Extracellular cathepsin S and intracellular caspase 1 activation are surrogate biomarkers of particulate-induced lysosomal disruption in macrophages.

BACKGROUND: Particulate matter has been shown to stimulate the innate immune system and induce acute inflammation. Therefore, while nanotechnology has the potential to provide therapeutic formulations with improved efficacy, there are concerns such pharmaceutical preparations could induce unwanted inflammatory side effects. Accordingly, we aim to examine the utility of using the proteolytic activity signatures of cysteine proteases, caspase 1 and cathepsin S (CTSS), as biomarkers to assess particulate-induced inflammation. METHODS: Primary peritoneal macrophages and bone marrow-derived macrophages from C57BL/6 mice and ctss(-/-) mice were exposed to micro- and nanoparticulates and also the lysosomotropic agent, L-leucyl-L-leucine methyl ester (LLOME). ELISA and immunoblot analyses were used to measure the IL-1ß response in cells, generated by lysosomal rupture. Affinity-binding probes (ABPs), which irreversibly bind to the active site thiol of cysteine proteases, were then used to detect active caspase 1 and CTSS following lysosomal rupture. Reporter substrates were also used to quantify the proteolytic activity of these enzymes, as measured by substrate turnover. RESULTS: We demonstrate that exposure to silica, alum and polystyrene particulates induces IL-1ß release from macrophages, through lysosomal destabilization. IL-1ß secretion positively correlated with an increase in the proteolytic activity signatures of intracellular caspase 1 and extracellular CTSS, which were detected using ABPs and reporter substrates. Interestingly IL-1ß release was significantly reduced in primary macrophages from ctss(-/-) mice. CONCLUSIONS: This study supports the emerging significance of CTSS as a regulator of the innate immune response, highlighting its role in regulating IL-1ß release. Crucially, the results demonstrate the utility of intracellular caspase 1 and extracellular CTSS proteolytic activities as surrogate biomarkers of lysosomal rupture and acute inflammation. In the future, activity-based detection of these enzymes may prove useful for the real-time assessment of particle-induced inflammation and toxicity assessment during the development of nanotherapeutics.

Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.

RAGE regulates immune cell infiltration and angiogenesis in choroidal neovascularization.

PURPOSE: RAGE regulates pro-inflammatory responses in diverse cells and tissues. This study has investigated if RAGE plays a role in immune cell mobilization and choroidal neovascular pathology that is associated with the neovascular form of age-related macular degeneration (nvAMD). METHODS: RAGE null (RAGE-/-) mice and age-matched wild type (WT) control mice underwent laser photocoagulation to generate choroidal neovascularization (CNV) lesions which were then analyzed for morphology, S100B immunoreactivity and inflammatory cell infiltration. The chemotactic ability of bone marrow derived macrophages (BMDMs) towards S100B was investigated. RESULTS: RAGE expression was significantly increased in the retina during CNV of WT mice (p<0.001). RAGE-/- mice exhibited significantly reduced CNV lesion size when compared to WT controls (p<0.05). S100B mRNA was upregulated in the lasered WT retina but not RAGE-/- retina and S100B immunoreactivity was present within CNV lesions although levels were less when RAGE-/- mice were compared to WT controls. Activated microglia in lesions were considerably less abundant in RAGE-/- mice when compared to WT counterparts (p<0.001). A dose dependent chemotactic migration was observed in BMDMs from WT mice (p<0.05-0.01) but this was not apparent in cells isolated from RAGE-/- mice. CONCLUSIONS: RAGE-S100B interactions appear to play an important role in CNV lesion formation by regulating pro-inflammatory and angiogenic responses. This study highlights the role of RAGE in inflammation-mediated outer retinal pathology.

Age- and light-dependent development of localised retinal atrophy in CCL2(-/-)CX3CR1(GFP/GFP) mice.

Previous studies have shown that CCL2/CX3CR1 deficient mice on C57BL/6N background (with rd8 mutation) have an early onset (6 weeks) of spontaneous retinal degeneration. In this study, we generated CCL2(-/-)CX3CR1(GFP/GFP) mice on the C57BL/6J background. Retinal degeneration was not detected in CCL2(-/-)CX3CR1(GFP/GFP) mice younger than 6 months. Patches of whitish/yellowish fundus lesions were observed in 17∼60% of 12-month, and 30∼100% of 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice. Fluorescein angiography revealed no choroidal neovascularisation in these mice. Patches of retinal pigment epithelium (RPE) and photoreceptor damage were detected in 30% and 50% of 12- and 18-month CCL2(-/-)CX3CR1(GFP/GFP) mice respectively, but not in wild-type mice. All CCL2(-/-)CX3CR1(GFP/GFP) mice exposed to extra-light (∼800lux, 6 h/day, 6 months) developed patches of retinal atrophy, and only 20-25% of WT mice which underwent the same light treatment developed atrophic lesions. In addition, synaptophysin expression was detected in the outer nucler layer (ONL) of area related to photoreceptor loss in CCL2(-/-)CX3CR1(GFP/GFP) mice. Markedly increased rhodopsin but reduced cone arrestin expression was observed in retinal outer layers in aged CCL2(-/-)CX3CR1(GFP/GFP) mice. GABA expression was reduced in the inner retina of aged CCL2(-/-)CX3CR1(GFP/GFP) mice. Significantly increased Müller glial and microglial activation was observed in CCL2(-/-)CX3CR1(GFP/GFP) mice compared to age-matched WT mice. Macrophages from CCL2(-/-)CX3CR1(GFP/GFP) mice were less phagocytic, but expressed higher levels of iNOS, IL-1ß, IL-12 and TNF-α under hypoxia conditions. Our results suggest that the deletions of CCL2 and CX3CR1 predispose mice to age- and light-mediated retinal damage. The CCL2/CX3CR1 deficient mouse may thus serve as a model for age-related atrophic degeneration of the RPE, including the dry type of macular degeneration, geographic atrophy.

