DNA Aptamer Raised against Advanced Glycation End Products Improves Sperm Concentration, Motility, and Viability by Suppressing Receptors for Advanced Glycation End Product-Induced Oxidative Stress and Inflammation in the Testes of Diabetic Mice.

Type 2 diabetes mellitus (T2DM) is a risk factor for male infertility, but the underlying molecular mechanisms remain unclear. Advanced glycation end products (AGEs) are pathogenic molecules for diabetic vascular complications. Here, we investigated the effects of the DNA aptamer raised against AGEs (AGE-Apt) on testicular and sperm abnormalities in a T2DM mouse model. KK-Ay (DM) and wild-type (non-DM) 4- and 7-week-old male mice were sacrificed to collect the testes and spermatozoa for immunofluorescence, RT-PCR, and histological analyses. DM and non-DM 7-week-old mice were subcutaneously infused with the AGE-Apt or control-aptamer for 6 weeks and were then sacrificed. Plasma glucose, testicular AGEs, and Rage gene expression in 4-week-old DM mice and plasma glucose, testicular AGEs, oxidative stress, and pro-inflammatory gene expressions in 7-week-old DM mice were higher than those in age-matched non-DM mice, the latter of which was associated with seminiferous tubular dilation. AGE-Apt did not affect glycemic parameters, but it inhibited seminiferous tubular dilation, reduced the number of testicular macrophages and apoptotic cells, and restored the decrease in sperm concentration, motility, and viability of 13-week-old DM mice. Our findings suggest that AGEs-Apt may improve sperm abnormality by suppressing AGE-RAGE-induced oxidative stress and inflammation in the testes of DM mice.

SMTP-44D Inhibits Atherosclerotic Plaque Formation in Apolipoprotein-E Null Mice Partly by Suppressing the AGEs-RAGE Axis.

SMTP-44D has been reported to have anti-oxidative and anti-inflammatory reactions, including reduced expression of receptor for advanced glycation end products (RAGE) in experimental diabetic neuropathy. Although activation of RAGE with its ligands, and advanced glycation end products (AGEs), play a crucial role in atherosclerotic cardiovascular disease, a leading cause of death in diabetic patients, it remains unclear whether SMTP-44D could inhibit experimental atherosclerosis by suppressing the AGEs-RAGE axis. In this study, we investigated the effects of SMTP-44D on atherosclerotic plaque formation and expression of AGEs in apolipoprotein-E null (Apoe-/-) mice. We further studied here whether and how SMTP-44D inhibited foam cell formation of macrophages isolated from Apoe-/- mice ex vivo. Although administration of SMTP-44D to Apoe-/- mice did not affect clinical or biochemical parameters, it significantly decreased the surface area of atherosclerotic lesions and reduced the atheromatous plaque size, macrophage infiltration, and AGEs accumulation in the aortic roots. SMTP-44D bound to immobilized RAGE and subsequently attenuated the interaction of AGEs with RAGE in vitro. Furthermore, foam cell formation evaluated by Dil-oxidized low-density lipoprotein (ox-LDL) uptake, and gene expression of RAGE, cyclin-dependent kinase 5 (Cdk5) and CD36 in macrophages isolated from SMTP-44D-treated Apoe-/- mice were significantly decreased compared with those from saline-treated mice. Gene expression levels of RAGE and Cdk5 were highly correlated with each other, the latter of which was also positively associated with that of CD36. The present study suggests that SMTP-44D may inhibit atherosclerotic plaque formation in Apoe-/- mice partly by blocking the AGEs-RAGE-induced ox-LDL uptake into macrophages via the suppression of Cdk5-CD36 pathway.

Complex Formation of Heme Oxygenase-2 with Heme Is Competitively Inhibited by the Cytosolic Domain of Caveolin-1.

