Collagen Glycation Research Articles

Focal Therapeutic Irradiation Induces an Early Transient Increase in Bone Glycation

Advanced glycation end products (AGEs) are an abnormal modification of the collagenous matrix in bone, and their accumulation contributes to alteration of mechanical properties. Using a mouse model of focal external radiotherapy, we quantified the time-dependent changes in the glycation of bone collagen after 4 daily fractions of 5 Gy exposure to unilateral hindlimb. Fluorometric analysis of decalcified femurs demonstrated a significant and transient increase in the quantity of pentosidine, pyridinolines and nonspecific AGEs per unit of collagen at one week postirradiation. These differences did not persist at 4, 8, 12 or 26 weeks postirradiation. Radiation had no effect on bone collagen content. We hypothesize that following the transient increase in glycation products, these crosslinks are then removed as a result of increased postirradiation osteoclast activity and continued mineralization of the bone.

The role of collagen crosslinks in ageing and diabetes - the good, the bad, and the ugly

The non-enzymatic reaction of proteins with glucose (glycation) is a topic of rapidly growing importance in human health and medicine. There is increasing evidence that this reaction plays a central role in ageing and disease of connective tissues. Of particular interest are changes in type-I collagens, long-lived proteins that form the mechanical backbone of connective tissues in nearly every human organ. Despite considerable correlative evidence relating extracellular matrix (ECM) glycation to disease, little is known of how ECM modification by glucose impacts matrix mechanics and damage, cell-matrix interactions, and matrix turnover during aging. More daunting is to understand how these factors interact to cumulatively affect local repair of matrix damage, progression of tissue disease, or systemic health and longevity. This focused review will summarize what is currently known regarding collagen glycation as a potential driver of connective tissue disease. We concentrate attention on tendon as an affected connective tissue with large clinical relevance, and as a tissue that can serve as a useful model tissue for investigation into glycation as a potentially critical player in tissue fibrosis related to ageing and diabetes.

Collagen advanced glycation inhibits its Discoidin Domain Receptor 2 (DDR2)-mediated induction of lysyl oxidase in osteoblasts

Diabetes increases the risk of bone fracture. Organic and inorganic bone extracellular matrix components determine bone strength. Previous studies indicate that in diabetes, glycation of collagen causes abnormal arrangements of collagen molecules and fragile bones. Diabetic bone fragility is additionally attributed to reduced levels of lysyl oxidase enzyme-dependent collagen cross-links. The mechanism underlying the presence of lower enzymatic collagen cross-links in diabetic bone has not been directly investigated. Here we determine in primary osteoblast cultures the regulation of lysyl oxidase protein by type I collagen and collagen modified by carboxymethylation (CML-collagen), a form of advanced glycation endproducts. Data indicate that non-glycated collagen up-regulates lysyl oxidase levels both in primary non-differentiated and in differentiating mouse and rat osteoblast cultures, while CML-collagen fails to regulate lysyl oxidase in these cells. Collagen binding to Discoidin Domain Receptor-2 (DDR2) mediates lysyl oxidase increases, determined in DDR2 shRNA knockdown studies. DDR2 binding and activation were disrupted by collagen glycation, pointing to a mechanism for the diminished levels of lysyl oxidase and consequently low lysyl oxidase-derived cross-links in diabetic bone. Our studies indicate that collagen–integrin interactions may not play a major role in up-regulating lysyl oxidase. Furthermore, non-collagenous ligands for the receptor for advanced glycation end products (RAGE) failed to alter lysyl oxidase levels. Taken together with published studies a new understanding emerges in which diabetes- and age-dependent inhibition of normal collagen-stimulated DDR2- and integrin-signaling, and independent advanced glycation-stimulated RAGE-signaling, each contributes to different aspects of diabetic osteopenia.

