Malignancies Masquerading as Uveitis

Malignancy presenting as uveitis can be symptomatically mild and difficult to diagnose, often leading to delayed treatment and poor outcomes. In this chapter, we describe the range of malignancies reported to present with uveitis, both primary ocular malignancies and systemic malignancies which metastasize to the eye. We describe the range of clinical features and corresponding complaints that may be encountered with specific malignancies and provide a thorough review of diagnostic tools available to aid in diagnosis. The primary goal of this chapter is to aid in the diagnosis of intraocular malignancy to reduce treatment delays and improve patient outcomes

Bone Marrow Transplantation Transfers Age-Related Susceptibility to Neovascular Remodeling in Murine Laser- Induced Choroidal Neovascularization

Citation: Espinosa-Heidmann DG, Malek G, Mettu PS, et al. Bone marrow transplantation transfers age-related susceptibility to neovascular remodeling in murine laser-induced choroidal neovascularization. Invest Ophthalmol Vis Sci. 2013;54:7439-7449. DOI:10.1167/iovs.13-12546 PURPOSE. Neovascular remodeling (NVR), the progression of small capillaries into large-caliber arterioles with perivascular fibrosis, represents a major therapeutic challenge in neovascular age-related macular degeneration (AMD). Neovascular remodeling occurs after laser-induced choroidal neovascularization (CNV) in aged but not young mice. Additionally, bone marrowderived cells, including macrophages, endothelial precursor cells, and mesenchymal precursor cells, contribute to CNV severity. In this study, we investigated the impact of aged bone marrow transplantation (BMT) on the degree of fibrosis, size, and vascular morphology of CNV lesions in a mouse model of laser-induced CNV. METHODS. Young (2 months) and old (16 months) mice were transplanted with green fluorescent protein (GFP)-labeled bone marrow isolated from either young or old donors. Laser CNV was induced 1 month following transplant, and eyes were analyzed via choroidal flat mounts and immunohistochemistry 1 month postlaser. The identity of cells infiltrating CNV lesions was determined using specific markers for the labeled transplanted cells (GFPþ), macrophages (F4/80þ), perivascular mesenchymal-derived cells (smooth muscle actin, SMAþ), and endothelial cells (CD31þ). RESULTS. Bone marrow transplantation from aged mice transferred susceptibility to NVR into young recipients. Inversely, transplantation of young marrow into old mice prevented NVR, preserving small size and minimal fibrosis. Mice with NVR demonstrated a greater relative contribution of marrow-derived SMAþ perivascular mesenchymal cells as compared to other cells. CONCLUSIONS. Our findings indicate that the status of bone marrow is an important determining factor of neovascular severity. Furthermore, we find that perivascular mesenchymal cells, rather than endothelial cells, derived from aged bone marrow may contribute to increased CNV severity in this murine model of experimental neovascularization

Macrophages Inhibit Neovascularization in a Murine Model of Age-Related Macular Degeneration

BACKGROUND: Age-related macular degeneration (AMD) is the leading cause of blindness in people over 50 y of age in at least three continents. Choroidal neovascularization (CNV) is the process by which abnormal blood vessels develop underneath the retina. CNV develops in 10% of patients with AMD but accounts for up to 90% of the blindness from AMD. Although the precise etiology of CNV in AMD remains unknown, the macrophage component of the inflammatory response, which has been shown to promote tumor growth and support atherosclerotic plaque formation, is thought to stimulate aberrant angiogenesis in blinding eye diseases. The current theory is that macrophage infiltration promotes the development of neovascularization in CNV. METHODS AND FINDINGS: We examined the role of macrophages in a mouse model of CNV. IL-10 (−/−) mice, which have increased inflammation in response to diverse stimuli, have significantly reduced CNV with increased macrophage infiltrates compared to wild type. Prevention of macrophage entry into the eye promoted neovascularization while direct injection of macrophages significantly inhibited CNV. Inhibition by macrophages was mediated by the TNF family death molecule Fas ligand (CD95-ligand). CONCLUSIONS: Immune vascular interactions can be highly complex. Normal macrophage function is critical in controlling pathologic neovascularization in the eye. IL-10 regulates macrophage activity in the eye and is an attractive therapeutic target in order to suppress or inhibit CNV in AMD that can otherwise lead to blindness

