Retinal Iron Overload Research Articles

Protective benefits of salvianic acid A against retinal iron overload by inhibition of ferroptosis

BackgroundBoth the accumulation of reactive oxygen species (ROS) and iron overload are significant variables that enhance the incidence of photoreceptor cell death and retinal degeneration. The discovery of ferroptosis, which is characterized by iron-dependent lipid peroxidation, has led to a new perspective on how retinal degeneration develops. As a natural phenolic acid, salvianic acid A (SAA) from Salvia miltiorrhiza has promise in treating eye diseases. The purpose of this research was to learn more about SAA and its function in the development of iron-overload-induced retinal degeneration. MethodsModels of iron overload in Kunming mice and the murine photoreceptor cell line 661 W were established, then the protective and antiferroptotic properties of SAA were assessed in vivo and in vitro. ResultsBiochemical and histopathological findings on the retina confirmed that SAA successfully alleviated retinal injury. In photoreceptor cells, iron overload caused cell death, mitochondrial dysfunction, ROS generation, and iron deposition. Salvianic acid A relieved lipid peroxidation and decreased iron accumulation by modulating Acyl-CoA synthetase long-chain family member 4, glutathione peroxidase 4, solute carrier family 7 member 11, and iron-metabolism-related proteins. The mitochondrial morphology suggests that the retinal protective effect of SAA is mediated via antiferroptotic action. ConclusionFerroptosis plays an important role in the pathogenesis of iron-overload-induced retinal degeneration. New roles of SAA in ferroptosis prevention via iron deposit inhibition, lipid peroxidation inhibition, and mitochondrial dysfunction reduction, were identified.

Minimal effect of conditional ferroportin KO in the neural retina implicates ferrous iron in retinal iron overload and degeneration

Iron-induced oxidative stress can cause or exacerbate retinal degenerative diseases. Retinal iron overload has been reported in several mouse disease models with systemic or neural retina-specific knockout (KO) of homologous ferroxidases ceruloplasmin (Cp) and hephaestin (Heph). Cp and Heph can potentiate ferroportin (Fpn) mediated cellular iron export. Here, we used retina-specific Fpn KO mice to test the hypothesis that retinal iron overload in Cp/Heph DKO mice is caused by impaired iron export from neurons and glia. Surprisingly, there was no indication of retinal iron overload in retina-specific Fpn KO mice: the mRNA levels of transferrin receptor in the retina were not altered at 7–10-months age. Consistent with this, levels and localization of ferritin light chain were unchanged. To “stress the system”, we injected iron intraperitoneally into Fpn KO mice with or without Cp KO. Only mice with both retina-specific Fpn KO and Cp KO had modestly elevated retinal iron levels. These results suggest that impaired iron export through Fpn is not sufficient to explain the retinal iron overload in Cp/Heph DKO mice. An increase in the levels of retinal ferrous iron caused by the absence of these ferroxidases, followed by uptake into cells by ferrous iron importers, is most likely necessary.

Ameliorative effects and mechanisms of salvianic acid A on retinal iron overload in vivo and in vitro

Excessive iron can be accumulated in the retina and lead to retinal iron overload. Salvianic acid A (SAA) has a variety of pharmacologic effects, but there is only a limited understanding of its benefits for retinal iron overload. The aim of this study was to examine the protective effects and latent mechanisms of SAA on retinal iron overload. SAA reduced iron in the serum and retina, attenuated pathophysiological changes, and reduced retinal iron deposition in the retinas of iron-overloaded mice. It also reduced intracellular iron in ARPE-19 cells by regulating iron-handling proteins and chelating with iron. It also significantly inhibited cellular oxidative and inflammatory damage by increasing the nuclear translocation of nuclear erythroid 2-related factor 2 (Nrf2) while decreasing nuclear factor-kappa B (NF-κB), protecting the ARPE-19 cells from apoptosis by suppressing the Bax/Bcl-2 ratio, cytochrome c release, caspase activation, and poly ADP-ribose polymerase cleavage. The ability of SAA to inhibit apoptosis, increase nuclear Nrf2 expression, and decrease nuclear NF-κB expression was further confirmed in the retinas of iron-overloaded mice. This study demonstrates that SAA shows significant protective effects against retinal iron overload; its mechanisms might be associated with iron chelation; regulation of iron-handling proteins; and inhibition of oxidative stress, inflammation and apoptosis.

