Ernst J. Menzel

Calcium antagonists as inhibitors ofin vitro low density lipoprotein oxidation and glycation

The peroxidation step in lipid transformation is considered to be essential in the pathogenesis of atherosclerosis. Calcium antagonists (CA) appear to have antioxidant effects in addition to their potent vasorelaxant properties. In the present study, we compared the antioxidative efficacy of CA (amlodipine, lacidipine, nifedipine, isradipine, diltiazem, and semotiadil) in the copper-catalysed oxidation of low-density lipoprotein (LDL) with that of glycated(g)/glycoxidated(go) LDL. This issue is of great importance when considering the potential therapeutic use of antioxidant drugs in diabetes-associated vasculopathy. Oxidation of native LDL was inhibited most efficiently (>90%) by lacidipine and semotiadil in the concentration range 10(-4)-10(-3) M. We found, however, a dramatic decrease in antioxidant activity towards g/goLDL as compared to native LDL in all the CA tested. Only lacidipine significantly inhibited copper-mediated oxidation of g/goLDL in the whole concentration range tested (10(-5) M-10(-3) M). This probably resulted from the increased auto-oxidative potential introduced by early and advanced glycation end products (AGE) into the g/goLDL. We noted that coincubation of LDL with 10(-3) M CA and 0.5 M glucose under oxidative/non-oxidative conditions partially or fully restored the antioxidant capacity of the different CA to inhibit the subsequent copper-catalysed oxidation of the modified LDL. This is a clear indication that CA inhibit glycative or glycoxidative LDL changes during the preceding long-term glycation period. The notion that both oxidative changes and long-term glycation effects were reduced by CA was corroborated by fluorescence analysis, AGE-ELISA, quantitation of lipid peroxidation, and thiobarbituric acid reactive substance (TBARS) measurement of long-term g/goLDL. The strongest antioxidative effects during long-term glycation of LDL were seen with isradipine, lacidipine, nifedipine, and semotiadil. Diltiazem was the only CA that could not prevent TBARS formation in LDL during the long-term glycation period. In contrast, Amadori product formation, as measured by the generation of fructosamines, was not significantly reduced by any CA tested. Thus CA, like other antioxidants, significantly retard AGE formation, while initial glycation reactions, such as Amadori product formation, are only weakly inhibited.

Differential scanning calorimetry, biochemical, and biomechanical analysis of human skin from individuals with diabetes mellitus

The aim of this work was to compare biochemical, two‐dimensional biomechanical and calorimetric parameters of diabetic skin vs. control skin. Skin specimens taken from the palms and backs of the hands of aged persons with non‐insulin‐dependent diabetes mellitus (NIDDM) and of controls (CO) were compared (age range 68–85 years). Only skin specimens from individuals with diabetes mellitus (DM) showed an increased fluorescence specific for the formation of advanced glycation end‐products (AGEs) and the presence of tissue AGEs, such as Ne‐(Carboxymethyl)lysine (CML). Differential scanning calorimetry (DSC) revealed an elevation of the heat flow per unit mass during collagen denaturation in diabetic skin samples. However, the temperatures of the heat flow maximum and the onset of the phase transformation were not uniformly altered. Young′s moduli were found to be increased in diabetic skin and correlated with AGE‐fluorescence and tissue AGEs. The ratio between the Youngs moduli, which defines a measure for the degree of anisotropy, was higher for dorsal skins from hands. In dorsal skin specimens from diabetic subjects the degree of anisotropy was more pronounced than in healthy controls. In general, neither of the measured parameters showed any correlation with age. However, E1 moduli were clearly associated with the duration of diabetes. Anat Rec 259:327–333, 2000.

Comparison of the Effect of Different Inhibitors on the Non-Enzymatic Glycation of Rat Tail Tendons and Bovine Serum Albumin

The biomechanical and biochemical properties of collagen are changed by non-enzymatic glycation culminating in increased cross-linking. We have previously shown that dibasic amino acids such as L-arginine inhibit in vitro the non-enzymatic glycation of soluble proteins and insoluble connective tissue macromolecules. In the present in vitro study we obtained evidence that the nucleophilic hydrazine derivative aminoguanidine and the non-steroidal antirheumatic drug ibuprofen inhibit the formation of fluorescent advanced glycation end products (AGEs) to a comparable extent, while arginine is ineffective as a consequence of its tendency to form AGEs itself. Periodic replacement of glycated arginine in the rat tail tendon system, however, engendered an inhibition of fluorescence similar to that obtained by the other inhibitors. Long-term glycation of rat tail tendons caused a significant increase in Youngs modulus, which could also be inhibited by periodically renewed arginine. In contrast to ibuprofen, aminoguanidine and arginine-lysine inhibited the marked increase in maximum contraction force of long-term glycated rat tail tendons. As opposed to other inhibitors, aminoguanidine also reduced the thermal contraction force of native tendons, shifted the maximum contraction temperature to markedly lower values and solubilized a significant part of the rat tail tendon collagen. These findings indicate that the in vitro alterations of rat tail tendon collagen induced by non-enzymatic glycation can be prevented by arginine, arginine-lysine and aminoguanidine. However, collagen structure is seriously affected by aminoguanidine.

