Maria G. Kotoula

Systems biology-based analysis implicates a novel role for vitamin D metabolism in the pathogenesis of age-related macular degeneration

Vitamin D has been shown to have anti-angiogenic properties and to play a protective role in several types of cancer, including breast, prostate and cutaneous melanoma. Similarly, vitamin D levels have been shown to be protective for risk of a number of conditions, including cardiovascular disease and chronic kidney disease, as well as numerous autoimmune disorders such as multiple sclerosis, inflammatory bowel diseases and type 1 diabetes mellitus. A study performed by Parekh et al. was the first to suggest a role for vitamin D in age-related macular degeneration (AMD) and showed a correlation between reduced serum vitamin D levels and risk for early AMD. Based on this study and the protective role of vitamin D in diseases with similar pathophysiology to AMD, we examined the role of vitamin D in a family-based cohort of 481 sibling pairs. Using extremely phenotypically discordant sibling pairs, initially we evaluated the association of neovascular AMD and vitamin D/sunlight-related epidemiological factors. After controlling for established AMD risk factors, including polymorphisms of the genes encoding complement factor H (CFH) and age-related maculopathy susceptibility 2/HtrA serine peptidase (ARMS2/HTRA1), and smoking history, we found that ultraviolet irradiance was protective for the development of neovascular AMD (p = 0.001). Although evaluation of serum vitamin D levels (25-hydroxyvitamin D [25(OH)D]) was higher in unaffected individuals than in their affected siblings, this finding did not reach statistical significance.Based on the relationship between ultraviolet irradiance and vitamin D production, we employed a candidate gene approach for evaluating common variation in key vitamin D pathway genes (the genes encoding the vitamin D receptor [VDR]; cytochrome P450, family 27, subfamily B, polypeptide 1 [CYP27B1]; cytochrome P450, family 24, subfamily A, polypeptide 1 [CYP24A1]; and CYP27A1) in this same family-based cohort. Initial findings were then validated and replicated in the extended family cohort, an unrelated case-control cohort from central Greece and a prospective nested case-control population from the Nurses Health Study and Health Professionals Follow-Up Studies, which included patients with all subtypes of AMD for a total of 2,528 individuals. Single point variants in CYP24A1 (the gene encoding the catabolising enzyme of the vitamin D pathway) were demonstrated to influence AMD risk after controlling for smoking history, sex and age in all populations, both separately and, more importantly, in a meta-analysis. This is the first report demonstrating a genetic association between vitamin D metabolism and AMD risk. These findings were also supplemented with expression data from human donor eyes and human retinal cell lines. These data not only extend previous biological studies in the AMD field, but further emphasise common antecedents between several disorders with an inflammatory/immunogenic component such as cardiovascular disease, cancer and AMD.

Blood velocity pulse quantification in the human conjunctival pre-capillary arterioles.

Axial red blood cell velocity pulse was quantified throughout its period by high speed video microcinematography in the human eye. In 30 conjunctival precapillary arterioles (6 to 12 microm in diameter) from 15 healthy humans, axial velocities ranged from 0.4 (the minimum of all the end diastolic values) to 5.84 mm/s (the maximum of all the peak systolic values). With the velocity pulse properly quantified, two parameters can be estimated: (1) the average velocity of the pulse during a cardiac cycle AVV (average velocity value) and (2) the magnitude of the pulsation using Pourcelots resistive index RI. These parameters are important for the estimation of other hemodynamic parameters such as the average volume flow and the average shear stress. The results of this study revealed that the AVV in the human precapillary arterioles ranged between 0.52 and 3.26 mm/s with a mean value for all microvessels of 1.66 mm/s+/-0.11(SE). The RI ranged between 35.5% and 81.8% with a mean value of 53.1%+/-2.2. Quantitative information was obtained for the first time on the velocity pulse characteristics just before the human capillary bed.

