Patricia Fernández-Robredo

Measurement of Factor H Variants in Plasma Using Variant-Specific Monoclonal Antibodies: Application to Assessing Risk of Age-Related Macular Degeneration

PURPOSE The Y402H polymorphism in the complement regulator factor H (fH) is strongly associated with age-related macular degeneration (AMD) across diverse populations. Persons homozygous for histidine at this position have up to 12-fold greater risk for AMD than those homozygous for tyrosine. Knowledge of fH-Y402H status is, therefore, valuable in predicting risk and focusing preventive measures in the elderly. This knowledge requires genetic analysis, which is unavailable in most laboratories and which provides no information about the levels of fH protein, a putative linked determinant of disease risk. METHODS The authors describe novel monoclonal antibodies that distinguish the two fH allelic variants in plasma. ELISA with these antibodies not only reliably identifies the fH-Y402H status, confirmed by genotyping, but also quantifies the concentration of total fH and the fH-Y402 and fH-H402 variants. RESULTS In young adult control subjects, mean fH concentration was 233 mg/L. In elderly control subjects, mean fH concentration was 269 mg/L, whereas in a matching AMD cohort, mean fH concentration was 288 mg/L. Total fH concentration was similar in each subgroup of young and elderly control subjects; however, in the AMD group, fH concentration was significantly higher in the heterozygous subgroup. Measurement of the two variants in this subgroup showed that both were elevated to a similar degree. CONCLUSIONS The novel monoclonal antibody MBI-7 was used to develop a robust assay for measurement of fH and the variants in plasma. The simplicity of the assay means that it may be used by any clinical laboratory to identify polymorphic status and to quantify plasma levels in persons at risk for AMD.

Relevance of Complement Factor H–Related 1 (CFHR1) Genotypes in Age-Related Macular Degeneration

PURPOSE Age-related macular degeneration (AMD) has a strong genetic component with a major locus at 1q31, including the complement factor H (CFH) gene. Detailed analyses of this locus have demonstrated the existence of one SNP haplotype block, carrying the CFH 402His allele, which confers increased risk for AMD, and two protective SNP haplotypes, one of them carrying a deletion of the CFHR1 and CFHR3 genes (ΔCFHR3-CFHR1). The purpose of these studies was to evaluate the contribution of newly described CFHR1 alleles to the association of the 1q31 locus with AMD. METHODS Two hundred fifty-nine patients and 191 age-matched controls of Spanish origin were included in a transversal case-control study using multivariate logistic regression analysis and ROC (receiver operating characteristic) statistics to generate and test models predictive of the development of AMD. RESULTS This study showed for the first time that a particular CFHR1 allotype, CFHR1*A, is strongly associated with AMD (odds ratio, 2.08; 95% confidence interval, 1.59-2.73; P<0.0001) and illustrate a peculiar genotype-phenotype correlation between the CFHR1 alleles and different diseases that may have important implications for understanding the pathophysiology of AMD. It also shows that CFHR1*A is in strong linkage disequilibrium with the CFH 402His allele, which provides additional candidate variants within the major risk haplotype at 1q31, promoting its association with AMD. Further, using the Spanish population as a model, the results showed that analysis of the CFHR1 genotypes provide sufficient information to delineate the individual risk of developing AMD. CONCLUSIONS The results support a relevant role of CFHR1 in the pathogenesis of AMD.

Optimization of 100 μm alginate-poly-L-lysine-alginate capsules for intravitreous administration.