The mechanism and physiological functions of heme oxygenase-2 (HO-2)-mediated carbon monoxide (CO) production, accompanied by heme metabolism, have been studied intensively in recent years. The enzymatic activity of constitutively expressed HO-2 must be strictly controlled in terms of the toxicity and chemical stability of CO. In this study, the molecular interaction between HO-2 and caveolin-1 and its effect on HO action were evaluated. An enzyme kinetics assay with residues 82-101 of caveolin-1, also called the caveolin scaffold domain, inhibited HO-2 activity in a competitive manner. Analytical ultracentrifugation and a hemin titration assay suggested that the inhibitory effect was generated by direct binding of caveolin-1 to aromatic residues, which were defined as components of the caveolin-binding motif in the HO-2 heme pocket. Herein, we developed a HO-2-based fluorescence bioprobe, namely EGFP-Δ19/D159H, which was capable of quantifying heme binding by HO-2 as the initial step in the CO production. The fluorescence of EGFP-Δ19/D159H decreased in accordance with 5-aminolevulinic acid-facilitated heme biosynthesis in COS-7 cells. In contrast, expression of the N-terminal cytosolic domain of caveolin-1 (residues 1-101) increased the probe fluorescence, suggesting that the cytosolic domain of caveolin-1 potently inhibits the binding of heme to the heme pocket of EGFP-Δ19/D159H. Taken together, our results suggest that caveolin-1 is a negative regulator of HO-2 enzymatic action. Moreover, our bioprobe EGFP-Δ19/D159H represents a powerful tool for use in future studies addressing HO-2-mediated CO production.

Glyceraldehyde-Derived Pyridinium Evokes Renal Tubular Cell Damage via RAGE Interaction.

Glyceraldehyde-derived advanced glycation end products (glycer-AGEs) contribute to proximal tubulopathy in diabetes. However, what glycer-AGE structure could evoke tubular cell damage remains unknown. We first examined if deleterious effects of glycer-AGEs on reactive oxygen species (ROS) generation in proximal tubular cells were blocked by DNA-aptamer that could bind to glyceraldehyde-derived pyridinium (GLAP) (GLAP-aptamer), and then investigated whether and how GLAP caused proximal tubular cell injury. GLAP-aptamer and AGE-aptamer raised against glycer-AGEs were prepared using a systemic evolution of ligands by exponential enrichment. The binding affinity of GLAP-aptamer to glycer-AGEs was measured with a bio-layer interferometry. ROS generation was evaluated using fluorescent probes. Gene expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). GLAP-aptamer bound to glycer-AGEs with a dissociation constant of 7.7 × 10-5 M. GLAP-aptamer, glycer-AGE-aptamer, or antibodies directed against receptor for glycer-AGEs (RAGE) completely prevented glycer-AGE- or GLAP-induced increase in ROS generation, MCP-1, PAI-1, or RAGE gene expression in tubular cells. Our present results suggest that GLAP is one of the structurally distinct glycer-AGEs, which may mediate oxidative stress and inflammatory reactions in glycer-AGE-exposed tubular cells. Blockade of the interaction of GLAP-RAGE by GLAP-aptamer may be a therapeutic target for proximal tubulopathy in diabetic nephropathy.

Advanced glycation end products evoke inflammatory reactions in proximal tubular cells via autocrine production of dipeptidyl peptidase-4.