Convergence of linkage, gene expression and association data demonstrates the influence of the RAR-related orphan receptor alpha (RORA) gene on neovascular AMD: A systems biology based approach

To identify novel genes and pathways associated with AMD, we performed microarray gene expression and linkage analysis which implicated the candidate gene, retinoic acid receptor-related orphan receptor alpha (RORA, 15q). Subsequent genotyping of 159 RORA single nucleotide polymorphisms (SNPs) in a family-based cohort, followed by replication in an unrelated case-control cohort, demonstrated that SNPs and haplotypes located in intron 1 were significantly associated with neovascular AMD risk in both cohorts. This is the first report demonstrating a possible role for RORA, a receptor for cholesterol, in the pathophysiology of AMD. Moreover, we found a significant interaction between RORA and the ARMS2/HTRA1 locus suggesting a novel pathway underlying AMD pathophysiology.

Influence of ROBO1 and RORA on risk of age-related macular degeneration reveals genetically distinct phenotypes in disease pathophysiology.

ROBO1 is a strong candidate gene for age-related macular degeneration (AMD) based upon its location under a linkage peak on chromosome 3p12, its expression pattern, and its purported function in a pathway that includes RORA, a gene previously associated with risk for neovascular AMD. Previously, we observed that expression of ROBO1 and RORA is down-regulated among wet AMD cases, as compared to their unaffected siblings. Thus, we hypothesized that contribution of association signals in ROBO1, and interaction between these two genes may be important for both wet and dry AMD. We evaluated association of 19 single nucleotide polymorphisms (SNPs) in ROBO1 with wet and dry stages of AMD in a sibling cohort and a Greek case-control cohort containing 491 wet AMD cases, 174 dry AMD cases and 411 controls. Association signals and interaction results were replicated in an independent prospective cohort (1070 controls, 164 wet AMD cases, 293 dry AMD cases). The most significantly associated ROBO1 SNPs were rs1387665 under an additive model (meta P = 0.028) for wet AMD and rs9309833 under a recessive model (meta P = 6×10−4) for dry AMD. Further analyses revealed interaction between ROBO1 rs9309833 and RORA rs8034864 for both wet and dry AMD (interaction P<0.05). These studies were further supported by whole transcriptome expression profile studies from 66 human donor eyes and chromatin immunoprecipitation assays from mouse retinas. These findings suggest that distinct ROBO1 variants may influence the risk of wet and dry AMD, and the effects of ROBO1 on AMD risk may be modulated by RORA variants.

Plasma and aqueous humour levels of ghrelin in open-angle glaucoma patients.

Background:  To compare aqueous humour and plasma levels of ghrelin, a peptide recently identified in human eyes, in patients with open‐angle glaucoma and controls.

Resolution of Macular Edema in Idiopathic Juxtafoveal Telangiectasis Using PDT

A 57-year-old woman was treated by photodynamic therapy for macular edema due to idiopathic juxtafoveal telangiectasis (presumed type 1A) without subretinal neovascularization. Initial visual acuity of the treated eye was 20/200 and it improved to 20/40 by 3 months after the photodynamic therapy session. Visual acuity remained stable 32 months after the treatment. Color photographs and fundus fluorescein angiography before and after photodynamic therapy revealed regression of hemorrhages, exudates, and fluorescein leakage. Photodynamic therapy has long-term benefits for the patient with idiopathic juxtafoveal telangiectasis, presumed type 1A, because it can improve visual acuity and macular edema.

Bimatoprost and Bimatoprost/Timolol Fixed Combination in Patients with Open-Angle Glaucoma and Ocular Hypertension

