Pigment epithelium-derived factor inhibits caveolin-induced interleukin-8 gene expression and proliferation of human prostate cancer cells.

Caveolin-1 (Cav), a primary protein component of caveolae, is overexpressed in prostate cancer, thereby promoting growth and metastasis of this tumor. By contrast, pigment epithelium-derived factor (PEDF) has been shown to inhibit tumor growth and metastasis, including that of prostate cancer, via its anti-angiogenic and anti-inflammatory effects. Although it was recently demonstrated that PEDF binds to Cav and blocks its pro-inflammatory actions in endothelial cells, it remains unclear whether PEDF also inhibits the tumor-promoting effects of Cav in cultured prostate cancer cells. The present study examined the effects of PEDF on cell growth, in addition to the gene expression of interleukin-8 (IL-8), which is involved in prostate cancer progression, in the PC-3 human prostate cancer cell line. Exogenous Cav led to a dose-dependent upregulation of the mRNA expression of IL-8 in PC-3 cells, which was blocked by treatment with 1 or 10 nM PEDF, or following the overexpression of small interfering RNAs directed against Cav. Cav (10 nM) increased DNA synthesis in PC-3 cells, which was again suppressed by the administration of 10 nM PEDF. The results of the present study indicated that PEDF may inhibit Cav-induced increases in IL-8 gene expression and proliferation of PC-3 cells. Therefore, the suppressive effects of PEDF in prostate cancer may, in part, be ascribed to its inhibitory actions on Cav.

DNA Aptamer Raised against Advanced Glycation End Products Prevents Abnormalities in Electroretinograms of Experimental Diabetic Retinopathy.

PURPOSE: Abnormalities in electroretinograms (ERG), such as reduced amplitudes and delayed implicit times of a- and b-wave and oscillatory potentials (OPs), are one of the earliest features of diabetic retinopathy prior to obvious vascular changes in diabetic retinas. We have previously shown that serum levels of advanced glycation end products (AGEs) are correlated with a delayed latency of OPs in type 2 diabetic rats. However, the pathological role of AGEs in ERG abnormalities remains unclear. We examined here whether high-affinity DNA aptamer directed against AGEs (AGE-aptamer) prevents ERG abnormalities in experimental type 1 diabetic retinopathy. METHODS: Streptozotocin-induced diabetic rats or control rats received continuous intraperitoneal infusion of either AGE-aptamer or control aptamer via an osmotic mini pump for 16 weeks. Anthropometric, metabolic, and hemodynamic variables were measured, and an ERG was performed. RESULTS: Although AGE-aptamer did not affect body weight, fasting and random blood glucose, HbA1c, blood pressure, or lipid parameters, it completely prevented the increase in serum AGE levels as well as the reduction of a- and b-wave and OP amplitudes in diabetic rats. CONCLUSION: The present study demonstrated for the first time that AGE-aptamer prevents abnormalities in ERG in experimental diabetic retinopathy probably by blocking the harmful effects of AGEs.

Dipeptidyl peptidase-4 deficiency protects against experimental diabetic nephropathy partly by blocking the advanced glycation end products-receptor axis.

Advanced glycation end products (AGEs) and their receptor (RAGE) have a role in diabetic nephropathy. We have recently found that linagliptin, an inhibitor of dipeptidyl peptidase-4 (DPP-4), could inhibit renal damage in type 1 diabetic rats by suppressing the AGE-RAGE axis. However, it remains unclear whether DPP-4 deficiency could also have beneficial effects on experimental diabetic nephropathy. To address the issue, we rendered wild-type F344/NSlc and DPP-4-deficient F344/DuCrl/Crlj rats diabetic by injection of streptozotocin, and then investigated whether DPP-4 deficiency could block the activation of AGE-RAGE axis in the diabetic kidneys and resultantly ameliorate renal injury in streptozotocin-induced diabetic rats. Compared with control rats at 9 and 11 weeks old, body weight and heart rates were significantly lower, while fasting blood glucose was higher in wild-type and DPP-4-deficient diabetic rats at the same age. There was no significant difference of body weight, fasting blood glucose and lipid parameters between the two diabetic rat strains. AGEs, 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine levels in the kidney, renal gene expression of RAGE and intercellular adhesion molecule-1, glomerular area, urinary excretion of 8-OHdG and albumin, and the ratio of renal to body weight were increased in wild-type diabetic rats at 9 and/or 11 weeks old compared with age-matched control rats, all of which except for urinary 8-OHdG levels at 11 weeks old were significantly suppressed in DPP-4-deficient diabetic rats. Our present study suggests that DPP-4 deficiency could exert beneficial actions on type 1 diabetic nephropathy partly by blocking the AGE-RAGE axis. DPP-4 might be a novel therapeutic target for preventing diabetic nephropathy.