We have previously shown that albuminuria and renal levels of advanced glycation end products (AGEs), receptor for AGEs (RAGE), and oxidative stress are suppressed in dipeptidyl peptidase-4 (DPP-4)-deficient diabetic rats, thus suggesting the crosstalk between AGE-RAGE axis and DPP-4 in experimental diabetic nephropathy. Therefore, we examined here the role of DPP-4 in AGE-evoked inflammatory reactions in human proximal tubular cells. Proteins were extracted from proximal tubular cells, and conditioned medium was collected, both of which were subjected to western blot analysis using anti-DPP-4 antibody. RAGE-aptamer was prepared using a systemic evolution of ligands by exponential enrichment. NF-κB p65 and monocyte chemoattractant protein-1 (MCP-1) gene expression was analyzed by reverse transcription-polymerase chain reaction. AGEs significantly increased DPP-4 expression and soluble DPP-4 production by tubular cells, the latter of which was attenuated by RAGE-aptamer or an anti-oxidant, N-acetylcysteine. AGEs or DPP-4 up-regulated NF-κB p65 or MCP-1 mRNA levels in tubular cells, which were suppressed by linagliptin, an inhibitor of DPP-4. AGEs stimulated NF-κB p65 gene expression in tubular cells isolated from control rats, but not from DPP-4-deficient rats. Our present results suggest that the AGE-RAGE-mediated oxidative stress could evoke inflammatory reactions in proximal tubular cells via autocrine production of DPP-4.

RAGE-aptamer Attenuates the Growth and Liver Metastasis of Malignant Melanoma in Nude Mice.

Epidemiological studies have suggested the link between cumulative diabetic exposure and cancer. Interaction of advanced glycation end products (AGEs) with their receptor (RAGE) may contribute to the phenomenon. We examined here the effects of DNA aptamer raised against RAGE (RAGE-aptamer) on growth and liver metastasis of G361 melanoma in nude mice. Malignant melanoma cells were intradermally injected into the upper flank region of nude mice, which received continuous administration of RAGE-aptamer (38.4 pmol/day/g body weight) or vehicle intraperitoneally by an osmotic pump up to 42 days. RAGE-aptamer significantly reduced levels of 8-hydroxy-2'-deoxy-guanosine, AGEs, RAGE, proliferating nuclear antigen, cyclin D1, vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), and CD31 and Mac-3, respective markers of endothelial cells and macrophages in tumors of nude mice and suppressed the proliferation and liver metastasis of malignant melanoma. Furthermore, RAGE-aptamer attenuated the AGE-induced oxidative stress generation, proliferation, and VEGF and MCP-1 gene expression in both G361 melanoma cells and endothelial cells. The present findings suggest that RAGE-aptamer could attenuate melanoma growth and liver metastasis in nude mice by suppressing the tumor angiogenesis and macrophage infiltration via inhibition of the AGE-RAGE system. RAGE-aptamer may be a novel therapeutic tool for the treatment of malignant melanoma.

Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase.

NADPH-cytochrome P450 oxidoreductase (CPR) supplies electrons to various heme proteins including heme oxygenase (HO), which is a key enzyme for heme degradation. Electrons from NADPH flow first to flavin adenine dinucleotide, then to flavin mononucleotide (FMN), and finally to heme in the redox partner. For electron transfer from CPR to its redox partner, the ''closed-open transition'' of CPR is indispensable. Here, we demonstrate that a hinge-shortened CPR variant, which favors an open conformation, makes a stable complex with heme-HO-1 and can support the HO reaction, although its efficiency is extremely limited. Furthermore, we determined the crystal structure of the CPR variant in complex with heme-HO-1 at 4.3-Å resolution. The crystal structure of a complex of CPR and its redox partner was previously unidentified. The distance between heme and FMN in this complex (6 Å) implies direct electron transfer from FMN to heme.

N-butanol extracts of Morinda citrifolia suppress advanced glycation end products (AGE)-induced inflammatory reactions in endothelial cells through its anti-oxidative properties.

