RPE-choroid Complex Research Articles

Tissue Kallikrein Attenuates Choroidal Neovascularization via Cleavage of Vascular Endothelial Growth Factor

To investigate the antiangiogenic properties of tissue kallikrein in a murine model of laser-induced choroidal neovascularization (CNV). CNV was induced in male C57BL/6J mice by laser photocoagulation. The animals received daily subcutaneous injections of tissue kallikrein (50 μg/kg) or vehicle control for 2 days before the laser photocoagulation, and this treatment continued until sample collection. Seven days after laser injury, the CNV size was quantified. The levels of monocyte chemoattractant protein (MCP)-1, intercellular adhesion molecule (ICAM)-1, and interleukin (IL)-6 were assessed by enzyme-linked immunosorbent assay 3 days after laser injury. Cleavage of mouse VEGF with tissue kallikrein was assessed in vivo and in vitro. The protein levels of bradykinin were assessed in the RPE-choroid complexes and hearts. A significant decrease in CNV size was observed in animals treated with tissue kallikrein (27,168.3 ± 2432.2 μm(2)) compared with vehicle-treated controls (36,374.6 ± 3204.1 μm(2), P < 0.05). Tissue kallikrein treatment significantly reduced MCP-1, ICAM-1, and IL-6 levels in RPE-choroid complexes. Furthermore, immunoblotting showed the bands, presumably corresponding to the fragmented VEGF(164) protein, in the samples of both mouse VEGF preincubated with tissue kallikrein and RPE-choroid complexes obtained from animals treated with tissue kallikrein. In addition, bradykinin was unchanged in the RPE-choroid complexes of animals treated with tissue kallikrein, whereas the level of bradykinin was increased in the heart obtained from these experimental animals. The current data indicate that kallikrein exhibits antiangiogenic properties by cleaving VEGF(164) in a laser-induced CNV model.

Suppression of Experimental Choroidal Neovascularization by Curcumin in Mice

PurposeTo investigate the effects of curcumin on the development of experimental choroidal neovascularization (CNV) with underlying cellular and molecular mechanisms.MethodsC57BL/6N mice were pretreated with intraperitoneal injections of curcumin daily for 3 days prior to laser-induced CNV, and the drug treatments were continued until the end of the study. The CNV area was analyzed by fluorescein-labeled dextran angiography of retinal pigment epithelium (RPE)-choroid flat mounts on day 7 and 14, and CNV leakage was evaluated by fluorescein angiography (FA) on day 14 after laser photocoagulation. The infiltration of F4/80 positive macrophages and GR-1 positive granulocytes were evaluated by immunohistochemistry on RPE-choroid flat mounts on day 3. Their expression in RPE-choroid complex was quantified by real-time PCR (F4/80) and Western blotting (GR-1) on day 3. RPE-choroid levels of vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule (ICAM)-1 were examined by ELISA on day 3. Double immunostaining of F4/80 and VEGF was performed on cryo-sections of CNV lesions on day 3. The expression of nuclear factor (NF)-κB and hypoxia-inducible factor (HIF)−1α in the RPE-choroid was determined by Western blotting.ResultsCurcumin-treated mice had significantly less CNV area (P<0.05) and CNV leakage (P<0.001) than vehicle-treated mice. Curcumin treatment led to significant inhibition of F4/80 positive macrophages (P<0.05) and GR-1 positive granulocytes infiltration (P<0.05). VEGF mainly expressed in F4/80 positive macrophages in laser injury sites, which was suppressed by curcumin treatment (P<0.01). Curcumin inhibited the RPE-choroid levels of TNF-α (P<0.05), MCP-1 (P<0.05) and ICAM-1 (P<0.05), and suppressed the activation of NF-κB in nuclear extracts (P<0.05) and the activation of HIF−1α (P<0.05).ConclusionCurcumin treatment led to the suppression of CNV development together with inflammatory and angiogenic processes including NF-κB and HIF−1α activation, the up-regulation of inflammatory and angiogenic cytokines, and infiltrating macrophages and granulocytes. This provides molecular and cellular evidence of the validity of curcumin supplementation as a therapeutic strategy for the suppression of age-related macular degeneration (AMD)-associated CNV.

