LRP2 Acts as SHH Clearance Receptor to Protect the Retinal Margin from Mitogenic Stimuli.

During forebrain development, LRP2 promotes morphogen signaling as an auxiliary SHH receptor. However, in the developing retina, LRP2 assumes the opposing function, mediating endocytic clearance of SHH and antagonizing morphogen action. LRP2-mediated clearance prevents spread of SHH activity from the central retina into the retinal margin to protect quiescent progenitor cells in this niche from mitogenic stimuli. Loss of LRP2 in mice increases the sensitivity of the retinal margin for SHH, causing expansion of the retinal progenitor cell pool and hyperproliferation of this tissue. Our findings document the ability of LRP2 to act, in a context-dependent manner, as activator or inhibitor of the SHH pathway. Our current findings uncovered LRP2 activity as the molecular mechanism imposing quiescence of the retinal margin in the mammalian eye and suggest SHH-induced proliferation of the retinal margin as cause of the large eye phenotype observed in mouse models and patients with LRP2 defects.

Latanoprost systemic exposure in pediatric and adult patients with glaucoma: a phase 1, open-label study.

OBJECTIVE: To evaluate short-term safety and steady-state systemic pharmacokinetics (PK) of latanoprost acid in pediatric subjects with glaucoma or ocular hypertension who received the adult latanoprost dose. DESIGN: Phase 1, open-label, multicenter study. PARTICIPANTS: Pediatric patients of 3 age groups (<3, 3-<12, and 12-<18 years) and adults (≥18 years) receiving latanoprost ophthalmic solution 0.005% once daily in 1 or both eyes for ≥2 weeks. INTERVENTION: Latanoprost was administered in both eyes each morning post-screening. Subjects returned 3 to 28 days later for evaluation of plasma concentrations, withholding morning latanoprost. At the clinic, a single drop of latanoprost ophthalmic solution was instilled into both eyes. Plasma latanoprost acid concentrations were collected predose and 5, 15, 30, and 60 minutes after administration. MAIN OUTCOME MEASURES: Latanoprost acid plasma exposure. RESULTS: The evaluable PK analysis set included data from 39 of 47 enrolled subjects. The median peak plasma concentration value was higher in the <3-year age group (166 pg/ml) versus other groups (49, 16, and 26 pg/ml for the 3-<12-year, 12-<18-year, and ≥18-year age groups, respectively). The median area under the concentration-time curve from zero to last measurable concentration value was also higher in the <3-year age group (2716 pg/min/ml) versus other groups (588, 106, and 380 pg/min/ml for the 3-<12-year, 12-<18-year, and ≥18-year age groups, respectively). Latanoprost acid was rapidly eliminated from the blood, with plasma concentrations undetectable within 10 to 30 minutes postdose in all but the <3-year age group. There were no discontinuations or dose reductions due to adverse events or treatment-emergent adverse events. CONCLUSIONS: Latanoprost acid systemic exposure was higher in younger children versus adolescents and adults, attributed primarily to lower body weight and smaller blood volume. Latanoprost acid was eliminated rapidly in all age groups and resulted in only a brief period of systemic exposure after once-daily dosing. Higher systemic exposure was not accompanied by adverse events, and on the basis of extrapolation of safety data from adults, this pilot study suggests an adequate safety margin for systemic adverse effects with use of the adult topical dose of latanoprost in pediatric patients. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosures may be found after the references.

Identification of differentially expressed proteins in the aqueous humor of primary congenital glaucoma.