Molecular insight of non enzymatic glycosylation of collagen-therapeutic perspective of dietary carbohydrates

Abstract Glycosylation is a significant post translational step in biosynthesis of collagen which plays a key role in fibrillogenesis and formation of advanced end glycation product (AEG). Diabetes leads to modification of collagen such as AEG and crosslinking which plays an important role in the pathogenesis of diabetes mellitus. In the present research, molecular strategy is chronicled to understand the in vitro non enzymatic glycation of collagen for assessing the influence of various dietary carbohydrates. Glycated collagen has been characterized by various physico-chemical techniques. Hydrogen bonding is more prevalent in collagen carbohydrate interaction which is indicated by lower shift in zeta potential value. Moreover, random aggregation has been observed in DLS study. The inherent structure of collagen has been retained with increase in stability in glycated state. The properties of non-enzymatic glycated collagen may help in understanding the prominence in diabetic complications.

Temperature-dependent FTIR spectra of collagen and protective effect of partially hydrolysed fucoidan

FTIR spectra of collagen (PC) and partially hydrolysed fucoidan (PHF) incorporated into collagen films were investigated at different temperatures between 20°C and 100°C. Changes within the bands of amide I, amide II and amide III may indicate stabilization of collagen by hydrogen bonds during its interaction with partially hydrolysed fucoidan. Spectroscopic studies revealed that partially hydrolysed fucoidan was bound to the collagen without affecting its triple helicity. Interactions of fucoidan with H2SO4 (mild acid hydrolysis), leading to changes of the sulphated band positions in the 800–590cm−1 region of IR spectra were observed. The effect of partially hydrolysed fucoidan on glucose-mediated collagen glycation and cross-linking of proteins in vitro was evaluated. It was observed that partially hydrolysed fucoidan incorporated into collagen films can be used as therapeutically active biomaterials that speed up the process of wound healing and may increase the anticancer activity of fucoidan.

Anti-glycation and Antioxidant Activities of Australian Centella species

The products of nonenzymatic glycation and oxidation of extracellular matrix contribute to the delayed healing of diabetic ulcers. Centella plant species have been used medicinally for various dermatological disorders including wound healing. The aim of this study was to evaluate the anti-glycation and antioxidant activities of the methanolic extract of two species of Australian Centella; Centella asiatica (L.) Urb. (Apiaceae/Umbelliferae) and Centella erecta (L.f.) Fern. The anti-glycation activity of the extracts was assessed by measuring the fluorescence intensity of advanced glycation end products (AGEs) formed after incubating collagen-type I and bovine serum albumin with methylglyoxal (MGO) for two and five days, respectively. The antioxidant activity was evaluated by interrogating the ability of each extract to scavenge DPPH free radicals. The phytochemical profile of both species was also examined by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) analysis. The methanolic extracts (100 µg/mL – 1200 µg/mL) of both species decreased significantly the formation of AGEs induced by MGO in a dose dependent manner. Free radical scavenging inhibition was 80.2 ± 4.4% for C. asiatica and 77.8 ± 3.9% for C. erecta. HPLC and TLC showed the presence of the major Centella saponins (asiaticoside, madecassoside) and their aglycones (asiatic and madecassic acids), phenolic acids (chlorogenic acid) and flavanoids (quercetin, isoquercetin and kaempferol) in both extracts. The results suggest that Centella species are not only capable of inhibiting the glycation of collagen but also exhibit substantial antioxidant activity, which may be clinically effective in impaired wound healing in diabetes.

Glycation of extracellular matrix proteins and its role in atherosclerosis

Glycation consists in formation of advanced glycation end-products (AGE) during non-enzymatic reaction between reducing sugars and proteins, lipids or nucleic acids. This review is focused mainly on glycation of collagen and its role in acceleration of vascular disease. Collagen is an extracellular matrix protein characterized by unique structure forming fibrils with great anti-tensile and anti-breaking strength. The protein builds the connective tissue and is responsible for biomechanical properties of blood vessels. It is reported that higher content of glycated collagen correlates with lower elasticity and greater toughness of the vessel walls and, as a consequence, a faster rate of atherosclerosis development. Numerous mechanisms connected with AGE formation are involved in atherogenesis, among others: receptor-mediated production of free radicals, triggering an inflammatory process, activation of leukocytes and thrombocytes, facilitation of LDL binding, change in level of growth factors, adhesion molecules, MMP and some other proteins' expression. The coverages allow the development of therapeutic strategies to prevent or slow down the pathological processes connected with glycation of collagen and other proteins in the artery wall. The main strategies are based on limitation of exogenous AGE, consumption of products which contain rutin, treatment with drugs which inhibit AGE formation, such as pyridoxamine, and chemicals which are able to cleave already formed AGE protein-protein crosslinks, such as ALT-711.

