Genetic analysis of typical wet-type age-related macular degeneration and polypoidal choroidal vasculopathy in Japanese population

Age-related macular degeneration (AMD) is a common cause of blindness in the elderly. Caucasian patients are predominantly affected by the dry form of AMD, whereas Japanese patients have predominantly the wet form of AMD and/or polypoidal choroidal vasculopathy (PCV). Although genetic association in the 10q26 (ARMS2/HTRA1) region has been established in many ethnic groups for dry-type AMD, typical wet-type AMD, and PCV, the contribution of the 1q32 (CFH) region seem to differ among these groups. Here we show a single nucleotide polymorphism (SNP) in the ARMS2/HTRA1 locus is associated in the whole genome for Japanese typical wet-type AMD (rs10490924: , OR = 4.16) and PCV (rs10490924: , OR = 2.72) followed by CFH (rs800292: , OR = 2.08; , OR = 2.00), which differs from previous studies in Caucasian populations. Moreover, a SNP (rs2241394) in complement component C3 gene showed significant association with PCV (, OR = 3.47). We conclude that dry-type AMD, typical wet-type AMD, and PCV have both common and distinct genetic risks that become apparent when comparing Japanese versus Caucasian populations.

Electronic supplementary material

The online version of this article (doi:10.1007/s12177-009-9047-1) contains supplementary material, which is available to authorized users.     

Genetic association of VEGF and PEDF polymorphisms with age-related macular degeneration in Korean

Age-related macular degeneration (AMD) causes progressive impairment of central vision and is the leading cause of vision loss in older individuals. Although the etiology of AMD has not been clearly elucidated, genetic and environmental factors have been implicated. Vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF), a major regulator of vascular permeability and angiogenesis, have been suggested to play an important role in the pathogenesis of AMD. This study was performed to determine whether VEGF and PEDF variations are associated with AMD in the Korean population. Four SNPs of both the VEGF gene the PEDF gene were used to screen for genetic variation. This analysis was performed using polymerase chain reaction–restriction fragment length polymorphism, direct sequencing and an allele-specific oligonucleotide analysis. The study investigated four SNPs in VEGF and PEDF in Korean patients with AMD. The frequency of the TT genotype of rs1413711 and the recessive VEGF allele significantly differed between the patient and control groups. The TT genotype of rs1136287 (M72T) in PEDF significantly differed between the patient and control groups. Six haplotypes in the VEGF gene and two haplotypes in the PEDF gene were significantly associated with AMD. In this study, rs1413711 of VEGF, rs1136287 of PEDF and haplotypes were identified as candidate variants associated with AMD in Korean patients.     

CD44 aptamer mediated cargo delivery to lysosomes of retinal pigment epithelial cells to prevent age-related macular degeneration

Age related macular degeneration (AMD) is a progressive, neurodegenerative disorder that leads to the severe loss of central vision in elderlies. The health of retinal pigment epithelial (RPE) cells is critical for the onset of AMD. Chronic oxidative stress along with loss of lysosomal activity is a major cause for RPE cell death during AMD. Hence, development of a molecule for targeted lysosomal delivery of therapeutic protein/drugs in RPE cells is important to prevent RPE cell death during AMD. Using human RPE cell line (ARPE-19 cells) as a study model, we confirmed that hydrogen peroxide (H₂O₂) induced oxidative stress results in CD44 cell surface receptor overexpression in RPE cells; hence, an important target for specific delivery to RPE cells during oxidative stress. We also demonstrate that the known nucleic acid CD44 aptamer - conjugated with a fluorescent probe (FITC) - is delivered into the lysosomes of CD44 expressing ARPE-19 cells. Hence, as a proof of concept, we demonstrate that CD44 aptamer may be used for lysosomal delivery of cargo to RPE cells under oxidative stress, similar to AMD condition. Since oxidative stress may induce wet and dry AMD, both, along with proliferative vitreoretinopathy, CD44 aptamer may be applicable as a carrier for targeted lysosomal delivery of therapeutic cargoes in ocular diseases showing oxidative stress in RPE cells.     

