Nessar Ahmed

Ageing of the human corneal stroma: structural and biochemical changes

High and low angle X-ray diffraction patterns from the corneal stroma give information about the mean intermolecular spacing of the collagen molecules and the mean interfibrillar spacing of the collagen fibrils, respectively. X-ray data were collected, using a high intensity synchrotron source, from human corneas and sclera at approximately physiological hydration. The spacings were measured as a function of tissue age. Between birth and 90 years there is an increase in the cross-sectional area associated with each molecule in corneal collagen from approx. 3.04 nm2 to 3.46 nm2, and an increase in scleral collagen from approx. 2.65 nm2 to 3.19 nm2. These changes may be due to an increase in the extent of non-enzymic cross-linking between collagen molecules over the age range. We have investigated this possibility by measuring collagen glycation using the thiobarbituric acid assay and the subsequent advanced glycation end-products (AGEs) using fluorescence emission. The results obtained have shown an age-related increase in glycation and AGEs in both tissues. We have also demonstrated a decrease in the interfibrillar spacing of corneal collagen with increasing age which may be related to changes in the proteoglycan composition of the interfibrillar matrix.

Antiglycation Properties of Aged Garlic Extract: Possible Role in Prevention of Diabetic Complications

Diabetes mellitus is a common endocrine disorder characterized by hyperglycemia and long-term complications affecting the eyes, nerves, blood vessels, skin, and kidneys. Increased glycation of proteins and accumulation of advanced glycation endproducts (AGEPs) have been implicated in the pathogenesis of diabetic complications. Glycation and AGEP formation are also accompanied by formation of free radicals via autoxidation of glucose and glycated proteins. Compounds with combined antiglycation and antioxidant properties may offer therapeutic potential. Recent studies suggest that aged garlic extract (AGE) inhibits formation of AGEPs in vitro and formation of glycation-derived free radicals. S-Allylcysteine, a key component of aged garlic, is a potent antioxidant and can inhibit AGEP formation. Aged garlic extract and S-allylcysteine deserve more attention and should be investigated to see whether they can reduce AGEPs in vivo.

Natural Products as Anti-glycation Agents: Possible Therapeutic Potential for Diabetic Complications

Diabetes mellitus is characterised by hyperglycaemia, lipidaemia and oxidative stress and predisposes affected individuals to long-term complications afflicting the eyes, skin, kidneys, nerves and blood vessels. Increased protein glycation and the subsequent build-up of tissue advanced glycation endproducts (AGEs) contribute towards the pathogenesis of diabetic complications. Protein glycation is accompanied by generation of free radicals through autoxidation of glucose and glycated proteins and via interaction of AGEs with their cell surface receptors (referred to as RAGE). Glycationderived free radicals can damage proteins, lipids and nucleic acids and contribute towards oxidative stress in diabetes. There is interest in compounds with anti-glycation activity as they may offer therapeutic potential in delaying or preventing the onset of diabetic complications. Although many different compounds are under study, only a few have successfully entered clinical trials but none have yet been approved for clinical use. Whilst the search for new synthetic inhibitors of glycation continues, little attention has been paid to anti-glycation compounds from natural sources. In the last few decades the traditional system of medicine has become a topic of global interest. Various studies have indicated that dietary supplementation with combined anti-glycation and antioxidant nutrients may be a safe and simple complement to traditional therapies targeting diabetic complications. Data for forty two plants/constituents studied for anti-glycation activity is presented in this review and some commonly used medicinal plants that possess anti-glycation activity are discussed in detail including their active ingredients, mechanism of action and therapeutic potential.

Effect of glycation on basic fibroblast growth factor induced angiogenesis and activation of associated signal transduction pathways in vascular endothelial cells: Possible relevance to wound healing in diabetes