Impact of carbamylation and glycation of collagen type I on migration of HT1080 human fibrosarcoma cells

Collagen type I is an abundant component of the extracellular matrix and due to its longevity, constitutes a prominent target of non-enzymatic post-translational in vivo modifications such as carbamylation and glycation. These protein modifications involved in aging, renal diseases and diabetes, are linked to elevated cancer risk. In this in vitro study, we investigated the impact of carbamylated and glycated collagen type I on the migratory behavior of the highly invasive HT1080 human fibrosarcoma cells. The proliferation of HT1080 on modified collagens did not differ from that on native form. The glycated collagen delayed the cell adhesion time whereas the carbamylated one had no effect. The migration ability of HT1080 was studied by quantifying single cell speed using videomicroscopy. Glycation strongly inhibited mean cell speed by 47% whereas carbamylation moderately affected it by 12%. In addition, the influence of these collagen modifications on actin and vinculin organization was studied. On the glycated collagen, 63% of cells revealed a dramatic loss of actin stress fibers vs. 28% on the carbamylated one. In these cells, disorganized F-actin was accompanied with a disturbance of vinculin and both proteins were localized at the rim of the cells. Concerning the focal adhesion kinase (FAK) expression, glycated collagen only induced a significant inhibition. Whereas, both collagen modifications provoked a differential inhibition of FAK phosphorylation state by 25% for carbamylation and 60% for glycation. In conclusion, our data suggest that, in vivo, collagen glycation and carbamylation may affect tumor cell metastasis. This suggestion is supported by clinical studies reporting less aggressive tumors in diabetic or uremic patients. Consequently, the impact of such post-translational modifications has to be taken into account in order to better understand the link between aging, diabetes or uremia and cancer progression.

Diabetes and Bone

Diabetes – both type 1 (T1D) and type 2 (T2D) has profound effects on the skeleton. Bone turnover is reduced, i.e. bone biopsies show a reduced bone formation with reduced mineralisation, and a reduced number of bone cells. Biochemical markers of bone turnover show a reduction in both formation and resoprtion. Bone mineral density (BMD) is reduced in T1D, whereas an increased BMD is seen in T2D. Despite this, an increased risk of hip fractures is seen in both T1D and T2D, the increase in risk of fractures being more pronounced in T1D than in T2D. This discrepancy between BMD and fracture risk along with evidence from animal studies of a reduced bone biomechanical competence in diabetes suggest that the bone tissue is weakened in patients with diabetes. This weakening may be related to glycation of collagen and formation of advanced glycation end-products (AGE), which together with their receptor (RAGE) lead to a decreased activity of the bone cells and thus a reduced turnover and a reduced de-novo formation of bone. The reduced competence despite normal BMD makes the diagnosis of osteoporosis difficult as standard bone scans (DXA) may not truly reflect bone strength. Regarding anti-diabetic treatment, most such drugs improve glucose control and thus reduce the detrimental effects of diabetes on the skeleton. However, the thiazolidinediones (rosiglitazone and pioglitazone) are associated with a decreased BMD and an increased fracture risk through an effect on the stem cells in the bone marrow leading to formation of adipocytes rather than osteoblasts. Anti-resoprtive treatment for osteoporosis seem equally effective in diabetes patients as in non-diabetics despite the reduced bone turnover in diabetes. In conclusion diabetes has many effects on bone and bone-turnover, and more research is needed.

