Introduction
Diabetes mellitus is associated with an increased risk of cardiovascular disease [
1]. Endothelial dysfunction is considered to be the primary cause in the pathogenesis of vascular disease in diabetes [
1]. Although the mechanism whereby diabetes leads to endothelial dysfunction is incompletely understood, a common feature of endothelial dysfunction is a decreased bioavailability of endothelium-derived nitric oxide (NO) and, as a consequence, impaired endothelium-dependent vasorelaxation [
1]. Several studies have demonstrated impaired endothelium-dependent vasorelaxation in diabetic patients [
2,
3] and in animal models of diabetes [
4,
5].
One hypothesis of how hyperglycaemia leads to endothelial dysfunction and vascular complications is the formation of AGEs [
6]. AGEs are a heterogeneous family of non-enzymatically modified proteins formed by carbohydrates, which are increased in patients with diabetes [
7‐
9]. The mechanisms by which AGEs contribute to vascular complications are multiple, including their interaction with the receptor for AGE (RAGE), the formation of cross-links in basal membranes and intracellular accumulation of glycated proteins [
6]. Although it has been demonstrated that AGEs are associated with impaired endothelium-dependent vasorelaxation in type 2 diabetes [
10] and can reduce endothelium-dependent vasorelaxation [
11], it is not known which specific AGEs are the culprits or which pathways are involved.
It is now apparent that especially intracellular sugars and their derivatives can participate in glycation and AGE formation [
12]. The reactive dicarbonyl methylglyoxal has received considerable attention as the most reactive AGE precursor in endothelial cells [
13]. Methylglyoxal is formed non-enzymatically by dephosphorylation of triose phosphates and is efficiently catabolised to
d-lactate by the glyoxalase (GLO) pathway, consisting of GLO-I, GLO-II and the co-factor glutathione [
14]. Methylglyoxal primarily reacts with arginine residues to form the major adduct 5-hydro-5-methylimidazolone (MG-H1) and with lysine to form
N
ε-(1-carboxyethyl)lysine (CEL) [
15,
16]. Plasma methylglyoxal levels are significantly increased in diabetic patients [
17] and in endothelial cells by hyperglycaemia [
18]. The importance of methylglyoxal and methylglyoxal-derived AGEs for the development of diabetic vascular complications and hypertension has only recently been recognised. Recent studies have demonstrated that increased methylglyoxal-derived AGE levels in diabetic patients are associated with diabetic complications such as nephropathy [
19] and retinopathy [
20], and with diabetes-related vascular disorders such as hypertension [
21‐
25]. As a major precursor of AGE formation, methylglyoxal can influence multiple aspects of cellular biology in diabetes [
26]. Methylglyoxal targets specific mitochondrial proteins accompanied by an increase in the formation of reactive oxygen species (ROS) [
27]. Since ROS production by mitochondria is responsible for major mechanisms involved in diabetic complications, methylglyoxal-induced ROS production may be an initial event in the pathogenesis of vascular complications.
We hypothesised that accumulation of methylglyoxal can lead to endothelial dysfunction by ROS formation and therefore evaluated the effect of hyperglycaemia, methylglyoxal and methylglyoxal-modified albumin on endothelium-dependent NO-mediated vasorelaxation, a measure of endothelial function, and tested the involvement of oxidative stress therein.
Methods
GLO-I (also known as
GLO1) transgenic rats were obtained from T. Miyata [
28]. Rat genomic DNA extracted from tail tissue was used to detect the transgene by PCR using specific primers for
GLO-I or pBsCAG-2 vector. Primers for cytomegalovirus enhancer, sense (5′-GTC GAC ATT GAT TAT TGA CTA G-3′) and antisense (5′-CCA TAA GGT CAT GTA CTG-3′), amplified a 350 bp fragment. Primers for the fragment containing human
GLO-I gene and 3′ junction of vector, sense (5′-GTA GTG TGG GTG ACT CCT CCG TTC CTT GGG-3′) and antisense (5′-TCG AGG GAT CTT CAT AAG AGA AGA G-3′), amplified a 1,200 bp fragment. PCR amplification was carried out with an initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C at 30 s, 55°C or 58°C at 30 s and 72°C at 30 or 75 s.
Wild-type rats (n = 9) and transgenic GLO-I rats (n = 8) were made diabetic by intravenous injection of streptozotocin (65 mg/kg body weight) in the tail vein. Weight- and age-matched control rats (n = 9) and transgenic GLO-I rats (n = 8) were not injected. Streptozotocin resulted in a fivefold increase of blood glucose levels, irrespective of GLO-I overexpression.