Bisretinoid-mediated Complement Activation on Retinal Pigment Epithelial Cells Is Dependent on Complement Factor H Haplotype

Age-related macular degeneration (AMD) is a common central blinding disease of the elderly. Homozygosity for a sequence variant causing Y402H and I62V substitutions in the gene for complement factor H (CFH) is strongly associated with risk of AMD. CFH, secreted by many cell types, including those of the retinal pigment epithelium (RPE), is a regulatory protein that inhibits complement activation. Recessive Stargardt maculopathy is another central blinding disease caused by mutations in the gene for ABCA4, a transporter in photoreceptor outer segments (OS) that clears retinaldehyde and prevents formation of toxic bisretinoids. Photoreceptors daily shed their distal OS, which are phagocytosed by the RPE cells. Here, we investigated the relationship between the CFH haplotype of human RPE (hRPE) cells, exposure to OS containing bisretinoids, and complement activation. We show that hRPE cells of the AMD-predisposing CFH haplotype (HH402/VV62) are attacked by complement following exposure to bisretinoid-containing Abca4^−/− OS. This activation was dependent on factor B, indicating involvement of the alternative pathway. In contrast, hRPE cells of the AMD-protective CFH haplotype (YY402/II62) showed no complement activation following exposure to either Abca4^−/− or wild-type OS. The AMD-protective YY402/II62 hRPE cells were more resistant to the membrane attack complex, whereas HH402/VV62 hRPE cells showed significant membrane attack complex deposition following ingestion of Abca4^−/− OS. These results suggest that bisretinoid accumulation in hRPE cells stimulates activation and dysregulation of complement. Cells with an intact complement negative regulatory system are protected from complement attack, whereas cells with reduced CFH synthesis because of the Y402H and I62V substitutions are vulnerable to disease.     

No evidence of association between variant rs2075650 in lipid metabolism-related locus APOE/TOMM40 and advanced age-related macular degeneration in Han Chinese population

Age-related macular degeneration (AMD) is a late-onset, neurodegenerative disease. Genes related to lipid metabolism are important in AMD pathogenesis. Recently, a variant rs2075650 located in lipid metabolism-related locus APOE/TOMM40 was identified to be associated with advanced AMD and early AMD, respectively, in two genome-wide association studies with European ancestry, while no association study between rs2075650 and overall advanced AMD in Chinese population has been conducted before. We evaluated the potential effect of this variant on advanced AMD in a Han Chinese cohort with 204 advanced AMD patients and 1536 healthy controls. The results suggested that rs2075650 was neither associated with advanced AMD in allele level (P = 0.348) nor in genotype level (P = 0.890 under additive model with age and sex adjusted). In conclusion, our study did not confirm the impact of rs2075650 on advanced AMD risk, indicating that rs2075650 is unlikely a superior marker for APOE/TOMM40 susceptible region with advanced AMD in Han Chinese population.     

Neovascular age-related macular degeneration risk based on CFH, LOC387715/HTRA1, and smoking

Background

Age-related macular degeneration (AMD) is the major cause of blindness in the elderly. Those with the neovascular end-stage of disease have irreversible loss of central vision. AMD is a complex disorder in which genetic and environmental factors play a role. Polymorphisms in the complement factor H (CFH) gene, LOC387715, and the HTRA1 promoter are strongly associated with AMD. Smoking also contributes to the etiology. We aimed to provide a model of disease risk based on these factors.

