A novel combination therapy with Cabozantinib and Honokiol effectively inhibits c-Met-Nrf2-induced renal tumor growth through increased oxidative stress.

Receptor tyrosine kinase (RTK), c-Met, is overexpressed and hyper active in renal cell carcinoma (RCC). Most of the therapeutic agents mediate cancer cell death through increased oxidative stress. Induction of c-Met in renal cancer cells promotes the activation of redox-sensitive transcription factor Nrf2 and cytoprotective heme oxygenase-1 (HO-1), which can mediate therapeutic resistance against oxidative stress. c-Met/RTK inhibitor, Cabozantinib, has been approved for the treatment of advanced RCC. However, acquired drug resistance is a major hurdle in the clinical use of cabozantinib. Honokiol, a naturally occurring phenolic compound, has a great potential to downregulate c-Met-induced pathways. In this study, we found that a novel combination treatment with cabozantinib + Honokiol inhibits the growth of renal cancer cells in a synergistic manner through increased production of reactive oxygen species (ROS); and it significantly facilitates apoptosis-and autophagy-mediated cancer cell death. Activation of c-Met can induce Rubicon (a negative regulator of autophagy) and p62 (an autophagy adaptor protein), which can stabilize Nrf2. By utilizing OncoDB online database, we found a positive correlation among c-Met, Rubicon, p62 and Nrf2 in renal cancer. Interestingly, the combination treatment significantly downregulated Rubicon, p62 and Nrf2 in RCC cells. In a tumor xenograft model, this combination treatment markedly inhibited renal tumor growth in vivo; and it is associated with decreased expression of Rubicon, p62, HO-1 and vessel density in the tumor tissues. Together, cabozantinib + Honokiol combination can significantly inhibit c-Met-induced and Nrf2-mediated anti-oxidant pathway in renal cancer cells to promote increased oxidative stress and tumor cell death.

Protocol for isolating human BCAM-positive corneal progenitor cells by flow cytometry and cell sorting.

BCAM-positive basal limbal epithelial cells are an early transit-amplifying cell population (TAC) capable of holoclone formation and corneal epithelial differentiation. Here, we present a protocol for isolating BCAM-positive cells from human donor corneas by flow cytometry and cell sorting. We describe steps for cell dissection and dissociation, antibody staining, and flow cytometry. We then detail procedures for culturing the purified BCAM-positive and BCAM-negative cells for holoclone and cell sheet formation assays to study the factors that regulate corneal regeneration. For complete details on the use and execution of this protocol, please refer to Sasamoto et al.1.

Epigenetic Regulation of Corneal Epithelial Differentiation by TET2.

Epigenetic DNA modification by 5-hydroxymethylcytosine (5hmC), generated by the Ten-eleven translocation (TET) dioxygenases, regulates diverse biological functions in many organ tissues, including the mammalian eye. For example, 5hmC has been shown to be involved in epigenetic regulation of retinal gene expression. However, a functional role of 5hmC in corneal differentiation has not been investigated to date. Here, we examined 5hmC and TET function in the human cornea. We found 5hmC highly expressed in MUC16-positive terminally differentiated cells that also co-expressed the 5hmC-generating enzyme TET2. TET2 knockdown (KD) in cultured corneal epithelial cells led to significant reductions of 5hmC peak distributions and resulted in transcriptional repression of molecular pathways involved in corneal differentiation, as evidenced by downregulation of MUC4, MUC16, and Keratin 12. Additionally, integrated TET2 KD RNA-seq and genome-wide Reduced Representation Hydroxymethylation Profiling revealed novel epigenetically regulated genes expressed by terminally differentiated cells, including KRT78, MYEOV, and MAL. In aggregate, our findings reveal a novel function of TET2 in the epigenetic regulation of corneal epithelial gene expression and identify novel TET2-controlled genes expressed in differentiated corneal epithelial cells. These results point to potential roles for TET2 induction strategies to enhance treatment of corneal diseases associated with abnormal epithelial maturation.

Limbal BCAM expression identifies a proliferative progenitor population capable of holoclone formation and corneal differentiation.

