Shira Hagbi-Levi

Impaired Cholesterol Efflux in Senescent Macrophages Promotes Age-Related Macular Degeneration

Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer, and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.

Chemokine receptor expression in peripheral blood monocytes from patients with neovascular age-related macular degeneration.

PURPOSE Chemokine signaling and monocytes/macrophages were implicated in the pathogenesis of AMD. We tested the association between chemokines involved in monocyte recruitment and AMD. METHODS Immunophenotyping for white blood cell (WBC) populations including CD14++CD16- and CD14+CD16+ monocytes, CD19+, CD3+, and CD16+ lymphocytes, and chemokine receptors CCR1, CCR2, CCR5, CX(3)CR1, and CXCR4 was performed on peripheral blood from treatment-naïve neovascular AMD (NV-AMD) patients and controls. The mRNA level of chemokine receptors in monocytes was measured with quantitative-PCR. Systemic levels of major chemokine ligands CCL2, CCL5, CCL3, and CXCL10 were evaluated by ELISA. Genotyping was performed for risk SNPs for AMD in the CFH, C3, and HTRA1 genes. RESULTS The percentage of WBC subpopulations tested was similar between NV-AMD patients (n = 18) and controls (n = 20). CD14+CD16+ monocyte subpopulation showed a 3.5-fold increased expression of CCR1 (P = 0.039; t-test) and a 2.2-fold increased expression of CCR2 (P = 0.027) in patients compared with controls. Increased CCR1 and CCR2 expression was correlated with each other in patients (R(2) = 0.64, P < 0.0001), but not controls (R(2) = 0.02, P = 0.57). Increased mRNA levels of CCR1 (1.6-fold, P = 0.037) and CCR2 (1.6-fold, P = 0.007) were found in monocytes from NV-AMD patients. Chemokine receptor expression was not correlated with the presence of risk SNPs, and was not associated with blood chemokine levels. CONCLUSIONS CCR1 and CCR2 are coupregulated on the CD14+CD16+ monocyte population in NV-AMD patients. These data implicate CD14+CD16+ monocytes and chemokine signaling in AMD. Additional investigation is needed to elucidate the role of these monocytes and their potential as a biomarker or therapeutic target for AMD.

The role of monocytes and macrophages in age-related macular degeneration.

White blood cells, particularly monocytes and their descendants, macrophages, have been implicated in age-related macular degeneration (AMD) pathology. In this minireview, we describe the current knowledge of monocyte and macrophage involvement in AMD. Chemokine receptors present on these cells such as CCR1, CCR2, and CX3CR1, and their roles in monocyte/macrophage recruitment to sites of injury and inflammation in the context of AMD will be reviewed. Mice models for perturbation of chemokine receptors that recapitulate some of the features of AMD are also described. The body of evidence from human and rodent studies at this point in time suggests that monocyte and macrophages may modulate the course of AMD.

Transcriptome Analysis on Monocytes from Patients with Neovascular Age-Related Macular Degeneration

Mononuclear phagocytes (MPs), including monocytes/macrophages, play complex roles in age-related macular degeneration (AMD) pathogenesis. We reported altered gene-expression signature in peripheral blood mononuclear cells from AMD patients, and a chemokine receptor signature on AMD monocytes. To obtain comprehensive understanding of MP involvement, particularly in peripheral circulation in AMD, we performed global gene expression analysis in monocytes. We separated monocytes from treatment-naïve neovascular AMD (nvAMD) patients (n = 14) and age-matched controls (n = 15), and performed microarray and bioinformatics analysis. Quantitative real-time PCR was performed on other sets of nvAMD (n = 25), atrophic AMD (n = 21), and controls (n = 28) for validation. This validated microarray genes (like TMEM176A/B and FOSB) tested, including differences between nvAMD and atrophic AMD. We identified 2,165 differentially-expressed genes (P < 0.05), including 79 genes with log2 fold change ≥1.5 between nvAMD and controls. Functional annotation using DAVID and TANGO demonstrated immune response alterations in AMD monocytes (FDR-P <0.05), validated by randomized data comparison (P < 0.0001). GSEA, ISMARA, and MEME analysis found immune enrichment and specific involved microRNAs. Enrichment of differentially-expressed genes in monocytes was found in retina via SAGE data-mining. These genes were enriched in non-classical vs. classical monocyte subsets (P < 0.05). Therefore, global gene expression analysis in AMD monocytes reveals an altered immune-related signature, further implicating systemic MP activation in AMD.