Technetium-99 Conjugated with Methylene Diphosphonate (99Tc-MDP) Inhibits Experimental Choroidal Neovascularization In Vivo and VEGF-Induced Cell Migration and Tube Formation In Vitro

To investigate the effects of (99)Tc-MDP, a decay product of (99m)Tc-MDP, on the development of choroidal neovascularization (CNV), together with its underlying mechanisms. C57BL/6J mice were used to induce CNV by laser photocoagulation. (99)Tc-MDP at the doses of 0.5 × 10(-1), 1 × 10(-1), and 2 × 10(-1) μg/kg or the same volume of PBS was intraperitoneally injected daily after photocoagulation until the end of the experiment. Seven days after laser injury, mice were perfused with fluorescein-labeled dextran, and areas of CNV were measured. Numbers of infiltrating macrophages, protein levels of VEGF, and inflammation-related molecules including intercellular adhesion molecule (ICAM)-1, tumor necrosis factor (TNF)-α, and matrix metalloproteinases (MMPs) in the RPE-choroid complex were detected 3 days after laser photocoagulation. Effects of (99)Tc-MDP on VEGF-induced endothelial cell migration and tube formation were also studied. Toxicity of (99)Tc-MDP was evaluated in vivo and in vitro. Areas of CNV were significantly suppressed by (99)Tc-MDP treatment without toxicity to the retina compared with PBS treatment in a dose-dependent manner: (99)Tc-MDP treatment of 0.5 × 10(-1) μg/kg (5698.60 ± 1037.70 μm(2)), 1 × 10(-1) μg/kg (3678.34 ± 1328.18 μm(2)), and 2 × 10(-1) μg/kg (2365.78 ± 923.80 μm(2)) suppressed the development of CNV by 36.12%, 58.76%, and 73.48%, respectively, compared with that in the PBS treatment group (8920.36 ± 1097.29 μm(2); P < 0.001). (99)Tc-MDP treatment led to significant inhibition of macrophages infiltrating to CNV together with downregulated protein expressions of VEGF, ICAM-1, TNF-α, and MMP-2. (99)Tc-MDP also showed an inhibitive effect on cell proliferation and VEGF-induced migration and capillary-like tube formation of endothelial cells. Anti-inflammatory treatment with (99)Tc-MDP has therapeutic potential for CNV-related diseases.

The pivotal role of the complement system in aging and age-related macular degeneration: Hypothesis re-visited

During the past ten years, dramatic advances have been made in unraveling the biological bases of age-related macular degeneration (AMD), the most common cause of irreversible blindness in western populations. In that timeframe, two distinct lines of evidence emerged which implicated chronic local inflammation and activation of the complement cascade in AMD pathogenesis. First, a number of complement system proteins, complement activators, and complement regulatory proteins were identified as molecular constituents of drusen, the hallmark extracellular deposits associated with early AMD. Subsequently, genetic studies revealed highly significant statistical associations between AMD and variants of several complement pathway-associated genes including: Complement factor H (CFH), complement factor H-related 1 and 3 (CFHR1 and CFHR3), complement factor B (CFB), complement component 2 (C2), and complement component 3 (C3). In this article, we revisit our original hypothesis that chronic local inflammatory and immune-mediated events at the level of Bruch's membrane play critical roles in drusen biogenesis and, by extension, in the pathobiology of AMD. Secondly, we report the results of a new screening for additional AMD-associated polymorphisms in a battery of 63 complement-related genes. Third, we identify and characterize the local complement system in the RPE-choroid complex - thus adding a new dimension of biological complexity to the role of the complement system in ocular aging and AMD. Finally, we evaluate the most salient, recent evidence that bears directly on the role of complement in AMD pathogenesis and progression. Collectively, these recent findings strongly re-affirm the importance of the complement system in AMD. They lay the groundwork for further studies that may lead to the identification of a transcriptional disease signature of AMD, and hasten the development of new therapeutic approaches that will restore the complement-modulating activity that appears to be compromised in genetically susceptible individuals.