A vicious cycle of bisretinoid formation and oxidation relevant to recessive Stargardt disease

The ability of iron to transfer electrons enables the contribution of this metal to a variety of cellular activities even as the redox properties of iron are also responsible for the generation of hydroxyl radicals (•OH), the most destructive of the reactive oxygen species. We previously showed that iron can promote the oxidation of bisretinoid by generating highly reactive hydroxyl radical (•OH). Now we report that preservation of iron regulation in the retina is not sufficient to prevent iron-induced bisretinoid oxidative degradation when blood iron levels are elevated in liver-specific hepcidin knockout mice. We obtained evidence for the perpetuation of Fenton reactions in the presence of the bisretinoid A2E and visible light. On the other hand, iron chelation by deferiprone was not associated with changes in postbleaching recovery of 11-cis-retinal or dark-adapted ERG b-wave amplitudes indicating that the activity of Rpe65, a rate-determining visual cycle protein that carries an iron-binding domain, is not affected. Notably, iron levels were elevated in the neural retina and retinal pigment epithelial (RPE) cells of Abca4−/− mice. Consistent with higher iron content, ferritin-L immunostaining was elevated in RPE of a patient diagnosed with ABCA4-associated disease and in RPE and photoreceptor cells of Abca4−/− mice. In neural retina of the mutant mice, reduced Tfrc mRNA was also an indicator of retinal iron overload. Thus iron chelation may defend retina when bisretinoid toxicity is implicated in disease processes.

Astragaloside IV protects against retinal iron overload toxicity through iron regulation and the inhibition of MAPKs and NF-κB activation

Iron overload toxicity has been implicated in retinal pigment epithelial cell injury in age-related macular degeneration. This study investigates the effects of astragaloside IV (AS-IV), a potential retinal protective agent, on the toxicity process of retinal iron overload in vivo and in vitro. AS-IV partially restored the retinal expression of rhodopsin and retinal pigment epithelium-specific 65 kDa protein, suppressed oxidative stress and inflammatory markers, and alleviated iron deposition and retinal pathological changes in vivo. Also, AS-IV inhibited the phosphorylation of p38 and ERK mitogen-activated protein kinases (MAPKs), as well as the nuclear translocation of nuclear factor-kappa B (NF-κB). Furthermore, AS-IV prevented cell death by decreasing the ratio of Bax/Bcl-2, caspase-3, and cleaved caspase-3 expression in vitro. Although there are no chelation effects between AS-IV and iron, AS-IV can reduce intracellular iron by regulating iron-handling proteins in ARPE-19 cells (Cav1.2, divalent metal transporter-1, transferrin receptor 1, and heavy-chain ferritin). In conclusion, the results show that AS-IV has significant protective effects against retinal iron overload toxicity and suggest that iron regulation and the inhibition of MAPKs and NF-κB activation might be mechanisms underlying the effects of AS-IV.

Fenofibrate prevents iron induced activation of canonical Wnt/\u03b2-catenin and oxidative stress signaling in the retina