Autoimmunity and T-cell subpopulations in old age

To investigate the interrelationship between T-cell-dependent immune functions and autoimmune phenomena in old age we determined T-cell subpopulations in 20 aged healthy individuals (80-96 years old) using monoclonal antibodies. These persons were also investigated as to humoral parameters such as antinuclear antibodies, rheumatoid factors (IgG-, IgA-, IgM-RF), antibodies to collagen types I-IV as well as autoantibodies to organ-specific antigens. In addition, immune complexes were determined. We found that aged individuals have an increased frequency of autoantibodies as compared to a young control population, each aged subject presenting with at least one autoantibody species. Immune complexes, however, were only rarely detected. Three individuals showed a slightly increased T-helper/T-suppressor cell ratio, four had a decreased ratio. An increased number of T-suppressor cells was significantly correlated with a lowered incidence of anticollagen antibodies. Other parameters tested by us: fibronectin, laminin, procollagen type III, C3 and C4 complement components, immunoglobulins and acid alpha 1-glycoprotein. Aged individuals have significantly higher serum levels of fibronectin, while laminin and procollagen concentrations are in the normal range. A large percentage of old individuals had increased serum levels of C3 and/or C4. The acute phase protein orosomucoid, however, was in the normal range.

Changes of Biochemical and Biomechanical Properties in Dupuytren Disease

BACKGROUND The major biochemical characteristic of Dupuytren disease is the progressive and irreversible deposition of excess fibrous collagen characterized by an enhanced type III collagen proportion. OBJECTIVE To investigate the influence of changes of the collagen spectrum on the biophysical properties of the palmar aponeurosis. DESIGN Variably affected palmar regions from 30 individuals with Dupuytren disease were classified according to histologic test results and clinical stage. Biochemical, biomechanical, and thermal contracture studies were performed. RESULTS The relative type III collagen content increased with increasing tissue involvement and was found to correlate with calorimetric and biomechanical properties with the exception of the Young modulus. In experiments on the thermal isometric contracture, the collagen denaturation temperature decreased with increasing type III collagen content, ie, increasing involvement. To study the dependence of biophysical properties from the collagen type distribution independent of structural changes, as seen in Dupuytren disease, we investigated rat skins from animals of an age range characterized by dramatic changes in type III collagen content (0-18 months). Biomechanical data also correlated significantly with type III collagen content in rat skin with the exception of the time constant of stress relaxation. CONCLUSION In light of these results, we suggest that structural changes, such as reduced collagen fibril diameters, associated with alterations in the type III collagen proportion may influence biophysical properties of connective tissues in the involved palmar aponeurosis in addition to alterations of the cross-linking pattern.

Comparison of palmar aponeuroses from individuals with diabetes Mellitus and Dupuytren's contracture

It is well known that Dupuytrens contracture is often associated with diabetes mellitus. Palmar fascia from individuals with diabetes mellitus and/or Dupuytrens contracture as well as controls were subjected to differential scanning calorimetry, biomechanical and biochemical analysis. The collagen denaturation temperature of the palmar aponeurosis from individuals with diabetes mellitus in the presence (71.0°C) or absence of Dupuytrens contracture (70.6°C) was increased as compared with controls (68.5°C), while this parameter was significantly reduced (about 3.5°C) in contracture bands of Dupuytrens contracture. Stress relaxation experiments revealed that the viscous fraction was slightly reduced in diabetes mellitus (6.5%) vs. controls (8.3%), whereas in Dupuytrens contracture, irrespective of additional diabetes mellitus, a pronounced increase of this parameter was seen (36.5% vs. 24.5%) in the presence of diabetes mellitus. The time constants were significantly elevated by both disorders, this increase being more pronounced in Dupuytrens contracture. Taken together, these changes can be explained by increased cross‐linking in diabetes mellitus, while in Dupuytrens contracture other structural changes, such as increased collagen type III content and loss of fascicular organization, play an additional role besides the finding of reduced cross linking. Anat Rec 255:401–406, 1999.

