Low Xanthophylls, Retinol, Lycopene, and Tocopherols in Grey and White Matter of Brains with Alzheimer's Disease.

BACKGROUND: Oxidative stress contributes to pathogenesis and progression of Alzheimer's disease (AD). Higher levels of the dietary antioxidants- carotenoids and tocopherols- are associated with better cognitive functions and lower risk for AD, and lower levels of multiple carotenoids are found in serum and plasma of patients with AD. Although brains donated by individuals with mild cognitive impairment had significantly lower levels of lutein and beta-carotene, previous investigators found no significant difference in carotenoid levels of brains with AD and cognitively normal brains. OBJECTIVE: This study tested the hypothesis that micronutrients are significantly lower in donor brains with AD than in healthy elderly brains. METHODS: Samples of donor brains with confirmed AD or verified health were dissected into grey and white matter, extracted with organic solvents and analyzed by HPLC. RESULTS: AD brains had significantly lower levels of lutein, zeaxanthin, anhydrolutein, retinol, lycopene, and alpha-tocopherol, and significantly increased levels of XMiAD, an unidentified xanthophyll metabolite. No meso-zeaxanthin was detected. The overlapping protective roles of xanthophylls, carotenes, α- and γ-tocopherol are discussed. CONCLUSION: Brains with AD had substantially lower concentrations of some, but not all, xanthophylls, carotenes, and tocopherols, and several-fold higher concentrations of an unidentified xanthophyll metabolite increased in AD (XMiAD).

Dietary Chitin Particles Called Mimetic Fungi Ameliorate Colitis in Toll-Like Receptor 2/CD14- and Sex-Dependent Manners.

Chitin is a natural N-acetylglucosamine polymer and a major structural component of fungal cell walls. Dietary chitin is mucoadhesive; anti-inflammatory effects of chitin microparticles (CMPs; 1- to 10-µm diameters) have been demonstrated in models of inflammatory bowel disease (IBD). The goals of this study were to assess (i) whether CMPs among various chitin preparations are the most effective against colitis in male and female mice and (ii) whether host chitin-binding Toll-like receptor 2 (TLR2) and CD14 are required for the anti-inflammatory effect of chitin. We found that colitis in male mice was ameliorated by CMPs and large chitin beads (LCBs; 40 to 70 µm) but not by chitosan (deacetylated chitin) microparticles, oligosaccharide chitin, or glucosamine. In fact, LCBs were more effective than CMPs. In female colitis, on the other hand, CMPs and LCBs were equally and highly effective. Neither sex of TLR2-deficient mice showed anti-inflammatory effects when treated with LCBs. No anti-inflammatory effect of LCBs was seen in either CD14-deficient males or females. Furthermore, an in vitro study indicated that when LCBs and CMPs were digested with stomach acidic mammalian chitinase (AMC), their size-dependent macrophage activations were modified, at least in part, suggesting reduced particle sizes of dietary chitin in the stomach. Interestingly, stomach AMC activity was greater in males than females. Our results indicated that dietary LCBs were the most effective preparation for treating colitis in both sexes; these anti-inflammatory effects of LCBs were dependent on host TLR2 and CD14.

Cell-specific gene therapy driven by an optimized hypoxia-regulated vector reduces choroidal neovascularization.

