Ferroptosis in retinal pigment epithelial cells: Current status and future developments

Abstract

Retinal pigment epithelial (RPE) cells play a crucial role in maintaining the normal function of the retina. In recent years, research on ferroptosis—a novel iron-dependent form of cell death—in RPE cells has gradually attracted attention. This article reviews the mechanisms of ferroptosis in RPE cells, including iron metabolism imbalance, lipid peroxidation, and functional impairment of the glutathione-glutathione peroxidase 4 (GSH-GPX4) system. It explores the association between ferroptosis in RPE cells and retinal diseases such as age-related macular degeneration and Stargardt disease. Additionally, it analyzes the future challenges in current research on ferroptosis in RPE cells, such as gaining in-depth understanding of complex regulatory networks and developing precise intervention strategies, so as to provide new insights and directions for the prevention and treatment of retinal diseases.

The retina is the innermost light-sensitive tissue of the eye wall. Through the layered structure and synergistic interaction of highly specialized cells, it accomplishes the conversion of “light signals → electrical signals → neural signals” and serves as a core link in visual formation. Structurally, it can be divided into two major functional regions from the outside to the inside: the retinal pigment epithelium (RPE) layer and the neuroretina layer. Cells in each layer have clear divisions of labor and work closely together. The neuroretina is a key component of retinal tissue, forming the inner layer of the retina.[1] It mediates phototransduction through the coordinated action of multiple cell types, including photoreceptor cells (rods and cones), bipolar cells, ganglion cells, amacrine cells, horizontal cells, and glial cells such as Müller cells and microglia. Among these, photoreceptor cells are the primary sensory cells of the neuroretina: Rods are responsible for visual perception in low-light conditions, while cones mediate color vision and high-acuity vision in bright light. The outer segments of photoreceptor cells are rich in light-sensitive opsin proteins, which serve as the molecular basis for capturing light photons.[2] When light irradiates the outer segments, it triggers a series of biochemical reactions and initiates the phototransduction cascade. This cascade ultimately causes changes in the membrane potential of photoreceptor cells, converting light signals into electrical signals. These electrical signals are then transmitted sequentially through bipolar cells and amacrine cells to retinal ganglion cells (RGCs). The axons of RGCs form the optic nerve, which conducts the signals to the visual cortex of the brain for processing, thereby generating visual perception.[3] RPE cells are specialized hexagonal epithelial cells located in the outer layer of the retina, between photoreceptors (PR) and Bruch’s membrane (BrM). Derived from neuroepithelial cells, they connect to PR via microvilli, and their development is accompanied by changes in morphology, gene and protein expression, and localization.[4] RPE cells play multiple crucial roles in maintaining the physiological functions of the retina. They not only provide nutritional support for photoreceptors, participate in the visual cycle, and phagocytose photoreceptor outer segments but also are essential for maintaining the homeostasis of the retinal internal environment or death of RPE cells is closely associated with a variety of severe retinal diseases,[5] among which age-related macular degeneration (AMD) is a common and highly detrimental one—approximately 200 million people worldwide suffer from some form of AMD.[6] In the United States, the number of affected individuals exceeds 10 million and is steadily increasing.[6] With aging, iron accumulation in RPE may be one of the important sources of oxidative stress leading to AMD.[7] A study compared the RPE/choroid of young and aged rodents and found that the iron content in the aged RPE/choroid increased significantly, and iron-regulating molecules changed. In vitro experiments also showed that increased intracellular iron reduced the phagocytic function and lysosomal activity of Adult Retinal Pigment Epithelial Cell Line-19 (ARPE-19). These findings suggest that age-related changes in RPE iron homeostasis may make tissues more susceptible to diseases such as AMD.[8]

Ferroptosis is a form of regulated cell death caused by lipid peroxidation, which differs from other types of cell death in terms of genetics, biochemistry, and morphology. A hallmark event of ferroptosis is uncontrolled phospholipid peroxidation, a process that relies on transition metal iron, reactive oxygen species (ROS), and phospholipids containing polyunsaturated fatty acid chains.[9] When the accumulation of intracellular phospholipid hydroperoxides (PLOOHs) exceeds a certain threshold, it causes damage to the cell membrane and ultimately leads to cell death.[9] Recent studies have shown that ferroptosis plays an important role in the pathological process of RPE cells, providing a new perspective for understanding the pathogenesis of retinal diseases and identifying new therapeutic targets.[10,11] In the evaluation of the effects of blood-derived components (hemoglobin and heme) on RPE cells, it was found that iron is one of the factors causing cell damage.[12] Under oxidative stress conditions, such as in the sodium iodate (NaIO₃)-induced RPE cell injury model, ferroptosis is the main mode of cell death. Studies have revealed that ARPE-19 cells and mouse mRPE cells treated with NaIO₃ exhibit typical characteristics of ferroptosis, including increased mitochondrial membrane density, reduced mitochondrial volume, and massive intracellular accumulation of ferrous ions and lipid ROS. Meanwhile, the expression of ferroptosis-related markers shows distinct changes: The protein expression of PTGS2 is upregulated, while that of GPX4 is downregulated. After treating the cells with ferroptosis inhibitors (Ferrostatin-1(Fer-1) and Deferoxamine (DFO)), cell death is significantly reduced, indicating that inhibiting ferroptosis can effectively rescue RPE cells.[11]

Mechanisms of Ferroptosis in Retinal Pigment Epithelial Cells

Iron metabolism imbalance

Iron is an essential element for the normal physiological activities of cells, but the imbalance of intracellular iron homeostasis can trigger a series of pathological processes, including ferroptosis. In RPE cells, the processes of iron uptake, storage, and release are finely regulated. The ferroxidases hephaestin (Heph) and ceruloplasmin (Cp) are crucial for retinal iron homeostasis. By constructing various conditional gene knockout mouse models, researchers have found that Heph exerts a cell-autonomous role in RPE cells. The specific deletion of Heph and Cp in RPE cells alone leads to iron accumulation and degeneration.[13,14] Heph and Cp are crucial for iron export from the retina but not essential for iron import. There are two pathways for iron import (from blood vessels to the retina)[15]: transferrin-bound iron (TBI) and non-transferrin-bound iron (NTBI). The TBI pathway is the fundamental pathway for retinal iron import, relying on endocytosis mediated by Tf-TfR. It mainly uptakes ferric iron (Fe³⁺) from the choroid (via TfR on the basolateral side of RPE cells) and retinal blood vessels (via TfR on vascular endothelial cells), providing a stable iron source for retinal physiological activities such as the visual cycle and neurotransmitter synthesis. The NTBI pathway is an important supplementary pathway during iron overload. It can mediate the transport of ferritin (Ft) across the blood-retinal barrier (BRB) via scavenger receptor class A member 5 (Scara5) and transport non-transferrin-bound iron via Zrt/Irt-like protein 8/14 (Zip8/Zip14). Particularly in Müller cells, it participates in the redistribution of iron in the retina to maintain iron homeostasis.[16,17] Iron export (from the retina to blood vessels) relies on an active transport mechanism mediated by the iron exporter protein Ferroportin (Fpn). During this process, the exported ferrous iron (Fe²⁺) must be oxidized to Fe³⁺ by two types of multi-copper ferroxidases, namely, Heph or Cp. Only then can the converted Fe³⁺ bind to Tf in the blood and ultimately be carried away by the blood circulation, completing the transport process of iron from the retina to blood vessels.[15] Fpn is currently the only known iron exporter in mammals and a key molecule for cells to transport iron out of the cytoplasm. In retinal tissue, it is mainly localized to specific membrane structures of three key cell types: retinal vascular endothelial cells (rVECs), RPE cells, and Müller cells. Defects in Fpn function or dysregulation of Fpn will directly lead to the disruption of retinal iron homeostasis.[18]

