Human Age-related Macular Degeneration Research Articles

Chronic photo-oxidative stress and subsequent MCP-1 activation as causative factors for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. Although pathogenic factors, such as oxidative stress, inflammation and genetics are thought to contribute to the development of AMD, little is known about the relationships and priorities between these factors. Here, we show that chronic photo-oxidative stress is an environmental factor involved in AMD pathogenesis. We first demonstrated that exposure to light induced phospholipid oxidation in the mouse retina, which was more prominent in aged animals. The induced oxidized phospholipids led to an increase in the expression of monocyte chemoattractant protein-1, which then resulted in macrophage accumulation, an inflammatory process. Antioxidant treatment prevented light-induced phospholipid oxidation and the subsequent increase of monocyte chemoattractant protein-1 (also known as C-C motif chemokine 2; CCL2), which are the beginnings of the light-induced changes. Subretinal application of oxidized phospholipids induced choroidal neovascularization, a characteristic feature of wet-type AMD, which was inhibited by blocking monocyte chemoattractant protein-1. These findings strongly suggest that a sequential cascade from photic stress to inflammatory processes through phospholipid oxidation has an important role in AMD pathogenesis. Finally, we succeeded in mimicking human AMD in mice with low-level, long-term photic stress, in which characteristic pathological changes, including choroidal neovascularization formation, were observed. Therefore, we propose a consecutive pathogenic pathway involving photic stress, oxidation of phospholipids and chronic inflammation, leading to angiogenesis. These findings add to the current understanding of AMD pathology and suggest protection from oxidative stress or suppression of the subsequent inflammation as new potential therapeutic targets for AMD.

Localization of Papillofoveal Bundles in Primates

Axons in the fovea are precisely organized to ensure accurate vision. We investigated the morphologic characteristics and localization of nerve bundles in the optic nerve in primates. Macaque eyes were studied for conventional and immunostaining, and also marmoset eyes for carbocyanine dye tracing. Locally confined lesions associated with similar findings to human age-related macular degeneration (ARMD) were also evaluated. Axons of retinal ganglion cells formed fasciculi near their origin, and these fasciculi formed bundles thereafter. In the retinal nerve fiber layer, ascending bundles assembled stratification adding proximal bundle underneath successively. Bundles in the arcuate zone displayed a characteristic fine, parallel arrangement, whereas those in the outside zone intermingled with undefined reticular bundles as they approached the optic nerve head. Macular bundles remained in groups and were distributed in the temporal wedge of the optic nerve head. Orthograde and retrograde tracing revealed that these bundles formed confined groups of various sizes and, ultimately, a specific group of small bundles located in the innermost row, near the central vessels. In addition, these bundles showed evidence of focal degenerative deterioration in eyes with ARMD. Papillomacular bundles have a characteristic alignment and configuration. Foveal bundles that compose the confined group closest to the optic trunk (which we term papillofoveal bundles) appear to have functional significance with respect to the isolated lesions that accompany central vision loss or preservation.

Upregulation of CCR3 by Age-Related Stresses Promotes Choroidal Endothelial Cell Migration via VEGF-Dependent and -Independent Signaling

To explore the molecular mechanisms by which the C-C chemokine receptor type 3 (CCR3) and chemokine (C-C motif) ligand 11 (CCL11) regulate choroidal endothelial cell (CEC) migration and the interactions with the vascular endothelial growth factor (VEGF) signaling pathway. Human retinal sections from young and aged donor normal eyes were immunolabeled. By real-time PCR, CCR3 mRNA was measured in retinal pigmented epithelium (RPE)/choroids obtained from young and aged human donor eyes and in cultured CECs exposed to hydrogen peroxide. CCR3 ligand and CCL11- or VEGF-stimulated CEC migration was also measured in the presence of the CCR3 inhibitor or control using fluorescence microscopy. Activation of Rac1, phosphorylated Akt as a readout for phosphoinositol 3-kinase signaling, and VEGFR2 activation were measured in CECs incubated with CCL11, VEGF, or combined CCL11/VEGF. CCR3 was expressed to a greater level in older compared with that in younger human retinas or RPE/choroids. Ligand-activated CCR3 increased CEC migration, which was inhibited by the CCR3 inhibitor. Rac1 activity, p-Akt, and p-VEGFR2 were significantly increased in CECs incubated with CCL11. The CCR3 inhibitor prevented VEGF-induced CEC migration and Rac1 activation in CECs. Rac1 activity was additively increased in CECs treated with CCL11 and VEGF compared with that in cells with CCL11 or VEGF treatment alone. Ligand-activated CCR3 caused VEGFR2 phosphorylation and coimmunoprecipitation of VEGFR2 and CCR3. Activated CCR3 promotes CEC migration and Rac1 activation and causes an association with and activation of VEGFR2. Cross-talk between CCR3 and VEGF signaling exists and may be important in choroidal neovascularization in human age-related macular degeneration.

