Atherosclerotic vascular disease is a major cause of cardiovascular (CV) morbidity and mortality and a significant driver of health care costs in patients with diabetes. Systemic abnormalities, including dyslipidemia, insulin resistance, hyperinsulinemia, hyperglycemia, oxidative stress, accentuated renin-angiotensin system, and tissue inflammation, have been proposed to play a role in mediating accelerated atherosclerosis in diabetes (1). Hyperglycemia not only predicts CV disease risk in individuals without known CV disease or diabetes (2), but atherosclerosis is also quite prevalent in prediabetes (3). Although difficult to achieve, near-normal glycemic control is not typically associated with further reduction in macrovascular events in patients with type 2 diabetes (4). Consequently, elucidating mechanisms by which hyperglycemia affects atherosclerosis is necessary to identify novel therapeutic targets.

Increased mitochondrial reactive oxygen species (ROS) has been identified as a common upstream event that mediates the atherogenic effects of hyperglycemia (5). ROS increases the formation of advanced glycation end products (AGEs), augments the expression of the receptor for AGEs (RAGE) and its ligands, activates protein kinase C (PKC) isoforms (primarily PKCβ isoform), and accentuates flux through the hexosamine and polyol pathways (5). Among these, the ligand-RAGE axis appears to play a major role in vascular dysfunction in diabetes. AGEs, S100/calgranulins, and high-mobility group box 1 are principal endogenous ligands for RAGE (6). Persistent activation of RAGE promotes atherosclerosis by activating a diverse array of intracellular signaling pathways that stimulate expression of cytokines, cellular adhesion molecules, growth factors, ROS, and vascular matrix metalloproteinases (6). Diabetes-induced atherosclerosis and vascular dysfunction are attenuated in RAGE-null mice confirming the pivotal role played by the ligand-RAGE axis (7,8).

AGEs are formed by rearrangement of Amadori products or as a result of reactions between dicarbonyls and amino acid residues (lysine, arginine, and cysteine) (9). Methylglyoxal (MG) is a highly reactive α-dicarbonyl and a major precursor of cellular and circulating AGEs (9). MG is primarily derived from triose phosphate intermediates formed during glycolysis (9). In humans, plasma/tissue MG concentrations are ∼1–5 μmol/L (10). More than 99% of cellular MG is metabolized by glyoxalase 1 (GLO1), GLO2, and reduced glutathione to d-lactate (11). Glycation of arginine residues by MG results in the formation of hydroimidazolones (MG-H1, MG-H2, and MG-H3) and that of lysine leads to Nɛ-(carboxymethyl) lysine (CML) and Nɛ-(carboxyethyl) lysine (CEL) (12). MG-H1 is the most prevalent MG-derived AGE with plasma concentration of ∼17 μmol/L in healthy individuals (12). Unlike CML/CELs, MG-Hs have a higher binding affinity to RAGE (KD, ∼40 nmol/L vs. 100 µmol/L) (13). Hyperglycemia and diabetes are associated with increased formation and decreased metabolism of MG (14,15). Thus, MG-Hs are capable of sustained activation of RAGE in the vasculature and circulating inflammatory cells (Figure 1).

Figure 1

Dicarbonyl stress and RAGE signaling. Increased flux through the glycolytic pathway increases MG formation. Reduced GLO1 activity in this setting decreases the degradation of MG, resulting in excess formation of MG-H1. MG-H1 activates RAGE. RAGE activation leads to increased ROS production that subsequently activates the transcription factor, nuclear factor-κΒ (NF-κB), to stimulate the expression of adhesion molecules and chemokines. P, phosphorylation.

Figure 1

Dicarbonyl stress and RAGE signaling. Increased flux through the glycolytic pathway increases MG formation. Reduced GLO1 activity in this setting decreases the degradation of MG, resulting in excess formation of MG-H1. MG-H1 activates RAGE. RAGE activation leads to increased ROS production that subsequently activates the transcription factor, nuclear factor-κΒ (NF-κB), to stimulate the expression of adhesion molecules and chemokines. P, phosphorylation.

