Mast cell infiltration of the choroid and protease release are early events in age-related macular degeneration associated with genetic risk at both chromosomes 1q32 and 10q26.

Age-related macular degeneration (AMD) is a leading cause of visual loss. It has a strong genetic basis, and common haplotypes on chromosome (Chr) 1 (CFH Y402H variant) and on Chr10 (near HTRA1/ARMS2) contribute the most risk. Little is known about the early molecular and cellular processes in AMD, and we hypothesized that analyzing submacular tissue from older donors with genetic risk but without clinical features of AMD would provide biological insights. Therefore, we used mass spectrometry­based quantitative proteomics to compare the proteins in human submacular stromal tissue punches from donors who were homozygous for high-risk alleles at either Chr1 or Chr10 with those from donors who had protective haplotypes at these loci, all without clinical features of AMD. Additional comparisons were made with tissue from donors who were homozygous for high-risk Chr1 alleles and had early AMD. The Chr1 and Chr10 risk groups shared common changes compared with the low-risk group, particularly increased levels of mast cell­specific proteases, including tryptase, chymase, and carboxypeptidase A3. Histological analyses of submacular tissue from donors with genetic risk of AMD but without clinical features of AMD and from donors with Chr1 risk and AMD demonstrated increased mast cells, particularly the tryptase-positive/chymase-negative cells variety, along with increased levels of denatured collagen compared with tissue from low­genetic risk donors. We conclude that increased mast cell infiltration of the inner choroid, degranulation, and subsequent extracellular matrix remodeling are early events in AMD pathogenesis and represent a unifying mechanistic link between Chr1- and Chr10-mediated AMD.

Deficit of human ornithine aminotransferase in gyrate atrophy: Molecular, cellular, and clinical aspects.

Gyrate Atrophy (GA) of the choroid and retina (MIM# 258870) is an autosomal recessive disorder due to mutations of the OAT gene encoding ornithine-delta-aminotransferase (OAT), associated with progressive retinal deterioration and blindness. The disease has a theoretical global incidence of approximately 1:1,500,000. OAT is mainly involved in ornithine catabolism in adults, thus explaining the hyperornithinemia as hallmark of the disease. Patients are treated with an arginine-restricted diet, to limit ornithine load, or the administration of Vitamin B6, a precursor of the OAT coenzyme pyridoxal phosphate. Although the clinical and genetic aspects of GA are known for many years, the enzymatic phenotype of pathogenic variants and their response to Vitamin B6, as well as the molecular mechanisms explaining retinal damage, are poorly clarified. Herein, we provide an overview of the current knowledge on the biochemical properties of human OAT and on the molecular, cellular, and clinical aspects of GA.

Retinal Distribution and Extracellular Activity of Granzyme B: A Serine Protease That Degrades Retinal Pigment Epithelial Tight Junctions and Extracellular Matrix Proteins.

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

Characterization of the gelatinase system of the laminar human optic nerve, and surrounding annulus of Bruch's membrane, choroid, and sclera.

PURPOSE: We determined the presence and levels of gelatinase matrix metalloproteinases (MMPs) in the optic nerve and surrounding rim region of the human fundus. METHODS: Samples of optic nerve, rim region, and Bruch's membrane-choroid from macular and peripheral regions were isolated from 9 pairs of human donor eyes. The MMPs were extracted and separated by gelatin zymography. Individual gelatinase species were identified by their respective molecular weights and levels quantified by standard densitometric techniques. Ratios of active/latent MMPs were calculated as representative indicators of the degree of proteolytic activity at each of the locations examined. RESULTS: All of the gelatinase species normally found in Bruch's membrane also were present in the optic nerve region. The presence of the high molecular weight MMP species (HMW1 and HMW2) was indicative of the age-related accumulation of polymerized MMPs 2 and 9. Level of activated MMPs was considerably raised in comparison with their latent forms at the optic nerve and surrounding region indicative of greater ongoing turnover of the matrix (P < 0.005). CONCLUSIONS: The components of the gelatinase pathway mediating matrix turnover in Bruch's membrane also were present in the optic nerve region. The presence of high levels of active MMPs 2 and 9 in comparison with the latent forms in the optic nerve and rim area is indicative of a high rate of matrix remodeling in these regions. Enhanced matrix turnover within the optic nerve region may represent an important mechanism for maintaining the plasticity of the lamina cribrosa.

