Background
Coronary artery atherosclerosis is very common in diabetes mellitus and its presence predicts future cardiovascular events. Endothelial dysfunction is a key early event in atherogenesis [
1,
2]. Normal endothelial cells constitutively express endothelial nitric oxide synthase (eNOS), which plays a critical role in maintaining the endothelial functions including regulation of vascular tone, barrier function, inhibition of coagulation and thrombosis, suppression of inflammatory cell adhesion and migration, and angiogenesis. Endothelium-derived NO, synthesized by eNOS, is a major mediator of endothelium-dependent vasorelaxation [
3]. Endothelial dysfunction has largely been assessed as alterations of endothelium-dependent vasorelaxation and gene expression. Many cardiovascular risk factors induce endothelial dysfunction through impairment of eNOS–NO system, which likely explains their promotion of atherogenesis.
Recently, researches suggest that AGEs, senescent macroprotein derivatives formed at an accelerated rate in diabetes, promotes the development of endothelial dysfunction in diabetic patient [
4,
5]. AGEs can bind to its receptor RAGE, triggering the production of several proinflammatory cytokines and chemokines, inducing oxidative stress via the activation of NF-kB [
6]. AGEs was able to reduce NO production and eNOS expression under the condition of high glucose [
5]. AGEs also increases ROS production by activation of NADPH oxidase [
7,
8]. AGE inhibitor, aminoguanidine, can improve endothelial function in diabetic animal models [
1,
4]. Despite accumulating evidence pointing to a causal role of AGEs-induced lesion in the pathogenesis of diabetic vascular disorder, the molecular mechanisms involved in endothelial dysfunction remains poorly understood.
Here we took advantage of HCAECs as a model to investigate the effects of AGEs on eNOS-NO system as well as its related mechanisms including oxidative stress, and MAPK activation involvement. The relationship between serum levels of AGE-p and flow-mediated vasodilatation (FMD) in diabetic patients with coronary artery atherosclerosis were also determined. This study may provide new insights into the mechanisms of AGEs interacting with endothelial cells that may contribute to the vascular lesion formation.
Methods
Cell culture
HCAECs and endothelial growth medium-2 were purchased from Cambrex BioWhittaker (Walkersville, MD). Cells were cultured at 37 °C in 5% carbon dioxide (CO
2) and detached by incubation with 0.25% trypsin–EDTA solution. Passages 5–6 were used in this study. AGEs were prepared as described previously [
9]. Briefly, 5 g of bovine serum albumin (BSA) was incubated with 9 g of
d-glucose in 100 ml sodium phosphate buffer (PBS) at 37 °C for 90 days, and finally was dialyzed against PBS. As a control, BSA was incubated in parallel without
d-glucose. No endotoxin was detectable in these preparations.
Subjects
15 type 2 diabetic patients (average age 63.6 years range 41–74 years; 11 men, 4 women) with coronary artery atherosclerosis scheduled for percutaneous coronary intervention at Zhongda hospital affiliated with the Southeast University were recruited for this study. 15 type 2 diabetic patients (average age 58.9 years range 43–77 years; 10 men, 5 women) without coronary artery atherosclerosis were enrolled as control. All patients received insulin injection to control blood glucose. Serum AGE-p was analyzed using flow injection assay. The protocol was approved by the Ethic Committee of the Zhongda Hospital affiliated with the Southeast University and the methods used in this study were carried out in accordance with the approved guidelines. The informed consent was obtained from all patients.
Measurements of endothelial function
Brachial artery FMD was used to test endothelial function. Methodology and reproducibility data have been described previously [
10]. Briefly, the brachial artery above the elbow was scanned in the supine position by use of high-resolution ultrasound at rest and 1 min after reactive hyperemia that was induced by 5-min cuff occlusion of forearm blood flow. The baseline diameter and maximum FMD diameter were measured from one media-adventitia interface to the other by commercially-available edge-detection software. Vasodilatarion was then calculated as the percent change in diameter over the baseline value.
