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.

daf-16/FOXO and glod-4/glyoxalase-1 are required for the life-prolonging effect of human insulin under high glucose conditions in Caenorhabditis elegans.

AIMS/HYPOTHESIS: The aim of this study was to determine the protective effects of human insulin and its analogues, B28Asp human insulin (insulin aspart) and B29Lys(ε-tetradecanoyl),desB30 human insulin (insulin detemir), against glucose-induced lifespan reduction and neuronal damage in the model organism Caenorhabditis elegans and to elucidate the underlying mechanisms. METHODS: Nematodes were cultivated under high glucose (HG) conditions comparable with the situation in diabetic patients and treated with human insulin and its analogues. Lifespan was assessed and neuronal damage was evaluated with regard to structural and functional impairment. Additionally, the activity of glyoxalase-1 and superoxide dismutase (SOD) and the formation of reactive oxygen species (ROS) and AGEs were determined. RESULTS: Insulin and its analogues reversed the life-shortening effect of HG conditions and prevented the glucose-induced neuronal impairment. Insulin treatment under HG conditions was associated with reduced concentration of glucose, as well as a reduced formation of ROS and AGEs, and increased SOD activity. These effects were dependent on the Forkhead box O (FOXO) homologue abnormal dauer formation (DAF)-16. Furthermore, glyoxalase-1 activity, which was impaired under HG conditions, was restored by human insulin. This was essential for the insulin-induced lifespan extension under HG conditions, as no change in lifespan was observed following either suppression or overexpression of glyoxalase-1. CONCLUSIONS/INTERPRETATION: Human insulin and its analogues prevent the reduction in lifespan and neuronal damage caused by HG conditions. The effect of human insulin is mediated by a daf-2/insulin receptor and daf-16/FOXO-dependent pathway and is mediated by upregulation of detoxifying mechanisms.

Systemic treatment with erythropoietin protects the neurovascular unit in a rat model of retinal neurodegeneration.

Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four-week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.

CD74 indicates microglial activation in experimental diabetic retinopathy and exogenous methylglyoxal mimics the response in normoglycemic retina.

Diabetes induces vasoregression, neurodegeneration and glial activation in the retina. Formation of advanced glycation endoproducts (AGEs) is increased in diabetes and contributes to the pathogenesis of diabetic retinopathy. CD74 is increased in activated microglia in a rat model developing both neurodegeneration and vasoregression. In this study, we aimed at investigating whether glucose and major AGE precursor methylglyoxal induce increased CD74 expression in the retina. Expression of CD74 in retinal microglia was analyzed in streptozotocin-diabetic rats by wholemount immunofluorescence. Nondiabetic mice were intravitreally injected with methylglyoxal. Expression of CD74 was studied by retinal wholemount immunofluorescence and quantitative real-time PCR, 48 h after the injection. CD74-positive cells were increased in diabetic 4-month retinas. These cells represented a subpopulation of CD11b-labeled activated microglia and were mainly located in the superficial vascular layer (13.7-fold increase compared to nondiabetic group). Methylglyoxal induced an 9.4-fold increase of CD74-positive cells in the superficial vascular layer and elevated gene expression of CD74 in the mouse retina 2.8-fold. In summary, we identified CD74 as a microglial activation marker in the diabetic retina. Exogenous methylglyoxal mimics the response in normoglycemic retina. This suggests that methylglyoxal is important in mediating microglial activation in the diabetic retina.

An epichromatin epitope: persistence in the cell cycle and conservation in evolution.

Interphase nuclear architecture is disrupted and rapidly reformed with each cell division cycle. Successive cell generations exhibit a "memory" of this nuclear architecture, as well as for gene expression. Furthermore, many features of nuclear and mitotic chromosome structure are recognizably species and tissue specific. We wish to know what properties of the underlying chromatin structure may determine these conserved features of nuclear architecture. Employing a particular mouse autoimmune anti-nucleosome monoclonal antibody (PL2-6), combined with deconvolution immunofluorescence microscopy, we present evidence for a unique epitope (involving a ternary complex of histones H2A and H2B and DNA) which is localized only at the exterior chromatin surface of interphase nuclei and mitotic chromosomes in mammalian, invertebrate and plant systems. As only the surface chromatin region is identified with antibody PL2-6, we have assigned it the name "epichromatin". We describe an "epichromatin hypothesis", suggesting that epichromatin may have a unique evolutionary conserved conformation which facilitates interaction with the reforming post-mitotic nuclear envelope and a rapid return of interphase nuclear architecture.

Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C. elegans.

Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo-3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo-3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo-3 significantly reduced life span: mean life span decreased from 18.5 +/- 0.4 to 15.4 +/- 0.1 days (P < 0.001) and maximum life span from 25.9 +/- 0.4 to 23.2 +/- 0.1 days (P = 0.001). Additional treatment of exo-3-suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep-1 in exo-3 RNAi-treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild-type animals, suppression of cep-1, involved in downregulation of exo-3, increased expression of exo-3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx-1 and trx-2, involved in the redox chaperone activity of exo-3, overrides the protective effect of cep-1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO-3, p53/CEP-1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function.

C. elegans as model for the study of high glucose- mediated life span reduction.

OBJECTIVE: Establishing Caenorhabditis elegans as a model for glucose toxicity-mediated life span reduction. RESEARCH DESIGN AND METHODS: C. elegans were maintained to achieve glucose concentrations resembling the hyperglycemic conditions in diabetic patients. The effects of high glucose on life span, glyoxalase-1 activity, advanced glycation end products (AGEs), and reactive oxygen species (ROS) formation and on mitochondrial function were studied. RESULTS: High glucose conditions reduced mean life span from 18.5 + or - 0.4 to 16.5 + or - 0.6 days and maximum life span from 25.9 + or - 0.4 to 23.2 + or - 0.4 days, independent of glucose effects on cuticle or bacterial metabolization of glucose. The formation of methylglyoxal-modified mitochondrial proteins and ROS was significantly increased by high glucose conditions and reduced by mitochondrial uncoupling and complex IIIQo inhibition. Overexpression of the methylglyoxal-detoxifying enzyme glyoxalase-1 attenuated the life-shortening effect of glucose by reducing AGE accumulation (by 65%) and ROS formation (by 50%) and restored mean (16.5 + or - 0.6 to 20.6 + or - 0.4 days) and maximum life span (23.2 + or - 0.4 to 27.7 + or - 2.3 days). In contrast, inhibition of glyoxalase-1 by RNAi further reduced mean (16.5 + or - 0.6 to 13.9 + or - 0.7 days) and maximum life span (23.2 + or - 0.4 to 20.3 + or - 1.1 days). The life span reduction by glyoxalase-1 inhibition was independent from the insulin signaling pathway because high glucose conditions also affected daf-2 knockdown animals in a similar manner. CONCLUSIONS: C. elegans is a suitable model organism to study glucose toxicity, in which high glucose conditions limit the life span by increasing ROS formation and AGE modification of mitochondrial proteins in a daf-2 independent manner. Most importantly, glucose toxicity can be prevented by improving glyoxalase-1-dependent methylglyoxal detoxification or preventing mitochondrial dysfunction.

Glyoxalase-1 prevents mitochondrial protein modification and enhances lifespan in Caenorhabditis elegans.

Studies of mutations affecting lifespan in Caenorhabditis elegans show that mitochondrial generation of reactive oxygen species (ROS) plays a major causative role in organismal aging. Here, we describe a novel mechanism for regulating mitochondrial ROS production and lifespan in C. elegans: progressive mitochondrial protein modification by the glycolysis-derived dicarbonyl metabolite methylglyoxal (MG). We demonstrate that the activity of glyoxalase-1, an enzyme detoxifying MG, is markedly reduced with age despite unchanged levels of glyoxalase-1 mRNA. The decrease in enzymatic activity promotes accumulation of MG-derived adducts and oxidative stress markers, which cause further inhibition of glyoxalase-1 expression. Over-expression of the C. elegans glyoxalase-1 orthologue CeGly decreases MG modifications of mitochondrial proteins and mitochondrial ROS production, and prolongs C. elegans lifespan. In contrast, knock-down of CeGly increases MG modifications of mitochondrial proteins and mitochondrial ROS production, and decreases C. elegans lifespan.

