Lipids, Lipoproteins, and Age-Related Macular Degeneration

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. While excellent treatment has emerged for neovascular disease, treatment for early AMD is lacking due to an incomplete understanding of the early molecular events. A prominent age-related change is the accumulation of neutral lipid in normal Bruch's membrane (BrM) throughout adulthood and also disease-related BrM accumulations called basal deposits and drusen. AMD lesion formation has thus been conceptualized as sharing mechanisms with atherosclerotic plaque formation, where low-density lipoprotein (LDL) retention within the arterial wall initiates a cascade of pathologic events. However, we do not yet understand how lipoproteins contribute to AMD. This paper explores how systemic and local production of lipoproteins might contribute to the pathogenesis of AMD

Role of the choroid in age-related macular degeneration: A current review

Age-related macular degeneration (AMD) is a major cause of vision loss in the developed world and its pathogenesis is a topic of active research. To date, much study has been focused on the role of the retinal pigment epithelium (RPE) and Bruch's membrane (BrM) in AMD pathogenesis, but the role of the choroid has also been investigated. In this review, we focus on recent advancements in research in the role of the choroid in AMD, beginning with an exploration of the histopathologic, cellular and molecular changes that occur in the choroid in AMD and concluding by discussing new choroidal imaging techniques and patterns seen on fluorescein angiography, indocyanine green angiography, spectral-domain optical coherence tomography and optical coherence tomography angiography. Exploring these domains will lead to a better understanding of the factors at play beyond the outer retina in this important disease

Abstract 1415: The Notch ligand Jag 2 promotes growth and invasion in uveal melanoma cells

Abstract Uveal melanoma is the most common primary intraocular cancer in the adults, and up to 50% of patients die from hematogenous visceral metastases which are extremely resistant to chemotherapy. Using oligonucleotide expression array analysis of mRNA from primary uveal melanomas, we found that the Jag 2 ligand was 1.9 fold more expressed in tumors which metastasized than those which did not. Other Notch pathway members were also significantly induced in the metastatic cohort. We then used quantitative RT PCR to analyze mRNA extracted from a second cohort of 30 snap-frozen primary tumors, and confirmed that elevated Jag 2 mRNA levels were significantly associated (p = 0.048) with the metastatic Class 2 signature as compared to non-metastatic Class 1 uveal melanoma. To directly examine the importance of Jag 2 in driving cellular growth and invasion, we introduced the ligand into a uveal melanoma cell line, Mel 290, which has low baseline levels of Notch activity as measured by expression of the pathway targets Hes1, Hey1 and Hey2. The Jag 2-GFP-MSCV-infected cells showed a 2.5 to 6 fold induction of Notch targets Hey 1 and Hes1, indicating that pathway activity had been induced. While overall growth of the Jag 2-GFP-MSCV infected cultures as measured by MTT increased only slightly, overexpression of Jag 2 induced an approximately 3 fold increase in clonogenic growth in soft agar. Finally, we assayed the migratory properties of the cells using a wound-healing (“scratch”) assay, as well as their ability to invade Matrigel. Jag 2 expressing cells showed increased motility in both of these tests, with a significant (p = 0.0003) approximately 2 fold induction of transwell invasion capacity. Our data suggest that expression of the Notch ligand Jag 2 may play an important role in uveal melanoma growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1415. doi:10.1158/1538-7445.AM2011-1415</jats:p

T cells and macrophages responding to oxidative damage cooperate in pathogenesis of a mouse model of age-related macular degeneration.

Age-related macular degeneration (AMD) is a major disease affecting central vision, but the pathogenic mechanisms are not fully understood. Using a mouse model, we examined the relationship of two factors implicated in AMD development: oxidative stress and the immune system. Carboxyethylpyrrole (CEP) is a lipid peroxidation product associated with AMD in humans and AMD-like pathology in mice. Previously, we demonstrated that CEP immunization leads to retinal infiltration of pro-inflammatory M1 macrophages before overt retinal degeneration. Here, we provide direct and indirect mechanisms for the effect of CEP on macrophages, and show for the first time that antigen-specific T cells play a leading role in AMD pathogenesis. In vitro, CEP directly induced M1 macrophage polarization and production of M1-related factors by retinal pigment epithelial (RPE) cells. In vivo, CEP eye injections in mice induced acute pro-inflammatory gene expression in the retina and human AMD eyes showed distinctively diffuse CEP immunolabeling within RPE cells. Importantly, interferon-gamma (IFN-γ) and interleukin-17 (IL-17)-producing CEP-specific T cells were identified ex vivo after CEP immunization and promoted M1 polarization in co-culture experiments. Finally, T cell immunosuppressive therapy inhibited CEP-mediated pathology. These data indicate that T cells and M1 macrophages activated by oxidative damage cooperate in AMD pathogenesis