BACKGROUND: Advanced glycation end products (AGEs), senescent macroprotein derivatives formed during a normal aging process and acceleratedly under diabetic conditions, play a role in atherosclerotic cardiovascular disease. AGEs cause endothelial cell (EC) damage, an initial trigger for atherosclerosis through the interaction with a receptor for AGEs (RAGE). We have previously shown that n-butanol extracts of Morinda citrifolia (noni), a plant belonging to the family Rubiaceae, block the binding of AGEs to RAGE in vitro. In this study, we examined the effects of n-butanol extracts of noni on reactive oxygen species (ROS) generation and inflammatory reactions on AGE-exposed human umbilical vein ECs (HUVECs). METHODS: HUVECs were treated with 100 µg/ml AGE-bovine serum albumin (AGE-BSA) or non-glycated BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni for 4 h. Then ROS generation and inflammatory and gene expression in HUVECs were evaluated by dihydroethidium staining and real-time reverse transcription-polymerase chain reaction analyses, respectively. THP-1 cell adhesion to HUVECs was measured after 2-day incubation of AGE-BSA or BSA in the presence or absence of 670 ng/ml n-butanol extracts of noni. RESULTS: N-butanol extracts of noni at 670 ng/ml significantly inhibited the AGE-induced ROS generation and RAGE, intercellular adhesion molecule-1 and plasminogen activator inhibitor-1 gene expressions in HUVECs. AGEs significantly increased monocytic THP-1 cell adhesion to HUVECs, which was also prevented by 670 ng/ml n-butanol extracts of noni. CONCLUSIONS: The present study demonstrated for the first time that N-butanol extracts of noni could suppress the AGE-induced inflammatory reactions in HUVECs through its anti-oxidative properties via blocking of the interaction of AGEs with RAGE. Inhibition of the AGE-RAGE axis by n-butanol extracts of noni may be a novel nutraceutical strategy for the treatment of cardiovascular disease.

Glyceraldehyde-derived pyridinium (GLAP) evokes oxidative stress and inflammatory and thrombogenic reactions in endothelial cells via the interaction with RAGE.

BACKGROUND: We have previously shown that serum levels of glyceraldehyde-derived advanced glycation end products (Gly-AGEs) are elevated under oxidative stress and/or diabetic conditions and associated with insulin resistance, endothelial dysfunction and vascular inflammation in humans. Further, Gly-AGEs not only evoke oxidative and inflammatory reactions in endothelial cells (ECs) through the interaction with a receptor for AGEs (RAGE), but also mimic vasopermeability effects of AGE-rich serum purified from diabetic patients on hemodialysis. These observations suggest that Gly-AGE-RAGE system might be a therapeutic target for vascular complications in diabetes. However, since incubation of glyceraldehyde with proteins will generate a large number of structurally distinct AGEs, it remains unclear what type of AGE structures could mediate the deleterious effects of Gly-AGEs on ECs. AIMS AND METHODS: Therefore, in this study, we examined (1) whether glyceraldehyde-derived pyridinium (GLAP), one of the Gly-AGEs generated by the incubation of lysine with glyceraldehyde, elicited reactive oxygen species (ROS) generation and inflammatory and thrombogenic gene expression in human umbilical vein ECs (HUVECs) via the interaction with RAGE and (2) if DNA aptamers raised against Gly-AGEs or GLAP (AGE-aptamer or GLAP-aptamer) inhibited the binding of GLAP to RAGE and subsequently suppressed the harmful effects of GLAP on HUVECs. RESULTS: GLAP stimulated ROS generation in a bell-shaped manner; GLAP at 10 µg/ml increased ROS generation in HUVECs by 40%, which was blocked by the treatment with RAGE-antibody (RAGE-Ab). Ten µg/ml GLAP significantly up-regulated mRNA levels of RAGE, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, vascular cell adhesion molecule-1 and plasminogen activator inhibitor-1 in HUVECs, which were also suppressed by RAGE-Ab. AGE-aptamer or GLAP-aptamer significantly blocked these deleterious effects of GLAP on HUVECs. Moreover, quartz crystal microbalance analyses revealed that GLAP actually bound to RAGE and that AGE-aptamer or GLAP-aptamer inhibited the binding of GLAP to RAGE. CONCLUSIONS: The present study suggests that GLAP might be a main glyceraldehyde-related AGE structure in Gly-AGEs that bound to RAGE and subsequently elicited ROS generation and inflammatory and thrombogenic reactions in HUVECs. Blockade of the GLAP-RAGE interaction by AGE-aptamer or GLAP-aptamer might be a novel therapeutic strategy for preventing vascular injury in diabetes.

