Glycosylation of proteins and lipoproteins can interfere with their normal function by disrupting molecular conformation, alter enzymatic activity, reduce degradative capacity, and interfere with receptor recognition (Table 1). Thus, changes in the normal physiology of proteins that are relevant to atherogenesis, may promote atherosclerosis in diabetic individuals.

Perhaps the most studied example is interference of the normal physiology of the low-density lipoprotein (LDL) particle. The glycosylation process occurs both on the apoprotein B 18 and phospholipid 19 components of LDL, leading to both functional alternations in LDL clearance and increased susceptibility to oxidative modifications.

Clinical studies have shown an increased level of AGEs on LDL obtained from diabetics compared with normal individuals 1920. Glycosylation of LDL apo B (the surface protein of LDL) occurs mainly on a positively charged lysine residues within the putative LDL receptor binding domain which are essential for the specific recognition of LDL by the LDL receptor 20. LDL glycosylation is increased in correlation with glucose levels, and AGE-ApoB levels are up to 4-fold higher in diabetic patients 1920. Glycosylation of ApoB results in a significant impairment of LDL-receptor-mediated uptake decreasing the in vivo clearance of LDL compared to native LDL 21 (Figure 2). Several studies have shown that degradation of glycated LDL is impaired in cultured human fibroblasts (which posses LDL receptor) compared with normal LDL, and that this impairment is proportional to the extent of glycation 21. In contrast to fibroblasts, human monocyte-derived macrophages recognize glycated LDL to a greater extent than native LDL 22. The uptake of glycated LDL by these cells, however, is not mediated by the LDL receptor pathway, but by a high-capacity, low-affinity receptor pathway 22. Thus, glycated LDL are poorly recognized by the specific LDL receptor and are preferentially recognized by a nonspecific (scavenger) receptor present on human macrophages. Because LDL glycosylation enhances its uptake by human aortic intimal cells 23 and monocyte-derived macrophages 22 with stimulation of foam cells formation, the recognition of glycated LDL by the scavenger receptor pathway is thought to promote intracellular accumulation of cholesteryl esters and promote atherosclerosis (Figure 2).

Another atherogenic effect of glycation is to confer increased susceptibility of LDL to oxidative modification. Oxidation reactions occur normally during glycation can oxidize the amine-containing phospholipids component of LDL, independently of transition metals or exogenous free radical-generating systems 19. Advanced glycosylation of an amine-containing phospholipids component of LDL is accompanied by progressive oxidative modification of unsaturated fatty acid residues 21. LDL oxidation following AGE-LDL formation occurs in direct proportion to glucose concentration and can be inhibited by the AGE formation inhibitor aminoguanidine 19. Thus, glycation confers increased susceptibility of LDL to oxidative modification 2124, which is considered a critical step in its atherogenicity.

Another example is the alterations in normal function of the complement regulatory protein. Deposition of the membrane attack complex of complement (MAC) in blood vessels stimulates proliferation of fibroblasts and smooth muscle cells, in part by releasing growth factors such as fibroblast growth factor and platelet derived growth factor from MAC-targeted endothelium 25. MAC deposition is normally restricted because cells express the regulatory membrane protein CD59, which limits complement activation and MAC formation. Glycation of the complement regulatory protein CD59 results in its inactivation 26 and may increased the sensitivity of the diabetic endothelium to MAC-induced release of growth factors and cytokines.

Glycosylation of matrix components such as collagen VI, laminin, and vitronectin decreased binding of anionic heparan sulfate, leading to greater turnover of heparan sulfate 10. The absence of HS is thought to stimulate a compensatory overproduction of other matrix components through altered partitioning of growth-regulatory factors between matrix bound proteoglycans and cells 10. AGEs on matrix also alter the normal interactions of transmembrane integrin receptors with three specific matrix liganeds. For example, modification of the cell binding domains of type IV collagen causes decreased endothelial cell adhesion 10.

The cellular interactions of AGEs are mediated through a specific receptor for AGE determinants on cell surfaces 16. The presence of the AGE receptor (RAGE), a member of the immunoglobulin superfamily of receptors 27, has been demonstrated in all cells relevant to the atherosclerotic process including monocyte-derived macrophages, endothelial cells, and smooth muscle cells 1628 (Table 2). The macrophage AGE receptor system is closely tied to AGE turnover, and is thought to represent a mechanism that responds to raising AGE levels with aging and degrade senescent proteins 29.

