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Life Extension Magazine November 2009


Reconstructed skin modified by glycation of the dermal equivalent as a model for skin aging and its potential use to evaluate anti-glycation molecules.

Glycation is a slow chemical reaction which takes place between amino residues in protein and a reducing sugar. In skin this reaction creates new residues or induces the formation of cross-links (advanced glycation end products or AGEs) in the extracellular matrix of the dermis. Formation of such cross-links between macromolecules may be responsible for loss of elasticity or modification of other properties of the dermis observed during aging. We had previously developed a reconstructed skin model which enabled us to study the consequences of matrix alteration by preglycation of the collagen and have reported several modifications of interest induced by glycation in the dermal and epidermal compartments of reconstructed skin as well as at the level of the dermal-epidermal junction. For example we showed that collagen IV and laminin were increased in the basement membrane zone and that alpha6 and beta1 integrins in epidermis were expanded to suprabasal layers. The aim of this new study was to look at the biological effects of glycation inhibitors like aminoguanidine in the skin model. Aminoguanidine was mixed with collagen in the presence of ribose as reducing sugar, and immunostaining was used to visualize its effects on AGE Products and biological markers. After aminoguanidine treatment, we found a low amount of AGE products and a possible return to the normal pattern of distribution of markers in skin constructs as compared to those treated with ribose only. Interestingly similar results were also obtained, although to a lesser extent, with a blueberry extract. In conclusion the glycation inhibitory effect has been functionally demonstrated in the reconstructed skin model and it is shown that this model can be used to assess anti-glycation agents.

Exp Gerontol. 2008 Jun;43(6):584-8

Glycation—a sweet tempter for neuronal death.

Glycation, one of the post-translational modifications of proteins, is a nonenzymatic reaction initiated by the primary addition of a sugar aldehyde or ketone to the amino groups of proteins. In the early stage of glycation, the synthesis of intermediates leading to the formation of Amadori compounds occurs. In the late stage, advanced glycation end products (AGE) are irreversibly formed after a complex cascade of reactions. Several AGEs have been characterized chemically, while other new compounds remain to be identified. To date, studies of the contribution of glycation to diseases have been primarily focused on its relationship to diabetes and diabetes-related complications. However, glucose-induced damage is not limited to diabetic patients. Although it does not cause rapid or remarkable cell damage, glycation advances slowly and accompanies every fundamental process of cellular metabolism. It has recently become clear that glycation also affects physiological aging and neurodegenerative diseases such as Alzheimer’s disease and amyotrophic lateral sclerosis. Glycation alters the biological activity of proteins and their degradation processes. Protein cross-linking by AGE results in the formation of detergent-insoluble and protease-resistant aggregates. Such aggregates may interfere with both axonal transport and intracellular protein traffic in neurons. In addition, glycation reactions lead to the production of reactive oxygen species. Conversely, glycation is promoted by oxidative stress. We speculate on the presence of synergism between glycation and oxidative stress. In this review, we provide an outline of glycation and propose some possible mechanisms of its cytotoxicity and defense systems against it.

Brain Res Brain Res Rev. 2003 Mar;41(2-3):306-23

The receptor for advanced glycation end products is highly expressed in the skin and upregulated by advanced glycation end products and tumor necrosis factor-alpha.

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, factor XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and keratinocytes and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nepsilon-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis factor-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue growth factor, transforming growth factor-beta (TGF-beta) 1, and procollagen-alpha1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, -2, -3, and -12 was unaffected. We conclude that in HFFs, AGE-RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression.

J Invest Dermatol. 2006 Feb;126(2):291-9

Effect of advanced glycation end-products on cell proliferation and cell death.

The effect of advanced glycation end products (AGE-s) was studied on the proliferation and cell death of human skin fibroblasts in culture. Several AGE-products were prepared from proteins, a peptide and amino acids, using Glucose or Fructose, with or without Fe2+. The AGE preparations increased cell death at the 7th day, after only 72 hours of incubation. Some of these glycation products modified also proliferation. This effect of AGE-s was even maintained without these products in fresh medium for a second period of incubation up to 10 days from the start of the experiment. In order to explore the role of AGE-receptors, especially of AGE-receptor and of growth factor receptors (fibroblast and epidermal growth factors receptors), antibodies to these receptors were added to cell cultures and their effect on both cell death and proliferation were determined as for the AGE-s. These anti-receptor antibodies imitated to some extent the results obtained with AGE-s, producing increase of cell death and proliferation, followed above a certain concentration of antibodies by a decrease and a new increase or plateau. This might correspond to the internalization of the receptors followed by a re-expression on the cell membrane. The role of receptor-mediated Reactive Oxygen Species-production was also explored using scavengers: N-acetyl-cysteine (NAC), L-Carnosine, superoxide dismutase (SOD) and Catalase. Several of these scavengers decreased cell death, suggesting that Reactive Oxygen Species-production is partially involved in the observed phenomena.