Elevation of Serum Levels of Advanced Glycation End Products in Patients With Non-B or Non-C Hepatocellular Carcinoma.

BACKGROUND: The prevalence of non-B or non-C hepatocellular carcinoma (NBNC-HCC) has been increasing all over the world. Advanced glycation end products (AGE) play a role in the pathogenesis of alcoholic liver injury or nonalcoholic steatohepatitis (NASH). METHODS: We examined here whether serum levels of AGE were elevated in NBNC-HCC patients compared with NASH subjects without HCC and investigated which anthropometric and clinical variables were independent determinants of AGE. RESULTS: Ninety NBNC-HCC, 56 NASH, and 27 control subjects underwent a complete history and physical examination, determination of blood chemistries, including AGE levels. Serum levels of AGE were significantly higher in NBNC-HCC patients compared with NASH and control subjects [9.1 ± 2.7, 5.2 ± 1.7, 3.5 ± 1.2 (U/ml), respectively, P < 0.05]. Univariate analysis showed that AGE levels were associated with male (P < 0.05), age (P < 0.01), aspartate aminotransferase (P < 0.05), γ-glutamyl transpeptidase (GGT) (P < 0.01), HDL-cholesterol (inversely, P < 0.01), fasting plasma glucose (P < 0.01), and HbA1c (P < 0.05). By the use of multiple stepwise regression analysis, age, GGT, and HDL-cholesterol (inversely) remained significant and were independently related to AGE levels (R(2) = 0.406). CONCLUSION: The present results suggest that AGE might be involved in the pathogenesis of NBNC-HCC, thereby being a biomarker that could discriminate NBNC-HCC from NASH.

Sulforaphane inhibits advanced glycation end product-induced pericyte damage by reducing expression of receptor for advanced glycation end products.

Advanced glycation end products (AGEs) not only inhibit DNA synthesis but also play a role in diabetic retinopathy by evoking apoptosis and inflammation in retinal pericytes via interaction with a receptor for AGE (RAGE). Similarly, sulforaphane, which is a naturally occurring isothiocyanate that is found in widely consumed cruciferous vegetables, protects against oxidative stress-induced tissue damage. Therefore, we hypothesized that sulforaphane could inhibit AGE-induced pericytes injury through its antioxidative properties. Advanced glycation end product stimulated superoxide generation as well as RAGE gene and protein expression in bovine-cultured retinal pericytes, and these effects were prevented by the treatment with sulforaphane. Antibodies directed against RAGE also blocked AGE-evoked reactive oxygen species generation in pericytes. Sulforaphane and antibodies directed against RAGE significantly inhibited the AGE-induced decrease in DNA synthesis, apoptotic cell death, and up-regulation of monocyte chemoattractant protein 1 messenger RNA levels in pericytes. For the first time, the present study demonstrates that sulforaphane could inhibit DNA synthesis, apoptotic cell death, and inflammatory reactions in AGE-exposed pericytes, partly by suppressing RAGE expression via its antioxidative properties. Blockade of the AGE-RAGE axis in pericytes by sulforaphane might be a novel therapeutic target for the treatment of diabetic retinopathy.

DNA aptamer raised against advanced glycation end products inhibits melanoma growth in nude mice.