Aberrant growth of blood vessels in the choroid layer of the eye, termed choroidal neovascularization (CNV), is the pathological hallmark of exudative age-related macular degeneration (AMD), causing irreversible blindness among the elderly. Co-localization of proangiogenic factors and hypoxia inducible factors (HIF) in neovascular membranes from AMD eyes suggests the role of hypoxia in pathogenesis of CNV. In order to utilize hypoxic conditions in RPE for therapeutic purposes, we developed an optimized hypoxia regulated, RPE cell-specific gene therapy to inhibit choroidal neovascularization. An adeno-associated virus (AAV2) vector comprising a RPE-specific promoter and HIF-1 response elements (HRE) was designed to regulate production of human endostatin (a powerful angiostatic protein) in RPE. The vector was tested in a mouse model of laser-induced CNV using subretinal delivery. Spectral domain optical coherence tomography (SD-OCT) images from live mice and confocal images from lectin stained RPE flat mount sections demonstrated reduction in CNV areas by 80% compared to untreated eyes. Quantitative real-time polymerase chain reaction (qPCR) confirmed exogenous endostatin mRNA expression from the regulated vector that was significantly elevated 3, 7, and 14 days following laser treatment, but its expression was completely shut off after 45 days. Thus, RPE-specific, hypoxia-regulated delivery of anti-angiogenic proteins could be a valuable therapeutic approach to treat neovascular AMD at the time and in the ocular space where it arises. KEY POINTS: An optimized gene therapy vector targeting hypoxia and tissue-specific expression has been designed. The inhibitory role of gene therapy vector was tested in a mouse model of laser-induced CNV. An 80% reduction in choroidal neovascularization was achieved by the optimized vector. The expression of endostatin was limited to retinal pigment epithelium and regulated by hypoxia.

Phagocytosis-mediated M1 activation by chitin but not by chitosan.

Chitin particles have been used to understand host response to chitin-containing pathogens and allergens and are known to induce a wide range of polarized macrophage activations, depending, at least in part, on particle size. Nonphagocytosable particles larger than a macrophage induce tissue repair M2 activation. In contrast, phagocytosable chitin microparticles (CMPs, 1-10 µm diameters) induce M1 macrophages that kill intracellular microbes and damage tissues. However, chitosan (deacetylated) microparticles (de-CMPs, 1-10 µm) induce poor M1 activation. Toll-like receptor 2 (TLR2) and associated coreceptors in macrophages appear to be required for the M1 activation. To understand the exact mechanism of phagocytosis-mediated M1 activation by chitin, we isolated macrophage proteins that bind to CMPs during early phagocytosis and determined that TLR1, TLR2, CD14, late endosomal/lysosomal adaptor MAPK and mechanistic target of rapamycin activator 1 (LAMTOR1), Lck/Yes novel tyrosine kinase (Lyn), and ß-actin formed phagosomal CMP-TLR2 clusters. These proteins were also detected in TLR2 phagosomal clusters in macrophages phagocytosing de-CMPs, but at relatively lower levels than in the CMP-TLR2 clusters. Importantly, CMP-TLR2 clusters further recruited myeloid differentiation primary response gene 88 (MyD88) and Toll-IL-1 receptor-containing adaptor protein (TIRAP) and phosphorylated Lyn, whereas neither the adaptors nor phosphorylated Lyn was detected in the de-CMP clusters. The results indicate that the acetyl group played an obligatory, phagocytosis-dependent role in the initiation of an integrated signal for TLR2-mediated M1 activation.

A hypoxia-responsive glial cell-specific gene therapy vector for targeting retinal neovascularization.

PURPOSE: Müller cells, the major glial cell in the retina, play a significant role in retinal neovascularization in response to tissue hypoxia. We previously designed and tested a vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) promoter to drive green fluorescent protein (GFP) expression in Müller cells in the murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of regulated and unregulated Müller cell delivery of endostatin in preventing neovascularization in the OIR model. METHODS: Endostatin cDNA was cloned into plasmids with hypoxia-regulated GFAP or unregulated GFAP promoters, and packaged into self-complementary adeno-associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on postnatal day (P)7, mice were given intravitreal injections of regulated or unregulated scAAV2, capsid, or PBS. Five days after return to room air, on P17, neovascular and avascular areas, as well as expression of the transgene and vascular endothelial growth factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. RESULTS: The hypoxia-regulated, glial-specific, vector-expressing endostatin reduced neovascularization by 93% and reduced the central vaso-obliteration area by 90%, matching the results with the unregulated GFAP-Endo vector. Retinas treated with the regulated endostatin vector expressed substantial amounts of endostatin protein, and significantly reduced VEGF protein. Endostatin production from the regulated vector was undetectable in retinas with undamaged vasculature. CONCLUSIONS: These findings suggest that the hypoxia-regulated, glial cell-specific vector expressing endostatin may be useful for treatment of neovascularization in proliferative diabetic retinopathy.

