways. Evidence from in vivo and in vitro studies using animal models and anterior segment explant and cell cultures indicates that the mechanism of IOP lowering by PGAs involves increased MMP expression in the TM and ciliary body, leading to tissue remodeling that enhances conventional and unconventional outflow. PGA effects on MMP expression are dependent on the identity and concentration of the PGA. An intracameral sustained-release PGA implant (Bimatoprost SR) in development for glaucoma treatment can reduce IOP for many months after expected intraocular drug bioavailability. We hypothesize that the higher concentrations of bimatoprost achieved in ocular outflow tissues with the implant produce greater MMP upregulation and more extensive, sustained MMP-mediated target tissue remodeling, providing an extended duration of effect

Differential response and withdrawal profile of glucocorticoid-treated human trabecular meshwork cells

The goal of the study was to examine secreted protein response and withdrawal profiles from cultured human trabecular meshwork (HTM) cells following short- and long-term glucocorticoid treatment. Primary cultures of five human HTM cell strains isolated from 5 different individual donor eyes were tested. Confluent HTM cells were differentiated in culture media containing 1% FBS for at least one week, and then treated with Dexamethasone (Dex, 100 nM) 3 times/week for 1 or 4 weeks. Cell culture supernatant was collected 3 times per week for 8 weeks. Secretion profiles of myocilin (MYOC), matrix metalloproteinase-2 (MMP2) and fibronectin (FN) were determined by Western blot analysis and MMP2 activity by zymography. Dex treatment reduced MMP2 expression and activity, returning to normal levels shortly after Dex withdrawal in 5 HTM cell strains. All five cell strains significantly upregulated MYOC in response to Dex treatment by an average of 17-fold, but recovery to basal levels after Dex withdrawal took vastly different periods of time depending on cell strain and treatment duration. Dex treatment significantly increased FN secretion in all strains but one, which decreased FN secretion in the presence of Dex. Interestingly, secretion of FN and MYOC negatively correlated during a 4 week recovery period following 4 weeks of Dex treatment. Taken together, the time course and magnitude of response and recovery for three different secreted, extracellular matrix-associated proteins varied greatly between HTM cell strains, which may underlie susceptibility to glucocorticoid-induced ocular hypertension

L-DOPA Is an Endogenous Ligand for OA1

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of β-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation

Localization of aquaporin CHIP in the human eye: implications in the pathogenesis of glaucoma and other disorders of ocular fluid balance

PURPOSE. The existence of integral membrane proteins that serve as selective water channels has been postulated to explain the movement of water across plasma membranes. Aquaporin CHIP (channel-forming integral membrane protein of 28 kd) is the first such channel to be characterized and is abundant in human erythrocytes and a variety of secretory and absorptive epithelia of the rat. Because disturbances in the movement of water characterize several ocular diseases, the distribution of CHIP in the human eye was studied. METHODS. Affinity-purified antibodies against purified CHIP protein were used for the indirect immunofluorescence localization of CHIP in human eye structures. Labeling was confirmed by immunoblot analyses of membrane preparations from eye structures. RESULTS. CHIP immunolabeling was found in the corneal endothelium, the lens epithelium, the nonpigmented epithelium of the ciliary process, the iris epithelium, and the endothelium of the trabecular meshwork and the canal of Schlemm. CONCLUSIONS. The presence of CHIP water channels in the secretory and absorptive tissues of the human eye provides a mechanism for transcellular water movement and may be important for understanding diseases of the eye that involve excess or insufficient movement of ocular fluid such as glaucoma, cataracts, and Fuch's dystrophy. In addition, the existence of CHIP in the outflow pathways of the human eye provides a novel explanation for the movement of water out of the eye

Distribution of Gold Nanoparticles in the Anterior Chamber of the Eye after Intracameral Injection for Glaucoma Therapy

In glaucoma therapy, nanoparticles (NPs) are a favorable tool for delivering drugs to the outflow tissues of the anterior chamber of the eye where disease development and progression take place. In this context, a prerequisite is an efficient enrichment of NPs in the trabecular meshwork with minimal accumulation in off-target tissues such as the cornea, lens, iris and ciliary body. We evaluated the optimal size for targeting the trabecular meshwork by using gold NPs of 5, 60, 80 and 120 nm with a bare surface (AuNPs) or coated with hyaluronic acid (HA-AuNPs). NPs were compared regarding their colloidal stability, distribution in the anterior chamber of the eye ex vivo and cellular uptake in vitro. HA-AuNPs demonstrated an exceptional colloidal stability. Even after application into porcine eyes ex vivo, the HA coating prevented an aggregation of NPs inside the trabecular meshwork. NPs with a diameter of 120 nm exhibited the highest volume-based accumulation in the trabecular meshwork. Off-target tissues in the anterior chamber demonstrated an exceptionally low gold content. Our findings are particularly important for NPs with encapsulated anti-glaucoma drugs because a higher particle volume would be accompanied by a higher drug payload

Visualization of conventional outflow tissue responses to netarsudil in living mouse eyes

AbstractVisual impairment due to glaucoma currently impacts 70 million people worldwide. While disease progression can be slowed or stopped with effective lowering of intraocular pressure, current medical treatments are often inadequate. Fortunately, three new classes of therapeutics that target the diseased conventional outflow tissue responsible for ocular hypertension are in the final stages of human testing. The rho kinase inhibitors have proven particularly efficacious and additive to current therapies. Unfortunately, non-contact technology that monitors the health of outflow tissue and its response to conventional outflow therapy is not available clinically. Using optical coherence tomographic (OCT) imaging and novel segmentation software, we present the first demonstration of drug effects on conventional outflow tissues in living eyes. Topical netarsudil (formerly AR-13324), a rho kinase/ norepinephrine transporter inhibitor, affected both proximal (trabecular meshwork and Schlemm’s Canal) and distal portions (intrascleral vessels) of the mouse conventional outflow tract. Hence, increased perfusion of outflow tissues was reliably resolved by OCT as widening of the trabecular meshwork and significant increases in cross-sectional area of Schlemm’s canal following netarsudil treatment. These changes occurred in conjunction with increased outflow facility, increased speckle variance intensity of outflow vessels, increased tracer deposition in conventional outflow tissues and decreased intraocular pressure. This is the first report using live imaging to show real-time drug effects on conventional outflow tissues and specifically the mechanism of action of netarsudil in mouse eyes. Advancements here pave the way for development of a clinic-friendly OCT platform for monitoring glaucoma therapy

Fasudil Loaded PLGA Microspheres as Potential Intravitreal Depot Formulation for Glaucoma Therapy

Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm's canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 mu m. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (similar to 2.4-fold), SC cells (similar to 1.4-fold) and fibroblasts (similar to 1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases