Glycation End-product Cross-link Research Articles

Pharmacological Modulation of Arterial Stiffness

Arterial stiffness has emerged as an important marker of cardiovascular risk in various populations and reflects the cumulative effect of cardiovascular risk factors on large arteries, which in turn is modulated by genetic background. Arterial stiffness is determined by the composition of the arterial wall and the arrangement of these components, and can be studied in humans non-invasively. Age and distending pressure are two major factors influencing large artery stiffness. Change in arterial stiffness with drugs is an important endpoint in clinical trials, although evidence for arterial stiffness as a therapeutic target still needs to be confirmed. Drugs that independently affect arterial stiffness include antihypertensive drugs, mostly blockers of the renin-angiotensin-aldosterone system, hormone replacement therapy and some antidiabetic drugs such as glitazones. While the quest continues for 'de-stiffening drugs', so far only advanced glycation endproduct cross-link breakers have shown promise.

Structural, biochemical, cellular, and functional changes in skeletal muscle extracellular matrix with aging

The extracellular matrix (ECM) of skeletal muscle is critical for force transmission and for the passive elastic response of skeletal muscle. Structural, biochemical, cellular, and functional changes in skeletal muscle ECM contribute to the deterioration in muscle mechanical properties with aging. Structural changes include an increase in the collagen concentration, a change in the elastic fiber system, and an increase in fat infiltration of skeletal muscle. Biochemical changes include a decreased turnover of collagen with potential accumulation of enzymatically mediated collagen cross-links and a buildup of advanced glycation end-product cross-links. Altered mechanotransduction, poorer activation of satellite cells, poorer chemotactic and delayed inflammatory responses, and a change in modulators of the ECM are important cellular changes. It is possible that the structural and biochemical changes in skeletal muscle ECM contribute to the increased stiffness and impairment in force generated by the contracting muscle fibers seen with aging. The cellular interactions provide and potentially coordinate an adaptation to mechanical loading and ensure successful regeneration after muscle injury. Some of the changes in skeletal muscle ECM with aging may be preventable with resistance or weight training, but it is clear that more human studies are needed on the topic.

Changes in the contents of enzymatic immature, mature, and non-enzymatic senescent cross-links of collagen after once-weekly treatment with human parathyroid hormone (1–34) for 18 months contribute to improvement of bone strength in ovariectomized monkeys

Improvements in total content of enzymatic cross-linking, the ratio of hydroxylysine-derived enzymatic cross-links, and non-enzymatic advanced glycation end product cross-link formation from once-weekly administration of hPTH(1-34) for 18months in OVX cynomolgus monkeys contributed to the improvement of bone strength. Parathyroid hormone (PTH) is used for the treatment of osteoporosis. To elucidate the contribution of material properties to bone strength after once-weekly treatment with hPTH(1-34) in an ovariectomized (OVX) primate model, the content of collagen and enzymatic immature, mature, and non-enzymatic cross-links, collagen maturity, trabecular architecture, and mineralization in vertebrae were simultaneously estimated. Adult female cynomolgus monkeys were divided into four groups (n = 18-20 each) as follows: SHAM group, OVX group, and OVX monkeys given once-weekly subcutaneous injections of hPTH(1-34) either at 1.2 or 6.0μg/kg (low- or high-PTH groups) for 18months. The content of collagen, enzymatic and non-enzymatic cross-linking pentosidine, collagen maturity, trabecular architecture, mineralization, and cancellous bone strength of vertebrae were analyzed. Low-PTH and high-hPTH treatments increased the content of enzymatic immature and mature cross-links, bone volume (BV/TV), and trabecular thickness, and decreased pentosidine, compared with the OVX group. Stepwise logistic regression analysis revealed that BV/TV, the content of total enzymatic cross-links, and calcium content independently affected ultimate load (model R (2) = 0.748, p < 0.001) and breaking energy (model R (2) = 0.702, p < 0.001). BV/TV was the most powerful and enzymatic cross-link content was the second powerful determinant of both ultimate load and breaking energy. The most powerful determinant of stiffness was the enzymatic cross-link content (model R (2) = 0.270, p < 0.001). Once-weekly preventive administration of hPTH(1-34) increased the total contents of immature and mature enzymatic cross-links, which contributed significantly to vertebral cancellous bone strength.

