Current Insights on the Photoprotective Mechanism of the Macular Carotenoids, Lutein and Zeaxanthin: Safety, Efficacy and Bio-Delivery.

Ocular health has emerged as one of the major issues of global health concern with a decline in quality of life in an aging population, in particular and rise in the number of associated morbidities and mortalities. One of the chief reasons for vision impairment is oxidative damage inflicted to photoreceptors in rods and cone cells by blue light as well as UV radiation. The scenario has been aggravated by unprecedented rise in screen-time during the COVID and post-COVID era. Lutein and Zeaxanthin are oxygenated carotenoids with proven roles in augmentation of ocular health largely by virtue of their antioxidant properties and protective effects against photobleaching of retinal pigments, age-linked macular degeneration, cataract, and retinitis pigmentosa. These molecules are characterized by their characteristic yellow-orange colored pigmentation and are found in significant amounts in vegetables such as corn, spinach, broccoli, carrots as well as fish and eggs. Unique structural signatures including tetraterpenoid skeleton with extensive conjugation and the presence of hydroxyl groups at the end rings have made these molecules evolutionarily adapted to localize in the membrane of the photoreceptor cells and prevent their free radical induced peroxidation. Apart from the benefits imparted to ocular health, lutein and zeaxanthin are also known to improve cognitive function, cardiovascular physiology, and arrest the development of malignancy. Although abundant in many natural sources, bioavailability of these compounds is low owing to their long aliphatic backbones. Under the circumstances, there has been a concerted effort to develop vegetable oil-based carriers such as lipid nano-emulsions for therapeutic administration of carotenoids. This review presents a comprehensive update of the therapeutic potential of the carotenoids along with the challenges in achieving an optimized delivery tool for maximizing their effectiveness inside the body.

Lutein and zeaxanthin are the two most abundant natural xanthophylls (oxygenated carotenoids) with a linear C40 tetraterpene/isoprenoid lycopene-based backbone.Presence of extensive conjugation (more than 10 double bonds) enable these molecules to act as accessory light harvesting pigments apart from chlorophyll.More importantly, the xanthophylls prevent photobleaching of the pigments and proteins in the Light Harvesting Complex (LHC) by sequestering the excess unutilized blue light and preventing triplet chlorophyll associated formation of Reactive Oxygen Species.In human eye, lutein, zeaxanthin along with mesozeaxanthin constitute the three macular pigments forming the so called "yellow spot" of the macula and are implicated in maintaining the redox balance, homeostasis and normal physiology of the eyes.However, unlike plants, xanthophylls must be acquired from dietary sources such as colored leafy vegetables and egg yolk.Increase in the number of eye diseases in the aging population coupled with insufficient bioavailability of xanthophylls has mandated the industrial production of supplements enriched in xanthophylls.The bioavailability and delivery of xanthophylls can be significantly enhanced by suspension in a blend of extra-virgin olive oil and other vegetable oils.

Lutein and zeaxanthin - radio- and chemoprotective properties. Mechanism and possible use.

Lutein and zeaxanthin are naturally occurring xanthophylls, mainly present in green, leafy vegetables and egg's yolk. Their presence is connected with blue spectrum light absorbance, including UV. This property, and fact, that these xanthophylls are accumulated by human eye's macula, leads to eye's protective functions of them including protection from age-related macular degeneration (AMD). Also, antioxidative features of lutein and zeaxanthin are boosting overall health of human body. Numerous studies proves anti-inflammatory and protective attributes of these compounds, based on many, different mechanisms. One of them is regulating redox potential in cells, and impact on expression of linked genes. In preventing of eye diseases, an important gene that is regulated by lutein and zeaxanthin is the Nrf2 gene, whose increased activity leads to optimizing the cellular response to reactive oxygen species (ROS) and preventing related diseases. Other research confirms antiproliferative properties of mentioned compounds in case of certain human cancer cell lines. There are e.g.: HepG2 (hepatitis cancer), MCF-7 (breast cancer), which treated in vitro with lutein solution showed reduction of cell growth. Lutein alone, during in vivo studies conducted on mice, exhibited also radioprotective properties, positively affecting the vitality of animals. Lutein provides also increasing of tolerance to UV radiation, reducing inflammatory processes in the skin and preventing oncogenesis. Low intake of lutein and zeaxanthin, associated with "western diet", rich in simple carbohydrates and processed food, common in developed countries, including Poland, is linked with diabetes and obesity incidence. Assuming, lutein and zeaxanthin significantly affect the well-being of the human body, and their appropriate amount in diet can help reduce risk of many diseases. For supplementation, the optimized dosage of these xanthophylls includes doses of 10 mg for lutein and 2 mg for zeaxanthin, and it is recommended to consume along with fats or meals rich in fats.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