Notch Signaling Promotes Growth and Invasion in Uveal Melanoma

PURPOSE: To determine whether uveal melanoma, the most common primary intraocular malignancy in adults, requires Notch activity for growth and metastasis. EXPERIMENTAL DESIGN: Expression of Notch pathway members was characterized in primary tumor samples and in cell lines, along with the effects of Notch inhibition or activation on tumor growth and invasion. RESULTS: Notch receptors, ligands, and targets were expressed in all five cell lines examined and in 30 primary uveal melanoma samples. Interestingly, the three lines with high levels of baseline pathway activity (OCM1, OCM3, and OCM8) had their growth reduced by pharmacologic Notch blockade using the γ-secretase inhibitor (GSI) MRK003. In contrast, two uveal melanoma lines (Mel285 and Mel290) with very low expression of Notch targets were insensitive to the GSI. Constitutively active forms of Notch1 and Notch2 promoted growth of uveal melanoma cultures and were able to rescue the inhibitory effects of GSI. MRK003 treatment also inhibited anchorage-independent clonogenic growth and cell invasion and reduced phosphorylation levels of STAT3 and extracellular signal-regulated kinase (Erk)1/2. Suppression of canonical Notch activity using short hairpin RNA targeting Notch2 or CBF1 was also able to reduce tumor growth and invasion. Finally, intraocular xenograft growth was significantly decreased by GSI treatment. CONCLUSION: Our findings suggest that Notch plays an important role in inducing proliferation and invasion in uveal melanoma and that inhibiting this pathway may be effective in preventing tumor growth and metastasis

CEP immunolabeling in normal healthy eyes and dry AMD eyes.

<p>(<b>A</b>) Macula from a 77 yo unaffected female with CEP immunolabeling (blue) in the ganglion cell layer (GCL), inner plexiform layer (IPL), inner nuclear layer (INL), and photoreceptor outer segments (POS). Minimal labeling is seen in the RPE. Thin immunolabeling is seen at the RPE basement membrane. (<b>B</b>) Magnified view of the region labeled with red asterisks in A. POS are prominently labeled, especially cones (c). CEP immunolabeling at the basal RPE in inner Bruch’s membrane is more obvious. (<b>C</b>) Macula from a 51 yo male with dry (non-neovascular) AMD with CEP immunolabeling in the nerve fiber layer and GCL, IPL, INL, and photoreceptors. Labeling in photoreceptors is more prominent in the inner regions than in the POS. The RPE is diffusely labeled. (<b>D</b>) Magnified view of the region labeled with red asterisks in C shows diffusely labeled RPE. A prominent drusen is minimally labeled for CEP. (<b>E</b>) 69 yo female with non-neovascular AMD. The RPE cells overlying a large drusen are diffusely labeled for CEP. The drusen has a speckled labeling pattern for CEP. (<b>F</b>) 83 yo female with dry AMD. The drusen has prominent CEP immunolabeling with diffuse labeling within the overlying RPE. (<b>G</b>) IgG control from a 61 yo male with non-neovascular AMD. Ch, choroid; Bar = 25 µm.</p

Proof of concept experiment showing that T cell inhibitory drugs Cyclosporin A and Rapamycin prevent CEP-induced retinal pathology.

<p>(<b>A</b>) WT BALB/c mice were immunized with CEP-MSA and treated every day (starting at day of immunization) with combined CsA+Rapa for either 21 or 40 days p.i. Control PBS injections were also performed. Serum was isolated at day 47 p.i. and anti-CEP titers were evaluated by ELISA. (<b>B</b>) Eyes were harvested at day 60 p.i. Retinal pathology scores for the indicated groups are shown (n = 3). ANOVA was used for statistical analysis (* denotes p<0.05). Results are representative of two independent experiments.</p

CEP directly activates macrophages <i>in</i><i>vitro</i>, leading to M1 polarization.

<p>(<b>A</b>) Bone marrow-derived macrophages (BMDM) from BALB/c mice were stimulated for 4 hrs with CEP-MSA (100 µg/ml), Sham-MSA (100 µg/ml) or left untreated. RNA was isolated and qPCR was used for gene expression analysis. Each treatment is represented as relative-expression (i.e., fold-expression over reference group), where the control (untreated) sample served as the reference with a set value of 1. CEP specifically induced the expression of M1 markers (iNOS, IL-1β, TNF-α and IL-12A) but had no effect on M2 or Mox marker genes (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0088201#pone.0088201.s001" target="_blank">Figure S1</a>). (<b>B</b>) BMDM from C57BL/6 mice were stimulated and analyzed as described above. Data from 4 independent experiments were pooled and two-tailed Student’s t tests were used for statistical analysis (* denotes p<0.05); error bars represent S.D.</p

Working model for the cooperation of antigen-specific T cells and M1 macrophages in the development of AMD.

<p>Oxidative damage in the outer retina initiates a cascade of events that activate both innate and adaptive immune responses that ultimately lead to retinal damage.</p