The serine protease matriptase-2 (encoded by the Transmembrane Protease, Serine 6 (Tmprss6) gene) is a membrane-bound serine protease that plays a crucial role in iron release.[19] Its primary function is to cleave membrane-bound hemojuvelin (HJV), thereby abolishing HJV’s ability to induce the production of hepcidin. Hepcidin, an iron-regulatory hormone, is a small peptide hormone composed of 25 amino acids. At physiological concentrations, its main function is to regulate iron homeostasis.[20] The target protein of hepcidin is FPN, the iron export transporter. Upon binding between hepcidin and FPN, FPN undergoes proteasomal degradation. This process reduces the delivery of dietary iron from intestinal cells into the bloodstream and decreases iron release from macrophages, ultimately regulating iron balance in the body. Elevated hepcidin levels lead to systemic iron deficiency; conversely, decreased hepcidin levels trigger iron overload.[20] Bone morphogenetic protein 6 (Bmp6) is a key activator of the BMP signaling pathway. It is expressed in RPE cells, and after secretion, it binds to receptors on the neural retina, inducing the phosphorylation of mothers against decapentaplegic homolog (Smad) proteins (e.g., Smad1, Smad5, and Smad8). These phosphorylated Smad proteins form complexes with Smad4, which then translocate into the cell nucleus to upregulate hepcidin expression in the neural retina.[21,22] Retinopathy in Bmp6 knockout mice exhibits age and sex differences[23]: Male mice show normal retinal morphology at 7 weeks of age, but by 41 weeks, approximately 50% of their retinas develop RPE cell hypertrophy and hyperplasia, accompanied by the loss of photoreceptor inner and outer segments and thinning of the outer nuclear layer. Female mice maintain normal retinal morphology throughout the observation period; Meanwhile, RPE cells in male Bmp6 knockout mice accumulate lipofuscin-like substances, whose autofluorescence spectrum shares a similar emission peak with that of RPE cells from patients with AMD. This suggests that iron-induced oxidative damage may lead to AMD-like molecular accumulation. In patients with early-stage AMD, Bmp6 levels in RPE cells are reduced; insufficient Bmp6 fails to effectively regulate iron metabolism, resulting in increased retinal iron accumulation—similar to the condition in Bmp6 knockout mice—which further exacerbates the disease. In patients with late-stage AMD, Bmp6 levels in the neural retina are elevated, potentially representing a compensatory response to iron accumulation, though the specific mechanism requires further investigation. These changes in Bmp6 levels indicate that it may play an important role in the development and progression of AMD, providing insights for understanding AMD pathogenesis and identifying therapeutic targets. However, studies have found that in the retinas of matriptase-2 knockout mice, hepcidin expression is upregulated—but this upregulation occurs via the interleukin-6 (IL-6) signaling pathway, which differs from the traditionally recognized HJV/BMP/Smad pathway. This finding revises the conventional understanding that “retinal iron metabolism depends solely on BMP signaling” and reveals the compensatory role of inflammatory signals (e.g., IL-6) in iron homeostasis. It suggests that the retina may possess a local regulatory mechanism independent of systemic iron metabolism.[24] Nevertheless, this view currently lacks sufficient evidence support, and the BMP signaling pathway remains central to maintaining systemic iron homeostasis.

Lipid peroxidation

Lipid peroxidation is one of the core characteristics of ferroptosis. Polyunsaturated fatty acids (PUFAs), especially arachidonic acid (AA) and adrenic acid (AdA), are important components of cell membrane lipids. They contain multiple double bonds and are extremely sensitive to oxidative stress.[25] Iron metabolism imbalance (such as Fe²⁺ accumulation) can directly catalyze the generation of ROS through the Fenton reaction. Among them, free radicals like hydroxyl radicals (・OH) attack PUFAs, triggering lipid peroxidation. Acyl-CoA synthetase long-chain family member 4 (ACSL4) plays a key role in this process: It specifically catalyzes PUFAs to form arachidonoyl-CoA (AA-CoA) and adrenoyl-CoA (AdA-CoA). These products further participate in phospholipid synthesis, generating phospholipids containing PUFAs (e.g., phosphatidylethanolamine (PE)). Cell membranes containing PUFA-PE are more prone to peroxidation under the action of ROS, forming lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE).[25] These peroxidation products are cytotoxic; they can damage the integrity of cell membranes, cause the leakage of intracellular substances, and ultimately induce the death of RPE cells.[10]

Dysfunction of the GSH-GPX4 system

The Glutathione-Glutathione Peroxidase 4(GSH-GPX4) system is a crucial intracellular antioxidant defense mechanism, playing a vital role in inhibiting ferroptosis. GPX4 is a selenium-containing enzyme that uses reduced GSH as a substrate to reduce lipid peroxides (e.g., L-OOH) to their corresponding alcohols (L-OH). This process blocks the lipid peroxidation chain reaction and protects cells from oxidative damage.[26] Studies have shown that GSH depletion induces ferroptosis, autophagy, and premature senescence in RPE cells: When ARPE-19 cells were treated with cystine-free medium, buthionine sulfoximine (BSO), or erastin to deplete GSH, the results revealed decreased cell viability, increased production of soluble and lipid ROS, downregulated GPX4 expression, and activated autophagy. Additionally, the cells exhibited characteristics of senescence, including an increase in senescence-associated β-galactosidase-positive cells, accumulation of senescence-associated heterochromatic foci (SAHF), and cell cycle arrest at the G1 phase. Notably, ferroptosis inhibitors, iron chelators, and autophagy inhibitors could prevent cell death caused by GSH depletion.[27] Interferon-γ (IFN-γ) increases intracellular Fe²⁺ levels by inhibiting the Fe²⁺ exporter SLC40A1 (Solute Carrier Family 40 Member 1). It also activates the Janus Kinase 1-2/Signal Transducer and Activator of Transcription 1/Solute Carrier Family 7 Member 11 (JAK1-2/STAT1/SLC7A11) signaling pathway, leading to GSH exhaustion and reduced GPX4 levels, thereby increasing cell sensitivity to ferroptosis. Therefore, reducing IFN-γ levels may be a potential target for the treatment of AMD.[28]