Simulated microgravity-induced aortic remodeling

We have previously shown that microgravity and simulated microgravity induce an increase in human and rat aortic stiffness. We attempted to elucidate the mechanism(s) responsible for this increase in stiffness. We hypothesize that an alteration in vessel wall collagen or elastin content or in extracellular matrix (ECM) cross-linking either individually or in a combination is responsible for the increased vessel stiffness. Rats underwent hindlimb unweighting (HLU) for a period of 7 days to simulate microgravity. The contribution of ECM cross-linking to the vessel wall stiffness was evaluated by measuring aortic pulse wave velocity following inhibition of the cross-linking enzymes lysyl oxidase (LOX) and transglutaminase (tTG) and the nonenzymatic advanced glycation end product cross-linking pathway during HLU. Aortic collagen and elastin content was quantified using established colorimetric assays. Collagen subtype composition was determined via immunofluorescent staining. The increase in aortic pulse wave velocity after HLU was significantly attenuated in the LOX and tTG inhibition groups compared with saline (1.13 +/- 0.11 vs. 3.00 +/- 0.15 m/s, LOX vs. saline, P < 0.001; 1.16 +/- 0.25 vs. 3.00 +/- 0.15 m/s, tTG vs. saline, P < 0.001). Hydroxyproline content, a measure of collagen content, was increased in all groups after HLU (2.01 +/- 0.62 vs. 3.69 +/- 0.68% dry weight, non-HLU vs. HLU, P = 0.009). Collagen subtype composition and aortic elastin content were not altered by HLU. Together, these data indicate that HLU-induced increases in aortic stiffness are due to both increased aortic collagen content and enzyme cross-linking activity.

Endothelial dysfunction and vascular inflammation in Type 2 diabetes: interaction of AGE/RAGE and TNF-α signaling

the receptor for advanced glycation end products (RAGE) interacts with a diverse range of endogenous ligands termed advanced glycation end products (AGEs), which are formed by the Maillard reaction, a nonenzymatic process linking reducing sugar groups to proteins, lipids, and nucleic acids. AGEs

Combination Therapy with the Advanced Glycation End Product Cross-Link Breaker, Alagebrium, and Angiotensin Converting Enzyme Inhibitors in Diabetes: Synergy or Redundancy?

Combination Therapy with the Advanced Glycation End Product Cross-Link Breaker, Alagebrium, and Angiotensin Converting Enzyme Inhibitors in Diabetes: Synergy or Redundancy?

AGE, HYPERTENSION AND ARTERIAL FUNCTION

1. Ageing exerts a marked effect on the cardiovascular system and, in particular, the large arteries. Using a variety of techniques to assess arterial stiffness, many cross-sectional studies have demonstrated a significant relationship between age and aortic stiffness, although the age-related changes observed in peripheral arteries appear to be less marked. 2. The relationship between arterial stiffness and hypertension is more complex. The distending, or mean arterial, pressure is an important confounder of measurements of arterial stiffness and, therefore, must be taken into consideration when assessing arterial stiffness in hypertensive subjects or investigating the effect of antihypertensive agents. Current methods for correcting for differences in distending pressure involve pharmacological manipulation, statistical correction or mathematical manipulation of stiffness indices. 3. Many studies have provided evidence that both peripheral (muscular) and central (elastic) arteries are stiffer in subjects with mixed (systolic/diastolic) hypertension compared with normotensive subjects. However, it is unclear to what extent differences in mean arterial pressure explain the observed differences in hypertensive subjects. In contrast, isolated systolic hypertension is associated with increased aortic, but not peripheral artery, stiffness, although the underlying mechanisms are somewhat unclear. 4. Traditional antihypertensive agents appear to reduce arterial stiffness, but mostly via an indirect effect of lowering mean pressure. Therefore, therapies that target the large arteries to reduce stiffness directly are urgently required. Agents such as nitric oxide donors and phosphodiesterase inhibitors may be useful in reducing stiffness via functional mechanisms. In addition, inhibitors or breakers of advanced glycation end-product cross-links between proteins, such as collagen and elastin, hold substantial promise.