High glucose and diabetes modulate cellular proteasome function: Implications in the pathogenesis of diabetes complications.

The precise link between hyperglycemia and its deleterious effects on retinal and kidney microvasculature, and more specifically loss of retinal perivascular supporting cells including smooth muscle cell/pericytes (SMC/PC), in diabetes are not completely understood. We hypothesized that differential cellular proteasome activity contributes to sensitivity of PC to high glucose-mediated oxidative stress and vascular rarefaction. Here we show that retinal endothelial cells (EC) have significantly higher proteasome peptidase activity compared to PC. High glucose treatment (HGT) increased the level of total ubiquitin-conjugated proteins in cultured retinal PC and EC, but not photoreceptor cells. In addition, in vitro proteasome activity assays showed significant impairment of proteasome chymotrypsin-like peptidase activity in PC, but not EC. The PA28-α/-ß and PA28-ß/-γ protein levels were also higher in the retina and kidney glomeruli of diabetic mice, respectively. Our results demonstrate, for the first time, that high glucose has direct biological effects on cellular proteasome function, and this modulation might be protective against cellular stress or damage induced by high glucose.

Antiangiogenic effects of axitinib, an inhibitor of vascular endothelial growth factor receptor tyrosine kinase, on laser-induced choroidal neovascularization in mice.

PURPOSE: To investigate the effects of axitinib, an inhibitor of vascular endothelial growth factor receptors, on choroidal neovascularization (CNV) in an animal model of neovascular age-related macular degeneration (AMD). METHODS: Experimental CNV lesions were induced in C57BL/6 mice by laser photocoagulation. Beginning 1 day after CNV induction, mice were treated with axitinib (5 mg/kg/day) or vehicle for 2 weeks. In other groups of mice, axitinib or vehicle treatment was started 7 days after the laser application to determine the effect of the drug on established CNV. Untreated mice were used as a baseline group. Two weeks after laser injury, the extent of CNV was assessed from choroidal flat mounts perfused with fluorescein-labeled dextran. Immunofluorescence staining with isolectin IB4 was also used to quantify the CNV lesions. RESULTS: Orally administered axitinib inhibited CNV growth in the laser-induced CNV model. Axitinib caused a 70.1% inhibition of CNV lesions compared to vehicle-treatment (p < 0.001). Axitinib also caused a significant regression of established CNV, reducing the area by 71.1% compared to vehicle treatment (p < 0.001). Moreover, immunofluorescence staining showed that the area of isolectin IB4 labeled vessels was smaller in the axitinib-treated group compared to the vehicle-treated group (p < 0.001). CONCLUSIONS: Axitinib effectively inhibits the progression of CNV in an experimental animal model. These results suggest that axitinib could constitute a therapeutic alternative for the treatment of neovascular AMD.

Vectorial release of matrix metalloproteinases (MMPs) from porcine RPE-choroid explants following selective retina therapy (SRT): towards slowing the macular ageing process.

The purpose of this study was to investigate release of matrix metalloproteinases (MMP) 2 and 9 during retinal pigment epithelium (RPE) wound healing after Selective Retina Therapy (SRT) with laser energy levels below and above the threshold of RPE cell death. Following exposure to SRT using a prototype pulsed Nd:YLF laser with energies of 80-180 mJ/cm(2) fresh porcine RPE-monolayers with Bruch's membrane and choroid were cultured in modified Ussing chambers which separate the apical (RPE-facing) and basal (choroid facing) sides of the RPE monolayer. Threshold energy for RPE cell death and wound healing were determined with calcein-AM viability test. Inactive and active forms of MMP 2 and 9 were quantified within tissue samples and in the culture medium of the apical and basal compartments of the Ussing chamber using gelatine zymography. Laser energies of 160-180 mJ/cm(2) resulted in cell death within 1 h while 120-140 mJ/cm(2) resulted in delayed death of exposed RPE cells. All cells survived 80 and 100 mJ/cm(2). Laser spots healed within 6 days after SRT accompanied by a transient vectorial increase of MMPs. SRT with 180 mJ/cm(2) increased active MMP 2 by 1.9 (p < 0.05) and 1.6 (p < 0.05) fold in tissue and basal compartments, respectively, without alterations in the apical compartment. Pro-MMP 2 levels were also significantly increased in all compartments (p < 0.05). Release of MMP 9 was not altered. Laser energy below the threshold of RPE cell death did not alter the release of MMP 2 or 9. The findings suggest that the release of active MMP 2 on the basal side of the RPE during wound healing following SRT may address age-related pathological changes of Bruch's membrane with a potential to slow degenerative macular ageing processes before irreversible functional loss has occurred.