The field of cell microencapsulation is advancing rapidly. Particle size plays a critical role in terms of biocompatibility and limits decisively its applicability. Producing reduced size microcapsules involves broadening the possibilities to employ this technology in the treatment of many disorders. Nervous system diseases (NSD) represent a clear example of that. This work describes the feasibility of reducing the size of alginate-poly-L-lysine-alginate (APA) microcapsules up to 100 μm in a highly monodisperse way using the novel Flow Focusing technique. C(2)C(12) myoblasts genetically engineered to express the triple reporter gene thymidine kinase-green fluorescent protein-luciferase (TGL) and secrete vascular endothelial growth factor soluble receptor 2 (VEGFR2, also known as KDR) were encapsulated for further characterization. Resulting new particles were assayed in vitro to explore whether their functionality might be affected due to the physicochemical changes arising from such dramatic size reduction. Not only were negative effects at this level not noticed in terms of cell viability, cell proliferation and KDR secretion, but once again the suitability of APA microcapsules was also reinforced against other microcapsule designs. Furthermore, the fully viable and functional biosystems were successfully administered in the intravitreous space of rats, where the activity of encapsulated cells was monitoring over 3 weeks.

Current Treatment Limitations in Age-Related Macular Degeneration and Future Approaches Based on Cell Therapy and Tissue Engineering

Age-related macular degeneration (AMD) is the leading cause of blindness in the Western world. With an ageing population, it is anticipated that the number of AMD cases will increase dramatically, making a solution to this debilitating disease an urgent requirement for the socioeconomic future of the European Union and worldwide. The present paper reviews the limitations of the current therapies as well as the socioeconomic impact of the AMD. There is currently no cure available for AMD, and even palliative treatments are rare. Treatment options show several side effects, are of high cost, and only treat the consequence, not the cause of the pathology. For that reason, many options involving cell therapy mainly based on retinal and iris pigment epithelium cells as well as stem cells are being tested. Moreover, tissue engineering strategies to design and manufacture scaffolds to mimic Bruchs membrane are very diverse and under investigation. Both alternative therapies are aimed to prevent and/or cure AMD and are reviewed herein.

Effect of Zeaxanthin and Antioxidant Supplementation on Vascular Endothelial Growth Factor (VEGF) Expression in Apolipoprotein-E Deficient Mice

Purpose: Apolipoprotein E-/- deficient (apoE-/-) mice develop hypercholesterolemia, atherosclerosis, and retinal alterations. We studied the oxidative status and vascular endothelial growth factor (VEGF) expression in murine retinal pigment epithelium-choroid (RPE) and Bruchs membrane (BM) ultrastructure and the effect of zeaxanthin. Methods: Ten 6-month-old C57BL/6 and 40 apoE-/- mice were divided into four groups (n = 10 each) and fed different diets for 12 weeks based on body weight: wild type (WT) and apoE-/- (AE-Con) mice standard rodent chow; apoE-/- mice (AES) standard rodent chow with ascorbate (800 mg/kg), tocopherol (1053 mg/kg), and zinc (135 mg/kg); and apoE-/- mice the last diet plus zeaxanthin with either 0.4 g/kg (AES-Z04) or 4 g/kg feed (AES-Z4). Results: Plasma total cholesterol (TC) and triglycerides (TG) and urine lipid peroxidation (isoprostanes) were measured. VEGF expression was determined in RPE-choroid homogenates. Zeaxanthin uptake was assessed in liver and retina by high-performance liquid chromatography; the retinal ultrastructure was analyzed by electron microscopy. AE-Con mice had higher plasma TC (p < 0.001) and TG (p < 0.001) values than WT mice. AE-Con mice had higher RPE-choroid-VEGF levels than WT mice (p < 0.05), BM thickness (p < 0.001) and presence of basal laminar deposits (BLamD). AES-Z4 resulted in lower urinary isoprostanes (p = 0.054) and lower VEGF expression in the RPE-choroid (p < 0.01). BM in the AES-Z4 animals had less confluent BLamD than AE-Con, AES, or AES-Z04 animals. Conclusions: We have reported that supplementation with zeaxanthin and antioxidants may delay or reverse alterations in the RPE and deposits in BM, and reduced VEGF expression observed in apoE-/- mice.

