Intervention With an Erythropoietin-Derived Peptide Protects Against Neuroglial and Vascular Degeneration During Diabetic Retinopathy.

OBJECTIVE:
Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy.
RESEARCH DESIGN AND METHODS:
After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 µg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 µg/kg pHBSP or control peptide).
RESULTS:
pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose.
CONCLUSIONS:
Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Diabetic retinopathy: quantitative variation in capillary basement membrane thickening in arterial or venous environments

Diabetes mellitus was induced in male beagles by a single injection of an alloxan and streptozotocin cocktail and fasting blood sugar levels maintained between 15 and 20 mmol/l. Five years after induction of diabetes, three diabetic animals were sacrificed, together with sex and age-matched controls, and the retinas fixed for either transmission electron microscopy (TEM) or trypsin digestion. In TEM specimens, capillaries in close proximity to the major vessels were designated as either AE (arterial environment) or VE (venous environment) and the thickness of their basement membranes (BMs) measured using an image analyser based two dimensional morphometric analysis system. Results show that the BMs of retinal capillaries from the diabetic dogs were significantly thicker than those from control dogs. Furthermore, within the diabetic group the AE capillaries had thicker BMs than VE capillaries (p less than or equal to 0.05). The controls, however, showed no significant difference in BM thickness between AE and VE capillaries. Although many of the capillaries designated as AE or VE would actually have been derived from the opposite side of the circulation, with respect to BM thickness, they conformed to values of their specific group. The conclusion is that diabetic capillaries are more vulnerable to BM thickening in an arterial environment than in a venous environment.

Diabetes-related adduct formation and retinopathy

The pathogenesis of diabetic retinopathy is complex, reflecting the array of systemic and tissue-specific metabolic abnormalities. A range of pathogenic pathways are directly linked to hyperglycaemia and dyslipidaemia, and the retina appears to be exquisitely sensitive to damage. Establishing the biochemical and molecular basis for this pathology remains an important research focus. This review concentrates on the formation of a range of protein adducts that form after exposure to modifying intermediates known to be elevated during diabetes. These so-called advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are thought to play an important role in the initiation and progression of diabetic retinopathy, and mechanisms leading to dysfunction and death of various retinal cells are becoming understood. Perspective is provided on AGE/ALE formation in the retina and the impact that such adducts have on retinal cell function. There will be emphasis placed on the role of the receptor for AGEs and how this may modulate retinal pathology, especially in relation to oxidative stress and inflammation. The review will conclude by discussion of strategies to inhibit AGE/ALE formation or harmful receptor interactions in order to prevent disease progression from the point of diabetes diagnosis to sight-threatening proliferative diabetic retinopathy and diabetic macular oedema.

OBSERVATIONS ON THE EFFECTS OF LONG-TERM WITHDRAWAL ON CARCASS COMPOSITION AND RESIDUE CONCENTRATIONS IN CLENBUTEROL-MEDICATED CATTLE

The detection of the illegal use of clenbuterol (CBL) as a growth promoter has relied on detecting residual concentrations of the drug in body fluids or tissues. Analysis of retinal extracts has recently been shown to considerably extend the detection period following withdrawal. The withdrawal periods required to eliminate residues from the liver and retina were investigated by medicating 20 cattle with CBL for 30 days; 6 control animals remained unmedicated. Residual concentrations were monitored throughout this period and for the subsequent 140 days. Concurrent changes in muscle areas and backfat thicknesses were recorded by ultrasound.

Isoprenylation of the G protein gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C

We have previously shown that isoprenylation and/or additional pest-translational processing of the G protein gamma(1) subunit carboxyl terminus is required for beta(1) gamma(1) subunit stimulation of phospholipase C-beta(2) (PLC beta(2)) [Dietrich, A., Meister, M., Brazil, D., Camps, M., & Gierschik, P. (1994) Eur. J. Biochem. 219, 171-178]. To examine whether isoprenylation of the gamma(1) subunit alone is sufficient for beta(1) gamma(1)-mediated PLC beta(2) stimulation or whether any of the two subsequent modifications, proteolytic removal of the carboxyl-terminal tripeptide and/or carboxylmethylation, is required for this effect, nonisoprenylated recombinant beta(1) gamma(1) dimers were produced in baculovirus-infected insect cells, purified to near homogeneity, and then isoprenylated in vitro using purified recombinant protein farnesyltransferase. Analysis of the beta(1) gamma(1) dimer after in vitro farnesylation by reversed phase high-performance liquid chromatography followed by delayed extraction matrix-assisted laser desorption/ionization mass spectrometry confirmed that the gamma(1) subunit was carboxyl-terminally farnesylated but not proteolyzed and carboxylmethylated. Functional reconstitution of in vitro-farnesylated beta(1) gamma(1) dimers with a recombinant PLC beta(2) isozyme revealed that farnesylation rendered recombinant nonisoprenylated beta(1) gamma(1) dimers capable of stimulating PLC beta(2) and that the degree of this stimulation was only approximately 45% lower for in vitro-farnesylated beta(1) gamma(1) dimers than for fully modified native beta(1) gamma(1) purified from bovine retinal rod outer segments. Taken together, these results suggest that isoprenylation of the gamma subunit is both necessary and sufficient for beta gamma dimer-mediated stimulation of phospholipase C.