Accumulating evidence strongly implicates iron in the pathogenesis of aging and disease. Iron levels have been found to increase with age in both the human and mouse retinas. We and others have shown that retinal diseases such as age-related macular degeneration and diabetic retinopathy are associated with disrupted iron homeostasis, resulting in retinal iron accumulation. In addition, hereditary disorders due to mutation in one of the iron regulatory genes lead to age dependent retinal iron overload and degeneration. However, our knowledge on whether iron toxicity contributes to the retinopathy is limited. Recently, we reported that iron accumulation is associated with the upregulation of retinal and renal renin–angiotensin system (RAS). Evidences indicate that multiple genes/components of the RAS are targets of Wnt/β-catenin signaling. Interestingly, aberrant activation of Wnt/β-catenin signaling is observed in several degenerative diseases. In the present study, we explored whether iron accumulation regulates canonical Wnt signaling in the retina. We found that in vitro and in vivo iron treatment resulted in the upregulation of Wnt/β-catenin signaling and its downstream target genes including renin–angiotensin system in the retina. We confirmed further that iron activates canonical Wnt signaling in the retina using TOPFlash T-cell factor/lymphoid enhancer factor promoter assay and Axin2-LacZ reporter mouse. The presence of an iron chelator or an antioxidant reversed the iron-mediated upregulation of Wnt/β-catenin signaling in retinal pigment epithelial (RPE) cells. In addition, treatment of RPE cells with peroxisome proliferator-activated receptor (PPAR) α-agonist fenofibrate prevented iron-induced activation of oxidative stress and Wnt/β-catenin signaling by chelating the iron. The role of fenofibrate, an FDA-approved drug for hyperlipidemia, as an iron chelator has potentially significant therapeutic impact on iron associated degenerative diseases.

Liver-Specific, but Not Retina-Specific, Hepcidin Knockout Causes Retinal Iron Accumulation and Degeneration

The liver secretes hepcidin (Hepc) into the bloodstream to reduce blood iron levels. Hepc accomplishes this by triggering degradation of the only known cellular iron exporter ferroportin in the gut, macrophages, and liver. We previously demonstrated that systemic Hepc knockout (HepcKO) mice, which have high serum iron, develop retinal iron overload and degeneration. However, it was unclear whether this is caused by high blood iron levels or, alternatively, retinal iron influx that would normally be regulated by retina-produced Hepc. To address this question, retinas of liver-specific and retina-specific HepcKO mice were studied. Liver-specific HepcKO mice had elevated blood and retinal pigment epithelium (RPE) iron levels and increased free (labile) iron levels in the retina, despite an intact blood-retinal barrier. This led to RPE hypertrophy associated with lipofuscin-laden lysosome accumulation. Photoreceptors also degenerated focally. In contrast, there was no change in retinal or RPE iron levels or degeneration in the retina-specific HepcKO mice. These data indicate that high blood iron levels can lead to retinaliron accumulation and degeneration. High blood iron levels can occur in patients with hereditary hemochromatosis or result from use of iron supplements or multiple blood transfusions. Our resultssuggest that high blood iron levels may cause or exacerbate retinal disease.

IRON TOXICITY AS A POTENTIAL FACTOR IN AMD

While it has been known for years that iron overload is associated with retinal degeneration in the context of ocular siderosis, intraocular hemorrhage, and the hereditary diseases aceruloplasminemia and pantothenate kinase associated neurodegeneration, recent evidence suggests that age-related macular degeneration (AMD) may also be exacerbated by retinal iron overload. In the retina, iron is necessary for normal cellular function. Iron overload, however, can cause retinal toxicity through the generation of oxygen free radicals. Histopathology of eyes with macular degeneration has shown elevated levels of iron in the retinal pigment epithelium, Bruch membrane, and within drusen, some of which was chelatable in vitro with deferoxamine. In this review, the authors summarize the evidence that iron overload may contribute to AMD pathogenesis. It is hoped that continued investigation of the role of iron and iron associated proteins in the retina will uncover clues to AMD pathogenesis and lead to new preventative or therapeutic options.

