[Use of artificial intelligence for recognition of biomarkers in intermediate age-related macular degeneration]. / Einsatz von künstlicher Intelligenz zur Erkennung von Biomarkern bei der intermediären altersabhängigen Makuladegeneration.

Advances in imaging and artificial intelligence (AI) have revolutionized the detection, quantification and monitoring for the clinical assessment of intermediate age-related macular degeneration (iAMD). The iAMD incorporates a broad spectrum of manifestations, which range from individual small drusen, hyperpigmentation, hypopigmentation up to early stages of geographical atrophy. Current high-resolution imaging technologies enable an accurate detection and description of anatomical features, such as drusen volumes, hyperreflexive foci and photoreceptor degeneration, which are risk factors that are decisive for prediction of the course of the disease; however, the manual annotation of these features in complex optical coherence tomography (OCT) scans is impractical for the routine clinical practice and research. In this context AI provides a solution by fully automatic segmentation and therefore delivers exact, reproducible and quantitative analyses of AMD-related biomarkers. Furthermore, the application of AI in iAMD facilitates the risk assessment and the development of structural endpoints for new forms of treatment. For example, the quantitative analysis of drusen volume and hyperreflective foci with AI algorithms has shown a correlation with the progression of the disease. These technological advances therefore improve not only the diagnostic precision but also support future targeted treatment strategies and contribute to the prioritized target of personalized medicine in the diagnostics and treatment of AMD.

CFH Haploinsufficiency and Complement Alterations in Early-Onset Macular Degeneration.

Purpose: Complement dysregulation is a key component in the pathogenesis of age-related macular degeneration (AMD) and related diseases such as early-onset macular drusen (EOMD). Although genetic variants of complement factor H (CFH) are associated with AMD risk, the impact of CFH and factor H-like protein 1 (FHL-1) expression on local complement activity in human retinal pigment epithelium (RPE) remains unclear. Methods: We identified a novel CFH variant in a family with EOMD and generated patient induced pluripotent stem cell (iPSC)-derived RPE cells. We assessed CFH and FHL-1 co-factor activity through C3b breakdown assays and measured complement activation by immunostaining for membrane attack complex (MAC) formation. Expression of CFH, FHL-1, local alternative pathway (AP) components, and regulators of complement activation (RCA) in EOMD RPE cells was determined by quantitative PCR, western blot, and immunostaining. Isogenic EOMD (cEOMD) RPE was generated using CRISPR/Cas9 gene editing. Results: The CFH variant (c.351-2A>G) resulted in loss of CFH and FHL-1 expression and significantly reduced CFH and FHL-1 protein expression (∼50%) in EOMD iPSC RPE cells. These cells exhibited increased MAC deposition upon exposure to normal human serum. Under inflammatory or oxidative stress conditions, CFH and FHL-1 expression in EOMD RPE cells paralleled that of controls, whereas RCA expression, including MAC formation inhibitors, was elevated. CRISPR/Cas9 correction restored CFH/FHL-1 expression and mitigated alternative pathway complement activity in cEOMD RPE cells. Conclusions: Identification of a novel CFH variant in patients with EOMD resulting in reduced CFH and FHL-1 and increased local complement activity in EOMD iPSC RPE supports the involvement of CFH haploinsufficiency in EOMD pathogenesis.

Topographic analysis of local OCT biomarkers which predict progression to atrophy in age-related macular degeneration.