Transforming Growth Factor-Beta Inhibition Reduces Progression of Early Choroidal Neovascularization Lesions in Rats: P17 and P144 Peptides

The purpose of this study was to assess the effects of transforming growth factor beta (TGF-β) inhibitor peptides (P17 & P144) on early laser-induced choroidal neovascularization (LI-CNV) lesions in rats, two weeks after laser CNV induction. Seventy-one Long Evans rats underwent diode laser application in an established LI-CNV model. Baseline fluorescein angiography (FA) was performed 14 days following laser procedure, and treatments were administered 16 days post-laser application via different administration routes. Intravenous groups included control (IV-Control), P17 (IV-17), and P144 (IV-144) groups, whereas intravitreal groups included P17 (IVT-17), P144 (IVT-144), and a mixture of both peptides (IVT-17+144) (with fellow eyes receiving vehicle alone). CNV evolution was assessed using FA performed weekly for four weeks after treatment. Following sacrifice, VEGF, TGF-β, COX-2, IGF-1, PAI-1, IL-6, MMP-2, MMP-9, and TNF-α gene expression was assessed using RT-PCR. VEGF and p-SMAD2 protein levels were also assessed by western-blot, while MMP-2 activity was assessed with gelatin zymography. Regarding the FA analysis, the mean CNV area was lower from the 3rd week in IVT-17 and IVT-144 groups, and also from the 2nd week in IVT-17+144. Biochemical analysis revealed that gene expression was lower for VEGF and COX-2 genes in IV-17 and IV-144 groups, VEGF gene in IVT-17+144 group and MMP-2 gene in IVT-17 and IVT-144 groups. VEGF protein expression was also decreased in IV-17, IV-144, IVT-17 and IVT-144, whereas pSMAD-2 levels were lower in IV-17, IV-144 and IVT-17+144 groups. Zymogram analysis revealed decreased MMP-2 activity in IV-17, IV-144, IVT-17 and IVT-144 groups. These data suggest that the use of TGF-β inhibitor peptides (P17 & P144) decrease the development of early CNV lesions by targeting different mediators than those typically affected using current anti-angiogenic therapies. Its potential role in the treatment of early CNV appears promising as a single therapy or adjuvant to anti-VEGF drugs.

Prevalence and causes of bilateral blindness and visual impairment among institutionalized elderly people in Pamplona, Spain.

Purpose. To estimate the prevalence and causes of bilateral blindness and visual impairment in an urban institutionalized population aged 65 years and older. Methods. A total of 392 nursing home residents completed a standardized eye examination, including measurement of visual acuity (VA), intraocular pressure, lens opacity grading, indirect ophthalmoscopy, and photography of the macular area. The major causes of vision loss identified for all participants were blindness and visual impairment. Results. The average subject age was 82 years (65–97); women outnumbered men 263 to 129. The prevalence of bilateral blindness (VA ≥1.0 logarithm of the minimum angle of resolution [logMAR]) was 14.9% (43/288); the prevalence of visual impairment (VA ≥0.5 and 1.0 logMAR) was 31.9% (92/288). Blindness and visual impairment increased significantly with age (p<0.05), odds ratio (OR) 1.047 and 1.088, respectively. Cataract was the most common cause of bilateral blindness and visual impairment (27.9% and 44.6%, respectively) followed by pathologic myopia (23.3%) and age-related macular degeneration (AMD) (20.9%) for blindness, and by AMD (27.2%) and pathologic myopia (12%) for visual impairment. Fifty percent of subjects with visual loss had the potential for improved vision with medical or surgical intervention. Conclusions. Although the prevalences were high, these data are important since it is difficult for epidemiologic studies to include aged, institutionalized individuals, although their numbers are increasing. Recognition of the predominant causes of visual loss dependent on age is fundamental for early diagnosis and treatment of ocular diseases. Many cases of low vision can be treated with appropriate ophthalmologic care.