Methods and Findings

We genotyped polymorphisms in CFH and LOC387715/HTRA1 in a case–control study of 401 patients with neovascular AMD and 266 controls without signs of disease, and used the data to produce genetic risk scores for the European-descent population based on haplotypes at these loci and smoking history. CFH and LOC387715/HTRA1 haplotypes and smoking status exerted large effects on AMD susceptibility, enabling risk scores to be generated with appropriate weighting of these three factors. Five common haplotypes of CFH conferred a range of odds ratios (ORs) per copy from 1 to 4.17. Most of the effect of LOC387715/HTRA1 was mediated through one detrimental haplotype (carriage of one copy: OR 2.83; 95% confidence interval [CI] 1.91–4.20), with homozygotes being at particularly high risk (OR 32.83; 95% CI 12.53–86.07). Patients with neovascular macular degeneration had considerably higher scores than those without disease, and risk of blinding AMD rose to 15.5% in the tenth of the population with highest predicted risk.

Conclusions

An individual''s risk of developing AMD in old age can be predicted by combining haplotype data with smoking status. Until there is effective treatment for AMD, encouragement to avoid smoking in those at high genetic risk may be the best option. We estimate that total absence of smoking would have reduced the prevalence of severe AMD by 33%. Unless smoking habits change or preventative treatment becomes available, the prevalence of AMD will rise as a consequence of the increasing longevity of the population.     

Anti-Human VEGF Repebody Effectively Suppresses Choroidal Neovascularization and Vascular Leakage

Age-related macular degeneration (AMD) is the leading cause of vision loss and blindness among people over the age of 60. Vascular endothelial growth factor (VEGF) plays a major role in pathological angiogenesis in AMD. Herein, we present the development of an anti- human VEGF repebody, which is a small-sized protein binder consisting of leucine-rich repeat (LRR) modules. The anti-VEGF repebody selected through a phage-display was shown to have a high affinity and specificity for human VEGF. We demonstrate that this repebody effectively inhibits in vitro angiogenic cellular processes, such as proliferation and migration, by blocking the VEGF-mediated signaling pathway. The repebody was also shown to have a strong suppression effect on choroidal neovascularization (CNV) and vascular leakage in vivo. Our results indicate that the anti-VEGF repebody has a therapeutic potential for treating neovascular AMD as well as other VEGF-involved diseases including diabetic retinopathy and metastatic cancers.     

The molecular genetic basis of age-related macular degeneration: an overview

Age-related macular degeneration (AMD) is a complex disorder of the eye and the third leading cause of blindness worldwide. With a multifactorial etiology, AMD results in progressive loss of central vision affecting the macular region of the eye in elderly. While the prevalence is relatively higher in the Caucasian populations, it has gradually become a major public health issue among the non-Caucasian populations (including Indians) as well due to senescence, rapidly changing demographics and life-style factors. Recent genome-wide association studies (GWAS) on large case-control cohorts have helped in mapping genes in the complement cascade that are involved in the regulation of innate immunity with AMD susceptibility. Genes involved with mitochondrial oxidative stress and extracellular matrix regulation also play a role in AMD pathogenesis. Majority of the associations observed in complement (CFH, CFB, C2 and C3) and other (ARMS2 and HTRA1) genes have been replicated in diverse populations worldwide. Gene-gene (CFH with ARMS2 and HTRA1) interactions and correlations with environmental traits (smoking and body mass index) have been established as significant covariates in AMD pathology. In this review, we have provided an overview on the underlying molecular genetic mechanisms in AMD worldwide and highlight the AMD-associated-candidate genes and their potential role in disease pathogenesis.     

CFH, VEGF, and PEDF genotypes and the response to intravitreous injection of bevacizumab for the treatment of age-related macular degeneration