Identification of RALDH2 as a visually regulated retinoic acid synthesizing enzyme in the chick choroid.

PURPOSE: All-trans-retinoic acid (atRA) has been implicated in the local regulation of scleral proteoglycan synthesis in vivo. The purpose of the present study was to identify the enzymes involved in the synthesis of atRA during visually guided ocular growth, the cells involved in modulation of atRA biosynthesis in the choroid, and the effect of choroid-derived atRA on scleral proteoglycan synthesis. METHODS: Myopia was induced in White leghorn chicks by form deprivation for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of atRA synthesizing enzymes was evaluated by semiquantitative qRT-PCR, in situ hybridization, and immunohistochemistry. atRA synthesis was measured in organ cultures of isolated choroids using LC-tandem MS quantification. Scleral proteoglycan synthesis was measured in vitro by the incorporation of (35)SO(4) in CPC-precipitable glycosaminoglycans. RESULTS; RALDH2 was the predominant RALDH transcript in the choroid (> 100-fold that of RALDH3). RALDH2 mRNA was elevated after 12 and 24 hours of recovery (60% and 188%, respectively; P < 0.01). The atRA concentration was significantly higher in cultures of choroids from 24-hour to 15-day recovering eyes than in paired controls (-195%; P < 0.01). Choroid conditioned medium from recovering choroids inhibited proteoglycan synthesis to 43% of controls (P < 0.02, paired t-test; n = 16) and produced a relative inhibition corresponding to a RA concentration of 7.20 × 10(-8) M. CONCLUSIONS: The results of this study suggest that RALDH2 is the major retinal dehydrogenase in the chick choroid and is responsible for increased atRA synthesis in response to myopic defocus.

Disturbed matrix metalloproteinase activity of Bruch's membrane in age-related macular degeneration.

PURPOSE: To evaluate the potential role of the matrix metalloproteinase (MMP) system of Bruch's membrane in the pathology of age-related macular degeneration. METHODS: Free and bound pools of gelatinase activity in Bruch's membrane-choroid preparations were isolated by phosphate-buffered saline (PBS) and sodium dodecyl sulfate (SDS) extraction, respectively. Individual MMP species were separated by gelatin-substrate zymography and the levels were quantified by densitometric techniques. Altogether, 13 control (age range, 71-99 years) and 6 AMD (age range, 71-95 years) donor eyes were used. RESULTS: All the gelatinase components normally present in control samples were also present in AMD tissue without any significant differences in their molecular masses. Total levels (bound plus free) of active MMP2 and -9 were significantly reduced in AMD donors (P < 0.05). The decrease in active MMP2 may be attributable to a similar reduction in the level of free pro-MMP2, the precursor to the active form. Reduction in active MMP9 occurred despite a nearly 3.5-fold increase in free pro-MMP9. The high-molecular-mass gelatinases denoted by HMW1 and -2 and comprising homo- and heteropolymers of pro-MMP2 and -9 were also raised in AMD (P < 0.05). The sequestration of free pro-MMP2 and -9 by these high-molecular-mass complexes may further contribute to reduced rates of activation of MMPs. CONCLUSIONS: The reduction in the levels of activated MMP2 and -9 may be responsible for impaired matrix degradation of Bruch's membrane, leading to the pathology associated with AMD. The degradation pathway is therefore a viable therapeutic target for future intervention.

Angiogenin in age-related macular degeneration.