Close modal

Overexpression of GLO1 reduces dicarbonyl stress and attenuates diabetes-induced endothelial dysfunction, impaired neovascularization, and nephropathy (1619). Similarly, knockdown of GLO1 in rodents is associated with increased MG-H1 levels in the kidney and, even in the face of normoglycemia, is associated with mesangial sclerosis and proteinuria, features typical of diabetic nephropathy (19). These studies suggest that dicarbonyl stress triggers renal pathology directly and independent of glycemia. Whether dicarbonyl stress similarly modulates atherosclerosis is unknown. In this issue of Diabetes, Tikellis et al. (20) examined the atherogenic effects of dicarbonyl and the potential role of RAGE in that process. To that end, they demonstrate that dicarbonyl stress due to exogenous MG supplementation or chemical inhibition of GLO1 in normoglycemic apolipoprotein E knockout (apoE−/−) mice is associated with increased atherosclerosis. Furthermore, they show that the atherogenic effects of dicarbonyl stress due to excess MG exposure are significantly abrogated in RAGE-deficient apoE−/− mice. These results suggest that excess MG initiates and augments atherosclerosis predominantly through RAGE activation and independent of glycemia.

Endothelial cells exposed to MG and aortas of MG-treated mice demonstrated increased functional adhesiveness and expression of genes associated with leukocyte adhesion and vascular inflammation (intercellular adhesion molecule-1 [ICAM-1], vascular cell adhesion molecule-1 [VCAM-1]) in wild-type mice. However, endothelial cells and aortas of RAGE/apoE−/− mice subjected to dicarbonyl stress had a significantly reduced leukocyte adhesion but a higher gene expression of VCAM-1, ICAM-1, and tetherin. Cellular mechanisms mediating these differential effects of RAGE activation and/or non-RAGE–mediated downstream pathways need further elucidation. Similarly, cell permeable superoxide dismutase mimetic prevented GLO1 inhibitor–induced expression of MCP-1, ICAM-1, and VCAM-1 in endothelium of RAGE knockout but not in wild-type mice. It is likely that MG activates distinct ROS-generating pathways in RAGE replete versus deficient endothelium.

BBGC, a chemical inhibitor of GLO1, was used to induce dicarbonyl stress in apoE−/− mice. However, a recent study examined if GLO1 overexpression decreased atherosclerosis in streptozotocin-treated apoE−/− mice, and if GLO1 knockdown can promote atherogenesis in nondiabetic apoE−/− mice (21). In contrast to the current report by Tikellis et al., despite a decrease in GLO1 activity (by ∼75%) and increased aortic MG-H1 content, Glo1 and apoE double knockout mice did not show increased atherosclerosis compared with apoE−/− mice. Furthermore, GLO1 overexpression prevented renal dysfunction but not atherosclerosis in streptozotocin-treated apoE−/− mice. Thus, chemical inhibition and gene ablation of GLO1 appear to have differential effects on atherosclerosis. As opposed to global inhibition of GLO1 with BBGC, reduced expression of GLO1 may be variable and tissue- or cell-specific (circulating inflammatory cells vs. resident vascular cells), which may explain the discrepant results. Nonetheless, future studies are needed to reconcile these seemingly contrasting results.

Glycation of LDL by MG promotes arterial atherogenicity by increasing small, dense LDL levels and modifying apoB100; both changes favor increased LDL retention in the vascular wall (22). In mouse models of atherosclerosis (apoE−/−), plasma cholesterol is transported in lipoprotein remnants, as opposed to LDL in humans (23). Thus, the magnitude of the effects of dicarbonyl stress on lipids may be different and species-dependent. In addition, mice do not exhibit coronary atherosclerosis or unstable plaques. Rupture-prone arterial plaques in humans are characterized by lower GLO1 expression and increased levels of MG-H1, inflammatory cytokines, and cellular apoptosis markers (24). Higher plasma levels of AGEs (CML, CEL, and pentosidine) increase the risk of incident CV events in individuals without prior CV events (25). Although the causal relationship cannot be ascertained, findings from these human studies suggest that AGEs may play an important role in atherosclerosis/plaque rupture and serve as a potential biomarker that helps improve CV risk reclassification.