Cholera toxin induces a shift from inactive to active cyclooxygenase 2 in alveolar macrophages activated by Mycobacterium bovis BCG.

Intranasal vaccination stimulates formation of cyclooxygenases (COX) and release of prostaglandin E(2) (PGE(2)) by lung cells, including alveolar macrophages. PGE(2) plays complex pro- or anti-inflammatory roles in facilitating mucosal immune responses, but the relative contributions of COX-1 and COX-2 remain unclear. Previously, we found that Mycobacterium bovis BCG, a human tuberculosis vaccine, stimulated increased release of PGE(2) by macrophages activated in vitro; in contrast, intranasal BCG activated no PGE(2) release in the lungs, because COX-1 and COX-2 in alveolar macrophages were subcellularly dissociated from the nuclear envelope (NE) and catalytically inactive. This study tested the hypothesis that intranasal administration of BCG with cholera toxin (CT), a mucosal vaccine component, would shift the inactive, NE-dissociated COX-1/COX-2 to active, NE-associated forms. The results showed increased PGE(2) release in the lungs and NE-associated COX-2 in the majority of COX-2(+) macrophages. These COX-2(+) macrophages were the primary source of PGE(2) release in the lungs, since there was only slight enhancement of NE-associated COX-1 and there was no change in COX-1/COX-2 levels in alveolar epithelial cells following treatment with CT and/or BCG. To further understand the effect of CT, we investigated the timing of BCG versus CT administration for in vivo and in vitro macrophage activations. When CT followed BCG treatment, macrophages in vitro had elevated COX-2-mediated PGE(2) release, but macrophages in vivo exhibited less activation of NE-associated COX-2. Our results indicate that inclusion of CT in the intranasal BCG vaccination enhances COX-2-mediated PGE(2) release by alveolar macrophages and further suggest that the effect of CT in vivo is mediated by other lung cells.

Role of PPARγ in COX-2 activation in mycobacterial pulmonary inflammation.

Preliminary studies show that intranasal (i.n.) administration of BCG in mice induces M1 activation of alveolar macrophages (M∅) that increase TNF-α production and cyclooxygenase-2 (COX-2) expression but reduce constitutive peroxisome proliferator-activated receptor gamma (PPARγ) expression. However, COX-2 is catalytically inactive for prostaglandin E(2) release, unlike COX-2 that is active in M1 activation in vitro by BCG. In this study, we determined the role of PPARγ for BCG-induced M1 activation in vivo and in vitro. We found that treatment of mice with GW9662, a PPARγ antagonist, prior to i.n. BCG, partially restored PPARγ expression, and decreased TNF-α production and COX-2 expression. But COX-2 was still inactive. The decreased effects on TNF-α and COX-2 were also observed when alveolar M∅ were treated in vitro with GW9662/BCG, but COX-2 was still active. Our results indicate that PPARγ upregulates M1 activation of alveolar M∅, but inactive COX-2 formation is independent of PPARγ in mycobacterial pulmonary inflammation.

Hypoxia-regulated retinal glial cell-specific promoter for potential gene therapy in disease.