The corneal epithelium is renowned for high regenerative potential, which is dependent on the coordinated function of its diverse progenitor subpopulations. However, the molecular pathways governing corneal epithelial progenitor differentiation are incompletely understood. Here, we identify a highly proliferative limbal epithelial progenitor subpopulation characterized by expression of basal cell adhesion molecule (BCAM) that is capable of holocone formation and corneal epithelial sheet generation. BCAM-positive cells can be found among ABCB5-positive limbal stem cells (LSCs) as well as among ABCB5-negative limbal epithelial cell populations. Mechanistically, we show that BCAM is functionally required for cellular migration and differentiation and that its expression is regulated by the transcription factor p63. In aggregate, our study identifies limbal BCAM expression as a marker of highly proliferative corneal epithelial progenitor cells and defines the role of BCAM as a critical molecular mediator of corneal epithelial differentiation.

Long-term survival in non-human primates of stem cell-derived, MHC-unmatched corneal epithelial cell sheets.

When corneal epithelial stem cells residing in the corneal limbus become dysfunctional, called a limbal stem cell deficiency (LSCD), corneal transparency is decreased, causing severe vision loss. Transplantation of corneal epithelial cell sheets (CEPS) derived from stem cells, including induced pluripotent stem cells, is a promising treatment for LSCD. However, the potential effect of human leukocyte antigen (HLA) concordance on CEPS transplantation has not been addressed. Here, we show that there is no difference in the immune response to CEPS between HLA-matched and -unmatched peripheral blood mononuclear cells in mixed lymphocyte reactions. CEPS transplantation in cynomolgus monkeys revealed that the immune response to major histocompatibility-unmatched CEPS was not strong and could be controlled by local steroid administration. Furthermore, programmed death ligand 1 was identified as an immunosuppressive molecule in CEPS under inflammatory conditions in vitro. Our results indicate that corneal epithelium has low immunogenicity and allogeneic CEPS transplantation requires mild immunosuppression.

High expression of SARS-CoV2 viral entry-related proteins in human limbal stem cells.

PURPOSE: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV2). While the ocular surface is considered one of the major SARS-CoV2 transmission routes, the specific cellular tropism of SARS-CoV2 is not fully understood. In the current study, we evaluated the expression and regulation of two SARS-CoV2 viral entry proteins, TMPRSS2 and ACE2, in human ocular epithelial cells and stem cells. METHODS: TMPRSS2 and ACE2 expression in ABCB5-positive limbal stem cells (LSCs) were assessed by RNAseq, flow cytometry and immunohistochemistry. PAX6, TMPRSS2, and ACE2 mRNA expression values were obtained from the GSE135455 and DRA002960 RNA-seq datasets. siRNA-mediated PAX6 knockdown (KD) was performed in limbal and conjunctival epithelial cells. TMPRSS2 and ACE2 expression in the PAX6 KD cells was analyzed by qRT-PCR and Western blot. RESULTS: We found that ABCB5-positive LSCs express high levels of TMPRSS2 and ACE2 compared to ABCB5-negative limbal epithelial cells. Mechanistically, gene knockout and overexpression models revealed that the eye transcription factor PAX6 negatively regulates TMPRSS2 expression. Therefore, low levels of PAX6 in ABCB5-positive LSCs promote TMPRSS2 expression, and high levels of TMPRSS2 and ACE2 expression by LSCs indicate enhanced susceptibility to SARS-CoV2 infection in this stem cell population. CONCLUSIONS: Our study points to a need for COVID-19 testing of LSCs derived from donor corneas before transplantation to patients with limbal stem cell deficiency. Furthermore, our findings suggest that expandable human ABCB5+ LSC cultures might represent a relevant novel model system for studying cellular SARS-CoV2 viral entry mechanisms and evaluating related targeting strategies.

Human iPS cells engender corneal epithelial stem cells with holoclone-forming capabilities.