GENOTYPE-PHENOTYPE ASSOCIATIONS IN NEOVASCULAR AGE-RELATED MACULAR DEGENERATION.

PURPOSE:To examine associations between recognized genetic susceptibility loci and angiographic subphenotypes of the neovascular variant of age-related macular degeneration (nvAMD).METHODS:Participants (247 nvAMD, 52 early age-related macular degeneration [AMD], and 103 controls) were genotyped (complement factor H and ARMS2/HTRA1). nvAMD participants were assigned to one of two subcategories: mainly classic or mainly occult (based on the proportions of classic and occult choroidal neovascularization). nvAMD and early AMD were reassigned to two groups based on the extent and severity of drusen (retinal pigment epithelium dysfunction or not). Univariate and multivariate analysis were used to examine for associations between participant characteristics and genetic loci after adjusting for age, smoking status, and history of cardiovascular disease.RESULTS:Univariate analysis confirmed the known significant associations between AMD stage and age, hypertension, and a history of cardiovascular disease. Those with retinal pigment epithelium dysfunction (F = 5.46; P = 0.02) or a positive smoking history (F = 3.89; P = 0.05) were more likely to have been classified as having mainly an occult rather than a mainly classic lesion. Multivariate analysis showed that significant associations were noted with the number of ARMS2/HTRA1 risk alleles (P

A novel GUCY2D mutation, V933A, causes central areolar choroidal dystrophy

Purpose: To identify the genetic cause of central areolar choroidal dystrophy (CACD) in a large Northern Irish family.
Methods: We previously reported linkage of the locus for CACD in this family to an interval of approximately 5 cM on chromosome 17p13 flanked by polymorphic markers D17S1810 and CHLC GATA7B03. We undertook sequence capture, massively-parallel sequencing and computational alignment, base-calling and annotation to identify a causative mutation. Conventional sequencing was used to confirm the results.
Results: Deep sequencing identified a single-base substitution in guanylate cyclase 2D, membrane (retina-specific) (GUCY2D). The novel mutation segregated with the disease phenotype and resulted in substitution of valine by alanine at position 933, within the catalytic domain of the protein. It altered a motif that is strongly conserved in a large number of distantly related proteins across several species, and was predicted to have a damaging effect on protein activity.
Conclusion: Mutations in GUCY2D have previously been associated with dominant cone rod dystrophies (CORD6) and recessive forms of Leber's congenital amaurosis (LCA). This is the first report of GUCY2D mutation causing CACD and adds to our understanding of genotype-phenotype correlation in this heterogeneous group of choroidoretinal dystrophies.

Visual function and dysfunction in early and late age-related maculopathy

Late age-related maculopathy (ARM) is responsible for the majority of blind registrations in the Western world among persons over 50 years of age. It has devastating effects on quality of life and independence and is becoming a major public health concern. Current treatment options are limited and most aim to slow progression rather than restore vision; therefore, early detection to identify those patients most suitable for these interventions is essential. In this work, we review the literature encompassing the investigation of visual function in ARM in order to highlight those visual function parameters which are affected very early in the disease process. We pay particular attention to measures of acuity, contrast sensitivity (CS), cone function, electrophysiology, visual adaptation, central visual field sensitivity and metamorphopsia. We also consider the impact of bilateral late ARM on visual function as well as the relationship between measures of vision function and self-reported visual functioning. Much interest has centred on the identification of functional changes which may predict progression to neovascular disease; therefore, we outline the longitudinal studies, which to date have reported dark-adaptation time, short-wavelength cone sensitivity, colour-match area effect, dark-adapted foveal sensitivity, foveal flicker sensitivity, slow recovery from glare and slower foveal electroretinogram implicit time as functional risk factors for the development of neovascular disease. Despite progress in this area, we emphasise the need for longitudinal studies designed in light of developments in disease classification and retinal imaging, which would ensure the correct classification of cases and controls, and provide increased understanding of the natural course and progression of the disease and further elucidate the structure-function relationships in this devastating disorder.

