Protective effect of autophagy on human retinal pigment epithelial cells against lipofuscin fluorophore A2E: implications for age-related macular degeneration

Age-related macular degeneration (AMD) is the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial (RPE) cells is a crucial causative factor responsible for the onset and progression of AMD. A2E, a major component of toxic lipofuscin implicated in AMD, is deposited in RPE cells with age. However, the mechanism whereby A2E may contribute to the pathogenesis of AMD remains unclear. We demonstrated that A2E was a danger signal of RPE cells, which induced autophagy and decreased cell viability in a concentration- and time-dependent manner. Within 15 min after the treatment of RPE with 25 μM A2E, the induction of autophagosome was detected by transmission electron microscopy. After continuous incubating RPE cells with A2E, intense punctate staining of LC3 and increased expression of LC3-II and Beclin-1 were identified. Meanwhile, the levels of intercellular adhesion molecule (ICAM), interleukin (IL)1β, IL2, IL-6, IL-8, IL-17A, IL-22, macrophage cationic peptide (MCP)-1, stromal cell-derived factor (SDF)-1, and vascular endothelial growth factor A (VEGFA) were elevated. The autophagic inhibitor 3-methyladenine (3-MA) and activator rapamycin were also used to verify the effect of autophagy on RPE cells against A2E. Our results revealed that 3-MA decreased the autophagosomes and LC3 puncta induced by A2E, increased inflammation-associated protein expression including ICAM, IL1β, IL2, IL-6, IL-8, IL-17A, IL-22, and SDF-1, and upregulated VEGFA expression. Whereas rapamycin augmented the A2E-mediated autophagy, attenuated protein expression of inflammation-associated and angiogenic factors, and blocked the Akt/mTOR pathway. Taken together, A2E induces autophagy in RPE cells at the early stage of incubation, and this autophagic response can be inhibited by 3-MA or augmented by rapamycin via the mTOR pathway. The enhancement of autophagy has a protective role in RPE cells against the adverse effects of A2E by reducing the secretion of inflammatory cytokines and VEGFA.Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among elderly people and is becoming a major public health issue.^{1, 2, 3} The pathological change in AMD is located in the macula, which is the central and posterior portion of the retina containing the retinal pigment epithelium (RPE) and photoreceptors. Central visual impairment caused by AMD results from the loss or damage of RPE cells and the photoreceptors.⁴ Currently, the etiology and pathogenesis of AMD is not fully understood and there is no effective treatment.^{5, 6} A chronic aberrant inflammatory response in RPE cells is considered to be one of the major factors contributing to the pathogenesis of AMD.^{7, 8}Lipofuscin is a complex aggregate of fluorescent material, formed in a variety of tissues but best studied in the eye.⁹ The buildup of lipofuscin in RPE cells has been identified as a byproduct of the visual cycle, and is derived from the ingestion of photoreceptor outer segments, which has been implicated in several retinal degenerations, including AMD.^{10, 11} As revealed by spectroscopic analyses, the bis-retinoid N-retinyl-N-retinylidene ethanolamine (A2E) is the first isolated, major fluorophore from RPE lipofuscin. Numerous in vitro and in vivo studies have found that toxicity effects associated with this compound, and A2E is involved in the pathological pathways of AMD, especially the inflammatory response.^{12, 13} Although several studies have suggested that A2E may induce cytokine production, activate inflammasomes or the complement system in RPE cells, and contribute to chronic inflammation in AMD,^{14, 15, 16} the exact mechanisms by which A2E exerts an effect on RPE cells remains unclear.Autophagy is an evolutionarily conserved cellular housekeeping process that removes damaged organelles and protein aggregates that are unnecessary or dysfunctional to the cells by delivering cytoplasmic substrates to lysosomes for degeneration.¹⁷ In addition to turnover of cellular components, autophagy is involved in development, differentiation, and tissue remodeling in various organisms.¹⁸ The failure of autophagy in aged postmitotic cells, including RPE cells, can result in the accumulation of aggregation-prone proteins, cellular degeneration, and finally the induction of cell death.^{19, 20} Currently, a large amount of evidence indicates that autophagy is associated with RPE damage and AMD pathology.^{21, 22, 23} In RPE cells, the preservation of autophagic activity, together with functional lysosomal enzymes, is a prerequisite to prevent detrimental intracellular accumulation of damaged molecules.²¹ A well-functioning proteolytic machine guarantees that there is sufficient capacity to handle damaged proteins and organelles.²⁴ In addition, Saadat KA et al.²⁵ have shown that RPE cell death is induced in the presence of A2E and the autophagic inhibitor 3-methyladenine (3-MA). Nevertheless, whether the autophagic pathway has effects on A2E-induced cell damage through the production of chemokines and cytokines remains unclear. Furthermore, the relationship between A2E and autophagy and how this interaction influences RPE cells'' inflammatory response requires further clarification.Therefore, the protective effect of autophagy on human RPE cells against lipofuscin fluorophore A2E-induced cell death and the inflammatory response were studied in the present article. This work facilitates our understanding of the role of autophagy in the survival and death of RPE cells accumulating excess lipofuscin and provides a new strategy in the treatment of AMD.     