Qihuang Granule protects the retinal pigment epithelium from oxidative stress via regulation of the alternative complement pathway.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of vision loss in elderly people, and dry AMD is the most common type of AMD. Oxidative stress and alternative complement pathway activation may play essential roles in the pathogenesis of dry AMD. There are no available drugs for dry AMD. Qihuang Granule (QHG) is an herbal formula for the treatment of dry AMD, and it achieves a good clinical effect in our hospital. However, its potential mechanism is unclear. Our study investigated the effects of QHG on oxidative stress-associated retinal damage to reveal its underlying mechanism. METHODS: Oxidative stress models were established using H2O2 and NaIO3 in ARPE-19 cells and C57BL/6 mice. Cell apoptosis and viability were assessed using phase contrast microscopy and flow cytometry, respectively. Alterations in the mouse retinal structure were evaluated using Masson staining and transmission electron microscopy (TEM). The expression of complement factor H (CFH), complement component 3a (C3a) and complement component 5a (C5a) in retinal pigment epithelium (RPE) cells and mice was measured using RT‒PCR, Western blot analysis and ELISA. RESULTS: Pretreatment with QHG significantly prevented cell apoptosis and disorder of the RPE and inner segment/outer segment (IS/OS) in H2O2-treated RPE cells and NaIO3-injected mice. QHG alleviated mitochondrial damage in mouse RPE cells, as shown by TEM. QHG also promoted CFH expression and inhibited the expression of C3a and C5a. CONCLUSIONS: The results suggest that QHG protects the retinal pigment epithelium from oxidative stress, likely by regulating the alternative complement pathway.

The glutamate/GABA system in the retina of male rats: effects of aging, neurodegeneration, and supplementation with melatonin and antioxidant SkQ1.

Glutamate and -aminobutyric acid (GABA) are the most abundant amino acids in the retina. An imbalance of the glutamate/GABA system is involved in the pathogenesis of various neurodegenerative disorders. Here we for the first time analyzed alterations of expression of glutamate- and GABA-synthesizing enzymes, transporters, and relevant receptors in the retina with age in Wistar rats and in senescence-accelerated OXYS rats who develop AMD-like retinopathy. We noted consistent age-dependent expression changes of GABAergic-system proteins (GAD67, GABA-T, and GAT1) in OXYS and Wistar rats: upregulation by age 3 months and downregulation at age 18 months. At a late stage of AMD-like retinopathy in OXYS rats (18 months), there was significant upregulation of glutaminase and downregulation of glutamine synthetase, possibly indicating an increasing level of glutamate in the retina. AMD-like-retinopathy development in the OXYS strain was accompanied by underexpression of glutamate transporter GLAST. Prolonged supplementation with both melatonin and SkQ1 (separately) suppressed the progression of the AMD-like pathology in OXYS rats without affecting the glutamate/GABA system but worsened the condition of the Wistar rat's retina during normal aging. We observed decreasing protein levels of glutamine synthetase, GLAST, and GABAAR1 and an increasing level of glutaminase in Wistar rats. In summary, both melatonin and mitochondrial antioxidant SkQ1 had different effect on the retinal glutamate / GABA in healthy Wistar and senescence-accelerated OXYS rats.

Cytochrome P450 oxidase 2J inhibition suppresses choroidal neovascularization in mice.