eNOS overexpression exacerbates vascular closure in the obliterative phase of OIR and increases angiogenic drive in the subsequent proliferative stage

Purpose: In ischemic retinopathies, the misdirection of reparative angiogenesis away from the hypoxic retina leads to pathologic neovascularization. Thus, therapeutic strategies that reverse this trend would be extremely beneficial. Nitric oxide (NO) produced by endothelial nitric oxide synthase (eNOS) is an important mediator of vascular endothelial growth factor (VEGF) function facilitating vascular growth and maturation. However, in addition to NO, eNOS can also produce superoxide (O), exacerbating pathology. Here, our aim was to investigate the effect of eNOS overexpression on vascular closure and subsequent recovery of the ischemic retina.

Methods: Mice overexpressing eNOS-GFP were subjected to oxygen-induced retinopathy (OIR) and changes in retinal vascularization quantified. Background angiogenic drive was assessed during vascular development and in aortic rings. NOS activity was measured by Griess assay or conversion of radiolabeled arginine to citrulline, nitrotyrosine (NT), and superoxide by immunolabeling and dihydroethidium fluorescence and VEGF by ELISA.

Results: In response to hyperoxia, enhanced eNOS expression led to increased NOS-derived superoxide and dysfunctional NO production, NT accumulation, and exacerbated vessel closure associated with tetrahydrobiopterin (BH) insufficiency. Despite worse vaso-obliteration, eNOS overexpression resulted in elevated hypoxia-induced angiogenic drive, independent of VEGF production. This correlated with increased vascular branching similar to that observed in isolated aortas and during development. Enhanced recovery was also associated with neovascular tuft formation, which showed defective NO production and increased eNOS-derived superoxide and NT levels.

Conclusions: In hyperoxia, reduced BH bioavailability causes overexpressed eNOS to become dysfunctional, exacerbating vaso-obliteration. In the proliferative phase, however, eNOS has important prorepair functions enhancing angiogenic growth potential and recovery in ischemia. © 2012 The Association for Research in Vision and Ophthalmology, Inc.

Microvascular lesions of diabetic retinopathy: clues towards understanding pathogenesis?

Retinopathy is a major complication of diabetes mellitus and this condition remains a leading cause of blindness in the working population of developed countries. As diabetic retinopathy progresses a range of neuroglial and microvascular abnormalities develop although it remains unclear how these pathologies relate to each other and their net contribution to retinal damage. From a haemodynamic perspective, evidence suggests that there is an early reduction in retinal perfusion before the onset of diabetic retinopathy followed by a gradual increase in blood flow as the complication progresses. The functional reduction in retinal blood flow observed during early diabetic retinopathy may be additive or synergistic to pro-inflammatory changes, leucostasis and vaso-occlusion and thus be intimately linked to the progressive ischaemic hypoxia and increased blood flow associated with later stages of the disease. In the current review a unifying framework is presented that explains how arteriolar dysfunction and haemodynamic changes may contribute to late stage microvascular pathology and vision loss in human diabetic retinopathy.

Heterogeneity in cytosolic calcium regulation among different microvascular smooth muscle cells of the rat retina

Rat retinae were dissociated to yield intact microvessels 7 to 42 microm in diameter. These were loaded with fura-2 AM and single fragments anchored down in a recording bath. Intracellular Ca(2+) levels from 20- to 30-microm sections of vessel were estimated by microfluorimetry. The vessels studied were identified as metarterioles and arterioles. Only the microvascular smooth muscle cells loaded with fura-2 AM and changes in the fluorescence signal were confined to these cells: Endothelial cells did not make any contribution to the fluorescence signal nor did they contribute to the actions of the drugs. Caffeine (10 mM) or elevated K(+) (100 mM) produced a transient rise in cell Ca(2+) in the larger vessels (diameters >18 microm) but had no effect on smaller vessels (diameters 30 min) on washing out the endothelin and the vessel failed to relax. These results demonstrate heterogeneity between smaller and larger retinal vessels with regard to Ca(2+) mobilisation and homogeneity with respect to the actions of vasoactive peptides.