Ineffectual wound healing in hyperglycaemic patients suffering from diabetes mellitus is characterised by a reduction in capillary reformation (angiogenesis). Basic fibroblast growth factor (FGF-2) is secreted by fibroblasts, macrophages and in particular endothelial cells (EC) in response to tissue injury and is important in promotion of neovascularisation. Recently, glycation of FGF-2 has been shown to significantly reduce its activity in vitro. We have examined the kinetics of FGF-2 glycation and compared its ability with that of native FGF-2 to activate mitogenesis, capillary formation and associated signal transduction in bovine aortic EC (BAEC). FGF-2 was exposed to 0.25 M glucose-6-phosphate (G-6-P) for 24–72 h and the degree of glycation determined by matrix assisted laser desorption ionisation mass spectrometry. Native FGF-2 was heterogeneous with Mw in the range 15,153.6–17,903 Da. After 24 h incubation with G-6-P there was evidence of glycation, and the mass increase corresponded to addition of 2.7 mol of G-6-P residues; after 48 h, 4 mol sugar was added and this increased to 8.7 after 72 h. Dimerisation of FGF-2 was observed after 72 h of treatment. Induction of mitogenesis in BAEC was significantly reduced by 25%–40% after treatment for 48–96 h with glycated (24 h) FGF-2 (gFGF-2;100 pg/ml–5 ng/ml; P < 0.05), whilst capillary tubule formation was significantly reduced by between 60% and 90% (100 pg/ml–1 ng/ml; P < 0.05) after 5 days compared to native FGF-2. Subsequent investigation of the signal transduction molecules associated with mitogenesis showed a reduction in FGF-2 induced tyrosine phosphorylated proteins of approximate Mw 20–150 kDa between 10 min and 24 h, in particular, mitogen activated protein kinase (MAPK)/early response kinase (ERK-1, ERK-2), after glycation. To determine the reason for reduced angiogenic activity of gFGF-2, we compared its binding characteristics to that of native FGF-2. Total binding of gFGF-2 to the cell surface was significantly reduced in BAEC analysed by FACS compared to native FGF-2 (P < 0.05). Further investigation using 125I-labelled differentially washed samples, demonstrated a significant reduction in gFGF-2 binding to the high affinity tyrosine kinase receptor (46%) compared to native FGF-2. In summary, glycation of FGF-2 in vitro occurs rapidly within 24 h in the presence of elevated levels of G-6-P. Glycation caused a significant reduction in the ability of FGF-2 to bind to the tyrosine kinase receptor and activate signal transduction pathways responsible for both mitogenesis and capillary formation in BAEC. These results could help to explain the mechanism behind impaired wound healing in patients with diabetes mellitus.

Antioxidant and anti–glycation activities correlates with phenolic composition of tropical medicinal herbs

OBJECTIVE To determine the contribution of total phenolic content (TPC) in glycation inhibitory activity of common tropical medicinal food and spices with potential antioxidative properties. METHODS In vitro glucose-bovine serum albumin (BSA) assay was used. Ethanolic extracts of ten common household condiments/herbs (Allium sativum, Zingiber officinale, Thymus vulgaris, Petroselinum crispum, Murraya koenigii Spreng, Mentha piperita L., Curcuma longa L., Allium cepa L., Allium fistulosum and Coriandrum sativum L.) were evaluated for antioxidative activity by 2,2-diphenyl-2-picrylhydrazyl (DPPH), and ferric reducing antioxidant power (FRAP) and the TPC, flavonoid and tannins content were determined. RESULTS Findings showed good correlation between TPC/DPPH (r = 0.8), TPC/FRAP (r = 0.8), TPC/anti-glycation (r = 0.9), DPPH/anti-glycation (r = 0.6), FRAP/anti-glycation (r = 0.9), Flavonoid/anti-glycation (r = 0.7) and Tannins/anti-glycation (r = 0.8) and relatively fair correlation for TPC/Flavonoids (r = 0.5) and TPC/Tannins (r = 0.5). Results imply that these plants are potential sources of natural antioxidants which have free radical scavenging activity and might be used for reducing oxidative stress. CONCLUSIONS The positive glycation inhibitory and antioxidative activities of these tropical herbs suggest a possible role in targeting ageing, diabetic complications and oxidative stress related diseases.

Aged garlic extract protects against doxorubicin-induced cardiotoxicity in rats.

Clinical uses of doxorubicin (DOX), a highly active anticancer agent, are limited by its severe cardiotoxic side effects associated with increased oxidative stress and apoptosis. In this study we investigated whether aged garlic has protective effects against doxorubicin-induced free radical production and cardiotoxicity in male rats. A single dose of doxorubicin (25mg/kg) caused increased both serum cardiac enzymes LDH and CPK activities and a significant increase malonyldialdehyde (MDA) in plasma. However, pretreatment of rats with aged garlic extract (250 mg/kg) for 27 days before doxorubicin therapy, reduced the activity of both enzymes, and significantly decreased of MDA production in plasma. Total antioxidant activity was increased after aged garlic extract administration. Histopathological examination of heart tissue showed that DOX treatment resulted in alteration of cardiac tissue structure in the form of peri arterial fibrosis and apoptotic changes in cardiomyocytes. Pretreatment with aged garlic extract for 27 days ameliorated the effect of DOX administration on cardiac tissue; cardiomyocytes looked more or less similar to those of control. However, still vascular dilatation, mild congestion and interstitial edemas were observed. Our results suggest that aged garlic extract is potentially protective against doxorubicin-induced cardiotoxicity.