INTRODUCTION: Choroidal neovascularization (CNV) in age-related macular degeneration (AMD) leads to blindness. It has been widely reported that increased intake of ω-3 long-chain polyunsaturated fatty acids (LCPUFA) diets reduce CNV. Of the three major pathways metabolizing ω-3 (and ω-6 LCPUFA), the cyclooxygenase and lipoxygenase pathways generally produce pro-angiogenic metabolites from ω-6 LCPUFA and anti-angiogenic ones from ω-3 LCPUFA. Howevehr, cytochrome P450 oxidase (CPY) 2C produces pro-angiogenic metabolites from both ω-6 and ω-3 LCPUFA. The effects of CYP2J2 products on ocular neovascularization are still unknown. Understanding how each metabolic pathway affects the protective effect of ω-3 LCPUFA on retinal neovascularization may lead to therapeutic interventions. OBJECTIVES: To investigate the effects of LCPUFA metabolites through CYP2J2 pathway and CYP2J2 regulation on CNV both in vivo and ex vivo. METHODS: The impact of CYP2J2 overexpression and inhibition on neovascularization in the laser-induced CNV mouse model was assessed. The plasma levels of CYP2J2 metabolites were measured by liquid chromatography and tandem mass spectroscopy. The choroidal explant sprouting assay was used to investigate the effects of CYP2J2 inhibition and specific LCPUFA CYP2J2 metabolites on angiogenesis ex vivo. RESULTS: CNV was exacerbated in Tie2-Cre CYP2J2-overexpressing mice and was associated with increased levels of plasma docosahexaenoic acids. Inhibiting CYP2J2 activity with flunarizine decreased CNV in both ω-6 and ω-3 LCPUFA-fed wild-type mice. In Tie2-Cre CYP2J2-overexpressing mice, flunarizine suppressed CNV by 33 % and 36 % in ω-6, ω-3 LCPUFA diets, respectively, and reduced plasma levels of CYP2J2 metabolites. The pro-angiogenic role of CYP2J2 was corroborated in the choroidal explant sprouting assay. Flunarizine attenuated ex vivo choroidal sprouting, and 19,20-EDP, a ω-3 LCPUFA CYP2J2 metabolite, increased sprouting. The combined inhibition of CYP2J2 with flunarizine and CYP2C8 with montelukast further enhanced CNV suppression via tumor necrosis factor-α suppression. CONCLUSIONS: CYP2J2 inhibition augmented the inhibitory effect of ω-3 LCPUFA on CNV. Flunarizine suppressed pathological choroidal angiogenesis, and co-treatment with montelukast inhibiting CYP2C8 further enhanced the effect. CYP2 inhibition might be a viable approach to suppress CNV in AMD.

Modeling of mitochondrial bioenergetics and autophagy impairment in MELAS-mutant iPSC-derived retinal pigment epithelial cells.

BACKGROUND: Mitochondrial dysfunction and mitochondrial DNA (mtDNA) damage in the retinal pigment epithelium (RPE) have been implicated in the pathogenesis of age-related macular degeneration (AMD). However, a deeper understanding is required to determine the contribution of mitochondrial dysfunction and impaired mitochondrial autophagy (mitophagy) to RPE damage and AMD pathobiology. In this study, we model the impact of a prototypical systemic mitochondrial defect, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), in RPE health and homeostasis as an in vitro model for impaired mitochondrial bioenergetics. METHODS: We used induced pluripotent stem cells (iPSCs) derived from skin biopsies of MELAS patients (m.3243A > G tRNA leu mutation) with different levels of mtDNA heteroplasmy and differentiated them into RPE cells. Mitochondrial depletion of ARPE-19 cells (p0 cells) was also performed using 50 ng/mL ethidium bromide (EtBr) and 50 mg/ml uridine. Cell fusion of the human platelets with the p0 cells performed using polyethylene glycol (PEG)/suspension essential medium (SMEM) mixture to generate platelet/RPE "cybrids." Confocal microscopy, FLowSight Imaging cytometry, and Seahorse XF Mito Stress test were used to analyze mitochondrial function. Western Blotting was used to analyze expression of autophagy and mitophagy proteins. RESULTS: We found that MELAS iPSC-derived RPE cells exhibited key characteristics of native RPE. We observed heteroplasmy-dependent impairment of mitochondrial bioenergetics and reliance on glycolysis for generating energy in the MELAS iPSC-derived RPE. The degree of heteroplasmy was directly associated with increased activation of signal transducer and activator of transcription 3 (STAT3), reduced adenosine monophosphate-activated protein kinase α (AMPKα) activation, and decreased autophagic activity. In addition, impaired autophagy was associated with aberrant lysosomal function, and failure of mitochondrial recycling. The mitochondria-depleted p0 cells replicated the effects on autophagy impairment and aberrant STAT3/AMPKα signaling and showed reduced mitochondrial respiration, demonstrating phenotypic similarities between p0 and MELAS iPSC-derived RPE cells. CONCLUSIONS: Our studies demonstrate that the MELAS iPSC-derived disease models are powerful tools for dissecting the molecular mechanisms by which mitochondrial DNA alterations influence RPE function in aging and macular degeneration, and for testing novel therapeutics in patients harboring the MELAS genotype.