In conclusion, the findings by Tikellis et al. (20) are novel and highlight the glucose-independent effects of dicarbonyl stress on atherosclerosis that are primarily mediated through RAGE activation. Whether GLO1 can be therapeutically targeted to reduce dicarbonyl stress and prevent atherosclerosis remains to be determined.

See accompanying article, p. 3915.

Funding. This work was supported by the Intramural Research Program of National Institute of Diabetes and Digestive and Kidney Diseases and the Extramural Research Program, National Heart, Lung, and Blood Institute, National Institutes of Health.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

1.
Bornfeldt
KE
.
2013 Russell Ross memorial lecture in vascular biology: cellular and molecular mechanisms of diabetes mellitus-accelerated atherosclerosis
.
Arterioscler Thromb Vasc Biol
2014
;
34
:
705
714
[PubMed]
2.
Di Angelantonio
E
,
Gao
P
,
Khan
H
, et al
Emerging Risk Factors Collaboration
.
Glycated hemoglobin measurement and prediction of cardiovascular disease
.
JAMA
2014
;
311
:
1225
1233
[PubMed]
3.
Kurihara
O
,
Takano
M
,
Yamamoto
M
, et al
.
Impact of prediabetic status on coronary atherosclerosis: a multivessel angioscopic study
.
Diabetes Care
2013
;
36
:
729
733
[PubMed]
4.
Gerstein
HC
,
Miller
ME
,
Genuth
S
, et al
ACCORD Study Group
.
Long-term effects of intensive glucose lowering on cardiovascular outcomes
.
N Engl J Med
2011
;
364
:
818
828
[PubMed]
5.
Giacco
F
,
Brownlee
M
.
Oxidative stress and diabetic complications
.
Circ Res
2010
;
107
:
1058
1070
[PubMed]
6.
Yan
SF
,
Ramasamy
R
,
Schmidt
AM
.
The RAGE axis: a fundamental mechanism signaling danger to the vulnerable vasculature
.
Circ Res
2010
;
106
:
842
853
[PubMed]
7.
Soro-Paavonen
A
,
Watson
AM
,
Li
J
, et al
.
Receptor for advanced glycation end products (RAGE) deficiency attenuates the development of atherosclerosis in diabetes
.
Diabetes
2008
;
57
:
2461
2469
[PubMed]
8.
Reiniger
N
,
Lau
K
,
McCalla
D
, et al
.
Deletion of the receptor for advanced glycation end products reduces glomerulosclerosis and preserves renal function in the diabetic OVE26 mouse
.
Diabetes
2010
;
59
:
2043
2054
[PubMed]
9.
Vistoli
G
,
De Maddis
D
,
Cipak
A
,
Zarkovic
N
,
Carini
M
,
Aldini
G
.
Advanced glycoxidation and lipoxidation end products (AGEs and ALEs): an overview of their mechanisms of formation
.
Free Radic Res
2013
;
47
(
Suppl. 1
):
3
27
[PubMed]
10.
Ahmed
N
,
Babaei-Jadidi
R
,
Howell
SK
,
Beisswenger
PJ
,
Thornalley
PJ
.
Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes
.
Diabetologia
2005
;
48
:
1590
1603
[PubMed]
11.
Thornalley
PJ
.
Glyoxalase I—structure, function and a critical role in the enzymatic defence against glycation
.
Biochem Soc Trans
2003
;
31
:
1343
1348
[PubMed]
12.
Thornalley
PJ
,
Battah
S
,
Ahmed
N
, et al
.
Quantitative screening of advanced glycation endproducts in cellular and extracellular proteins by tandem mass spectrometry