Primary Congenital Glaucoma (PCG) is an autosomal recessive disease caused by an abnormal development of the anterior chamber angle. Although, PCG has been linked to several genetic loci, the role that the genes at these loci or their encoded proteins play in the pathophysiology of PCG and development of the anterior chamber is not known. To identify proteins that may be altered in PCG and that may help in understanding the underlying pathophysiology of the disease, we took a global proteomics approach. Tryptic digests of the complex mixtures of proteins in aqueous humor were analyzed using Liquid Chromatography/Mass Spectrometry (LC-MS/MS). Proteins were identified by searching the data against the human subset of the UniProt database. The proteomes of aqueous humor in PCG (n = 7) and patients undergoing cataract surgery as control (n = 4) were compared based on the scan counts of comparable proteins. Using stringent filtering criteria, Apolipoprotein A-IV (APOA-IV), Albumin and Antithrombin 3 (ANT3) were detected at significantly higher levels in PCG AH compared to control, whereas Transthyretin (TTR), Prostaglandin-H2 D-isomerase (PTGDS), Opticin (OPT) and Interphotoreceptor Retinoid Binding Protein (IRBP) were detected at significantly lower levels. Many of these proteins play a role in retinoic acid (RA) binding/transport and have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's (AD). It is possible that similar to AD, the pathologic changes in PCG during development could be influenced by the availability of RA in the anterior chamber.

Anterior segment alterations and comparative aqueous humor proteomics in the buphthalmic rabbit (an American Ophthalmological Society thesis).

PURPOSE: To use an integrated proteohistologic approach to gain insight into the anterior segment alterations in the buphthalmic rabbit. METHODS: Eyes from 2- and 5-year-old buphthalmic and normal rabbits (n=20) were studied histologically. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) of aqueous humor (AH) was used to determine differential protein expression between animal groups. Western blot and immunohistochemistry were performed on selected differentially expressed proteins identified by LC-MS/MS. RESULTS: The buphthalmic rabbits manifested a mild clinical phenotype with typical angle anomalies that appeared progressive by histology. Significantly thickened Descemet's membrane (DM) and anterior lens capsule in all buphthalmic rabbits showed increased fibronectin and collagen-IV immunolabeling. LC-MS/MS applying stringent filtering criteria revealed significant differential expression of several AH proteins in these rabbits. The protein of interest in the 2-year-old group was histidine-rich glycoprotein, and those in the 5-year-old group included alpha-2-HS-glycoprotein, clusterin, apolipoprotein E, interphotoreceptor retinoid-binding protein, transthyretin, cochlin, gelsolin, haptoglobin, hemopexin, and beta-2 microglobulin. The proteomic data for selected proteins was validated by Western blot and immunohistochemistry. A wide range of functional groups were affected by the altered AH proteins. These included extracellular matrix modulation, regulation of apoptosis, oxidative stress, and protein transport. CONCLUSIONS: Multiple anterior segment alterations were histologically identified in the buphthalmic rabbits that showed progressive changes with age. The differentially expressed AH proteins in these rabbits suggest a multifunctional role for AH in modulating pathologic changes in DM, anterior lens capsule, and the angular meshwork in these animals.

VAV2 and VAV3 as candidate disease genes for spontaneous glaucoma in mice and humans.

BACKGROUND: Glaucoma is a leading cause of blindness worldwide. Nonetheless, the mechanism of its pathogenesis has not been well-elucidated, particularly at the molecular level, because of insufficient availability of experimental genetic animal models. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that deficiency of Vav2 and Vav3, guanine nucleotides exchange factors for Rho guanosine triphosphatases, leads to an ocular phenotype similar to human glaucoma. Vav2/Vav3-deficient mice, and to a lesser degree Vav2-deficient mice, show early onset of iridocorneal angle changes and elevated intraocular pressure, with subsequent selective loss of retinal ganglion cells and optic nerve head cupping, which are the hallmarks of glaucoma. The expression of Vav2 and Vav3 tissues was demonstrated in the iridocorneal angle and retina in both mouse and human eyes. In addition, a genome-wide association study screening glaucoma susceptibility loci using single nucleotide polymorphisms analysis identified VAV2 and VAV3 as candidates for associated genes in Japanese open-angle glaucoma patients. CONCLUSIONS/SIGNIFICANCE: Vav2/Vav3-deficient mice should serve not only as a useful murine model of spontaneous glaucoma, but may also provide a valuable tool in understanding of the pathogenesis of glaucoma in humans, particularly the determinants of altered aqueous outflow and subsequent elevated intraocular pressure.

Enlargement of the globe with ocular malformations in c-Myc transgenic mice.