Iron homeostasis and toxicity in retinal degeneration

Iron is essential for many metabolic processes but can also cause damage. As a potent generator of hydroxyl radical, the most reactive of the free radicals, iron can cause considerable oxidative stress. Since iron is absorbed through diet but not excreted except through menstruation, total body iron levels buildup with age. Macular iron levels increase with age, in both men and women. This iron has the potential to contribute to retinal degeneration. Here we present an overview of the evidence suggesting that iron may contribute to retinal degenerations. Intraocular iron foreign bodies cause retinal degeneration. Retinal iron buildup resulting from hereditary iron homeostasis disorders aceruloplasminemia, Friedreich's ataxia, and panthothenate kinase-associated neurodegeneration cause retinal degeneration. Mice with targeted mutation of the iron exporter ceruloplasmin have age-dependent retinal iron overload and a resulting retinal degeneration with features of age-related macular degeneration (AMD). Post mortem retinas from patients with AMD have more iron and the iron carrier transferrin than age-matched controls. Over the past 10 years much has been learned about the intricate network of proteins involved in iron handling. Many of these, including transferrin, transferrin receptor, divalent metal transporter-1, ferritin, ferroportin, ceruloplasmin, hephaestin, iron-regulatory protein, and histocompatibility leukocyte antigen class I-like protein involved in iron homeostasis (HFE) have been found in the retina. Some of these proteins have been found in the cornea and lens as well. Levels of the iron carrier transferrin are high in the aqueous and vitreous humors. The functions of these proteins in other tissues, combined with studies on cultured ocular tissues, genetically engineered mice, and eye exams on patients with hereditary iron diseases provide clues regarding their ocular functions. Iron may play a role in a broad range of ocular diseases, including glaucoma, cataract, AMD, and conditions causing intraocular hemorrhage. While iron deficiency must be prevented, the therapeutic potential of limiting iron-induced ocular oxidative damage is high. Systemic, local, or topical iron chelation with an expanding repertoire of drugs has clinical potential.

Iron Induced Oxidative Damage As a Potential Factor in Age-Related Macular Degeneration: The Cogan Lecture

Iron is a potent generator of oxidative damage whose levels increase with age, potentially exacerbating age-related diseases. Several lines of evidence suggest that iron accumulation may be a factor in age-related macular degeneration (AMD). AMD retinas have more iron within the photoreceptors, RPE, and drusen than do age-matched control retinas. Accelerated AMD-like maculopathy develops in patients with retinal iron overload from the hereditary disease aceruloplasminemia. Mice with retinal iron overload resulting from knockout of ceruloplasmin and its homologue hephaestin exhibit retinal degeneration with some features of AMD, including subretinal neovascularization, accumulation of RPE lipofuscin and sub-RPE deposits, and RPE/photoreceptor death. Increased understanding of the mechanisms of retinal iron homeostasis may help in the development of therapies to prevent iron overload. For example, herein it is shown that one regulator of systemic iron homeostasis, HFE, is expressed in the RPE. Thus, patients with the common disease hereditary hemochromatosis, which is often caused by an HFE mutation, may have retinal iron overload predisposing to AMD. Preliminary data suggest that iron chelation can reduce RPE iron overload in mice and protect them from degeneration, suggesting that iron-binding drugs may one day prove useful in reducing RPE oxidative stress and decreasing the risk of AMD progression.

The Iron Carrier Transferrin Is Upregulated in Retinas from Patients with Age-Related Macular Degeneration