Gene expression analysis
Total RNA was isolated from HCAECs with TRIzol reagent (Invitrogen Carlsbad, CA) according to the manufacturer’s instructions. 50 ng of RNA were converted to cDNA using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Glyceraldehyde-3-phosphatede hydrogenase was used as housekeeping gene to account for variations in mRNA loading. PCR amplification was performed using SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. To assess the mRNA stability or half-life of eNOS mRNA, HCAECs were treated with 5 μg/ml actionmycin D with or without AGEs (100 mmol/l).
Antibodies and immunoblotting
Equal amounts of endothelial proteins (6 g) were resolved using SDS–polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membranes according to standard procedures. Immunoblot analysis was carried out using antibodies directed against β-actin (Sigma), eNOS, phospho-eNOS Ser1177 (BD Biosciences, SanJose, CA), phospho- and total extracellular signal-regulated kinase (ERK)1/2, c-Jun NH2-terminal kinase (JNK), and p38 (RD systems, Minneapolis, MN).
eNOS enzyme activity
A fuorometric cell-associated NOS detection system (Sigma) was used to measure intracellular production of nitric oxide (NO) from supplemented l-arginine by a nonradiometric method.
Nitrite detection
NO levels in HCAECs supernatants were determined by measuring the levels of nitrite and nitrate, the stable degradation products of NO (Griess reaction NO assay kit; Calbiochem). Total amount of nitrite in HCAECs was determined and normalized to total proteins of HCAECs (pmol/mg protein).
Cellular NO levels and reactive oxygen species (ROS) production assay
HCAECs were harvested and adjusted to (1 × 106/ml) cells per each FACS tube. For cellular NO staining, treated cells were incubated with 4-amino-5-methylamino-2′, 7′-difluorofluorescein diacetate (DAF, 10 μM; Molecular Probes) at 37 °C for 30 min and then washed. Flow cytometry assay was used to measure the stained cells. ROS levels were studied with dihydroethidium (DHE, 5 μM; Molecular Probes) staining and flow cytometric analysis. Samples were analyzed using FACScan and Cell Quest software (Becton–Dickinson, Franklin Lakes, NJ). Mitochondrial membrane potential was determined with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanide iodine (JC-1, MitoScreen kit; BD Biosciences) staining and flow cytometric analysis. ATP levels in HCAECs were measured with an ATPLite kit (Perkin–Elmer, Waltham, MA) per manufacturer’s instructions. HCAECs were cultured and treated as previously described on 6 well plates for 24 h. The lysis and substrate solutions were added to each well. The luminescence was measured by the TopCount Microplate Scintillation and Luminescence Counter (Perkin-Elmer, Waltham, MA).
Cellular glutathione assay
GSH-Glo Glutathione assay (Promega, Madison, WI) measures a change in the redox state of the cell due to oxidants, which are downstream metabolites of O2
−.
Measurement of NAPDH oxidase, CAT and SOD activities
NAPDH oxidase was determined by lucigenin-enhanced chemiluminescence method (Promega, Madison, WI). CAT and SOD enzyme activities were measured with commercial enzyme assay kits (Cay-man Chemical, Ann Arbor, MI) according to the manufacturer’s protocols.
BioPlex immunoassay
HCAECs were cultured with 100 μg/ml of AGEs for 0, 5, 10, 20, 30, 45, 60, or 90 min. Cell lysate was prepared. Detection of phospho- and total extracellular signal-regulated kinase (ERK) 1/2, c-Jun NH2-terminal kinase (JNK), and p38 was performed by the BioPlex Luminex system 2200 (Bio-Rad).
Statistical analysis
Data are reported as mean ± SD of at least triplicate determinations. Statistical significance (P < 0.05) was determined by paired Student’s t test (Statview, Abacus Concepts, Berkeley, CA).
