E. J. Menzel

Autoantibodies to oxidised low density lipoproteins in IDDM are inversely related to metabolic control and microvascular complications

Summary Diabetes mellitus is associated with an increased risk of atherosclerosis. The oxidation of low-density lipoproteins (LDL) is considered a key event in the initiation of atherosclerosis. To investigate LDL oxidation in vivo we measured autoantibodies to oxidised LDL (oxLDL) in 94 patients with insulin-dependent diabetes mellitus (IDDM), compared to 27 age-matched, healthy control subjects. Patients and control subjects were screened for autoantibodies using a solid phase ELISA, comparing the binding to oxLDL with that to native LDL (nLDL). In patients with IDDM the oxLDL/nLDL antibody ratio was significantly higher than in control subjects (means ± SEM: 2.24 ± 0.26 vs 1.17 ± 0.17, p < 0.03). Antibody-negative patients had a longer diabetes duration (13.5 ± 1.3 vs 9.1 ± 1.1 years, p < 0.01) and higher actual and mean HbA1 c levels compared to antibody-positive patients (8.8 ± 0.2 vs 7.9 ± 0.2 %, p < 0.005 and 8.3 ± 0.2 vs 7.7 ± 0.2 %, p < 0.03; respectively). In patients with a high microangiopathy score, the antibody ratio was lower than in patients without complications (1.04 ± 0.10 vs 2.40 ± 0.29, p < 0.01). OxLDL specific immune complexes were found exclusively in antibody-negative as compared to antibody-positive patients (18.3 vs 0 %; p < 0.01). Our data demonstrate an inverse relationship between free oxLDL antibodies and the severity of the disease. This apparent paradox can be explained in part by our demonstration of oxLDL immune complexes, masking free antibodies. [Diabetologia (1998) 41: 350–356]

Alterations of biochemical and biomechanical properties of rat tail tendons caused by non-enzymatic glycation and their inhibition by dibasic amino acids arginine and lysine

SummaryThe influence of dibasic amino acids arginine and lysine on non-enzymatic glycation of tail tendon fibers from old (900-day-old) and young (61-day-old) rats was investigated in vitro. The biomechanical changes in tendon fibers of young rats after an incubation interval of 7 or 14 days in a glucose solution were abolished by the addition of arginine or lysine (molar ratio amino acid:glucose 1∶10). Glucose incorporation into rat tail tendon fibers as well as Amadori product formation was decreased significantly in the presence of the amino acids. The inhibitory effect of arginine was further confirmed by measurement of the amount of ketoamine formed during the glycation reaction using soluble albumin as a protein target. The effective inhibition of non-enzymatic glycation by arginine or lysine suggests their potential use in vivo as a means of controling protein over-glycation.

Alterations of biochemical and two-dimensional biomechanical properties of human skin in diabetes mellitus as compared to effects of in vitro non-enzymatic glycation.

OBJECTIVE The aim of this study was to evaluate whether multiaxial analysis of diabetes-specific biomechanical changes generated in vitro by non-enzymatic glycation of human skin samples from healthy subjects reflect the changes seen in skin from subjects with diabetes mellitus. DESIGN Descriptive study. BACKGROUND Non-enzymatic glycation of skin in vitro causes an increased stiffness comparable to in vivo changes seen in diabetic patients. These changes are probably due to increased cross-linking of collagen molecules. METHODS Skin specimens from 7 subjects with diabetes mellitus and 7 controls (age range: 74-90) were analyzed for biomechanical changes using a multiaxial tensile testing device. Control skins from healthy individuals in the age range of 50-65 yr were artificially glycated. One part of these samples was coincubated with the glycation inhibitor aminoguanidine. Glycation of tissues was determined by measuring fluorescence of solubilized samples. Multiaxial biomechanical analysis allows the determination of maximum (a(I)) and minimum elastic modulus (a(II)). These parameters describe the amplitude of the elastic stress response, which is exponentially related to strain. RESULTS Principal stresses, both maximum and minimum, were increased in skins from diabetic subjects as compared to controls. The increases of the principal stresses were comparable to those obtained by in vitro glycation of normal skins. CONCLUSION These results, which can be detected unequivocally with the multiaxial test mode, show that our in vitro model closely reflects changes in skin samples from individuals with diabetes mellitus. Aminoguanidine partially inhibited these as well as biochemical changes. RELEVANCE Multiaxial testing of in vitro glycated skin samples can be used as a model for in vivo changes caused by diabetes mellitus. In addition, therapeutical effects of aminoguanidine, an inhibitor of non-enzymatic glycation, can be monitored in this model.