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples.

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.

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.

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.

Laminin receptor mediates anti-inflammatory and anti-thrombogenic effects of pigment epithelium-derived factor in myeloma cells.

Pigment epithelium-derived factor (PEDF) has anti-inflammatory and anti-thrombogenic properties both in cell culture and animal models. Although adipose triglyceride lipase (ATGL) and laminin receptor (LR) are two putative receptors for PEDF, which receptor mainly mediates the beneficial effects of PEDF is largely unknown. In this study, we addressed the issue. siRNA raised against LR (siLR) and siATGL transfection dramatically decreased LR and ATGL levels in human cultured myeloma cells, respectively. Ten nM PEDF significantly reduced vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels in siCon- or siATGL-transfected myeloma cells, whereas PEDF increased rather than decreased these gene expressions in siLR-transfected cells. Neutralizing antibody directed against LR (LR-Ab) or LR antagonist actually bound to LR and reduced mRNA levels of VEGF, MCP-1, ICAM-1 and PAI-1 in myeloma cells. Further, pre-treatment of LR-Ab or LR antagonist suppressed the binding of PEDF to LR and resultantly blocked the effects of PEDF in myeloma cells. In addition, high concentration of LR agonist mimicked the actions of PEDF on these gene expressions in myeloma cells. This study indicates that PEDF causes anti-angiogenic, anti-inflammatory and anti-thrombogenic reactions in myeloma cells through the interaction with LR. Target domain of LR agonist and antagonist might be involved in the PEDF-signaling to gene suppression in myeloma cells.

EprS, an autotransporter protein of Pseudomonas aeruginosa, possessing serine protease activity induces inflammatory responses through protease-activated receptors.

PA3535 (EprS), an autotransporter (AT) protein of Pseudomonas aeruginosa, is predicted to contain a serine protease motif. The eprS encodes a 104.5 kDa protein with a 30-amino-acid-long signal peptide, a 51.2 kDa amino-terminal secreted passenger domain and a 50.1 kDa carboxyl-terminal outer membrane channel formed translocator. Although the majority of AT proteins have been reported to be virulence factors, little is known about the functions of EprS in the pathogenicity of P. aeruginosa. In this study, we performed functional analyses of recombinant EprS secreted by Escherichia coli. The proteolytic activity of EprS was markedly decreased by changing Ser to Ala at position 308 or by serine protease inhibitors. EprS preferred to cleave substrates that terminated with arginine or lysine residues. Thus, these results indicate that EprS, a serine protease, displays the substrate specificity, cleaving after basic residues. We demonstrated that EprS activates NF-κB-driven promoters through protease-activated receptor (PAR)-1, -2 or -4 and induces IL-8 production through PAR-2 in a human bronchiole epithelial cell line. Moreover, EprS cleaved the peptides corresponding to the tethered ligand region of PAR-1, -2 and -4 at a specific site with exposure oftheir tethered ligands. Collectively, these results suggest that EprS activates host inflammatory responses through PARs.

Evaluation of in vitro properties of predicted kinases that phosphorylate serine residues within nuclear localization signal 1 of high mobility group box 1.

Phosphorylation of high mobility group box 1 (HMGB1) is involved in the subcellular translocation of this protein and its subsequent secretion. Two nuclear localization signals (NLSs), NLS1 and NLS2, in this protein regulate its nucleocytoplasmic relocation, and phosphorylation of both NLSs strongly promotes HMGB1 mobilization. However, the phosphorylation properties of serine residues in NLS1 and the kinases involved are not well known. In the present study, we predicted kinases that phosphorylate serine residues in NLS1 and performed an in vitro kinase assay utilizing NLS1-derived phosphopeptides. Among the predicted kinases, protein kinase C phosphorylated Ser(46) of HMGB1-derived peptides, and a mutagenesis experiment confirmed that phosphorylation at this site could induce the translocation of the N-terminal region of NLS1-containing HMGB1 into the cytosol.