Advanced Glycation End-Products and Arterial Stiffness in Hypertension

The formation of advanced glycation end-products is associated with arterial stiffness in experimental models and alagebrium (formerly known as ALT-711), an advanced glycation end-product cross-link breaker, has been shown to reduce arterial stiffness in elderly subjects. We related plasma concentrations of advanced glycation end-products (AGEs), measured using a noncompetitive immunoassay, and markers of aortic stiffness-pulse wave velocity (PWV) and augmentation index (AIx), a measure of aortic wave reflection-in 46 subjects, aged 47 +/- 2 years, comprising 30 untreated hypertensive and 16 normotensive subjects. Results were analyzed using univariate and multiple logistic regression analysis. Plasma AGEs were significantly higher in hypertensive than in normotensive subjects (7.8 +/- 1 v 3 +/- 1 mug/ml; P < .0001). There was a significant relationship between plasma AGEs and aortic PWV (r = 0.49, P < .01), but not with AIx. In a stepwise regression model age, plasma AGE levels, smoking status, and total cholesterol explained 67% of the variability in PWV. For AIx, the only variables that entered the model were age, gender, and heart rate (R(2) = 0.53, P < .0001) with no contribution from plasma AGEs. Concentration of plasma AGEs is significantly higher in hypertensive than in normotensive subjects and related to aortic stiffness independent of age and blood pressure, with no relationship with aortic wave reflection. Plasma AGEs may play a blood pressure-independent role in large but not small vessel remodeling in essential hypertension.

Combination Therapy with the Advanced Glycation End Product Cross-Link Breaker, Alagebrium, and Angiotensin Converting Enzyme Inhibitors in Diabetes: Synergy or Redundancy?

Blockade of advanced glycation end product (AGE) accumulation with alagebrium with concomitant angiotensin converting enzyme inhibition was tested for effects on renal function and on other postulated mediators of diabetic renal disease including the renin-angiotensin system, AGEs, mitochondrial and cytosolic oxidative stress, and intracellular signaling molecules. Sprague Dawley rats were rendered diabetic with streptozocin and followed consecutively for 32 wk with nondiabetic controls. Groups were treated with ramipril (1 mg/kg.d; wk 0-32); alagebrium (10 mg/kg.d; wk 16-32); or a combination of both. Although individual treatments had significant effects on albuminuria, no further improvements were seen with combination therapy. Changes in urinary vascular endothelial growth factor excretion mirrored those seen in albuminuria. Diabetes was associated with suppression of circulating angiotensin II in the context of increased circulating and renal levels of the AGE, carboxymethyllysine. All treatments attenuated circulating but not renal carboxymethyllysine levels. The renal gene expression of AGE receptor 1 and soluble receptor for advanced glycation end products were markedly reduced by diabetes and normalized with alagebrium. Diabetes induced renal mitochondrial oxidative stress, which was reduced with alagebrium. In the cytosol, both therapies were equally effective in reducing reactive oxygen species production. Increases in membranous protein kinase C activity in diabetes were attenuated by all treatments, whereas diabetes-associated increases in nuclear factor-kappaB p65 translocation remained unaltered by any therapy. It is evident that renin-angiotensin system blockade and AGE inhibition have specific effects. However, many of their downstream effects appear to be similar, suggesting that their renoprotective benefits may ultimately involve common pathways and key points of convergence, which could be important targets for new therapies in diabetic nephropathy.

Advanced glycation end-product cross-link breakers: A novel approach to cardiovascular pathologies related to the aging process

Advanced glycation end product (AGE) formation that occurs with aging and diabetes leads to the cross-linking of proteins and subsequent changes in the physicochemical properties of tissues. Cellular responses to AGE that lead to either pathological conditions or removal of AGE are mediated by a number of receptors that have been identified on various cell types such as macrophages, endothelial cells, and smooth-muscle cells. Mechanisms by which AGE affect the cardiovascular system include AGE cross-linking of long-lived proteins such as collagen and elastin and altered cellular responses. Alagebrium (3-phenacyl-4,5-dimethylthiazolium chloride, ALT-711) is the first drug in a new class of thiazolium therapeutic agents that break established AGE cross-links between proteins. In animal studies, alagebrium was effective in reducing large artery stiffness, slowing pulse-wave velocity, enhancing cardiac output, and improving left ventricular diastolic distensibility. In human studies to determine safety and efficacy, alagebrium was safe and well tolerated. In the first phase 2 clinical study, alagebrium improved arterial compliance in elderly patients with vascular stiffening. In two subsequent phase 2 clinical studies, one addressing diastolic heart failure and the other addressing systolic hypertension, alagebrium was effective in improving cardiac function and uncontrolled systolic blood pressure, particularly in more severely affected patients. Additional clinical studies to determine the utility of alagebrium in treating cardiovascular disorders associated with aging are in progress.

