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Pro-inflammatory conditions induced by products of protein glycation in diabetes substantially enhance the risk of endothelial dysfunction and related vascular complications. Endothelial cell specific molecule-1 (ESM-1) or endocan has been demonstrated as a potential biomarker in cancer and sepsis. Its role in diabetes-induced pathologies remains unknown. The expression of ESM-1 gene is under cytokine regulation, indicating its role in endothelium-dependent pathological disorders. In this study, we investigated the effect of advanced glycated human serum albumin (AGE-HSA) on the production of ESM-1. We show that AGE-HSA exerts a modulating role on the expression of ESM-1 in human umbilical vein endothelial cells. It up-regulates expression of ESM-1 protein in a dose-dependent manner which correlates with its messenger RNA (mRNA) transcription. RAGE and galectin-3, both AGE receptors, show antagonistic action on its expression. While gene silencing of RAGE has down-regulatory effect, that of galectin-3 has up-regulatory effect on AGE-induced expression of ESM-1. Inhibition of MAPKKK and JNK pathways did not alter the expression. In contrast, phosphatidylinositol 3 kinase (PI3K) inhibition significantly up-regulated ESM-1 expression. In conclusion, these results suggest that AGE-induced activation of human umbilical vein endothelial cells promotes formation of endocan which is an endothelial dysfunction marker and may be related to vascular disease in diabetes., (© The Author(s) 2015.)

Objective: Alternative medicine or herbal therapies have been in use for blood glucose control in patients with diabetes for considerable time. Effect of garlic, more specifically its biologically active component s-allyl cysteine, on amelioration of hyperglycemia has also been reported. However, the cellular or molecular target of this compound is little known. This study was designed to determine the action of s-allyl cysteine sulfoxide (SACSO) at the cellular and molecular level in the widely reported HUVEC model when activated with advanced glycation end products (AGEs)., Materials and Methods: AGE-HSA was derived from non-enzymatic glycation of human serum albumin in the presence of 20 mM glucose. AGE-HSA induced expression of receptors of AGEs, namely RAGE and galectin-3 has been assayed. Activity of endothelial nitric oxide synthase (NOS) denoting normal endothelial function and expression of AGE-RAGE triggered inflammatory marker sICAM-1 is also evaluated in the presence or absence of SACSO., Results: In presence of SACSO, AGE-HSA induced expression of RAGE was down-regulated, galectin-3 was significantly up-regulated, NOS activity was enhanced and sICAM-1 expression was reduced., Conclusions: The data suggest that SACSO exerts an attenuating effect on 20 mM glucose derived AGE-HSA induced inflammation, by selectively inhibiting RAGE while stimulating galectin-3 expression. The former triggers inflammatory pathways while the latter sequesters AGE molecules and prevents AGE-RAGE engagement. This may form the basis for its therapeutic action.

The accelerated non-enzymatic modification of proteins by Maillard reaction during prolonged hyperglycemia is a key player in the diabetes associated pathology. In addition, hypoxia has been implicated in the recent past as a modulating factor. Therefore we have examined the interaction of glycation modified human serum albumin (AGE-HSA) and deferoxamine (DFO) mimicked hypoxia on the expression of hypoxia inducible factor 1α (HIF-1α), and the role of RAGE (receptor for AGE) signaling in up-regulation of HIF-1α. Expression of VEGF (a downstream target of HIF-1α) and sICAM-1 (inflammatory marker) was also detected. When HUVEC were subjected to hypoxia, highest expression of HIF-1α was observed. When treated with AGE-HSA at two concentrations, higher expression was found vis-a-vis control, with 0.2 mg ml(-1) than 2.0 mg ml(-1) which was mediated in part by RAGE as determined by RAGE silencing. However, when the cells were exposed to a combination treatment of hypoxia and AGE-HSA, a biphasic effect at the two different concentrations was observed as compared to the individual treatments. VEGF was synergistically up-regulated by hypoxia and AGE-HSA. On the other hand sICAM-1 was up-regulated by AGE-HSA but down-regulated by hypoxia. These results show that AGE-HSA functions as a non-hypoxic factor which modulates the expression of HIF-1α in a concentration dependent manner in the range studied. It can be concluded that glycated serum proteins may activate HIF-1α independently in diabetes. Further, when both glycated proteins and hypoxic conditions are present, they act in opposition in regulation of HIF-1α.