Human induced pluripotent stem cells (hiPSCs) can generate a multiplicity of organoids, yet no compelling evidence currently exists as to whether or not these contain tissue-specific, holoclone-forming stem cells. Here, we show that a subpopulation of cells in a hiPSC-derived corneal epithelial cell sheet is positive for ABCB5 (ATP-binding cassette, sub-family B, member 5), a functional marker of adult corneal epithelial stem cells. These cells possess remarkable holoclone-forming capabilities, which can be suppressed by an antibody-mediated ABCB5 blockade. The cell sheets are generated from ABCB5+ hiPSCs that first emerge in 2D eye-like organoids around six weeks of differentiation and display corneal epithelial immunostaining characteristics and gene expression patterns, including sustained expression of ABCB5. The findings highlight the translational potential of ABCB5-enriched, hiPSC-derived corneal epithelial cell sheets to recover vision in stem cell-deficient human eyes and represent the first report of holoclone-forming stem cells being directly identified in an hiPSC-derived organoid.

Investigation of factors associated with ABCB5-positive limbal stem cell isolation yields from human donors.

PURPOSE: To identify factors associated with isolation yields of ATP-binding cassette (ABC) superfamily member B5 (ABCB5)-positive limbal stem cells (LSCs) from human cadaveric donor eyes. METHODS: Whole eye globes were obtained from the Saving Sight eye bank, Kansas City, MO and the CorneaGen eye bank, Seattle, WA. ABCB5-positive LSCs were sorted by flow cytometry upon anti-ABCB5 monoclonal antibody staining within one week after donor death. The yields of live limbal epithelial cells in their entirety and of isolated pure ABCB5-positive LSC subsets were correlated with variables contained in the eye donors' medical information. RESULTS: The mean isolation yield of live limbal epithelial cells and ABCB5-positive LSCs per donor eye was (340,000 ±â€¯160,000 and 2,608 ±â€¯1,842 respectively, mean ±â€¯SD). Stepwise regression analysis showed that cardiac disease-related death was the strongest negative predictor of the ABCB5-positive LSC isolation yield (p = 0.01). While we observed a trend for an age-related decline in the yield of ABCB5-positive LSCs, a statistically significant association could not be established (2% decrease/year, p = 0.11). Additionally, despite a trend for decreased isolation yields of total live limbal epithelial cells isolated from single donors with a longer time between death and tissue processing (p = 0.04), this did not affect the yields of purified ABCB5-positive LSC, which was independent of increasing time between death and tissue processing (p = 0.50). CONCLUSIONS: Our study identifies cardiac disease-related death as a donor variable significantly associated with lower ABCB5-positive LSC isolation yields.

KLF4 prevents epithelial to mesenchymal transition in human corneal epithelial cells via endogenous TGF-ß2 suppression.

INTRODUCTION: Krüppel-like factor 4 (KLF4) is considered one of the Yamanaka factors, and recently, we and others have shown that KLF4 is one of the transcription factors essential for reprogramming non-human corneal epithelial cells (HCECs) into HCECs. Since epithelial to mesenchymal transition (EMT) suppression is vital for homeostasis of HCECs via regulation of transcription factors, in this study, we aimed to investigate whether KLF4 prevents EMT in HCECs and to elucidate the underlying mechanism within the canonical TGF-ß signalling pathway, which is involved in corneal epithelial wound healing. METHODS: HCECs were collected from cadaver donors and cultivated. We generated KLF4-knockdown (KD) HCECs using siRNA transfection and analysed morphology, gene or protein expression, and endogenous TGF-ß secretion. KLF4 was overexpressed using lentiviral KLF4 expression vectors and underwent protein expression analyses after TGF-ß2 treatment. RESULTS: KLF4-KD HCECs showed a fibroblastic morphology, downregulation of the epithelial markers, keratin 12 and keratin 14, and upregulation of the mesenchymal markers, fibronectin 1, vimentin, N-cadherin, and SLUG. Although E-cadherin expression remained unchanged in KLF4-KD HCECs, immunocytochemical analysis showed that E-cadherin-positive adherens junctions decreased in KLF4-KD HCECs as well as the decreased total protein levels of E-cadherin analysed by immunoblotting. Moreover, within the TGF-ß canonical signalling pathway, TGF-ß2 secretion by HCECs increased up to 5 folds, and several TGF-ß-associated markers (TGFB1, TGFB2, TGFBR1, and TGFBR2) were significantly upregulated up to 6 folds in the KLF4-KD HCECs. SMAD2/3, the main signal transduction molecules of the TGF-ß signalling pathway, were found to be localised in the nucleus of KLF4-KD HCECs. When KLF4 was overexpressed, cultivated HCECs showed upregulation of epithelial markers, keratin 14 and E-cadherin, indicating the contributory role of KLF4 in the homeostasis of human corneal epithelium in vivo. In addition, KLF4 overexpression in HCECs resulted in decreased SMAD2 phosphorylation and altered nuclear localisation of SMAD2/3, even after TGF-ß2 treatment. CONCLUSIONS: These results show that KLF4 prevents EMT in HCECs and suggest a novel role of KLF4 as an endogenous TGF-ß2 suppressor in the human corneal epithelium, thus highlighting the potential of KLF4 to prevent EMT and subsequent corneal fibrotic scar formation by attenuating TGF-ß signalling.

