Review
Molecular mechanisms of ageing in connective tissues

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Abstract

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.

Introduction

The outward manifestations of ageing are obvious concerns to us all — wrinkled skin, stiffened joints and shortening stature — but similar changes are taking place within the internal organs, principally the heart, vascular system and lungs. Consequently, there is a wealth of clinical data available in the literature on age-related changes in the functioning of all these tissues. However, these deleterious effects of ageing all involve, to a greater or lesser degree, the two structural proteins supporting these tissues, namely, collagen and elastin. Both proteins turn over slowly in tissues, and are therefore susceptible to age-related changes. The turnover of collagen in mature tissue varies considerably from months to years compared with days for the non-structural proteins. The changes in physical properties have been known for several decades (Verzar, 1964, Viidik, 1982), but the molecular mechanisms are only now being unravelled. It is therefore necessary to focus on the molecular events occurring in these structural proteins during ageing if we are ultimately to understand the mechanisms necessary to alleviate at least some of the damage.

The overall shape and function of the animal body depend on the basic framework of collagen molecules. Collagen is an ancient protein and is involved in binding cells together from the simplest animal, the sponge, on up to man. Indeed, the vertebrate fibrillar collagens are believed to be derived from a common ancestral α-chain of multiples of a 54 bp exon, although recent studies have included duplication of a (54 bp+45 bp) exon (Exposito et al., 2000).

Collagenous tissues exist as a diverse array of structures from parallel fibres in tendon, through laminated structures in bone, skin and cornea, to the non-fibrous transparent membranes of the lens capsule of the eye. The biological diversity of collagenous tissues can be accounted for by selective combination of one or more of 20 genetically distinct collagen molecules (Kielty et al., 1993, Comper, 1996). All are triple helical molecules based on polypeptide chains with the repeating unit Gly–X–Y, which polymerises into a variety of supramacromolecular structures. However, the major supporting collagens are the fibrous types (I, II, III) (Fig. 1), in which the monomeric molecules polymerise to form striated fibres that are stabilized by intermolecular cross-links, thereby preventing the long rod-like collagen molecules from sliding past each other under stress (Bailey et al., 1980). As a consequence, they form virtually inextensible fibres, thus providing exceptional tensile strength and resistance to external mechanical forces. Type IV forms a non-fibrous structure as a support for a more flexible basement membrane. Others have specific functions; for example, type VII binds basement membrane to underlying fibres, but at the present time, there is little information on the functional role of the other minor collagens. However, there is some in-vitro evidence of their regulating fibre diameter.

In contrast, elastin imparts extensibility and elastic recoil to particular collagenous tissues, characteristics crucial for their physiological function. It is a major constitutent of the large blood vessels and some ligaments (40–60%) and is present to a lesser extent in skin and lung (2–5%) (Ciba Foundation Symposium 192, 1995). In complete contrast to collagen, the fibres are capable of reversible extension to about double their length and possess a basically amorphous structure, as revealed by electron microscopy and X-ray diffraction studies. The elastic fibre actually comprises two distinct components. In developing elastic tissue, fine microfibrils are initially present, but with increasing maturity, elastin becomes the major component. The microfibrils, which are mainly fibrillin (Sakai et al., 1986) together with the EMILINS (Columbatti et al., 2000), act as a template organizing the elastic tissue. However, they become minor components, and it is generally believed that they do not play a role in the mechanical properties of elastic tissue, although recent studies have demonstrated the elastic properties of fibrillin in zonal filaments (Wright et al., 1999).

The third major components of connective tissues are the proteoglycans. Age-related changes have been reported to occur in the amount and composition of the chondroitin sulphate and keratan in articular cartilage, but the changes vary considerably with different tissues. Proteoglycans, being polyhydroxy in character, hold large amounts of water, and so changes in their composition could lead to mild dehydration and some loss of function. However, no overall pattern has as yet emerged. This review will therefore focus on the molecular changes in the structural components collagen and elastin.

Section snippets

Changes in molecular composition of tissues

The biological diversity of collagenous tissues can be accounted for by the family of collagen molecules, which are, to some extent, tissue-specific. For example, bone and tendon are predominantly type I, the vascular system type III and cartilage type II, whilst the non-fibrous basement membranes possess type IV in a network structure. Most collagenous tissues contain small amounts of one or more of the other minor collagens. Variations in the type of collagen in a tissue with age have been

Intermolecular cross-linking

Perhaps the most important changes in collagen and elastin with age involve the formation of intermolecular cross-links. These cross-links are initially formed to provide an optimum function but can subsequently over-stiffen the fibres when present in excess (Fig. 3). There are two major cross-link processes:

  • 1.

    the initial stabilisation of the fibres through lysyl oxidase to provide the characteristic functional properties during development and maturation;

  • 2.

    a second process based on the reaction

Collagen

The mechanism of enzyme-derived cross-linking in which the N- and C-terminal lysines are oxidatively deaminated by lysyl oxidase to form lysyl aldehyde is now well established (Bailey et al., 1998), and only recent studies need be emphasised. The nature of the cross-links depends primarily on the extent of the hydroxylation of the telopeptide lysine. In skin, where this particular lysine is not significantly hydroxylated, the major cross-link in immature tissue is the divalent

Non-enzymic glycosylation-derived cross-links (glycation)

This second mechanism is known as glycation and plays a central role in the pathogenesis of ageing (Cerami et al., 1987, Paul and Bailey, 1996). As would be expected, glycation reactions are enhanced in diabetic subjects due to hyperglycaemia, but the processes are the same and have led to diabetes being described as a process of ‘accelerated ageing’. The process occurs non-enzymically and can ultimately lead to intermolecular cross-links. The most serious late complications of ageing are the

Glycation-modified cell–matrix interactions

Modifications of the amino acid side-chains by reaction with glucose, such as lysine to form carboxymethyl-lysine, or arginine with methylglyoxal to form imidazolones clearly alter the charge profile of the molecules, leading to changes in the cell–collagen interactions and the subsequent remodelling of the collagen. The modification of arginine is particularly important because the well known binding site Arg–Gly–Asp (RGD) is involved in the recognition site for the two matrix integrins, α1β1

Inhibition of glycation cross-linking

Considerable efforts have been made to inhibit the formation or to cleave existing cross-links and thereby alleviate the dramatic changes in body tissue with age. The acceleration of the changes in diabetes has stimulated these inhibition studies.

Aminoguanidine has been shown to inhibit the formation of AGEs and reduce the characteristic damage to tissues (Brownlee, 1994), but clinical trials in the USA have recently been suspended. Its mechanism of preventing AGE formation is controversial: it

Photoageing

Repeated exposure to the sun's UV radiation causes premature skin ageing, which is manifest as wrinkles, modified pigmentation, abnormal elastin bundles and fragmented elastin, and a loss of collagen (Scharffetter-Kochanek et al., 2000). The epidermal–dermal basement membrane may also be damaged (Craven et al., 1997). Damage may result from absorption by chromophores in the skin, which eventually lead to biological effects, or through the formation of reactive free radicals from the water

Future prospects

The study of the molecular processes of ageing of connective tissues has a long but rather unsuccessful history, reports generally being concerned with changes in the physical properties rather than mechanisms. However, there is now considerable evidence that glycation and the formation of AGEs play a significant role in the ageing of connective tissues. Glycation may result in the formation of cross-links, thereby altering the physical properties of the connective tissue, and at the same time

Acknowledgements

I am indebted to Drs R.G. Paul and D.A. Slatter for reading and commenting on the manuscript.

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