PURPOSE: To define optical coherence tomography (OCT) biomarkers that precede the development of complete retinal pigment epithelium and outer retinal atrophy (cRORA) at that location in eyes with age-related macular degeneration (AMD). METHODS: In this retrospective case-control study, patients with dry AMD who had evidence of cRORA and OCT data available for 4 years (48 ± 4 months) prior to the first visit with evidence of cRORA were included. The visit 4 years prior to the development of cRORA was defined as the baseline visit, and the region on the OCT B-scans of future cRORA development was termed the case region. A region in the same eye at the same distance from the foveal center as the case region that did not progress to cRORA was selected as the control region. OCT B-scans at the baseline visit through both the case and control regions were evaluated for the presence of soft and cuticular drusen, drusen with hyporeflective cores (hcD), drusenoid pigment epithelial detachments (PED), subretinal drusenoid deposits (SDD), thick and thin double-layer signs (DLS), intraretinal hyperreflective foci (IHRF), and acquired vitelliform lesions (AVL). RESULTS: A total of 57 eyes of 41 patients with dry AMD and evidence of cRORA were included. Mean time from the baseline visit to the first visit with cRORA was 44.7 ± 6.5 months. The presence of soft drusen, drusenoid PED, AVL, thin DLS, and IHRF at the baseline visit was all associated with a significantly increased risk of cRORA at that location. Multivariable logistic regression revealed that IHRF (OR, 8.559; p < 0.001), drusenoid PED (OR, 7.148; p = 0.001), and a thin DLS (OR, 3.483; p = 0.021) were independent predictors of development of cRORA at that location. CONCLUSIONS: IHRF, drusenoid PED, and thin DLS are all local risk factors for the development of cRORA at that same location. These findings would support the inclusion of these features within a more granular staging system defining specific steps in the progression from early AMD to atrophy.

Calpain-specific breakdown fragment in human drusen.

PURPOSE: With aging and age-related macular dystrophy (AMD), proteolytic fragments are deposited in extracellular drusen located between the RPE and Bruch's membrane. Localized hypoxia may be a risk factor for AMD. Our hypothesis is that following hypoxia, activation of proteolytic enzymes called calpains may cause proteolysis/degeneration of retinal cells and RPE. No direct evidence has yet demonstrated activation of calpains in AMD. The purpose of the present study was to identify calpain-cleaved proteins in drusen. METHODS: Seventy-six (76) drusen were analyzed in human eye sections from six normal and twelve AMD human donor eyes. The sections were subjected to immunofluorescence for the calpain-specific 150 kDa breakdown product from α-spectrin, SBDP150 - a marker for calpain activation, and for recoverin - a marker for photoreceptor cells. RESULTS: Among 29 nodular drusen, 80% from normal eyes and 90% from AMD eyes stained positive for SBDP150. Among 47 soft drusen, mostly from AMD eyes, 72% stained positive for SBDP150. Thus, the majority of both soft and nodular drusen from AMD donors contained SBDP150. CONCLUSIONS: SBDP150 was detected for the first time in soft and nodular drusen from human donors. Our results suggest that calpain-induced proteolysis participates in the degeneration of photoreceptors and/or RPE cells during aging and AMD. Calpain inhibitors may ameliorate AMD progression.

Spatial Dissociation of Subretinal Drusenoid Deposits and Impaired Scotopic and Mesopic Sensitivity in AMD.

Purpose: Subretinal drusenoid deposits (SDD) first appear in the rod-rich perifovea and can extend to the cone-rich fovea. To refine the spatial relationship of visual dysfunction with SDD burden, we determined the topography of mesopic and scotopic light sensitivity in participants with non-neovascular AMD with and without SDD. Methods: Thirty-three subjects were classified into three groups: normal (n = 9), AMD-Drusen (with drusen and without SDD; n = 12), and AMD-SDD (predominantly SDD; n = 12). Mesopic and scotopic microperimetry were performed using 68 targets within the Early Treatment Diabetic Retinopathy Study grid, including points at 1.7° from the foveal center (rod:cone ratio, 0.35). Age-adjusted linear regression was used to compare mesopic and scotopic light sensitivities across groups. Results: Across the entire Early Treatment Diabetic Retinopathy Study grid and within individual subfields, the three groups differed significantly for mesopic and scotopic light sensitivities (all P < 0.05). The AMD-SDD group exhibited significantly decreased mesopic and scotopic sensitivity versus both the normal and the AMD-Drusen groups (all P < 0.05), while AMD-Drusen and normal eyes did not significantly differ (all P > 0.05). The lowest relative sensitivities were recorded for scotopic light levels, especially in the central subfield, in the AMD-SDD group. Conclusions: SDD-associated decrements in rod-mediated vision can be detected close to the foveola, and these deficits are proportionately worse than functional loss in the rod-rich perifovea. This finding suggests that factors other than the previously hypothesized direct cytotoxicity to photoreceptors and local transport barrier limitations may negatively impact vision. Larger prospective studies are required to confirm these observations.

