Introduction. Even though ocular refractive state is highly heritable and under strong genetic control, the identification of susceptibility genes remains a challenge. Several HGF (hepatocyte growth factor) gene variants have been associated with ocular refractive errors and corneal pathology. Purpose. Here, we assess the association of an HGF gene variant, previously reported as associated with hyperopia, and ocular biometric parameters in a multicenter Spanish cohort. Methods. An observational prospective multicenter cross-sectional study was designed, including a total of 403 unrelated subjects comprising 188 hyperopic children (5 to 17years) and 2 control groups: 52 emmetropic adolescents (13 to 17years) and 163 emmetropic young adults (18 to 28years). Each individual underwent a comprehensive eye examination including cycloplegic refraction, and topographic and ocular biometric analysis. Genomic DNA was extracted from oral swabs. HGF single nucleotide polymorphism (SNP) rs12536657 was genotyped. Genotypic, allelic, and logistic regression analyses were performed comparing the different groups. A quantitative trait association test analyzing several biometric parameters was also performed using generalized estimating equations (GEEs) adjusting for age and gender. Results. No association between rs12536657 and hyperopia was found through gender-adjusted logistic regression comparing the hyperopic children with either of the two control groups. Significant associations between mean topographic corneal curvature and rs12536657 for G/A (slope=+0.32; CI 95%: 0.04-0.60; p=0.023) and A/A (slope=+0.76; CI 95%: 0.12-1.40; p=0.020) genotypes were observed with the age- and gender-adjusted univariate GEE model. Both flat and steep corneal topographic meridians were also significantly associated with rs12536657 for the G/A and A/A genotypes. No association was found between rs12536657 and any other topographic or biometric measurements. Conclusions. Our results support a possible role for HGF gene variant rs12536657 in corneal curvature in our population. To our knowledge, this is the first multicenter quantitative trait association study of HGF genotypes and ocular biometric parameters comprising a pediatric cohort.

Corneal neovascularization (CNV) is associated with different ocular pathologies, including infectious keratitis, trachoma or corneal trauma. Pharmacological treatments based on the topical application of anti-VEGF therapies have been shown to be effective in the treatment and prevention of CNV. The aim of this work was to evaluate the effect of bevacizumab-loaded albumin nanoparticles in a rat model of CNV. Bevacizumab-loaded nanoparticles, either "naked" (B-NP) or coated with PEG 35,000 (B-NP-PEG), were administered once a day in the eyes of animals (10 mu L, 4 mg/mL every 24 h) during 7 days. Bevacizumab and dexamethasone were employed as controls and administered at the same dose every 12 h. At the end of the study, the area of the eye affected by neovascularization was about 2-times lower for animals treated with B-NP than with free bevacizumab. In the study, dexamethasone did not demonstrate an inhibitory effect on CNV at the employed dose. All of these results were confirmed by histopathological analysis, which clearly showed that eyes treated with nanoparticles displayed lower levels of fibrosis, inflammation and edema. In summary, the encapsulation of bevacizumab in human serum albumin nanoparticles improved its efficacy in an animal model of CNV.

PurposeWe sought to determine if specific genetic single nucleotide polymorphisms (SNPs) influence vascular endothelial growth factor inhibition response to ranibizumab in neovascular age-related macular degeneration (AMD). MethodsA total of 403 Caucasian patients diagnosed with exudative AMD were included. After a three-injection loading phase, a pro re nata regimen was followed. Nine SNPs from six different genes (CFH, CFB, ARMS2, SERPINF1, VEGFR1, VEGF) were genotyped. Non-genetic risk factors (gender, smoking habit and hypertension) were also assessed. Patients were classified as good or poor responders (GR or PR) according to functional (visual acuity), anatomical (foveal thickness measured by OCT) and fluid criteria (fluid/no fluid measured by OCT). ResultsHypertension was the environmental factor with the strongest poor response association with ranibizumab in the anatomical measure after the loading phase (p=0.0004; OR 3.7; 95% CI, 2.4-5.8) and after 12months of treatment (p=10(-5); OR 2.3; 95% CI, 1.5-3.4). The genetic variants rs12614 (CFB), rs699947 (VEGFA) and rs7993418 (VEGFR1) predisposed patients to a good response, while rs12603486 and rs1136287 (SERPINF1) were associated with a poor response. The protective genotype of rs800292 variant (CFH) was also associated with a poor anatomical response (p 0.0048). ConclusionAll these data suggest that genetics play an important role in treatment response in AMD patients.

