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Sir,Szalecksy and colleagues raise an interesting series of points on the enzymatic antioxidant defence system and its role in the pathogenesis of diabetic complications when its function of free-radical clearance is impaired.1 It is conceivable that one of the ways in which the scavenging function of the antioxidant enzymes may be reduced is by glycation of their free amino terminals to produce the advanced glycated end-product (AGE) variants of these enzymes. High blood glucose is known to cause glucose to bind covalently and nonenzymatically to various proteinsin vivo to generate AGEs. Glycation can alter the structure as well as the function of physiological proteins. Therefore, hyperglycaemia may modify the properties of the antioxidant enzymatic defence proteins and increase the susceptibility of the patient with diabetes mellitus to vascular damage from the resulting excess of free-radical species.
The levels of AGE are known to be elevated in the serum of patients with diabetes. AGEs have been linked with retinopathy, nephropathy and neuropathy, as well as being implicated in large vessel disease. The mechanisms leading to micro- and macro-angiopathy are thought to occur by an interaction of AGE with a receptor (known as RAGE) expressed on the cell surface membrane of vascular endothelial cells. The agonist–receptor interaction appears to be responsible for eliciting fundamental lesions in the vessel wall that promote atherosclerosis and diabetic microangiopathy in the animal model. In human retinae with non-proliferative and proliferative diabetic retinopathy, AGEs have been closely associated with the expression of vascular endothelial growth factor,2 which is known to induce new vessel formation and thus AGE may play a crucial role in the pathogenesis of retinopathy by elevating vascular endothelial growth factor levels in the microenvironment of the diabetic retina. Ultrastructural studies of human diabetic retinopathy have shown AGE to be localised in the nodular lesions of sclerosing glomeruli, where it is believed to disrupt the normal architecture of mesangial matrix proteins. In another set of experiments, elevated AGE levels in the sciatic nerve of diabetic rats were associated with a significantly reduced nerve conduction velocity when compared with age-matched control animals, suggesting a role of AGE in diabetic nephropathy.3
It is therefore interesting to speculate that an inhibition of AGE synthesis, or its antagonism at the level of the RAGE receptor, could be important in preventing the genesis of vascular diabetic complications. For example, it has been shown in a Type 2 diabetes animal model that decreasing serum AGE levels prevents the progression of diabetic glomerular sclerosis.4 Inhibition of AGE with aminoguanidine has been observed to prevent or improve retinopathy and neuropathy in animals. Furthermore, prevention of AGE synthesis with aminoguanidine in mice reduces the development of diabetes and in addition preserves pancreatic structure and function.5 In the quest for AGE-suppressing compounds, aspirin has also been found to prevent the formation of AGE by competing with glucose for selected amino terminal binding sites on proteins. With regard to large vessel disease, the use of particular compounds to degrade the AGE formed in connective tissue has been observed to decrease the accelerated arterial wall stiffness found in experimental diabetes. When the binding of AGE to its vascular endothelial receptor was prevented in diabetic mice by using a soluble moiety of the receptor, the process of atherosclerosis was suppressed completely.6
Since AGE species are chemically irreversible and correlate with the level of glycaemia, they may potentially serve as a biochemical marker of diabetes mellitus in a similar way to the use of glycated haemoglobin to monitor metabolic control, in addition to providing a direct index of the severity of micro- and macrovascular damage. Furthermore, a slowing of the progression of micro- and macrovascular disease by reducing the production or physiological effectiveness of AGEs by drug treatment may herald an intended intervention at a molecular level in diabetic therapeutics.
This letter was shown to the authors who responded as follows:
Sir,Drs Gandhi and Patel, in their valuable letter related to our article, refer to the fact that research in recent years has proven that levels of the late products of non-enzymatic glycosylation, the so-called advanced glycated end products (AGEs), are elevated in the sera of diabetic patients. Moreover, it is also accepted that binding of these molecules to their receptors (RAGEs) expressed on vascular endothelial cells plays a basic role in the development of diabetic micro- and macro-angiopathy. Thus, AGE can play a role in diabetic retino-, nephro- and neuropathy.
These data lead to the possibility, as reviewed in Dr Gandhi's letter, that by inhibiting AGE production (eg, by aminoguanidine or aspirin) or blocking receptor binding, we may be able to reduce the risk of late diabetic complications and thereby improve life quality and expectancy in diabetic patients. Since AGE species are irreversibly modified and their concentrations are correlated with the severity of glycaemia, it has been suggested that they could be used as a diabetic marker, or as an index of micro- and macro-angiopathy.
Two stages of non-enzymatic protein glycosylation are known.1-1 1-2 Early glycation events are mainly detectable on short-lived proteins such as haemoglobin enzymes. In this case a Schiff base is produced via the reaction between glucose and free amino groups. In a few weeks time a more stable but still reversible Amadori product is formed, a molecule with a ketoamine bond. This process is directly correlated with the serum glucose concentration and is selective to protein structures, the latter being responsible for tissue selectivity. Amadori products go through further structural changes (dehydration, chemical cross-linkage, conformational change) to yield stable species of AGE, the late products of glycation. AGEs are capable of further reacting with other proteins. AGE-modified soluble and matrix proteins possess a chemotactic activity, inducing monocyte migration, they inactivate NO, and induce production of adhesion molecules. Monocytes phagocytose AGEs into molecules with lower molecular weight, which are more mobile and able to activate smooth muscle cells deeper in the vessel wall. The physicochemical properties of the collagen matrix also suffer changes. Free radicals released during these processes promote degradation and polymerization of proteins. Taken together, glycation starts a cascade of pathophysiological reactions which form the basis of atherosclerosis.
AGEs are removed by the liver, by macrophages via scavenger receptors (the latter also take up glycated and modified lipids), and by RAGEs. RAGEs are expressed at higher levels in certain pathological states (diabetes, inflammation, etc) in some organs (eg, kidneys) and allow the uptake and integration of AGE into the tissues and their excretion via the kidneys. Immunohistochemical studies can demonstrate these processes,1-4 but these techniques are not yet commonly available. AGEs can also be detected in body fluids by competitive ELISA and immunofluorescence.1-5
It would therefore seem reasonable that inhibition of the development of early and late glycation products may prevent the premature atherosclerosis of diabetic patients. The most potent and important means of preventing diabetic complications is the strict control of glucose metabolism, as demonstrated by the Diabetes Control and Complications Trial in 1993.1-5 The trial proved that good metabolic control, as assessed by HbA1c levels, not only prevents the development of complications but also stops further deterioration.
In summary, we conclude that there are, at least theoretically, several points and ways of intervening with the production, uptake and elimination of AGEs. As the formation of this species is a relatively long-term process influenced by numerous factors acting at different time points (actual glucose control, protein structures, diseases, exogenous factors such as smoking, etc), we think that the presence of AGEs is primarily a global marker of an individual's pathological history, thence micro- and macrovascular damage, and not only of the metabolic changes of diabetes.
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