Extracellular vesicles released by human retinal pigment epithelium mediate increased polarised secretion of drusen proteins in response to AMD stressors.

Age-related macular degeneration (AMD) is a leading cause of blindness worldwide. Drusen are key contributors to the etiology of AMD and the ability to modulate drusen biogenesis could lead to therapeutic strategies to slow or halt AMD progression. The mechanisms underlying drusen biogenesis, however, remain mostly unknown. Here we demonstrate that under homeostatic conditions extracellular vesicles (EVs) secreted by retinal pigment epithelium (RPE) cells are enriched in proteins associated with mechanisms involved in AMD pathophysiology, including oxidative stress, immune response, inflammation, complement system and drusen composition. Furthermore, we provide first evidence that drusen-associated proteins are released as cargo of extracellular vesicles secreted by RPE cells in a polarised apical:basal mode. Notably, drusen-associated proteins exhibited distinctive directional secretion modes in homeostatic conditions and, differential modulation of this directional secretion in response to AMD stressors. These observations underpin the existence of a finely-tuned mechanism regulating directional apical:basal sorting and secretion of drusen-associated proteins via EVs, and its modulation in response to mechanisms involved in AMD pathophysiology. Collectively, our results strongly support an active role of RPE-derived EVs as a key source of drusen proteins and important contributors to drusen development and growth.

Integrative analysis of the choroid by quantifying Haller vessel and choriocapillaris parameters in different drusen subtypes.

This study aimed to quantify the Haller vessel and choriocapillaris (CC) parameters in drusen subtypes in nonexudative age-related macular degeneration (AMD) and pachydrusen. Ninety-five eyes of 80 patients and 28 control eyes were categorized into soft drusen, subretinal drusenoid deposit (SDD), soft drusen plus SDD, pachydrusen, and control groups. The diameter, length and intersections of Haller vessels and the total area, size and number of CC flow voids were quantified using en face optical coherence tomography (OCT) or OCT angiography. The pachydrusen group showed the largest Haller vessel area and diameter and shortest total length but similar CC parameters to those in the control group. The soft drusen plus SDD group showed the largest CC flow void area and size, while the Haller parameters were similar to those in the control group. The area and size of the flow voids in the SDD group were smaller than those in the soft drusen plus SDD group. Based on unsupervised machine learning, the eyes were classified into 4 clusters-the control, pachydrusen, soft drusen plus SDD and soft drusen plus SDD groups. Cluster 3 showed a larger diameter and shorter total length of the Haller vessels than cluster 4.

Descriptive analysis of Fibulin-3 and the extracellular vesicle marker, Alix, in drusen from a small cohort of postmortem human eyes.

Fibulin-3 (Fib3) is a secreted glycoprotein that is expressed in the retina and has been associated with drusen formation in age-related macular degeneration (AMD). The purpose of this study was to assess whether Fib3 is associated with extracellular vesicles (EVs) in drusen from non-diseased and AMD human donors. De-identified sections of human eyes were received from the National Disease Research Institute (NDRI, Philadelphia). Donor eyes were either non-diseased (no known ocular pathology) or had been diagnosed with AMD. Retinal cryostat sections were labeled with primary antibodies targeted to Fib3, Apolipoprotein E (ApoE; a drusen marker), and ALG-2 interacting protein X (Alix, an EV marker) for confocal imaging (Leica TCS SP8). Fib3-positive (Fib3+) puncta were detected on the apical region of the RPE layer and within large AMD drusen. Alix-positive (Alix+) puncta were also detected in a single AMD druse, where a number were Fib3+ and the remaining were Fib3-negative. Similarly, there were Fib3+ puncta that were Alix-negative. Fib3 and Alix also showed a degree of colocalization in the photoreceptor outer segments of the neural retina. Our data suggest that the Alix+ puncta are EV-rich populations that accumulate, together with Fib3, within the drusen matrix during AMD. The EV population is likely heterogeneous, such that there are sub-populations with different cargo content.

