Allen J. Bailey

Collagen cross-links in mineralizing tissues: A review of their chemistry, function, and clinical relevance

Bone collagen cross-links are now widely used to assess bone resorption levels in many metabolic bone diseases. The post-translational modifications of bone and other mineralizing collagens are significantly different from those of other type I collagen matrices, a fact that has been exploited during recent advances in the development of biochemical markers of bone resorption. The enzymatic collagen cross-linking mechanism is based upon aldehyde formation from specific telopeptide lysine or hydroxylysine residues. The immature ketoimine cross-links in bone form via the condensation of a telopeptide aldehyde with a helical lysine or hydroxylysine. Subsequent maturation to the pyridinoline and pyrrole cross-links occur by further reaction of the ketoimines with telopeptide aldehydes. In mineralizing tissues, a relatively low level of lysyl hydroxylation results in low levels of hydroxylysyl pyridinoline, and the occurrence of the largely bone specific lysyl pyridinoline and pyrrolic cross-links. The collagen post-translational modifications appear to play an integral role in matrix mineralization. The matrix of the turkey tendon only mineralizes after a remodeling of the collagen and the subsequent formation of a modified matrix more typical of bone than tendon. Further, disturbances in the post-translational modification of collagen can also affect the mineralization density and crystal structure of the tissue. In addition to their use as a convenient measure of matrix degradation, collagen cross-links are of significant importance for the biomechanical integrity of bone. Recent studies of osteoporotic bone, for example, have demonstrated that subtle perturbations in the pattern of lysine hydroxylation result in changes in the cross-link profile. These alterations, specifically changes in the level of the pyrrolic cross-link, also correlate with the strength of the bone. Further research into the biochemistry of bone collagen cross-links may expand current understanding and their clinical application in metabolic bone disease. This review also demonstrates the potential for further study into this area to provide more subtle information into the mechanisms and etiology of disease and aging of mineralizing tissues.

Mechanisms of maturation and ageing of collagen

The deleterious age-related changes in collagen that manifest in the stiffening of the joints, the vascular system and the renal and retinal capillaries are primarily due to the intermolecular cross-linking of the collagen molecules within the tissues. The formation of cross-links was elegantly demonstrated by Verzar over 40 years ago but the nature and mechanisms are only now being unravelled. Cross-linking involves two different mechanisms, one a precise enzymically controlled cross-linking during development and maturation and the other an adventitious non-enzymic mechanism following maturation of the tissue. It is this additional non-enzymic cross-linking, known as glycation, involving reaction with glucose and subsequent oxidation products of the complex, that is the major cause of dysfunction of collagenous tissues in old age. The process is accelerated in diabetic subjects due to the higher levels of glucose. The effect of glycation on cell-matrix interactions is now being studied and may be shown to be an equally important aspect of ageing of collagen. An understanding of these mechanisms is now leading to the development of inhibitors of glycation and compounds capable of cleaving the cross-links, thus alleviating the devastating effects of ageing.

Molecular mechanisms of ageing in connective tissues

The outward manifestations of tissue ageing occurring in the elderly primarily involve the two major structural proteins of the body, collagen and elastin. The changes in these proteins are associated with intermolecular cross-linking and side-chain modifications. Cross-linking involves two different mechanisms, a precise enzymic process during development and maturation, and a subsequent non-enzymic adventitious reaction with glucose during ageing. The latter glycation reactions are the major cause of tissue dysfunction in the elderly due to cross-linking, which stiffens the tissues, and to side-chain modification, which alters normal cell-matrix interactions. Photoageing by UV involves two competing reactions, chain cleavage and cross-linking, the former predominating on long-term exposure. The molecular mechanisms involved are gradually being unravelled and will lead to the development of inhibitors of these deleterious effects of ageing.

Changes in metabolism of collagen in genitourinary prolapse

BACKGROUND Genitourinary prolapse is a common problem, the pathophysiology of which is unknown. METHODS We analysed vaginal-epithelial tissue from premenopausal women with genitourinary prolapse and compared them with controls. FINDINGS We found that genitourinary prolapse is associated with a reduction in total collagen content and a decrease in collagen solubility. Both intermediate intermolecular cross-links and advanced glycation cross-links were increased in prolapse tissue. Collagen turnover, as indicated by matrix metalloproteinase activity, was up to four times higher in prolapse tissue. Collagen-type ratios, mature cross-link pyridinoline and total elastin content were similar in both prolapse and control tissues. Increased collagenolytic activity causes loss of collagen from prolapse tissue. INTERPRETATION Based on these findings, we have identified a probable mechanism for genitourinary prolapse. Development of agents to inhibit collagenolytic activity may help in the treatment of this condition.

Glycation of collagen: the basis of its central role in the late complications of ageing and diabetes

The most serious late complication of ageing and diabetes mellitus follow similar patterns in the dysfunction of retinal capillaries, renal tissue, and the cardiovascular system. The changes are accelerated in diabetic patients owing to hyerglycaemia and are the major cause of premature morbidity and mortality. These tissues and their optimal functioning are dependent on the integrity of their supporting framework of collagen. It is the modification of the properties by glycation that results in many of the damaging late complications. Initially glycation affects the interactions of collagen with cells and other matrix components, but the most damaging effects are caused by the formation of glucose-mediated intermolecular cross-links. These cross-links decrease the critical flexibility and permeability of the tissues and reduce turnover. In contrast to the renal and retinal tissue, the cardiovascular system also contains a significant proportion of other fibrous connective tissue protein elastin, and its properties are similarly modified by glycation. The nature of these glycation cross-links is now being unravelled and this knowledge is crucial in any attempt to inhibit these deleterious glycation reactions.