Breaking barriers: insight into the pathogenesis of neovascular age-related macular degeneration.

Neovascular age-related macular degeneration (AMD) is a leading cause of central visual acuity loss in a growing segment of the population, those over the age of 60 years. Treatment has improved over the last decade, with the availability of agents that inhibit the bioactivity of vascular endothelial growth factor (VEGF), but it is still limited, because of tachyphylaxis and potential risk and toxicity of anti-VEGF agents. The authors have sought to understand the mechanisms of choroidal endothelial cell (CEC) activation and transmigration of the retinal pigment epithelium (RPE) and of RPE barrier dysfunction, events preceding vision-threatening neovascular AMD. The authors developed physiologically relevant human RPE and CEC coculture and transmigration models that have been important in helping to understand causes of events in human neovascular AMD. The authors can control for interactions between these cells and can separately assess activation of signaling pathways in each cell type relevant during CEC transmigration. Using these models, it was found that VEGF, particularly the cell-associated VEGF splice variant VEGF189, accounts for about 40% of CEC transmigration across the RPE. This percentage is in the range of similar reports following clinical inhibition of VEGF in neovascular AMD. RPE VEGF189 working through CEC VEGF receptor 2 activates the small guanosine triphosphatase (GTPase) of the Rho family, Rac1, in CECs, which in turn facilitates CEC transmigration. Conversely, inhibition of Rac1 activity prevents CEC transmigration. Once activated, Rac1 aggregates with subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, resulting in the generation of reactive oxygen species. Activated NADPH oxidase increases choroidal neovascularization in animal models of laser-induced injury. Rac1 is also downstream of the eotaxin-CCR3 pathway, another pathway important in human neovascular AMD. Studies also suggest that active Ras-related protein 1 (Rap1), another small GTPase, in RPE can strengthen the RPE barrier integrity and can resist CEC transmigration of the RPE, suggesting Rap1 activation may be another potential target for preventing neovascular AMD.

Advanced Glycation Endproduct Changes to Bruch's Membrane Promotes Lipoprotein Retention by Lipoprotein Lipase

Lipoprotein particles accumulate in Bruch's membrane before the development of basal deposits and drusen, two histopathologic lesions that define age-related macular degeneration (AMD). We therefore, sought to determine which molecules could participate in lipoprotein retention. Wild-type or lipoprotein lipase-deficient mice were injected with low-dose D-galactose or PBS subcutaneously for 8 weeks to induce advanced glycation endproduct (AGE) formation. Some mice were also injected with the AGE breaker phenacylphiazolium bromide and D-galactose. Rhodamine-labeled low-density lipoproteins were injected into mice, and the fluorescence was measured up to 72 hours later. AGEs, proteoglycans, and other lipid-retaining molecules were evaluated by IHC. Lipoprotein lipase distribution was assessed in AMD samples by IHC. D-galactose-treated mice retained lipoproteins in the retinal pigment epithelial and Bruch's membrane to a greater extent than either PBS- or phenacylphiazolium bromide/D-galactose-treated mice at 24 and 72 hours after injection (P ≤ 0.04). Immunolabeling for carboxymethyllysine, biglycan, and lipoprotein lipase was found in D-galactose-treated mice only. Mice deficient for lipoprotein lipase treated with D-galactose did not retain lipoproteins to any measureable extent. Human AMD samples had lipoprotein lipase labeling within drusen, basal deposits, and the choroid. Mice treated with D-galactose to induce AGE formation in Bruch's membrane retain intravenously injected lipoproteins. Our results suggest that lipoprotein retention in Bruch's membrane is mediated by lipoprotein lipase.

Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

Age-Related Retinopathy in NRF2-Deficient Mice

BackgroundCumulative oxidative damage is implicated in the pathogenesis of age-related macular degeneration (AMD). Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that plays key roles in retinal antioxidant and detoxification responses. The purposes of this study were to determine whether NRF2-deficient mice would develop AMD-like retinal pathology with aging and to explore the underlying mechanisms.Methods and FindingsEyes of both wild type and Nrf2−/− mice were examined in vivo by fundus photography and electroretinography (ERG). Structural changes of the outer retina in aged animals were examined by light and electron microscopy, and immunofluorescence labeling. Our results showed that Nrf2−/− mice developed age-dependent degenerative pathology in the retinal pigment epithelium (RPE). Drusen-like deposits, accumulation of lipofuscin, spontaneous choroidal neovascularization (CNV) and sub-RPE deposition of inflammatory proteins were present in Nrf2−/− mice after 12 months. Accumulation of autophagy-related vacuoles and multivesicular bodies was identified by electron microcopy both within the RPE and in Bruch's membrane of aged Nrf2−/− mice.ConclusionsOur data suggest that disruption of Nfe2l2 gene increased the vulnerability of outer retina to age-related degeneration. NRF2-deficient mice developed ocular pathology similar to cardinal features of human AMD and deregulated autophagy is likely a mechanistic link between oxidative injury and inflammation. The Nrf2−/− mice can provide a novel model for mechanistic and translational research on AMD.

Evidence for enhanced tissue factor expression in age-related macular degeneration

Tissue factor (TF) is the primary initiator of blood coagulation. In addition to hemostasis, TF can initiate intracellular signaling and promote inflammation and angiogenesis, the key processes underlying the pathogenesis of age-related macular degeneration (AMD). AMD, the leading cause of irreversible blindness among the elderly, involves many genetic and environmental risk factors, including oxidative stress and inflammation. In this study, TF expression was examined in human AMD tissue and in the eyes of a model of AMD, the Ccl2−/−/Cx3cr1−/− (DKO) mouse, as well as in the ARPE-19 cell line after lipopolysaccharide (LPS) and H2O2 stimulation. Total RNA was extracted from tissue samples and further analyzed by real-time RT-PCR. Immunohistochemistry was performed to evaluate TF protein expression. In the human retina, a 32-fold increase of TF mRNA expression was detected in AMD macular lesions compared with normal maculae. TF protein expression was also enhanced in human AMD maculae. Similarly, TF transcript and protein expression were moderately increased in retinal lesions, neuroretinal tissue, and cultured RPE cells of DKO mice compared with age-matched wild-type mice. TF expression level correlated with age in both wild-type and DKO mice. In order to better understand how AMD might lead to enhanced TF expression, 1, 5, and 10 μg/ml LPS as well as 100 and 200 μM H2O2 were used to stimulate ARPE-19 cells for 24 and 2 h, respectively. LPS treatment consistently increased TF transcript and protein expression. H2O2 alone or in combination with LPS also moderately enhanced TF expression. These results indicate that upregulated TF expression may be associated with AMD, and inflammatory and oxidative stress may contribute to TF expression in AMD eyes.

Toll-like Receptor 3 Is Required for Development of Retinopathy Caused by Impaired All-trans-retinal Clearance in Mice

Chronic inflammation is an important component that contributes to many age-related neurodegenerative diseases, including macular degeneration. Here, we report a role for toll-like receptor 3 (TLR3) in cone-rod dystrophy (CORD) of mice lacking ATP-binding cassette transporter 4 (ABCA4) and retinol dehydrogenase 8 (RDH8), proteins critical for all-trans-retinal clearance in the retina. Increased expression of toll-like receptor-signaling elements and inflammatory changes were observed in Rdh8(-/-)Abca4(-/-) eyes by RNA expression analysis. Unlike 3-month-old Rdh8(-/-)Abca4(-/-) mice that developed CORD, 6-month-old Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice did not evidence an abnormal retinal phenotype. Light-induced retinal degeneration in Tlr3(-/-)Rdh8(-/-)Abca4(-/-) mice was milder than that in Rdh8(-/-)Abca4(-/-) mice, and a 2-fold increased TLR3 expression was detected in light-illuminated retinas of Rdh8(-/-)Abca4(-/-) mice compared with nonilluminated retinas. Poly(I-C), a TLR3 ligand, caused caspase-8-independent cellular apoptosis. Whereas poly(I-C) induced retinal cell death in Rdh8(-/-)Abca4(-/-) and WT mice both in vivo and ex vivo, this was not seen in mice lacking Tlr3. Far fewer invasive macrophage/microglial cells in the subretinal space and weaker activation of Muller glial cells were exhibited by Tlr3(-/-)Rdh8(-/-) Abca4(-/-) mice compared with Rdh8(-/-)Abca4(-/-) mice at 3 and 6 months of age, indicating that loss of TLR3 inhibits local inflammation in the retina. Both poly(I-C) and endogenous products emanating from dying/dead retinal cells induced NF-κB and IRF3 activation. These findings demonstrate that endogenous products from degenerating retina stimulate TLR3 that causes cellular apoptosis and retinal inflammation and that loss of TLR3 protects mice from CORD.