PURPOSE To investigate the intraocular pressure (IOP) lowering effect of bimatoprost (BIM) 0.03% and the potential additional effect of the BIM 0.03%/timolol 0.5% fixed combination (BTFC) in eyes with ocular hypertension, primary open-angle glaucoma, or exfoliation glaucoma. METHODS Following an appropriate washout period that varied with previous medication, participants with ocular hypertension, primary open-angle glaucoma, or exfoliation glaucoma were treated with evening-dosed BIM for 5 weeks. They were then given evening-dosed BTFC for another 5 weeks. One randomly selected eye was evaluated. Goldmann applanation tonometry was performed by the same investigator at 8 a.m., 12 noon, 4 p.m., and 8 p.m. at baseline and at the end of each treatment period. RESULTS Thirty-three participants completed the study. Three patients discontinued because of local adverse effects during the BIM treatment period. The mean diurnal IOP (mean ± SD) at baseline, on BIM, and on BTFC were 24.8 ± 5.4, 17.3 ± 3.5, and 14.9 ± 3.1 mmHg, respectively (repeated measures analysis of variance, P < 0.001 for all pairwise comparisons). The individual time-point IOP values showed similar significant reductions. The percentage of IOP reduction from baseline was 30.2% for BIM and 39.9% for the BTFC. The mean ± SD diurnal fluctuation at baseline was 6.8 ± 3.2 mmHg, which decreased to 4.0 ± 3.1 and 2.9 ± 1.4 mmHg on BIM and BTFC, respectively (P < 0.05 for both treatments versus baseline). CONCLUSIONS Both BIM 0.03% and the BTFC were effective in lowering IOP in eyes with ocular hypertension and open-angle glaucoma. However, the fixed combination provided an additional statistically significant reduction in IOP compared with BIM 0.03%.

FLT1 Genetic Variation Predisposes to Neovascular AMD in Ethnically Diverse Populations and Alters Systemic FLT1 Expression

PURPOSE Current understanding of the genetic risk factors for age-related macular degeneration (AMD) is not sufficiently predictive of the clinical course. The VEGF pathway is a key therapeutic target for treatment of neovascular AMD; however, risk attributable to genetic variation within pathway genes is unclear. We sought to identify single nucleotide polymorphisms (SNPs) associated with AMD within the VEGF pathway. METHODS Using a tagSNP, direct sequencing and meta-analysis approach within four ethnically diverse cohorts, we identified genetic risk present in FLT1, though not within other VEGF pathway genes KDR, VEGFA, or VASH1. We used ChIP and ELISA in functional analysis. RESULTS The FLT1 SNPs rs9943922, rs9508034, rs2281827, rs7324510, and rs9513115 were significantly associated with increased risk of neovascular AMD. Each association was more significant after meta-analysis than in any one of the four cohorts. All associations were novel, within noncoding regions of FLT1 that do not tag for coding variants in linkage disequilibrium. Analysis of soluble FLT1 demonstrated higher expression in unaffected individuals homozygous for the FLT1 risk alleles rs9943922 (P = 0.0086) and rs7324510 (P = 0.0057). In silico analysis suggests that these variants change predicted splice sites and RNA secondary structure, and have been identified in other neovascular pathologies. These data were supported further by murine chromatin immunoprecipitation demonstrating that FLT1 is a target of Nr2e3, a nuclear receptor gene implicated in regulating an AMD pathway. CONCLUSIONS Although exact variant functions are not known, these data demonstrate relevancy across ethnically diverse genetic backgrounds within our study and, therefore, hold potential for global efficacy.

Detection of Macular Photocoagulation Scars with Confocal Infrared Reflection Imaging

BACKGROUND AND OBJECTIVE To study the diagnostic reliability (specificity and sensitivity) of confocal infrared reflection in detecting threshold argon laser photocoagulation scars in the macula. PATIENTS AND METHODS Fifty-six maculae with diabetic macular edema were evaluated by biomicroscopic slit-lamp fundus examination, digital color fundus photography, digital fundus fluorescein angiography, and digital infrared reflection images. Three examiners evaluated whether the eye had undergone any laser photopexy in the macula. RESULTS Sensitivity, specificity, and false-positive and false-negative results were calculated for each method. Fluorescein fundus angiography and infrared imaging, although using different approaches, both detect pigment epithelium changes such as laser scars. It seems that both methods are of equal specificity. On the other hand, both biomicroscopic fundus examination and digital color photography showed poor reliability. CONCLUSION Infrared reflection imaging is an easy, noninvasive method with excellent sensitivity and specificity in detecting photocoagulation scars from previous threshold laser treatment. It may be useful in estimating photocoagulated areas, especially if threshold treatment has been applied.