PURPOSE: Age-related macular degeneration (AMD) is a common blinding disease in the elderly population. AMD is frequently complicated by choroidal neovascularization, causing irreversible losses in visual acuity. Proteins that induce pathologic angiogenesis in other systems include angiogenin, a small protein involved in angiogenesis in tumor metastases. Our goal was to determine if angiogenin participates in angiogenesis during choroidal neovascular membrane formation in AMD. METHODS: The expression of angiogenin in the human retina and retinal pigment epithelium (RPE)-choroid was determined using reverse-transcription (RT)-PCR and immunoblotting. Localization of angiogenin in human control eyes and in eyes with choroidal neovascularization was determined using immunohistochemistry. Potential angiogenin-mediated effects on endothelial cell migration, as well as angiogenin internalization by Rf/6a cells, were determined. RESULTS: Angiogenin was synthesized by the human choroid and retina and localized to normal and pathologic vasculature. Angiogenin did not change the migratory behavior of Rf/6a chorioretinal endothelial cells; however, these cells did internalize exogenous angiogenin in culture. CONCLUSIONS: Chorioretinal endothelial cells bind and internalize angiogenin, a protein localized to the choroid in normal eyes, as well as in some drusen and in neovascular membranes in AMD eyes. Angiogenin has been shown to participate in angiogenesis in other tissues. Although angiogenin does not increase the migratory behavior of these cells, it may play a role in other aspects of endothelial cell activation in neovascular AMD.

Loss of tubedown expression as a contributing factor in the development of age-related retinopathy.

PURPOSE: Tubedown (Tbdn), a cortactin-binding acetyltransferase subunit, regulates retinal vascular permeability and homeostasis in adulthood. Here the authors explore whether Tbdn loss during aging might contribute to the mechanisms underlying age-related neovascular retinopathy. METHODS: A conditional endothelial-specific transgenic model of Tbdn loss was compared with aged mouse and human specimens from 5- to 93-year-old individuals. Specimens were analyzed by morphometric measurements and for functional markers using immunohistochemistry and Western blot analysis. RESULTS: An age-dependent decrease in Tbdn expression in endothelial cells of the posterior pole of the eye correlated with pathologic changes in choroidal and retinal tissues of aged mice. In humans, aged specimens without eye disease exhibited a moderate decrease in retinal and choroidal endothelial Tbdn expression compared with younger persons, whereas a greater decrease in choroid vascular Tbdn expression was observed in patients with age-related macular degeneration. In mice, Tbdn loss resulting from old age or conditional Tbdn knockdown was associated with retinal lesions showing significant extravascularly localized albumin and correlated with increased activity of senescence-associated ß-galactosidase in the retinal pigment epithelium. A range of abnormalities in RPE, Bruch's membrane, and choriocapillaris observable at the ultrastructural level in Tbdn-knockdown eyes recapitulate those present in human AMD. CONCLUSIONS: This work provides evidence that the marked decrease in the level of expression of Tbdn in the retinal and choroidal vasculature during aging contributes to the multifactorial process that leads to the development of age-related retinopathy and choroidopathy.

Ecto-5'-nucleotidase-positive cells in the choroid and ciliary body of the rat eye.

Adenosine is known to exert multiple functions within the eye. The aim of this report was to find out if adenosine can be produced locally in the choroid and ciliary body. Therefore, I investigated the distribution of ecto-5'-nucleotidase (5'-NT), the key enzyme for the production of extracellular adenosine. This report provides evidence that 5'-NT is expressed in the choroid and in the ciliary body (and its processes) of the rat eye, predominantly in endothelial cells. These locations of 5'-NT indicate strategically important production sites of adenosine regulating choroid and ciliary body functions (e.g., blood flow, aqueous fluid production, and immune response).

Low nitric oxide synthases (NOSs) in eyes with age-related macular degeneration (AMD).

