Heterozygous triplication of upstream regulatory sequences leads to dysregulation of matrix metalloproteinase 19 in patients with cavitary optic disc anomaly.

Patients with a congenital optic nerve disease, cavitary optic disc anomaly (CODA), are born with profound excavation of the optic nerve resembling glaucoma. We previously mapped the gene that causes autosomal-dominant CODA in a large pedigree to a chromosome 12q locus. Using comparative genomic hybridization and quantitative PCR analysis of this pedigree, we report identifying a 6-Kbp heterozygous triplication upstream of the matrix metalloproteinase 19 (MMP19) gene, present in all 17 affected family members and no normal members. Moreover, the triplication was not detected in 78 control subjects or in the Database of Genomic Variants. We further detected the same 6-Kbp triplication in one of 24 unrelated CODA patients and in none of 172 glaucoma patients. Analysis with a Luciferase assay showed that the 6-Kbp sequence has transcription enhancer activity. A 773-bp fragment of the 6-Kbp DNA segment increased downstream gene expression eightfold, suggesting that triplication of this sequence may lead to dysregulation of the downstream gene, MMP19, in CODA patients. Lastly, immunohistochemical analysis of human donor eyes revealed strong expression of MMP19 in optic nerve head. These data strongly suggest that triplication of an enhancer may lead to overexpression of MMP19 in the optic nerve that causes CODA.

The Antiangiogenic Effect and Ocular Pharmacology of Novel Modified Nonsteroidal Anti-Inflammatory Drugs in the Treatment of Oxygen-Induced Retinopathy.

Purpose: To evaluate the hypothesis that 3 novel compounds, OXT-328, Q-922, and CL-717 show efficacy in the treatment of oxygen-induced retinopathy (OIR) and whether or not their route of administration is intravitreal, topical, or systemic. Methods: The OIR mouse model, characterized by an avascular area (AVA) and a neovascular area (NVA) of the retina, was used to study retinopathy of prematurity and other retinal diseases characterized by abnormal vessel growth. We measured the effect of our compounds on both the AVA and NVA in whole mounts of mouse retinal tissue. We also evaluated their ability to prevent new vessel formation in chicken chorioallantoic membranes (CAMs). Finally, we measured the in vitro uptake and biodistribution of topically applied CL-717 in human eye explants. Results: In mice with OIR, compared to controls, a single intravitreal administration of Q-922 or OXT-328 significantly reduced both AVA and NVA. CL-717 administered as eye drops over 5 days also reduced AVA and NVA, whereas OXT-328 eye drops had no effect. Q-922 given intraperitoneal (150 mg/kg/day × 5 days) reduced AVA and NVA. Remarkably, explanted human eyes bathed in CL-717 show rapid uptake and biodistribution in ocular tissues. In the chicken CAM model, all 3 compounds reduced the formation of new blood vessels by about one-third. No side effect in mice was observed, except for mild ocular surface irritation with Q-922. Conclusions: Systemic administration of Q-922 or topical administration of CL-717 holds particular promise for a simplified treatment of proliferative retinopathies without the necessity of intravitreal injections.

Animal models of dry eye disease: Useful, varied and evolving (Review).

Dry eye disease (DED), which is a prevalent disease that still lacks successful treatment options, remains a major challenge in ophthalmology. Multiple animal models of DED have been used to decipher its pathophysiology and to develop novel treatments. These models use mice, rats, rabbits, cats, dogs and non-human primates. Each model assesses aspects of DED by focusing on elements of the lacrimal functional unit, which controls the homeostasis of the tear film. The present review outlines representative DED animal models and assesses their contribution to the study of DED. Murine models are the most extensively used, followed by rabbit models; the latter offer the advantage of larger eyes, a favorable biochemical profile for drug studies, experimental ease and relatively low cost, contrasting with non-human primates, which, although closer to humans, are not as accessible and are expensive. No comprehensive 'ideal' animal model encompassing all aspects of human DED exists nor is it feasible. Investigators often choose an animal model based on their experimental needs and the following four features of a given model: The size of the eye, its biochemical composition, the available research reagents and cost. As research efforts in DED expand, more refined animal models are needed to supplement the enormous contribution made to date by existing models.

Phospho-Sulindac (OXT-328) Inhibits Dry Eye Disease in Rabbits: A Dose-, Formulation- and Structure-Dependent Effect.

