Mechanism for the selective uptake of macular carotenoids mediated by the HDL cholesterol receptor SR-BI.

The macular carotenoids lutein and zeaxanthin are taken up from the bloodstream into the human retina through a selective process, for which the HDL cholesterol receptor scavenger receptor BI (SR-BI) in the cells of retinal pigment epithelium (RPE) is thought to be a key mediator. However, the mechanism of SR-BI-mediated selective uptake of macular carotenoids is still not fully understood. Here, we investigate possible mechanisms using biological assays and cultured HEK293 cells, a cell line without endogenous SR-BI expression. Binding affinities between SR-BI and various carotenoids were measured by surface plasmon resonance (SPR) spectroscopy, which shows that SR-BI cannot bind lutein or zeaxanthin specifically. Overexpression of SR-BI in HEK293 cells results in more lutein and zeaxanthin taken up than ß-carotene, and this effect can be eliminated by an SR-BI mutant (C384Y) whose cholesterol uptake tunnel is blocked. Next, we determined the effects of HDL and hepatic lipase (LIPC), SR-BI's partners in HDL cholesterol transport, on SR-BI-mediated carotenoid uptake. HDL addition dramatically reduced lutein, zeaxanthin, and ß-carotene in HEK293 cells expressing SR-BI, but the cellular lutein and zeaxanthin are higher than ß-carotene. LIPC addition increases the uptake of all three carotenoids in HDL-treated cells, and promotes the transport of lutein and zeaxanthin better than ß-carotene. Our results suggest that SR-BI and its HDL cholesterol partner HDL and LIPC may be involved in the selective uptake of macular carotenoids.

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.

Retinal bioavailability and functional effects of a synthetic very-long-chain polyunsaturated fatty acid in mice.

Rare, nondietary very-long-chain polyunsaturated fatty acids (VLC-PUFAs) are uniquely found in the retina and a few other vertebrate tissues. These special fatty acids play a clinically significant role in retinal degeneration and development, but their physiological and interventional research has been hampered because pure VLC-PUFAs are scarce. We hypothesize that if Stargardt-3 or age-related macular degeneration patients were to consume an adequate amount of VLC-PUFAs that could be directly used in the retina, it may be possible to bypass the steps of lipid elongation mediated by the retina's ELOVL4 enzyme and to delay or prevent degeneration. We report the synthesis of a VLC-PUFA (32:6 n-3) in sufficient quantity to study its bioavailability and functional benefits in the mouse retina. We acutely and chronically gavage fed wild-type mice and Elovl4 rod-cone conditional knockout mice this synthetic VLC-PUFA to understand its bioavailability and its role in visual function. VLC-PUFA-fed wild-type and Elovl4 conditional knockout mice show a significant increase in retinal VLC-PUFA levels in comparison to controls. The VLC-PUFA-fed mice also had improvement in the animals' visual acuity and electroretinography measurements. Further studies with synthetic VLC-PUFAs will continue to expand our understanding of the physiological roles of these unique retinal lipids, particularly with respect to their potential utility for the treatment and prevention of retinal degenerative diseases.

Imaging lutein and zeaxanthin in the human retina with confocal resonance Raman microscopy.

Lutein and zeaxanthin are xanthophyll carotenoids that are highly concentrated in the human macula, where they protect the eye from oxidative damage and improve visual performance. Distinguishing lutein from zeaxanthin in images of the human retina in vivo or in donor eye tissues has been challenging because no available technology has been able to reliably differentiate between these two carotenoids, which differ only in the position of one C = C bond. Here, we report the differential distributions of lutein and zeaxanthin in human donor retinas mapped with confocal resonance Raman microscopy. Zeaxanthin is highly concentrated in the fovea, extending from the inner to the outer limiting membranes, with especially high concentrations in the outer plexiform layer, while lutein is much more diffuse at relatively lower concentration. Our results imply that zeaxanthin may play a more important role than lutein in human macular health and disease.

Expression of AHI1 Rescues Amyloidogenic Pathology in Alzheimer's Disease Model Cells.

A hallmark of Alzheimer's disease (AD) pathogenesis is the accumulation of extracellular plaques mainly composed of amyloid-ß (Aß) derived from amyloid precursor protein (APP) cleavage. Recent reports suggest that transport of APP in vesicles with huntingtin-associated protein-1 (HAP1) negatively regulates Aß production. In neurons, HAP1 forms a stable complex with Abelson helper integration site-1 (AHI1), in which mutations cause neurodevelopmental and psychiatric disorders. HAP1 and AHI1 interact with tropomyosin receptor kinases (Trks), which are also associated with APP and mediate neurotrophic signaling. In this study, we hypothesize that AHI1 participates in APP trafficking and processing to rescue AD pathology. Indeed, AHI1 was significantly reduced in mouse neuroblastoma N2a cells expressing human Swedish and Indiana APP (designed as AD model cells) and in 3xTg-AD mouse brain. The AD model cells as well as Ahi1-knockdown cells expressing wild-type APP-695 exhibited a significant reduction in viability. In addition, the AD model cells were reduced in neurite outgrowth. APP C-terminal fragment-ß (CTFß) and Aß42 were increased in the AD cell lysates and the culture media, respectively. To investigate the mechanism how AHI1 alters APP activities, we overexpressed human AHI1 in the AD model cells. The results showed that AHI1 interacted with APP physically in mouse brain and transfected N2a cells despite APP genotypes. AHI1 expression facilitated intracellular translocation of APP and inhibited APP amyloidogenic process to reduce the level of APP-CTFß in the total lysates of AD model cells as well as Aß in the culture media. Consequently, AHI1-APP interactions enhanced neurotrophic signaling through Erk activation and led to restored cell survival and differentiation.

n-3 PUFA Supplementation Alters Retinal Very-Long-Chain-PUFA Levels and Ratios in Diabetic Animal Models.