Retinal Pigment Epithelium in Human Donor Eyes Contains Higher Levels of Bisretinoids Including A2E in Periphery than Macula.

Purpose: With age, human retinal pigment epithelium (RPE) accumulates bisretinoid fluorophores that may impact cellular function and contribute to age-related macular degeneration (AMD). Bisretinoids are comprised of a central pyridinium, dihydropyridinium, or cyclohexadiene ring. The pyridinium bisretinoid A2E has been extensively studied, and its quantity in the macula has been questioned. Age-changes and distributions of other bisretinoids are not well characterized. We measured levels of three bisretinoids and oxidized A2E in macula and periphery in human donor eyes of different ages. Methods: Eyes (N = 139 donors, 61 women and 78 men, aged 40-80 years) were dissected into 8 mm diameter macular and temporal periphery punches. Using liquid chromatography - electrospray ionization - mass spectrometry (LC-ESI-MS) and an authentic synthesized standard, we quantified A2E (ng). Using LC-ESI-MS and a 50-eye-extract of A2E, we semiquantified A2E and 3 other compounds (eye extract equivalent units [EEEUs): A2-glycerophosphoethanolamine (A2GPE), dihydropyridine phosphatidyl ethanolamine (A2DHPE), and monofuranA2E (MFA2E). Results: A2E quantities in ng and EEEUs were highly correlated (r = 0.97, P < 0.001). From 262 eyes, 5 to 9-fold higher levels were observed in the peripheral retina than in the macula for all assayed compounds. A2E, A2DHPE, and MFA2E increased with age, whereas A2GPE remained unaffected. No significant right-left or male-female differences were detected. Conclusions: Significantly higher levels were observed in the periphery than in the macula for all assayed compounds signifying biologic differences between these regions. Levels of oxidized A2E parallel native A2E and not the distribution of retinal illuminance. Data will assist with the interpretion of clinical trial outcomes of agents targeting bisretinoid-related pathways.

COVID-19 and retinal degenerative diseases: Promising link "Kaempferol".

Coronavirus disease (COVID-19) outbreak has caused unprecedented global disruption since 2020. Approximately 238 million people are affected worldwide where the elderly succumb to mortality. Post-COVID syndrome and its side effects have popped up with several health hazards, such as macular degeneration and vision loss. It thus necessitates better medical care and management of our dietary practices. Natural flavonoids have been included in traditional medicine and have also been used safely against COVID-19 and several other diseases. Kaempferol is an essential flavonoid that has been demonstrated to influence several vital cellular signaling pathways involved in apoptosis, angiogenesis, inflammation, and autophagy. In this review, we emphasize the plausible regulatory effects of Kaempferol on hallmarks of COVID-19 and macular degeneration.

Citrulline protects human retinal pigment epithelium from hydrogen peroxide and iron/ascorbate induced damages.

Oxidative stress plays an important role in the ageing of the retina and in the pathogenesis of retinal diseases such as age-related macular degeneration (ARMD). Hydrogen peroxide is a reactive oxygen species generated by the photo-excited lipofuscin that accumulates during ageing in the retinal pigment epithelium (RPE), and the age-related accumulation of lipofuscin is associated with ARMD. Iron also accumulates with age in the RPE that may contribute to ARMD as an important source of oxidative stress. The aim of this work was to investigate the effects of L-Citrulline (CIT), a naturally occurring amino acid with known antioxidant properties, on oxidative stressed cultured RPE cells. Human RPE (ARPE-19) cells were exposed to hydrogen peroxide (H2 O2 ) or iron/ascorbate (I/A) for 4 h, either in the presence of CIT or after 24 h of pretreatment. Here, we show that supplementation with CIT protects ARPE-19 cells against H2 O2 and I/A. CIT improves cell metabolic activity, decreases ROS production, limits lipid peroxidation, reduces cell death and attenuates IL-8 secretion. Our study evidences that CIT is able to protect human RPE cells from oxidative damage and suggests potential protective effect for the treatment of retinal diseases associated with oxidative stress.