Controversial behavior of aminoguanidine in the presence of either reducing sugars or soluble glycated bovine serum albumin

The elucidation of the controversial inhibitory effect of aminoguanidine (AG) on the cross-linking and fluorescent advanced glycation end products (AGEs) formation during long-term in vitro glycation of type I collagen with 250 mM reducing sugars or 0.5 mg/ml soluble glycated bovine serum albumin (AGE-BSA) was researched. Chromatographic and SDS–PAGE analyses revealed the formation of aggregates during collagen glycation. AG at all concentrations (5–80 mM) prevented the cross-linking of collagen peptides with monosaccharides but an increase in fluorescence with a maximum value at 10 mM AG was noticed. In the presence of AGE-BSA, AG prevented the cross-linking process and decreased the fluorescence levels in a concentration-dependent manner. Our results suggest that AG is an efficient inhibitor of collagen cross-linking and the highest increase in fluorescence due to reducing sugars and AG can be explained by the competition between guanidine group of AG and arginine residues of some protein-bound dideoxyosones, which could form fluorescent compounds.

Glycated collagen alters endothelial cell actin alignment and nitric oxide release in response to fluid shear stress

People with diabetes suffer from early accelerated atherosclerosis, which contributes to morbidity and mortality from myocardial infarction, stroke, and peripheral vascular disease. Atherosclerosis is thought to initiate at sites of endothelial cell injury. Hyperglycemia, a hallmark of diabetes, leads to non-enzymatic glycosylation (or glycation) of extracellular matrix proteins. Glycated collagen alters endothelial cell function and could be an important factor in atherosclerotic plaque development. This study examined the effect of collagen glycation on endothelial cell response to fluid shear stress. Porcine aortic endothelial cells were grown on native or glycated collagen and exposed to shear stress using an in vitro parallel plate system. Cells on native collagen elongated and aligned in the flow direction after 24 h of 20 dynes/cm 2 shear stress, as indicated by a 13% decrease in actin fiber angle distribution standard deviation. However, cells on glycated collagen did not align. Shear stress-mediated nitric oxide release by cells on glycated collagen was half that of cells on native collagen, which correlated with decreased endothelial nitric oxide synthase (eNOS) phosphorylation. Glycated collagen likely inhibited cell shear stress response through altered cell–matrix interactions, since glycated collagen attenuated focal adhesion kinase activation with shear stress. When focal adhesion kinase was pharmacologically blocked in cells on native collagen, eNOS phosphorylation with flow was reduced in a manner similar to that of glycated collagen. These detrimental effects of glycated collagen on endothelial cell response to shear stress may be an important contributor to accelerated atherosclerosis in people with diabetes.

Abstract 1383: Impact of carbamylation and glycation of collagen type I on HT1080 cell migration

Abstract Carbamylation and glycation are well known as nonenzymatic posttranslational modifications that occur throughout the lifespan of proteins in vivo. Due to its longevity, collagen type I, largely present in connective tissues, becomes a prominent target for such modifications. Protein carbamylation and glycation have been implicated in normal aging and in a number of pathologies like diabetes, renal diseases and cancer. In addition, recent studies suggest a possible link between increased cancer risk and diabetes or end-stage renal disease. Here, we investigate the impact of carbamylation and glycation of collagen on the proliferation and migration capacities of the highly invasive human fibrosarcoma cell line HT1080. For this purpose, collagen type I extracted from rat tail tendons was used for in vitro carbamylation with cyanate or glycation with ribose. The amount of carbamylation and glycation was evaluated respectively via HPLC and spectrofluorimetry, and only the maximum levels of carbamylated and glycated collagens were considered. Tumor cell behavior was then evaluated on coatings of native, carbamylated or glycated collagen. Our results show that the proliferation of HT1080 cells on modified collagens did not differ from that on native form after 1, 2 and 3 days of culture. Concerning tumor cell adhesion, the glycated collagen delayed the adhesion time of seeded cells whereas the carbamylated form had no effect. Using time-lapse videomicroscopy, the migration ability of HT1080 was studied by quantifying single cell speed. Only, the glycated collagen strongly inhibited cell speed migration. We next investigated the effect of these collagen modifications on the organization of actin and vinculin, two proteins involved in cell locomotion. On glycated collagen, cells revealed a dramatic loss of actin stress fibers where disorganized F-actin was principally localized at the rim of the cell. Disturbance of actin integrity was accompanied with a disorganization of vinculin that was also localized at the cell periphery. On the other hand, the cells on carbamylated collagen maintained a fully organized actin network. The impact on the focal adhesion kinase (FAK), which plays a crucial role in focal adhesion formation, was also evaluated. Only glycated collagen induced a significant inhibition of the expression level of FAK, whereas both collagen modifications provoke a differential inhibition of its phosphorylation state that is mainly taking place with glycation. In conclusion, only glycation is demonstrated as an important factor affecting tumor cell migration. This impact has to be certainly considered in order to better understand the link between diabetes, aging and cancer incidence. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1383. doi:10.1158/1538-7445.AM2011-1383