Autophagy regulates death of retinal pigment epithelium cells in age-related macular degeneration

Age-related macular degeneration (AMD) is an eye disease underlined by the degradation of retinal pigment epithelium (RPE) cells, photoreceptors, and choriocapillares, but the exact mechanism of cell death in AMD is not completely clear. This mechanism is important for prevention of and therapeutic intervention in AMD, which is a hardly curable disease. Present reports suggest that both apoptosis and pyroptosis (cell death dependent on caspase-1) as well as necroptosis (regulated necrosis dependent on the proteins RIPK3 and MLKL, caspase-independent) can be involved in the AMD-related death of RPE cells. Autophagy, a cellular clearing system, plays an important role in AMD pathogenesis, and this role is closely associated with the activation of the NLRP3 inflammasome, a central event for advanced AMD. Autophagy can play a role in apoptosis, pyroptosis, and necroptosis, but its contribution to AMD-specific cell death is not completely clear. Autophagy can be involved in the regulation of proteins important for cellular antioxidative defense, including Nrf2, which can interact with p62/SQSTM, a protein essential for autophagy. As oxidative stress is implicated in AMD pathogenesis, autophagy can contribute to this disease by deregulation of cellular defense against the stress. However, these and other interactions do not explain the mechanisms of RPE cell death in AMD. In this review, we present basic mechanisms of autophagy and its involvement in AMD pathogenesis and try to show a regulatory role of autophagy in RPE cell death. This can result in considering the genes and proteins of autophagy as molecular targets in AMD prevention and therapy.     

Apolipoprotein B-containing lipoproteins in retinal aging and age-related macular degeneration

The largest risk factor for age-related macular degeneration (ARMD) is advanced age. With aging, there is a striking accumulation of neutral lipids in Bruch''s membrane (BrM) of normal eye that continues through adulthood. This accumulation has the potential to significantly impact the physiology of the retinal pigment epithelium (RPE). It also ultimately leads to the creation of a lipid wall at the same locations where drusen and basal linear deposit, the pathognomonic extracellular, lipid-containing lesions of ARMD, subsequently form. Here, we summarize evidence obtained from light microscopy, ultrastructural studies, lipid histochemistry, assay of isolated lipoproteins, and gene expression analysis. These studies suggest that lipid deposition in BrM is at least partially due to accumulation of esterified cholesterol-rich, apolipoprotein B-containing lipoprotein particles produced by the RPE. Furthermore, we suggest that the formation of ARMD lesions and their aftermath may be a pathological response to the retention of a sub-endothelial apolipoprotein B lipoprotein, similar to a widely accepted model of atherosclerotic coronary artery disease (Tabas, I., K. J. Williams, and J. Borén. 2007. Subendothelial lipoprotein retention as the initiating process in atherosclerosis: update and therapeutic implications. Circulation. 116:1832–1844). This view provides a conceptual basis for the development of novel treatments that may benefit ARMD patients in the future.     

Oxidative stress in retinal pigment epithelium impairs stem cells: a vicious cycle in age-related macular degeneration

Molecular and Cellular Biochemistry - Aging, chronic oxidative stress, and inflammation are major pathogenic factors in the development and progression of age-related macular degeneration (AMD)...     

Fluorescent pigments of the retinal pigment epithelium and age-related macular degeneration.

The major hydrophobic fluorophore of the retinal pigment epithelium (RPE) is A2E, a pyridinium bis-retinoid derived from all-trans-retinal and phosphatidyl-ethanolamine. The accumulation of fluorophores such as A2E is implicated in the pathogenesis of age-related macular degeneration (AMD), a disease associated with the deterioration of central vision and a leading cause of blindness in the elderly. Recent chemical and biological studies have provided insight into the synthesis and biosynthesis of A2E, the spectroscopic properties of this pigment, and the role of A2E and RPE cell death.     