HDL is the primary transporter for carotenoids from liver to retinal pigment epithelium in transgenic ApoA-I-/-/Bco2-/- mice.

Supplementation with antioxidant carotenoids is a therapeutic strategy to protect against age-related macular degeneration (AMD); however, the transport mechanism of carotenoids from the liver to the retina is still not fully understood. Here, we investigate if HDL serves as the primary transporter for the macular carotenoids. ApoA-I, the key apolipoprotein of HDL, was genetically deleted from BCO2 knockout (Bco2-/-) mice, a macular pigment mouse model capable of accumulating carotenoids in the retina. We then conducted a feeding experiment with a mixed carotenoid chow (lutein:zeaxanthin:ß-carotene = 1:1:1) for one month. HPLC data demonstrated that the total carotenoids were increased in the livers but decreased in the serum, retinal pigment epithelium (RPE)/choroids, and retinas of ApoA-I-/-/Bco2-/- mice compared to Bco2-/- mice. In detail, ApoA-I deficiency caused a significant increase of ß-carotene but not lutein and zeaxanthin in the liver, decreased all three carotenoids in the serum, blocked the majority of zeaxanthin and ß-carotene transport to the RPE/choroid, and dramatically reduced ß-carotene and zeaxanthin but not lutein in the retina. Furthermore, surface plasmon resonance spectroscopy (SPR) data showed that the binding affinity between ApoA-I and ß-carotene â‰« zeaxanthin > lutein. Our results show that carotenoids are transported from the liver to the eye mainly by HDL, and ApoA-I may be involved in the selective delivery of macular carotenoids to the RPE.

Epithelial phenotype restoring drugs suppress macular degeneration phenotypes in an iPSC model.

Age-related Macular Degeneration (AMD), a blinding eye disease, is characterized by pathological protein- and lipid-rich drusen deposits underneath the retinal pigment epithelium (RPE) and atrophy of the RPE monolayer in advanced disease stages - leading to photoreceptor cell death and vision loss. Currently, there are no drugs that stop drusen formation or RPE atrophy in AMD. Here we provide an iPSC-RPE AMD model that recapitulates drusen and RPE atrophy. Drusen deposition is dependent on AMD-risk-allele CFH(H/H) and anaphylatoxin triggered alternate complement signaling via the activation of NF-κB and downregulation of autophagy pathways. Through high-throughput screening we identify two drugs, L-745,870, a dopamine receptor antagonist, and aminocaproic acid, a protease inhibitor that reduce drusen deposits and restore RPE epithelial phenotype in anaphylatoxin challenged iPSC-RPE with or without the CFH(H/H) genotype. This comprehensive iPSC-RPE model replicates key AMD phenotypes, provides molecular insight into the role of CFH(H/H) risk-allele in AMD, and discovers two candidate drugs to treat AMD.

Goji Berry Intake Increases Macular Pigment Optical Density in Healthy Adults: A Randomized Pilot Trial.

Age-related macular degeneration (AMD) is the third leading cause of blindness worldwide. Macular pigment optical density (MPOD), a biomarker for AMD, is a non-invasive measure to assess risk. The macula xanthophyll pigments lutein (L) and zeaxanthin (Z) protect against blue light and provide oxidant defense, which can be indexed by MPOD. This study examined the effects of Z-rich goji berry intake on MPOD and skin carotenoids in healthy individuals. A randomized, unmasked, parallel-arm study was conducted with 27 participants, aged 45-65, who consumed either 28 g of goji berries or a supplement containing 6 mg L and 4 mg Z (LZ), five times weekly for 90 days. After 90 days, MPOD was significantly increased in the goji berry group at 0.25 and 1.75 retinal eccentricities (p = 0.029 and p = 0.044, respectively), while no changes were noted in the LZ group. Skin carotenoids were significantly increased in the goji berry group at day 45 (p = 0.025) and day 90 (p = 0.006), but not in the LZ group. Regular intake of goji berries in a healthy middle-aged population increases MPOD may help prevent or delay the development of AMD.