Two-dimensional stress-relaxation behavior of human skin as influenced by non-enzymatic glycation and the inhibitory agent aminoguanidine

In order to simulate the in vivo stress alterations of diabetic skin in an in vitro model, we examined the viscoelastic properties of long-term glycated human skin samples. Since skin is subjected to biaxial tension, we used two-dimensional multiaxial testing which better reflects the in vivo situation than the uniaxial testing mode. For native skin samples from the abdominal region we found a direction-dependent elastic stress strain behavior. The viscous stress component was separated from the elastic stress component by relaxation tests at consecutive incremental steps of radial strains. We hypothesize that glycation-induced changes in the tissue stiffness are generated in a direction-dependent mode. A marked increase of the direction-dependent stiffness was found upon long-term incubation with glucose-6-phosphate. This increase was statistically significant for the maximum principal elastic stress component which was highly correlated with the degree of non-enzymatic collagen modification. The viscous fractions obtained from two-dimensional relaxation tests at consecutive radial strains were inversely correlated with non-enzymatic modification. Only at 30% radial strain a significant decrease of the viscous fraction engendered by glucose-6-phosphate was observed together with a direction-dependent significant increase of the expectation value of the time constant. The biomechanical and biochemical effects of long-term glycation could be partially reversed by aminoguanidine, a potential therapeutic agent for patients with diabetes mellitus. Our findings suggest that additional cross-links generated by long-term glycation cause two-dimensional biomechanical alterations in human skin, which can be unequivocally detected by multiaxial testing.

Biomechanical properties of normal tendons, normal palmar aponeuroses, and tissues from patients with Dupuytren's disease subjected to elastase and chondroitinase treatment

Normal tendons, normal palmar aponeuroses and palmar aponeuroses from patients with Dupuytrens disease were subjected to elastase or chondroitinase treatment. Youngs modulus was derived from the linear portion of stress-strain graph. It showed the lowest value for the apparently normal palmar aponeuroses and the highest value for tendon samples. Elastase treatment caused an increase of extensibility and a reduction of Youngs modulus of normal palmar aponeuroses and tendons, but not of contracture bands. In normal tendons, normal palmar aponeuroses and apparently normal palmar aponeuroses residual strain and hysteresis loop increased significantly as a linear function of the amount of digested elastin. In contrast these biomechanical parameters were not affected significantly in contracture bands. In normal and apparently normal areas incubation with chondroitinase ABC resulted in a significant increase of residual strain and, as opposed to elastase, a decrease of normalized hysteresis loop. In contracture bands, however, these biomechanical parameters remained unchanged. RELEVANCE: The increasing evidence of a correlation between morphological changes of palmar elastin and ground substance with the progress of Dupuytrens disease emphasizes the need to determine the relative importance of these connective tissue components for the pathogenesis of Dupuytrens disease.

Inhibition of Cytokine Production and Adhesion Molecule Expression by Ibuprofen is Without Effect on Transendothelial Migration of Monocytes

The present study focusses on the effects of ibuprofen and its enantiomers on cytokine production by peripheral blood monocytes and endothelial cells as well as on the potential modulation of ADM-expression by human umbilical vein endothelial cells and the concomitant effects on monocyte transendothelial migration as measured by a cell migration assay system. This consists of an endothelial cell monolayer on a solid collagen substrate, i.e. an artificial vessel wall construct. We observed a significant inhibition by 100 μg/ml ibuprofen of VCAM-1 expression by endothelial cells while ELAM-1 and ICAM-1 expression was not influenced. However, we could not see any concomitant inhibitory effects on the spontaneous migration of monocytes after preincubating the endothelial cell monolayer with ibuprofen up to concentrations of 100 μg/ml and activating with suboptimal and optimal concentrations of TNF-α. Our monocyte transendothelial migration system reflects very sensitively endothelial cell-activation even by very low TNF-α concentrations. (S)- and (R)-ibuprofen were equal in their inhibitory/activating effects on cytokine production, with the exception of stronger IL-8 induction in endothelial cells by (R)-ibuprofen as compared to its chiral analogue.