Epidemiological studies have suggested that diabetes is associated with an increased risk of cancer. However, the underlying molecular mechanism remains unclear. We investigated here whether DNA aptamer directed against advanced glycation end products (AGE-aptamer) inhibited melanoma growth in nude mice. G361 melanoma cells were injected intradermally into the upper flank of athymic nude mice. Mice received continuous intraperitoneal infusion (0.136 µg/day) of either AGE-aptamer (n=9) or Control-aptamer (n=8) by an osmotic mini pump. Tumor volume was measured at 4-day interval, and G361 melanoma was excised at day 43 after the aptamer treatment. We further examined the effects of AGE-aptamer on proliferation of AGE-exposed endothelial cells and G361 cells. AGE-aptamer significantly inhibited the in vivo-tumor growth of G361 melanoma. Immunohistochemical and western blotting analyses of G361 melanoma revealed that AGE-aptamer decreased expression levels of proliferating nuclear antigen, CD31 and Mac-3, markers of endothelial cells and macrophages, respectively. AGE-aptamer significantly decreased the number of tumor-associated vessels. AGE, receptor for AGE (RAGE) and vascular endothelial growth factor levels were also reduced in AGE-aptamer-treated G361 melanoma. AGE-aptamer inhibited the AGE-induced proliferation and tube formation of endothelial cells as well as the growth of G361 cells in vitro. The present findings suggest that AGE-aptamer could inhibit the AGE-RAGE axis in G361 melanoma and resultantly suppress the tumor growth in nude mice by blocking the angiogenesis. AGE-aptamer might be a novel therapeutic strategy for preventing the progression of malignant melanoma in diabetes.

Pigment epithelium-derived factor improves metabolic derangements and ameliorates dysregulation of adipocytokines in obese type 2 diabetic rats.

Oxidative stress and inflammation in the adipose tissues contribute to the metabolic syndrome. Pigment epithelium-derived factor (PEDF) inhibits vascular inflammation through its anti-oxidative properties. However, it remains unclear whether PEDF could suppress adipocyte inflammation. We investigated the effects of long-term administration or suppression of PEDF on adipocyte inflammation and metabolic derangements in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes with insulin resistance. Circulating and adipose tissue PEDF levels were increased as OLETF rats became more obese and insulin resistant. Long-term administration of PEDF improves metabolic parameters, ameliorates dysregulation of adipocytokines, and suppresses NADPH oxidase-induced oxidative stress and macrophage infiltration in the adipose tissues of OLETF rats, whereas these variables are exacerbated by the knockdown of PEDF by administering siRNAs. Our study suggests that PEDF could improve metabolic derangements by suppressing the inflammatory and oxidative reactions in adipose tissues of OLETF rats. PEDF levels may be elevated as a countersystem against obesity-related metabolic derangements.

DNA aptamer raised against advanced glycation end products inhibits neointimal hyperplasia in balloon-injured rat carotid arteries.

BACKGROUND: Advanced glycation end products (AGE) and their receptor (RAGE) interaction elicit inflammatory and proliferative reactions in arteries, thus playing a role in cardiovascular disease. We have recently found that high-affinity DNA aptamer directed against AGE (AGE-aptamer) prevents the progression of experimental diabetic nephropathy by blocking the harmful actions of AGEs in the kidney. However, effects of AGE-aptamer on vascular injury remain unknown. In this study, we examined whether and how AGE-aptamer inhibits neointimal hyperplasia in balloon-injured rat carotid arteries. METHODS: Male Wistar rats (weighting ca. 400 g at 11 weeks old) were anesthetized with sodium pentobarbital. The left common carotid artery was balloon-injured 3 times with 2F Fogaty catheter inserted through the femoral artery. Then the rats received continuous intraperitoneal infusion (3 µg/day) of either AGE-aptamer or control-aptamer by an osmotic mini pump for 2 weeks. 14 days after the procedure, the left common carotid arteries were excised for morphometric, immunohistochemical and western blot analyses. RESULTS: Compared with control-aptamer, AGE-aptamer significantly suppressed neointima formation after balloon injury and reduced AGE accumulation, oxidative stress generation, proliferation cell nuclear antigen-positive area, macrophage infiltration, RAGE and platelet-derived growth factor-BB (PDGF-BB) expression levels in balloon-injured carotid arteries. CONCLUSION: The present study suggests that AGE-aptamer could prevent balloon injury-induced neointimal hyperplasia by reducing PDGF-BB and macrophage infiltration via suppression of the AGE-RAGE-mediated oxidative stress generation. AGE-aptamer might be a novel therapeutic strategy for suppressing neointima formation after balloon angioplasty.