PERIPHERAL RETINAL DRUSEN-LIKE DEPOSITS IN GUCY2C CONGENITAL SECRETORY DIARRHEA SYNDROME.

PURPOSE: To report the presence of drusen in infancy, in a patient with Type 1 retinopathy of prematurity and a rare congenital sodium diarrhea secondary to a sporadic GUCY2C mutation. METHODS: A case report generated by review of clinical course, with imaging of 1 patient and literature review. RESULTS: A 1.075-kg infant born at gestation age 27 weeks was admitted to our institution with respiratory distress and secretory diarrhea. During screening for retinopathy of prematurity, peripheral drusen-like subretinal deposits were identified. There were no similar findings in either parent or family history of ocular pathologies. Their distribution is atypical for that seen in other causes of early onset drusen such as autosomal dominant drusen or Sorsby fundus dystrophy. Retinopathy of prematurity was identified, which progressed to Type 1, and was treated with bilateral indirect peripheral retinal photocoagulation at gestational age of 40 weeks. Fluorescein angiography was performed and was consistent with peripheral drusen. Optical coherence tomography of the central macula and an awake electroretinogram at 6 months were normal. Serial examinations confirmed no progression in the drusen-like deposits or in retinopathy of prematurity, with clinically appropriate visual development observed during close follow-up. CONCLUSION: We identify a unique ocular phenotype of retinal drusen-like deposits in an infant with a rare, sporadic GUCY2C mutation.

NATURAL HISTORY OF QUANTITATIVE AUTOFLUORESCENCE IN INTERMEDIATE AGE-RELATED MACULAR DEGENERATION.

PURPOSE: To investigate differences in quantitative autofluorescence (qAF) imaging measurements between eyes with and without large drusen, and whether qAF measurements change over time in the eyes with large drusen. METHODS: Eighty-five eyes from participants with bilateral large drusen and 51 eyes from healthy participants underwent qAF imaging at least once, and the age-related macular degeneration participants were reviewed 6-monthly. Normalized grey values at 9° to 11° eccentricity from the fovea were averaged to provide a summary measure of qAF values (termed qAF8). RESULTS: In a multivariable model, qAF8 measurements were not significantly different between age-related macular degeneration eyes with large drusen and healthy eyes (P = 0.130), and qAF8 measurements showed a decline over time in the age-related macular degeneration eyes (P = 0.013). CONCLUSION: These findings add to the body of evidence that qAF levels are not increased in eyes with large drusen compared with healthy eyes, and qAF levels show a significant decline over time in the age-related macular degeneration eyes. These findings highlight how the relationship between qAF levels and retinal pigment epithelium health does not seem to be straightforward. Further investigation is required to better understand this relationship, especially if qAF levels are to be used as an outcome measure in intervention trials.

Complement Activation Products and Cytokines in Pachychoroid Neovasculopathy and Neovascular Age-Related Macular Degeneration.

Purpose: To investigate the characteristics of complement activation products and angiogenic cytokines in the aqueous humor in eyes with pachychoroid neovasculopathy (PNV) and neovascular age-related macular degeneration (nAMD). Methods: This was a prospective, comparative, observational study. All patients with choroidal neovascularization were classified as PNV without polyps, PNV with polyps (polypoidal choroidal vasculopathy [PCV]), or drusen-associated nAMD according to the presence or absence of pachychoroid features and soft drusen. This study included a total of 105 eyes. Aqueous humor samples were collected from 25 eyes with PNV without polyps, 23 eyes with PCV, and 24 eyes with drusen-associated nAMD before intravitreal anti-vascular endothelial growth factor (VEGF) injection and cataract surgery in 33 control eyes. Clinical samples were measured for complement component 3a (C3a), C4a, C5a, VEGF, and macrophage chemoattractant protein 1 (MCP-1) using a bead-based immunoassay. Results: C3a and MCP-1 levels were significantly higher in PCV (P = 0.032 and P = 0.039, respectively) and drusen-associated nAMD (P = 0.01 for both comparisons) than in controls, and no difference was seen in C3a and MCP-1 levels between PNV and controls (P = 0.747 and P = 0.294, respectively). VEGF levels were significantly higher in PNV (P = 0.016), PCV (P = 0.009), and drusen-associated nAMD (P = 0.043) than in controls. In PNV, the VEGF levels elevated without elevated C3a and MCP-1. Conclusions: PNV, PCV, and drusen-associated nAMD had significantly distinct profiles of complement activation products and cytokines in the aqueous humor.