PURPOSE: Retinal Müller cells span the retina and secrete several trophic factors and represent the functional link between blood vessels and neurons, making them attractive targets for gene therapy. Therefore, a hypoxia-regulated, retinal glial cell-specific vector was constructed and tested for its response to hypoxia. METHODS: A hybrid promoter containing domains of human glial fibrillary acidic protein (GFAP) and several hypoxia-responsive and aerobically silenced elements (HRSE) was incorporated separately into plasmid vectors for generation of self-complementary adeno-associated virus. Müller cells trasfected with plasmids or virus were compared with other cell lines using standard METHODS: The mouse model of oxygen-induced retinopathy (OIR) was used to analyze retinas from mice exposed to high oxygen or room air to evaluate the induction of the regulated promoter. RESULTS: The regulated promoter was silenced under aerobic conditions in comparison with unregulated promoter in Müller cells. Hypoxia induced a 12-fold and 16-fold increase in promoter activity in primary Müller cells and human Müller cell lines, respectively. In the OIR model, intravitreal injection of the regulated promoter at postnatal day 7 (P7) resulted in high levels of green fluorescent protein expression only in retinal Müller cells at P17. GFP expression was absent in retinas of mice only exposed to room air. In vivo studies confirm normoxia silencing, hypoxic induction, and cell specificity of the regulated promoter in the mouse retina. CONCLUSIONS: This hypoxia-regulated, retinal glial cell-specific AAV vector provides a platform for gene therapy within regions of retinal hypoxia which are found in diabetic retinopathy and age-related macular degeneration.

Chitin particles induce size-dependent but carbohydrate-independent innate eosinophilia.

Murine Mϕ that phagocytose CMP develop into M1; this response depends on the size and the chemical composition of the particles. In contrast, recent studies concluded that chitin particles induce M2 and eosinophil migration, promoting acquired Th2 immune responses against chitin-containing microbes or allergens. This study examined whether these apparently inconsistent responses to chitin could be induced by variation in the size and chemical composition of the chitin particles. We compared the responses of Mϕ with CMP, LCB, and Sephadex G-100 beads (>40 µm). Beads were given i.p. to WT mice and to mice deficient in a CRTH2, a receptor for the eosinophil chemoattractant PGD(2). In contrast to the M1 activation induced by CMP, i.p. administration of LCB or Sephadex beads induced within 24 h a CRTH2-dependent peritoneal eosinophilia, as well as CRTH2-independent activation of peritoneal Mϕ that expressed Arg I, an M2 phenotype. LCB-induced Mϕ exhibited elevated Arg I and a surface MR, reduced surface TLR2 levels, and no change in the levels of CHI3L1 or IL-10 production. Our results indicate that the effects of chitin in vivo are highly dependent on particle size and that large, nonphagocytosable beads, independent of their chemical composition, induce innate eosinophilia and activate Mϕ expressing several M2, but not M1, phenotypes.

Expression of the diabetes risk gene wolframin (WFS1) in the human retina.

Wolfram syndrome 1 (WFS1, OMIM 222300), a rare genetic disorder characterized by optic nerve atrophy, deafness, diabetes insipidus and diabetes mellitus, is caused by mutations of WFS1, encoding WFS1/wolframin. Non-syndromic WFS1 variants are associated with the risk of diabetes mellitus due to altered function of wolframin in pancreatic islet cells, expanding the importance of wolframin. This study extends a previous report for the monkey retina, using immunohistochemistry to localize wolframin on cryostat and paraffin sections of human retina. In addition, the human retinal pigment epithelial (RPE) cell line termed ARPE-19 and retinas from both pigmented and albino mice were studied to assess wolframin localization. In the human retina, wolframin was expressed in retinal ganglion cells, optic axons and the proximal optic nerve. Wolframin expression in the human retinal pigment epithelium (RPE) was confirmed with intense cytoplasmic labeling in ARPE-19 cells. Strong labeling of the RPE was also found in the albino mouse retina. Cryostat sections of the mouse retina showed a more extended pattern of wolframin labeling, including the inner nuclear layer (INL) and photoreceptor inner segments, confirming the recent report of Kawano et al. [Kawano, J., Tanizawa, Y., Shinoda, K., 2008. Wolfram syndrome 1 (Wfs1) gene expression in the normal mouse visual system. J. Comp. Neurol. 510, 1-23]. Absence of these cells in the human specimens despite the use of human-specific antibodies to wolframin may be related to delayed fixation. Loss of wolframin function in RGCs and the unmyelinated portion of retinal axons could explain optic nerve atrophy in Wolfram Syndrome 1.

Selective degeneration of central photoreceptors after hyperbaric oxygen in normal and metallothionein-knockout mice.