Bone marrow-derived cells in ocular neovascularization: contribution and mechanisms

Ocular neovascularization often leads to severe vision loss. The role of bone marrow-derived cells (BMCs) in the development of ocular neovascularization, and its significance, is increasingly being recognized. In this review, we discuss their contribution and the potential mechanisms that mediate the effect of BMCs on the progression of ocular neovascularization. The sequence of events by which BMCs participate in ocular neovascularization can be roughly divided into four phases, i.e., mobilization, migration, adhesion and differentiation. This process is delicately regulated and liable to be affected by multiple factors. Cytokines such as vascular endothelial growth factor, granulocyte colony-stimulating factor and erythropoietin are involved in the mobilization of BMCs. Studies have also demonstrated a key role of cytokines such as stromal cell-derived factor-1, tumor necrosis factor-α, as well as vascular endothelial growth factor, in regulating the migration of BMCs. The adhesion of BMCs is mainly regulated by vascular cell adhesion molecule-1, intercellular adhesion molecule-1 and vascular endothelial cadherin. However, the mechanisms regulating the differentiation of BMCs are largely unknown at present. In addition, BMCs secrete cytokines that interact with the microenvironment of ocular neovascularization; their contribution to ocular neovascularization, especially choroidal neovascularization, can be aggravated by several risk factors. An extensive regulatory network is thought to modulate the role of BMCs in the development of ocular neovascularization. A comprehensive understanding of the involved mechanisms will help in the development of novel therapeutic strategies related to BMCs. In this review, we have limited the discussion to the recent progress in this field, especially the research conducted at our laboratory

Chronic Cigarette Smoke Causes Oxidative Damage and Apoptosis to Retinal Pigmented Epithelial Cells in Mice

The purpose of this study was to determine whether mice exposed to chronic cigarette smoke develop features of early age-related macular degeneration (AMD). Two month old C57Bl6 mice were exposed to either filtered air or cigarette smoke in a smoking chamber for 5 h/day, 5 days/week for 6 months. Eyes were fixed in 2.5% glutaraldehyde/2% paraformaldehyde and examined for ultrastructural changes by transmission electron microscopy. The contralateral eye was fixed in 2% paraformaldehyde and examined for oxidative injury to the retinal pigmented epithelium (RPE) by 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-OHdG) immunolabeling and apoptosis by TUNEL labeling. Mice exposed to cigarette smoke had immunolabeling for 8-OHdG in 85±3.7% of RPE cells counted compared to 9.5±3.9% in controls (p<0.00001). Bruch membrane was thicker in mice exposed to smoke (1086±332 nm) than those raised in air (543±132 nm; p = 0.0069). The two most pronounced ultrastructural changes (severity grading scale from 0–3) seen were a loss of basal infoldings (mean difference in grade = 1.98; p<0.0001), and an increase in intracellular vacuoles (mean difference in grade = 1.7; p<0.0001). Ultrastructural changes to Bruch membrane in cigarette-smoke exposed mice were smaller in magnitude but consistently demonstrated significantly higher grade injury in cigarette-exposed mice, including basal laminar deposits (mean difference in grade = 0.54; p<0.0001), increased outer collagenous layer deposits (mean difference in grade = 0.59; p = 0.002), and increased basal laminar deposit continuity (mean difference in grade = 0.4; p<0.0001). TUNEL assay showed a higher percentage of apoptotic RPE from mice exposed to cigarette smoke (average 8.0±1.1%) than room air (average 0±0%; p = 0.043). Mice exposed to chronic cigarette smoke develop evidence of oxidative damage with ultrastructural degeneration to the RPE and Bruch membrane, and RPE cell apoptosis. This model could be useful for studying the mechanism of smoke induced changes during early AMD