Effect of Lutein and Antioxidant Supplementation on VEGF Expression, MMP-2 Activity, and Ultrastructural Alterations in Apolipoprotein E-Deficient Mouse

Oxidative stress is involved in the pathogenesis of several diseases such as atherosclerosis and age-related macular degeneration (AMD). ApoE-deficient mice (apoE−/−) are a well-established model of genetic hypercholesterolemia and develop retinal alterations similar to those found in humans with AMD. Thus supplementation with lutein or multivitamin plus lutein and glutathione complex (MV) could prevent the onset of these alterations. ApoE−/− mice (n = 40, 3 months old) were treated daily for 3 months with lutein (AE-LUT) or MV (two doses): AE-MV15 (15 mg/kg/day) and AE-MV50 (50 mg/kg/day) and were compared to controls with vehicle (AE-C). Wild-type mice (n = 10) were also used as control (WT-C). ApoE−/− mice showed higher retinal lipid peroxidation and increased VEGF expression and MMP-2 activity, associated with ultrastructural alterations such as basal laminar deposits, vacuoles, and an increase in Bruchs membrane thickness. While lutein alone partially prevented the alterations observed in apoE−/− mice, MV treatment substantially reduced VEGF levels and MMP-2 activity and ameliorated the retinal morphological alterations. These results suggest that oxidative stress in addition to an increased expression and activity of proangiogenic factors could participate in the onset or development of retinal alterations of apoE−/− mice. Moreover, these changes could be prevented by efficient antioxidant treatments.

Antioxidant effects of vitamins C and E, multivitamin-mineral complex and flavonoids in a model of retinal oxidative stress: the ApoE-deficient mouse.

The aim of the current study was to investigate the biochemical changes in the plasma and retina of apolipoprotein E deficient (apoE-/-) mice supplemented with various antioxidants. Ten wild type (WT-Con, C57BL/6) and 10 apoE-/- (AE-Con) mice received drinking water. Another 40 apoE-/- animals were divided into four groups of 10 mice each and received either chromocarbe diethylamine (AE-CD, 50mg/kg), cyaninosides chloride (AE-CC, 50mg/kg), multivitamin complex (AE-MC, 50mg/kg), or vitamins C and E (AE-CE, 100mg/kg and 200IU/kg). Cholesterol, triglycerides, and lipid peroxidation (thiobarbituric acid reactive substances [TBARS]) were measured in plasma, and TBARS and nitric oxide metabolites (NOx) concentration were determined in retinal homogenates. Transmission electron microscopy was performed to examine the retinal ultrastructure. AE-Con mice had significantly (P<0.05) increased oxidative stress in the plasma and retina with augmented production of retinal NOx compared with WT-Con mice. Retinal TBARS decreased in the AE-MC and AE-CE animals compared with the AE-Con group (P<0.05 and P<0.01, respectively). Only AE-CE treatment significantly (P<0.01) lowered retinal NOx. Morphologic retinal changes in the AE-Con group decreased in the AE-CE and AE-MC groups. There were no significant changes in the biochemical and structural parameters in the AE-CD and AE-CC groups. AE-Con mice had increased systemic and retinal oxidative stress compared with WT-Con animals. Vitamins C and E and the multivitamin-mineral complex reduced oxidative stress and ultrastructural retinal changes in this murine model of hypercholesterolemia.

Oxidative Stress and Histological Changes in a Model of Retinal Phototoxicity in Rabbits

Photochemical damage occurs after an exposure to high energy radiation within the visible spectrum of light, causing morphological changes in the retina and the formation of superoxide anion. In this study we created a model of phototoxicity in rabbits. Animals were exposed to a light source for 120 minutes and were sacrificed immediately or one week after exposure. Outer nuclear layer and neurosensory retina thickness measurements and photoreceptor counting were performed. Caspase-1 and caspase-3 were assessed by immunohistochemistry. Dihydroethidium was used to evaluate in situ generation of superoxide and thiobarbituric acid reactive substances were measured in retinal homogenates as indicators of lipid peroxidation. The total antioxidant capacity and oxidative ratio were also determined. Retinas from rabbits exposed to light showed higher levels of lipid peroxidation than the unexposed animals and a decrease in outer nuclear layer and neurosensory retina thickness. Our study demonstrates that light damage produces an increase in retinal oxidative stress immediately after light exposure that decreases one week after exposure. However, some morphological alterations appear days after light exposure including apoptotic phenomena. This model may be useful in the future to study the protective effect of antioxidant substances or new intraocular lenses with yellow filters.