Discussion
In the present study, we found that plasma AGEs level was inversely correlated with endothelial function in type 2 diabetic patients with coronary artery atherosclerosis. The findings have extended the previous observations showing that serum AGEs level was correlated with endothelial dysfunction in diabetes. Moreover, we also present evidence that AGEs are able to induce endothelial dysfunction in HCAECs. Specifically, AGEs significantly reduce eNOS expression level and NOS activity as well as NO bioavailability in HCAECs. In addition, AGEs directly induce oxidative stress and activation of p38 and ERK MAPKs in HCAECs. This study provides insight into the biological functions and molecular mechanisms of AGEs in the vascular system. Consistent with previous reports [
11], we observed increased serum level of AGEs in diabetes mellitus with coronary artery atherosclerotic stenosis.
Our previous study demonstrates that AGEs can increase the expression of its receptor RAGE on human umbilical vein endothelial cells (HUVECs) and rat vascular smooth muscle cells (VSMCs) through which AGEs could enhance its biologic functions in the vascular system. AGEs showed a decrease of eNOS mRNA, protein levels in HUVECs, and the eNOS enzyme activity was also decreased.
eNOS plays a critical role in maintaining the endothelial functions. Normal endothelial cells constitutively express eNOS, which catalyzes the production of NO from
l-arginine. NO directly mediates vasorelaxation and many other biological processes [
12]. However, many cardiovascular risk factors induce eNOS dysfunctional or decrease eNOS expression [
13]. These changes of eNOS not only impair endothelium-dependent vasorelaxation but also accelerate the atherosclerotic lesion formation [
14]. Growing evidence from preclinical and clinical studies has implicated that accumulation of advanced glycation end products, reactive oxygen species overproduction, and endoplasmic reticulum stress may cause the vascular structure changes through vascular endothelial growth factor A/phosphoinositide 3′ kinase/AKT/endothelial nitric oxide synthase and in the activation of antiangiogenic signals [
15]. Matsui et al. reported that AGEs could elicit ROS generation and inflammatory and thrombogenic reactions in HUVECs [
16]. Jo-Watanabe et al. demonstrates that age-related endothelial glycative altered phosphorylation of eNOS, and attenuated endothelial dysfunction through modulation of endothelial nitric oxide synthase phosphorylation [
17].
In the present study, we demonstrated a direct effect of AGEs on cultured HCAECs. AGEs at high plasma concentrations in diabetic individuals could repress eNOS expression and activity in a time- and concentration-dependent manner, which also confirmed by anti-RAGE antibody blocking experiment. LY-83583 (O2
− generating molecule) was also used as a positive control in this study to confirm the role of O2
− in eNOS expression. Indeed, treatment with LY-83583 led to a decrease in NO levels in HCAECs, which was similar to that seen in AGEs-treated cells. These data demonstrated that one of mechanisms of AGEs-induced eNOS down-regulation is the decrease of eNOS mRNA stability in AGEs-treated human endothelial cells.
Cardiovascular disease is a multifactorial disease. One of the major reasons is endothelial dysfunction that is characterized by a decrease in NO bioactivity, with a concomitant increase in superoxide formation, despite the observation that eNOS mRNA and protein levels are maintained or even increased. For example, endothelial function was impaired whereas eNOS protein expression was increased in response to hyperglycemia [
18,
19] or advanced age [
17,
20]. eNOS overexpression in apolipoprotein E knockout mice was reported to accelerate the development of atherosclerosis [
21]. These findings indicate that alteration of eNOS itself can be a significant cause for endothelial dysfunction, and sufficient expression of eNOS protein alone does not guarantee bioavailability of NO. Madamanchi et al. demonstrated that ROS was the key mediators for vascular inflammation and atherogenesis [
22]. Human investigations support the oxidative stress hypothesis of atherogenesis [
22,
23]. This is further supported by impaired vascular function and enhanced atherogenesis in animal models that have deficiencies in internal antioxidant enzymes [
24]. In the present study, we found that AGEs induces a significant increase of O
2
− in HCAECs, which could be one of the mechanisms for AGEs-induced eNOS dysfunction.