Caveolin-1 interacts with protein phosphatase 5 and modulates its activity in prostate cancer cells.

Caveolin-1 is highly expressed in prostate cancer cells, and is implicated in disease progression. Here, we identified protein phosphatase 5 (PP5) as a novel cellular binding partner of caveolin-1 using a pull-down approach in combination with mass spectrometry-based proteomic analyses. In situ proximity ligation assays demonstrated co-localization and physical interaction of caveolin-1 and PP5 in the cytoplasm of PC-3 human prostate cancer cells. Using yeast two-hybrid analysis, we found that caveolin-1 interacted with the catalytic domain of PP5. We also found that PP5 activity was elevated about 1.7-fold in the presence of 2 µM caveolin-1, and that the scaffolding domain of caveolin-1 is required for this activation. Our results suggest that caveolin-1 is a novel physiological activator of PP5.

Pigment epithelium-derived factor (PEDF) binds to caveolin-1 and inhibits the pro-inflammatory effects of caveolin-1 in endothelial cells.

Pigment epithelium-derived factor (PEDF) exerts atheroprotective effects both in cell culture and animal models through its anti-oxidative and anti-inflammatory properties. Caveolin-1 (Cav), a major protein component of caveolae in endothelial cells (ECs), plays a role in the progression of atherosclerosis. However, effects of PEDF on Cav-exposed ECs remain unknown. In this study, we examined whether and how PEDF could inhibit the Cav-induced inflammatory and thrombogenic reactions in human umbilical vein ECs (HUVECs). Surface plasmon resonance revealed that PEDF bound to Cav at the dissociation constant of 7.36×10(-7) M. Further, one of the major Cav-interacting proteins in human serum was identified as PEDF by peptide mass fingerprinting analysis using BIAcore 1000 combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Exogenously added Cav was taken up into the membrane fraction of HUVECs and dose-dependently increased monocyte chemoattractant protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1) and plasminogen activator inhibitor-1 (PAI-1) mRNA levels, all of which were blocked by the simultaneous treatment with 10nM PEDF. Small interfering RNAs directed against Cav decreased endogenous Cav levels and suppressed gene expression of MCP-1, VCAM-1 and PAI-1 in HUVECs. This study indicates that PEDF binds to Cav and could block the inflammatory and thrombogenic reactions in Cav-exposed HUVECs. Our present study suggests that atheroprotective effects of PEDF might be partly ascribed to its Cav-interacting properties.

Advanced glycation end products evoke endothelial cell damage by stimulating soluble dipeptidyl peptidase-4 production and its interaction with mannose 6-phosphate/insulin-like growth factor II receptor.

BACKGROUND: Advanced glycation end products (AGEs) and receptor RAGE interaction play a role in diabetic vascular complications. Inhibition of dipeptidyl peptidase-4 (DPP-4) is a potential therapeutic target for type 2 diabetes. However, the role of DPP-4 in AGE-induced endothelial cell (EC) damage remains unclear. METHODS: In this study, we investigated the effects of DPP-4 on reactive oxygen species (ROS) generation and RAGE gene expression in ECs. We further examined whether an inhibitor of DPP-4, linagliptin inhibited AGE-induced soluble DPP-4 production, ROS generation, RAGE, intercellular adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1) gene expression in ECs. RESULTS: DPP-4 dose-dependently increased ROS generation and RAGE gene expression in ECs, which were prevented by linagliptin. Mannose 6-phosphate (M6P) and antibodies (Ab) raised against M6P/insulin-like growth factor II receptor (M6P/IGF-IIR) completely blocked the ROS generation in DPP-4-exposed ECs, whereas surface plasmon resonance revealed that DPP-4 bound to M6P/IGF-IIR at the dissociation constant of 3.59 x 10⁻5 M. AGEs or hydrogen peroxide increased soluble DPP-4 production by ECs, which was prevented by N-acetylcysteine, RAGE-Ab or linagliptin. Linagliptin significantly inhibited the AGE-induced ROS generation, RAGE, ICAM-1 and PAI-1 gene expression in ECs. CONCLUSIONS: The present study suggests that AGE-RAGE-induced ROS generation stimulates the release of DPP-4 from ECs, which could in turn act on ECs directly via the interaction with M6P/IGF-IIR, further potentiating the deleterious effects of AGEs. The blockade by linagliptin of positive feedback loop between AGE-RAGE axis and DPP-4 might be a novel therapeutic target for vascular injury in diabetes.