Repairing the corneal epithelium using limbal stem cells or alternative cell-based therapies.

INTRODUCTION: The corneal epithelium is maintained by limbal stem cells (LSCs) that reside in the basal epithelial layer of the tissue surrounding the cornea termed the limbus. Loss of LSCs results in limbal stem cell deficiency (LSCD) that can cause severe visual impairment. Patients with partial LSCD may respond to conservative therapies designed to rehabilitate the remaining LSCs. However, if these conservative approaches fail or, if complete loss of LSCs occurs, transplantation of LSCs or their alternatives is the only option. While a number of clinical studies utilizing diverse surgical and cell culture techniques have shown favorable results, a universal cure for LSCD is still not available. Knowledge of the potential risks and benefits of current approaches, and development of new technologies, is essential for further improvement of LSCD therapies. AREAS COVERED: This review focuses on cell-based LSCD treatment approaches ranging from current available clinical therapies to preclinical studies of novel promising applications. EXPERT OPINION: Improved understanding of LSC identity and development of LSC expansion methods will influence the evolution of successful LSCD therapies. Ultimately, future controlled clinical studies enabling direct comparison of the diverse employed approaches will help to identify the most effective treatment strategies.

Factors Limiting the Visual Outcome After Descemet Stripping Automated Endothelial Keratoplasty: Comprehensive Analysis Including the Graft Position and Irregularity.

PURPOSE: To investigate factors affecting best postoperative corrected distance visual acuity (CDVA) in patients who have undergone Descemet stripping automated endothelial keratoplasty (DSAEK). METHODS: Forty-two eyes of 42 patients with predominantly non-Fuchs endothelial corneal dystrophy after DSAEK were retrospectively evaluated. All patients were examined using anterior segment optical coherence tomography when their best postoperative CDVA had been achieved. We analyzed the relationship between best postoperative CDVA and preoperative, intraoperative, and postoperative factors, including corneal higher-order aberrations (HOAs), corneal backward scattering, graft irregularity, and graft decentration. Variables with P < 0.2 in univariate analysis were entered into a multivariate linear regression model to determine the factors affecting best postoperative CDVA. RESULTS: Mean CDVA improved from 1.16 ± 0.46 preoperatively to 0.22 ± 0.19 logarithm of the minimum angle of resolution equivalents postoperatively (P < 0.0001). Best postoperative CDVA was achieved 11.6 ± 7.7 months after DSAEK. Multivariate analysis showed that anterior corneal coma-like HOAs and venting incisions inside the pupil area were significantly correlated with best postoperative CDVA (t = 2.90, P = 0.006; t = 2.62, P = 0.01, respectively). No significant relationship was found between the visual outcome and any other factor, including posterior corneal HOAs, corneal backward scattering, graft decentration, and graft irregularity. CONCLUSIONS: Irregularity of the anterior corneal surface and venting incisions inside the pupil area are important factors that impair best postoperative CDVA in patients who have undergone DSAEK. In contrast, our results indicate that the graft position and irregularity might not have a strong influence on the visual outcome after DSAEK.

Limbal stem cells: identity, developmental origin, and therapeutic potential.