Nitric oxide (NO) production by vascular endothelium is important in regulation of blood flow. Reduced production of NO can adversely affect blood flow and other vascular functions. We investigated the expression of three nitric oxide synthase (NOS) isoforms in retina and choroid of aged human eyes and eyes with AMD. Alkaline phosphatase immunohistochemistry was performed using antibodies against inducible (iNOS), neuronal (nNOS), and endothelial (eNOS) NOSs on cryopreserved sections from aged control donor eyes (n = 13) and eyes with AMD (n = 22). CD34 antibody was used as an endothelial cell (EC) marker. Three independent masked observers scored the intensity of the immunohistochemical reaction product. Mean scores from the aged control and AMD eyes were statistically compared. In aged control retinas, nNOS was in ganglion cells (RGCs) and neurons of both nuclear layers. In choroid, perivascular nerve fibers and retinal pigment epithelial (RPE) cells were nNOS+. eNOS and iNOS were confined to the retinal and choroidal vascular ECs. Some cells presumably melanocytes or dendritic cells in choroid were also eNOS+. In AMD eyes, nNOS was significantly lower in RGCs, neurons, retinal vessels and RPE (p < or = 0.05) compared to the aged control eyes. iNOS and eNOS showed no significant differences between aged control and AMD eyes except that there was significantly less eNOS in choroidal arteries (p = 0.006) and choroidal cells (p = 0.03) of AMD eyes. Although NO was not measured directly, these findings suggest that there is less NO produced in AMD eyes. The decrease in retinal nNOS in AMD eyes is probably related to neuronal degeneration. The decrease in nNOS and eNOS in AMD choroid could be associated with vasoconstriction and hemodynamic changes.

Lysyl oxidase activity in the ocular tissues and the role of LOX in proliferative diabetic retinopathy and rhegmatogenous retinal detachment.

PURPOSE: Lysyl oxidase (LOX) cross-links the side chain of collagen and elastin and thereby contributes to extracellular matrix (ECM) integrity. ECM remodeling is seen in various ocular diseases. Until now, there have been no reports on the LOX enzyme's activity in ocular tissues. The purpose of this study was to estimate LOX activity and expression in human donor ocular tissues and to measure the specific activity of LOX in the vitreous of proliferative diabetic retinopathy (PDR) and rhegmatogenous retinal detachment (RRD). METHOD: Human donor eyeballs obtained from an eye bank were used to study tissue distribution of LOX. Human vitreous specimens were obtained during vitreoretinal surgery from PDR (n = 16) and RRD (n = 10). LOX activity was estimated by N-acetyl-3,7-dihydroxyphenoxazine assay, immunohistochemistry, and real-time polymerase chain reaction (RT-PCR). Matrix metalloprotease (MMP)-2 and -9 were quantified in the vitreous by sandwich enzyme-linked immunosorbent assay (ELISA). RESULTS: The specific activity of LOX in ocular tissues was on the order of vitreous, iris ciliary body, lens, choroid RPE, and retina, which were comparable by mRNA expression and immunolocalization. The vitreous level of LOX activity decreased significantly in PDR and RRD, with an increase in total MMP-2 and -9 levels compared with normal donor vitreous. CONCLUSIONS: LOX activity showed a statistically significant decrease in the vitreous of PDR and RRD relative to control specimens. This effect can contribute to the inadequate collagen cross-linking that causes the ECM changes that occur in these diseases.

Abnormal vessel formation in the choroid of mice lacking tissue inhibitor of metalloprotease-3.

PURPOSE: Tissue inhibitor of metalloprotease (TIMP)-3 is an inhibitor of matrix metalloprotease (MMP) and regulates angiogenesis. In the eye, TIMP3 is tightly associated with Bruch's membrane. In this study, the authors analyzed mice lacking TIMP3 for retinal abnormalities. METHODS: Mice with targeted disruption of the Timp3 gene were generated (Timp3(-/-)) and bred into C57/Bl6 and CD1 backgrounds. Eyes were analyzed by light and electron microscopy. Vasculature was examined by scanning laser ophthalmoscopy, corrosion casts, and whole mount preparations. MMP activity was assessed by in situ zymography, angiogenic potential was evaluated by tube formation, and aortic ring assays and signaling pathways were studied by immunoblotting. RESULTS: TIMP3-deficient mice develop abnormal vessels with dilated capillaries throughout the choroid. Enhanced MMP activity in the choroid region of Timp3(-/-) eyes was detected when compared with controls. Timp3(-/-)-derived tissue showed an increased angiogenic activity over wild-type, an effect that could specifically be inhibited by recombinant TIMP3. Moreover, the antiangiogenic property of TIMP3 was demonstrated to reside within the C-terminal domain. When VEGFR2 inhibitor was added to Timp3(-/-) aortic explants, endothelial sprout formation was markedly reduced, which provided evidence for an unbalanced VEGF-mediated angiogenesis in Timp3(-/-) animals. Finally, angiogenic signaling pathways are activated in Timp3(-/-)-derived cells. CONCLUSIONS: These findings suggest that the distinct choroidal phenotype in mice lacking TIMP3 may be the result of a local disruption of extracellular matrix and angiogenic homeostasis, and they support an important role of TIMP3 in the regulation of choroidal vascularization.

