Tf

(Thrombogenesis)

Figure 4 NF-KB-mediated pathways leading to a thrombogenic transformation of the vessel wall.

suggesting that the ROI generation is necessary for the synthesis of AGE products. On the other hand, there is some evidence that endothelium starts to produce ROI as soon as the receptor for AGE products becomes occupied (40,41). Although the exact intracellular signaling is not yet known, there is some evidence that binding of AGE products to its receptors causes an activation of NADPH oxidases (41). This AGE-mediated production of ROI is prevented by antioxidants and inhibitors of NADPH oxidases but not inhibitors of NO synthases, cyclo- and lipoxygenases, or xanthin oxidase.

Thus, the available evidence suggests that activation of NADPH oxidase by glucose or AGE is a key step for the generation of ROI by endothelium. Whether the generated ROI are transformed to peroxynitrite depends on the type of cell and the concomitant reactions. If, as in HUVECs, NO-synthase becomes activated simultaneously, peroxynitrite may be formed and represent the key mediator for the subsequent transformation of endothelium. In the absence of NO synthase activation or insufficient amounts of NO, superoxide anions may directly act as signal mediators and exert the deleterious cytotoxic effects of hyperglycemia and AGE on endothelium and other vascular cells.

There are two open questions. What are the mechanisms for the hyper-glycemia-mediated increase in intracellular calcium? The formation of vascular endothelial growth factor (VEGF) as a consequence of the generation of superoxide anions is one interesting mechanism, especially because it has been shown that AGE are able to induce the expression of VEGF (42). Do changes in the cytosolic NADH/NAD ratio contribute to the formation of ROI in addition to the activation of NADPH oxidases? It has been suggested that the NADH/NADPH ratio is elevated in diabetes because more glucose is metabolized by the sorbitol pathway and that the increased NADH/NAD ratio causes the formation of superoxide anions by various mechanisms (43). We do not believe that this mechanism is working in endothelium, because the formation of ROI was not inhibited by inhibitors of the sorbitol dehydrogenase, either in HUVEC or in porcine endothelial cells (35).

In summary, there is good evidence that the vasculature and more specifically the endothelium is one important source for the generation of ROIs. The formation of ROIs is specifically related to the diabetic state because it is stimulated by glucose and advanced glycation end products in a dose-dependent manner. It is interesting to note that similar observations have been reported for the vasculature in hypertension and hypercholesterolemia, suggesting that the initial processes and stimuli might be different, but that the three pathophysiological conditions finally result in an enhanced oxidative stress.

III. WHAT ARE THE CONSEQUENCES OF OXIDANT STRESS IN DIABETES?

There is a lot of evidence that ROI and especially peroxynitrite are involved in activation of transcription factors such as NF-kB. NF-kB is responsible for a variety of reactions contributing to the thrombogenic transformation of endothelium (Fig. 4), (44-48): release of tumor necrosis factor a and interleu-kin 1 (i (proinflammatory), release of growth factors (M-CSF [monocyte colony stimulating factor], GM-CSF [granulocyte-monocyte colony stimulating factor], c-myc), activation of the monocyte chemoattractant protein MCP-1 (chemotaxis); expression of adhesion proteins (VCAM-1 [vascular cell adhesion molecule-1], ICAM-1 [intercellular adhesion molecule-1]); and expression of tissue factor (thrombogenesis).