In summary, iron metabolism imbalance can catalyze ROS generation through the Fenton reaction, triggering lipid peroxidation. Lipid peroxidation products, in turn, deplete GSH and inhibit GPX4 activity, resulting in dysfunction of the GSH-GPX4 system. This dysfunction impairs the ability to scavenge lipid peroxides, further exacerbating iron-driven oxidative damage. These three factors form a cascading pathological chain of “iron metabolism imbalance → lipid peroxidation → GSH-GPX4 system dysfunction,” which collectively promotes the progression of ferroptosis and other oxidative stress-related diseases. Furthermore, mitochondria play a central role in the initiation and progression of ferroptosis by regulating iron metabolism, ROS production, energy metabolism, and the autophagy-lysosome system.[29,30] Damaged mitochondria are cleared through mitophagy; during this process, released iron-sulfur clusters (4Fe-4S) and mitochondrial ferritin enter lysosomes. After degradation, they release free Fe²⁺, which serves as a critical iron source for ferroptosis.[29] Meanwhile, mitochondria are important sites for GSH synthesis—abnormal mitochondrial function reduces GSH production and decreases GPX4 activity, failing to block the lipid peroxidation chain reaction and ultimately exacerbating ferroptosis.[31] Within mitochondria, key enzymes of the tricarboxylic acid (TCA) cycle (e.g., aconitase 2) and electron transport chain complexes (I, II, III) rely on iron-sulfur clusters (4Fe-4S) to maintain their activity. The assembly of these iron-sulfur clusters requires intracellular iron supply. If iron-sulfur cluster synthesis is impaired (e.g., due to oxidative stress damage), excess free Fe²⁺ leaks into the cytoplasm, becoming a key driver of ferroptosis.[32,33,34] Excessive mitophagy leads to excessive lysosomal load, causing increased lysosomal membrane permeability (LMP). The released hydrolases and Fe²⁺ further promote intracellular oxidative stress and lipid peroxidation, forming a vicious cycle of “mitochondrial damage → mitophagy → lysosomal rupture → exacerbated ferroptosis”.[35,36,37] Additionally, the mitochondrial functional status of RPE cells may indirectly affect the ferroptosis process by regulating the inflammatory differentiation phenotype of monocytes.[38] When RPE cells are rich in mitochondria, their normal oxidative phosphorylation (OXPHOS) metabolism can inhibit the differentiation of co-cultured THP-1 monocytes into a pro-inflammatory phenotype by secreting anti-inflammatory metabolites or signaling molecules (characterized by decreased expression of inflammatory factors such as IL6 and IL8). Under this condition, the expression of anti-ferroptotic genes (e.g., ACSL3, GPX4) in monocytes dominates, iron storage remains stable, and the accumulation of free Fe²⁺ is reduced, thereby indirectly inhibiting ferroptosis. In contrast, when RPE cells lack mitochondria, their metabolic pattern shifts to inefficient glycolysis. They may secrete pro-inflammatory metabolites (e.g., lactic acid) or abnormal ROS, which induce the differentiation of monocytes into an inflammatory phenotype (manifested by increased expression of IL-6 and IL-8). These inflammatory monocytes then upregulate pro-ferroptotic genes (e.g., ACSL4) and downregulate iron storage-related genes (e.g., Ferritin heavy chain (FTH1)), promoting the release of free Fe²⁺ and lipid peroxidation, which in turn indirectly exacerbates ferroptosis [Fig. 1].[38]

Figure 1.

In RPE cells, ferroptosis is coordinately regulated by iron metabolism, oxidative stress, and lipid peroxidation pathways: TfR1/2 mediates iron uptake, FPN is responsible for iron export, and hepcidin inhibits FPN via the SMAD pathway, leading to iron accumulation. System Xc ¯ takes up cystine to synthesize GSH, and GPX4 relies on GSH to scavenge lipid peroxides (PLOOH). If this system is inhibited, iron catalyzes lipid peroxidation through the Fenton reaction, and meanwhile, mitochondria generate ROS to exacerbate oxidative damage, ultimately triggering ferroptosis in RPE cells. The nuclear factor erythroid 2-related factor 2 (Nrf2) pathway can exert a compensatory protective effect by activating antioxidant genes such as heme oxygenase-1 (HO-1)

Association between Iron Accumulation in RPE Cells and Retinal Diseases

In recent years, research on the expression of genes related to retinal iron homeostasis regulation has been ongoing. Researchers have found that the abnormal expression of some genes disrupts the iron metabolism balance in RPE cells, thereby affecting the occurrence and development of ferroptosis.[39,40] Moreover, changes in the expression of these genes exhibit certain specificity in different types of retinal diseases. Here, we summarize retinal diseases associated with iron accumulation and the corresponding changes in their gene expression, aiming to identify new targets and insights for the diagnosis and treatment of retinal diseases.

AMD

AMD is one of the leading causes of vision loss in the elderly, and it is classified into dry AMD and wet AMD. Dry AMD is characterized by irreversible degeneration of RPE cells and photoreceptors, which further leads to atrophy of retinal tissue—this type of atrophy is referred to as geographic atrophy, accompanied by progressive vision loss. In contrast, wet AMD is primarily characterized by the formation of choroidal neovascularization; these newly formed blood vessels are prone to leakage and hemorrhage, causing severe visual impairment. Studies have found that iron accumulation in RPE cells is highly significant in both dry and wet AMD.[41] The ferroptosis process triggered by iron accumulation impairs the function of RPE cells or even leads to their death, disrupting the normal structure and function of the retina. Through sophisticated high-throughput sequencing and biochemical analysis, it was discovered that in the NaIO3-induced oxidative stress model, the main pathological process of RPE degeneration is ferroptosis regulated by HO-1, and this process is controlled by the Nrf2/SLC7A11/HO-1 hierarchical pathway.[11] As an iron-dependent, lipid peroxidation-regulated form of cell death, ferroptosis drives the degeneration of RPE and photoreceptor cells through mechanisms such as the System Xc⁻-GPX4 axis, iron metabolism imbalance, lipid peroxidation, and mitochondrial dysfunction.[42] A study used machine learning algorithms to screen five ferroptosis-related diagnostic genes: VEGFA, SLC2A1, hepcidin antimicrobial peptide (HAMP), heat shock protein beta 1 (HSPB1), and fatty acid desaturase 2 (FADS2). Experimental verification showed that the expression of SLC2A1 and FADS2 is increased in AMD models, suggesting that these two genes are promising diagnostic biomarkers and therapeutic targets for AMD.[43] However, further research is still needed to fully clarify the specific role of ferroptosis in AMD, for example, how the various mechanisms of ferroptosis synergize with each other, and whether the dominant mechanism of ferroptosis changes in different stages of AMD. Only by gaining in-depth insights into these issues can we better utilize the research findings related to ferroptosis and achieve substantial breakthroughs in the diagnosis and treatment of AMD. Meanwhile, considering the multifactorial pathogenic characteristics of AMD,[44] comprehensively investigating the relationships between ferroptosis and factors such as oxidative stress, inflammatory responses, and complement system activation will help fully reveal the pathogenesis of AMD and lay a foundation for the development of more effective interventional strategies.