SCOPE: Long-chain (LC)-PUFAs act as precursors for the special class of retinal lipids known as very-long-chain (VLC)-PUFAs and the effect of diabetes on retinal VLC-PUFA levels is unexplored. In order to understand the supplemental effect of omega-3 (n-3) LC-PUFAs on decreasing levels of VLC-PUFAs due to diabetes, Nile rats, which develop diabetes spontaneously, and Akita mouse, a genetic diabetes model, are chosen. METHODS AND RESULTS: Human retinal punches from donors are collected from an eye bank; lipids are extracted and analyzed to study the alterations in VLC-PUFAs and their omega-3/omega-6 (n-3/n-6) ratios. Nile rats are fed a high-fat diet to induce hyperglycemia, and then an n-3 PUFA-rich diet is fed to the experimental group for 2 months. Diabetic male Akita mice and WT mice are fed with 5% fish-oil mixed in with their chow for 2 months to observe the effect of n-3 PUFAs. Results indicate that VLC-PUFA levels are lower in human diabetic and retinopathic retinal punches compared to age-matched controls. With supplementation of n-3 PUFAs, there is a significant increase in n-3/n-6 VLC-PUFA ratios in both animal models compared to diabetic controls. CONCLUSION: Dietary supplementation with n-3 LC-PUFAs helps to prevent progression of diabetes and associated retinopathy.

Supplementation with macular carotenoids improves visual performance of transgenic mice.

Carotenoid supplementation can improve human visual performance, but there is still no validated rodent model to test their effects on visual function in laboratory animals. We recently showed that mice deficient in ß-carotene oxygenase 2 (BCO2) and/or ß-carotene oxygenase 1 (BCO1) enzymes can accumulate carotenoids in their retinas, allowing us to investigate the effects of carotenoids on the visual performance of mice. Using OptoMotry, a device to measure visual function in rodents, we examined the effect of zeaxanthin, lutein, and ß-carotene on visual performance of various BCO knockout mice. We then transgenically expressed the human zeaxanthin-binding protein GSTP1 (hGSTP1) in the rods of bco2-/- mice to examine if delivering more zeaxanthin to retina will improve their visual function further. The visual performance of bco2-/- mice fed with zeaxanthin or lutein was significantly improved relative to control mice fed with placebo beadlets. ß-Carotene had no significant effect in bco2-/- mice but modestly improved cone visual function of bco1-/- mice. Expression of hGSTP1 in the rods of bco2-/-mice resulted in a 40% increase of retinal zeaxanthin and further improvement of visual performance. This work demonstrates that these "macular pigment mice" may serve as animal models to study carotenoid function in the retina.

Development and characterization of eucalyptol microemulsions for topic delivery of curcumin.

Microemulsions have received great attention for applications in transdermal drug delivery. The use of curcumin for treating various skin diseases like scleroderma, psoriasis, and skin cancer was extensively reported. The solubility of curcumin in various oils, surfactants, and cosurfactants was studied herein in order to find the optimal components for a transdermal delivery vehicle. Microemulsion systems composed of eucalyptol, polysorbate 80, ethanol, and water were developed as transdermal delivery vehicles for curcumin. Effects of the microemulsion composition on transdermal curcumin delivery were studied using Franz diffusion cells. The transdermal curcumin flux, permeability coefficient, and enhancement ratio were analyzed to evaluate the effects of eucalyptol/water ratios in the microemulsions. Pseudo-ternary phase diagrams of the eucalyptol microemulsions with various surfactant/cosurfactant ratios (1:1-1:3) were constructed to investigate their phase behaviors. Conductivity, interfacial tension, size, and viscosity data of the microemulsions were used to characterize the physicochemical properties of transdermal vehicles. The influence of the microemulsions on skin histology and on the delivery route was analyzed using hematoxylin/eosin staining and confocal laser scanning microscopy. In conclusion, microemulsions were successfully developed for transdermal curcumin delivery after screening various components and adjusting the oil/water ratios. The curcumin permeation rate of the microemulsion developed was 15.7-fold higher than that of the control (eucalyptol only). These results indicate that an eucalyptol microemulsion system is a promising tool for the percutaneous delivery of curcumin.

Terpene microemulsions for transdermal curcumin delivery: effects of terpenes and cosurfactants.

Microemulsion systems composed of terpenes, polysorbate 80, cosurfactants, and water were investigated as transdermal delivery vehicles for curcumin. Pseudoternary phase diagrams of three terpenes (limonene, 1,8-cineole, and α-terpineol) at a constant surfactant/cosurfactant ratio (1:1) were constructed to illustrate their phase behaviors. Limonene combined with cosurfactants like ethanol, isopropanol, and propylene glycol were employed as microemulsion ingredients to study their potential for transdermal curcumin delivery. The transdermal delivery efficacy and skin retention of curcumin were evaluated using neonate pig skin mounted on a Franz diffusion cell. The curcumin permeation rates in the limonene microemulsion studied were 30- and 44-fold higher than those of 1,8-cineole and α-terpineol microemulsions, respectively. Significant effects on the skin permeation rates were observed from microemulsions containing different limonene/water contents. Histological examination of treated skin was performed to investigate the change of skin morphologies. Characteristics such as droplet size, conductivity, interfacial tension, and viscosity were analyzed to understand the physicochemical properties of the transdermal microemulsions. In conclusion, microemulsions loaded with curcumin were successfully optimized for transdermal delivery after screening various terpenes, cosurfactants, and limonene/water ratios. These results indicate that the limonene microemulsion system is a promising tool for the percutaneous delivery of curcumin.