PURPOSE: Metallothioneins (MTs) in the brain and retina are believed to bind metals and reduce free radicals, thereby protecting neurons from oxidative damage. This study was undertaken to investigate whether retinal photoreceptor (PR) cells lacking MTs are more susceptible to hyperbaric oxygen (HBO)-induced cell death in vivo. METHODS: Wild-type (WT) and MT-knockout (MT-KO) mice lacking metallothionein (MT)-1 and MT-2 were exposed to three atmospheres of 100% oxygen for 3 hours, 3 times per week for 1, 3, or 5 weeks. The control animals were not exposed. Histologic analysis of PR viability was performed by counting rows of nuclei in the outer nuclear layer (ONL). Ultrastructure studies verified PR damage. RESULTS: HBO exposure produced a major loss of PR cells in the central retinas of WT and MT-KO mice, with no effect on the peripheral retina even at the longest (5 weeks) exposures. The degree of PR damage and cell death increased with duration of HBO exposure. One week of HBO exposure was insufficient to cause PR death, but tissue damage was observed in the inner and outer segments. At 3 weeks, the rows of PR nuclei in the central retina were significantly reduced by 38% in WT and 28% in MT-KO animals. At 5 weeks, PR loss was identical in WT (34%) and MT-KO (34%) animals and was comparable to that in WT at 3 weeks. CONCLUSIONS: The data suggest that MT-1 and -2 alone are not sufficient for protecting PRs against HBO-induced cell death. The selective degeneration of central PRs may provide clues to mechanisms of oxidative damage in retinal disease.

Robust hypoxia-selective regulation of a retinal pigment epithelium-specific adeno-associated virus vector.

PURPOSE: To develop an hypoxia-regulated retinal pigment epithelium (RPE)-specific adeno-associated virus (AAV) gene transfer platform that exploits hypoxia as a physiologic trigger for an early antiangiogenic treatment strategy directed at arresting neovascularization in the eye. METHODS: Tissue-specific and hypoxia-regulated expression vectors were constructed with tandem combinations of hypoxia responsive elements and aerobically silenced elements (HRSE) that together induce gene expression in hypoxia and suppress it in normoxia. For RPE-specific expression, the HRSE and a (6x) HRE (hypoxia responsive element) oligomer were ligated upstream of the Rpe65 promoter in a pGL3 firefly luciferase plasmid (pGL3-HRSE-6xHRE-Rpe65). The cell specificity of this novel hybrid promoter was tested in human RPE (ARPE-19), human glioblastoma, rat C6 glioma, mouse hippocampal neurons, and human embryonic kidney cell lines. Expression of all cell types in normoxia, and following 40 h hypoxia, was analyzed by dual luciferase assay. After confirmation of its tissue-specificity and hypoxia-inducibility, the hybrid promoter construct was integrated into a replication-deficient AAV delivery system and tested for cell-selectivity and hypoxia-inducible green fluorescent protein (GFP) reporter expression. RESULTS: The HRSE-6xHRE-Rpe65 promoter was highly selective for RPE cells, strongly induced in hypoxia, and similar in activation strength to the cytomegalovirus (CMV) promoter. The AAV.HRSE.6xHRE.Rpe65 vector induced robust GFP expression in hypoxic ARPE-19 cells, but elicited no GFP expression in hypoxia in other cell types or in normoxic ARPE-19 cells. CONCLUSIONS: The hypoxia-regulated, aerobically-silenced RPE-targeting vector forms a platform for focal autoregulated delivery of antiangiogenic agents in hypoxic regions of the RPE. Such autoinitiated therapy provides a means for early intervention in choroidal neovascularization, when it is most sensitive to inhibition.

Dietary modulation of lens zeaxanthin in quail.