PURPOSE: To study the ocular development in transgenic mice carrying the mouse c-myc gene under the control of the Mx gene promoter (Mx-c-myc). METHODS: Transgenic mice were generated by standard techniques. For histological studies, the tissues were fixed with 10% buffered formalin, embedded in paraffin according to the standard procedure and sliced in 4-microm sections. c-Myc expression was investigated by reverse transcriptase-polymerase chain reaction and Southern blot analysis. RESULTS: A line of the Mx-c-myc mice displayed progressive enlargement of the globe with other ocular malformations. Histologically, the enlarged eyes exhibited closed cornea-iris angle, microphakia, corneal epithelial disorders, and attenuation of the inner retinal layers. Developmental analysis of eyes from these Mx-c-myc mice revealed irregular development of the iris and ciliary body at embryonic day 15.5 and the closed angle at 1 week of age. Leaky exogenous c-myc expression was detected in cornea, iris, lens, and retina from the Mx-c-myc mice by reverse transcriptase-polymerase chain reaction and Southern blot analysis. No other developmental abnormalities were observed in the Mx-c-myc mice. The anterior segment of the enlarged eyes showed the closed angle with elongation of the iris and ciliary body. There was no attenuation in the outer retinal layers from the outer plexiform layer to the retinal pigment epithelium. CONCLUSIONS: We conclude that the buphthalmos and accompanying changes were not due to expression of the exogenous c-myc in cornea and retina but may be the secondary changes of elevated intraocular pressure. We suggest that Mx-c-myc mice can serve as a useful model for investigating the development of the anterior segment and the genesis of buphthalmos.

Morphologic studies of uveoscleral outflow in normotensive and glaucomatous beagles with fluorescein-labeled dextran.

Aqueous humor leaves the anterior chamber through 2 pathways: the trabecular meshwork of the iridocorneal angle and the unconventional uveoscleral route. In the latter route, aqueous humor leaves the anterior chamber, passes caudally through the trabecular meshwork and sclerociliary cleft to enter the supraciliary and suprachoroidal spaces. The fluid is absorbed by the choroidal and scleral circulation. Fluorescein-labeled dextran was slowly infused into the posterior chamber of healthy and glaucomatous Beagles for 30 minutes. The eyes were fixed in a mixture of 70% alcohol and formalin, placed in epoxy resin for histologic evaluation, and examined by fluorescent microscopy. Fluorescence was detected in the healthy dogs throughout the uveoscleral pathway. In the glaucomatous dogs, the uveoscleral outflow was decreased or completely absent.

Aqueous ascorbate concentration in hereditary buphthalmic rabbits.

Some aqueous humor constituents, including ascorbate, protein, and sodium, potassium, and calcium ions, were analyzed in three groups of adult buphthalmic (bu/bu) rabbits from strain AXBU/J and in control rabbits of the closely related strain AX/J. Aqueous humor ascorbate concentration of the normal strain was 22.5/"2.3mg/100ml. The buphthalmic rabbits that had no meaningful clinical signs of glaucoma at the time of sample collection had lower values (11.7/"1.7mg/100ml). Much lower values were observed in the bu/bu mutant rabbits that showed mild to severe glaucoma: 8.9/"3.8mg/100 ml and 1.9"/0.4mg/100ml, respectively. It was concluded that reduction of aqueous ascorbate is a biochemical change associated with the pathological development of buphthalmia in rabbits of the AXBU/J strain. However, the exact mechanism is still unclear.

Calcium oxalate crystals localized in the eye. Subretinal and retinal deposits, including deposits in the pigment epithelium.

Calcium oxalate crystals, identified morphologically, chemically and histochemically, were found in the eyes of two patients, one with congenital buphthalmia and one with proliferative diabetic retinopathy. The deposits had not been observed clinically or by macroscopic examination during sectioning of the eyeballs. Generalized oxalosis could be ruled out as a source of the oxalates. The crystals were localized in the detached, degenerated retina, subretinally and in the retinal pigment epithelium. Previously, deposition in the last-named layer has been reported only once and only in a case of generalized oxalosis. The literature is reviewed, and suggestions concerning the formation of localized oxalate crystals are advanced. It is concluded that the pigment epithelium is probably involved in the production, perhaps through a changed metabolism.