Stargardt disease

Stargardt disease is an autosomal recessive hereditary macular dystrophy, among which STGD1 caused by ATP-binding cassette subfamily A member 4 (ABCA4) gene mutation accounts for more than 95% of all Stargardt disease patients.[45] The ABCA4 gene encodes an ATP-binding cassette (ABC) transporter protein located on the outer segment disc membrane of photoreceptors. Its main function is to transport all-trans retinol (atRAL) from photoreceptors to RPE cells and participate in the visual cycle. When the ABCA4 gene is mutated, a large amount of atRAL accumulates in photoreceptors and then enters RPE cells through diffusion.[46] AtRAL is cytotoxic and can induce ferroptosis in RPE cells through multiple pathways. On one hand, atRAL can damage the glutamate/cystine transporter system in RPE cells, leading to reduced cystine uptake and blocked GSH synthesis. This further results in decreased activity of GPX4, which fails to effectively inhibit lipid peroxidation. On the other hand, atRAL disrupts iron homeostasis in RPE cells, increasing the intracellular Fe²⁺ level. Through the Fenton reaction, ROS are generated, which promote lipid peroxidation and trigger ferroptosis.[46] The ferroptosis of RPE cells leads to the loss of their supportive function for photoreceptors, ultimately causing progressive degeneration of photoreceptor cells in the macular region of Stargardt disease patients and severe visual impairment.[47]

Hemochromatosis

Hemochromatosis is a disease characterized by disorders in iron metabolism within the body, which in turn leads to iron overload. It can be divided into two main types: hereditary and acquired. Based on differences in genetic defects and pathological mechanisms, hereditary hemochromatosis (HH) is mainly classified into the following five types:[48] HFE (histocompatibility leucocyte antigen class I-like protein involved in iron homeostasis)-associated hemochromatosis (Type 1): Caused by mutations in the HFE gene, with the common mutation being C282Y.[49] It is most prevalent in the Caucasian population. The HFE protein is primarily expressed in RPE cells, and its expression is restricted to the basolateral membrane.[50] As a key protein regulating iron homeostasis, its localization on the basolateral membrane of RPE cells may enable it to directly sense iron signals in the choroidal blood circulation and participate in regulating iron transmembrane transport in RPE cells. Juvenile hemochromatosis (Type 2): Subdivided into Type 2A and Type 2B. Type 2A is caused by mutations in the HJV gene. Type 2B results from mutations in the HAMP gene. The HAMP gene is expressed in RPE cells, Müller cells, and photoreceptor cells.[51] It inhibits intracellular iron export by binding to ferroportin and inducing its degradation, thereby synergistically maintaining iron homeostasis across various retinal cell types. HJV is significantly expressed in RPE cells, Müller cells, photoreceptor cells, and RGCs. In RPE cells, its expression is restricted to the apical membrane. Acting as a co-receptor for bone morphogenetic proteins (BMPs), it induces hepcidin secretion. The expression of HJV on the apical membrane of RPE cells may mediate the regulation of iron metabolism by local signals.[52] Transferrin Receptor 2-Related Hemochromatosis (Type 3): It is caused by mutations in the transferrin receptor 2 (TFR2) gene. TFR2 is expressed throughout the retina, cooperates with HFE to regulate the expression of hepcidin, and is widely distributed in cells of all retinal layers—this suggests it plays a role in the overall regulation of iron uptake in the retina.[51] Ferroportin disease (Type 4): Classified into Type 4A (ferroportin dysfunction subtype) and Type 4B (hepcidin resistance subtype).[51] In Type 4A, iron release from macrophages is impaired, leading to iron deposition in macrophages (e.g., in the spleen). In Type 4B, ferroportin is insensitive to hepcidin, resulting in excessive iron absorption. Aceruloplasminemia: CP is an iron oxidase. It interacts with the cellular iron export protein ferroportin to oxidize Fe²⁺ to Fe³⁺, which then bind to plasma transferrin for transport—thus facilitating intracellular iron excretion. Ferroportin is expressed on the basolateral membrane of RPE cells and the inner segments of photoreceptor cells, and it is the only iron export transporter.[53] In patients with aceruloplasminemia, iron overload causes RPE depigmentation, atrophy and hypertrophy, drusen (nodular, diffuse, and subretinal deposits), and complement activation (positive for C5b-9 and vitronectin). These changes are highly similar to those in AMD.[54] This also suggests that abnormal iron metabolism may be a key pathogenic factor in AMD. Therefore, regulating CP-mediated iron export or inhibiting iron-induced oxidative stress could be potential strategies for intervening in retinal iron overload-related diseases (e.g., AMD).[14] Acquired hemochromatosis is caused by hemolysis or multiple blood transfusions. Iron first deposits in reticuloendothelial cells, and once these cells become iron-saturated, systemic manifestations similar to those of hereditary hemochromatosis occur.[55] All these iron metabolism abnormalities lead to iron accumulation in the body, laying hidden risks for the development of retinal diseases.

MITF mutations

As a key transcription factor for the development and function of RPE, microphthalmia-associated transcription factor (MITF) mutations are associated with various diseases such as albinism, microphthalmia, and retinal degeneration.[56] In RPE cells, it is involved in melanin production, the expression of neurotrophic factors (e.g., Pigment Epithelium-Derived Factor (PEDF)), the regulation of cell proliferation, antioxidant function (via peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1α)), and the visual cycle (regulating genes such as retinol dehydrogenase 5 (RDH5) and retinaldehyde binding protein 1 (RLBP1)).[57] Mitf^viᵗ/^viᵗ (Vitiligo mutant) and Mitfᵐⁱ/ᵐⁱ (Microphthalmia mutant) are two core recessive homozygous mutants of the MITF gene in mice. Both mutants disrupt the structure or function of MITF, leading to abnormal regulation of downstream target genes. Specifically, Mitfᵐⁱ/ᵐⁱ is classified as a “complete loss-of-function” mutation, while Mitf^viᵗ/^viᵗ is categorized as a “hypomorphic” (partial loss-of-function) mutation.[58] Complete loss of MITF (e.g., in Mitf^mi/mi mice) leads to abnormal development of microvilli and structural disorganization of the basolateral membrane in RPE cells;[56] MITF mutations impair the function of tight junctions in RPE cells.[57] In summary, MITF can maintain the polarized structure of RPE cells, ensuring the efficient directional flow of iron ions within RPE cells along the “uptake-transport-output” pathway, thereby preventing abnormal local accumulation of iron in the retina.[59] PUFAs are core substrates for iron-dependent lipid peroxidation, and phosphatidylethanolamine-arachidonic acid (PE-AA) is even a critical precursor for lipid ROS generation during ferroptosis. By maintaining cells in a state enriched with saturated/monounsaturated fatty acids (S/MUFAs), MITF reduces the accumulation of ferroptosis-sensitive substrates, thereby indirectly decreasing the susceptibility of cells to iron-dependent oxidative damage.[60] Additionally, through the secreted product (secreted apolipoprotein E (sApoE)) of its target gene APOE, MITF reduces the accumulation of PUFA-containing lysophospholipids (PUFAs-LysoPL) in invasive melanoma cells (INV cells) and upregulates the expression of GPX4, which in turn impairs iron-dependent lipid peroxidation.[60] Genome-wide association study (GWAS) has shown that individuals carrying low-frequency genetic variants of MITF exhibit increased iron-related parameters, including blood erythrocyte count, blood hemoglobin concentration, and hematocrit value. MITF-A, a member of the MITF family, is a novel activator of hepcidin transcription, which can directly promote the transcriptional activation of hepcidin by binding to the E-box (5′-CATGTG-3′) spanning nt-645 to nt-640 of the murine hepcidin promoter.[61] It is suggested that MITF may affect systemic iron homeostasis by regulating hepcidin. In Mitf mutants (such as Mitf^vit/vit and Mitf^mi/mi), Mitf mutation disrupts iron homeostasis in RPE cells and triggers oxidative stress by regulating the expression of the iron transporter natural resistance-associated macrophage protein 1 (Nramp1) (Slc11a1) and other iron metabolism-related genes.[58] Specifically, the upregulated expression of Nramp1 in Mitf^vit/vit mutants may promote more iron ions (Fe²⁺) to enter cells or subcellular structures, while the downregulated expression of Nramp1 in Mitf^mi/mi mutants may inhibit the normal transport of iron. Meanwhile, the expression of transferrin receptor (TfRc) related to iron uptake is downregulated in Mitf^vit/vit, and the expressions of Cp and Ferroportin (Slc40a1) related to iron export are upregulated. These changes indicate that cells attempt to compensate for iron overload but fail. Excessive Fe²⁺ generates reactive oxygen free radicals through the Fenton reaction, causing oxidative damage such as lipid peroxidation. In addition, Mitf mutation leads to the downregulated expression of melanin synthesis-related genes (e.g., glycoprotein NMB, melan A), which reduces the production of melanin with antioxidant effects and further impairs the antioxidant capacity of cells. Eventually, this results in the imbalance of iron homeostasis and the aggravation of oxidative stress in RPE cells, promoting the occurrence and development of retinal diseases.

Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease associated with abnormal iron metabolism. Iron accumulation in FRDA patients primarily occurs in mitochondria, which is caused by the trinucleotide (GAA) repeat expansion in the FXN gene—this gene encodes Frataxin, a mitochondrial iron-binding protein. The expansion leads to reduced Frataxin expression; as a key protein involved in the synthesis of iron-sulfur clusters (4Fe-4S), Frataxin deficiency directly impairs the assembly of iron-sulfur clusters.[62] Iron-sulfur clusters are critical components of mitochondrial electron transport chain complexes and tricarboxylic acid cycle enzymes (e.g., aconitase). Impaired synthesis of these clusters triggers a feedback mechanism that increases cellular iron uptake while decreasing the effective utilization of iron. Ultimately, this results in mitochondrial iron accumulation, which catalyzes the production of ROS—particularly ・OH, and further damages mitochondrial function and cellular structure.[63] These pathological processes can lead to the accumulation of melanolipofuscin in RPE cells, accompanied by cell hypertrophy or atrophy and abnormal melanin content. Additionally, they cause photoreceptor degeneration, loss of the inner and outer segments of photoreceptors, and destruction of the outer retinal structure.[64]

Diabetic retinopathy

Diabetic retinopathy (DR) is a specific microvascular complication of diabetes mellitus and a major cause of visual impairment in working-age adults. Its prevalence and severity increase with the age of patients with diabetes. Hyperglycemia synergistically induces ferroptosis in endothelial cells and RPE cells through multiple pathophysiological mechanisms.[65] On one hand, the hyperglycemic environment promotes the upregulation of angiotensin II (Ang-II), which stimulates the expression of TfRc and divalent metal transporter 1, thereby increasing iron uptake.[66] On the other hand, it activates the ferritinophagy pathway mediated by nuclear receptor coactivator 4 (NCOA4), releasing free Fe²⁺ and leading to intracellular iron overload. Excess Fe²⁺ generates large amounts of ROS and ・OH through the Fenton reaction, causing oxidative damage.[67] Furthermore, hyperglycemia upregulates the expression of acyl-CoA synthetase 4 (ACSL4) and lysophosphatidylcholine acyltransferase 3 (LPCAT3), promoting the incorporation of PUFAs into membrane phospholipids (PUFA-PLs). Under the catalysis of lipoxygenase (LOX) or nonenzymatic oxidation, PUFA-PLs generate lipid peroxides (e.g., MDA, 4-hydroxynonenal (4-HNE)), disrupting the integrity of membrane structures.[68] The hyperglycemic environment also impairs the antioxidant defense system: It inhibits the function of the system Xc⁻, reducing cystine uptake and GSH synthesis;[69,70,71] meanwhile, it weakens the cell’s ability to clear lipid peroxides by promoting the ubiquitin-mediated degradation of GPX4 via tripartite motif-containing protein 46 (TRIM46).[72] Persistent hyperglycemia can activate inflammatory pathways such as nuclear factor kappa B (NF-κB), promoting the release of inflammatory factors including IL-6 and tumor necrosis factor-alpha (TNF-α). Senescent cells secrete pro-inflammatory factors and vascular endothelial growth factor (VEGF) through the senescence-associated secretory phenotype (SASP), further exacerbating oxidative stress and ferroptosis.[73] In addition, hyperglycemia causes abnormalities in the mitochondrial electron transport chain (ETC), leading to massive ROS leakage; at the same time, it induces excessive activation of mitophagy, releasing free iron and ROS and forming a vicious cycle.[74] Hyperglycemia also promotes the formation of advanced glycation end products (AGEs), which activate NADPH oxidase by binding to their receptors (RAGEs), increasing ROS production and indirectly promoting ferroptosis.[75] These mechanisms collectively induce ferroptosis in endothelial cells and RPE cells, disrupting the structure and function of the BRB and ultimately accelerating the pathological progression of DR.

Future Challenges

In-depth understanding of the complex regulatory network

Although some understanding of the basic mechanisms underlying ferroptosis in RPE cells has been achieved, the intracellular regulatory network of ferroptosis is extremely complex, involving numerous signaling pathways and molecular interactions. For instance, in addition to the classical signaling pathways related to iron metabolism, lipid peroxidation, and the GSH-GPX4 system, other signaling pathways—such as the JAK-STAT signaling pathway and the Nrf2 antioxidant signaling pathway—also engage in crosstalk with ferroptosis. For example, studies have shown that corosolic acid (COR) inhibits high glucose-induced damage to retinal tight junction proteins and ferroptosis by activating Nrf2. After Nrf2 knockdown or knockout, the regulatory effects of COR on epithelial tight junction proteins, iron ion levels, oxidative stress indicators, and ferroptosis-related proteins are significantly attenuated. This indicates that the protective effect of COR against DR depends on Nrf2 activation.[76] In oxidative stress-induced RPE degeneration, ferroptosis is regulated by the Nrf2/SLC7A11/HO-1 pathway.[11] Nrf2 can downregulate the expression of TfR1, reducing cellular iron uptake and thereby decreasing intracellular Fe²⁺ concentration.[77] Additionally, Nrf2 can upregulate the expression of FPN1 to promote intracellular iron efflux, further reducing intracellular iron load and mitigating lipid peroxidation damage caused by iron overload. Nrf2 can activate the expression of SLC7A11;[78] as the light chain subunit of System Xc⁻, SLC7A11 facilitates cystine uptake, providing raw materials for GSH synthesis. This enhances the ability of GPX4 to reduce lipid peroxides and inhibits ferroptosis.[79,80] Nrf2 can also directly regulate the transcription of GPX4 to scavenge lipid peroxides and maintain cell membrane stability.[81]