A novel source of methylglyoxal and glyoxal in retina: implications for age-related macular degeneration

Aging of retinal pigment epithelial (RPE) cells of the eye is marked by accumulations of bisretinoid fluorophores; two of the compounds within this lipofuscin mixture are A2E and all-trans-retinal dimer. These pigments are implicated in pathological mechanisms involved in some vision-threatening disorders including age-related macular degeneration (AMD). Studies have shown that bisretinoids are photosensitive compounds that undergo photooxidation and photodegradation when irradiated with short wavelength visible light. Utilizing ultra performance liquid chromatography (UPLC) with electrospray ionization mass spectrometry (ESI-MS) we demonstrate that photodegradation of A2E and all-trans-retinal dimer generates the dicarbonyls glyoxal (GO) and methylglyoxal (MG), that are known to modify proteins by advanced glycation endproduct (AGE) formation. By extracellular trapping with aminoguanidine, we established that these oxo-aldehydes are released from irradiated A2E-containing RPE cells. Enzyme-linked immunosorbant assays (ELISA) revealed that the substrate underlying A2E-containing RPE was AGE-modified after irradiation. This AGE deposition was suppressed by prior treatment of the cells with aminoguanidine. AGE-modification causes structural and functional impairment of proteins. In chronic diseases such as diabetes and atherosclerosis, MG and GO modify proteins by non-enzymatic glycation and oxidation reactions. AGE-modified proteins are also components of drusen, the sub-RPE deposits that confer increased risk of AMD onset. These results indicate that photodegraded RPE bisretinoid is likely to be a previously unknown source of MG and GO in the eye.     

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

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

Mechanism of all-trans-retinal toxicity with implications for stargardt disease and age-related macular degeneration

Compromised clearance of all-trans-retinal (atRAL), a component of the retinoid cycle, increases the susceptibility of mouse retina to acute light-induced photoreceptor degeneration. Abca4(-/-)Rdh8(-/-) mice featuring defective atRAL clearance were used to examine the one or more underlying molecular mechanisms, because exposure to intense light causes severe photoreceptor degeneration in these animals. Here we report that bright light exposure of Abca4(-/-)Rdh8(-/-) mice increased atRAL levels in the retina that induced rapid NADPH oxidase-mediated overproduction of intracellular reactive oxygen species (ROS). Moreover, such ROS generation was inhibited by blocking phospholipase C and inositol 1,4,5-trisphosphate-induced Ca(2+) release, indicating that activation occurs upstream of NADPH oxidase-mediated ROS generation. Because multiple upstream G protein-coupled receptors can activate phospholipase C, we then tested the effects of antagonists of serotonin 2A (5-HT(2A)R) and M(3)-muscarinic (M(3)R) receptors and found they both protected Abca4(-/-)Rdh8(-/-) mouse retinas from light-induced degeneration. Thus, a cascade of signaling events appears to mediate the toxicity of atRAL in light-induced photoreceptor degeneration of Abca4(-/-)Rdh8(-/-) mice. A similar mechanism may be operative in human Stargardt disease and age-related macular degeneration.     

Anti-neovascularization effects of DMBT in age-related macular degeneration by inhibition of VEGF secretion through ROS-dependent signaling pathway

Molecular and Cellular Biochemistry - Choroidal neovascularization (CNV) is the hallmark of late-staged wet age-related macular degeneration (AMD). Vascular endothelial growth factor (VEGF) is a...     