On the Orthogonal Anisotropy of Human Skin as a Function of Anatomical Region

Skin samples were obtained from 8 anatomical sites of 6 human deceased at ages ranging from 30 to 80 years 24 hours post mortem. As shown by biochemical analysis the collagen content varied between 71% and 78% depending on the anatomical location of the skin samples. The content of collagen type III was in the range of 19.2% to 22.2% of the total collagen concentration. As to the biomechanical analysis the axes of minimum and maximum shrinkage after excision were determined and correlated with Langer cleavage line drawn on the specimen with a marker after incision. Two-dimensional biomechanical tests were conducted with a multiaxial tensile testing device consisting of 12 loading axes. The in vivo configuration was a circle with 30 mm diameter. The in vivo stresses were determined by restoring the original shape of the specimen. According to the nonlinear stress-strain relationship incremental strains were applied to the sample with the in vivo configuration and states of uniform extension as reference. The corresponding stresses were recorded after stress relaxation was completed and the equilibrium stresses were regarded as the elastic contribution to the viscoelastic biomechanical behavior. The elastic parameters as a function of the initial strain level were calculated using a set of different incremental strains and stresses. The highest in vivo stresses were found in patella, and upper and lower back. The maximum deviation of the direction of maximum in vivo stress from the Langer cleavage line was found in upper back, the volar part of thigh, and sternum. In vivo orthogonal anisotropy was most pronounced in patella and hollow of the knee.

Differential expression of receptors for advanced glycation end products on monocytes in patients with IDDM

Summary Accelerated modification of proteins by glucose terminating in the formation of advanced glycation endproducts (AGEs) is one of the main pathogenetic mechanisms of diabetes-associated complications. One pathway by which AGEs may exert their effects is by interaction with specific receptors initially identified on macrophages, monocytes and endothelial cells. As AGE-induced autocrine upregulation of AGE receptors has been observed in vitro, we hypothesized that AGE-binding might be enhanced in diabetic patients to compensate for the elevated levels of circulating AGEs. We therefore examined the expression of AGE-binding sites on peripheral monocytes, serum levels of AGEs and AGE-induced cytokine production in patients with insulin-dependent diabetes mellitus (IDDM) compared to age-matched, healthy control subjects. In patients, AGE-binding capacity was significantly increased and there was only one class of binding sites, as revealed by Scatchard analysis (1.8 × 105 vs 1.4 × 105 binding sites per cell). Affinity of binding was, however, similar (Ka 1.5 × 106 vs 1.4 × 106 mol− 1). Saturation of binding was reached at 2.0–3.0 μmol/l with AGE-bovine serum albumin (BSA) as ligand. In contrast, cytometry using fluorescein isothiocyanate-labelled AGE-proteins showed no saturability and reversibility of AGE-binding up to 80 μmol/l, indicating non-specific binding in this concentration range. Again, this non-specific binding was significantly higher in IDDM patients. In addition, we found much higher levels of circulating AGEs in patients as compared to controls and studied possible functional consequences of increased AGE binding in vitro, monocyte stimulation by AGEs triggering cytokine release to a similar extent in patients and controls, i. e. independently of the AGE-binding capacity. Our finding of an enhanced overall AGE-binding capacity of peripheral monocytes in IDDM could be instrumental in limiting the plasma concentration of AGEs, the non-specific binding coming into play after saturation of specific binding sites by higher plasma AGE-levels. Both binding strategies may act in concert as “damage limitation mechanisms” in the development of AGE-dependent diabetic complications. [Diabetologia (1998) 41: 674–680]

Biomechanical Properties of Elastase Treated Palmar Aponeuroses

Human palmar aponeurosis was treated with elastase in the presence or absence of soybean trypsin inhibitor. The removal of elastic fibers was complete as proved by electron microscopy. Cyclic loading was performed at a constant strain rate. Residual strain was measured and the stiffness and the fraction of dissipated energy of strain energy was calculated and compared to that of untreated samples of palmar aponeurosis. Residual strain and dissipated energy showed a dramatic increase after enzyme treatment, both in presence and absence of soybean trypsin inhibitor. Stiffness, on the other hand was reduced. The remaining collagen fibers show a more viscous behavior. Our results support the hypothesis that the elastin fibers are responsible for the elastic recovery, observed on specimens of untreated palmar aponeurosis.

Comparison of Normal and in vitro Aging by Non-Enzymatic Glycation as Verified by Differential Scanning Calorimetry

The biomechanical parameters of rat tail tendons (RTTs) from 35-, 64-, 180- and 900-day-old animals, corresponding to the early maturation phase, the mature and the senescent state were determined. The increase of maximum stiffness, ultimate stress and the elastic fraction of stress was most pronounced in the maturation phase. Differential scanning calorimetry (DSC) experiments were performed showing an almost linear increase of the collagen denaturation temperature in the age range 35–139 days. After 14 days incubation in glucose, we observed a marked increase of the biomechanical parameters in the young, an increase of maximum stiffness in mature, and only slight alterations of the biomechanical behavior in senescent RTTs. Both glucose incorporation and formation of advanced glycation end products were most prominent in 35-day-old RTTs. These biochemical findings were in excellent agreement with the enhancement of the collagen denaturation temperature after the incubation phase. Results suggest that the validity of the term ‘accelerated aging’ depends on the experimental approach, i.e. biomechanical tests, thermal isometric contraction or DSC.