Non-Enzymatic Glycation of Collagen: Role in Metabolic Disease and Inflammation

Diabetes can lead to high levels of sugar in the blood and that sugar reacts with the proteins in our tissues, causing significant disruption of the tissues and impairment of their function. For this reason, diabetics suffer a range of problems like poor kidney function, so-called hardening of the arteries and are also at high risk of osteoporosis and severe osteoarthritis. The sugar that is considered primarily responsible for that process is glucose, as this is the sugar that has high, uncontrolled blood levels in diabetics. However, we have discovered that a sugar polymer called poly(ADP ribose), that is produced by cells under stress, is released into the surrounding tissue if the cell dies - which it may if it continues to be stressed - and that this polymeric sugar is very reactive with tissue proteins like collagen. The type of situation where a cell might be stressed is where there is chronic inflammation, for instance, or following a cut-off of oxygen. The high blood glucose levels in diabetics means that their tissues suffer significantly from chronic inflammation; however, there are many diseases and conditions in non-diabetics which result in chronic inflammation as well, of either specific tissues or the body's tissues in general. We know that chronic inflammation often results in impairment of the tissue's function and/ or stiffening of the tissue, for instance, chronic inflammation in joints results in osteoarthritis through degradation of the joint cartilage; chronic inflammation in arteries leads to the so-called "hardening of the arteries" - and we know that although these conditions are common in diabetics, they are also common in non-diabetics. This project will explore whether chronic inflammation in bones and blood vessels - for whatever reason - leads to poly(ADP ribose) in those tissues, which then reacts with the tissue proteins and causes the sort of tissue deterioration we see in those tissues after chronic inflammation. We think the high blood glucose levels causing chronic tissue inflammation throughout the body in diabetics, may lead to poly(ADP ribose) in the affected tissues and that it the poly(ADP ribose), which causes the damage to the tissue, rather than glucose directly. We think the primary role of the high glucose levels in diabetics is to cause the chronic inflammation conditions which lead to poly(ADP ribose) being released into the tissue. In this project, we will determine under what conditions cells might die and leave poly(ADP ribose) in the tissue around them and how this reacts with the tissue proteins, particularly collagen. We will then find out how the reaction of poly(ADP ribose) with the tissue proteins affects both the mechanical flexibility of the tissue and how the remaining live cells in the tissue behave once the proteins around them have reacted with poly(ADP ribose). We think that damage to collagen, the main protein in our tissues, caused by poly(ADP ribose) may be responsible for the onset of osteoporosis, at least in some cases, so we will also investigate how different the calcification of bone is after bone tissue has been disrupted by reaction with poly(ADP ribose). If we can identify what is causing the damage to tissues in chronic inflammation, then we can begin to devise strategies to control or perhaps even reverse that damage. This project will specifically look at bone and blood vessels as there are many diseases and conditions for which chronic inflammation is a feature, but if indeed poly(ADP ribose) is responsible for significant damage, then we will look at other conditions where chronic inflammation and cell necrosis are factors, such as neurodegenerative diseases like Alzheimers disease.

We have discovered that necrosis of bone osteoblasts and vascular smooth muscle cells (VSMCs) results in poly(ADP ribose) (PAR) in the extracellular matrix (ECM). The ribose moiety at the end of a free PAR molecule is a potentially much more reactive sugar species for glycation than the sugar normally considered to be responsible for the majority of AGEs, namely glucose. Moreover, the finding of PAR in the ECM implied that damaging glycation could potentially occur in other pathologies where there is uncontrolled cell necrosis, not just in diabetic tissues.
PAR is generated in cells from NAD+ by PAR polymerase (PARP) enzymes. Over-activation of PARPs, as happens as a result of cellular damage due to oxidative stress for instance, leads to cellular ATP and NAD+ depletion and drives cell necrosis. In other words, the oxidative stress that goes hand-in-hand with chronic inflammation and that can result in cell necrosis, may result in PAR - a potentially powerful glycator - in the surrounding ECM.
In our pilot work, we found that as expected, ribose-based species react much faster to glycate proteins than does glucose, leading (amongst other things) to rapid formation of irreversible collagen cross-links which severely disrupt the collagen molecular structure, even for fibrillar collagen. As a result of this pilot work, we have formed a new hypothesis: that the most damaging glycating agent in oxidatively-stressed tissues is PAR and its degradation products, rather than glucose - the role of glucose is to create the oxidative stress conditions that drive the formation of PAR and cell necrosis. We will investigate this in in vitro oxidative stress models for bone and vascular tissue.