Cardiac aging – a review

BACKGROUND: Physiology of aging includes numerous modifications at different levels of the cardiovascular system, resulting in adverse remodeling of the heart and blood vessels. METHODS: The present review aims to summarize the different mechanisms in detail during the process of aging with special emphasis on the neurohumoral regulation, autophagy, and remodeling of the extracellular matrix (ECM). RESULTS: Age-associated cellular and molecular mechanisms involve cardiomyocyte function, alterations of neurohormonal regulation, as well as changes in the ECM. Moreover, interactions between the distinct territories involved in the age-related modifications increase the complexity of senescence-associated cardiovascular remodeling. Altered adrenergic and renin-angiotensin systems control neurohormonal regulation of age-associated cardiovascular function. Increased oxidative stress induces enhanced production of cellular waste material, and facilitates the detrimental effects of aging on senescent cardiomyocytes, which are unable to maintain their homeostasis and redox equilibrium due to altered autophagic capacity. Increased collagen and advanced glycation endproducts (AGEs) deposition in the ECM further worsen global function of the aging heart. CONCLUSIONS: During the aging process, several modifications in the heart and blood vessels occur at different levels of the cardiovascular system. This adverse remodeling is further influenced by a number of cross-talk actions between the separate mechanisms. Further investigations are needed to elucidate the exact cause-effect relations among the underlying mechanisms, and to clarify their hierarchical position during the course of the aging process.

English

Non-enzymatic glycation of proteins, leading to chemical modification and cross-linking are of importance in the pathology of diabetic complications. In our early reported we showed that, camel milk possesses antihyperglycemic, antihyperlipidemic and exhibit beneficial role on membrane-bound ATPases in streptozotocin-diabetic rats after 45 days of treatment of camel milk at the optimum dose of 250 ml/day. In the present study, the effect of camel milk on the glycation and cross-linking of collagen from tail tendon of streptozotocin (STZ)-diabetic rats. Diabetes was induced in adult male albino rats of the Wistar strain, weighing 180 to 200 g, by administration of streptozotocin (STZ) (40 mg/kg) intraperitoneally. The animals were randomly divided into five groups of six animals each. Rats of groups II and IV were fed 250 ml of raw camel milk daily through watering bottle instead of water. Whereas animals in groups I, III and V were given tap water, and rats of group V were given 600 μg/kg body weight of glibenclamide orally, once in a day in the morning for 45 days. The levels of hydroxylproline and total collagen content elevated in the tail tendon of the diabetic control. The levels of extent of glycation and fluorescence of collagen increased while decreased levels of acid, neutral and pepsin soluble collagens were observed in the tail tendon of diabetic rats. These changes were alleviated by the simultaneous ingestion of camel milk. Our results demonstrate that intake of camel milk has a positive influence on tail tendon collagen glycation and other variables in STZ-diabetic rats and its effect was comparable with glibenclamide.   Key words: Camel milk, glycation, collagen, streptozotocin, diabetes.