Parallel findings in age-related macular degeneration and Alzheimer’s disease

Age is a common risk factor for Alzheimer's disease (AD) and age-related macular degeneration (AMD). Because of the increasing age of the population, these two age-related diseases have recently received a great deal of attention. In addition to age as a risk factor, AD and AMD have many characteristics in common. An important characteristic common to both diseases is the presence of amyloid β (Aβ) in the senile plaques of the AD brain and in the drusen of AMD patients. We have focused on the role of Aβ as a key regulator of the progression from drusen to AMD, and our results have shown that Aβ causes an imbalance of angiogenesis-related factors in the retinal pigment epithelial (RPE) cells. Mice that lack the Aβ-degrading enzyme neprilysin develop RPE degeneration, and the sub-RPE deposits that are formed have features similar to those of AMD in humans. These data suggest that a common pathogenic mechanism might exist between AMD and AD. Thus, therapeutic approaches that have targeted Aβ in patients with AD can also be applied to AMD. In this review, we summarise recent findings on the shared characteristics and perspectives between AMD and AD, beginning with the mechanism of Aβ deposition and including a discussion of Aβ-targeted therapeutic approaches for both AD and AMD.

Alterations of retinal pigment epithelium cause AMD-like retinopathy in senescence-accelerated OXYS rats

Pathogenesis of age-related macular degeneration (AMD), the leading cause of blindness in the world, remains poorly understood. This makes it necessary to create animal models for studying AMD pathogenesis and to design new therapeutic approaches. Here we showed that retinopathy in OXYS rats is similar to human AMD according to clinical signs, morphology, and vascular endothelium growth factor (VEGF) and pigment epithelium-derived factor (PEDF) genes expression. Clinical signs of retinopathy OXYS rats manifest by the age 3 months against the background of significantly reduced expression level of VEGF and PEDF genes due to the decline of the amount of retinal pigment epithelium (RPE) cells and alteration of choroidal microcirculation. The disruption in OXYS rats' retina starts at the age of 20 days and appears as reduce the area of RPE cells but does not affect their ultrastructure. Ultrastructural pathological alterations of RPE as well as develop forms of retinopathy are observed in OXYS rats from age 12 months and manifested as excessive accumulation of lipofuscin in RPE regions adjacent to the rod cells, whirling extentions of the basement membrane into the cytoplasm. These data suggest that primary cellular degenerative alterations in the RPE cells secondarily lead to choriocapillaris atrophy and results in complete loss of photoreceptor cells in the OXYS rats' retina by the age of 24 months.

Progressive focal retinal degeneration in Ccl2/Cx3cr1 double deficient mice with pathological features of human age-related macular degeneration

Progressive focal retinal degeneration in Ccl2/Cx3cr1 double deficient mice with pathological features of human age-related macular degeneration

Retinal ultrastructure of murine models of dry age-related macular degeneration (AMD)