Iron can cause oxidative stress, and elevated iron levels have been associated with several neurodegenerative diseases including age-related macular degeneration (AMD). Transferrin, an iron transport protein, is expressed at high levels in the retina. The purpose of this study was to assess transferrin involvement in AMD by determining the expression profile of transferrin in retinas with AMD compared with retinas without evidence of disease. Postmortem retinas were obtained from AMD and non-AMD eyes. Expression of transferrin was assessed in a microarray dataset from 33 retinas of unaffected donors and 12 retinas of patients with AMD (six with neovascular AMD and six with non-neovascular AMD). Quantitative real-time RT-PCR (QPCR) was used to confirm the microarray results. Transferrin protein expression was assessed by semiquantitative Western blot analysis and immunohistochemistry. In comparison to unaffected retinas, mean transferrin mRNA levels, as measured by microarray analysis were elevated 3.5- and 2.1-fold in non-neovascular and neovascular AMD retinas, respectively. Semiquantitative Western blot analysis demonstrated a 2.1-fold increase in transferrin protein in AMD eyes. Immunohistochemistry showed more intense and widespread transferrin label in AMD maculas, particularly in large drusen, Müller cells, and photoreceptors. These data demonstrate that transferrin expression is increased in the retinas of patients with AMD relative to those of healthy control patients of comparable age. Along with previous studies that have demonstrated elevated iron levels in AMD retinas, early onset drusen formation in a patient with retinal iron overload resulting from aceruloplasminemia, and retinal degeneration with some features of macular degeneration in the iron-overloaded retinas of ceruloplasmin/hephestin knockout mice, the present study suggests that altered iron homeostasis is associated with AMD.

Macular Degeneration in a Patient with Aceruloplasminemia, a Disease Associated with Retinal Iron Overload

To provide the first ophthalmic case report of a Caucasian patient with the rare autosomal recessive disease aceruloplasminemia, which results in iron overload in the retina, brain, and pancreas. Single observational case report. Perls' staining of a conjunctival biopsy was used to detect elevated iron levels in the conjunctival epithelium. Fundus photography, fluorescein angiography, and electroretinography were used to document retinal appearance and function. Unlike a report of a Japanese patient with aceruloplasminemia, who had midperipheral retinal pigment epithelium (RPE) cell atrophy and yellowish discoloration of the fundus, our Caucasian patient had a maculopathy. Beginning at age 47, he had development and progression of multiple subretinal yellowish-white lesions and RPE cell atrophy. To confirm tissue iron overload in our patient, we took the novel approach of a conjunctival biopsy, which showed Perls' Prussian blue-positive epithelial cells. Given our recent finding of elevated iron levels in the RPE of patients with age-related macular degeneration (AMD), it is interesting that retinal iron overload in aceruloplasminemia is associated with a maculopathy that clinically resembles AMD. This finding supports the hypothesis that retinal iron homeostasis is essential for normal retinal function. Disruption of iron homeostasis could contribute to the pathogenesis of AMD.

Disruption of cereuloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Hahn P, Qian Y, Dentchev T, Chen L, Beard J, Harris ZL, Dunaief JL. PNAS 2004;101:13850–13855

Disruption of cereuloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration. Hahn P, Qian Y, Dentchev T, Chen L, Beard J, Harris ZL, Dunaief JL. PNAS 2004;101:13850–13855

Disruption of ceruloplasmin and hephaestin in mice causes retinal iron overload and retinal degeneration with features of age-related macular degeneration.

Mechanisms of brain and retinal iron homeostasis have become subjects of increased interest after the discovery of elevated iron levels in brains of patients with Alzheimer's disease and retinas of patients with age-related macular degeneration. To determine whether the ferroxidase ceruloplasmin (Cp) and its homolog hephaestin (Heph) are important for retinal iron homeostasis, we studied retinas from mice deficient in Cp and/or Heph. In normal mice, Cp and Heph localize to Müller glia and retinal pigment epithelium, a blood-brain barrier. Mice deficient in both Cp and Heph, but not each individually, had a striking, age-dependent increase in retinal pigment epithelium and retinal iron. The iron storage protein ferritin was also increased in Cp-/-Heph-/Y retinas. After retinal iron levels had increased, Cp-/-Heph-/Y mice had age-dependent retinal pigment epithelium hypertrophy, hyperplasia and death, photoreceptor degeneration, and subretinal neovascularization, providing a model of some features of the human retinal diseases aceruloplasminemia and age-related macular degeneration. This pathology indicates that Cp and Heph are critical for CNS iron homeostasis and that loss of Cp and Heph in the mouse leads to age-dependent retinal neurodegeneration, providing a model that can be used to test the therapeutic efficacy of iron chelators and antiangiogenic agents.