A Potential New Role for Zinc in Age-Related Macular Degeneration through Regulation of Endothelial Fenestration.

Age-related macular degeneration (AMD) is a common blinding disease in the western world that is linked to the loss of fenestration in the choriocapillaris that sustains the retinal pigment epithelium and photoreceptors in the back of the eye. Changes in ocular and systemic zinc concentrations have been associated with AMD; therefore, we hypothesized that these changes might be directly involved in fenestrae formation. To test this hypothesis, an endothelial cell (bEND.5) model for fenestrae formation was treated with different concentrations of zinc sulfate (ZnSO4) solution for up to 20 h. Fenestrae were visualized by staining for Plasmalemmal Vesicle Associated Protein-1 (PV-1), the protein that forms the diaphragms of the fenestrated endothelium. Size and distribution were monitored by transmission electron microscopy (TEM). We found that zinc induced the redistribution of PV-1 into areas called sieve plates containing ~70-nm uniform size and typical morphology fenestrae. As AMD is associated with reduced zinc concentrations in the serum and in ocular tissues, and dietary zinc supplementation is recommended to slow disease progression, we propose here that the elevation of zinc concentration may restore choriocapillaris fenestration resulting in improved nutrient flow and clearance of waste material in the retina.

Environmental and Nutritional Determinants of Macular Pigment in a Mexican Population.

Purpose: The carotenoids lutein (L), zeaxanthin (Z), and meso-zeaxanthin deposit at the macula as macular pigment (MP) and provide visual benefits and protection against macular diseases. The present study investigated MP, its nutritional and environmental determinants, and its constituent carotenoids in serum from a Mexican sample, in healthy participants and with metabolic diseases. Additionally, we compared these variables with an Irish sample. Methods: MP was measured in 215 subjects from a rural community in Mexico with dual-wavelength autofluorescence imaging reported as MP optical volume (MPOV). Dietary intake and serum concentrations of L and Z were evaluated. Results: The mean MPOV was 8429 (95% confidence interval, 8060-8797); range. 1171-15,976. The mean L and Z serum concentrations were 0.25 ± 0.15 µmol/L and 0.09 ± 0.04 µmol/L, respectively. The MPOV was positively correlated with L and Z serum concentrations (r = 0.347; P < 0.001 and r = 0.311; P < 0.001, respectively), but not with L + Z dietary estimates. Subjects with daily sunlight exposure of more than 50% were found to have significantly higher MPOV than those with less than 50% (P = 0.005). MPOV and serum concentrations of L and Z were significantly higher in the Mexican sample compared with the Irish sample, but this difference was not reflected in dietary analysis. Conclusions: These new data from a Mexican sample provide evidence of the multifactorial interactions and environmental determinants of MP such as sunlight exposure and dietary patterns. These findings will be essential for future studies in Mexico for eye health, visual function, and ocular pathology.

Serum and Macular Carotenoids in Relation to Retinal Vessel Caliber Fifteen Years Later, in the Second Carotenoids in Age-Related Eye Disease Study.