Aminosalicylic acid reduces the antiproliferative effect of hyperglycaemia, advanced glycation endproducts and glycated basic fibroblast growth factor in cultured bovine aortic endothelial cells: Comparison with aminoguanidine

Hyperglycaemia reduces proliferation of bovine aortic endothelial cells in vitro. A similar effect in vivo may contribute to longterm complications of diabetes such as impaired wound-healing and retinopathy.

Increased whole blood manganese concentrations observed in children with iron deficiency anaemia

A prospective observational study was carried out at Alder Hey Childrens Hospital, Liverpool, England, UK on children aged 1-6 years attending the pathology department for routine blood tests (n=225). Whole blood manganese concentrations were measured plus the following markers of iron status; haemoglobin, MCV, MCH, RBC count, ferritin, transferrin saturation and soluble transferrin receptors. Multiple regression analysis was performed, with blood manganese as the dependent variable and factors of iron status, age and gender as independent variables. A strong relationship between blood manganese and iron deficiency was demonstrated (adjusted R(2)=34.3%, p<0.001) and the primary contributing factors to this relationship were haematological indices and soluble transferrin receptors. Subjects were categorised according to iron status using serum ferritin, transferrin saturation and haemoglobin indices. Children with iron deficiency anaemia had higher median blood manganese concentrations (16.4 μg/L, range 11.7-42.4, n=20) than children with iron sufficiency (11 μg/L, range 5.9-20.9, n=59, p<0.001). This suggests that children with iron deficiency anaemia may be at risk from manganese toxicity (whole blood manganese >20 μg/L), and that this may lead to neurological problems. Treatment of iron deficiency in children is important both to improve iron status and to reduce the risk of manganese toxicity.

A microassay for protein glycation based on the periodate method

The periodate assay for glycated protein has been adapted for use with a microplate reader. Up to 94 samples can be read in 40 s, and sensitivity has been improved so that only 2-40 nmol of protein-bound sugar are needed per well. Yields have been increased to over 90%, allowing in vitro glycation of human serum albumin to be assayed on 0.1-mg aliquots.

Advanced glycation endproducts increase proliferation, migration and invasion of the breast cancer cell line MDA-MB-231.

Diabetic patients have increased likelihood of developing breast cancer. Advanced glycation endproducts (AGEs) underlie the pathogenesis of diabetic complications but their impact on breast cancer cells is not understood. This study aims to determine the effects of methylglyoxal-derived bovine serum albumin AGEs (MG-BSA-AGEs) on the invasive MDA-MB-231 breast cancer cell line. By performing cell counting, using wound-healing assay, invasion assay and zymography analysis, we found that MG-BSA-AGEs increased MDA-MB-231 cell proliferation, migration and invasion through Matrigel™ associated with an enhancement of matrix metalloproteinase (MMP)-9 activities, in a dose-dependent manner. Using Western blot and flow cytometry analyses, we demonstrated that MG-BSA-AGEs increased expression of the receptor for AGEs (RAGE) and phosphorylation of key signaling protein extracellular signal-regulated kinase (ERK)-1/2. Furthermore, in MG-BSA-AGE-treated cells, phospho-protein micro-array analysis revealed enhancement of phosphorylation of the ribosomal protein 70 serine S6 kinase beta 1 (p70S6K1), which is known to be involved in protein synthesis, the signal transducer and activator of transcription (STAT)-3 and the mitogen-activated protein kinase (MAPK) p38, which are involved in cell survival. Blockade of MG-BSA-AGE/RAGE interactions using a neutralizing anti-RAGE antibody inhibited MG-BSA-AGE-induced MDA-MB-231 cell processes, including the activation of signaling pathways. Throughout the study, non-modified BSA had a negligible effect. In conclusion, AGEs might contribute to breast cancer development and progression partially through the regulation of MMP-9 activity and RAGE signal activation. The up-regulation of RAGE and the concomitant increased phosphorylation of p70S6K1 induced by AGEs may represent promising targets for drug therapy to treat diabetic patients with breast cancer.