MRP4-mediated regulation of intracellular cAMP and cGMP levels in trabecular meshwork cells and homeostasis of intraocular pressure

PURPOSE. Multidrug, resistance-associated protein-4 (MRP4) is a membrane transporter that regulates the cellular efflux of cyclic nucleotides (cAMP and cGMP) involved in various physiologic responses. This study examined the expression and distribution of MRP4 in the trabecular meshwork (TM) cells and its role in homeostasis of IOP. METHODS. Expression and distribution of MRP4 in human TM (HTM) cells and aqueous humor (AH) outflow pathway was determined by RT-PCR, immunoblotting, and immunofluorescence. Effects of inhibiting MRP4 activity and suppression of MRP4 expression on cAMP and cGMP levels, myosin light chain (MLC) phosphorylation, actin filament organization and activity of protein kinase G (PKG), protein kinase A (PKA), Rho guanosine triphosphatase (GTPase), and MLC phosphatase was monitored in HTM cells using ELISA, siRNA, biochemical, and immunofluorescence analyses. Topical application of the MRP4 inhibitor MK571 was tested to assess changes in IOP in rabbits. RESULTS. RT-PCR, immunoblot, and immunofluorescence analyses confirmed the expression of MRP4 in HTM cells and distribution in human AH outflow pathway. Inhibition of MRP4 in HTM cells by MK571 or probenecid resulted in cell shape changes and decreases in actin stress fibers and MLC phosphorylation. Levels of intracellular cAMP and cGMP in HTM cells were increased significantly under these conditions. MK571-induced HTM cell relaxation appeared to be mediated predominantly via activation of the cGMP-dependent PKG signaling pathway. Topical application of MK571 significantly decreased IOP in Dutch-Belted rabbits. CONCLUSIONS. These observations reveal that cyclic nucleotide efflux controlling transporter-MRP4 plays a significant role in IOP homeostasis potentially by regulating the relaxation characteristics of AH outflow pathway cells. (Invest Ophthalmol Vis Sci. 2013;54:1636-1649) DOI:10.1167/iovs.12-11107 G laucoma is an optic neuropathy accounting for the second leading cause of blindness in the world. Global estimates indicate that over 60 million people currently suffer from glaucomatous neuropathy, which, if not treated adequately and in a timely manner, can result in irreversible blindness in many of these patients. 1 POAG, which is the most prevalent type of glaucoma, is commonly associated with elevated IOP caused by impaired drainage of aqueous humor (AH). 2,3 Importantly, elevated IOP is a primary risk factor for POAG. 2,3 IOP is determined by the balance between secretion of AH by the nonpigmented ciliary epithelium and its drainage from the eye anterior chamber via both the conventional and nonconventional routes. 2,3 The conventional outflow pathway consists of the trabecular meshwork (TM) and Schlemm's canal (SC) and accounts for over 80% of total AH drainage. 2-4 It is generally believed that impaired AH outflow through the conventional pathway is the main cause for elevated IOP in glaucoma patients, 2-4 however, the molecular and cellular basis for increased resistance to AH outflow remains to be clarified. Therefore, identifying and characterizing molecular mechanisms regulating AH outflow is important and necessary to support the development of novel and targeted therapies for treatment of elevated IOP in glaucoma patients. 4,5 The contractile and relaxation characteristics, and adhesive interactions of TM cells with the extracellular matrix (ECM), together with the tissue material properties of TM, are considered to be attributes that influences AH outflow via the conventional pathway. 5-10 Support for this speculation derives from observations indicating that activation and inhibition of contractile activity of TM cells by actomyosin cytoskeletal integrity, myosin II phosphorylation, and ECM organization reciprocally influence AH outflow and IOP in various model systems. 5,7-10 Additionally, various intracellular signaling responses mediated by protein kinase C, Rho/Rho kinase, myosin light chain (MLC) kinase, extracellular signalregulated kinase (ERK kinase), Wnt and calcium have also been demonstrated to modulate AH outflow and IOP. 21,23 TM cells and tissues of the AH outflow pathway have been demonstrated to express both the cyclases and phosphodiesterases and they have been reported to participate in modulation of AH outflow in different species

Current Best Clinical Practices—Management of Retinal Vein Occlusion

Retinal vein occlusion (RVO) is the second most common cause of vision loss from retinal vascular diseases in adults in the United States. Visual loss arises as a result of a host of factors, including macular ischemia and macular edema. Primary antivascular endothelial growth factor therapy is the current standard of care, with level I evidence demonstrating sustained visual gains up to 2 years after treatment in both branch and central RVO. Prompt antivascular endothelial growth factor therapy is important because delays in treatment yield lesser visual gains. Steroid therapy also improves visual outcomes in RVO but with higher rates of adverse effects, including cataract formation and ocular hypertension. Although the treatment burden can be high, these drugs have collectively revolutionized treatment outcomes in this disease state, providing improved visual outcomes over previous laser therapies