Purpose: To explore the relationship between plasma C-reactive protein (CRP) levels, the main ARMS2 gene single nucleotide polymorphism (SNP), and gender in patients with neovascular age-related macular degeneration (wet AMD). Methods: Our study included 131 patients with wetAMD [age-related eye disease study (AREDS) category 4] and 153 control participants (AREDS category 1) from two Spanish retinal units. CRP levels were determined on blood samples by high-sensitivity ELISA assay. According to their CRP level, subjects were categorized into three well-established CRP categories: low (< 1.00 mg/L, L-CRP), moderate (1-2.99 mg/L, M-CRP), and high (> 3.00 mg/L, H-CRP). Genomic DNA was extracted from oral swabs using QIAcube (Qiagen, Hilden, Germany) and the A69S; rs10490924 of ARMS2 gene was genotyped by allelic discrimination with validated TaqMan assays (Applied Biosystems, Foster City, CA, USA). Univariate and multivariate logistic regression adjusted for age was used to analyze the genomic frequencies and to calculate odds ratio (OR) using SNPStats software. Results: Considering CRP risk categories, H-CRP group showed a significant [OR 4.0 (1.9-8.3)] association with wetAMD compared to L-CRP group. The risk genotypes of A69S (TT) SNPs showed an association with wetAMD risk [OR 14.0 (4.8-40.8)]. Interestingly, the gender stratification of the CRP categories showed a significant increase in CRP levels in wetAMD women compared with control women [OR 6.9 (2.2-22.3)] and with wetAMD men [OR 4.6 (1.3-16.9)]. In addition, the subgroup analysis of CRP within A69S genotype and gender showed a link in women between the A69S and CRP levels in the AMD group compared to controls [OR 4.2 (1.4-12.6)]. Conclusion: Our study shows, for the first time, that a different genetic association related with gender could contribute to AMD risk. As a consequence, the risk of female gender in the different CRP levels and A69S SNP frequencies could be taken into consideration to the established risk relationship of high levels of CRP and its association with risk A69S genotype.

Pigment epithelium derived factor (PEDF) is a potent antiangiogenic, neurotrophic, and neuroprotective molecule that is the endogenous inhibitor of vascular endothelial growth factor (VEGF) in the retina. An ex vivo gene therapy approach based on transgenic overexpression of PEDF in the eye is assumed to rebalance the angiogenic-antiangiogenic milieu of the retina, resulting in growth regression of choroidal blood vessels, the hallmark of neovascular age-related macular degeneration. Here, we show that rat pigment epithelial cells can be efficiently transfected with the PEDF-expressing non-viral hyperactive Sleeping Beauty transposon system delivered in a form free of antibiotic resistance marker miniplasmids. The engineered retinal and iris pigment epithelium cells secrete high (141 ± 13 and 222 ± 14 ng) PEDF levels in 72 hr in vitro. In vivo studies showed cell survival and insert expression during at least 4 months. Transplantation of the engineered cells to the subretinal space of a rat model of choroidal neovascularization reduces almost 50% of the development of new vessels.

We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.

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.

Purpose: Macrolide antibiotics are known to have various anti-inflammatory effects in addition to their antimicrobial activity, but the mechanisms are still unclear. The effect of azithromycin on inflammatory molecules in the lipopolysaccharide-induced rat conjunctivitis model was investigated. Methods: Twenty-four Wistar rats were divided into two groups receiving topical ocular azithromycin (15 mg/g) or vehicle. In total, six doses (25 mu l) were administered as one dose twice a day for three days before subconjunctival lipopolysaccharide injection (3 mg/ml). Before the rats were euthanized, mucus secretion, conjunctival and palpebral edema and redness were evaluated. Real-time polymerase chain reaction was used to determine gene expression for interleukin-6, cyclooxygenase-2, tumor necrosis factor-a, matrix metalloproteinase (MMP)-2, and MMP-9. Interleukin-6 was determined with enzyme-linked immunosorbent assay, nuclear factor-kappa B with western blot, and MMP-2 activity with gelatin zymogram. Four eyes per group were processed for histology and subsequent periodic acid-Schiff staining and CD68 for immunofluorescence. The Student t test or the Wilcoxon test for independent samples was applied (SPSS v.15.0). Results: Azithromycin-treated animals showed a significant reduction in all clinical signs (p<0.05) compared to controls. Interleukin-6 (p<0.05), nuclear factor-kappa B protein expression (p<0.01), and MMP-2 activity (p<0.05) in conjunctival homogenates were significantly reduced compared with the control animals. MMP-2 gene expression showed a tendency to decrease in the azithromycin group (p=0.063). Mucus secretion by goblet cells and the macrophage count in conjunctival tissue were also decreased in the azithromycin group (p<0.05). Conclusions: These results suggest that azithromycin administration ameliorates induced inflammation effects in a rat model of acute conjunctivitis.

We studied the macular pigment ocular density (MPOD) in patients with early age macular degeneration (AMD) before and 1 year after nutritional supplementation with lutein and docosahexaenoic acid (DHA). Forty-four patients with AMD were randomly divided into two groups that received placebo (n = 21) or a nutritional supplement (n = 23, 12 mg of lutein and 280 mg of DHA daily). Heterochromatic flicker photometry was used to determine the MPOD. At baseline, the MPOD in AMD patients with placebo was 0.286 +/- 0.017 meanwhile in AMD patients with supplementation it was 0.291 +/- 0.016. One year later, the mean MPOD had increased by 0.059 in the placebo group and by 0.162 in patients receiving lutein and DHA. This difference between groups was significant (p < 0.05). Lutein and DHA supplementation is effective in increasing the MPOD and may aid in prevention of age related macular degeneration.

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

TGF-ß inhibition with these peptides represents a promising new therapeutic line for CNV targeting a different pathway than current therapies. More studies are needed to assess this effect on early CNV, alone or in combination with anti-VEGF.