Intervention with an erythropoietin-derived peptide protects against neuroglial and vascular degeneration during diabetic retinopathy.

OBJECTIVE: Erythropoietin (EPO) may be protective for early stage diabetic retinopathy, although there are concerns that it could exacerbate retinal angiogenesis and thrombosis. A peptide based on the EPO helix-B domain (helix B-surface peptide [pHBSP]) is nonerythrogenic but retains tissue-protective properties, and this study evaluates its therapeutic potential in diabetic retinopathy. RESEARCH DESIGN AND METHODS: After 6 months of streptozotocin-induced diabetes, rats (n = 12) and age-matched nondiabetic controls (n = 12) were evenly split into pHBSP and scrambled peptide groups and injected daily (10 µg/kg per day) for 1 month. The retina was investigated for glial dysfunction, microglial activation, and neuronal DNA damage. The vasculature was dual stained with isolectin and collagen IV. Retinal cytokine expression was quantified using real-time RT-PCR. In parallel, oxygen-induced retinopathy (OIR) was used to evaluate the effects of pHBSP on retinal ischemia and neovascularization (1-30 µg/kg pHBSP or control peptide). RESULTS: pHBSP or scrambled peptide treatment did not alter hematocrit. In the diabetic retina, Müller glial expression of glial fibrillary acidic protein was increased when compared with nondiabetic controls, but pHBSP significantly reduced this stress-related response (P < 0.001). CD11b+ microglia and proinflammatory cytokines were elevated in diabetic retina responses, and some of these responses were attenuated by pHBSP (P < 0.01-0.001). pHBSP significantly reduced diabetes-linked DNA damage as determined by 8-hydroxydeoxyguanosine and transferase-mediated dUTP nick-end labeling positivity and also prevented acellular capillary formation (P < 0.05). In OIR, pHBSP had no effect on preretinal neovascularization at any dose. CONCLUSIONS: Treatment with an EPO-derived peptide after diabetes is fully established can significantly protect against neuroglial and vascular degenerative pathology without altering hematocrit or exacerbating neovascularization. These findings have therapeutic implications for disorders such as diabetic retinopathy.

Differential modulation of angiogenesis by erythropoiesis-stimulating agents in a mouse model of ischaemic retinopathy.

BACKGROUND: Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models. METHODOLOGY/PRINCIPAL FINDINGS: The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1-20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFalpha and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001). CONCLUSIONS: This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs.

Reduced nitro-oxidative stress and neural cell death suggests a protective role for microglial cells in TNFalpha-/- mice in ischemic retinopathy.

PURPOSE: Neovascularization occurs in response to tissue ischemia and growth factor stimulation. In ischemic retinopathies, however, new vessels fail to restore the hypoxic tissue; instead, they infiltrate the transparent vitreous. In a model of oxygen-induced retinopathy (OIR), TNFalpha and iNOS, upregulated in response to tissue ischemia, are cytotoxic and inhibit vascular repair. The aim of this study was to investigate the mechanism for this effect. METHODS: Wild-type C57/BL6 (WT) and TNFalpha(-/-) mice were subjected to OIR by exposure to 75% oxygen (postnatal days 7-12). The retinas were removed during the hypoxic phase of the model. Retinal cell death was determined by TUNEL staining, and the microglial cells were quantified after Z-series capture with a confocal microscope. In situ peroxynitrite and superoxide were measured by using the fluorescent dyes DCF and DHE. iNOS, nitrotyrosine, and arginase were analyzed by real-time PCR, Western blot analysis, and activity determined by radiolabeled arginine conversion. Astrocyte coverage was examined after GFAP immunostaining. RESULTS: The TNFalpha(-/-) animals displayed a significant reduction in TUNEL-positive apoptotic cells in the inner nuclear layer of the avascular retina compared with that in the WT control mice. The reduction coincided with enhanced astrocytic survival and an increase in microglial cells actively engaged in phagocytosing apoptotic debris that displayed low ROS, RNS, and NO production and high arginase activity. CONCLUSIONS: Collectively, the results suggest that improved vascular recovery in the absence of TNFalpha is associated with enhanced astrocyte survival and that both phenomena are dependent on preservation of microglial cells that display an anti-inflammatory phenotype during the early ischemic phase of OIR.

Nanoceria have no genotoxic effect on human lens epithelial cells.

There are no treatments for reversing or halting cataract, a disease of the structural proteins in the eye lens, that has associations with other age-related degenerative conditions such as Alzheimer's disease. The incidence of cataract and associated conditions is increasing as the average age of the population rises. Protein folding diseases are difficult to assess in vivo as proteins and their age-related changes are assessed after extraction. Nanotechnology can be used to investigate protein changes in the intact lens as well as for a potential means of drug delivery. Nanoparticles, such as cerium oxide (CeO(2)) which have antioxidant properties, may even be used as a means of treating cataract directly. Prior to use in treatments, nanoparticle genotoxicity must be tested to assess the extent of any DNA or chromosomal damage. Sister chromatid exchanges were measured and DNA damage investigated using the alkaline COMET assay on cultured human lens epithelial cells, exposed to 5 and 10 microg ml(-1) of CeO(2) nanoparticles (nanoceria). Nanoceria at these dosages did not cause any DNA damage or significant increases in the number of sister chromatid exchanges. The absence of genotoxic effects on lens cells suggests that nanoceria, in the doses and exposures tested in this study, are not deleterious to the eye lens and have the potential for use in studying structural alterations, in developing non-surgical cataract treatments and in investigating other protein folding diseases.