Gene expression profiling of human endometrial-trophoblast interaction in a coculture model.

Investigating the interaction of human endometrium and trophoblast during implantation is difficult in vitro and impossible in vivo. This study was designed to analyze the effect of trophoblast on endometrial stromal cells during implantation by comprehensive gene profiling. An in vitro coculture system of endometrial stromal cells with first-trimester trophoblast explants was established. Trophoblast and endometrial stromal cells were separated after 24 h. Gene expression of endometrial stromal cells after coculture was compared with the gene expression of endometrial stromal cells cultured alone by microarray analysis. We confirmed the expression of distinct genes using real-time PCR. Genes up-regulated included those for inflammatory response, immune response, and chemotaxis (pentraxin-related gene 3, chemokine ligands, IL-8, IL-1 receptors, IL-18 receptor, IL-15, IL-15 receptor, TNF-alpha-induced protein 6, and IL-6 signal transducer), regulators of cell growth (IGF-binding proteins 1 and 2) and signal transduction. Also up-regulated were genes for growth and development, glucose metabolism, and lipid metabolism: DKK-1, WISP, IGF-II, hydroxysteroid 11beta-dehydrogenase 1, hydroxyprostaglandin dehydrogenase 15, prostaglandin E synthase, prostaglandin F receptor, aldehyde dehydrogenase 1 family, member A3 and phosphatidic acid phosphatase type 2B. Other genes included genes for cell-cell signaling (pre-B-cell colony-enhancing factor 1), proteolysis, calcium ion binding, regulation of transcription, and others. Down-regulated genes included genes for proteolysis (MMP-11 and mitochondrial intermediate peptidase), genes for cell death (caspase 6, death-associated protein kinase 1, and histone deacetylase 5), transcription factors (sex determining region Y-box 4, dachshund homolog 1, ets variant gene 1, and zinc finger protein 84 and 435), and genes for humoral immune response (CD24 antigen). Trophoblast has a significant impact on endometrial stromal cell gene expression. Some of the genes regulated by trophoblast in endometrial stromal cells are already known to be regulated by progesterone and show the endocrine function of trophoblast during pregnancy. Others are genes so far unknown to play a role in endometrial-trophoblast interaction and open a wide field of investigation.

Functional polymorphisms of UCP2 and UCP3 are associated with a reduced prevalence of diabetic neuropathy in patients with type 1 diabetes.

OBJECTIVE: We studied the association between polymorphisms in the UCP genes and diabetes complications in patients with type 1 diabetes. RESEARCH DESIGN AND METHODS: We analyzed 227 patients with type 1 diabetes using PCR and subsequent cleavage by restriction endonucleases for the promoter variants A-3826G in the UCP1 gene, G-866A in the UCP2 gene, and C-55T in the UCP3 gene. RESULTS: No effect of the A-3826G polymorphism in the UCP1 gene on diabetes complications was found. Patients who were heterozygous or homozygous for the G-866A polymorphism in the UCP2 gene or the C-55T polymorphism in the UCP3 gene had a significantly reduced prevalence of diabetic neuropathy (UCP2: odds ratio 0.44 [95% CI 0.24-0.79], P = 0.007; UCP3: 0.48 [0.25-0.92], P = 0.031), whereas there was no association with other diabetes complications. This effect was stronger when G-866A and C-55T occurred in a cosegregatory manner (UCP2 and UCP3: 0.28 [0.12-0.65], P = 0.002). Furthermore, a multiple logistic regression model showed an age- and diabetes duration-independent effect of the cosegregated polymorphisms on the prevalence of diabetic neuropathy (P = 0.013). CONCLUSIONS: Our data indicate that both the G-866A polymorphism in the UCP2 gene and the C-55T polymorphism in the UCP3 gene are associated with a reduced risk of diabetic neuropathy in type 1 diabetes. Thus, the results presented here support the hypothesis that higher expression of uncoupling protein might prevent mitochondria-mediated neuronal injury and, ultimately, diabetic neuropathy.