Visual Function and Macular Carotenoid Changes in Eyes with Retinal Drusen-An Open Label Randomized Controlled Trial to Compare a Micronized Lipid-Based Carotenoid Liquid Supplementation and AREDS-2 Formula.

PURPOSE: To compare the changes in visual and ocular parameters in individuals with retinal drusen who were treated with two commercially available nutritional supplements. METHODS: An open-label, single-center, randomized, parallel-treatment with an observational control group design was utilized. The treatment groups included individuals with fine retinal drusen sub-clinical age-related macular degeneration (AMD), while the control group consisted of ocular normal individuals. The treatment groups were randomly assigned to the micronized lipid-based carotenoid supplement, Lumega-Z (LM), or the PreserVision Age-Related Eye Disease Study 2 (AREDS-2) soft gel (PV). Visual performance was evaluated using the techniques of visual acuity, dark adaptation recovery and contrast sensitivity, at baseline, three months, and six months. Additionally, the macular pigment optical density (MPOD) was measured. The control group was not assigned any carotenoid supplement. The right eye and left eye results were analyzed separately. RESULTS: Seventy-nine participants were recruited for this study, of which 68 qualified and 56 participants had useable reliable data. Of the individuals who completed this study, 25 participants belonged to the LM group, 16 belonged to the PV group, and 15 to the control group. The LM group demonstrated statistically significant improvements in contrast sensitivity function (CSF) in both eyes at six months (p < 0.001). The LM group displayed a positive linear trend with treatment time in CSF (p < 0.001), with benefits visible after just three months of supplementation. Although there was a trend showing improvement in CSF in the PV group, the change was not significant after a Bonferroni-corrected p-value of p < 0.00625. Visual acuity, dark adaptation recovery and MPOD did not significantly improve in either treatment groups. CONCLUSION: The LM group demonstrated greater and faster benefits in visual performance as measured by CSF when compared to the PV group. This trial has been registered at clinicaltrials.gov (NCT03946085).

Novel Programmed Cell Death as Therapeutic Targets in Age-Related Macular Degeneration?

Age-related macular degeneration (AMD) is a leading cause of severe visual loss among the elderly. AMD patients are tormented by progressive central blurring/loss of vision and have limited therapeutic options to date. Drusen accumulation causing retinal pigment epithelial (RPE) cell damage is the hallmark of AMD pathogenesis, in which oxidative stress and inflammation are the well-known molecular mechanisms. However, the underlying mechanisms of how RPE responds when exposed to drusen are still poorly understood. Programmed cell death (PCD) plays an important role in cellular responses to stress and the regulation of homeostasis and diseases. Apart from the classical apoptosis, recent studies also discovered novel PCD pathways such as pyroptosis, necroptosis, and ferroptosis, which may contribute to RPE cell death in AMD. This evidence may yield new treatment targets for AMD. In this review, we summarized and analyzed recent advances on the association between novel PCD and AMD, proposing PCD's role as a therapeutic new target for future AMD treatment.

Activation of C-reactive protein proinflammatory phenotype in the blood retinal barrier in vitro: implications for age-related macular degeneration.