Collagen glycation has already been implicated in a wide range of tissue pathologies, particularly those associated with ageing and diabetes mellitus, but the currently held belief is that collagen glycation is largely due to glycation from glucose. Our pilot project suggests that ribose phosphate species derived from poly(ADP ribose) (PAR) could be a significant source of glycation. Ribose phosphate species react with collagen in a matter of days, whilst glucose reactions occur over months. PAR is produced by cells under conditions of oxidative stress. Glycation with glucose triggers the receptor for advanced glycation endproducts, RAGE, and sets in train chronic inflammation with associated oxidative stress. This in turn is likely to lead to PAR formation and cell necrosis, which in turn would result in PAR glycation in the extracellular matrix (ECM) of the chronically inflamed tissue, where it can participate in glycation reactions with collagen and other ECM proteins.. If the resulting glycation products also trigger RAGE, a rapid downhill spiral of the tissue mechanical properties primarily mediated by PAR, and very likely, altered cellular function, would ensue.
Vascular stiffening and bone and joint pathologies, such as osteoarthritis and osteoporosis have already been shown to have contributions from advanced glycation endproducts (AGEs) assumed to arise from glucose glycation. This project will determine the biological, structural and mechanical effects on in vitro vascular and bone ECM of PAR glycation compared with glucose glycation, and the relative timescales of the respective reactions. If PAR is found to have a significant effect on any of these three properties of the ECM, the next step would be to verify the presence of PAR glycation products in vivo, in man. Our Pathways to Impact document details how we plan to do this. If PAR glycation is a significant factor in vascular, bone or joint pathologies, then PARP inhibitors, some of which are already licensed drugs, represent a possible therapeutic strategy, and again, our Pathways to Impact document describes how we will pursue therapeutic options.
Other pathologies where chronic inflammation and cell necrosis are a major feature include cancer and neurodegenerative diseases. In both these cases, the biological consequences of ECM glycation are likely to be the important factors. In cancer tissue, ECM stiffness is known to affect the biological properties of the cancerous tissue and it is plausible that ECM glycation may influence cancer cell metastasis, cell invasion etc. Neurodegenerative diseases such as Alzheimers involve upregulation of PAR synthesis, chronic inflammation and cell necrosis - all the features necessary for PAR glycation to occur. Factors to consider are whether glycated ECM could act as a nidus for amyloid plaque formation, whether glycated proteins are more likely to form stable plaques, how glycated proteins affect cellular function in e.g. brain. Details of our plans in this area are in the Pathways to Impact document.
This project will consider the relevance of PAR glycation in models of chronic inflammation, but acute inflammation can also cause cell necrosis and it is possible that uncontrolled PAR production for even the relatively short time period when acute inflammation might exist, could result in significant glycation, because of the rapid rate at which free PAR and its degradation products can react with proteins (and other biomolecules) in our model systems. We are mindful that scar tissue formed as a result of acute inflammation such as in spinal cord injury and after surgery, can often not be remodelled by the tissue's cells. This is a significant factor in repairing spinal cord injuries, for instance. We are planning for the possibility of the lack of tissue remodelling being due to glycation of the repairing tissue, with PAR and PAR degradation products formed in the extreme inflammatory conditions.