In mature animals, RAGE expression on these cells is low 28. However, under certain pathological circumstances, sustained upregulation of RAGE occurs. In pathological lesions, abundance of RAGE expressing cells is usually associated with sites of accumulated RAGE ligands. In diabetic vasculature, cells expressing high levels of RAGE are often proximal to areas in which AGEs are abundant 1730.

AGE interaction with RAGE on endothelial cells results in the induction of oxidative stress and consequently of the transcription factor NF-κB 3132 and VCAM-1 33 (Figure 3). In addition, engagement of AGEs with their specific receptors results in reduced endothelial barrier function 3334. With increased permeability of endothelial cell monolayers 3435. Thus, the interaction of AGEs with RAGE-bearing endothelial cells can mediate initiating events in atherogenesis. For example, increased endothelial permeability can lead to increased lipid entry into the subendothelium. Enhancement of adhesive interactions of monocytes with the endothelial surface can subsequently result in transendothelial migration (Figure 4).

Binding of soluble AGEs to RAGE-bearing monocytes induces chemotaxis 36, followed by mononuclear infiltration through an intact endothelial monolayer 3738. Pathological studies of human atherosclerotic plaques showed infiltration of RAGE-expressing cells in the expanded intima 28. Monocyte-macrophage interaction with AGEs results also in the production of mediators such as interleukin-1, tumor necrosis factor-α, platelet-derived growth factor, and insulin growth factor-I 373940, which have a pivotal role in the pathogenesis of atherosclerosis 41.

In smooth muscle cells, binding of AGE-modified proteins to RAGE is associated with increased cellular proliferation 42. Although the precise mechanism of this response is unknown, the growth-promoting effects mediated by the RAGE are likely to be cytokine or growth-factor mediated. Thus, under conditions of enhanced tissue AGE deposition, receptor-mediated interaction of AGE-proteins with vascular wall cells facilitate the migration of inflammatory cells into the lesion with the subsequent release of growth-promoting cytokines.

The potential role of RAGE in the atherogenic process in diabetes has been demonstrated by Park and associates 43. In the model of atherosclerosis-prone mice due to homozygous deletion of apolipoprotein E (apoE) gene, the induction of diabetes using streptozotocin resulted in atherosclerosis of increased severity compared to euglycemic apoE controls. The development of vascular was more rapid with the formation of more complex lesions (fibrous caps, extensive monocyte infiltration, etc) and atherosclerosis extending distally in the aorta and major arteries. Increased expression of RAGE and the presence of AGEs in the vessel wall, especially at sites of vascular lesions were also evident. Blockade of AGE-RAGE interaction using a truncated soluble extracellular domain of RAGE resulted in a striking suppression of lesions in diabetic mice, with lesions largely arrested at the fatty streak stage and a large reduction in complex lesions. These effects were independent of glucose and lipid levels 43.

Some of the individual advanced glycosylation products such as N^ε-carboxymethyl)lysine (CML) and pentosidine are formed in reactions of protein with glucose only under oxidative conditions 15646566. Thus, some AGEs are produced by combined processes of glycation and oxidation and have been termed glycoxidation products 56. Each AGE structure has its own formation mechanism and thus its own dependence on oxidative stress. However, since glycoxidation products on proteins are irreversible, it has been suggested that they may be an integrative biomarker for the accumulated oxidative stress the respective tissue has been exposed to 1455.

As previously discussed, the interaction between AGE epitopes and the cell surface AGE receptor upregulate oxidative stress response genes 31 and release oxygen radicals 67. Thus, hyperglycemia simultaneously enhances both AGEs formation and oxidative stress, and the mutual facilitatory interactions between glycation and oxidation chemistry can contribute sinergistically to the formation of AGEs, oxidative stress, and diabetic complications (Figure 5). Indeed, there are strong correlations between levels of glycoxidation products in skin collagen and the severity of diabetic retinal, renal, and vascular disease 6869.

Oxidative stress may also be involved in the activation of DAG-PKC in vascular tissue 7. Oxidants produced in the setting of hyperglycemia can activate PKC 70. Furthermore, several studies have shown that antioxidants such as vitamin E can inhibit PKC activation probably by decreasing DAG levels 7717273.