Suppression and Regression of Choroidal Neovascularization in Mice by a Novel CCR2 Antagonist, INCB3344

PURPOSE: To investigate the effect of an intravitreally administered CCR2 antagonist, INCB3344, on a mouse model of choroidal neovascularization (CNV). METHODS: CNV was induced by laser photocoagulation on Day 0 in wild type mice. INCB3344 or vehicle was administered intravitreally immediately after laser application. On Day 14, CNV areas were measured on retinal pigment epithelium (RPE)-choroid flat mounts and histopathologic examination was performed on 7 µm-thick sections. Macrophage infiltration was evaluated by immunohistochemistry on RPE-choroid flat mounts and quantified by flow cytometry on Day 3. Expression of vascular endothelial growth factor (VEGF) protein in RPE-choroid tissue was examined by immunohistochemistry and ELISA, VEGF mRNA in sorted macrophages in RPE-choroid tissue was examine by real-time PCR and expression of phosphorylated extracellular signal-regulated kinase (p-ERK 1/2) in RPE-choroid tissue was measured by Western blot analysis on Day 3. We also evaluated the efficacy of intravitreal INCB3344 to spontaneous CNV detected in Cu, Zn-superoxide dismutase (SOD1) deficient mice. Changes in CNV size were assessed between pre- and 1week post-INCB3344 or vehicle administration in fundus photography and fluorescence angiography (FA). RESULTS: The mean CNV area in INCB3344-treated mice decreased by 42.4% compared with the vehicle-treated control mice (p<0.001). INCB3344 treatment significantly inhibited macrophage infiltration into the laser-irradiated area (p<0.001), and suppressed the expression of VEGF protein (p = 0.012), VEGF mRNA in infiltrating macrophages (p<0.001) and the phosphorylation of ERK1/2 (p<0.001). The area of spontaneous CNV in Sod1⁻/⁻ mice regressed by 70.35% in INCB3344-treated animals while no change was detected in vehicle-treated control mice (p<0.001). CONCLUSIONS: INCB3344 both inhibits newly forming CNV and regresses established CNV. Controlling inflammation by suppressing macrophage infiltration and angiogenic ability via the CCR-2/MCP-1 signal may be a useful therapeutic strategy for treating CNV associated with age-related macular degeneration

Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina

INTRODUCTION Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.Funding provided by Australian Research Council Centres of Excellence Program Grant (CE0561903)

Cigarette Smoke-Related Hydroquinone Dysregulates MCP-1, VEGF and PEDF Expression in Retinal Pigment Epithelium in Vitro and in Vivo

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD

Ocular Delivery of Compacted DNA-Nanoparticles Does Not Elicit Toxicity in the Mouse Retina

Subretinal delivery of polyethylene glycol-substituted lysine peptide (CK30PEG)-compacted DNA nanoparticles results in efficient gene expression in retinal cells. This work evaluates the ocular safety of compacted DNA nanoparticles. CK30PEG-compacted nanoparticles containing an EGFP expression plasmid were subretinally injected in adult mice (1 µl at 0.3, 1.0 and 3.0 µg/µl). Retinas were examined for signs of inflammation at 1, 2, 4 and 7 days post-injection. Neither infiltration of polymorphonuclear neutrophils or lymphocytes was detected in retinas. In addition, elevation of macrophage marker F4/80 or myeloid marker myeloperoxidase was not detected in the injected eyes. The chemokine KC mRNA increased 3–4 fold in eyes injected with either nanoparticles or saline at 1 day post-injection, but returned to control levels at 2 days post-injection. No elevation of KC protein was observed in these mice. The monocyte chemotactic protein-1, increased 3–4 fold at 1 day post-injection for both nanoparticle and saline injected eyes, but also returned to control levels at 2 days. No elevations of tumor necrosis factor alpha mRNA or protein were detected. These investigations show no signs of local inflammatory responses associated with subretinal injection of compacted DNA nanoparticles, indicating that the retina may be a suitable target for clinical nanoparticle-based interventions