The retinal pigment epithelium (RPE) is considered one of the main targets of age-related macular degeneration (AMD), the leading cause of irreversible vision loss among the ageing population worldwide. Persistent low grade inflammation and oxidative stress eventually lead to RPE dysfunction and disruption of the outer blood-retinal barrier (oBRB). Increased levels of circulating pentameric C-reactive protein (pCRP) are associated with higher risk of AMD. The monomeric form (mCRP) has been detected in drusen, the hallmark deposits associated with AMD, and we have found that mCRP induces oBRB disruption. However, it is unknown how mCRP is generated in the subretinal space. Using a Transwell model we found that both pCRP and mCRP can cross choroidal endothelial cells and reach the RPE in vitro and that mCRP, but not pCRP, is able to cross the RPE monolayer in ARPE-19 cells. Alternatively, mCRP can originate from the dissociation of pCRP in the surface of lipopolysaccharide-damaged RPE in both ARPE-19 and primary porcine RPE lines. In addition, we found that the proinflammatory phenotype of mCRP in the RPE depends on its topological localization. Together, our findings further support mCRP contribution to AMD progression enhancing oBRB disruption.

Unstimulated, Serum-free Cultures of Retinal Pigment Epithelium Excrete Large Mounds of Drusen-like Deposits.

Purpose: A hallmark of age-related macular degeneration is the accumulation of deposits of lipids and proteins, called drusen, in Bruch's membrane. Several culture models of retinal pigment epithelia (RPE) develop drusen-like deposits. We examined whether prolonged culture of RPE with a retina-like tissue affected the number or size of these deposits. Methods: RPE and retinal progenitor cells (RPC) were differentiated from induced pluripotent stem cells derived from fetal tissue and maintained in serum-free medium containing the B27 supplement. RPE was cultured on Transwell filter inserts, and RPC were cultured on a planar matrix composed of gelatin, hyaluronic acid, and chondroitin sulfate. After seeding the filter, RPC were layered on top of the RPE. RPE ± RPC were cultured for six months. The function of RPE tight junctions was assessed by the transepithelial electrical resistance. Cultures were stained for actin, neutral lipids, APOE, TIMP3, vitronectin, and calcium deposits. Morphometric analysis was used to determine the number and volume of the "druse". Results: After six months, the TER was greater for the co-cultures (304 ± 11 Ω× cm2 vs 243 ± 7 Ω× cm2, p < .01). RPE formed mounds of druse-like deposits that contained, vitronectin, APOE, TIMP3 and calcium deposits, but lipids were undetected. The mounds overlay areas of the filter where no lipid was detected in the pores, and the RPE overlying the mounds was often thin. The number of "druse"/100,000 µm2 was 5.0 ± 0.4 (co-cultures) vs 2.3 ± 0.1 (monocultures) (p < .05). The total volume of "drusen"/100,000 µm3 was 15,133 ± 1544 (co-cultures) vs 5,993 ± 872 (monocultures) (p < .05). There was no statistical difference between the size-distribution of druse-like particles formed by each culture. Conclusions: Covering the apical membrane of RPE with a thick tissue increased the number of druse-like deposits. The apparent size limitation of the deposits may reflect the apparent interruption of the of lipid cycle found at the basal membrane of the RPE.

Loss-of-Function Mutations in the CFH Gene Affecting Alternatively Encoded Factor H-like 1 Protein Cause Dominant Early-Onset Macular Drusen.