Beneficial effects of vildagliptin on retinal injury in obese type 2 diabetic rats.

BACKGROUND/AIMS: Vildagliptin is an oral inhibitor of dipeptidyl peptidase-4, an enzyme mainly responsible for inactivating incretins, and one of the widely used drugs for the treatment of type 2 diabetes. However, effects of vildagliptin on retinal injury in diabetes remain unclear. We examined here whether oral administration of vildagliptin inhibited gene expression of inflammatory and thrombogenic parameters in Otsuka Long-Evans Tokushima Fatty rats (OLETF rats), an animal model of obese type 2 diabetes. METHODS: OLETF rats at 22 weeks of age were given vehicle or 3 mg/kg of vildagliptin for another 10 weeks. Gene expression was analyzed in quantitative real-time reverse transcription-polymerase chain reaction. RESULTS: Vildagliptin significantly inhibited the increase in body weight and decreased average fasting blood glucose in the OLETF rats. Compared with 22-week-old OLETF rats, gene expression levels of vascular endothelial growth factor, intercellular adhesion molecule-1, plasminogen activator inhibitor-1 and pigment epithelium-derived factor were significantly increased in the retinas of OLETF rats at 32 weeks of age, all of which were inhibited by treatment with vildagliptin. CONCLUSIONS: The present study demonstrated for the first time that vildagliptin inhibited inflammatory and thrombogenic reactions in the retinas of obese type 2 diabetic rats. Vildagliptin may play a protective role against diabetic retinopathy.

Glucagon-like peptide-1 receptor agonist inhibits asymmetric dimethylarginine generation in the kidney of streptozotocin-induced diabetic rats by blocking advanced glycation end product-induced protein arginine methyltranferase-1 expression.

Advanced glycation end products (AGEs) and their receptor (RAGE) play a role in diabetic nephropathy. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, contributes to diabetic nephropathy. We have found that glucagon-like peptide-1 (GLP-1) inhibits the AGE-induced inflammatory reactions in endothelial cells. However, effects of GLP-1 on the AGE-RAGE-ADMA axis are unknown. This study examined the effects of GLP-1 on reactive oxygen species (ROS) generation, gene expression of protein arginine methyltransfetase-1 (PRMT-1), an enzyme that mainly generates ADMA, and ADMA levels in human proximal tubular cells. Streptozotocin-induced diabetic rats received continuous i.p. infusion of 0.3 µg of vehicle or 1.5 µg of the GLP-1 analog exendin-4 per kilogram of body weight for 2 weeks. We further investigated whether and how exendin-4 treatment reduced ADMA levels and renal damage in streptozotocin-induced diabetic rats. GLP-1 inhibited the AGE-induced RAGE and PRMT-1 gene expression, ROS, and ADMA generation in tubular cells, which were blocked by small-interfering RNAs raised against GLP-1 receptor. Exendin-4 treatment decreased gene expression of Rage, Prmt-1, Icam-1, and Mcp-1 and ADMA level; reduced urinary excretions of 8-hydroxy-2'-deoxyguanosine and albumin; and improved histopathologic changes of the kidney in diabetic rats. Our present study suggests that GLP-1 receptor agonist may inhibit the AGE-RAGE-mediated ADMA generation by suppressing PRMT-1 expression via inhibition of ROS generation, thereby protecting against the development and progression of diabetic nephropathy.

Glucagon-like peptide-1 inhibits angiotensin II-induced mesangial cell damage via protein kinase A.