Advanced glycation end products (AGEs) are known to be involved in the pathogenesis of several diseases, in particular diabetes, via signaling through their receptor. Numerous studies have been carried out on protein-sugar interactions at very high concentrations of the latter. The objective of this investigation was to determine the effects of nonenzymatic glycation induced by reducing sugars on the secondary structure of human serum albumin (HSA) under different physiological conditions and to correlate that with expression of RAGE (receptor for advanced glycation end products) on HUVECs (human umbilical vein endothelial cells) in a controlled hemodynamic environment. Our results indicate that RAGE expression is shear stress modulated and that glycated HSA enhances the expression further. The secondary structure of AGE-HSA derived from glucose at 20 mM contains higher α-helical content and elicits maximum expression of the receptor. The effect of shear stress at 10 dynes cm(-2) is independent of AGE-HSA., (This journal is © The Royal Society of Chemistry 2011)

Human umbilical vein endothelial cells (HUVEC) were cultured in two different media, viz. the commonly used M199 containing 20% fetal bovine serum (FBS) and endothelial cell growth factor and a defined media EGM-2 containing 2% FBS along with growth supplements in known concentrations. The purpose of this study was to determine the effect of different media on the growth potential and cell morphology in subsequent passages. We have established that a dual coating of gelatin and human fibronectin extracellular matrix provides optimal cell attachment. Growth rate for primary culture was almost double in defined media. For secondary culture a two fold higher proliferation rate was observed in defined EGM-2 media. Histological studies were done using phase contrast, confocal and scanning electron microscopy which showed that cells cultured in M199 started losing their morphological characteristic from 3rd passage and after 6th passage appeared to come in senescent stage, while in case of defined media there was no change observed in the cells up to 10th passage. A significant difference was found in the expression of soluble intracellular adhesion molecule-1 (sICAM-1) which is an endothelial cell marker on cells cultured in different media. Additionally it was observed that exposure duration to trypsin-EDTA during cell detachment also plays an important role in maintaining cell morphological characteristics. These results show that significant morphological changes appear in higher order passages if cells are grown in routine medium for a long time and therefore may not be suitable for cell signaling experiments., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Accurate estimation of non-transferrin bound iron (NTBI) is an important tool in monitoring effects of chemotherapy and iron chelation therapy in various conditions of iron overload and transfusion related thalassemias. A key factor in its estimation, is its extraction from putative ligands in the serum, barring transferrin and ferritin and blocking of unsaturated binding sites of the same. The molecular interactions between azotobactin and blocking agents Co(3+) and Ga(3+) were studied by UV/visible spectrophotometry. The role of different mobilizing agents in modulating Fe(3+) binding to Azotobactin was monitored with fluorescence emission studies. The fluorescence spectrum of Azotobactin is Exc(lambda) 380 nm/Em(lambda) 490 nm. In the presence of Ga(3+), the emission peak underwent a blue shift to 465 nm with a significant decrease in the intensity, whereas, Co(3+) did not show any shift or decrease in the fluorescence emission spectrum. With the addition of EDTA to the azotobactin-Fe (Az-Fe) complex, there is an immediate regain in the fluorescence of azotobactin whereas addition of nitrilotriacetate (NTA) did not show any regain in the fluorescence. Results illustrate that the citrate complex of cobalt and NTA are suitable blocking and mobilizing agents in the azotobactin assay of NTBI in biological fluids like human serum, since they do not affect either the spectroscopic properties of azotobactin or the binding kinetics of azotobactin and Fe(3+).

We report the synthesis of gold and silver nanostructures embedded in different dielectric matrices by atom beam co-sputtering, a novel technique. We have synthesized gold-silicon core shell nanostructures and Au-ZnO nanocomposite with tunable surface plasmon resonance (SPR) by atom beam co-sputtering and subsequent annealing. The Au-ZnO nanocomposite shows significant enhancement in intensity of Raman modes of fullerene molecules and therefore can help in surface enhanced Raman spectroscopy investigation of organic molecules. The synthesized Ag-polymer nanocomposite thin films show excellent features of broad SPR absorption extending upto IR region and a narrow transmission of light in UV region approximately 320 nm which could be of technological interest in solar absorbers and UV light filters respectively. The Ag-silica nanocomposite thin films show their utility in glucose sensing. The gold-silica nanocomposite thin films exhibit their possible use in detection of human ovarian cancer cells in a preliminary study. The shift in SPR peak of Au nanoparticles (NPs) present at the surface of silica synthesized by thermal evaporation and annealing, after attachment of biological molecules like proteins has been studied.

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe3+ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 microM (0.375+/-0.028 microM [means+/-SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson's correlation coefficients were found to be 0.6074 (P<0.0001) and 0.6102 (P<0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.