Purpose: Inflammation of the ocular surface is central to dry eye disease (DED). The anti-inflammatory agent phospho-sulindac (PS) at a high dose was efficacious against DED in a rabbit model. We assessed the dose, formulation and structure dependence of PS's effect. Methods: In rabbits with concanavalin A-induced DED we evaluated a range of PS concentrations (0.05%-1.6%) and dosing frequencies, assessed the duration of its effect with PS in 2 solution formulations and one emulsion formulation, and compared the efficacy of PS to that of sulindac, and of the structurally similar phospho-ibuprofen amide. We determined tear breakup time (TBUT) (tear stability), Schirmer's tear test (tear production), and by esthesiometry corneal sensitivity (symptoms). We also determined the biodistribution in the eye of topically applied PS. Results: PS in a solution formulation, given as eye drops q.i.d. was efficacious starting at a dose of 0.1%. The effect was apparent after 2 days of treatment and lasted at least 8 days after the last dose. Both signs (evidenced by TBUT and Schirmer's test) and symptoms (measured by corneal sensitivity) improved significantly. The best formulation was the solution formulation; a cyclodextrin-based formulation was also successful but the emulsion formulation was not. PS and its metabolites were essentially restricted to the anterior chamber of the eye. Sulindac and phospho-ibuprofen amide had no efficacy on DED. Conclusions: PS is efficacious against DED. Its effect, encompassing signs, and symptoms, are dose, formulation, and structure dependent. PS has therapeutic promise and merits further development.

A Rabbit Model of Aqueous-Deficient Dry Eye Disease Induced by Concanavalin A Injection into the Lacrimal Glands: Application to Drug Efficacy Studies.

Dry eye disease (DED), a multifactorial inflammatory disease of the ocular surface, affects 1 in 6 humans worldwide with staggering implications for quality of life and health care costs. The lack of informative animal models that recapitulate its key features impedes the search for new therapeutic agents for DED. Available DED animal models have limited reproducibility and efficacy. A model is presented here in which DED is induced by injecting the mitogen concanavalin A (Con A) into the orbital lacrimal glands of rabbits. Innovative aspects of this model are the use of ultrasound (US) guidance to ensure optimal and reproducible injection of Con A into the inferior lacrimal gland; injection of Con A into all orbital lacrimal glands that limits compensatory production of tears; and use of periodic repeat injections of Con A that prolong the state of DED at will. DED and its response to test agents are monitored with a panel of parameters that assess tear production, the stability of the tear film, and the status of the corneal and conjunctival mucosa. They include tear osmolarity, tear break-up time, Schirmer's tear test, rose bengal staining, and tear lactoferrin levels. The induction of DED and the monitoring of its parameters are described in detail. This model is simple, robust, reproducible, and informative. This animal model is suitable for the study of tear physiology and of the pathophysiology of DED as well as for the assessment of the efficacy and safety of candidate agents for the treatment of DED.

Establishment of a Severe Dry Eye Model Using Complete Dacryoadenectomy in Rabbits.

Dry eye disease (DED) is a complex disease with multiple etiologies and variable symptoms, having ocular surface inflammation as its key pathophysiologic step. Despite advances in our understanding of DED, significant knowledge gaps remain. Advances are limited in part due to the lack of informative animal models. The authors recently reported on a method of DED induced by injecting all orbital lacrimal gland (LG) tissues with the lectin concanavalin A. Here, we report a novel model of aqueous-deficient DED based on the surgical resection of all orbital LG (dacryoadenectomy) tissues. Both methods use rabbits because of their similarity to human eyes in terms of the size and structure of the ocular surface. One week after removal of the nictitating membrane, the orbital superior LG was surgically removed under anesthesia, followed by removal of the palpebral superior LG, and finally removal of the inferior LG. Dacryoadenectomy induced severe DED, evidenced by a marked reduction in the tear break up time test and the Schirmer's tear test, and significantly increased tear osmolarity and rose bengal staining. Dacryoadenectomy-induced DED lasted at least eight weeks. There were no complications and animals tolerated the procedure well. The technique can be mastered relatively easily by those with adequate surgical experience and appreciation of the relevant rabbit anatomy. Since this model recapitulates the features of human aqueous-deficient DED, it is suitable for studies of ocular surface homeostasis, DED, and candidate therapeutics.

Phosphosulindac is efficacious in an improved concanavalin A-based rabbit model of chronic dry eye disease.