PURPOSE: To characterize the molecular mechanism underpinning early-onset macular drusen (EOMD), a phenotypically severe subtype of age-related macular degeneration (AMD), in a subgroup of patients. DESIGN: Multicenter case series, in vitro experimentation, and retrospective analysis of previously reported variants. PARTICIPANTS: Seven families with apparently autosomal dominant EOMD. METHODS: Patients underwent a comprehensive ophthalmic assessment. Affected individuals from families A, B, and E underwent whole exome sequencing. The probands from families C, D, F, and G underwent Sanger sequencing analysis of the complement factor H (CFH) gene. Mutant recombinant factor H like-1 (FHL-1) proteins were expressed in HEK293 cells to assess the impact on FHL-1 expression and function. Previously reported EOMD-causing variants in CFH were reviewed. MAIN OUTCOME MEASURES: Detailed clinical phenotypes, genomic findings, in vitro characterization of mutation effect on protein function, and postulation of the pathomechanism underpinning EOMD. RESULTS: All affected participants demonstrated bilateral drusen. The earliest reported age of onset was 16 years (median, 46 years). Ultra-rare (minor allele frequency [MAF], ≤0.0001) CFH variants were identified as the cause of disease in each family: CFH c.1243del, p.(Ala415ProfsTer39) het; c.350+1G→T het; c.619+1G→A het, c.380G→A, p.(Arg127His) het; c.694C→T p.(Arg232Ter) het (identified in 2 unrelated families in this cohort); and c.1291T→A, p.(Cys431Ser). All mutations affect complement control protein domains 2 through 7, and thus are predicted to impact both FHL-1, the predominant isoform in Bruch's membrane (BrM) of the macula, and factor H (FH). In vitro analysis of recombinant proteins FHL-1R127H, FHL-1A415f/s, and FHL-1C431S demonstrated that they are not secreted, and thus are loss-of-function proteins. Review of 29 previously reported EOMD-causing mutations found that 75.8% (22/29) impact FHL-1 and FH. In total, 86.2% (25/29) of EOMD-associated variants cause haploinsufficiency of FH or FHL-1. CONCLUSIONS: Early-onset macular drusen is an underrecognized, phenotypically severe subtype of AMD. We propose that haploinsufficiency of FHL-1, the main regulator of the complement pathway in BrM, where drusen develop, is an important mechanism underpinning the development of EOMD in a number of cases. Understanding the molecular basis of EOMD will shed light on AMD pathogenesis given their pathologic similarities.

On the origin of proteins in human drusen: The meet, greet and stick hypothesis.

Retinal drusen formation is not only a clinical hallmark for the development of age-related macular degeneration (AMD) but also for other disorders, such as Alzheimer's disease and renal diseases. The initiation and growth of drusen is poorly understood. Attention has focused on lipids and minerals, but relatively little is known about the origin of drusen-associated proteins and how they are retained in the space between the basal lamina of the retinal pigment epithelium and the inner collagenous layer space (sub-RPE-BL space). While some authors suggested that drusen proteins are mainly derived from cellular debris from processed photoreceptor outer segments and the RPE, others suggest a choroidal cell or blood origin. Here, we reviewed and supplemented the existing literature on the molecular composition of the retina/choroid complex, to gain a more complete understanding of the sources of proteins in drusen. These "drusenomics" studies showed that a considerable proportion of currently identified drusen proteins is uniquely originating from the blood. A smaller, but still large fraction of drusen proteins comes from both blood and/or RPE. Only a small proportion of drusen proteins is uniquely derived from the photoreceptors or choroid. We next evaluated how drusen components may "meet, greet and stick" to each other and/or to structures like hydroxyapatite spherules to form macroscopic deposits in the sub-RPE-BL space. Finally, we discuss implications of our findings with respect to the previously proposed homology between drusenogenesis in AMD and plaque formation in atherosclerosis.

Quantitative proteomic analysis of aqueous humor from patients with drusen and reticular pseudodrusen in age-related macular degeneration.

BACKGROUND: To identify novel biomarkers related to the pathogenesis of dry age-related macular degeneration (AMD), we adopted a human retinal pigment epithelial (RPE) cell culture model that mimics some features of dry AMD including the accumulation of intra- and sub-RPE deposits. Then, we investigated the aqueous humor (AH) proteome using a data-independent acquisition method (sequential window acquisition of all theoretical fragment ion mass spectrometry) for dry AMD patients and controls. METHODS: After uniformly pigmented polarized monolayers of human fetal primary RPE (hfRPE) cells were established, the cells were exposed to 4-hydroxy-2-nonenal (4-HNE), followed by Western blotting, immunofluorescence analysis and ELISA of cells or conditioned media for several proteins of interest. Data-dependent acquisition for identification of the AH proteome and SWATH-based mass spectrometry were performed for 11 dry AMD patients according to their phenotypes (including soft drusen and reticular pseudodrusen [RPD]) and 2 controls (3 groups). RESULTS: Increased intra- and sub-RPE deposits were observed in 4-HNE-treated hfRPE cells compared with control cultures based on APOA1, cathepsin D, and clusterin immunoreactivity. Additionally, the differential abundance of proteins in apical and basal chambers with or without 4-HNE treatment confirmed the polarized secretion of proteins from hfRPE cells. A total of 119 proteins were quantified in dry AMD patients and controls by SWATH-MS. Sixty-five proteins exhibited significantly altered abundance among the three groups. A two-dimensional principal component analysis plot was generated to identify typical proteins related to the pathogenesis of dry AMD. Among the identified proteins, eight proteins, including APOA1, CFHR2, and CLUS, were previously considered major components or regulators of drusen. Three proteins (SERPINA4, LUM, and KERA proteins) have not been previously described as components of drusen or as being related to dry AMD. Interestingly, the LUM and KERA proteins, which are related to extracellular matrix organization, were upregulated in both RPD and soft drusen. CONCLUSIONS: Differential protein expression in the AH between patients with drusen and RPD was quantified using SWATH-MS in the present study. Detailed proteomic analyses of dry AMD patients might provide insights into the in vivo biology of drusen and RPD.