Increased mitochondrial DNA damage and down-regulation of DNA repair enzymes in aged rodent retinal pigment epithelium and choroid.

PURPOSE: In the central nervous system (CNS), increased mitochondrial DNA (mtDNA) damage is associated with aging and may underlie, contribute to, or increase the susceptibility to neurodegenerative diseases. Because of the focus on the retinal pigment epithelium (RPE) and choroid as tissue relevant to age-related macular degeneration (AMD), we examined young and aged RPE and choroid, harvested from rodent eyes, for DNA damage and for changes in selected DNA repair enzymes. METHODS: Immunohistochemical labeling and quantitative ELISA for the oxidative DNA damage marker, 8-hydroxy-2'-deoxy-guanosine (8-OHdG), were measured in young and aged rodent RPE and choroid. mtDNA and nuclear DNA (nDNA) damage was determined by quantitative polymerase chain reaction (PCR) by comparing the relative amplification of small and large DNA fragments. Expression of several DNA repair enzymes was measured using real-time quantitative reverse transcription -PCR (qRT-PCR) and immunoblot. RESULTS: Immunohistochemical labeling for 8-OHdG increased in aged rodent RPE and choroid. Quantitative ELISA confirmed increased levels of 8-OHdG. Measurements of nDNA and mtDNA lesions indicated that DNA damage is primarily in mtDNA in aged RPE and choroid. Using qRT-PCR, we found that gene expression of DNA repair enzymes, 8-oxoguanine-DNA glycosylase 1 (OGG1), mutY homolog (MYH), and thymine DNA glycosylase were decreased in an age-dependent pattern in RPE and choroid. However, endonuclease III homolog 1 was not significantly changed in aged RPE and choroid. Using immunoblots, we found that protein levels of OGG1 and MYH were decreased in aged RPE and choroid. CONCLUSIONS: Our results show that there is increased mtDNA damage in aged RPE and choroid, which is likely due to decreased DNA repair capability. mtDNA damage in the RPE and choroid may be a susceptibility factor that underlies the development of AMD.

Time-course of changes to nitric oxide signaling pathways in form-deprivation myopia in guinea pigs.

The aim of this study was to investigate the time-course change of nitric oxide synthase (NOS) activity and cyclic GMP (cGMP) concentration in the posterior retina, choroid and sclera after differing periods of form-deprivation in guinea pigs. Three groups of guinea pigs were subjected to monocular FD for 7, 14 or 21 days. NOS activity and cGMP concentrations in ocular tissues of FD eyes and control eyes were analyzed by radioimmunoassay. The presence of NOS isoforms was detected by immunohistochemistry. Guinea pigs presented with considerable myopia after 14 days of FD. Retinal NOS activity in the FD group was lower than in the control group after 7 days of FD and was higher than in the control group after 14 and 21 days of FD. The choroidal and scleral NOS activities in the FD groups were higher than in the control groups after 21 days. The cGMP concentrations in the FD groups were higher than in the control groups at 21 days of the retinal, choroidal, and scleral tissues. Furthermore, the retinal cGMP concentration in the FD group was also significantly elevated at 14 days relative to the control group. We detected expression of three NOS isoforms in guinea pig ocular tissues. Our main observations were a change in NOS activity and an up-regulation in cGMP concentrations in posterior ocular tissues during the development of myopia. The function of elevated NOS activity may be mediated by cGMP.

Vascular changes in the posterior eye segment of secondary angle-closure glaucoma: cause or consequence?