Furthermore, the JAK-STAT signaling pathway is also involved in ferroptosis of RPE cells. After IFN-γ binds to its receptor on the RPE cell membrane, it activates JAK1/2, which in turn phosphorylates signal transducer and STAT1. Phosphorylated STAT1 translocates into the nucleus to regulate the expression of downstream genes. Once activated, STAT1 directly inhibits the transcription of SLC7A11, preventing cells from scavenging lipid peroxides and ultimately triggering ferroptosis.[28] In RPE cells, the occurrence of ferroptosis is also closely associated with the phospholipase D (PLD) signaling pathway. As key enzymes in this pathway, PLD1 and PLD2 can directly promote ferroptosis in RPE cells by regulating iron metabolism, the antioxidant system, and lipid peroxidation. Activation of phospholipase D1/2 (PLD1/2) downregulates the expression of FPN1, blocking Fe²⁺ efflux and leading to intracellular iron overload; it also downregulates the expression of FTH1, reducing iron storage and increasing free Fe²⁺ levels. Additionally, PLD1/2 can inhibit the expression of SLC7A11 via the JAK1/2/STAT1 signaling pathway, blocking cystine uptake and resulting in decreased GSH synthesis.[82] How these signaling pathways synergistically regulate ferroptosis in RPE cells, and how their regulatory patterns change under different pathological conditions, remains incompletely understood. Future in-depth studies are needed to map a more detailed and accurate regulatory network of RPE cell ferroptosis, providing a theoretical basis for precise intervention.

Development of precise and effective intervention strategies

Based on research on the mechanism of ferroptosis in RPE cells, effectively regulating iron metabolism balance, reducing iron ion concentration, and thereby alleviating oxidative stress damage is a key direction for the prevention and treatment of retinal diseases.[83] In practical treatment, targeted iron chelators can be developed, such as deferiprone (DFP),[83,84] deferoxamine (DFO),[85] deferasirox (DFS),[86] and salicylaldehyde isonicotinoyl hydrazone.[87,88] These chelators can specifically bind to iron ions, reducing the harm caused by iron ion overload, and have been clinically used in the treatment of hereditary iron overload (e.g., hemochromatosis) and transfusion-related iron overload (e.g., thalassemia). DFP is a low-molecular-weight compound that can easily penetrate cells, be absorbed by the human body after oral administration, and cross both the blood-brain barrier and the BRB. It mainly binds to excess iron ions in the body through chelation to form stable complexes, thereby regulating the concentration of iron ions in the body and reducing the toxicity caused by iron overload. In retinal-related research, it has been found that long-term oral administration of DFP can significantly protect the retina and RPE from degeneration induced by chronic systemic iron overload by reducing iron levels in the retina and RPE of hepcidin knockout (Hepc KO) mice and alleviating oxidative stress.[84] Compared with other commonly used iron chelators, DFP has unique advantages. For example, although DFO exerts a protective effect against retinal photodamage and ischemia-reperfusion injury, it has retinal toxicity, which limits its clinical application;[30] DFS lacks evidence of significantly reducing iron levels in the brain or retina, restricting its use in the treatment of neurodegenerative diseases. In some studies, DFP has not shown obvious retinal toxicity and can effectively reduce brain iron levels.[84] Treatment with DFP can reduce iron-catalyzed all-trans retinoic acid oxidation. All-trans retinoic acid is photosensitive and can generate ROS through energy and electron transfer, undergoing photooxidation and photolysis to produce harmful degradation products. These products are associated with a variety of retinal diseases.[89,90] However, approximately 1–5% of patients treated with DFP may develop agranulocytosis, requiring regular monitoring of complete blood counts; in addition, it may cause joint pain and abnormal liver function.[91] Therefore, in terms of drug development, although some iron chelators and ferroptosis inhibitors—such as 2,2′-bipyridine (BP)[12] and Fer-1[92]—have shown inhibitory effects on RPE cell ferroptosis in in vitro experiments and animal models, the safety and efficacy of these drugs still need further verification in human clinical trials. Furthermore, how to precisely deliver these drugs to retinal RPE cells while avoiding adverse reactions to other intraocular tissues is a major challenge that drug delivery systems need to address. Some experiments have investigated the application of dissolving microneedles (OcMNs) loaded with DFS nanosuspensions (DFS-NS) in ocular drug delivery. In the study, DFS-NS was prepared by wet media milling and then loaded into polymeric dissolving microneedles. The results showed that this formulation increased solubility by 4 times, had good stability and mechanical strength, and could effectively penetrate porcine sclera. The drug deposition rate reached 64% within 5 minutes (approximately 5 times that of pure DFS-loaded OcMNs), and it exhibited good biocompatibility with ARPE-19. This is a promising minimally invasive technology for delivering DFS to the posterior segment of the eye and deserves further research.[93] As a potential therapeutic strategy, gene therapy also faces issues such as vector selection, gene editing accuracy, and long-term safety. For example, in the editing of key genes related to RPE cell ferroptosis, how to ensure precise regulation of gene expression and avoid off-target effects is an urgent technical challenge to be solved [Table 1].

Table 1.

Latest strategies targeting iron dysregulation in RPE cells

Type		Name		Mechanism		Efficacy
Potential Phytomedicines		Astragaloside IV (AS-IV)[94,95]		Activate the Nrf2 signaling pathway to alleviate ferroptosis in retinal cells induced by iron overload		Protect the retina from iron overload-induced toxicity
		Puerarin[96]		Reduce iron levels in the serum and retina of iron-overloaded mice, increase the expression levels of rhodopsin and Rpe65, and regulate iron-handling proteins		Alleviate retinal pathological changes
		Salidroside[97]		Activate the Nrf2/SLC7A11/GPX4 Axis		Alleviate the thinning of the outer nuclear layer of the retina and the disruption of RPE tight junctions induced by ferric ammonium citrate (FAC), and maintain the integrity of the retinal structure
		The traditional Chinese medicine Qihuang Granule and its main components Salvia miltiorrhiza and Fructus Lycii[98]		Upregulate the expression of ferroptosis-related proteins GPX4 and SLC7A11		Regulate ferroptosis in AMD
		Salvianic Acid A (SAA)[99,100]		It directly chelates Fe2+ and Fe3+ to reduce free iron levels, and can also regulate the expression of iron-handling proteins to decrease iron uptake, while downregulating ferritin to reduce intracellular iron storage. Through these mechanisms, it lowers iron content in the retina and ARPE-19 cells, thereby alleviating damage caused by iron overload.		Alleviate retinal structural damage
Potential Target		Pigment Epithelium-Derived Factor (PEDF)[101]		Upregulate the expression of GPX4 and FTH1, and inhibit lipid peroxidation and RPE ferroptosis		Improve retinal dysfunction in mice induced by Sodium Iodate (SI)
		Lipocalin 2 (LCN2) Monoclonal Antibody (Clone #6 mAb)[102]		Neutralize LCN2 monomers and homodimers		Improve retinal function in crystallin beta A1 knockout mice and alleviate abnormalities in autophagy, inflammasome activation, and lipid peroxidation
		Ceria Nanoparticles (CNP)[103]		Scavenge ROS to thereby reduce ROS-induced cellular damage and maintain intracellular redox balance		Alleviate ferro-oxidative toxicity in ARPE-19
		Knock down ZIP8[17,104]		Knocking down ZIP8 can directly reduce the entry of iron ions into cells through the non-transferrin-bound iron (NTBI) pathway, decrease free iron levels, and thereby inhibit iron-dependent Fenton reactions and the production of ROS		Protect the integrity of retinal structure and function
		Calcium-doped Prussian blue analogue nanoparticles (CaPB)[105]		It binds iron ions efficiently, downregulates the ferroptosis pathway, upregulates GPX4, and downregulates the iron transporter SLC39A14 as well as the iron-responsive protein IREB2		Maintain the integrity of retinal structure and improve visual function
		N-tert-butyl hydroxylamine (NtBHA)[106]		Reduce intracellular iron content and oxidative stress, and partially restore the activity of Complex IV and the content of GSH		Exerts a protective effect on RPE cells with iron overload
		Inhibit the isoforms PLD1 and PLD2 of PLD[82]		Alleviate oxidative stress induced by oxygen-glucose deprivation/reoxygenation (OGD/R) and reduce intracellular iron ion levels and lipid peroxidation		Inhibit ferroptosis in RPE cells
		Transferrin[107]		Bind free iron and reduce iron-mediated oxidative stress reactions		Protect retinal structure and function
		Deuterated Docosahexaenoic Acid (D-DHA)[108]		Inhibit DHA oxidation through the isotope effect and reduce the production of toxic products (e.g., carboxyethylpyrrole)		Protect photoreceptors and RPE, and also provide long-term prevention of iron-induced geographic atrophy
		Concanavalin A-Modified Melanin-Like Nanoparticles (ConA - MelNPs)[109]		Specifically target damaged RPE cells and surrounding tissues, and enhance their effects of chelating iron ions, alleviating oxidative stress and inhibiting ferroptosis		Alleviate retinal degeneration and visual loss, and improve visual behavior
		Phosphoethanolamine/Phosphocholine Phosphatase 1 (PHOSPHO1)[110]		Reduce the level of phosphatidylethanolamine (PE) in the endoplasmic reticulum, limit PE-derived lipid peroxidation; inhibit autophagy and ferritinophagy, and reduce the accumulation of intracellular free iron		Protect RPE cells from ferroptotic damage