Purpose: We investigated whether dietary carotenoids lutein and zeaxanthin (L/Z) in the serum and macula were associated with central retinal arteriole and venule calibers in a follow-up ancillary study among older women in the Women's Health Initiative. Methods: Among 390 women who participated in Carotenoids in Age-Related Eye Disease Study 2 (CAREDS2) (2016-2019), we investigated associations between serum L/Z at Women's Health Initiative baseline (1994-1998), and macular pigment optical density (MPOD) at CAREDS baseline (2001-2004), with central retinal vessel caliber in CAREDS2. MPOD was measured using heterochromatic flicker photometry (0.5° from the foveal center) in CAREDS baseline and CAREDS2. Vessel calibers were measured from fundus photographs (CAREDS2). We also explored associations in women with stable MPOD (±0.10 optical density units) over 15 years (n = 106), given the long-term increases in MPOD related to diet patterns and supplement use. Associations were investigated using linear modeling. Results: In the full sample (n = 390), higher serum L/Z (tertile 3 vs. 1) was positively associated with arteriole caliber (mean ± SE, 145.0 ± 1.4 µm vs. 140.8 ± 1.4 µm; P = 0.05) and venule caliber (214.6 ± 2.2 µm vs. 207.5 ± 2.2 µm; P = 0.03). MPOD was also associated with wider vessel calibers (tertile 3 vs. 1), but the trend was only statistically significant for venules (144.4 ± 1.4 µm vs. 141.1 ± 1.4 µm [P = 0.12] and 213.3 ± 2.1 µm vs. 206.0 ± 2.1 µm [P = 0.02], respectively.) Most associations were strengthened in women with stable MPOD over 15 years, including between MPOD and arteriole caliber (149.8 ± 2.6 µm vs.135.8 ± 3.0 µm; P = 0.001). Conclusions: Higher L/Z status in serum and retina was associated with larger central retinal vessel calibers. Prospective studies and clinical trials are needed to elucidate whether L/Z supplementation prevents vision loss through increasing blood flow.

Lutein and zeaxanthin reduce A2E and iso-A2E levels and improve visual performance in Abca4-/-/Bco2-/- double knockout mice.

Accumulation of bisretinoids such as A2E and its isomer iso-A2E is thought to mediate blue light-induced oxidative damage associated with age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1). We hypothesize that increasing dietary intake of the macular carotenoids lutein and zeaxanthin in individuals at risk of AMD and STGD1 can inhibit the formation of bisretinoids A2E and iso-A2E, which can potentially ameliorate macular degenerative diseases. To study the beneficial effect of macular carotenoids in a retinal degenerative diseases model, we used ATP-binding cassette, sub-family A member 4 (Abca4-/-)/ß,ß-carotene-9',10'-oxygenase 2 (Bco2-/-) double knockout (KO) mice that accumulate elevated levels of A2E and iso-A2E in the retinal pigment epithelium (RPE) and macular carotenoids in the retina. Abca4-/-/Bco2-/- and Abca4-/- mice were fed a lutein-supplemented chow, zeaxanthin-supplemented chow or placebo chow (~2.6 mg of carotenoid/mouse/day) for three months. Visual function and electroretinography (ERG) were measured after one month and three months of carotenoid supplementation. The lutein and zeaxanthin supplemented Abca4-/-/Bco2-/- mice had significantly lower levels of RPE/choroid A2E and iso-A2E compared to control mice fed with placebo chow and improved visual performance. Carotenoid supplementation in Abca4-/- mice minimally raised retinal carotenoid levels and did not show much difference in bisretinoid levels or visual function compared to the control diet group. There was a statistically significant inverse correlation between carotenoid levels in the retina and A2E and iso-A2E levels in the RPE/choroid. Supplementation with retinal carotenoids, especially zeaxanthin, effectively inhibits bisretinoid formation in a mouse model of STGD1 genetically enhanced to accumulate carotenoids in the retina. These results provide further impetus to pursue oral carotenoids as therapeutic interventions for STGD1 and AMD.

The Role of Oxidative Stress and Autophagy in Blue-Light-Induced Damage to the Retinal Pigment Epithelium in Zebrafish In Vitro and In Vivo.