BACKGROUND: To study the role of choroidal and retinal vessels in the pathology of secondary angle-closure glaucoma. METHODS: DBA/2NNia and non-glaucomatous C57BL/6J mice over the age range 2-20 months were investigated. Corrosion cast preparations of the vasculature were studied using scanning electron microscopy. Whole mounts of the retina and choroid were stained enzyme-histochemically for NADPH diaphorase as an indicator for nitric oxide synthase activity. Semi- and ultra-thin sections of the posterior eye segment were performed and evaluated. RESULTS: DBA/2NNia mice showed loss of choroidal pigmentation and a decrease in choriocapillary density already at 4 months of age. In animals 9 months and older, a decrease of choroidal NADPH-diaphorase positive nerve fibers was evident. The retinal vasculature showed only mild changes in NADPH-diaphorase staining, even in the oldest animals. The ultrastructural appearance of the retinal vessels was similar in both mouse strains and for all ages investigated. CONCLUSIONS: Choroidal changes in the DBA/2NNia mouse are similar to that seen in other glaucoma models. The lack of retinal vasculature changes in adult and senescent DBA/2NNia mice suggests a normal blood supply of the retina during the progress of secondary angle-closure glaucoma in these animals.

Phosphatase enzyme histochemistry for studying vascular hierarchy, pathology, and endothelial cell dysfunction in retina and choroid.

Phosphatase enzymes cleave an inorganic phosphate from a substrate. Phosphatase enzyme histochemistry followed by flat-embedding in glycol methacrylate is extremely useful in studying retinal and choroidal vascular development and loss, since only viable blood vessels have these enzyme activities. Sites of occlusion and remodeling can be identified and analysed, resulting in new insights into the cause of occlusion. The phosphatase activities are elevated in neovascularization making possible high resolution analysis of neovascularization, the feeder vessels, and the retinal milieu in which angiogenesis occurs. Adenosine diphosphatase (ADPase) catalyzes ADP to an inorganic phosphate plus adenosine monophosphate, preventing accumulation of ADP, one of the most potent stimuli for platelet aggregation. The ADPase technique can be used in any species but this report highlights its use in dog and human retinas. The ADPase technique has yielded important insights into vaso-occlusive and vasoproliferative processes in retinopathy of prematurity, sickle cell and diabetic retinopathies. The alkaline phosphatase flatembedding technique is useful in evaluating dog, cat, and human choroidal vasculatures. It has permitted quantification of the loss of choriocapillaris in diabetic choroidopathy and of the RPE and choriocapillaris in geographic atrophy and exudative age-related macular degeneration.

Mitochondrial fatty acid beta-oxidation in the human eye and brain: implications for the retinopathy of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

The retinal pigment epithelium (RPE) and the choriocapillaris are affected early in the retinopathy associated with long-chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency. RPE in culture possesses the machinery needed for mitochondrial fatty acid beta-oxidation in vitro. To further elucidate pathogenesis of LCHAD retinopathy, we performed immunohistochemistry of the human eye and brain with antibodies to beta-oxidation enzymes. Human eye and brain sections were stained with antibodies to medium-chain (MCAD) and very long-chain acyl-CoA dehydrogenase (VLCAD), short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), and mitochondrial trifunctional protein (MTP) harboring LCHAD. Antibodies to 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) and cytochrome c oxidase subunit I (COX I) were used as a reference. VLCAD, MTP, MCAD, SCHAD, MHBD, and COX I antibodies labeled most retinal layers and tissues of the human eye actively involved in oxidative metabolism (extraocular and intraocular muscle, the RPE, the corneal endothelium, and the ciliary epithelium). MTP and COX I antibodies labeled the inner segments of photoreceptors. The choriocapillaris was labeled only with SCHAD and MCAD antibodies. In the brain, the choroid plexus and nuclei of the brain stem were most intensely labeled with beta-oxidation antibodies, whereas COX I antibodies strongly labeled neurons in several regions of the brain. Mitochondrial fatty acid beta-oxidation likely plays a role in ocular energy production in vivo. The RPE rather than the choriocapillaris could be the critical affected cell layer in LCHAD retinopathy. Reduced energy generation in the choroid plexus may contribute to the cerebral edema observed in patients with beta-oxidation defects.