Theo G. M. F. Gorgels

The dynamic nature of Bruch's membrane.

Bruchs membrane (BM) is a unique pentalaminar structure, which is strategically located between the retinal pigment epithelium (RPE) and the fenestrated choroidal capillaries of the eye. BM is an elastin- and collagen-rich extracellular matrix that acts as a molecular sieve. BM partly regulates the reciprocal exchange of biomolecules, nutrients, oxygen, fluids and metabolic waste products between the retina and the general circulation. Accumulating evidence suggests that the molecular, structural and functional properties of BM are dependent on age, genetic constitution, environmental factors, retinal location and disease state. As a result, part of the properties of BM are unique to each human individual at a given age, and therefore uniquely affect the development of normal vision and ocular disease. The changes occurring in BM with age include increased calcification of elastic fibres, increased cross-linkage of collagen fibres and increased turnover of glycosaminoglycans. In addition, advanced glycation end products (AGEs) and fat accumulate in BM. These age-related changes may not only influence the normal age-related health of photoreceptor cells, but also the onset and progression of diseases like retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Undoubtedly, BM is the site of drusen development. Confluent drusen and uncontrolled activation of the complement cascade are most likely the first signs of AMD. Furthermore, the nature of adhesive interactions between the RPE and BM are instrumental in the development of retinal detachments and proliferative retinal disease. Finally, BM is passively or actively involved in a range of other retinal disorders such as Pseudoxanthoma elasticum (PXE), Sorsbys Fundus Dystrophy and Malattia Leventinese. Here, we review the dynamic nature of Bruchs membrane, from molecule to man, during development, aging and disease. We propose a simple and straightforward nomenclature for BM deposits. Finally, we attempt to correlate recently published mRNA expression profiles of the RPE and choroid with molecular, structural and functional properties of BM. Our review may shed light on the complex involvement of BM in retinal pathology, notably age-related macular degeneration.

A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14

Refractive errors are the most common ocular disorders worldwide and may lead to blindness. Although this trait is highly heritable, identification of susceptibility genes has been challenging. We conducted a genome-wide association study for refractive error in 5,328 individuals from a Dutch population-based study with replication in four independent cohorts (combined 10,280 individuals in the replication stage). We identified a significant association at chromosome 15q14 (rs634990, P = 2.21 × 10−14). The odds ratio of myopia compared to hyperopia for the minor allele (minor allele frequency = 0.47) was 1.41 (95% CI 1.16–1.70) for individuals heterozygous for the allele and 1.83 (95% CI 1.42–2.36) for individuals homozygous for the allele. The associated locus is near two genes that are expressed in the retina, GJD2 and ACTC1, and appears to harbor regulatory elements which may influence transcription of these genes. Our data suggest that common variants at 15q14 influence susceptibility for refractive errors in the general population.

The vast complexity of primary open angle glaucoma : Disease genes, risks, molecular mechanisms and pathobiology

Primary open angle glaucoma (POAG) is a complex progressive optic nerve neuropathy triggered by both environmental and genetic risk factors. Several ocular tissues, including the ciliary body, trabecular meshwork and optic nerve head, and perhaps even brain tissues, are involved in a chain of pathological events leading to POAG. Genetic risk evidence for POAG came from family linkage-studies implicating a small number of disease genes (MYOC, OPTN, WDR36). Recent Genome Wide Association Studies (GWAS) identified a large number of new POAG loci and disease genes, such as CAV1, CDKN2B and GAS7. In the current study, we reviewed over 120 family and GWA studies. We selected in total 65 (candidate) POAG disease genes and proceeded to assess their function, mRNA expression in POAG relevant eye tissues and possible changes in disease state. We found that the proteins corresponding to these 65 (candidate) POAG disease genes take part in as few as four common functional molecular networks. Functions attributed to these 4 networks were developmental (dys)function, lipid metabolism, and inflammatory processes. For the 65 POAG disease genes, we reviewed the available (transgenic) mouse models of POAG, which may be useful for future functional studies. Finally, we showed that the 65 (candidate) POAG genes substantially increased the specificity and sensitivity of a discriminative POAG risk test. This suggests that personal risk assessment and personalized medicine for POAG are on the horizon. Taken together, the data presented are essential to comprehend the role of genetic variation in POAG, and may provide leads to understand the pathophysiology of POAG as well as other neurodegenerative disorders, such as Alzheimers disease.