There is a growing body of evidence that renin-angiotensin system plays a role in diabetic nephropathy. Recently, we have found that glucagon-like peptide-1 (GLP-1), one of the incretins, a gut hormone secreted from L cells in the intestine in response to food intake, inhibits advanced glycation end product-induced monocyte chemoattractant protein-1 gene expression in mesangial cells thorugh the interaction with the receptor of GLP-1. However, effects of GLP-1 on angiotensin II-exposed mesangial cells are unknown. This study investigated whether and how GLP-1 blocked the angiotensin II-induced mesangial cell damage in vitro. GLP-1 completely blocked the angiotensin II-induced superoxide generation, NF-κB activation, up-regulation of mRNA levels of intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 in mesangial cells, all of which were prevented by the treatments with H-89, an inhibitor of protein kinase A. The present results demonstrated for the first time that GLP-1 blocked the angiotensin II-induced mesangial cell injury by inhibiting superoxide-mediated NF-κB activation via protein kinase C pathway. Our present study suggests that strategies to enhance the biological actions of GLP-1 may be a promising strategy for the treatment of diabetic nephropathy.

Participation of CD11b and F4/80 molecules in the conjunctival eosinophilia of experimental allergic conjunctivitis.

BACKGROUND: CD11b and F4/80 are macrophage surface markers. How these molecules participate in allergic eosinophil infiltration remains unclear. We examined the roles CD11b and F4/80 play in the conjunctival eosinophil infiltration associated with experimental allergic conjunctivitis. METHODS: Ragweed-immunized BALB/c mice were challenged with ragweed in eye drops to induce conjunctival eosinophil infiltration. The effect of challenge on conjunctival CD11b+ and F4/80+ cell numbers was determined by immunohistochemistry. In the same model, blocking anti-CD11b and anti-F4/80 Abs were injected intraperitoneally during the induction or the effector phase, or subconjunctivally 2 h before challenge, to determine their effect on challenge-induced conjunctival eosinophilia. To examine whether eosinophils express CD11b and F4/80 molecules, splenocytes from IL-5 gene-electroporated mice were subjected to flow cytometric analysis. To clarify the involvement of CD11b and F4/80 in conjunctival eosinophil infiltration, mice were intraperitoneally injected with anti-CD11b and anti-F4/80 Abs and then subconjunctivally injected with eotaxin to induce conjunctival eosinophilia. RESULTS: Ragweed challenge elevated conjunctival CD11b+ and F4/80+ cell numbers. Systemic anti-CD11b and anti-F4/80 Ab treatments during the effector phase, but not in either the induction phase or the local injection of Ab, suppressed conjunctival eosinophil infiltration in ragweed-induced conjunctivitis. Most splenic eosinophils from IL-5 gene-introduced mice expressed CD11b and F4/80. Systemic anti-CD11b and anti-F4/80 Ab treatment suppressed conjunctival eosinophilia induced by subconjunctival eotaxin injection. CONCLUSIONS: CD11b and F4/80 appear to participate in conjunctival eosinophil infiltration in allergic conjunctivitis. Their involvement in conjunctival eosinophilia appears to be due to their expression on eosinophils rather than on macrophages.

Notch ligand Delta-like4 inhibits the development of murine experimental allergic conjunctivitis.