Drusen prevalence and pigmentary changes in Caucasians aged 18-54 years

Aims and Purpose The aim of this study was to describe the prevalence and characteristics of drusen and pigmentary changes in a middle-aged population.

Methods Retinal images from 500 individuals aged 18–54 years were included. The source of participants was two UK optometry practices. Retinal images were graded using the Wisconsin Age-Related
Maculopathy Grading System. However, owing to the relatively young age of the population studied, a new category of drusen of smaller size (o31.5mm) was introduced.

Results Drusen were identi?ed within the central macular grid in 91.48% of all gradable eyes and in 444 subjects. Drusen sized o31.5mm were present in 89.7% of eyes, drusen sized 431.5mm and o63mm were present in 45.9% of all eyes and drusen 463mm and o125mm were present in only 1.7% of eyes. No eye had drusen larger or equal to 125mm. Very few eyes (1.2%) showed pigmentary changes within the grid. Drusen load increased with increasing age, P o0.001.

Conclusions The frequency of drusen in a younger Caucasian population aged 18–54 years is high, with 91.48% of all gradable eyes having drusen. The most frequent drusen subtype was hard distinct drusen o31.5mm. No druse greater or equal in size to 125mm was seen. Pigmentary changes are rare. Eye(2012) 26, 1357–1362; doi:10.1038/eye.2012.165; published online 17 August 2012

A genome-wide association study provides evidence for association of chromosome 8p23 (MYP10) and 10q21.1 (MYP15) with high myopia in the French population.

PURPOSE. Myopia is a complex trait affected by both genetic and environmental factors. High myopia is associated with increased risk of sight-threatening eye disorders such as retinal detachment. The purpose of this genome-wide association study was to identify susceptibility genes contributing to high myopia in the French population. METHODS. High myopic cases were genotyped using Affymetrix SNP 6.0 chips and population controls were selected from the GABRIEL French dataset in which samples were genotyped by Illumina Human610 quad array. The association study was conducted using 152,234 single nucleotide polymorphisms that were present on both manufacturers' chips in 192 high myopic cases and 1064 controls to identify associated regions. Imputation was performed on peak regions. RESULTS. Associations were found at known myopia locus MYP10 on chromosome 8p23 and MYP15 on chromosome 10q21.1. Rs189798 (8p23) and rs10825992 (10q21.1) showed the strongest associations in these regions (P=6.32x10-7 and P=2.17x10-5, respectively). The imputed results at 8p23 showed 2 peaks of interest. The first spanned 30kb including rs189798 between MIR4660 and PPP1R3B with the most significant association at rs17155227 (P=1.07x10-10). The second novel peak was 4kb in length, encompassing MIR124-1 and the MSRA gene, with the strongest association at rs55864141 (P=1.30x10-7). The peak of imputed data at 10q21.1 was 70kb in length between ZWINT and MIR3924, with rs3107503 having the lowest P value (P=1.54x10-7). CONCLUSION. We provide evidence for the association of MYP10 at 8p23 and MYP15 at 10p21.1 with high myopia in the French population and refine these regions of association.

Good news-bad news:the Yin and Yang of immune privilege in the eye

The eye and the brain are prototypical tissues manifesting immune privilege (IP) in which immune responses to foreign antigens, particularly alloantigens are suppressed, and even completely inhibited. Explanations for this phenomenon are numerous and mostly reflect our evolving understanding of the molecular and cellular processes underpinning immunological responses generally. IP is now viewed as a property of many tissues and the level of expression of IP varies not only with the tissue but with the nature of the foreign antigen and changes in the limited conditions under which privilege can operate as a mechanism of immunological tolerance. As a result, IP functions normally as a homeostatic mechanism preserving normal function in tissues, particularly those with highly specialized function and limited capacity for renewal such as the eye and brain. However, IP is relatively easily bypassed in the face of a sufficiently strong immunological response, and the privileged tissues may be at greater risk of collateral damage because its natural defenses are more easily breached than in a fully immunocompetent tissue which rapidly rejects foreign antigen and restores integrity. This two-edged sword cuts its swathe through the eye: under most circumstances, IP mechanisms such as blood-ocular barriers, intraocular immune modulators, induction of T regulatory cells, lack of lymphatics, and other properties maintain tissue integrity; however, when these are breached, various degrees of tissue damage occur from severe tissue destruction in retinal viral infections and other forms of uveoretinal inflammation, to less severe inflammatory responses in conditions such as macular degeneration. Conversely, ocular IP and tumor-related IP can combine to permit extensive tumor growth and increased risk of metastasis thus threatening the survival of the host.