Although higher dietary intake of lutein/zeaxanthin has been associated with reduced risk for cataracts, the impact of dietary supplements on lens lutein (L) or zeaxanthin (Z) has not been examined. If higher lens carotenoids do reduce risk for cataract, it would be essential to know whether dietary carotenoids can elevate carotenoids in the adult vertebrate lens. In this study, a covey of Japanese quail were hatched and raised 6 months on carotenoid-deficient diet, then switched to deficient diet supplemented with low or high 3R,3R'-zeaxanthin (5 or 35 mgkg(-1) food) or beta-carotene (50 mgkg(-1) food). Controls included a group of covey-mates that remained on the deficient diet and another raised from birth on the high Z (35 mg Zkg(-1)) diet. At 1 year of age, carotenoids and tocopherols in the lens and in the serum were analysed by HPLC, and compared by analysis of variance. Serum Z was significantly elevated in deficient birds fed the lower or higher Z supplement for 6 months (P<0.0001 for each). Serum Z in birds maintained on the higher Z supplement for 1 year was much higher than that in deficient birds (P<0.0001), but not different from deficient birds given the higher Z supplement. As in humans, the predominant lens carotenoids were lutein (L) and zeaxanthin (Z), and the total carotenoid concentration was of lower magnitude than the concentration of alpha-tocopherol. Responses to Z supplementation were sex-related. Female quail had 5-10 times higher serum concentrations of both Z and L than males (P<0.0001, <0.001), and they also had higher lens Z concentrations than males (P<0.0006); possible effects of estrogen on lens carotenoids are discussed. Lens Z concentration was strongly and positively correlated with serum Z in females (r=0.77; P<0.002). Deficient adult females supplemented with the 35 mgkg(-1) dose of Z for 6 months had a mean lens Z concentration (0.252+/-0.06 microgg(-1) protein) close to that in females fed with the supplement from birth (0.282+/-0.15 microgg(-1) protein). Birds fed with the higher dietary Z supplement for 6 or 12 months had significantly higher lens Z than birds fed lower or no dietary Z (P<0.0001). Lens L was not altered by dietary supplementation with either Z or beta-carotene. beta-Carotene supplements did not result in detectable lens beta-carotene, and had no effect on lens Z. Neither Z nor beta-carotene supplementation had a significant effect on serum or lens tocopherol concentrations. These studies in quail provide the first experimental evidence that lens carotenoids in adult vertebrates can be manipulated by dietary Z supplements.

Adenoviral-mediated gene transfer to retinal explants during development and degeneration.

Naturally occurring mutations of the beta subunit of the cyclic guanosine monophosphate (cGMP) phosphodiesterase (beta-PDE) gene in rod photoreceptors of mice and dogs are similar to one of the inherited retinal degenerations termed retinitis pigmentosa in humans. Defects in the rod beta-PDE gene leading to photoreceptor cell degeneration in retinal degenerative (rd) mice can be corrected by transfer of a wild type beta-PDE gene. However, the rapid photoreceptor degeneration in this mutant makes the study of gene therapy difficult. Since the retinal degeneration is slowed in vitro, we have employed retinal explants from rd mice to study factors influencing viral transduction. Retinal explants provide a rapid, efficient method to compare the transduction efficiency of adenoviral vector-mediated reporter gene delivery at different ages in normal and rd mice. Retinal explants from postnatal day (P)2 to P28 control (C57BL/6J) and P2-P42 rd mice were exposed for 20 hr to 2.5 x 10(8) plaque forming units (pfu) ml(-1) of adenoviral vector with a beta-galactosidase (Lac Z) reporter gene (Ad-CMV-Lac Z). After incubation in vector-free media for an additional 3 days, the explants were fixed and histochemically stained for beta-galactosidase to reveal Lac Z gene expression. The explants were also embedded and sectioned for light microscopic observation. Transduction efficiency was higher in rd mice than in controls on all postnatal days examined. In normal retinal explants, expression of the Lac Z gene increased from P2 to a peak around P7-P8, then decreased at subsequent ages; little transduction could be found after P17. In rd mice transduction efficiency of Ad-CMV-Lac Z increased from P2 to P7, decreased by P10 and increased again after P10. The most dramatic increase in the transduction efficiency occurred in the rd retina between P10 and P15 when Lac Z was intensely expressed throughout the retina. Microscopic examination of retinal sections revealed the types and distribution of Lac Z-positive cells responsible for the deep blue staining in the retinal whole mount. In normal and rd mice, Lac Z-positive cells were located throughout the retina. However, larger numbers of Lac Z-positive cells were present at all ages examined in retinal explants from rd mice compared to normal mice. These data indicate a difference in transduction efficiency between normal and rd mice, especially after P12, and suggest efficient adenovirus-mediated gene transfer is more attainable in developing or degenerating retina. Thus, transduction efficiency in rd mice depends on the relationship between development, maturation and the degenerative state of the photoreceptor cells.