Degeneration Modulates Retinal Response to Transient Exogenous Oxidative Injury

Purpose Oxidative injury is involved in retinal and macular degeneration. We aim to assess if retinal degeneration associated with genetic defect modulates the retinal threshold for encountering additional oxidative challenges. Methods Retinal oxidative injury was induced in degenerating retinas (rd10) and in control mice (WT) by intravitreal injections of paraquat (PQ). Retinal function and structure was evaluated by electroretinogram (ERG) and histology, respectively. Oxidative injury was assessed by immunohistochemistry for 4-Hydroxy-2-nonenal (HNE), and by Thiobarbituric Acid Reactive Substances (TBARS) and protein carbonyl content (PCC) assays. Anti-oxidant mechanism was assessed by quantitative real time PCR (QPCR) for mRNA of antioxidant genes and genes related to iron metabolism, and by catalase activity assay. Results Three days following PQ injections (1 µl of 0.25, 0.75, and 2 mM) the average ERG amplitudes decreased more in the WT mice compared with the rd10 mice. For example, following 2 mM PQ injection, ERG amplitudes reduced 1.84-fold more in WT compared with rd10 mice (p = 0.02). Injection of 4 mM PQ resulted in retinal destruction. Altered retina morphology associated with PQ was substantially more severe in WT eyes compared with rd10 eyes. Oxidative injury according to HNE staining and TBARS assay increased 1.3-fold and 2.1-fold more, respectively, in WT compared with rd10 mice. At baseline, prior to PQ injection, mRNA levels of antioxidant genes (Superoxide Dismutase1, Glutathione Peroxidase1, Catalase) and of Transferrin measured by quantitative PCR were 2.1–7.8-fold higher in rd10 compared with WT mice (p<0.01 each), and catalase activity was 1.7-fold higher in rd10 (p = 0.0006). Conclusions This data suggests that degenerating rd10 retinas encounter a relatively lower degree of damage in response to oxidative injury compared with normal retinas. Constitutive up-regulation of the oxidative defense mechanism in degenerating retinas may confer such relative protection from oxidative injury.

Retinal Phenotype following Combined Deletion of the Chemokine Receptor CCR2 and the Chemokine CX3CL1 in Mice

Purpose: Conflicting data were reported with respect to the retinal phenotype of mice with dual perturbation of the CCL2 and CX3CR1 genes. We report the generation and retinal phenotype of mice with a reverse CCR2/CX3CL1 gene deficiency as a suggested model for age-related macular degeneration (AMD). Methods: Crossing of single-deficient mice generated CCR2/CX3CL1 DKO mice. DKO mice were compared with age-matched C57BL6J mice. Evaluation included color fundus photographs, electroretinography (ERG), histology and morphometric analysis. Immunohistochemistry for CD11b in retinal cross-sections and retinal pigment epithelium (RPE)-choroid flat mounts was performed to assess microglia and macrophage recruitment. Results: A minority of DKO mice showed yellowish subretinal deposits at 10 months. ERG recordings showed reduced cone sensitivity in young, but not older DKO mice. Compared to wild-type mice, DKO mice exhibited 11% reduction in the number of outer nuclear layer nuclei. Old DKO mice had an increased number of CD11b-positive cells across the retina, and on RPE-choroid flat mounts. Conclusions: In the absence of the rd8 allele, deficiency of CCR2 and CX3CL1 in mice leads to a mild form of retinal degeneration which is associated with the recruitment of macrophages, particularly to the subretinal space. This model enables to assess consequences of perturbed chemokine signaling, but it does not recapitulate cardinal AMD features.

Characterising the phenotype and progression of sporadic adult-onset foveomacular vitelliform dystrophy

Background/aims Adult-onset foveomacular vitelliform dystrophy (AFVD) is a relatively common macular degeneration which might lead to substantial visual loss. Our purpose was to describe the natural course of genetically evaluated patients with sporadic AFVD. Methods A retrospective, consecutive, cohort study included 95 eyes of 51 patients. Mutations in genes previously associated with AFVD (PRPH2, BEST1, IMPG-1 and IMPG-2) were evaluated. Demographics, clinical characteristics, and spectral domain optical coherence tomography features were analysed. Main outcome measures were changes in the best corrected visual acuity (BCVA) and lesion morphology during the follow-up. Results The mean age (±SD) at diagnosis was 73.8±10.7 years. Mean (±SD) follow-up period was 30.4±16.3 months (range 0–44 months; median 25 months). All patients were genotyped negative for the evaluated mutations. Fifty-three of the eyes were followed for at least 36 months. At baseline these eyes had a mean BCVA (±SD) of 0.27±0.35 LogMAR, and at 36-months BCVA decreased to 0.38±0.35 (p=0.02). At baseline, 23 of these 53 eyes (43.4%) had the vitelliform stage, while only 10 eyes (18.9%) remained at this stage at 36 months (p=0.01). Ellipsoid zone alterations progressed during the follow-up (n=53 eyes) and showed correlation with BCVA reduction (Pearsons correlation coefficient=0.7, p=0.03). Conclusions Sporadic AFVD is a slowly progressing macular degeneration of older people. It is associated with visual decline at the rate of approximately one ETDRS line during 3 years. Patients with sporadic AFVD are usually negative for the known mutations previously associated with this phenotype, and present at an age that is higher than described for monogenic AFVD.