Early glycation products of collagen: a solid-state NMR spectrscopy and ultrastructural study of biological and health implications

Collagen is the principal component of the body's tissues. Like many proteins in the body, it is glycosylated, that is, it is decorated with sugar molecules. The function of these is not clear, although there is evidence that they assist in lining up the collagen molecules to form the strong network that is needed for the structural integrity of a tissue. Over a lifetime, the body's collagen accumulates more sugar molecules and these can and do undergo various chemical reactions that result in permanent cross links between collagen molecules - so-called advanced glycation endproducts (AGEs). These stiffen the collagen matrix and are, at least in part, responsible for hardening of the arteries (which in turn causes high blood pressure and various other vascular issues). They are also important in ageing of skin and other tissues and they accumulate much faster if a person's blood sugar level tends to be higher than normal, i.e. in diabetic patients, where they are responsible for many of the chronic issues that diabetics suffer, such as reduced kidney function.

Little is known about the early stages of sugar accumulation in collagen and the effects it may have on the health of a tissue. Not only is it likely to affect the material strength and integrity of the tissue, it is likely to affect how the cells that live and grow in the tissue behave, so that for instance, the cells may act to cause chronic inflammation in the tissue. Thus while the current focus is on drugs to reverse the cross linking process, there is significant evidence that removing the cross links does not of itself restore the tissue's health. Although the AGE cross links undoubtedly do damage in tissues, it is not clear how much of the damage is caused by the AGEs themselves and how much by the initial sugar binding, and the sugar which remains bound even after AGEs have formed.

This project will use state-of-the-art methods to determine how the sugar that initially binds to collagen is actually bound to the collagen and the effect it has on the tissue strength and the behaviour of cells. We will use a form of spectroscopy known as solid-state nuclear magnetic resonance (NMR) to determine detailed structures of the collagen and collagen-bound sugar and electron microscopy to determine how the sugar affects the way collagen molecules align themselves together. We will use similar techniques to find out if the sugar binding affects the way that bone mineralises, which is relevant to diseases such as osteoporosis or whether it might encourage calcification of arteries, a process that causes very significant hardening of the arteries and all the health implications that that has.

Determining the effect of the early stages of sugar binding on collagen will eventually translate into early detection methods for those at risk, such as diabetic patients, and will allow us to determine how important it is to find methods to prevent or reverse this early stage sugar binding as well as the endstages. Part of the project will involve developing cell cultures which mimic the sugar binding process that happens in the body and these can be used to assess potential therapies for preventing or reversing the sugar binding.

The results of this project will particularly benefit diabetic patients, in eventually allowing therapies which will prevent or alleviate the debilitating chronic effects of the disease, but will also assist in devising healthy ageing strategies for all.

This project will examine the biomaterial and pathological implications of the early stages of non-enzymatic glycation of type I collagen in bones and vascular tissue (medial layer).

1. To determine in vitro the structure of EGPs in type I collagen, including the presence or otherwise of non-covalently bound sugar species.
2. To determine any effects of EGPs (and any additional collagen-bound sugar species identified and water) on collagen molecular structure and organisation, nanoarchitecture of the collagen matrix and material properties of the collagen matrix.
3. To determine if the in vitro results from objectives 1 and 2 are representative of the situation in vivo in mouse models.
4. To determine the effect of EGPs on the pathology of calcification processes in (i) vascular and (ii) bone contexts (in vitro and in animal models).

The principal structural tools will be solid-state nuclear magnetic resonance (NMR) spectroscopy and electron microscopy. The project will use in vitro and mouse models, with animals being fed 13-C, 15-N enriched diets to allow advanced NMR methods to be used in examining their tissues. Results will be compared to those from (unenriched) human tissues to validate the mouse models.

This project will develop solid-state NMR and EM methodologies to detect and quantify early glycation products (EGPs) of collagen in intact tissues. The project will also determine if EGPs are relevant in pathological calcification and impaired calcification of bone. If they are, then clearly it would be highly beneficial to the ageing population and those with diabetes (or at risk of diabetes) if these methodologies could be translated into clinically-measurable parameters and definition of risk factors allowing much earlier intervention to stem the progression of the chronic implications of diabetes, for instance. Not only would this benefit patients, it could represent a significant cost saving to the NHS and UK industry by keeping diabetics (currently more than 4 % of the UK population) healthier for longer; chronic implications of disease are costly in terms of drugs, because of the length of use and in terms of loss of members of the UK workforce and their subsequent dependence on state services etc.

There may be nutritional factors in addition to the obvious control of blood glucose levels that can discourage collagen EGPs and their subsequent development into cross-linked advanced glycation endproducts (AGEs), but these cannot be investigated without methods for assessing EGPs in collagen. Likewise, there may well be genetic factors which pre-dispose to collagen EGP formation, but again, these are difficult to investigate without methods to assess the degree and nature of EGPs in collagenous tissues. This project will provide that methodology and translate into research in these crucial areas, which ultimately could significantly benefit society in terms of better nutritional information, especially for those genetically at risk of EGP formation.

In addition to methodology to detect and quantify EGPs in tissues, this project will develop in vitro models of extracellular matrices with collagenous EGPs. These would represent a relatively cheap and quick model for screening therapies for possible use in removing EGPs or protecting collagen from glycation in the first place which would be of huge benefit to the pharmaceutical industry.