ABCC6 prevents ectopic mineralization seen in pseudoxanthoma elasticum by inducing cellular nucleotide release

Significance Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding the transporter ABCC6. PXE is a metabolic disease caused by the absence of an unknown factor in the circulation. Our study indicates that the factor that normally prevents PXE is pyrophosphate, which is provided to the circulation in the form of nucleoside triphosphates via an ABCC6-dependent mechanism. Our findings provide leads for the treatment of this intractable disease. Pseudoxanthoma elasticum (PXE) is an autosomal recessive disease characterized by progressive ectopic mineralization of the skin, eyes, and arteries, for which no effective treatment exists. PXE is caused by inactivating mutations in the gene encoding ATP-binding cassette sub-family C member 6 (ABCC6), an ATP-dependent efflux transporter present mainly in the liver. Abcc6−/− mice have been instrumental in demonstrating that PXE is a metabolic disease caused by the absence of an unknown factor in the circulation, the presence of which depends on ABCC6 in the liver. Why absence of this factor results in PXE has remained a mystery. Here we report that medium from HEK293 cells overexpressing either human or rat ABCC6 potently inhibits mineralization in vitro, whereas medium from HEK293 control cells does not. Untargeted metabolomics revealed that cells expressing ABCC6 excrete large amounts of nucleoside triphosphates, even though ABCC6 itself does not transport nucleoside triphosphates. Extracellularly, ectonucleotidases hydrolyze the excreted nucleoside triphosphates to nucleoside monophosphates and inorganic pyrophosphate (PPi), a strong inhibitor of mineralization that plays a pivotal role in several mineralization disorders similar to PXE. The in vivo relevance of our data are demonstrated in Abcc6−/− mice, which had plasma PPi levels <40% of those found in WT mice. This study provides insight into how ABCC6 affects PXE. Our data indicate that the factor that normally prevents PXE is PPi, which is provided to the circulation in the form of nucleoside triphosphates via an as-yet unidentified but ABCC6-dependent mechanism.

Retinal Degeneration and Ionizing Radiation Hypersensitivity in a Mouse Model for Cockayne Syndrome

ABSTRACT Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csbm/m mice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csbm/m retina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csbm/m mice (as well as Csa−/− mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csbm/m retina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging.

The Action Spectrum of Photochemical Damage to the Retina: A Review of Monochromatic Threshold Data

Photochemical damage to the retina occurs for prolonged exposures of intense light. Two action spectra exist for this phenomenon. In rat an action spectrum matching the absorption spectrum of rhodopsin was found. In macaque, the susceptibility for photochemical damage decreased continuously from the UV to long visible wavelengths. Later, such a spectrum was also found in rat. In search for critical parameters that determine the shape of the spectrum we gathered all available data on the damage threshold dose for monochromatic radiation and noted the experimental conditions. The rhodopsin action spectrum was found in two sources; the other 16 sources adhered to the short wavelength spectrum. Comparing the conditions we conclude that the critical parameters for the generation of either action spectrum remain elusive. Experiments are suggested to resolve this issue and fill a few gaps in our knowledge.