Discussion
In this study we have shown that hyperglycaemia-induced impairment of endothelium-dependent NO-mediated vasorelaxation of mesenteric arteries is mediated by the major AGE precursor methylglyoxal. The effect of methylglyoxal occurs intracellularly and is most likely to be caused by decreased bioavailability of NO due to a methylglyoxal-induced increase of oxidative stress. These data provide a mechanistic link between hyperglycaemia, the formation of the AGE precursor methylglyoxal and vascular complications.
Endothelial dysfunction is characterised by impaired vascular response to endothelium-derived NO and is associated with the development of micro- and macrovascular complications in diabetes [
33]. To gain insight into the mechanism by which hyperglycaemia alters endothelial function, we used acetylcholine, which stimulates the release of NO from endothelial cells, leading to relaxation of the underlying smooth muscle cells. Experiments were performed in the presence of indometacin and elevated K
+ concentration to rule out involvement of endothelium-derived prostaglandins and hyperpolarising factors, and thus to focus on NO-mediated relaxation. Treatment of arteries with high concentrations of glucose significantly decreased acetylcholine-induced NO-mediated vasorelaxation (Fig.
1), which is in agreement with a previous study performed in mesenteric arteries [
34]. We also found that the effect of high glucose was prevented in arteries with
GLO-I overexpression and that the impaired relaxation in diabetic rats was improved by
GLO-I overexpression. In addition, incubation with exogenous methylglyoxal resulted in dose-dependent impairment of acetylcholine-induced vasorelaxation (Fig.
3). These data from ex vivo and in vivo experiments provide strong evidence that the impairment of endothelium-dependent NO-mediated vasorelaxation in diabetes is mediated by the major AGE precursor methylglyoxal.
Since the arterial contractile responses to K
+ and relaxing responses to the NO-donor SNP were not modified by methylglyoxal, our data indicate that short exposure to methylglyoxal leads to an endothelium-dependent effect on vasorelaxation without a direct effect of methylglyoxal on arterial smooth muscle function. In accordance, Mukohda et al. showed that methylglyoxal had no effects on K
+-induced contraction [
35]. Although not tested in our study, chronic exposure to exogenous methylglyoxal may also lead to impaired endothelium-independent vasorelaxation [
36].
We did not find any effects of extracellularly added methylglyoxal-modified albumin on vascular reactivity (Fig.
2). In accordance with this result, we had previously demonstrated that there is also no binding and biological effect of methylglyoxal-modified albumin on endothelial cells [
37]. In contrast, Xu et al. [
11] showed that other extracellularly formed AGEs can impair endothelium-dependent vasorelaxation, but these data were obtained with AGE-albumin, which was prepared with a degree of modification that is much higher than that ever seen in diabetes. A recent paper by Gao et al. [
38] described the importance of S100b-induced RAGE activation in endothelial dysfunction and impaired vasoreactivity. We confirmed the finding that the RAGE ligand S100b reduces acetylcholine-induced NO-mediated vasorelaxation (Fig.
2e). Combined, these data provide strong evidence that extracellular methylglyoxal-derived AGEs are not involved in diabetic arterial dysfunction and that other ligands for RAGE, such as CML and S100b, impair endothelium-dependent vasorelaxation.
Another important finding of our study is that the effect of hyperglycaemia and methylglyoxal on NO-mediated vasorelaxation in rat mesenteric arteries is completely abolished in rat mesenteric arteries with overexpression of
GLO-I, indicating that the effect of hyperglycaemia and exogenous methylglyoxal is due to intracellular effects of methylglyoxal in endothelial cells. We recently found that endothelial cells incubated with high glucose concentrations display an increase in methylglyoxal [
18] and we have now demonstrated that methylglyoxal leads to intracellular accumulation of the major methylglyoxal-induced AGE, MG-H1. This is in agreement with a study showing that methylglyoxal, given intraperitoneally to rats for 7 weeks, induces an increase in vascular AGE content [
36]. In our study, MG-H1 appeared to be localised in the cytoplasm and in the nucleus of endothelial cells. The latter is in accordance with data of two recent papers about methylglyoxal modification of nuclear proteins [
39,
40]. Furthermore, Schlotterer et al. [
41] recently reported that hyperglycaemia leads to methylglyoxal-modified mitochondrial proteins and reduced lifespan in
C. elegans. This oxidative stress-dependent process was inhibited by
GLO-I overexpression, thereby linking methylglyoxal to the formation of ROS.