BINDING OF LONG-TERM GLYCATED LOW DENSITY LIPOPROTEIN AND AGE- ALBUMIN BY PERIPHERAL MONOCYTES AND ENDOTHELIAL CELLS

Modification of low density lipoprotein (LDL) and plasma or tissue proteins by non-enzymatic glycation culminating in the formation of advanced glycation endproducts (AGEs) is one of the essential pathomechanisms leading to diabetes-associated long-term complications. We compared binding of glycated, glycoxidated and oxidated LDL by peripheral monocytes in activated and quiescent form. Interaction via specific receptors was different for glycated as compared to (glyc)oxidated LDL-modifications. In addition, binding of glycated LDL to quiescent and activated human umbilical vein endothelial cells was studied. In patients with insulin-dependent diabetes mellitus (IDDM), AGE-binding was significantly increased as compared to healthy individuals. Specific and non-specific monocyte binding mechanisms were detected, and both were significantly increased in IDDM patients. Specific and non-specific binding strategies possibly act in concert to eliminate circulating AGEs, which are instrumental in the development and progress of microangiopathic and macroangiopathic complications of diabetes mellitus.

Induction of anti-pepsin antibodies after immunization with pepsin-extracted collagen

Immunization with pepsin-extracted human type II collagen purified by different precipitation steps, although not showing any contamination with the enzyme on SDS-polyacrylamide gel electrophoresis, resulted in the generation of antibodies to the enzyme in addition to an immune response to collagen. These antibodies could be removed by immune absorption on a pepsin affinity column, leaving reactivity to type II collagen unaltered. High performance liquid chromatography on hydroxylapatite columns indicated that pepsin remained associated with the collagen molecules even after repeated precipitation and coeluted with a fraction of the collagen preparation. These results demonstrate that pepsin-extracted collagens may contain minimal amounts of the enzyme. On immunization, these impurities may induce the formation of unwanted antibodies, which might simulate a false specificity of the antibody preparation.

Troglitazone-binding to LDL and its glycated modifications: its role in cell-catalysed and Cu-mediated LDL-oxidation.

Troglitazone (T), an anti-diabetic drug improving insulin resistance, was studied as to its inhibition of copper ion-catalysed oxidation of native, glycated and glycoxidated low-density lipoprotein (LDL). A dose-dependent inhibition was noted in the concentration range 40-160 microg/ml. An almost complete inhibition of oxidation (2-8 h), as monitored by the formation of thiobarbituric acid-reactive substances, was observed for both native and glycated LDL at a concentration of 160 microg/ml T, while the maximal inhibition for glycoxidated LDL amounted only to 60% at this concentration of the drug. This is reflected by differences in the affinity of the drug for the different types of LDL modification: While the binding of T both to native or glycated LDL increased linearly with increasing T concentration and was not saturable in the concentration range tested (0-160 microg/ml), binding of the drug to glycoxidated LDL was already nearly saturated at 10 microg/ml. The nearly complete inhibitory action of T towards oxidation of native and glycated LDL was lost, however, upon increasing the total oxidation time to 24 h. In human umbilical vein endothelial cell-mediated oxidation of LDL, T at a concentration of 20 microg/ml significantly reduced formation of oxidation-dependent fluorescent chromophores and liberation of 8-epi-PGF2alpha. In contrast, generation of thiobarbituric acid-reactive substances was not significantly inhibited. As opposed to copper-mediated LDL-oxidation, different binding of T to LDL-modifications does not govern inhibition of human umbilical vein endothelial cell-mediated LDL-oxidation.

The Influence of L-Arginine on Biomechanical Parameters of Native and Nonenzymatically Glycated Rat Tail Tendons

Rat tail tendons from animals of an age range from 35 days to 900 days were glycated using different glucose concentrations. After nonenzymatic glycation the biomechanical properties of tendons from young rats approached that of old tendons. In tendons from old animals the glucose incorporation was significantly lower than in young rat tail tendons. Addition of L-arginine to the glucose solution reduced the incorporation of glucose especially in young rats. The concentration of early glycation products and of advanced glycation end products were measured via affinity chromatography on boronic acid agarose and relative fluorescence per collagen content respectively. Both parameters were significantly reduced by an addition of 10 mmol/l arginine. Biomechanical changes due to glycation were partially reversed. Incubation of rat tail tendons in L-arginine in the absence of glucose caused a dose dependent binding of the amino acid mainly to the proteoglycan matrix. High concentrations of L-arginine induced pronounced biomechanical alterations contrary to the action of glucose. The biomechanical effect of L-arginine is compared to the action of Na+ and Ca2+ and discussed on the basis of a structural model of the proteoglycan matrix. Incubation of already glycated rat tail tendons with L-arginine caused a reduction of the elastic stress component This effect was diminished by increasing the preincubation interval with glucose. The equilibrium values of the elastic fraction were achieved after approximately five days incubation with the amino acid.