Attenuation of Extracellular Matrix Accumulation in Diabetic Nephropathy by the Advanced Glycation End Product Cross-Link Breaker ALT-711 via a Protein Kinase C-α−Dependent Pathway

This study investigated the role of advanced glycation end products (AGEs) in mediating protein kinase C (PKC) isoform expression in diabetic nephropathy. In vitro, vascular smooth muscle cells incubated in a high-glucose (25-mmol/l) medium demonstrated translocation and increased expression of PKC-alpha as compared with those from a low-glucose (5-mmol/l) environment. Coincubation with the cross-link breaker ALT-711 and, to a lesser extent, with aminoguanidine, an inhibitor of AGE formation, attenuated the increased expression and translocation of PKC-alpha. Streptozotocin-induced diabetic rats were randomized to no treatment, treatment with ALT-711, or treatment with aminoguanidine. Diabetes induced increases in PKC-alpha as well as in the -betaI, -betaII, and -epsilon isoforms. Treatment with ALT-711 and aminoguanidine, which both attenuate renal AGE accumulation, abrogated these increases in PKC expression. However, translocation of phosphorylated PKC-alpha from the cytoplasm to the membrane was reduced only by ALT-711. ALT-711 treatment attenuated expression of vascular endothelial growth factor and the extracellular matrix proteins, fibronectin and laminin, in association with reduced albuminuria. Aminoguanidine had no effect on VEGF expression, although some reduction of fibronectin and laminin was observed. These findings implicate AGEs as important stimuli for the activation of PKC, particularly PKC-alpha, in the diabetic kidney, which can be directly inhibited by ALT-711.

Glycation end-product cross-link breaker reduces collagen and improves cardiac function in aging diabetic heart

Aging and diabetes mellitus (DM) both affect the structure and function of the myocardium, resulting in increased collagen in the heart and reduced cardiac function. As part of this process, hyperglycemia is a stimulus for the production of advanced glycation end products (AGEs), which covalently modify proteins and impair cell function. The goals of this study were first to examine the combined effects of aging and DM on hemodynamics and collagen types in the myocardium in 12 dogs, 9-12 yr old, and second to examine the effects of the AGE cross-link breaker phenyl-4,5-dimethylthazolium chloride (ALT-711) on myocardial collagen protein content, aortic stiffness, and left ventricular (LV) function in the aged diabetic heart. The alloxan model of DM was utilized to study the effects of DM on the aging heart. DM induced in the aging heart decreased LV systolic function (LV ejection fraction fell by 25%), increased aortic stiffness, and increased collagen type I and type III protein content. ALT-711 restored LV ejection fraction, reduced aortic stiffness and LV mass with no reduction in blood glucose level (199 +/- 17 mg/dl), and reversed the upregulation of collagen type I and type III. Myocardial LV collagen solubility (%) increased significantly after treatment with ALT-711. These data suggest that an AGE cross-link breaker may have a therapeutic role in aged patients with DM.

Cleavage of in vitro and in vivo formed lens protein cross-links by a novel cross-link breaker.

The purpose of this study was to investigate the effect of N-phenacyl-4,5-dimethylthiazolium bromide (DMPTB), an advanced glycation end product (AGE) cross-link breaker, on lens protein cross-links formed in vitro and in vivo. DMPTB was synthesized and its structure confirmed by its NMR spectrum. To show whether DMPTB can inhibit AGE cross-linking, recombinant human alphaA-crystallin was glycated with glucose-6-phosphate (G6P) in the presence and absence of DMPTB. Reversal of the already formed cross-links was studied by treating pre-glycated alphaA-crystallin with DMPTB. The ability of DMPTB to cleave in vivo formed cross-links was ascertained by treating water-insoluble protein fractions from diabetic human lenses with this compound. Glycation of alphaA-crystallin with G6P showed several high molecular weight (HMW) protein bands on the SDS-PAGE gel; DMPTB inhibited the formation of these HMW proteins. Molecular sieve HPLC confirmed the inhibition of formation of larger aggregates not separated by SDS-PAGE. Treatment of pre-glycated alphaA-crystallin with DMPTB gave evidence for the degradation of the already formed cross-linked HMW aggregates. Both molecular sieve HPLC and reverse-phase HPLC of the water-insoluble protein fractions from two diabetic human lenses showed that DMPTB could degrade a major portion of the cross-linked HMW aggregates to lower molecular weight proteins. This suggests that the cross-linked proteins in human lenses are formed predominantly by the advanced glycation process and cross-link breakers like DMPTB may have application for the intervention of protein cross-linking in the eye lens.