.
Biochem J
2003
;
375
:
581
592
[PubMed]
13.
Xue
J
,
Ray
R
,
Singer
D
, et al
.
The receptor for advanced glycation end products (RAGE) specifically recognizes methylglyoxal-derived AGEs
.
Biochemistry
2014
;
53
:
3327
3335
[PubMed]
14.
Yao
D
,
Brownlee
M
.
Hyperglycemia-induced reactive oxygen species increase expression of the receptor for advanced glycation end products (RAGE) and RAGE ligands
.
Diabetes
2010
;
59
:
249
255
[PubMed]
15.
Ahmed
N
,
Babaei-Jadidi
R
,
Howell
SK
,
Thornalley
PJ
,
Beisswenger
PJ
.
Glycated and oxidized protein degradation products are indicators of fasting and postprandial hyperglycemia in diabetes
.
Diabetes Care
2005
;
28
:
2465
2471
[PubMed]
16.
Brouwers
O
,
Niessen
PM
,
Ferreira
I
, et al
.
Overexpression of glyoxalase-I reduces hyperglycemia-induced levels of advanced glycation end products and oxidative stress in diabetic rats
.
J Biol Chem
2011
;
286
:
1374
1380
[PubMed]
17.
Brouwers
O
,
Niessen
PM
,
Haenen
G
, et al
.
Hyperglycaemia-induced impairment of endothelium-dependent vasorelaxation in rat mesenteric arteries is mediated by intracellular methylglyoxal levels in a pathway dependent on oxidative stress
.
Diabetologia
2010
;
53
:
989
1000
[PubMed]
18.
Vulesevic
B
,
McNeill
B
,
Geoffrion
M
, et al
.
Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice
.
Cardiovasc Res
2014
;
101
:
306
316
[PubMed]
19.
Giacco
F
,
Du
X
,
D’Agati
VD
, et al
.
Knockdown of glyoxalase 1 mimics diabetic nephropathy in nondiabetic mice
.
Diabetes
2014
;
63
:
291
299
[PubMed]
20.
Tikellis C, Pickering RJ, Tsorotes D, et al. Dicarbonyl stress in the absence of hyperglycemia increases endothelial inflammation and atherogenesis similar to that observed in diabetes. Diabetes 2014;63:3915–3925
21.
Geoffrion M, Du X, Irshad Z, et al. Differential effects of glyoxalase 1 overexpression on diabetic atherosclerosis and renal dysfunction in streptozotocin-treated, apolipoprotein E-deficient mice. Physiol Rep 2014;2:e12043
22.
Rabbani
N
,
Godfrey
L
,
Xue
M
, et al
.
Glycation of LDL by methylglyoxal increases arterial atherogenicity: a possible contributor to increased risk of cardiovascular disease in diabetes
.
Diabetes
2011
;
60
:
1973
1980
[PubMed]
23.
Getz
GS
,
Reardon
CA
.
Animal models of atherosclerosis
.
Arterioscler Thromb Vasc Biol
2012
;
32
:
1104
1115
[PubMed]
24.
Hanssen
NM
,
Wouters
K
,
Huijberts
MS
, et al
.
Higher levels of advanced glycation endproducts in human carotid atherosclerotic plaques are associated with a rupture-prone phenotype
.
Eur Heart J
2014
;
35
:
1137
1146
[PubMed]
25.
Hanssen
NM
,
Beulens
JW
,
van Dieren
S
, et al
.
Plasma advanced glycation end products are associated with incident cardiovascular events in individuals with type 2 diabetes: a case-cohort study with a median follow-up of 10 years (EPIC-NL)
.
Diabetes.
21 May
2014 [Epub ahead of print]