The cornea is our window to the world and our vision is critically dependent on corneal clarity and integrity. Its epithelium represents one of the most rapidly regenerating mammalian tissues, undergoing full-turnover over the course of approximately 1-2 weeks. This robust and efficient regenerative capacity is dependent on the function of stem cells residing in the limbus, a structure marking the border between the cornea and the conjunctiva. Limbal stem cells (LSC) represent a quiescent cell population with proliferative capacity residing in the basal epithelial layer of the limbus within a cellular niche. In addition to LSC, this niche consists of various cell populations such as limbal stromal fibroblasts, melanocytes and immune cells as well as a basement membrane, all of which are essential for LSC maintenance and LSC-driven regeneration. The LSC niche's components are of diverse developmental origin, a fact that had, until recently, prevented precise identification of molecularly defined LSC. The recent success in prospective LSC isolation based on ABCB5 expression and the capacity of this LSC population for long-term corneal restoration following transplantation in preclinical in vivo models of LSC deficiency underline the considerable potential of pure LSC formulations for clinical therapy. Additional studies, including genetic lineage tracing of the developmental origin of LSC will further improve our understanding of this critical cell population and its niche, with important implications for regenerative medicine. WIREs Dev Biol 2018, 7:e303. doi: 10.1002/wdev.303 This article is categorized under: Adult Stem Cells, Tissue Renewal, and Regeneration > Stem Cells and Disease Adult Stem Cells, Tissue Renewal, and Regeneration > Tissue Stem Cells and Niches Adult Stem Cells, Tissue Renewal, and Regeneration > Regeneration.

High-resolution promoter map of human limbal epithelial cells cultured with keratinocyte growth factor and rho kinase inhibitor.

An in vitro model of corneal epithelial cells (CECs) has been developed to study and treat corneal disorders. Nevertheless, conventional CEC culture supplemented with epidermal growth factor (EGF) results in a loss of CEC characteristics. It has recently been reported that limbal epithelial cells (LECs) cultured with keratinocyte growth factor (KGF) and the rho kinase inhibitor Y-27632 could maintain the expression of several CEC-specific markers. However, the molecular mechanism underlying the effect of culture media on LECs remains to be elucidated. To elucidate this mechanism, we performed comprehensive gene expression analysis of human LECs cultured with EGF or KGF/Y-27632, by cap analysis of gene expression (CAGE). Here, we found that LECs cultured with KGF and Y-27632 presented a gene expression profile highly similar to that of CECs in vivo. In contrast, LECs cultured with EGF lost the characteristic CEC gene expression profile. We further discovered that CEC-specific PAX6 promoters are highly activated in LECs cultured with KGF and Y-27632. Our results provide strong evidence that LECs cultured with KGF and Y-27632 would be an improved in vitro model in the context of gene expression. These findings will accelerate basic studies of CECs and clinical applications in regenerative medicine.

Coordinated generation of multiple ocular-like cell lineages and fabrication of functional corneal epithelial cell sheets from human iPS cells.

We describe a protocol for the generation of a functional and transplantable corneal epithelium derived from human induced pluripotent stem (iPS) cells. When this protocol is followed, a proportion of iPS cells spontaneously form circular colonies, each of which is composed of four concentric zones. Cells in these zones have different morphologies and immunostaining characteristics, resembling neuroectoderm, neural crest, ocular-surface ectoderm, or surface ectoderm. We have named this 2D colony a 'SEAM' (self-formed ectodermal autonomous multizone), and previously demonstrated that cells within the SEAM have the potential to give rise to anlages of different ocular lineages, including retinal cells, lens cells, and ocular-surface ectoderm. To investigate the translational potential of the SEAM, cells within it that resemble ocular-surface epithelia can be isolated by pipetting and FACS sorting into a population of corneal epithelial-like progenitor cells. These can be expanded and differentiated to form an epithelial layer expressing K12 and PAX6, and able to recover function in an animal model of corneal epithelial dysfunction after surgical transplantation. The whole protocol, encompassing human iPS cell preparation, autonomous differentiation, purification, and subsequent differentiation, takes between 100 and 120 d, and is of potential use to researchers with an interest in eye development and/or ocular-surface regeneration. Experience with human iPS cell culture and sorting via FACS will be of benefit for researchers performing this protocol.