Chronic hyperglycaemia is associated with increased intracellular levels of methylglyoxal [
18,
42], oxidative stress [
43,
44] and an impairment of NO synthesis pathways [
45]. Our results link these studies by showing an oxidative stress-dependent effect of methylglyoxal on NO bioavailability. This impairment was prevented by superoxide dismutase mimetics and by the antioxidant NAC (Fig.
6). Moreover, the impaired relaxation could also be prevented by apocynin, but because of the lack of specificity of this NAD(P)H inhibitor [
46], the exact mechanism by which methylglyoxal induces the formation of ROS is not completely clear. Although not addressed in this study, methylglyoxal modifications of mitochondrial membrane proteins [
27], antioxidant enzymes [
47] and GAPDH [
48] may increase oxidative stress and, as a consequence, impair endothelium-dependent vasorelaxation.
Because AGEs are known to inactivate NO by a direct chemical reaction, the impaired endothelium-dependent vasodilatation in diabetes may be mediated by NO quenching and NO depletion by methylglyoxal-derived AGE accumulation [
49]. Indeed, we observed quenching of NO by methylglyoxal, but not by methylglyoxal-derived AGEs. Methylglyoxal reduced the half-life of NO in buffer from 4 to approximately 1 min. However, in vivo the very fast reaction of NO with numerous bio-molecules, such as haemoglobin, is one of the reasons why the half-life of NO in biological systems is very short, i.e. less than 1 s [
50], and not susceptible to being shortened by methylglyoxal. Therefore, the physiological relevance of quenching of NO by methylglyoxal, for example in methylglyoxal-induced impairment of endothelium-dependent vasodilatation, is very limited. Methylglyoxal and methylglyoxal-derived AGEs may also decrease NO availability by decreasing NO synthase activity, but in our previous study we already ruled out the possibility that free methylglyoxal and the methylglyoxal-derived AGEs argpyrimidine and MG-H1, which have some structural homology with the NOS inhibitor asymmetric dimethylarginine, directly inhibit eNOS activity [
51]. Together, these findings indicate that the effects of methylglyoxal on vasoreactivity are thus caused by methylglyoxal-induced oxidative stress and the quenching of NO by oxidative stress, rather than direct interaction of methylglyoxal-adducts with NO or eNOS activity. The question of whether methylglyoxal or methylglyoxal-induced AGEs are involved in impaired eNOS activation by oxidative-stress-related interference with eNOS phosphorylation needs to be further investigated.
For the ex vivo and in vitro experiments, we used methylglyoxal in a range of 10 to 1,000 µmol/l, a range that caused effects in a concentration-dependent manner. The in vivo concentration of methylglyoxal is under debate; plasma methylglyoxal levels are estimated to be about 0.5 µmol/l in healthy individuals and can increase twofold in diabetes [
52], while others have demonstrated that plasma methylglyoxal concentration in poorly controlled human diabetic patients is about 400 µmol/l [
53]. Nevertheless, cells produce large amounts of methylglyoxal [
54] and therefore intracellular levels are probably much higher than plasma levels [
55]. Although the maximum concentration of methylglyoxal used in the present study appears to be higher than levels observed in vivo, this concentration was also used to increase intracellular concentrations and to mimic life time exposure to elevated levels of methylglyoxal such as in diabetes and in hypertension. Under our experimental conditions, exposure to elevated methylglyoxal levels did not lead to cytotoxic effects.
What are the clinical implications of these observations? Because higher plasma levels of methylglyoxal have been demonstrated under postprandial conditions [
36] and in plasma of diabetic patients [
17], as well as in endothelial cells incubated with high glucose concentrations [
18,
43], we propose here that methylglyoxal plays a crucial role in impaired endothelium-dependent vasorelaxation under postprandial conditions and in chronic hyperglycaemia, and in the long term in development of vascular complications.
In summary, we found that hyperglycaemia-induced impairment of endothelium-dependent NO-mediated vasorelaxation is prevented by GLO-I overexpression and that methylglyoxal decreased NO-mediated vasorelaxation due to an increase in oxidative stress. We conclude that hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation is mediated by intracellular methylglyoxal levels in an oxidative stress-dependent pathway. These data provide a new mechanistic link between hyperglycaemia, the formation of methylglyoxal-derived AGEs and vascular complications.