It is well established that hyperglycemia is a major causative factor for micro-vascular disease. In addition, recent epidemiological reports have suggested that postprandial hyperglycemia is an important risk factor for cardiovascular diseases, although the pathological role of chronic hyperglycemia is still controversial. Several well-researched theories have been proposed to explain how chronic hyperglycemia or postprandial hyperglycemia causes the micro-and macro-vascular derangements. These theories include increased polyol pathway flux, increased advanced glycation end-product (AGE) formation, activation of protein kinase C (PKC) and increased oxidative stress. Among them, oxidative stress has been paid increasing attention to as a common mediator of these biochemical abnormalities. Among many possible sources for oxidative stress, recent reports including ours have shown that a PKC-dependent activation of NAD(P)H oxidase may be a major source for increased oxidative stress in vascular tissues of diabetes and insulin resistant state. In this article, we show that this mechanism may be an effective therapeutic target for preventing diabetic vascular complications.

A number of in vitro and in vivo studies have demonstrated that many protein, lipid and DNA markers of oxidative stress are increased in vascular tissues from animals and patients with diabetes [1-4]. We also showed the increase in reactive oxygen species (ROS) production in diabetic animal models by in vivo electron spin resonance method that is non-invasive and more specific than above-mentioned oxidative stress markers [5]. Several mechanisms for increased oxidative stress have been postulated. In the process of mitochondrial respiration, several percentages of oxygen consumed is converted into superoxide. High glucose level was reported to increase such a mitochondrial superoxide production in vascular endothelial cells [6]. High glucose level also can increase ROS production through glucose auto-oxidation, increased non-enzymatic glycation of proteins and activation of xanthine oxidase. Among possible various sources, increasing evidence has indicated that NAD(P)H oxidase may be the most important source for ROS production in diabetic vascular tissues. We first reported that high glucose level stimulated ROS production in cultured vascular cells through a PKC-dependent activation of NAD(P)H oxidase [7]. The non-phagocytic NAD(P)H oxidase comprises of a membrane-associated cytochrome b558 composed of Nox family proteins (gp91phox, Nox1, and Nox4) and p22phox, and several cytosolic regulatory components, p47phox, p67phox and small GTP binding protein Rac 1 or Rac 2. Our in vitro evidence has been supported by accumulating evidence showing that the activity of NAD(P)H oxidase, in parallel with the increased level of its subunit proteins, is increased in vascular tissues form animal models and diabetic patients [5,8-11]. We also reported that the expression of essential subunits of Nox4 and p22phox in the kidney of diabetic animal models [12]. Activation of Rac-1 and translocation of p47phox and p67phox, both of which are mediated by PKC activation, are supposed to be the mechanism underlying high glucose level-induced NAD(P)H oxidase activation [13,14]. The superoxide production from NAD(P)H oxidase is controlled by its expression dynamics as well as by its activation. The mechanism for increased expression of NAD(P)H oxidase subunits in diabetic vascular tissues remained to be well established, since the regulatory mechanism of the expression of NAD(P)Oxidase subunits is generally not fully understood. In the knockout mouse of the PKC β isoform, the normalization of increased expression of Nox4, oxidative stress, proteinuria and tissue abnormalities in diabetic kidney was observed [15]. These findings also support the role of PKC activation in the increased expression of NAD(P)H oxidase subunits. The detailed molecular mechanism should be clarified in the further studies.