Age-related macular degeneration (AMD) is the most prevalent form of irreversible blindness worldwide in the elderly population. The pathology of dry AMD consists of macular degeneration of photoreceptors and the RPE, lipofuscin (A2E) accumulation, and drusen formation. Mice have been widely used for generating models that simulate human AMD features for investigating the pathogenesis, treatment and prevention of the disease. Although the mouse has no macula, focal atrophy of photoreceptors and RPE, lipofuscin accumulation, and increased A2E can develop in aged mouse eyes. However, drusen are rarely seen in mice because of their simpler Bruch's membrane and different process of lipofuscin extrusion compared with humans. Thus, analyzing basal deposits at the ultrastructural level and understanding the ultrastructural pathologic differences between various mouse AMD models are critical to comprehending the significance of research findings and response to possible therapeutic options for dry AMD. Based on the multifactorial pathogenesis of AMD, murine dry AMD models can be classified into three groups. First, genetically engineered mice that target genes related to juvenile macular dystrophies are the most common models, and they include abcr(-/-) (Stargardt disease), transgenic ELOVL4 (Stargardt-3 dominant inheritary disease), Efemp1(R345W/R345W) (Doyne honeycomb retinal dystrophy), and Timp3(S156C/S156C) (Sorsby fundus dystrophy) mice. Other murine models target genes relevant to AMD, including inflammatory genes such as Cfh(-/-), Ccl2(-/-), Ccr2(-/-), Cx3cr1(-/-), and Ccl2(-/-)/cx3cr1(-/-), oxidative stress associated genes such as Sod1(-/-) and Sod2 knockdown, metabolic pathway genes such as neprilysin(-/-) (amyloid beta), transgenic mcd/mcd (cathepsin D), Cp(-/-)/Heph(-/Y) (ferroxidase ceruloplasmin/hepaestin, iron metabolism), and transgenic ApoE4 on high fat and high cholesterol diet (lipid metabolism). Second, mice have also been immunologically manipulated by immunization with carboxyethylpyrrole (CEP), an oxidative fragment of DHA found in drusen, and found to present with dry AMD features. Third, natural mouse strains such as arrd2/arrd2 (Mdm gene mutation) and the senescence accelerated mice (SAM) spontaneously develop features of dry AMD like photoreceptor atrophy and thickening of Bruch's membrane. All the aforementioned models develop retinal lesions with various features that simulate dry AMD lesions: focal photoreceptor degeneration, abnormal RPE with increased lipofuscin, basal infolding, decreased melanosomes and degeneration. However, Bruch's membrane changes are less common. Most mice develop retinal lesions at an older age (6-24 months, depending on the models), while the Ccl2(-/-)/cx3cr1(-/-) mice develop lesions by 4-6 weeks. Although murine models present various degrees of retinal and/or RPE degeneration, classical drusen is extremely rare. Using electron microscopy, small drusenoid deposits are found between RPE and Bruch's membrane in a few models including Efemp1(R345W/R345W), Ccl2(-/-)/cx3cr1(-/-), neprilysin(-/-), transgenic mcd/mcd, and ApoE4 transgenic mice on a high fat diet. High A2E levels are measured in the retinas of abcr(-/-), transgenic ELOVL4, and Ccl2(-/-)/cx3cr1(-/-) mice. In summary, murine models provide useful tools for studying AMD pathogenesis and evaluating novel therapies for this disease. This review compares the major dry AMD murine models and discusses retinal pathology at the ultrastructural level.

Choroidal Neovascular Membranes Express Toll-Like Receptor 3

Background: Recent evidence has suggested a role for toll-like receptor 3 (TLR3) in experimental models of age-related macular degeneration (AMD). To date, however, few data exist about TLR3 in human AMD. The purpose of this study was to investigate the expression of TLR3 in human choroidal neovascular (CNV) membranes. Methods: Immunostaining for TLR3 was performed on sections of CNV membranes from 8 AMD patients and eyes from 4 donors without CNV. Results: All CNV membranes expressed TLR3 in retinal pigment epithelial (RPE) cells. One was classified as having strong intensity, 5 as having moderate intensity and 2 as having weak intensity. All cases had ≧30% of the RPE cells staining for TLR3, ranging from 30 to 90%. No expression of TLR3 was observed in vascular endothelial cells or fibroblasts in any CNV membrane. In the donor eyes, the RPE cells near the ora serrata stained stronger than those at the posterior pole, where no staining was observed in 3 out of 4 cases. Conclusion: TLR3 was found in all CNV membranes and was expressed exclusively in RPE cells. The observed difference in RPE staining for TLR3 in donor eyes and CNV membranes suggests a possible role for this receptor in human neovascular AMD.