Dry eye disease (DED) currently has no satisfactory treatment partly because of the lack of informative animal models. We evaluated the anti-inflammatory phosphosulindac (PS) for the treatment of DED using a new rabbit model of DED based on the concanavalin A (Con A) acute DED model: we injected all lacrimal glands with Con A weekly under ultrasound guidance, which prolonged DED to >3 weeks, and thoroughly assessed efficacy with tear break-up time (TBUT), tear osmolarity, Schirmer test, and tear lactoferrin levels. Rabbits with DED (n = 8-10 eyes per group) were treated topically with PS or vehicle 3×/day for 21days. PS restored TBUT, tear osmolarity, and lactoferrin levels (P < 0.0001-0.04) to normal but did not significantly improve the results of the Schirmer test. PS showed no side effects and was much more efficacious than cyclosporine or lifitegrast. In the cornea, PS suppressed the activation of nuclear factor kappa-B, the levels of transforming growth factor beta, interleukin-1 beta, interleukin-6, and interleukin-8, and the levels of matrix metalloproteinase (MMP)-1 and MMP-9, and MMP activity. Levels of prostaglandin E2 (PGE2) in tears and cornea were preserved in PS-treated rabbits. Ketorolac and diclofenac, two ophthalmic NSAIDs causing corneal melt, nearly completely suppressed PGE2 levels but had no effect on MMPs. The effects of PS on PGE2 and MMPs likely account for its apparent ocular safety. Our results establish an animal model for acute and chronic DED suitable for drug efficacy studies and indicate that PS merits evaluation for DED.

Familial cavitary optic disk anomalies: clinical features of a large family with examples of progressive optic nerve head cupping.

PURPOSE: To describe a multigenerational family with autosomal dominant inheritance of cavitary optic nerve head (ONH) anomalies and abnormal ONH vasculature. DESIGN: Description of a single family with inherited eye disease. METHODS: A four-generation pedigree was investigated. Examination included visual acuity, slit-lamp biomicroscopy, intraocular pressure (IOP) measurement, and ophthalmoscopy. Visual fields and fundus photography were obtained when possible. RESULTS: Seventeen clinically affected individuals and two obligate carriers were identified. Most (64.7%) affected persons had bilateral involvement. Visual acuity in affected eyes ranged from 20/20 to no light perception. Although the appearance of affected nerves varied greatly, most lacked a well-formed central retinal artery and instead had multiple radial cilioretinal arteries. Prominent cupping was seen in most affected nerves. Four individuals for whom information was available were treated for glaucoma, but none had documented elevated IOP. Four eyes of two patients demonstrated progressive ONH cupping at normal IOPs. Nine (56.3%) of the 16 individuals for whom we had data had evidence of serous macular detachments; five of these had bilateral macular disease. CONCLUSIONS: A large family with autosomal dominant inheritance of cavitary ONH anomalies and abnormal vasculature is presented. Clinical phenotypes varied markedly. Progressive ONH cupping was documented in four eyes of two patients. Genetic linkage analysis of this family has identified the chromosomal location of a gene responsible for ONH development. This may provide insight into the pathogenesis of glaucomatous ONH damage.

Familial cavitary optic disk anomalies: identification of a novel genetic locus.

PURPOSE: To identify the chromosomal location of the gene involved in the pathogenesis of cavitary optic disk anomalies in a large pedigree with autosomal dominant inheritance of disease. DESIGN: Linkage analysis of a pedigree affected with cavitary optic disk anomalies. METHODS: Optic disk photographs were examined for the presence of cavitary optic disk anomalies. Sixteen affected family members and one obligate carrier were identified and studied with linkage analysis using both microarrays of single nucleotide polymorphisms (SNPs) and short tandem repeat polymorphism (STRP) markers. RESULTS: Multipoint linkage analysis of SNP genotypes yielded a maximum nonparametric logarithm of the odds (LOD) score of 21.7 with markers located on chromosome 12q. Linkage was confirmed with 16 STRP markers in the 12q region. A maximum two-point LOD score of 4.06 (theta = 0) was obtained with marker D12S1700. The disease interval defined by observed recombinants is 9.1 cM, which corresponds to 13.5 Mbp. Three candidate genes (GDF-11, NEUROD4, and WIF1) in the chromosome 12q locus were evaluated as possible disease-causing genes. No mutations were detected in the coding sequence of these genes. CONCLUSIONS: The discovery of the chromosomal location of a gene responsible for cavitary optic disk anomalies is a key step in identifying the genetic basis of this condition and ultimately may provide important insight into the pathogenesis of more common optic nerve diseases such as normal-tension glaucoma and primary open-angle glaucoma (POAG).

Vitreous amino acid concentrations in patients with glaucoma undergoing vitrectomy.