1P-0273 Attenuation of diabetes-associated atherosclerosis with the advanced glycation end product cross-link breaker, ALT-711

1P-0273 Attenuation of diabetes-associated atherosclerosis with the advanced glycation end product cross-link breaker, ALT-711

Levels of Mature Cross-Links and Advanced Glycation End Product Cross-Links in Human Vitreous

Purpose: To determine the levels of pyridinoline and deoxypyridinoline, two mature enzymatic cross-links, and pentosidine, an advanced glycation end product (AGE) cross-link, in the human vitreous, and to investigate the correlations among the cross-links and the effects of aging and diabetes mellitus (DM) on the levels of cross-links. Methods: Forty-five vitreous samples were collected from 32 patients (32 eyes) undergoing vitrectomy for diabetic retinopathy (DM group) and from 13 patients (13 eyes) (control group) who were age- and sex-matched patients with idiopathic macular hole or epiretinal membrane with no systemic conditions. The levels of the cross-links were determined using high-performance liquid chromatography after acid hydrolysis and pretreatment with SP-Sephadex. Results: The levels of pentosidine, pyridinoline, and deoxypyridinoline were 27.3 ± 23.1 (mean ± SD) pmol/mL (detectable in 45 of 45 specimens), 79.0 ± 40.2 ng/mL (43 of 45 specimens), and 54.0 ± 9.5 (32 of 45 specimens) ng/mL, respectively. When the vitreous samples from the DM and the control groups were compared, a significant difference (P < .05) was found in the pentosidine level but not in the levels of pyridinoline or deoxypyridinoline. No significant correlations were found between age and the cross-links. Significant correlations ( P < .01) were found among the cross-links. Conclusions: The results indicate that mature cross-link substances exist in the human vitreous. The results also suggest that glycation may occur in the vitreous after mature cross-links form and result in the formation of AGE cross-links. In human vitreous from patients with DM, increased levels of AGE cross-links may stabilize the formation of mature cross-links, but they did not increase the mature cross-links.

Levels of Mature Cross-links and Advanced Glycation End Product Cross-links in Human Vitreous

Levels of Mature Cross-links and Advanced Glycation End Product Cross-links in Human Vitreous

An advanced glycation endproduct cross-link breaker can reverse age-related increases in myocardial stiffness.

Decreased elasticity of the cardiovascular system is one of the hallmarks of the normal aging process of mammals. A potential explanation for this decreased elasticity is that glucose can react nonenzymatically with long-lived proteins, such as collagen and lens crystallin, and link them together, producing advanced glycation endproducts (AGEs). Previous studies have shown that aminoguanidine, an AGE inhibitor, can prevent glucose cross-linking of proteins and the loss of elasticity associated with aging and diabetes. Recently, an AGE cross-link breaker (ALT-711) has been described, which we have evaluated in aged dogs. After 1 month of administration of ALT-711, a significant reduction ( approximately 40%) in age-related left ventricular stiffness was observed [(57.1 +/- 6.8 mmHg x m(2)/ml pretreatment and 33.1 +/- 4.6 mmHg x m(2)/ml posttreatment (1 mmHg = 133 Pa)]. This decrease was accompanied by improvement in cardiac function.

Interactions of LDL and modified LDL with mesangial cells and matrix.

Hyperlipidemia may play a role in the progression of diabetic and other renal diseases. Low density lipoprotein (LDL) and other proteins including extracellular matrix components undergo nonenzymatic glycation in vivo. We examined the effects of glycation of LDL as occurs in diabetes (4 to 8%) on binding and uptake by mesangial cells and their proliferation. The glycation of LDL (g-LDL) significantly decreased its binding and uptake by mesangial cells by 15 to 20%, indicating that glycated LDL binds to the LDL receptor, but with lower affinity than LDL. Both LDL and g-LDL modestly stimulated [3H] thymidine incorporation into mesangial cells at 5 to 10 micrograms/ml. Native, oxidized (Ox-LDL) and glycated LDL all bound to the extracellular matrix generated by rat mesangial cells in culture. The binding of LDL, Ox-LDL and g-LDL to mesangial matrix was two to four times higher than to mesangial cells. Binding of LDL and g-LDL was significantly higher to glycolaldehyde modified matrix, which serves as an in vitro model for nonenzymatic glycation end-product cross-linking of matrix which occurs in long-standing diabetes. Based on these findings, we propose that glycation of LDL decreases its binding and uptake by the LDL receptor of mesangial cells and may slow its catabolism. Furthermore, LDL bound to extracellular mesangial matrix can undergo oxidation and generate cytotoxic LDL components. This process may be further enhanced by advanced glycation of the mesangial matrix in diabetes, contributing to glomerular pathology.