Abnormal cancellous bone collagen metabolism in osteoarthritis.

Biochemical investigations into the pathogenesis of osteoarthritis have, for the last two decades, concentrated on the mechanisms involved in the destruction of the articular cartilage. Although bone changes are known to occur, the biochemistry of the collagenous matrix within osteoarthritic bone has received scant attention. We report that bone collagen metabolism is increased within osteoarthritic femoral heads, with the greatest changes occurring within the subchondral zone. Collagen synthesis and its potential to mineralize were determined by the carboxy-terminal propeptide content and alkaline phosphatase activity, respectively. These data supported elevated new matrix formation. Our finding of a three- to fourfold increase in TGF-beta in osteoarthritic bone indicates that this might represent a stimulus for the increased collagen synthesis observed. Of additional significance is the hypomineralization of deposited collagen in the subchondral zone of osteoarthritic femoral heads, supporting a greater proportion of osteoid in the diseased tissue. The cross-linking of collagen was similar to that observed for controls. In addition, the degradative potential of osteoarthritic bone was considerably higher as demonstrated by increased matrix metalloproteinase 2 activity, and again the greater activity was associated with the subchondral bone tissue. The polarization exhibited in the metabolism of bone collagen from osteoarthritic hips might exacerbate the processes involved in joint deterioration by altering joint morphology. This in turn may alter the distribution of mechanical forces to the various tissues, to which bone is a sensitive responder. Bone collagen metabolism is clearly an important factor in the pathogenesis of osteoarthritis and certainly warrants further biochemical study.

The location of three collagen types in skeletal muscle

Collagen is the major supporting tissue of skeletal muscles and these collagenous tissues have been classified on anatomical and histological grounds. Thus, the epimysium forms the outer sheath of the muscle, the perimysium surrounds the muscle fibre bundles and the endomysium surrounds each individual muscle fibre [l] . Histologically the periand endomysium are composed of fine fibres and are often referred to as reticulin. Further, the endomysium appears from electron microscope studies to be mainly an amorphous basement membrane structure PI. The existence of several chemically and genetically distinct collagens has now been established [3] . Skin, tendon and bone consist mainly of Type I collagen, hyaline cartilage Type II, foe&l skin and blood vessels mainly Type III and basement membrane Type IV collagen. Our recent chemical studies have indicated the presence of collagen Types I, III and a third form, believed to be Type IV, in bovine muscle [4] . It is possible that there is a correlation between the histological classification and these genetic types of collagen. In this study we have therefore attempted firstly, to confirm the identity of the ‘Type IV’ as basement membrane and secondly to determine the tissue-specific location of these collagens by immunofluorescence using antibodies that react specifically with the three polymorphic forms of collagen.

Mechanical properties of adult vertebral cancellous bone: correlation with collagen intermolecular cross-links.

Although the mechanical strength of cancellous bone is well known to depend on its apparent density, little is known about the influence of other structural or biochemical parameters. This study specifically investigates the cross‐linking of the collagen in human vertebral bone samples and its potential influence on their mechanical behavior. Multiple cylindrical samples were cored vertically in the vertebral bodies of nine subjects (aged 44–88 years). Three spinal levels (T9, T12 or L1, and L4) and three sample sites within a vertebral body (anterior, posterior, and lateral) were used, for a total of 68 samples. The density was measured with peripheral quantitative computed tomography (pQCT) and all cylinders were mechanically tested in compression. After mechanical testing, they were unmounted and used for biochemical analysis. The amount of collagen (wt/wt of bone) and its content in reduced immature cross‐links, that is, hydroxylysinonorleucine (HLNL, mol/mol of collagen) and dihydroxylysinornorleucine (DHLNL), as well as stable mature cross‐links, that is, hydroxylysyl‐pyridinoline (HP), lysyl‐pyridinoline (LP), and pyrrole cross‐link were determined for each cylinder. None of the biochemical parameters correlated to the density. On multiple linear regression, the prediction of the mechanical properties was improved by combining density data with direct collagen cross‐link assessment. The HP/LP ratio appeared as a significant predictor to the strength (r = 0.40; p = 0.001) and stiffness (r = 0.47; p < 0.001) samples with a high HP/LP ratio being stronger and stiffer. Additionally, the ultimate strain correlated to the HP or LP concentration (r = 0.38 or 0.49; p < 0.01). Different subjects had different HP/LP ratios and different HP or LP concentrations in their vertebral bone samples, and the location of origin within a subject had no influence on the concentration. These observations suggest that the nature of the organic matrix in adult vertebral bone is variable and that these variations influence its mechanical competence.

The rôle of epimysial, perimysial and endomysial collagen in determining texture in six bovine muscles

Epimysium, perimysium and endomysium were purified in high yield by a new extraction method from six bovine muscles of increasing toughness. In each case, the mean perimysial collagen fibre diameter, the total collagen content, the relative proportions of the major collagen types (I and III) and the collagen cross-link content were measured. Correlations were found between both collagen fibre diameter and collagen content of perimysial and endomysial connective tissue and meat toughness. No meaningful link could be made between textural quality of the meat and the relative content of types I and III collagen. Significant differences were seen, however, in both the total heat-stable cross-link content and the proportion of heat-stable collagen cross-links relative to heat-labile cross-links present in epimysium, perimysium and endomysiuim and the relative toughness of each muscle. The results are discussed with respect to the rôle of collagen in determining meat texture and toughness.

Analysis of collagen status in premenopausal nulliparous women with genuine stress incontinence.

Objective To determine if differences exist in the collagen status of premenopausal nulliparous women with genuine stress incontinence compared with continent controls.