Pyridoxamine prevents age-related aortic stiffening and vascular resistance in association with reduced collagen glycation

An increase in oxidative chemical modifications of tissue proteins by advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) has been implicated in normal aging. Pyridoxamine (PM), one of the three natural forms of vitamin B 6, has been identified as an inhibitor of AGE/ALE products formed during the autoxidation of carbohydrates and peroxidation of lipids. The current study seeks to determine whether PM intervention could prevent the age-related aortic stiffening and vascular resistance through its ability to inhibit the pathogenic cross-linking of glycated collagen. Male Fisher 344 rats at 15 months were treated daily with PM (1 g l − 1 in drinking water) for 5 months and compared with the age-matched, untreated controls at 20 months. Pulsatile aortic pressure and flow signals were measured to perform the vascular impedance analysis. The anti-AGE antibody 6D12 was used to detect glycation-derived modification of aortic collagen, using protein blotting analysis. PM therapy attenuated the age-related increase in total peripheral resistance. An increase in wave transit time and aortic compliance by PM indicated that the drug improved aortic distensibility of the aged vasculature. This paralleled its reduction of AGE-collagen cross-links on aortas. Treatment of the old animals with PM also prevented the age-induced augmentation in vascular load imposed on the heart, as evidenced by an increased wave transit time and a decreased wave reflection factor. These findings suggest a partial role of PM in improving arterial mechanics by targeting the pathogenic formation of AGE-induced aortic collagen cross-links in old rats.

<i>N<sup>ω</sup></i>-(carboxymethyl)arginine Accumulates in Glycated Collagen and Klotho-deficient Mouse Skin

Objective: Advanced glycation end products (AGEs) accumulate in tissues due to aging, diabetic complications, and atherosclerosis. The acid lability of Nω-carboxymethylarginine (CMA) present in glycated collagen has hampered detailed studies on its function and in vivo localization. In the present study, we analyzed the effects of collagen glycation on human dermal fibroblast (HDF) function. We also took advantage of Klotho-deficient mice ( kl/kl), which undergo accelerated senescence, to determine glycated collagen’s tissue localization. Methods: Bovine type I collagen was incubated with ribose, and CMA formation was measured by enzyme-linked immunosorbent assay (ELISA). We measured the contraction of 3-dimensional matrix gels (3D gel), consisting of either native or glycated collagens, after culture with HDFs. CMA accumulation in Klotho-deficient mouse skin was measured by immunohistochemical staining. Results: When collagen was incubated with ribose, CMA levels increased with time. In our HDF culture system, gels prepared with native, but not glycated collagen, contracted with time. In Klotho-deficient mice, CMA localized to the extracellular dermal matrix. Conclusions: Here we show that CMA may provide a marker for collagen glycation, which may adversely affect HDFs’ growth and survival. Therefore, treatment with AGE inhibitors might help prevent pathologies associated with AGE formation.

Effects of nonenzymatic glycation on mechanical properties of demineralized bone matrix under compression

Effects of in vitro induced nonenzymatic glycation of bone collagen on stiffness and fracture of demineralized bone matrix in unconfined compression were investigated. Regular specimens from mid-shaft of bovine femur were grouped in pairs. One sample (R) from each pair was incubated in ribose solution, control samples (C) were incubated in a buffer solution. Samples were then demineralized in formic acid. Demineralized samples were axially compressed to failure (0.033/s). Direction of compression was along the longitudinal axis of femur (L) or perpendicular (transverse) to that (T). Mechanical behavior of demineralized samples was characterized in terms of secant modulus, stress, and strain at fracture and work to failure. The development of damage was examined in terms of acoustic emission (AE) signal recorded during loading. In L direction, strain at fracture following glycation was lower than in the controls (p=0.038); secant modulus and ultimate stress were not significantly different in R and C. In the transverse direction, strain at fracture in R was higher than in C (p=0.053), as well as work to failure (p=0.020). Anisotropy of bone matrix, defined in terms of a ratio of the parameters in two perpendicular directions, decreased markedly in ribosylated samples. Both the number of AE events and cumulative AE energy during deformation were significantly higher in ribosylated samples than in the control for both directions of compression. The study demonstrated that nonenzymatic glycation plays a significant role in modifying organic matrix properties in cortical bone.