Long term dietary supplementation with zeaxanthin reduces photoreceptor death in light-damaged Japanese quail.

The purpose of these studies was to evaluate the effects of light damage on Japanese quail whose retinal carotenoids had been experimentally manipulated through altered diets. The birds were raised 6 months on a commercial turkey diet (T), on a custom carotenoid-deficient diet (C-) containing 90% less carotenoid than the T diet, or on Z+ diet [the C- diet supplemented with natural zeaxanthin (35mgkg(-1) food)]. Equal numbers of males and females on each diet were exposed to nine intervals (1hr on, 2hr off) of 3200lux cool white light, then placed in the dark for 14hr before tissue collection. One retina was immediately frozen for HPLC analysis; the other eye was immediately fixed and processed for microscopy. There were no significant differences in the retinal carotenoid concentrations in hatch-mates that were and were not exposed to light. Supplementation resulted in three- to four-fold increases in retinal zeaxanthin and no change in retinal lutein or alpha-tocopherol, but the C- diet did not reduce the retinal carotenoid concentration in C- birds below that in T birds. The light-exposed retinas had significant numbers of apoptotic photoreceptors and photoreceptor ghosts. The number of ghosts was negatively correlated with the number of dying photoreceptors (P<0.05), and with retinal concentrations of zeaxanthin, alpha-tocopherol or gamma-tocopherol (P<0.04, 0.02, 0.04, respectively), but not with lutein. The number of dying photoreceptors was positively correlated with alpha-tocopherol and the sum alpha-tocopherol plus zeaxanthin (P<0.1; P0.04). Photoreceptor death was semi-quantitatively scored, assuming that ghosts were formed by removal of apoptotic photoreceptors with nuclear condensation. Stepwise regression produced a good model (r(2)=0.67;P <0.0001) for predicting death scores from retinal concentrations of zeaxanthin (Standard Coefficient=-0.76) and lutein (Standard Coefficients=+0.43). Absence of lutein in gender-specific analyses suggests lutein served as surrogate marker for gender. Combined analysis of the C- and T birds also demonstrated that dying photoreceptors were negatively correlated with retinal zeaxanthin. These data confirm our previous report that retinal carotenoids prevent photoreceptor cell death, and provide the first direct evidence that retinal zeaxanthin protects photoreceptors from light-induced death.

Elevated retinal zeaxanthin and prevention of light-induced photoreceptor cell death in quail.

PURPOSE: Inferential evidence indicates that macular pigments (lutein and zeaxanthin) protect photoreceptors and/or retard age-related macular degeneration. These experiments tested the hypothesis that retinal zeaxanthin prevents light-induced photoreceptor cell death. METHODS: Retinal damage was assessed in quail fed a carotenoid-deficient (C-) diet for 6 months. Groups of 16 birds (8 male, 8 female) were fed a C- diet supplemented with 35 mg 3R,3'R-zeaxanthin for 1, 3, or 7 days; one group was continued on C- diets. Half of each group was exposed to intermittent 3200-lux white light (10 1-hour intervals separated by 2 hours in dark). After 14 additional hours in the dark, one retina of each quail was collected for HPLC analysis, and the contralateral retina was embedded in paraffin for counts of apoptotic nuclei. RESULTS: After 7 days' supplementation, concentrations of zeaxanthin in serum, liver, and fat had increased by factors of 50.8, 43.2, and 6.5, respectively (all P < 0.001). In contrast, retinal zeaxanthin fluctuated significantly upward on day 3, but there was no net change on day 7. The number of apoptotic rods and cones in light-damaged eyes correlated significantly and inversely with zeaxanthin concentration in the contralateral retina (r = -0.61; P < 0.0001 and r = -0.54; P < 0.002), but not with serum zeaxanthin. Similar correlations were observed with retinal lutein, which correlated strongly with retinal zeaxanthin (r = 0.95; P < 0.0001). CONCLUSIONS: Retinal zeaxanthin dose dependently reduced light-induced photoreceptor apoptosis; elevated serum levels did not. These data provide the first experimental evidence that xanthophyll carotenoids protect photoreceptors in vivo.

