N(epsilon)-(Carboxymethyl)lysine (CML), a major product of oxidative modification of glycated proteins, has been suggested to represent a general marker of oxidative stress and long-term damage to proteins in aging, atherosclerosis, and diabetes. To investigate the occurrence and distribution of CML in humans an antiserum specifically recognizing protein-bound CML was generated. The oxidative formation of CML from glycated proteins was reduced by lipoic acid, aminoguanidine, superoxide dismutase, catalase, and particularly vitamin E and desferrioxamine. Immunolocalization of CML in skin, lung, heart, kidney, intestine, intervertebral discs, and particularly in arteries provided evidence for an age-dependent increase in CML accumulation in distinct locations, and acceleration of this process in diabetes. Intense staining of the arterial wall and particularly the elastic membrane was found. High levels of CML modification were observed within atherosclerotic plaques and in foam cells. The preferential location of CML immunoreactivity in lesions may indicate the contribution of glycoxidation to the processes occurring in diabetes and aging. Additionally, we found increased CML content in serum proteins in diabetic patients. The strong dependence of CML formation on oxidative conditions together with the increased occurrence of CML in diabetic serum and tissue proteins suggest a role for CML as endogenous biomarker for oxidative damage.

Objective. To investigate the appearance of hypertrophic chondrocytes in osteoarthritic (OA) cartilage, using type X collagen as a specific marker.Methods. The biosynthesis of type X collagen was examined by metabolic labeling of freshly isolated articular chondrocytes with ³H‐proline, immunoprecipitation, and sodium dodecyl sulfate–polyacrylamide gel electrophoresis of the synthesized collagens. Extracellular deposition of types X and II collagen was analyzed immunohistochemically.Results. Immunostaining revealed an irregular distribution of type X collagen, which was localized around chondrocyte clusters in fibrillated OA cartilage, but was absent from the noncalcified region of normal articular cartilage. Freshly isolated OA chondrocytes synthesized predominantly type X collagen, while control chondrocytes synthesized mostly type II collagen.Conclusion. Our findings indicate focal premature chondrocyte differentiation to hypertrophic cells in OA cartilage.

Previous studies revealed that exposure of mesangial cells to high glucose concentration induces the production of matrix proteins mediated by TGF-beta1. We tested if structural analogues of D-glucose may mimic the high glucose effect and found that D-glucosamine was strikingly more potent than D-glucose itself in enhancing the production of TGF-beta protein and subsequent production of the matrix components heparan sulfate proteoglycan and fibronectin in a time- and dose-dependent manner. D-Glucosamine also promoted conversion of latent TGF-beta to the active form. Therefore, we suggested that the hexosamine biosynthetic pathway (the key enzyme of which is glutamine:fructose-6-phosphate amidotransferase [GFAT]) contributes to the high glucose-induced TGF-beta1 production. Inhibition of GFAT by the substrate analogue azaserine or by inhibition of GFAT protein synthesis with antisense oligonucleotide prevented the high glucose-induced increase in cellular glucosamine metabolites and TGF-beta1 expression and bioactivity and subsequent effects on mesangial cell proliferation and matrix production. Overall, our study indicates that the flux of glucose metabolism through the GFAT catalyzed hexosamine biosynthetic pathway is involved in the glucose-induced mesangial production of TGF-beta leading to increased matrix production.