We used two different approaches to test whether hyperglycemia causes an activation of NF-kB: the electromobility shift assay (EMSA), measuring the DNA-binding activity of nuclear proteins to an NF-KB-specific oligonucleotide (48), and a histochemical approach using a fluorescence-labeled antibody coupled to the NF-kb-specific oligonucleotide (49). Using both methods we can show that hyperglycemia causes a dose- and time-dependent activation of NF-kB. The maximum of activation by high glucose is achieved after 4 h; after 10-12 h NF-kB is again completely inactivated (Fig. 5). This activation is inhibited by antioxidants (tocopherol 10 |0.g/mL, thioctic acid 0.5 |0.M) and

Figure 5 Time dependence of NF-kB activation by hyperglycemia in human endothelial cells. HUVECs were incubated with low glucose (5 mM), high glucose (30 mM), and 3-O-methyl-d-glucose (3-OMG, 25 + 5 mM glucose). After a 2-, 4-, 6-, and 12-h incubation (37 °C), the cells were fixed with paraformaldehyde and stained by the specific fluorescein isothiocyanate (FITC)-labeled consensus sequence for NF-kB as described. Osmotic controls (25 mM mannitol + 5 mM glucose) did not show staining above the background. Controls; high glucose; 3-OMG.

Time (hrs)

Figure 5 Time dependence of NF-kB activation by hyperglycemia in human endothelial cells. HUVECs were incubated with low glucose (5 mM), high glucose (30 mM), and 3-O-methyl-d-glucose (3-OMG, 25 + 5 mM glucose). After a 2-, 4-, 6-, and 12-h incubation (37 °C), the cells were fixed with paraformaldehyde and stained by the specific fluorescein isothiocyanate (FITC)-labeled consensus sequence for NF-kB as described. Osmotic controls (25 mM mannitol + 5 mM glucose) did not show staining above the background. Controls; high glucose; 3-OMG.

by the NO synthase inhibitor l-nitroarginine (100 |j.M), whereas the modulation of protein kinase C was without any influence. These data suggest that the short-term activation of NF-kB by hyperglycemia is caused by peroxynitrite. It is an open question whether the recently reported long-term activation of NF-kB by AGE products (48) was caused by a similar mechanism. In any case, hyperglycemia seems to cause an activation of NF-kB by different signaling cascades: High glucose leads to a short-term activation, which might be important for the transformation of immediate and short-term variations in blood glucose into vascular reactions. Such a mechanism would also explain why not only the long-term elevation of blood glucose is cytotoxic for the vessel wall but also the spikes in blood glucose that are often observed even in patients with an overall near normoglycemic metabolic control. Activation of NF-kB by AGE products, on the other hand, would induce a long-term modulation of vascular functions and might be especially of importance for angio-genesis.

In addition to activation of NF-kB, the formation of peroxynitrite may have several other consequences that may contribute to the development of vascular complications in diabetes. First, the induction of apoptosis. We have recently reported (50) that high glucose induces the programmed cell death in HUVECs. This process was inhibited by antioxidants (thioctic acid and tocopherol), but also by inhibitors of NO synthases. The underlying mechanism is not yet fully understood at this time, but there are several lines of evidence that the induction of apoptosis is independent of the activation of NF-kB. The induction of apoptosis can be understood as an indicator of damage of endothelium by high glucose, as an attempt of the vasculature to get rid of damaged endothelial cells. Such a loss of endothelium would be associated with induction of angiogenesis, a process typically observed in the eye and the kidney of many diabetic patients (51,52). On the other hand, loss of endothelium would lead to an exposition of thrombogenic structures (subendothel-ial matrix) to the bloodstream and thereby cause an increased thrombotic risk.

Another consequence is the oxidation of low-density lipoproteins. It has been reported that peroxynitrite is a strong prooxidant and accelerates the oxidation of low-density lipoproteins (53). Because oxidized low-density lipoproteins are themselves cytotoxic for endothelium, the formation of peroxynitrite would reinforce the oxidant stress by constituting a deleterious vicious cycle. Finally, the activation of metalloproteinases has been reported to become activated by peroxynitrite in vivo and in vitro (54). Such an activation of metalloproteinases at the edge of an atherosclerotic plaque is assumed to cause a destabilization of the plaque, enhance plaque rupture, and finally a thrombotic event that is the most common cause for myocardial infarction, angina pectoris, and cardiac death (30,31).

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