We determined whether there is an association between complement factor H (CFH), high-temperature requirement A-1 (HTRA1), vascular endothelial growth factor (VEGF), and pigment epithelium-derived factor (PEDF) genotypes and the response to treatment with a single intravitreous injection of bevacizumab for age-related macular degeneration (AMD). Eighty-three patients with exudative AMD treated by bevacizumab injection were genotyped for three single nucleotide polymorphisms (SNPs; rs800292, rs1061170, rs1410996) in the CFH gene, a rs11200638-SNP in the HTRA1 gene, three SNPs (rs699947, rs1570360, rs2010963) in the VEGF gene, and four SNPs (rs12150053, rs12948385, rs9913583, rs1136287) in the PEDF gene using a TaqMan assay. The CT genotype (heterozygous) of CFH-rs1061170 was more frequently represented in nonresponders in vision than TT genotypes (nonrisk allele homozygous) at the time points of 1 and 3 months, while there was no CC genotype (risk allele homozygous) in our study cohort (p = 7.66 × 10⁻³, 7.83 × 10⁻³, respectively). VEGF-rs699947 was also associated with vision changes at 1 month and PEDF-rs1136287 at 3 months (p = 5.11 × 10⁻³, 2.05 × 10⁻², respectively). These variants may be utilized for genetic biomarkers to estimate visual outcomes in the response to intravitreal bevacizumab treatment for AMD.     

Silencing of miR-23a attenuates hydrogen peroxide (H2O2) induced oxidative damages in ARPE-19 cells by upregulating GLS1: an in vitro study

BackgroundOxidative damages contributes to age-related macular degeneration (AMD) caused vision blindness, but the molecular mechanisms are still largely unknown.ObjectivesThis study managed to investigate this issue by conducting in vitro experiments.MethodsOxidative stress were evaluated by L-012 dye, DHE staining and MDA assay. CCK-8 and colony formation assay were conducted to examine cell proliferation. Cell death was evaluated by trypan blue staining and Annexin V-FITC/PI double staining method through flow cytometry (FCM). The binding sites of miR-23a and GLS1 mRNA were predicted by online miRDB database and validated by dual-luciferase reporter gene system. Real-Time qPCR for miR-23a levels and Western Blot for protein expressions.ResultsThe retinal pigment epithelial (RPE) cells (ARPE-19) were subjected to hydrogen peroxide (H₂O₂) stimulation to simulate AMD progression in vitro, and we identified a novel miR-23a/glutaminase-1 (GLS1) pathway that regulated H₂O₂ induced oxidative damages in ARPE-19 cells. Mechanistically, H₂O₂ induced oxidative stress, inhibited cell proliferation and induced cell death in ARPE-19 cells in a dose- and time-dependent manner. Also, H₂O₂ stimulation hindered cell invasion, migration and glutamine uptake in ARPE-19 cells. Interestingly, we proved that H₂O₂ increased miR-23a levels, while downregulated glutaminase-1 (GLS1) in ARPE-19 cells, and miR-23a targeted 3′ untranslated region (3′UTR) of GLS1 mRNA for GLS1 degradation. Finally, our data suggested that silencing miR-23a upregulated GLS1 to reverse the detrimental effects of H₂O₂ treatment on ARPE-19 cells.ConclusionsIn general, analysis of the data suggested that miR-23a ablation upregulated GLS1 to attenuate H₂O₂ stimulation induced oxidative damages in ARPE-19 cells in vitro, and this study broadened our knowledge in this field, which might help to provide novel theranostic signatures for AMD.     

Phosphorylation/inactivation of PTEN by Akt-independent PI3K signaling in retinal pigment epithelium

Retinal pigment epithelium (RPE) plays a critical role in vertebrate vision by providing functional and structural support to the retina. Degeneration of RPE by cumulative oxidative stresses or acute injury frequently results in retinal degenerative diseases, notably age-related macular degeneration (AMD). Moreover, it has been shown that phosphorylation-mediated inactivation of PTEN (phosphatase and tensin homolog) in RPE is closely linked to AMD-like retinal degeneration in mice [1]. In this study, we used AMD mouse models, in which chemokine (C–C motif) ligand 2 (Ccl2) or chemokine (C–C motif) receptor 2 (Ccr2) were genetically ablated, to examine mechanisms linking reactive oxygen species (ROS) to phosphorylation/inactivation of PTEN in RPE. We found that ROS levels were increased in these RPE cells in association with phosphorylation/inactivation of PTEN. Both PTEN phosphorylation/inactivation and consequent Akt activation in the RPE of AMD model mice were inhibited by antioxidant treatment, indicating a functional role for elevated intracellular ROS. We further discovered that PTEN phosphorylation in oxidatively stressed RPE was repressed by a phosphoinositide 3-kinase (PI3K) inhibitor, but not by an Akt inhibitor. Taken together, these results suggest that ROS-activated PI3K potentiates AMD-related RPE pathogenesis through phosphorylation/inactivation of PTEN.     