Immunohistochemical study of tumor necrosis factor α expression in autopsy and surgical specimens of human lumbar intervertebral discs. To investigate the occurrence and localization of tumor necrosis factor α in intervertebral disc tissue and to correlate its expression with age and the degree of disc degeneration. The source and origin of discogenic pain are as yet unknown. Recently identified changes of the cellular phenotype during senescence and disc pathology with partly phagocytic properties suggest an ‘inflammatory’ phenotype. Tumor necrosis factor α is one of the most potent proinflammatory cytokines possibly modulating cellular phenotypes. It may also promote pain induction. Very little is known about the occurrence and localization of tumor necrosis factor α in intervertebral disc tissue of defined age and degree of histologic tissue degeneration. The study population comprised 20 cross-sections of the complete motion segment of human lumbar vertebrae (age range 0–86 years) obtained at autopsy and 28 surgical disc specimens of individuals undergoing lumbar surgical interventions for various reasons. The temporospatial distribution of tumor necrosis factor α-positive cells using a polyclonal antibody was correlated with a histologic degeneration score. Tumor necrosis factor α is expressed substantially in (nonsymptomatic) autopsy material in fetal/infantile and older adult nucleus pulposus, whereas it is sparsely expressed in adolescent and young adult nucleus pulposus. In the anulus fibrosus, tumor necrosis factor α is not found in young adults (<25 years), but then significantly increases in extent. In contrast, symptomatic nucleus pulposus and anulus fibrosus (surgical material) contain substantially more tumor necrosis factor α-positive cells. A significant positive correlation of tumor necrosis factor α expression and disc degeneration (histologic degeneration score) was found for the anulus fibrosus in both sample groups. In the surgical material, an additional significant positive correlation was identified for nuclear tumor necrosis factor α, disc degeneration, and age. Tumor necrosis factor α is substantially expressed in disc material of symptomatic patients (surgical specimens) in comparison to samples taken at autopsy. The expression of tumor necrosis factor α in early fetal/infantile nucleus pulposus may indicate ‘physiologic’ tissue disarrangement with closure of the blood vessel canals. The expression of tumor necrosis factor α in adult discs, in contrast, is statistically associated with disc degeneration. Its occurrence in adults of more advanced age suggests that tumor necrosis factor α is not involved in the initiation of disc degeneration, but may be associated with further promotion of degenerative disarrangement and pain induction.

Study Design. The authors performed a correlative macroscopic, histologic, and immunohistochemical investigation on human lumbar intervertebral discs using complete motion segment slices, including all age groups and stages of degeneration. Objectives. To identify markers for age-related changes of human lumbar intervertebral discs. In particular, to investigate changes in the distribution pattern of collagen Types I, II, III, IV, V, VI, IX, and X. In addition, to study posttranslational protein modification by the immunolocalization of N-(carboxylmethyl)lysine (CML), which is regarded as a biomarker for oxidative stress. Summary of Background Data. Data on a correlation of age-related changes in disc morphology and disc matrix composition is sparse. So far, no comprehensive analysis considered a correlation of macroscopic, histologic, and biochemical age-related alterations using complete sections of intervertebral discs (i.e., including nucleus pulposus, anulus fibrosus, endplates, and vertebral bodies). In addition, there is need for specific markers for these disc changes to allow for a better correlation with disc function. Methods. After photodocumentation of the macroscopic appearance, 229 sagittal lumbar motion segments obtained from 47 individuals (fetal to 86 years) during routine autopsy were processed for histologic and immunohistochemical analysis. All slices were investigated for histologic alterations of disc degeneration. A randomly selected subset of these specimens (n = 45) was used for a correlative analysis of interstitial collagens and molecular modifications of matrix proteins. Results. The presence of CML-modification of extracellular matrix proteins, mainly collagen, was observed first in the nucleus pulposus of a 13-year-old individual and increased significantly with age. In elderly people, both the nucleus pulposus and the anulus fibrosus showed extensive CML deposition. This CML deposition was accentuated in areas of macroscopic and histologic disc degeneration. After the occurrence of CML in the nucleus pulposus, we found a change in the collagen type pattern. An initial increase in nuclear collagen Types II, III, and VI staining was followed by a loss of collagen Type II, the occurrence of collagen Type I, and the persistence of high collagen Type III and VI levels, which were finally decreased again. The nuclear chondrocytes revealed significant changes in their immediate pericellular matrix, indicating phenotypic changes. Thus, exclusively in the nucleus pulposus of adolescents and young adults a significant proportion of cells positively stained for the basement membrane collagen Type IV. Collagen Type X was expressed by nuclear chondrocytes at a higher age and was associated with advanced degenerative disc alterations. Conclusions. The authors present the first study in which age-related changes are correlated on a macroscopic, histologic, and molecular level using complete sections of lumbar motion segments. They reconfirm the notion that disc degeneration starts as early as in the second decade of life. Therefore, only early prevention of disc damage may inhibit disc degeneration and its sequelae. Phenotypic alterations of nuclear chondrocytes as monitored by collagen Type IV in young adults with minor lesions and collagen Type X in advanced lesions indicate distinct cellular reactions, possibly as a reaction to enhanced oxidative stress. The degree of this oxidative stress is reflected by the CML-staining pattern which, in turn, indicates that the disc undergoes an accumulative stress, possibly leading to altered properties of the collagen fibrils and, thereby, tissue destruction. The deposition of CML proved to be the best marker for ongoing age-related changes in the intervertebral disc.