Antecedents of Soft Drusen, the Specific Deposits of Age-Related Macular Degeneration, in the Biology of Human Macula.

AMD pathobiology was irreversibly changed by the recent discovery of extracellular cholesterol-containing deposits in the subretinal space, between the photoreceptors and retinal pigment epithelium (RPE), called subretinal drusenoid deposits (SDDs). SDDs strikingly mirror the topography of rod photoreceptors in human macula, raising the question of whether an equivalent process results in a deposition related to foveal cones. Herein we propose that AMD's pathognomonic lesion-soft drusen and basal linear deposit (BLinD, same material, diffusely distributed)-is the leading candidate. Epidemiologic, clinical, and histologic data suggest that these deposits are most abundant in the central macula, under the fovea. Strong evidence presented in a companion article supports the idea that the dominant ultrastructural component is large apolipoprotein B,E-containing lipoproteins, constitutively secreted by RPE. Lipoprotein fatty acids are dominated by linoleate (implicating diet) rather than docosahexaenoate (implicating photoreceptors); we seek within the retina cellular relationships and dietary drivers to explain soft druse topography. The delivery of xanthophyll pigments to highly evolved and numerous Müller cells in the human fovea, through RPE, is one strong candidate, because Müller cells are the main reservoir of these pigments, which replenish from diet. We propose that the evolution of neuroglial relations and xanthophyll delivery that underlie exquisite human foveal vision came with a price, that is, soft drusen and sequela, long after our reproductive years.

Soft Drusen in Age-Related Macular Degeneration: Biology and Targeting Via the Oil Spill Strategies.

AMD is a major cause of legal blindness in older adults approachable through multidisciplinary research involving human tissues and patients. AMD is a vascular-metabolic-inflammatory disease, in which two sets of extracellular deposits, soft drusen/basal linear deposit (BLinD) and subretinal drusenoid deposit (SDD), confer risk for end-stages of atrophy and neovascularization. Understanding how deposits form can lead to insights for new preventions and therapy. The topographic correspondence of BLinD and SDD with cones and rods, respectively, suggest newly realized exchange pathways among outer retinal cells and across Bruch's membrane and the subretinal space, in service of highly evolved, eye-specific physiology. This review focuses on soft drusen/BLinD, summarizing evidence that a major ultrastructural component is large apolipoprotein B,E-containing, cholesterol-rich lipoproteins secreted by the retinal pigment epithelium (RPE) that offload unneeded lipids of dietary and outer segment origin to create an atherosclerosis-like progression in the subRPE-basal lamina space. Clinical observations and an RPE cell culture system combine to suggest that soft drusen/BLinD form when secretions of functional RPE back up in the subRPE-basal lamina space by impaired egress across aged Bruch's membrane-choriocapillary endothelium. The soft drusen lifecycle includes growth, anterior migration of RPE atop drusen, then collapse, and atrophy. Proof-of-concept studies in humans and animal models suggest that targeting the "Oil Spill in Bruch's membrane" offers promise of treating a process in early AMD that underlies progression to both end-stages. A companion article addresses the antecedents of soft drusen within the biology of the macula.