Pathol Biol (Paris). 2006 Sep;54(7):396-404

Glycation induces expansion of the molecular packing of collagen.

Exposure of rat tail tendon to a reducing sugar results in covalent attachment of the sugar to collagen, a process termed glycation, and leads to the formation of stable intermolecular cross-links. We have used X-ray diffraction to study the changes in the crystalline unit cell of rat tail tendon collagen brought about by glycation. Ribose was selected as a model compound for most of the study because its reaction with proteins is faster than that of glucose, and therefore more convenient for laboratory studies, but glucose and glyceraldehyde were used as well. A kinetic model describing the process of glycation by ribose and subsequent cross-link formation has been developed. Glycation resulted in an expansion by more than 12% of the unit cell that describes the three-dimensional structure of rat tail tendon collagen. The expansion was in a direction perpendicular to the axes of the rod-shaped molecules, indicating that the intermolecular spacing of the collagen increased. Thus, the structure of collagen in rat tail tendon is significantly altered by glycation in vitro. The expansion was not isotropic, but was directed parallel to the (120) planes, one of the three major planes of the quasi-hexagonal structure that is densely populated by collagen molecules. It is hypothesized that this expansion is brought about by the formation of one, or at most a few, specific intermolecular cross-links in the overlap zone that act to push the molecules apart. It is likely that similar structural changes in collagenous tissues are caused by glycation in vivo during the natural course of aging, and that these changes are accelerated in chronic hyperglycemia such as that associated with diabetes. Analysis of the structure of glycated rat tail tendon potentially can give us new insight into the detailed molecular structure of collagen.

J Mol Biol. 1988 Sep 20;203(2):495-505

Advanced glycation end products induce crosslinking of collagen in vitro.

We have investigated the effect of advanced glycation end products (AGEs) on the crosslinking of collagen. The potential pathological significance of AGEs and the altered metabolism of ascorbic acid (ASA) in diabetes have prompted us to investigate the role of ASA in the crosslinking and advanced glycation of collagen. Rat tail tendons were incubated with ASA and dehydroascorbic acid (DHA) under physiological conditions of temperature and pH, and the crosslinking and the level of AGEs were analyzed. Analysis of crosslinking was conducted by pepsin solubility and cyanogen bromide digestion. Level of AGEs was estimated by enzyme-linked immunosorbent assay (ELISA) using antibodies raised against AGE-ribonuclease. It was noted that ASA and DHA induced crosslinking of collagen and stimulated the formation of AGEs. It was also noted that these pathways were dependent on oxidative conditions. Similarly incubation of collagen with AGEs, prepared by the in vitro incubation of bovine serum albumin (BSA) with glucose, also resulted in increased crosslinking. The extent of crosslinking was dependent on the duration of incubation. The novel finding of this study, which is in contrast to the earlier reports on glucose-induced crosslinking of collagen, was that AGEs-induced crosslinking of collagen was not inhibited by radical scavengers and the metal chelator. EDTA, whereas glucose-induced crosslinking of collagen was almost completely prevented by free radical scavengers. The increased fluorescence intensity observed in collagen incubated with AGEs was also not prevented by radical scavengers. Estimation of AGEs by ELISA revealed an increased accumulation of AGEs in collagen incubated with AGE-BSA. The inhibitory effect of aminoguanidine and aspirin on AGEs-induced modification of collagen, strongly suggests that the amino-carbonyl interaction between AGEs and collagen may play a key role in the crosslinking process. The results obtained in this study indicate that soluble AGEs can directly induce crosslinking of collagen and this process is independent of oxidative conditions. From these results it may be hypothesized that glucose, under oxidative conditions, reacts with proteins to form potentially reactive end products called AGEs. These AGEs, once formed, could induce crosslinking of collagen even in the absence of both glucose and oxygen.

Biochim Biophys Acta. 1998 Sep 30;1407(3):215-24

Advanced glycation end products enhance expression of pro-apoptotic genes and stimulate fibroblast apoptosis through cytoplasmic and mitochondrial pathways.