Age-related macular degeneration (AMD) is the progressive degeneration of the retinal pigment epithelium (RPE), retina, and choriocapillaris among elderly individuals and is the leading cause of blindness worldwide. Thus, a better understanding of the underlying mechanisms in retinal tissue activated by blue light exposure is important for developing novel treatment and intervention strategies. In this study, blue-light-emitting diodes with a wavelength of 440 nm were applied to RPE cells at a dose of 3.7 ± 0.75 mW/cm2 for 24 h. ARPE-19 cells were used to investigate the underlying mechanism induced by blue light exposure. A trypan blue exclusion assay was used for the cell viability determination. Flow cytometry was used for apoptosis rate detection and autophagy analysis. An immunofluorescence microscopy analysis was used to investigate cellular oxidative stress and DNA damage using DCFDA fluorescence staining and an anti-γH2AX antibody. Blue light exposure of zebrafish larvae was established to investigate the effect on retinal tissue development in vivo. To further demonstrate the comprehensive effect of blue light on ARPE-19 cells, next-generation sequencing (NGS) was performed for an ingenuity pathway analysis (IPA) to reveal additional related mechanisms. The results showed that blue light exposure caused a decrease in cell proliferation and an increase in apoptosis in ARPE-19 cells in a time-dependent manner. Oxidative stress increased during the early stage of 2 h of exposure and activated DNA damage in ARPE-19 cells after 8 h. Furthermore, autophagy was activated in response to blue light exposure at 24-48 h. The zebrafish larvae model showed the unfavorable effect of blue light in prohibiting retinal tissue development. The RNA-Seq results confirmed that blue light induced cell death and participated in tissue growth inhibition and maturation. The current study reveals the mechanisms by which blue light induces cell death in a time-dependent manner. Moreover, both the in vivo and NGS data uncovered blue light's effect on retinal tissue development, suggesting that exposing children to blue light could be relatively dangerous. These results could benefit the development of preventive strategies utilizing herbal medicine-based treatments for eye diseases or degeneration in the future.

Vitamin Analysis Comparison Study.

PURPOSE: We compared and analyzed the concentrations of vitamin C, vitamin E, zinc, and copper in both national and regional brands of dietary supplements recommended for patients who are at risk for macular degeneration. DESIGN: Prospective cross-sectional study. METHODS: National brand name and generic multivitamin formulations for age-related macular degeneration were obtained. Comparative analysis of the vitamin C and vitamin E content was performed by gas chromatography-mass spectrometry and the zinc and copper content was analyzed by atomic absorption spectroscopy in an institutional chemistry laboratory. RESULTS: All national brand name vitamins, both tablet and gel capsule formulations, and generic brands in tablet form were relatively accurate in their product labeling. For most of the samples tested, the measured quantities of vitamin C, vitamin E, zinc, and copper were slightly higher than labeled but not to an amount that would cause any systemic toxicity if taken at the recommended dosages. CONCLUSIONS: Physicians may recommend national brand name vitamins and generic brands in tablet form to their patients with some confidence; however, the content may have some inaccuracies regarding labeling.

Ferroptosis drives photoreceptor degeneration in mice with defects in all-trans-retinal clearance.

The death of photoreceptor cells in dry age-related macular degeneration (AMD) and autosomal recessive Stargardt disease (STGD1) is closely associated with disruption in all-trans-retinal (atRAL) clearance in neural retina. In this study, we reveal that the overload of atRAL leads to photoreceptor degeneration through activating ferroptosis, a nonapoptotic form of cell death. Ferroptosis of photoreceptor cells induced by atRAL resulted from increased ferrous ion (Fe2+), elevated ACSL4 expression, system Xc- inhibition, and mitochondrial destruction. Fe2+ overload, tripeptide glutathione (GSH) depletion, and damaged mitochondria in photoreceptor cells exposed to atRAL provoked reactive oxygen species (ROS) production, which, together with ACSL4 activation, promoted lipid peroxidation and thereby evoked ferroptotic cell death. Moreover, exposure of photoreceptor cells to atRAL activated COX2, a well-accepted biomarker for ferroptosis onset. In addition to GSH supplement, inhibiting either Fe2+ by deferoxamine mesylate salt (DFO) or lipid peroxidation with ferrostatin-1 (Fer-1) protected photoreceptor cells from ferroptosis caused by atRAL. Abca4-/-Rdh8-/- mice exhibiting defects in atRAL clearance is an animal model for dry AMD and STGD1. We observed that ferroptosis was indeed present in neural retina of Abca4-/-Rdh8-/- mice after light exposure. More importantly, photoreceptor atrophy and ferroptosis in light-exposed Abca4-/-Rdh8-/- mice were effectively alleviated by intraperitoneally injected Fer-1, a selective inhibitor of ferroptosis. Our study suggests that ferroptosis is one of the important pathways of photoreceptor cell death in retinopathies arising from excess atRAL accumulation and should be pursued as a novel target for protection against dry AMD and STGD1.