Aggregation and self assembly of non-enzymatic glycation of collagen in the presence of amino guanidine and aspirin: An in vitro study

Non-enzymatic glycation of collagen has been used in modern biomaterials science. This paper deals with in vitro studies on the effects of amino guanidine (AG) and aspirin in the non-enzymatic glycation (NEG) of collagen using thermal, conformational, fluorescence, turbidity and powder XRD measurements. There is no significant change in the fluorescence emission spectra for different concentrations of AG treated NEG of collagen whereas the emission intensity decreases as the concentration of aspirin increases. Circular dichroism (CD) revealed the disappearance of the positive peak at 220 nm for glycated collagen in the presence of amino guanidine and aspirin suggesting the collapse of triple helical configuration. Nearly 15 °C decrease is observed in shrinkage temperature of glycated rat tail tendon (RTT) collagen fibres in the presence of aspirin. Powder XRD of glycated collagen nano-fibrils in the presence of amino guanidine reveals high crystalline nature and the enhancement of self assembly processes when compared to aspirin. To the best of our knowledge, this is the first report of powder XRD of the self assembly of collagen nano-fibrils without mineralization. Our experimental results suggest that in the non-enzymatic glycation of collagen both AG and aspirin play a pivotal role in the aggregation and self assembly processes. From the present study, it is possible to conclude that while AG significantly influences the self assembly processes, aspirin facilitates the aggregation processes.

Methylglyoxal-modified collagen promotes myofibroblast differentiation

Fibrosis is a frequent complication of diabetes mellitus in many organs and tissues but the mechanism of how diabetes-induced glycation of extracellular matrix proteins impacts the formation of fibrotic lesions is not defined. As fibrosis is mediated by myofibroblasts, we investigated the effect of collagen glycation on the conversion of human cardiac fibroblasts to myofibroblasts. Collagen glycation was modeled by the glucose metabolite, methylglyoxal (MGO). Cells cultured on MGO-treated collagen exhibited increased activity of the α-smooth muscle actin promoter and enhanced expression of α-smooth muscle actin, ED-A fibronectin and cadherin, which are markers for myofibroblasts. In cells remodeling floating or stress-relaxed collagen gels, MGO treatment promoted more contraction (p<0.025) than vehicle controls, which was MGO dose-dependent. Transwell assays showed that cell migration was increased by MGO-treated collagen (p<0.025). In shear-force detachment assays, cells on MGO-treated collagen were less adherent than untreated collagen, and the formation of high affinity, β1 integrin-dependent adhesions was inhibited. MGO-collagen-induced expression of SMA was dependent on TGF-β but not on Rho kinase. We conclude that collagen glycation augments the formation and migration of myofibroblasts, critical processes in the development of fibrosis in diabetes.

Open tubular capillary electrochromatography: A useful microreactor for collagen I glycation and interaction studies with low-density lipoprotein particles

Diabetes, a multifunctional disease and a major cause of morbidity and mortality in the industrialized countries, strongly associates with the development and progression of atherosclerosis. One of the consequences of high level of glucose in the blood circulation is glycation of long-lived proteins, such as collagen I, the most abundant component of the extracellular matrix (ECM) in the arterial wall. Glycation is a long-lasting process that involves the reaction between a carbonyl group of the sugar and an amino group of the protein, usually a lysine residue. This reaction generates an Amadori product that may evolve in advanced glycation end products (AGEs). AGEs, as reactive molecules, can provoke cross-linking of collagen I fibrils. Since binding of low-density lipoproteins (LDLs) to the ECM of the inner layer of the arterial wall, the intima, has been implicated to be involved in the onset of the development of an atherosclerotic plaque, collagen modifications, which can affect the affinity of native and oxidized LDL for collagen I, can promote the entrapment of LDLs in the intima and accelerate the progression of atherosclerosis. In this study, open tubular capillary electrochromatography is proposed as a new microreactor to study in situ glycation of collagen I. The kinetics of glycation was first investigated in a fused silica collagen I-coated capillary. Dimethyl sulphoxide, injected as an electroosmotic flow marker, gave information about the charge of coating. Native and oxidized LDL, and selected peptide fragments from apolipoprotein B-100, the protein covering LDL particles, were injected as marker compounds to clarify the interactions between LDLs and the glycated collagen I coating. The method proposed is simple and inexpensive, since only small amounts of collagen and LDL are required. Atomic force microscopy images complemented our studies, highlighting the difference between unmodified and glycated collagen I surfaces.