Effect of dietary zeaxanthin on tissue distribution of zeaxanthin and lutein in quail.

PURPOSE: The xanthophyll carotenoids (lutein and zeaxanthin) are hypothesized to delay progression of age-related macular degeneration. The quail has a cone-dominant retina that accumulates carotenoids. The purpose of these experiments was to characterize the carotenoid composition of retina, serum, liver, and fat in quail and to determine whether dietary enrichment with zeaxanthin alters zeaxanthin or lutein concentrations in these tissues. METHODS: Quail were fed for 6 months with a commercial turkey diet (T group; n = 8), carotenoid-deficient diet (C- group; n = 8), or a carotenoid-deficient diet supplemented with 35 mg 3R,3'R-zeaxanthin per kilogram of food, (Z+ group; n = 8). Zeaxanthin was derived from Sphingobacterium multivorum (basonym Flavobacterium). Carotenoids in serum, retina, liver, and fat were analyzed by HPLC. RESULTS: As in the primate fovea, the retina accumulated zeaxanthin, lutein, and cryptoxanthin, and preferentially absorbed zeaxanthin (P < 0.005). In contrast, lutein was preferentially absorbed by liver (P < 0.01) and fat (P < 0.0001). In supplemented females, zeaxanthin increased approximately 4-fold in retina, and 74-, 63- and 22-fold in serum, liver, and fat, respectively. In males, zeaxanthin was elevated approximately 3-fold in retina, and 42-, 17-, and 12-fold in serum, liver, and fat, respectively. Birds fed the Z+ diet absorbed a higher fraction of dietary lutein into serum, but lutein was reduced in the retina (P < 0.05). CONCLUSIONS: Xanthophyll profiles in quail mimic those in primates. Dietary supplements of zeaxanthin effectively increased zeaxanthin concentrations in serum, retina, liver, and fat. The robust response to zeaxanthin supplementation identifies the quail as an animal model for exploration of factors regulating delivery of dietary carotenoids to the retina.

Lipid hydroperoxide-induced tumor necrosis factor (TNF)-alpha, vascular endothelial growth factor and neovascularization in the rabbit cornea: effect of TNF inhibition.

Lipid hydroperoxides (LHP) at high concentrations are cytotoxic, but at sublethal concentration, they induce synthesis of cytokine vascular growth factors. Intracorneal injections of 30 µg LHP placed 5 mm from the superior limbus stimulated early vasodilation of limbal vasculature and a rapidly developing, sustained neovascularization. Under these conditions, vessels grew at the rate of 0.3 mm/day to a total length of 7.5 mm, 25 days after injection. Cholesterol peroxides were less effective. Developing vessels were oriented towards the stimulus. Around the developing vessel there was dissolution of the stromal extracellular matrix. The most distal endothelial cells displayed prominent endoplasmic reticulum, a lack of basement membrane or tight junction complexes and leakage of fluorescein dye. Both the injection site and superior quadrant showed increased levels of tumor necrosis factor (TNF)-alpha and vascular endothelial growth factor after exposure to LHP. The neovascular response was inhibited by simultaneous administration of TNF-alpha antibody or pentoxifylline, an inhibitor of TNF-alpha synthesis. This corneal model of peroxide-induced neovascularization should prove useful for temporal studies of events in the initiation and propagation of signals leading to neovascularization, and for evaluating effects of treatment on neovascular growth.