BACKGROUND: To investigate the involvement of Notch ligands in the development of experimental allergic conjunctivitis (EC) in mice. METHODS: To induce EC, wild-type (WT) or IFN-gamma-deficient (GKO) BALB/c mice were immunized with ragweed (RW) in alum followed by RW challenge in eye drops. Twenty-four hours after RW challenge, the conjunctivas, spleens and blood were harvested to evaluate conjunctival eosinophil infiltration, RW-specific cytokine production and serum Ig levels, respectively. Abs against Notch ligands (anti-Jagged1, anti-Jagged2, anti-Delta-like (Dll)1 and anti-Dll4) were injected intraperitoneally into EC-developing mice during the induction or effector phase. As a control, normal hamster IgG (nhIgG) was injected. RESULTS: Treatment with anti-Dll4 Ab but not the other Abs during the induction phase significantly augmented the severity of EC, as measured by the conjunctival eosinophil infiltration. Anti-Dll4 Ab treatment also significantly upregulated RW-recall IL-4 production and suppressed serum IgE and IgG1 levels. However, anti-Dll4 Ab treatment during the induction phase did not significantly affect the severity of EC in GKO mice. None of the Abs significantly affected the severity of EC, splenocyte cytokine production, or serum Ig levels when administered during the effector phase. CONCLUSIONS: These observations suggest that Dll4, a Notch ligand, plays a role in suppressing the development of EC, possibly by providing a negative signal for Th2 development during the induction phase. In addition, IFN-gamma may participate in the augmentation of EC by anti-Dll4 Ab treatment.

CD27 and CD70 do not play a critical role in the development of experimental allergic conjunctivitis in mice.

CD27, which belongs to the TNF receptor family, is a costimulatory molecule that participates in T-cell activation. Unlike costimulatory molecules such as OX40 and 4-1BB, little is known about the role CD27 plays a role in the development of experimental diseases. We asked whether CD27 and its ligand CD70 participate in the development of experimental allergic conjunctivitis (EC) in BALB/c mice, which is generated by immunization with ragweed (RW) in alum and challenged 10 days later with RW in eye drops. The roles of CD27 and CD70 were tested by intraperitoneally injecting the mice with anti-CD27, anti-CD70 or a control Ab during the induction or effector phase. Twenty-four hours after challenge, the conjunctivas, blood and spleens were harvested for histological analysis, measuring Ig levels and cytokine analysis, respectively. Regardless of when the mice were treated, anti-CD27 or anti-CD70 Ab treatment did not significantly affect the severity of EC as evaluated by conjunctival eosinophil numbers. However, anti-CD27 or anti-CD70 Ab treatment during the induction phase did significantly modulate systemic humoral and cellular immune responses. In vitro treatment of RW-primed splenocytes with anti-CD27 or anti-CD70 Ab did not affect the EC-inducing capability of the splenocytes. Taken together, CD27 and CD70 do not play a critical role in the development of EC.

Depletion of thymus-derived CD4+CD25+ T cells abrogates the suppressive effects of alpha-galactosylceramide treatment on experimental allergic conjunctivitis.

BACKGROUND: We showed previously that alpha-galactosylceramide (alpha-GalCer) treatment elevated splenic CD4+CD25+Foxp3+ T-cell numbers and suppressed the development of experimental allergic conjunctivitis (EC). Here, we investigated whether CD4+CD25+Foxp3+ T cells mediate the suppressive effects of alpha-GalCer treatment on EC. METHODS: To deplete CD4+CD25+Foxp3+ T cells, neonatal mice were thymectomized and intraperitoneally injected with anti-CD25 Ab. At 6 weeks of age, these mice were immunized with ragweed (RW) in aluminum hydroxide. Ten days later, the mice were challenged with RW in eye drops and 24 hours later, the conjunctivas and spleens were harvested for histological and flow cytometric analyses, respectively. alpha-GalCer or vehicle was injected 2 hours prior to RW challenge. In addition, alpha-GalCer was injected into thymus-intact EC-developing mice that had not been treated with anti-CD25 Ab. RESULTS: alpha-GalCer treatment significantly suppressed EC in the thymus-intact mice that had not been treated with anti-CD25 Ab. In contrast, alpha-GalCer treatment of thymectomized and anti-CD25 Ab-treated mice did not affect the severity of EC or splenic CD4+CD25+Foxp3+ T-cell numbers. However, alpha-GalCer treatment did significantly increase splenic CD4+CD25+Foxp3+ T-cell numbers in thymectomized mice that had not received anti-CD25 Ab. CONCLUSIONS: alpha-GalCer treatment during the effector phase of EC increased CD4+CD25+Foxp3+ T-cell numbers, which in turn suppressed the development of EC.