Peroxisome Proliferator-Activated Receptor Expression in Murine Models and Humans with Age-related Macular Degeneration

Peroxisome proliferator-activated receptors (PPARs) play a role in oxidative stress and VEGF regulation, which are closely related to age-related macular degeneration (AMD). PPAR γ expression and its downstream molecules were examined in fat-1 mice (transgenic mice that convert n-6 to n-3 fatty acids), Ccl2(-/-)/Cx3cr1(-/-) mice (an AMD model), ARPE19 cells (a human retinal pigment epithelial cell line, RPE, a cell type with a critical role in AMD), and human eyes with and without AMD. PPAR α, β, and γ, VEGF and receptors were determined by immunohistochemistry in the mice models, humans, and ARPE19 cells. Transcripts of PPARs, VEGF, MMP-9 and HO-1 were determined by RQ-PCR. PPARs were constitutively expressed in normal neuroretina and RPE of humans and mice. PPAR γ expression was increased in fat-1 and Ccl2(-/-)/Cx3cr1(-/-) mice. VEGF was decreased in fat-1 mice but increased in Ccl2(-/-)/Cx3cr1(-/-) mice. VEGF receptors were stable. VEGF, MMP9 and HO-1 transcript levels were increased in ARPE19 cells under H(2)O(2) - induced oxidative stress. Human AMD retinas exhibited higher PPAR γ. The findings of increased expression of PPAR γ and its downstream proteins (VEGF, MMP9, and HO-1) in H(2)O(2)-treated ARPE19 cells, Ccl2(-/-)/Cx3cr1(-/-) mice, and human AMD eyes, but decreased VEGF in fat-1 mice, suggest that PPAR γ may play a role in AMD.

Aptamers: from bench side research towards patented molecules with therapeutic applications

Background: RNA and DNA aptamers recognize their targets with high specificity and affinity. These aptamers can be developed against almost any target protein through iterative cycles of in vitro screening of a combinatorial oligonucleotide library for target binding. Aptamer sequences from the final pool of in vitro selection are screened for pharmacological activity and possible medical applications. Methods: Chemical modifications and improvements of the identification of aptamer selection procedures made aptamers rival antibodies in diagnostic and therapeutic applications. This article reviews recent literature and patents and discusses the properties of aptamers as high-affinity and specificity target binders as well as their stability in biological fluids that turns them into therapeutic agents. Conclusion: The development of aptamers into compounds with therapeutic and diagnostic compounds has resulted in patents protecting the sequences and the use of these oligonucleotides. Several of these patented aptamers are currently being tested in Phase I or II clinical trials. Moreover, an anti-VEGF aptamer has already been approved by the FDA for treatment of age-related macular degeneration in humans.

Preclinical Evaluation of the Novel Small-Molecule Integrin α5β1 Inhibitor JSM6427 in Monkey and Rabbit Models of Choroidal Neovascularization

To evaluate the pharmacologic activity and tolerability of JSM6427, a potent and first selective small-molecule inhibitor of integrin alpha5beta1, in monkey and rabbit models of choroidal neovascularization (CNV). JSM6427 selectivity for alpha5beta1 was evaluated by in vitro binding assays while the ability of JSM6427 to inhibit CNV was investigated in a laser-induced monkey model and a growth factor-induced rabbit model. Intravitreal injections of JSM6427 (100, 300, or 1000 microg) or vehicle were administered immediately after the CNV induction procedure and at weekly intervals for 4 weeks. Fluorescein angiography was performed weekly. Ocular tolerability was evaluated ophthalmoscopically and histologically in both models; additional assessments in monkeys included electroretinography, biomicroscopy, pathological examination, and analysis of JSM6427 pharmacokinetics. JSM6427 was highly selective for the alpha5beta1-fibronectin interaction. Weekly intravitreal injections of JSM6427 resulted in a statistically significant dose-dependent inhibition of CNV in laser-induced and growth factor-induced models without any ocular JSM6427-related adverse effects. JSM6427 was cleared through the systemic circulation with no evidence of systemic accumulation. Intravitreal JSM6427 provided dose-dependent inhibition of CNV in monkey and rabbit experimental models. JSM6427 may provide a new approach for the treatment of ocular neovascular diseases such as age-related macular degeneration in humans.