Excess Lead in the Neural Retina in Age-Related Macular Degeneration

To measure lead and cadmium in retinal tissues of human donor eyes with and without age-related macular degeneration (AMD). Laboratory investigation. Lead and cadmium concentrations in retinal tissues (neural retina and retinal pigment epithelium [RPE]-choroid complex) in 25 subjects with AMD (50 donor eyes) and 36 normal subjects (72 donor eyes) were determined by using inductively coupled plasma-mass spectrometry. Severity of AMD was graded by using color fundus photographs and the Minnesota Grading System. Differences in metal concentrations were compared by using Wilcoxon rank-sum tests. The neural retinas of subjects with AMD had increased lead concentrations (median, 12.0 ng/g; 25% to 75% interquartile range, 8 to 18 ng/g; n = 25) compared with normal subjects (median, 8.0 ng/g; 25% to 75% interquartile range, 0 to 11 ng/g; P = .04; n = 36). There was no difference in lead concentration in the RPE-choroid complex between subjects with AMD (median, 198 ng/g; 25% to 75% interquartile range, 87 to 381 ng/g) and normal subjects (median, 172 ng/g; 25% to 75% interquartile range, 100 to 288 ng/g; P = .25). Cadmium concentration in the neural retina (median, 0.9 microg/g; 25% to 75% interquartile range, 0.7 to 1.8 microg/g) and RPE-choroid complex (median, 2.2 microg/g; 25% to 75% interquartile range, 1.8 to 3.7 microg/g) in subjects with AMD was not different from concentrations in the neural retina (median, 0.9 microg/g; 25% to 75% interquartile range, 0.7 to 1.4 microg/g; P = .32) and RPE-choroid complex (median, 1.5 microg/g; 25% to 75% interquartile range, 0.9 to 2.5 microg/g; P = .12) of normal subjects. AMD is associated with excess lead in the neural retina, and this relationship suggests that metal homeostasis in AMD eyes is different from normal.

Interleukin-6 Receptor-Mediated Activation of Signal Transducer and Activator of Transcription-3 (STAT3) Promotes Choroidal Neovascularization

Interleukin (IL)-6, a potent proinflammatory cytokine, is suggested to be a risk factor for choroidal neovascularization (CNV) because of its increased levels in the serum of patients with age-related macular degeneration; however, the role of IL-6 in CNV has not been defined. The present study reveals the critical contribution of IL-6 signaling and its downstream STAT3 pathway to the murine model of laser-induced CNV. CNV induction by laser treatment stimulated IL-6 expression in the retinal pigment epithelium-choroid complex, and antibody-based blockade of IL-6 receptor or genetic ablation of IL-6 led to significant suppression of CNV. CNV generation was accompanied by STAT3 activation in choroidal endothelial cells and macrophages, and IL-6 receptor blockade resulted in selectively inhibited phosphorylation of STAT3 but not extracellular signal-regulated kinase 1/2. Consistently, pharmacological blockade of STAT3 pathway also suppressed CNV. In addition, IL-6 receptor neutralization led to significant inhibition of the in vivo and in vitro expression of inflammation-related molecules including monocyte chemotactic protein, intercellular adhesion molecule-1, and vascular endothelial growth factor, and of macrophage infiltration into CNV. These results indicate the significant involvement of IL-6 receptor-mediated activation of STAT3 inflammatory pathway in CNV generation, suggesting the possibility of IL-6 receptor blockade as a therapeutic strategy to suppress CNV associated with age-related macular degeneration.