OBJECTIVE: To measure vitreous concentrations of glutamate and other amino acids in patients with glaucoma undergoing vitrectomy. METHODS: Undiluted vitreous samples were collected from patients undergoing vitrectomy at the University of Iowa (Iowa City) between 1997 and 1998 (n = 69). Vitreous concentrations of 16 amino acids, including glutamate, were determined using high-pressure liquid chromatography. Patients with a history of diabetes mellitus were excluded from the analysis. The study group consisted of those with a history of glaucoma (n = 8), and the control group included those with an epiretinal membrane and/or macular hole with no history of glaucoma (n = 17). Comparison of amino acid concentrations between the 2 groups was performed using a multifactor main effects model that adjusted for the effect of 10 selected covariates. Power analysis was done to determine the level of significant difference in amino acid concentrations. RESULTS: The glaucoma group comprised vitreal specimens from patients with primary open-angle (n = 3) and angle-closure glaucomas that included aqueous misdirection (n = 2), uveitis with secondary angle-closure (n = 2), and Axenfeld Rieger syndrome (n = 1). Indications for vitrectomy in this group included epiretinal membrane, retinal detachment, aqueous misdirection, and uveitis. The control group included specimens from patients with a macular hole (n = 11) and epiretinal membrane (n = 7), with 1 eye having both. Surgical indications in controls were macular hole, retinal detachment, and epiretinal membrane. The mean +/- SD levels of vitreous glutamate, glycine, gamma-aminobutyric acid, and alanine were 6.1 +/- 2.4, 16.3 +/- 7.5, 0.8 +/- 0.3, and 260.5 +/- 101.9 microM, respectively, in glaucoma and 5.2 +/- 2.3, 8.5 +/- 2.5, 0.6 +/- 0.2, and 159.5 +/- 54.9 microM in controls (P >.05 for all). None of the 16 amino acid concentrations measured showed a statistically significant difference between glaucoma and controls (P values between.06 and >.99). A power analysis indicated that a 1.8-fold elevation in the glutamate level was needed to reach significance. MAIN OUTCOME MEASURES: Vitreous amino acid concentrations. CONCLUSIONS: None of the 16 amino acids measured, including glutamate, were significantly elevated in the vitreous of glaucomatous eyes compared with controls. Our results are not consistent with the simple hypothesis of glutamate excitotoxicity in glaucoma. Instead, our findings indicate the dynamic nature of extracellular glutamate, whose concentration is dependent on complex mechanisms not yet fully understood. Further studies are needed to fully elucidate the role of glutamate in the pathogenesis of glaucoma.

A family with Axenfeld-Rieger syndrome and Peters Anomaly caused by a point mutation (Phe112Ser) in the FOXC1 gene.

PURPOSE: Mutations of the forkhead transcription factor gene FOXC1 result in anterior segment anomalies. No description of the spectrum of defects resulting from a single point mutation of this gene exists in the ophthalmology literature. We have screened all available patients with Axenfeld-Rieger genes (PITX2 and FOXC1). In this report, we clinically characterize the spectrum of ocular and systemic manifestations in one family resulting from a previously reported point mutation (Phe112Ser) in FOXC1. DESIGN: Observational case series. METHODS: Ten members of a multigenerational family were examined for signs of glaucoma, anterior segment abnormalities, and systemic features of Axenfeld-Rieger syndrome. The examinations were performed in an ophthalmology examination room or in the patients' homes. Blood was obtained from 10 members and screened for mutations in FOXC1 using direct DNA sequencing. RESULTS: A single mutation causing a T to C change in codon 112 (Phe112Ser) of FOXC1 was present in six members of the family. Five of these six patients were examined and all demonstrated anterior segment anomalies. One patient had Axenfeld anomaly, one had Rieger syndrome, and one had both Axenfeld anomaly and Peters anomaly. Additionally, some members demonstrated cardiac abnormalities, which may be secondary to their FOXC1 mutation. CONCLUSIONS: A wide spectrum of clinical phenotypes can result from a single point mutation of FOXC1. This report confirms that Rieger syndrome (with dental and facial abnormalities) can be caused by a mutation in FOXC1. It is also the first report of Peters anomaly being caused by a FOXC1 mutation.

Ischemic central retinal vein occlusion and neovascular glaucoma as a result of optic nerve head drusen.

PURPOSE: The purpose of this study was to report a case of ischemic central retinal vein occlusion (CRVO) caused by optic nerve head drusen. METHODS: Case report and review of the literature. RESULTS: A healthy 13-year-old boy was diagnosed with bilateral optic nerve head drusen. Two years later, he developed an ischemic CRVO and secondary neovascular glaucoma. A full medical workup was negative. Despite treatment, his vision eventually declined to no light perception from a funnel detachment. CONCLUSION: Nonischemic CRVO or venous stasis retinopathy is a well-known entity associated with optic nerve head drusen. The authors report a case of ischemic CRVO in a patient with no underlying risks for this other than the observed drusen. This report clearly shows that optic nerve drusen may not be entirely benign and that they can precipitate ischemic CRVO.