Autophagy and Exosomes in the Aged Retinal Pigment Epithelium: Possible Relevance to Drusen Formation and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is a major cause of loss of central vision in the elderly. The formation of drusen, an extracellular, amorphous deposit of material on Bruch''s membrane in the macula of the retina, occurs early in the course of the disease. Although some of the molecular components of drusen are known, there is no understanding of the cell biology that leads to the formation of drusen. We have previously demonstrated increased mitochondrial DNA (mtDNA) damage and decreased DNA repair enzyme capabilities in the rodent RPE/choroid with age. In this study, we found that drusen in AMD donor eyes contain markers for autophagy and exosomes. Furthermore, these markers are also found in the region of Bruch''s membrane in old mice. By in vitro modeling increased mtDNA damage induced by rotenone, an inhibitor of mitochondrial complex I, in the RPE, we found that the phagocytic activity was not altered but that there were: 1) increased autophagic markers, 2) decreased lysosomal activity, 3) increased exocytotic activity and 4) release of chemoattractants. Exosomes released by the stressed RPE are coated with complement and can bind complement factor H, mutations of which are associated with AMD. We speculate that increased autophagy and the release of intracellular proteins via exosomes by the aged RPE may contribute to the formation of drusen. Molecular and cellular changes in the old RPE may underlie susceptibility to genetic mutations that are found in AMD patients and may be associated with the pathogenesis of AMD in the elderly.     

Microglia in the Mouse Retina Alter the Structure and Function of Retinal Pigmented Epithelial Cells: A Potential Cellular Interaction Relevant to AMD

Background

Age-related macular degeneration (AMD) is a leading cause of legal blindness in the elderly in the industrialized word. While the immune system in the retina is likely to be important in AMD pathogenesis, the cell biology underlying the disease is incompletely understood. Clinical and basic science studies have implicated alterations in the retinal pigment epithelium (RPE) layer as a locus of early change. Also, retinal microglia, the resident immune cells of the retina, have been observed to translocate from their normal position in the inner retina to accumulate in the subretinal space close to the RPE layer in AMD eyes and in animal models of AMD.

Methodology/Principal Findings

In this study, we examined the effects of retinal microglia on RPE cells using 1) an in vitro model where activated retinal microglia are co-cultured with primary RPE cells, and 2) an in vivo mouse model where retinal microglia are transplanted into the subretinal space. We found that retinal microglia induced in RPE cells 1) changes in RPE structure and distribution, 2) increased expression and secretion of pro-inflammatory, chemotactic, and pro-angiogenic molecules, and 3) increased extent of in vivo choroidal neovascularization in the subretinal space.

Conclusions/Significance

These findings share similarities with important pathological features found in AMD and suggest the relevance of microglia-RPE interactions in AMD pathogenesis. We speculate that the migration of retinal microglia into the subretinal space in early stages of the disease induces significant changes in RPE cells that perpetuate further microglial accumulation, increase inflammation in the outer retina, and fosters an environment conducive for the formation of neovascular changes responsible for much of vision loss in advanced AMD.     