Establish a more precise disease model

Most existing studies rely on in vitro cell culture models and animal models to explore the relationship between RPE cell ferroptosis and retinal diseases. However, in vitro cell culture models often lack the complex in vivo microenvironment and cannot fully simulate the cell-cell interactions and physiological/pathological processes in retinal tissues. Although animal models can reflect disease characteristics to a certain extent, there are differences in retinal structure and function between different animal species and humans, which leads to limitations in the translation of research results. Therefore, establishing more precise retinal disease models—such as three-dimensional retinal organoid models constructed from RPE cells differentiated from human induced pluripotent stem cells (iPSC), or gene-edited non-human primate models—is of great significance for in-depth study of the mechanism of RPE cell ferroptosis in retinal diseases and evaluation of the effectiveness of intervention measures [Table 2].

Table 2.

Methods for constructing retinal iron dysregulation

		Subjects		Methods		Results
In vivo		C57BL/6 mice[76]		Intraperitoneal injection of streptozotocin (STZ, 55 mg/kg for 6 consecutive days), and Nrf2 gene knockout can exacerbate high glucose-induced ferroptosis		The RPE is separated from the choroid, the expression of tight junction proteins ZO-1 and occludin is decreased, and the expression of ferroptosis-related proteins is altered—specifically, the expression of GPX4 and xCT is reduced, the expression of TfR and FTH-1 is increased, and iron deposition is enhanced simultaneously.
		CD1 mice[111]		Knockout of the hepcidin gene		Iron metabolism regulation imbalance, systemic and retinal iron overload
		CD1 mice[23]		Knockout of the Bmp6 gene		Increased intracellular iron storage and decreased iron uptake
		BALB/cJ mice[23]		BALB/cJ mice were exposed to intense light of 10,000 lux for 18 hours (photodamage model)		Downregulation of Bmp6 indirectly induces iron dysregulation
		C57BL/6 mice[112]		Intravitreal injection of 500 μM and 1000 μM FeCl₃		Abnormal accumulation of iron ions in the retina to establish a chronic model of AMD
		C57BL/6 mice[113]		1 μl of 0.5 mM FAC intravitreal injection		Abnormal accumulation of iron ions in the retina to establish a chronic model of AMD
		C57BL/6 mice[114]		Intravenous injection of iron sucrose via the tail vein		Increased local iron concentration in the retina
		C57BL/6 mice[11]		Intravenous injection of NaIO₃ (35 mg/kg) via the tail vein		Oxidative stress induced by NaIO₃ can activate RPE ferroptosis through the Nrf2-SLC7A11-HO-1 pathway
		C57BL/6 mice[46]		Abca4⁻/⁻Rdh8⁻/⁻ double knockout mice (with impaired atRAL clearance) were used to induce intracellular atRAL overload in the retina via light exposure (10,000 lx LED light irradiation for 2 hours)		Elevated levels of atRAL in the mouse retina are accompanied by upregulated expression of ferroptosis markers (such as COX2 and ACSL4) and increased lipid peroxidation products
		Kunming mice[99,100]		Intraperitoneal injection of iron dextran		Increased retinal iron deposition, elevated MDA content, and decreased GSH content
		129/SvEvTac (129/S) mice[115]		HJV gene knockout		HJV deficiency leads to blockage of the BMP6-hepcidin pathway, reduced cellular iron export, and iron overload
		Albino Fischer rats[116]		Intravitreal injection of 20 mmol/L ferrous sulfate (3 μl)		Iron-induced accumulation of lipofuscin-like fluorescent pigments in RPE
		C57BL/6 mice[117]		Intravitreal injection of cigarette smoke extract		Ferroptosis of RPE cells and retinal injury
In vitro		ARPE - 19[76]		30 mM D-(+)-glucose		The intracellular iron ion content increases, the level of lipid peroxidation rises, and the ferroptosis-related proteins change
		ARPE-19[23,118]		Treatment with 250 μmol/L FAC for 16 hours and 4 days, respectively		Long-term iron ion overload mimics the chronic pathological features of AMD
		ARPE-19[23]		ARPE-19 cells were treated with 75 μmol/L H₂O₂ for 16 hours		Downregulation of Bmp6 indirectly induces iron dysregulation
		ARPE-19 and mouse photoreceptor cell line 661W[112]		Add 100 μM, 500 μM, or 1000 μM FeCl₃ to the culture medium		Long-term iron ion overload mimics the chronic pathological features of AMD
		661W[119]		SI		Disruption of system Xc⁻ depletes GSH, triggers lipid peroxidation, and simultaneously increases intracellular Fe²⁺ and ROS levels to promote ferroptosis
		ARPE-19[11]		Cells were treated with 30 mM NaIO₃ for 24 hours		Simulating the oxidative stress-induced injury and ferroptosis-dominated degeneration process of RPE in AMD
		ARPE-19[11]		Treatment with Erastin (5 μM)		Reduced GSH synthesis leads to decreased GPX4 activity, which in turn triggers lipid peroxidation and iron-dependent cell death
		ARPE-19[12]		ARPE-19 cells were treated with 25 μM hemoglobin or heme for 3 hours		A large accumulation of intracellular free Fe²⁺
		661W[46]		AtRAL (2.5–20 μM) was incubated in the culture medium for 3–6 hours		Abnormal iron metabolism, lipid peroxidation, and mitochondrial damage, thereby inducing ferroptosis
		ARPE-19[100,103]		Expose the cells to a 1 μM FeSO₄ solution for 24 hours		The level of Fe²⁺ is significantly increased, leading to damage of RPE cells
		ARPE-19[117]		Cells were exposed to cigarette smoke extract for 24 hours		Induces ferroptosis in RPE cells