Experimental and clinical trials have suggested that blockade of the renin-angiotensin system (RAS) with angiotensin 1-converting enzyme (ACE) inhibitors or angiotensin II (ATII) receptor blocker (ARB) have some protective effects on diabetic nephropathy and cardiovascular events, which appear to be independent of its antihypertensive effect. ATII was reported to mediate NAD(P)H oxidase-dependent superoxide production via PKC activation. We reported the improvement of Nox4 expression and oxidative stress in the kidney of diabetic mice by administration of ARB [16]. ACE is involved in the production of tissue ATII, but it is recently suggested that chymase has more efficient tissue ATII-forming activities than ACE in several species including human and hamsters. In addition, several reports have shown that chymase expression may be up-regulated in diabetic renal tissues [17]. We have confirmed that chymase expression increases in renal tissues, aorta and cardiac tissues of streptozotocin-induced diabetic hamster and examined the effect of chymase-specific inhibitor [18]. Oral administration of the chymase-specific inhibitors protected against elevated intra-renal ATII concentrations, Nox4 expression, oxidative stress, and renal dysfunction including proteinuria and mesangial expansion in streptozotocin-induced diabetic hamsters [18]. It also normalized oxidative stress, Nox4 expression and myocardial fibrosis in diabetic hamsters [19]. Because low-grade inflammation is known to occur in diabetic vascular tissues, including renal tissues, infiltrated inflammatory cells, such as mast cells, may be the source of the increased chymase levels. High glucose is the initiator of increased oxidative stress and inflammation, but the subsequent increases in chymase expression and local ATII production may further accelerate ROS production and inflammation, ultimately forming a vicious cycle of oxidative stress and inflammation, although this hypothesis should be evaluated in future studies.

Although the involvement of oxidative stress in diabetic vascular complications has been demonstrated in vitro and in animal studies, the effectiveness of antioxidant therapy is still controversial because there have been very few human studies indicating the beneficial effects of antioxidants on diabetic vascular complications. Bilirubin has been recognized as an important endogenous antioxidant [20] and also reported to have an inhibitory effect on the activity of NAD(P)H oxidase [21]. We therefore compared the prevalence of vascular complications in patients with diabetes and Gilbert syndrome (a congenital hyperbilirubinemia) and in patients with diabetes without Gilbert syndrome [22]. The adjusted odds ratio for the association of Gilbert syndrome with retinopathy was 0.22 (P<001); with macroalbuminuria, 0.20 (P=0.01); with coronary artery disease, 0.21 (P=0.04); and with cerebrovascular disease, 0.50 (P=0.22) (Table 1). This study showed for the first time the lower prevalence of vascular complications in patients with diabetes and Gilbert syndrome, as well as reduced markers of oxidative stress and inflammation (Fig. 1). These findings strongly suggest that antioxidant therapy may be useful for preventing the development of diabetic vascular complications. Furthermore, serum bilirubin concentrations were demonstrated to be negatively associated with albuminuria in patients with type 2 diabetes [23]. Another group of Japanese investigators reported in their cross-sectional study on more than 3,000 participants, that compared to subjects with in the lowest bilirubin quartile, those with the highest bilirubin levels had a four times lower prevalence of diabetic retinopathy [24]. Interestingly, our cross-sectional study of 12,949 general persons revealed an inverse relation between serum bilirubin level and the prevalence of type 2 diabetes [25]. In a large Korean cross-sectional study on almost 94,000 subjects, high serum bilirubin was also found to be associated with the reduced risk of diabetes mellitus and diabetic nephropathy [26]. Consistent with these data, low prevalence of metabolic syndrome in subjects with phenotypic Gilbert syndrome was described in a recent large Korean study on more than12,000 participants [27]. Antioxidant therapy may be useful for inhibiting the onset of diabetes itself as well as inhibiting diabetic complications.

We have proposed an antioxidant therapy using the drugs or chemicals which target the mechanism for PKC-NAD(P)H oxidase activation for preventing diabetic vascular complications [28]. The possible mechanism underlying PKC-NAD(P)H oxidase activation and drugs or chemicals targeting its mechanism are shown in Fig. 2.

In this review, the experimental and clinical studies on the role of oxidative stress in the development of diabetic vascular complication and the molecular mechanism underlying oxidative stress are discussed. These studies strongly show that antioxidant therapy is potentially effective to protect against diabetic vascular complications and one of the therapeutic targeting mechanism is activation or up-regulation of PKC-NAD(P)H oxidase. In this article, the effectiveness of several drugs or chemicals which can inhibit this mechanism is shown. These drug or chemicals are expected to be clinically effective for preventing diabetic vascular complications, but the development of more effective drugs for clinical use is also expected.