The authors have demonstrated, as have others, that hyperglycemic conditions in vitro lead to increased PKC activity, AGE formation and polyol pathway activity. As shown in Figure 1, prevention of increased superoxide formation by the above inhibitors or molecular mechanisms completely prevents the increased activity of these three pathways at 30 mmol/l glucose. Finally, it is pointed out that PKC activation also activates nuclear factor kB (NFkB). As with PKC activation, prevention of superoxide accumulation also prevented the activation of NFkB at an elevated medium glucose concentration in the BAEC preparation.
Thus the authors conclude that in the cell culture system examined, increased activity of the putative complication-inducing biochemical pathways under hyperglycemic conditions appears to have a unifying theme. Prevention of superoxide formation will prevent increased activity of all three pathways (PKC, AGE, polyol). Therefore, inhibitors of superoxide formation open a novel and potentially exciting approach to the prevention of the chronic complications of diabetes.

Comment
The neuropathic, micro- and macrovascular complications of diabetes occur in tissues in which transport of glucose is not rate-limiting and thus intracellular glucose concentrations in these tissues approximate those of the extracellular space. Many groups, utilizing a number of tissue preparations, have shown that hyperglycemia increases PKC activity, sorbitol accumulation and AGE formation.
Over the past 30–40 years, experimental studies have shown that inhibition of each of these pathways will prevent the development of one or more of the chronic complications. However, use of specific inhibitors in humans has largely failed to reproduce animal experiments. Therefore, to date, an effective approach to the prevention of complications has not been demonstrated except for the rigorous achievement of near-normoglycemia over prolonged periods (Diabetes Control and Complications Trial and United Kingdom Prospective Diabetes Study).
One can postulate that all of these pathways along with others such as hexosamine formation and osmolites are operative, and therefore inhibition of just one pathway in vivo will not suppress overactivity of others in the diabetic condition. This may help to explain the poor clinical results seen to date with individual pathway inhibitors.
Nishikawa et al., from Brownlee’s group, present a unifying hypothesis with some very preliminary data. They argue that hyperglycemia increases mitochondrial electron transport and superoxide formation. By preventing ROS accumulation, overactivity of the biochemical pathways examined is prevented. They thus postulate a non-specific ‘upstream’ inhibition at the TCA cycle level but they also present some early data on the more specific ‘downstream’ effect of prevention of increased PKC activity blocking NFkB activation. It would appear that the former mechanism would be more plausible at present because of the broad ramifications of reducing superoxide accumulation.
Thus far, only one cell type (BAEC) has been utilized and it is known that endothelial cells can behave very differently in the aorta, retina, kidney, etc. Therefore replication of the present results in other systems and cell types must be shown. In addition, we have been tantalized many times over several decades by exciting in vitro or animal data but disappointing clinical correlates. Nevertheless, it will be very important to pursue these observations in other systems and hopefully in humans.
These caveats do not detract in the least from these very exciting findings and the authors are to be congratulated for their innovative approach to a most important area of diabetes research.

Summary and Comment:
Anthony D. Morrison, Tampa, FL, USA