Exploring biomarkers for early diagnosis and prediction

For retinal diseases associated with ferroptosis of RPE cells, such as AMD and DR, early diagnosis and disease progression prediction are crucial for timely intervention and improvement of patient prognosis. For instance, in DR research, the potential association between ferroptosis and the pathogenesis of DR was analyzed for the first time. Through Weighted Gene Co-expression Network Analysis (WGCNA) and machine learning methods, six core diagnostic biomarkers for DR related to ferroptosis were identified, namely, thymosin beta 4 X-linked (TMSB4X), NADPH oxidase 4 (NOX4), poly (ADP-ribose) polymerase 14 (PARP14), solute carrier family 1 member 5 (SLC1A5), tumor protein P53 (TP53), and cyclin-dependent kinase inhibitor 2A (CDKN2A). These genes were all upregulated in DR patients. Receiver operating characteristic (ROC) curve analysis showed high diagnostic accuracy (area under the curve [AUC] ≥0.9 for all, with AUC = 1 for NOX4), and they were closely associated with immune cell infiltration (e.g., CDKN2A and PARP14 were positively correlated with multiple immune cell types). Additionally, DR patients could be classified into three molecular subtypes based on these six genes, providing new evidence for the diagnosis and targeted treatment of DR.[120] In healthy populations, relatively higher serum ferritin levels are associated with the progression of AMD and may serve as an important predictive indicator for AMD, especially its early subtypes. Studies have found a significant association between serum ferritin levels and AMD, even when the diagnostic criteria for hyperferritinemia are not met. The prevalence of early AMD increases gradually with the elevation of serum ferritin quintiles, showing a dose-dependent relationship.[121] A study conducted on a healthy Danish population investigating the association between HFE genotypes (wild-type wt/wt, H63D, C282Y, and their combinations) and iron status markers revealed that compared with the wt/wt genotype, all other HFE genotype combinations had a significant impact on iron status markers.[122] Among the six genotypes sorted by population frequency (wt/wt → H63D/wt → C282Y/wt → H63D/H63D → C282Y/H63D → C282Y/C282Y), serum iron and transferrin saturation increased gradually, while serum transferrin decreased gradually. This trend was observed in both men and women. Regarding gender differences, serum ferritin in men showed a gradient increase with genotype, whereas ferritin levels in women were barely affected by genotype—this may be related to physiological iron loss caused by menstruation and pregnancy.[122] The C282Y/C282Y genotype had the most significant impact on iron status and the strongest association with iron overload in the body. This suggests that HFE genotype testing can assist in assessing the risk of iron overload, with particular emphasis on monitoring the C282Y/C282Y genotype. Moreover, women may need enhanced monitoring of iron status after menopause. Beyond known factors such as age, genetics, and gender, the environment may also be a potentially important pathogenic factor for ferroptosis of RPE cells. For example, exposure to cigarette smoke extract (as mentioned earlier) affects ferroptosis of RPE cells.[117] Additionally, studies have shown that blue light plays a crucial role in promoting ferroptosis in RPE cells mediated by N-retinylidene-N-retinylethanolamine (A2E). Blue light irradiation increases the level of Fe²⁺ in A2E-loaded RPE cells and at the same time inhibits the solute SLC7A11-GSH-GPX4 axis (downregulating the protein expression of SLC7A11 and GPX4). This leads to GSH depletion and GPX4 inactivation, which in turn induces the generation of ROS through Fe²⁺ overload and exacerbates lipid peroxidation, ultimately promoting ferroptosis in RPE cells.[47] This implies that more attention should be paid to the complex relationship between environmental factors and intracellular iron metabolism, and efforts should be made to reduce the pathogenic risk of RPE cell ferroptosis in the population through lifestyle interventions. Currently, there is a lack of early diagnostic and predictive biomarkers in clinical practice that can directly and specifically reflect the ferroptosis status of RPE cells. In the future, large-scale clinical studies and multi-omics technologies are needed to screen and validate specific biomarkers associated with RPE cell ferroptosis, thereby providing a basis for the early diagnosis, disease monitoring, and personalized treatment of retinal diseases.

Conclusions

Research on ferroptosis in RPE cells is gradually revealing its core role in retinal diseases. Through a variety of animal models and cell experiments, researchers have observed a close connection between abnormal iron metabolism and retinal damage. For example, in different mouse models, iron overload not only increases iron deposition in the retina but also triggers oxidative stress and lipid peroxidation reactions, further exacerbating damage to RPE cells. Additionally, in vitro experiments have also confirmed the toxic effect of iron ion overload on the ARPE-19 cell line and 661W cell line, which is manifested by a significant upregulation of ferroptosis markers and a decrease in antioxidant capacity.

Notably, ferroptosis not only is a pathological feature of retinal diseases but may also become a new target for therapeutic intervention. Some compounds such as Fer-1 and DFP have been shown to effectively inhibit ferroptosis and protect retinal cells from damage.[47] These findings provide a theoretical basis for the development of new therapeutic strategies, especially for intractable diseases such as AMD and DR. However, current research is still in its preliminary stage, and more clinical trials are needed to verify the safety and effectiveness of these intervention methods.

In the future, with the development of multi-omics technologies and the advancement of bioinformatics analysis methods, scientists are expected to more comprehensively analyze the molecular mechanism of ferroptosis in RPE cells and screen out reliable biomarkers for early diagnosis and personalized treatment. At the same time, combining lifestyle interventions and environmental factor management may further reduce the risk of retinal diseases and bring better prognostic outcomes to patients.

Authors' contributions

All authors agree with the content of the manuscript. Each author has participated in the work as described in the following. W.L. – manuscript preparation, design, data acquisition, and manuscript editing and manuscript review. W.W.L., D.W– literature search. P.J.W, J.Y.J– concept, definition of intellectual content. Z.M.L. – resources, project administration. X.D.C. – validation, funding acquisition, and final approval of the version to be published.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Financial support and sponsorship: This research was funded by Foundation for Innovative Research Groups of the National Natural Science Foundation of China, 82374525; The project of Hunan Provincial Administration of Traditional Chinese Medicine, B2024045; The project of Hunan Provincial Administration of Traditional Chinese Medicine, B2024048; The key research project of Hunan Provincial Administration of Traditional Chinese Medicine, C2023013; Hunan Provincial Clinical Medical Research Center for Ophthalmic Diseases (Traditional Chinese Medicine), 2023SK4038; The Natural Science Foundation of Hunan Province of China, 2025JJ90006; Key Project of Postgraduate Scientific Research Innovation Program of Hunan Provincial Department of Education, CX20251151; Project for Improving Clinical Evidence-based Capacity of Advantageous Diseases Treated with Traditional Chinese Medicine, JBGS-A1-02; The key project of the University-Hospital Joint Fund of Hunan University of Chinese Medicine, 2025XYLH002.