Evaluation of Potential Therapies for a Mouse Model of Human Age-Related Macular Degeneration Caused by Delayed all-trans-Retinal Clearance

Evaluate the efficacy of potential therapeutics in Rdh8(-/-)Abca4(-/-) mice, a rodent model of human age-related macular degeneration (AMD). Therapeutic efficacy of several antioxidant agents (ascorbic acid, alpha-lipoic acid, alpha-tocopherol, Mn(III)-tetrakis(4-benzoic acid)-porphyrin, and butylated hydroxytoluene), an immunosuppressive agent with antivascular endothelial growth factor (VEGF) activity (sirolimus, also known as rapamycin), a retinoid cycle inhibitor (retinylamine), and an artificial chromophore (9-cis-retinyl acetate) were evaluated side by side in a recently described murine model of AMD, the Rdh8(-/-)Abca4(-/-) mouse. This animal exhibits a retinopathy caused by delayed all-trans-retinal clearance resulting from the absence of both ATP-binding cassette transporter 4 (Abca4) and retinol dehydrogenase 8 (Rdh8) activities. Drug efficacy was evaluated by retinal histologic analyses and electroretinograms (ERGs). All tested agents partially prevented atrophic changes in the Rdh8(-/-)Abca4(-/-) retina with retinylamine demonstrating the greatest efficacy. A significant reduction of complement deposition on Bruch's membrane was observed in sirolimus-treated mice, although the severity of retinal degeneration was similar to that observed in antioxidant- and 9-cis-retinyl acetate-treated mice. Sirolimus treatment of 6-month-old Rdh8(-/-)Abca4(-/-) mice for 4 months prevented choroidal neovascularization without changing retinal VEGF levels. Mechanism-based therapy with retinylamine markedly attenuated degenerative retinopathy in Rdh8(-/-)Abca4(-/-) mice. Further understanding of pathogenic mechanisms involved in AMD is needed to develop more effective therapeutics.

A human apoB100 transgenic mouse expresses human apoB100 in the RPE and develops features of early AMD

apoB100 lipoprotein particles have been found to accumulate in Bruch membrane prior to the development of age-related macular degeneration (AMD). This work was performed to determine whether mice that overexpress apoB100 in the RPE/choroid and liver develop landmarks of early AMD over time. Mice transgenic for a human genomic fragment encoding the full length human apoB (“apoB100” mice) and litter-mate control mice were given a normal chow or high-fat diet for 12 months. Mice were evaluated for human apoB mRNA expression in the RPE/choroid and liver by RT-qPCR. Phenotypic changes associated with early AMD were evaluated by ultrastructural analysis using transmission electron microscopy. Changes were semi-quantified using linear regression analysis. Both the RPE/choroid and liver of apoB100 mice expressed both human and mouse apoB mRNA. Transmission electron microscopy showed ultrastructural changes consistent with early human AMD including loss of basal infoldings and accumulation of cytoplasmic vacuoles in the RPE, and basal laminar deposits containing long-spacing collagen and heterogeneous debris in Bruch membrane of apoB100 mice. In apoB100 mice given a high-fat diet, basal linear-like deposits were identified in 12-month-old mice. Linear regression analysis showed that the genotype (human apoB transgene) was a stronger influencing factor than high-fat diet in producing AMD-like lesions used in this study. Human apoB100 transgenic mice overexpress apoB in RPE and, with time, develop validated phenotypic changes that are seen in early human AMD. The phenotypic changes were aggravated by feeding a high-fat diet. The apoB100 mouse model could be valuable in determining the role of apoB-containing lipoproteins in triggering the onset of early AMD.

Complement factor H related proteins in immune diseases

The complement system is a powerful part of the host innate immune defense and is aimed to damage and eliminate microbes and modified self-cells. To protect host cells and biological surfaces from damage mediated by complement activation products a tight control of the complement system is necessary. Imbalances in complement regulation contribute to tissue injury and can result in autoimmune diseases such as hemolytic uremic syndrome (HUS), membranoproliferative glomerulonephritis (MPGN) or age related macular degeneration (AMD). Disease associated mutations have been identified in several complement regulators or components, such as members of the factor H protein family. This group includes the major alternative pathway regulator, complement factor H (CFH) and five complement factor H related proteins (CFHR). Homozygous chromosomal deletion of a genomic 84kb, chromosomal fragment which includes the genes CFHR1/CFHR3 is a risk factor for hemolytic uremic syndrome (HUS) at young age and is predominantly associated with the generation of autoantibodies to CFH, leading to a specific type of HUS, called DEAP (deficiency of CFHR and autoantibody positive)—HUS. The same deletion however is protective to the development of age related macular degeneration (AMD) in elderly people. Thus CFHR1 and CFHR3 proteins, and likely also the other members of this gene family are linked to human diseases. We here summarize the current knowledge about the role or association of CFHR1 and CFHR3 in the human diseases HUS and AMD.