VEGF Expression and Receptor Activation in the Choroid during Development and in the Adult

Previous studies have demonstrated a role for the retinal pigment epithelium (RPE) in the development and maintenance of the choroidal vasculature, suggesting that RPE serves a trophic role for the choroidal vessels. The goal of this study was to determine the expression pattern of vascular endothelial growth factor (VEGF) and its receptors and their activation status in embryonic and adult choroid, with the purpose of providing cues regarding the role of VEGF in development and stabilization of the choroidal vasculature. Transgenic VEGF-LacZ mice were used to examine VEGF expression in embryonic and adult eyes. Expression of VEGF isoforms and receptors in the RPE-choroid complex was assessed by RT-PCR and real-time PCR. VEGF receptor 2 expression was assessed by immunohistochemistry and its activation state was examined by immunoprecipitation followed by phosphotyrosine blot. VEGF is expressed by RPE throughout the choroidal vascular development and in the adult. The major VEGF isoforms detected in adult RPE were VEGF120 and VEGF164, with almost no detectable VEGF188. RT-PCR analysis showed expression of VEGF receptors and coreceptors in the RPE-choroid complex. VEGFR2 was detected in the choriocapillaris underlying the RPE. Immunoprecipitation and phosphotyrosine blot of this receptor revealed that VEGFR2 is activated in adult mouse and bovine choroids. The observations suggest that VEGF signaling is involved, not only in choroidal vessel formation, but perhaps also in the maintenance of the choriocapillaris.

Proteins modified by malondialdehyde, 4-hydroxynonenal, or advanced glycation end products in lipofuscin of human retinal pigment epithelium.

Lipofuscin (LF) accumulation in the retinal pigment epithelium (RPE) is associated with age and various retinal diseases. Toxic LF compounds may interfere with normal RPE function. Oxidative modification of proteins was determined in LF granules from human eyes. LF was isolated from the RPE-choroid complex of 10 pairs of donor eyes by gradient ultracentrifugation. Protein compounds were separated by two-dimensional (2-D) gel electrophoresis and screened by Western blot analysis for lipid peroxidation- or glucoxidation-induced damage-in particular, by malondialdehyde (MDA), 4-hydroxynonenal (HNE), and advanced glycation end products (AGEs). Identity of the immunostained proteins was revealed using 2-D software for comparison of the spot position with Coomassie-stained 2-D gels of the same samples. By comparing the results taken from the authors' previous proteome analysis of RPE LF with an immunoblot analysis of the same samples, this study shows that a variety of LF-associated proteins were damaged by aberrant covalent modifications of MDA, 4-HNE, and AGEs. Several proteins were altered by two or three different modification types. Modified mitochondrial proteins indicated that autophagy of altered proteins also contributed to lipofuscin formation. The identification of lipid peroxidation and glucoxidation products in proteinaceous LF components in human RPE supports the hypothesis that these compounds are involved in lipofuscinogenesis and may contribute to the cytotoxic effects of LF in retinal diseases such as age-related macular degeneration and Stargardt disease. Their identification may help to identify potential future treatment targets.

Choroideremia: A Clinical, Electron Microscopic, and Biochemical Report

An asymptomatic 19-year-old male with choroideremia had diffuse loss of retinal pigment epithelium (RPE) and choroid except for the periphery and macula. Fluorescein angiography of the arteriovenous phase showed absence of retinal pigment epithelium and exaggerated visualization of choroidal vessels in involved areas. The mother was a typical carrier with pigment stippling of the midperipheral retina. Histopathologic examination of affected areas of one eye showed marked degeneration of the outer and midretina with loss of retinal pigment epithelium and Bruch's membrane, absence of choriocapillaris, chorioretinal adhesions and gliosis. Atrophy of inner and mid-choroid was also observed. Pigmented macrophage-like cells had migrated into the outer and midretinal layers. Electron microscopy disclosed macrophage-like cells with trilaminar structures and photoreceptor phagosomes in the RPE and outer retina. Remnants of photoreceptor outer segments were adherent to the plasma membranes of the macrophage-like cells. Biochemical analysis of retinal tissue samples for interphotoreceptor retinoid-binding protein (IRBP) showed marked reduction in the 146K bands in the equator and posterior pole in the patient compared to controls. Cyclic nucleotide content was altered in the retinal equator. Cyclic AMP was several-fold higher in the RPE-choroid complex of the affected eye than in the control