Unexplained choroidal embolisation: remember the antiphospholipid syndrome

In January, 2005, a 58-year-old woman presented complaining of bilateral sudden painless loss of vision, followed by perception of fl oaters. She had a history of colon carcinoma 2 years previously, and had been diseasefree since her treatment. Her visual acuity was 20/100 in the right eye and 20/40 in the left. Fundus examination showed scattered multiple choroidal infarcts bilaterally in the posterior pole surrounding the macular area (fi gure A). A 2+ vitreal fl are was also seen bilaterally. Fluorescein angiography showed hypofl uoresence at the infarcted sites in early stages and hyperfl uoresence at the late stages. Physical examination showed only a grade 2/6 systolic murmur of the heart. Two-dimensional transoesophageal echocardiography showed mobile vegetations of the mitral valve. Extensive laboratory blood tests, including tumour markers, C-reactive protein, fi brinogen, rheumatoid factor, antinuclear antibodies, anti-double-stranded DNA antibodies, antineutrophil cytoplasmic antibodies, and C3 and C4 complement components, showed only a mild prolongation of activated partial thromboplastin time (37 s). Serological tests for bacterial and viral infections were negative. Long-duration incubation of blood and aqueoushumour cultures were negative. Abdominal ultrasonography, CT of the chest and abdomen, brain MRI, carotid doppler tests, and colonoscopy showed no abnormalities. Further testing gave a positive result for IgG anticardiolipin antibodies (IgG-aCL), with a binding index of 370 (index cut-off =100, which corresponds to the cut-off of 15–20 GPL units of the commercially available assays) on a highly sensitive, specifi c, and inter nationally accepted ELISA. She was also positive for IgM-aCL and lupus anticoagulant (ratio: 1·65; normal <1·2) by the diluted Russell’s viper venom test (HemosIL, Instrumentation Laboratory Company, Lexington, MA, USA). The IgG-aCL titre remained positive 6 weeks and 12 weeks after the fi rst test (binding index 456 and 379, respectively, which correspond to more than 80 GPL units with the conventional assays). Antiphospholipid syndrome (APS) accom panied by non-bacterial thrombotic endocarditis was considered, and anticoagulant treatment with acenocoumarol was started with target INR 3·0–3·5. After 4 weeks, her visual acuity had improved (right eye 20/50; left eye 20/25). Vitreal fl are disappeared 6 weeks after presentation. 8 weeks after her intitial presentation, transoesophageal echocardiography and cardiac MRI showed no abnormalities. At a 5-month follow-up appoint ment, her visual acuity had improved further (right eye 20/25; left eye 20/20). 1 year after her initial presentation, the patient’s fundus (fi gure B), visual acuity, and general health have remained stable. When last seen in May, 2006, she remained well. APS is an autoimmune disorder characterised by arterial or venous thrombosis, recurrent fetal loss, and circulating antiphospholipid antibodies. It has been implicated in a range of ocular disorders. The clinical features of ocular involvement seem to be diff erent in primary and secondary APS—vasculopathic eye disease involving retinal and choroidal vessels seems to be typical in the primary form, whereas retinal thrombosis is more common in APS associated with systemic lupus erythematosus. In our patient, APS was the main mechanism leading to choroidal infarction. Non-bacterial thrombotic endocarditis can accompany malignant disorders whether or not they are associated with APS. In our case, we were not able to detect a relapse of cancer, which is in accordance with a prospective study showing high aCL titres in some cancer patients even during clinical remission of the disease. These associations suggest the need for thorough followup for thrombotic events in aCL-positive cancer. Additionally, unexplained ocular manifestations should alert physicians for APS presence because the ophthalmological symptoms can be the fi rst clinically evident manifestations of this disorder, and anticoagulant treatment should be started as soon as possible.