ABCC6 and pseudoxanthoma elasticum

ABCC6 belongs to the adenosine triphosphate-binding cassette (ABC) gene subfamily C. This protein family is involved in a large variety of physiological processes, such as signal transduction, protein secretion, drug and antibiotic resistance, and antigen presentation [Kool et al. (1999) 59:175–182; Borst and Elferink (2002) 71:537–592]. ABCC6 is primarily and highly expressed in the liver and kidney [Kool et al. (1999) 59:175–182; Bergen et al. (2000) 25:228–2231]. The precise physiological function and natural substrate(s) transported by ABCC6 are unknown, but the protein may be involved in active transport of intracellular compounds to the extracellular environment [Kool et al. (1999) 59:175–182] [Scheffer et al. (2002) 82:515–518]. Recently, it was shown that loss of function mutations in ABCC6 cause pseudoxanthoma elasticum (PXE) [Bergen et al. (2000) 25:228–2231; Le Saux et al. (2000) 25:223–227]. PXE is an autosomal recessively inherited multi-organ disorder [Goodman et al. (1963) 42:297–334; Lebwohl et al. (1994) 30:103–107]. PXE is primarily associated with the accumulation of mineralized and fragmented elastic fibers of the connective tissue in the skin [Neldner (1988) 6:1–159], Bruch’s membrane in the retina [Hu et al. (2003) 48:424–438], and vessel walls [Kornet et al. (2004) 30:1041–1048]. PXE patients usually have skin lesions and breaks in Bruch’s membrane of the retina (angioid streaks). Also, a variety of cardiovascular complications has been observed [Hu et al. (2003) 48:424–438]. Recently, a mouse model for PXE was created by targeted disruption of Abcc6 [Gorgels et al. (2005) 14:1763–1773; Klement et al. (2005) 25:8299–8310], which may be useful to elucidate the precise function of Abcc6 and to develop experimental therapies.

Temporal sequence of changes in rat retina after UV-A and blue light exposure.

Two spectral types of retinal light damage were induced in pigmented rats by irradiating small retinal patches at either 380 or 470 nm. The temporal sequence of changes in the retina was followed for up to 2 months by funduscopy and histology. For both damage types, fundus changes were best visible after 3 days. Histology showed that 380 nm specifically damaged photoreceptor cells, particularly the rods. All cell compartments of the rods, including the nucleus were affected already after 3 h. In the next days, damaged rods degenerated. At high doses (2.5 x the funduscopic threshold dose) all rods in the irradiated area were lost, resulting in a local photoreceptor lesion, which was still present at 2 months after the irradiation. At 470 nm, damage occurred both in the photoreceptor layer and in the pigment epithelium. Acute changes, at 1 h after irradiation, consisted mainly of damaged mitochondria in these layers. Next, the pigment epithelium showed swelling, an altered melanin distribution and, at high doses (2.5 x threshold), interruptions of the monolayer. Degeneration of photoreceptor cells was initially limited to a few scattered cells, but 3 days after high doses focal areas of massive degeneration were seen. At late stages, the cells of the pigment epithelium recovered and the photoreceptor layer showed a loss of cells. The results show that the spectral damage types are distinct in the early phases, indicating that different mechanisms are involved. Yet, the end effect of both damage types after exposure at doses up to 2.5 x the funduscopic threshold is remarkably similar and consists of local photoreceptor lesions.

Spectral transmittance of the rat lens

The spectral transmittance of the isolated rat lens for radiation of wavelengths from 300 to 700 nm was measured using a spectrophotometer. Transmittance decreased from 93-95% at 700 nm, to 70-80% at 400 nm, 50-60% at 360 nm, 4-18% at 320 nm, and to < 2% at 310-300 nm. These results indicate that the rat lens is remarkably transparent for UVA radiation, which has consequences for rat vision, and for interpretation of earlier studies on retinal light damage.

The effects of two stereoisomers of N-acetylcysteine on photochemical damage by UVA and blue light in rat retina.

Abstract High doses of light can cause damage to the retina, e.g. during intraocular surgery. Previously, thiols have been demonstrated to protect against retinal damage in various damage models. Such protection is very promising for clinical practice. Retinal light damage can be caused by a relatively short exposure to high irradiance levels. These conditions occur during intraocular surgery. In the current study we therefore investigated whether the thiol N‐acetylcysteine protects against retinal light damage under high irradiance conditions in the rat retina. Two stereoisomers of this thiol were tested for protection against two spectrally denned types of retinal light damage. Shortly after administration N‐acetyl‐L‐cysteine in doses of 270–1000 mg/kg intraperitoneal protected against 380 nm (UVA) light but not against 470 nm (blue) light. Two hours after injection the protection had diminished. We observed no protection by the stereoisomer N‐acetyl‐D‐cysteine. From this study we conclude that N‐acetyl‐L‐cysteine protects stereospecifically against retinal damage in the UV but not in the visible part of the spectrum. This limits the possible applications.