Modelling the genetic risk in age-related macular degeneration

Late-stage age-related macular degeneration (AMD) is a common sight-threatening disease of the central retina affecting approximately 1 in 30 Caucasians. Besides age and smoking, genetic variants from several gene loci have reproducibly been associated with this condition and likely explain a large proportion of disease. Here, we developed a genetic risk score (GRS) for AMD based on 13 risk variants from eight gene loci. The model exhibited good discriminative accuracy, area-under-curve (AUC) of the receiver-operating characteristic of 0.820, which was confirmed in a cross-validation approach. Noteworthy, younger AMD patients aged below 75 had a significantly higher mean GRS (1.87, 95% CI: 1.69-2.05) than patients aged 75 and above (1.45, 95% CI: 1.36-1.54). Based on five equally sized GRS intervals, we present a risk classification with a relative AMD risk of 64.0 (95% CI: 14.11-1131.96) for individuals in the highest category (GRS 3.44-5.18, 0.5% of the general population) compared to subjects with the most common genetic background (GRS -0.05-1.70, 40.2% of general population). The highest GRS category identifies AMD patients with a sensitivity of 7.9% and a specificity of 99.9% when compared to the four lower categories. Modeling a general population around 85 years of age, 87.4% of individuals in the highest GRS category would be expected to develop AMD by that age. In contrast, only 2.2% of individuals in the two lowest GRS categories which represent almost 50% of the general population are expected to manifest AMD. Our findings underscore the large proportion of AMD cases explained by genetics particularly for younger AMD patients. The five-category risk classification could be useful for therapeutic stratification or for diagnostic testing purposes once preventive treatment is available.     

Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration

Introduction  

The aim of this study was to compare the effects of tumour necrosis factor-alpha (TNF-α) and interleukin-1-beta (IL-1β) on protease and catabolic cytokine and receptor gene expression in normal and degenerate human nucleus pulposus cells in alginate culture.     

New therapies for the treatment of age-related macular degeneration]

Age-related macular degeneration has a natural progression from the precursors (the drusen) towards atrophic or neovascular complications. Choroidal neovascularization is undoubtedly the aspect of the disease that benefits most from new therapeutical approaches. Destructive photocoagulation based on fluorescein angiography has demonstrated since 20 years its efficiency on choroidal neovascularization. The same approach based on indocyanine green (ICG) angiography would increase the number of patients available to therapy. Very recently photodynamic therapy has demonstrated its efficiency to stabilize visual acuity at least at two years in patients with choroidal new vessels predominantly well defined. Other treatment developments are considered, such as refinement of photocoagulation techniques or of surgery. Until now, none has demonstrated its efficiency although they raise justified hopes. The future approaches rely upon the progress of the research both in physiopathology of the disease and on the angiogenic process requiring a constant interaction with all thematics of research. Finally, palliative treatments will be required before heading up to a preventive treatment.     

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

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

Transformation of the proteasome with age-related macular degeneration

The proteasome mediates pathways associated with oxidative stress and inflammation, two pathogenic events correlated with age-related macular degeneration (AMD). In human donor eyes corresponding to four stages of AMD, we found the proteasomal chymotrypsin-like activity increased in neurosensory retina with disease progression. Increased activity correlated with a dramatic increase in the inducible subunits of the immunoproteasome, which was not due to an increase in CD45 positive immune cells in the retina. The novel observation of proteasome transformation may reflect retinal response to local inflammation or oxidative stress with AMD.     

Morin hydrate attenuates CSE-induced lipid accumulation,ER stress,and oxidative stress in RPE cells: implications for age-related macular degeneration

Oxidative stress has a key role in the pathogenesis of age-related macular degeneration (AMD). Cigarette smoking is known to the one of the main risk factors of AMD through oxidative stress-mediated endoplasmic reticulum (ER) stress and lipid accumulation in human retinal pigment epithelium (RPE) cells. A number of studies have investigated the benefits of antioxidants in the AMD. However, previous studies have not shown that efficacy of antioxidant in the treatment of AMD. Recent studies demonstrated that morin hydrate (MH) has antioxidant properties, anti-inflammatory, and antiapoptosis effects, however, the protective effects of MH against cigarette smoke extract (CSE)-induced AMD have not been studied in detail. We tested the potential effect of MH against the CSE-induced lipid accumulation in RPE cells and mice RPE layer. Herein, we observed that expose of RPE cells to CSE reduced cell viability, increased the lipid accumulation, ER stress, and oxidative stress. Concomitantly, CSE treatment to mice induced AMD associated histopathological changes, lipid accumulation, ER stress and oxidative stress in RPE layer. MH significantly attenuated cytotoxicity, lipid accumulation, ER stress, and oxidative stress via activated AMPK-Nrf2 signaling pathway in RPE cells and mice RPE layer. In addition, AMPK inhibition reversed MH-induced RPE cell protection against CSE. Thus, we conclude that MH protects RPE cells from CSE through reduced oxidative stress, ER stress, and lipid accumulation via activated AMPK-Nrf2-HO-1 signaling pathway. These findings suggest that MH treatment may be exploited in effective strategy against CSE-induced AMD.