Both aging and diabetes are characterized by the formation of advanced glycation end products (AGEs). Both exhibit other similarities including deficits in wound healing that are associated with higher rates of fibroblast apoptosis. In order to investigate a potential mechanism for enhanced fibroblast apoptosis in diabetes and aged individuals, experiments were carried out to determine whether the predominant advanced glycation end product in skin, N-epsilon-(carboxymethyl) lysine (CML)-collagen, could induce fibroblast apoptosis. In vivo experiments established that CML-collagen but not unmodified collagen induced fibroblast apoptosis and that apoptosis was dependent upon caspase-3, -8, and -9 activity. In vitro experiments demonstrated that CML-collagen but not control collagen induced a time- and dose-dependent increase in fibroblast apoptosis. By use of blocking antibodies, apoptosis was shown to be mediated through receptor for AGE signaling. AGE-induced apoptosis was largely dependent on the effector caspase, caspase-3, which was activated through both cytoplasmic (caspase-8-dependent) and mitochondrial (caspase-9) pathways. CML-collagen had a global effect of enhancing mRNA levels of pro-apoptotic genes that included several classes of molecules including ligands, receptors, adaptor molecules, mitochondrial proteins, and others. However, the pattern of expression was not identical to the pattern of apoptotic genes induced by tumor necrosis factor alpha.

J Biol Chem. 2005 Apr 1;280(13):12087-95

Decrease in skin collagen glycation with improved glycemic control in patients with insulin-dependent diabetes mellitus.

Glycation, oxidation, and nonenzymatic browning of protein have all been implicated in the development of diabetic complications. The initial product of glycation of protein, fructoselysine (FL), undergoes further reactions, yielding a complex mixture of browning products, including the fluorescent lysine-arginine cross-link, pentosidine. Alternatively, FL may be cleaved oxidatively to form N(epsilon)-(carboxymethyl)lysine (CML), while glycated hydroxylysine, an amino-acid unique to collagen, may yield N(epsilon)-(carboxymethyl)hydroxylysine (CMhL). We have measured FL, pentosidine, fluorescence (excitation = 328 nm, emission = 378 nm), CML, and CMhL in insoluble skin collagen from 14 insulin-dependent diabetic patients before and after a 4-mo period of intensive therapy to improve glycemic control. Mean home blood glucose fell from 8.7 +/- 2.5 (mean +/- 1 SD) to 6.8 +/- 1.4 mM (P less than 0.005), and mean glycated hemoglobin (HbA1) from 11.6 +/- 2.3% to 8.3 +/- 1.1% (P less than 0.001). These changes were accompanied by a significant decrease in glycation of skin collagen, from 13.2 +/- 4.3 to 10.6 +/- 2.3 mmol FL/mol lysine (P less than 0.002). However, levels of browning and oxidation products (pentosidine, CML, and CMhL) and fluorescence were unchanged. These results show that the glycation of long-lived proteins can be decreased by improved glycemic control, but suggest that once cumulative damage to collagen by browning and oxidation reactions has occurred, it may not be readily reversed. Thus, in diabetic patients, institution and maintenance of good glycemic control at any time could potentially limit the extent of subsequent long-term damage to proteins by glycation and oxidation reactions.

J Clin Invest. 1991 Jun;87(6):1910-5

Dysfunction of dermal fibroblasts induced by advanced glycation end-products (AGEs) and the contribution of a nonspecific interaction with cell membrane and AGEs.

Advanced glycation end-products (AGEs) have been reported to accumulate in the dermal skin. However, it remains unknown whether the AGEs interact with the dermal fibroblasts and influence their function. Previously, we demonstrated that AGEs hastened photoaging of the skin by means of active oxygen species such as *O(2)(-), H(2)O(2), and *OH, generated during UVA irradiation. The purpose of the present study was to clarify the influence of AGEs on the functions of dermal fibroblasts under physiological conditions. It was found that AGEs decreased both hyaluronic acid (HA) synthesis and activity of elastase-type matrix metalloproteinase (ET-MMP). Because the reactions of both HA synthesis and ET-MMP were found to take place at the cell membrane region, it appeared that AGEs modulated cellular dysfunction through an interaction with the cell membrane. To clarify the mechanisms of these dysfunction in relation to AGEs, we examined the interaction between AGEs and cell membranes, and obtained the following results: (1) AGEs associated with the cell membranes and liposomal membrane prepared with phosphatidyl choline; (2) AGEs hydrophobically modified the circumstances of the cell membrane and liposome membrane as evaluated by experiments using a fluorescence probe; (3) AGEs increased the fluidity of the cell membrane and liposomal membrane as estimated by ESR spin-labeling using 5-doxylstearic acid; and (4) AGEs accelerated lactate dehydrogenase (LDH) leakage from the cells. On the basis of these experimental results, we proposed that AGEs modulated cell function through a nonspecific interaction with the membranes of dermal fibroblasts.

J Dermatol Sci. 2002 Sep;29(3):171-80