康柏西普与雷珠单抗治疗年龄相关性黄斑变性的临床研究

目的:探讨康柏西普联合雷珠单抗治疗年龄相关性黄斑变性(AMD)的临床疗效。方法:选择2015年6月到2016年10月我院收治的60例AMD患者,按随机数字表法分为对照组和治疗组。对照组患者给予康柏西普治疗,治疗组患者给予康柏西普联合雷珠单抗治疗,两组患者均治疗3个月。评价并比较两组患者临床疗效。统计并比较两组患者治疗后的视网膜渗漏总改善率。观察并比较两组患者治疗前后最佳矫正视力和黄斑视网膜厚度。结果:治疗后,治疗组患者的视力提高率为80.00%,明显高于对照组的55.00%,差异具有统计学意义(X~2=4.104,P=0.043)。治疗后,治疗组患者的视网膜渗漏总改善率为92.50%,明显高于对照组的70.00%,差异具有统计学意义(X~2=5.294,P=0.021)。治疗前,两组患者最佳矫正视力、黄斑视网膜厚度比较差异无统计学意义(P0.05);治疗后,两组患者最佳矫正视力均明显大于治疗前,黄斑视网膜厚度均明显小于治疗前,并且治疗组均明显优于对照组,差异均具有统计学意义(P0.05)。结论:康柏西普联合雷珠单抗治疗AMD的临床疗效显著,能够明显提高视力,减轻视网膜渗漏,值得在临床上推广应用。     

雷珠单抗联合复方血栓通胶囊对年龄相关性黄斑变性患者血液流变学和血清VEGF、PDGF的影响

摘要 目的：观察雷珠单抗联合复方血栓通胶囊对年龄相关性黄斑变性（AMD）患者血液流变学和血清血管内皮生长因子（VEGF）、血小板衍生生长因子（PDGF）的影响。方法：选取我院2019年6月~2021年1月收治的AMD患者60例（96眼）,按照随机数字表法分别为对照组（n=30,46眼）和实验组（n=30,50眼）,对照组给予雷珠单抗治疗,实验组给予雷珠单抗联合复方血栓通胶囊治疗,对比两组眼部相关指标、血液流变学指标和血清VEGF、PDGF水平变化情况,观察两组治疗期间不良反应发生情况。结果：实验组治疗3个月后眼内压（IOP）、黄斑中央区厚度（CMT）、脉络新生血管（CNV）面积低于对照组,视力高于对照组（P<0.05）。实验组治疗3个月后全血低切/中切/高切黏度、血浆黏度低于对照组（P<0.05）。实验组治疗3个月后血清VEGF、PDGF水平低于对照组（P<0.05）。两组不良反应发生率对比无差异（P>0.05）。结论：雷珠单抗联合复方血栓通胶囊可改善AMD临床症状,促进视力恢复,其作用机制可能与调节机体血液流变学、血清VEGF、PDGF水平有关。     

Differential DNA methylation identified in the blood and retina of AMD patients

Age-related macular degeneration (AMD) is a major cause of blindness in the western world. While genetic studies have linked both common and rare variants in genes involved in regulation of the complement system to increased risk of development of AMD, environmental factors, such as smoking and nutrition, can also significantly affect the risk of developing the disease and the rate of disease progression. Since epigenetics has been implicated in mediating, in part, the disease risk associated with some environmental factors, we investigated a possible epigenetic contribution to AMD. We performed genome-wide DNA methylation profiling of blood from AMD patients and controls. No differential methylation site reached genome-wide significance; however, when epigenetic changes in and around known GWAS-defined AMD risk loci were explored, we found small but significant DNA methylation differences in the blood of neovascular AMD patients near age-related maculopathy susceptibility 2 (ARMS2), a top-ranked GWAS locus preferentially associated with neovascular AMD. The methylation level of one of the CpG sites significantly correlated with the genotype of the risk SNP rs10490924, suggesting a possible epigenetic mechanism of risk. Integrating genome-wide DNA methylation analysis of retina samples with and without AMD together with blood samples, we further identified a consistent, replicable change in DNA methylation in the promoter region of protease serine 50 (PRSS50). These methylation changes may identify sites in novel genes that are susceptible to non-genetic factors known to contribute to AMD development and progression.