Co-ordinated ocular development from human iPS cells and recovery of corneal function.

The eye is a complex organ with highly specialized constituent tissues derived from different primordial cell lineages. The retina, for example, develops from neuroectoderm via the optic vesicle, the corneal epithelium is descended from surface ectoderm, while the iris and collagen-rich stroma of the cornea have a neural crest origin. Recent work with pluripotent stem cells in culture has revealed a previously under-appreciated level of intrinsic cellular self-organization, with a focus on the retina and retinal cells. Moreover, we and others have demonstrated the in vitro induction of a corneal epithelial cell phenotype from pluripotent stem cells. These studies, however, have a single, tissue-specific focus and fail to reflect the complexity of whole eye development. Here we demonstrate the generation from human induced pluripotent stem cells of a self-formed ectodermal autonomous multi-zone (SEAM) of ocular cells. In some respects the concentric SEAM mimics whole-eye development because cell location within different zones is indicative of lineage, spanning the ocular surface ectoderm, lens, neuro-retina, and retinal pigment epithelium. It thus represents a promising resource for new and ongoing studies of ocular morphogenesis. The approach also has translational potential and to illustrate this we show that cells isolated from the ocular surface ectodermal zone of the SEAM can be sorted and expanded ex vivo to form a corneal epithelium that recovers function in an experimentally induced animal model of corneal blindness.

PAX6 Isoforms, along with Reprogramming Factors, Differentially Regulate the Induction of Cornea-specific Genes.

PAX6 is the key transcription factor involved in eye development in humans, but the differential functions of the two PAX6 isoforms, isoform-a and isoform-b, are largely unknown. To reveal their function in the corneal epithelium, PAX6 isoforms, along with reprogramming factors, were transduced into human non-ocular epithelial cells. Herein, we show that the two PAX6 isoforms differentially and cooperatively regulate the expression of genes specific to the structure and functions of the corneal epithelium, particularly keratin 3 (KRT3) and keratin 12 (KRT12). PAX6 isoform-a induced KRT3 expression by targeting its upstream region. KLF4 enhanced this induction. A combination of PAX6 isoform-b, KLF4, and OCT4 induced KRT12 expression. These new findings will contribute to furthering the understanding of the molecular basis of the corneal epithelium specific phenotype.

Methodology for setting risk-based concentrations of contaminants in soil and groundwater and application to a model contaminated site.

In Japan, environmental standards for contaminants in groundwater and in leachate from soil are set with the assumption that they are used for drinking water over a human lifetime. Where there is neither a well nor groundwater used for drinking, the standard is thus too severe. Therefore, remediation based on these standards incurs excessive effort and cost. In contrast, the environmental-assessment procedure used in the United States and the Netherlands considers the site conditions (land use, existing wells, etc.); however, a risk assessment is required for each site. Therefore, this study proposes a new framework for judging contamination in Japan by considering the merits of the environmental standards used and a method for risk assessment. The framework involves setting risk-based concentrations that are attainable remediation goals for contaminants in soil and groundwater. The framework was then applied to a model contaminated site for risk management, and the results are discussed regarding the effectiveness and applicability of the new methodology.

Clinical outcomes and changes in aqueous vascular endothelial growth factor levels after intravitreal bevacizumab for iris neovascularization and neovascular glaucoma: a retrospective two-dose comparative study.