Characterization of Cytoskeleton-Enriched Protein Fraction of the Trabecular Meshwork and Ciliary Muscle Cells

This study reveals that the activity of non-muscle myosin II, a critical molecule of cellular contraction, was found to be regulated differentially in TM and CM cells by the Rho kinase and the MLCK pathways despite their compositional similarity in cytoskeletal protein profile

AIDS-related Cryptococcus neoformans choroiditis

We describe a case of Cryptococcal choroiditis in a person with advanced HIV/AIDS. A 29-year-old male with AIDS presented with fever, photophobia, and ataxia secondary to cryptococcal and toxoplasma meningoencephalitis. Dilated fundoscopic examination revealed bilateral and multifocal posterior infiltrates consistent with cryptococcal choroiditis. Treatment with parenteral and intravitreal liposomal amphotericin B, oral flucytosine, and oral trimethoprim-sulfamethoxazole led to resolution of his symptoms and improvement in his vision. Our case documents a rare, intraocular opportunistic infection and highlights the importance of ophthalmologic examination in immunocompromised hosts with visual symptoms and invasive fungal infection. We discuss diagnostic and treatment considerations in cryptococcal choroiditis

Randomized trial of bilateral gene therapy injection for m.11778G>A MT-ND4 Leber optic neuropathy

Leber hereditary optic neuropathy (LHON) is an important example of mitochondrial blindness with the m.11778G>A mutation in the MT-ND4 gene being the most common disease-causing mtDNA variant worldwide. The REFLECT phase 3 pivotal study is a randomized, double-masked, placebo-controlled trial investigating the efficacy and safety of bilateral intravitreal injection of lenadogene nolparvovec in patients with a confirmed m.11778G>A mutation, using a recombinant adeno-associated virus vector 2, serotype 2 (rAAV2/2-ND4). The first-affected eye received gene therapy; the fellow (affected/not-yet-affected) eye was randomly injected with gene therapy or placebo. The primary end point was the difference in change from baseline of best-corrected visual acuity (BCVA) in second-affected/not-yet-affected eyes treated with lenadogene nolparvovec versus placebo at 1.5 years post-treatment, expressed in logarithm of the minimal angle of resolution (LogMAR). Forty-eight patients were treated bilaterally and 50 unilaterally. At 1.5 years, the change from baseline in BCVA was not statistically different between second-affected/not-yet-affected eyes receiving lenadogene nolparvovec and placebo (primary end point). A statistically significant improvement in BCVA was reported from baseline to 1.5 years in lenadogene nolparvovec-treated eyes: -0.23 LogMAR for the first-affected eyes of bilaterally treated patients (P < 0.01); and -0.15 LogMAR for second-affected/not-yet-affected eyes of bilaterally treated patients and the first-affected eyes of unilaterally treated patients (P < 0.05). The mean improvement in BCVA from nadir to 1.5 years was -0.38 (0.052) LogMAR and -0.33 (0.052) LogMAR in first-affected and second-affected/not-yet-affected eyes treated with lenadogene nolparvovec, respectively (bilateral treatment group). A mean improvement of -0.33 (0.051) LogMAR and -0.26 (0.051) LogMAR was observed in first-affected lenadogene nolparvovec-treated eyes and second-affected/not-yet-affected placebo-treated eyes, respectively (unilateral treatment group). The proportion of patients with one or both eyes on-chart at 1.5 years was 85.4% and 72.0% for bilaterally and unilaterally treated patients, respectively. The gene therapy was well tolerated, with no systemic issues. Intraocular inflammation, which was mostly mild and well controlled with topical corticosteroids, occurred in 70.7% of lenadogene nolparvovec-treated eyes versus 10.2% of placebo-treated eyes. Among eyes treated with lenadogene nolparvovec, there was no difference in the incidence of intraocular inflammation between bilaterally and unilaterally treated patients. Overall, the REFLECT trial demonstrated an improvement of BCVA in LHON eyes carrying the m.11778G>A mtDNA mutation treated with lenadogene nolparvovec or placebo to a degree not reported in natural history studies and supports an improved benefit/risk profile for bilateral injections of lenadogene nolparvovec relative to unilateral injections