Blockade by phosphorothioate aptamers of advanced glycation end products-induced damage in cultured pericytes and endothelial cells.

Advanced glycation end products (AGEs) not only inhibit DNA synthesis of retinal pericytes, but also elicit vascular hyperpermeability, pathological angiogenesis, and thrombogenic reactions by inducing vascular endothelial growth factor (VEGF) and plasminogen activator inhibitor-1 (PAI-1) through the interaction with the receptor for AGEs (RAGE), thereby being involved in the pathogenesis of diabetic retinopathy. In this study, we screened novel phosphorothioate-modified aptamers directed against AGEs (AGEs-thioaptamers) using a combinatorial chemistry in vitro, and examined whether these aptamers could inhibit the AGE-induced damage in both retinal pericytes and human umbilical vein endothelial cells (HUVECs). We identified 11 AGEs-thioaptamers; among them, clones #4, #7s and #9s aptamers had higher binding affinity to AGEs-human serum albumin (HSA) than the others. Surface plasmon resonance analysis revealed that KD values of #4s, #7s and #9s were 0.63, 0.36, and 0.57nM, respectively. Furthermore, these 3 clones dose-dependently restored the decrease in DNA synthesis in AGE-exposed pericytes. AGEs significantly increased RAGE, VEGF and PAI-1 mRNA levels in HUVEC, all of which were completely blocked by the treatment with 20nM clone #4s aptamer. Quartz crystal microbalance analysis confirmed that #4s aptamer dose-dependently inhibited the binding of AGEs-HSA to RAGE. Our present study demonstrated that AGEs-thioaptamers could inhibit the harmful effects of AGEs in pericytes and HUVEC by suppressing the binding of AGEs to RAGE. Blockade by AGEs-thioaptamers of the AGEs-RAGE axis might be a novel therapeutic strategy for diabetic retinopathy.

Development of a heme sensor using fluorescently labeled heme oxygenase-1.

Free heme, the protein-unbound form of heme, has both toxic and regulatory effects on cells. To detect free heme at low concentrations, we developed a heme sensor using fluorescently labeled heme oxygenase-1 (HO-1), an enzyme that catalyzes oxidative heme degradation and has a high affinity for heme. The response of the heme sensor is based on the fluorescence quenching that occurs when heme binds to the enzyme. Each of the three fluorescently labeled HO-1s exhibits a 1:1 binding stoichiometry and an absorption spectrum similar to that of the heme complex of the wild-type HO-1. Titration of the labeled proteins with hemin resulted in fluorescence quenching in a hemin concentration-dependent manner, presumably due to an energy transfer from the fluorophore to the heme bound to HO-1. The sensor showed a potent affinity for heme with a dissociation constant in the low nanomolar range and a high selectivity for heme. Based on the linear response of the sensor to heme, we performed a fluorometric microplate assay. The sensor was able to selectively detect free heme but did not respond to heme bound to native hemoglobin. This assay will be a useful tool for determination of free heme in biological samples containing protein-bound heme.