PURPOSE: To evaluate the clinical outcomes and biologic effects on the aqueous humor concentrations of vascular endothelial growth factor (VEGF) in patients with neovascular glaucoma (NVG) treated with intravitreal bevacizumab (IVB). METHODS: Twenty-nine consecutive patients (35 eyes) treated with 1.0- or 0.1-mg injections of IVB for NVG between January and December 2009 were enrolled in this retrospective, interventional pilot study. The visual prognosis and changes in intraocular pressure (IOP) were followed for >6 months after the initial injection. Aqueous humor samples were obtained at the initial IVB injection from all study eyes and 1 week after the first injection in eyes undergoing a second intervention to measure the VEGF concentration. RESULTS: The VEGF concentrations in the 35 eyes significantly correlated (r=0.535, P<0.001) with the pretreatment IOP. The mean reductions of the VEGF levels 1 week after IVB did not differ significantly between the 1.0- and 0.1-mg groups (P=0.738). Despite more repeated injections in the 0.1-mg group and additional medical or surgical interventions in both groups, both dosages inhibited the neovascular activity. The ability to control the IOP after IVB did not differ significantly between groups at 1 week (P=0.625) and 6 months (P>0.99). Visual improvements also did not differ significantly between groups during the 6-month follow-up (P=0.437). CONCLUSIONS: Aqueous humor levels of VEGF were significantly correlated with the IOP. Low-dose (0.1 mg) IVB was as effective as the currently used higher dose (1.0 mg) for treating NVG within at least 6 months after the initial injection.

Effect of 1-year lutein supplementation on macular pigment optical density and visual function.

BACKGROUND: Although it is known that antioxidants including lutein can affect macular pigment optical density (MPOD) and visual function, we still have much to learn about their effect. Our aim was to assess the 1-year changes in MPOD and visual function in response to supplementation containing lutein. METHODS: We prospectively measured the MPOD level of those who received a supplement containing 6 mg of lutein daily for 1 year. MPOD level was measured every 3 months by using autofluorescence spectrometry with the two-wavelength method. Other examinations, including contrast sensitivity and retinal sensitivity were also measured every 3 or 6 months. Stepwise regression analysis was performed to determine the factors that correlated with the changes observed in those examinations. RESULTS: Forty-three eyes of 43 Japanese subjects, including five normal eyes, five fellow eyes with central serous chorioretinopathy (CSC), and 33 fellow eyes with age-related macular degeneration (AMD) were enrolled. The higher baseline MPOD level was correlated with the eye with a clear intraocular lens (IOL). Although no time-dependent changes in the MPOD level were obtained in any area, subjects without cardiovascular diseases showed higher increase in the MPOD level. We observed significant increases in the contrast sensitivity at 1 year (p = 0.0124) and in the retinal sensitivity at 6 months (p < 0.0001) and 1 year (p < 0.0001). Stepwise regression analysis showed that nonsmokers had increased contrast sensitivity (p = 0.0173), and the fellow eye of those with CSC had less of an increase in retinal sensitivity (p = 0.0491). CONCLUSIONS: Daily supplementation with 6 mg of lutein did not affect the MPOD level for 1 year, suggesting that 6 mg of lutein may be insufficient to increase the MPOD level. However, supplementation seems to improve visual functions such as contrast sensitivity and retinal sensitivity.

Effect of cataract in evaluation of macular pigment optical density by autofluorescence spectrometry.

PURPOSE: To assess the effect of cataract on the evaluation of macular pigment optical density (MPOD) in aged patients. METHODS: MPOD was prospectively measured using autofluorescence spectrometry before and after cataract surgery. The Lens Opacities Classification System III was used to grade the cataracts at baseline. RESULTS: Forty-five eyes of 41 subjects, who had no ocular disorders or fundus autofluorescence abnormalities except for age-related nuclear cataract, were included. Preoperative MPOD was 0.350 ± 0.117 density unit (DU). Regression analysis showed that a higher nuclear color score correlated with lower MPOD (t = -2.90, P = 0.0063). The preoperative MPOD prediction formula was MPOD = 0.545 - 0.069 × nuclear color score. A higher nuclear color score correlated significantly with failure to measure the MPOD (χ(2) = 5.08, P = 0.0242). The mean postoperative MPOD was 0.600 DU (95% confidence interval [CI], 0.562-0.637), which was significantly (P < 0.0001) higher than the preoperative level of 0.350 DU (95% CI, 0.313-0.388). Regression analysis showed that higher preoperative MPOD correlated with higher postoperative MPOD (t = 2.91, P = 0.0061). CONCLUSIONS: Cataract, especially its nuclear component, affects MPOD measured by autofluorescence spectrometry. Care should be taken when using this method in eyes with age-related macular maculopathy and age-related macular degeneration and in older patients who may develop these diseases.