To explore where increased O2
− originated from in AGEs-treated cells, we verified that the decrease of mitochondrial membrane potential could cause the increase of O2
− in AGEs-treated cells. In the meantime, this result suggests that AGEs may induce mitochondrial dysfunction, which in turn, may be partially responsible for the increase in O2
− production detected in AGEs-treated cells. Interestingly, treatment with TTFA, a mitochondrial inhibitor, effectively blocked AGEs-induced the increase of O2 production and the decrease of eNOS. Clearly, treatment with AGEs substantially increases NOX activity, whereas it decreases CAT and SOD activities. This may impair the internal cellular response to oxidative stress in AGEs treated cells. Thus AGEs-increased O2
− production may result from mitochondrial dysfunction and compromised cellular redox enzymes. Similar results were obtained from the antioxidants SeMet and Specific O2
− scavenger MnTBAP, which blocked AGEs’s effects on O2
− production in AGEs-treated cells.
Beside above reason on O
2
− production, xanthine oxidase and lipoxygenases. SOD facilitate the decrease of O
2
− by converting O
2
− to of H
2O
2 [
25], and then CAT [
26] and glutathione peroxidase (GPX) [
27] coordinate the conversion of H
2O
2 to water. SeMet is the form reported to be the major component of dietary selenium, and undergoes an intramolecular transsulfuration reaction to form selenocysteine [
28]. It is able to directly interact with some oxidant molecules or oxidant-generating ions, which, in turn, increases the activity of internal antioxidant enzymes GPX and thioredoxin reductase [
29,
30]. These two antioxidants effectively blocked AGEs-induced eNOS downregulation and ROS production. Thus, our findings provide a suggestion that antioxidant therapy may be an effective strategy in treating vascular diseases related to AGEs.
Inflammatory also play an important role in the development of vascular damage associated to hyperglycemia [
31,
32]. During inflammation, inducible nitric oxide synthase activation generates an overabundance of NO in the circulation and induced the cardiovascular dysfunction. High levels of NO availability without inflammatory stress can promote insulin resistance [
33]. Nair M et al. shown that endoplasmic reticulum stress induced by glyLDL is possibly involved in eNOS downregulation [
34]. In addition, insulin plays an important role in the regulation of vascular homeostasis and maintenance of endothelial function. Insulin signaling might cause activation of two separate and parallel pathways: PI3K/AKT/eNOS and Ras/Raf/MAPK pathways [
35]. AKT phosphorylates eNOS at Ser1177, resulting in increased nitric oxide production and vasodilation. The MAPK pathway results in endothelin-1 production and vasoconstriction and mitogenic effects [
19].
MAPKs are important in regulating cell growth, migration, and differentiation in response to various extracellular stimuli [
11]. The pattern of MAPK activation in response to oxidative stress varies depending on the oxidant strength and cell type. There is increasing evidence that the engagement of AGEs by RAGE mainly recruits MAPKs including p44/42 MAPK, p38 MAPK, and Jun N-terminal kinase (JNK) and a downstream activation of the transcription factor NF-kappaB for induction of proinflammatory and procoagulatory gene expression [
36,
37]. In the study, we demonstrated that p38 and ERKl/2 were activated in response to AGEs stimulation, and that the p38 inhibitor SB-239063 and the ERKl/2 inhibitor PD-98059 effectively blocked AGEs-induced eNOS downregulation and ROS production in HCAECs.
Although different clinical studies reported different serum or plasma levels of AGEs due to different measurement procedures and conditions, human AGEs levels usually increase in patients with diabetes. Our data demonstrated that compared with healthy individuals, AGEs levels significantly increased in diabetic patients. In the current study, we have used a much higher concentration (100 or 200 μg/l) for in vitro experiments than those in human serum or plasma levels. The major reason for this choice is considering that similar concentrations of AGEs are used in the previous discoveries of AGEs functions and underlying mechanisms from in vitro experiments in endothelial cells [
38,
39].
Thus, the data gene rated from the current study is comparable with those in the previous publications. In addition, local concentrations of AGEs at the vascular lesion site may be much higher than those in patients’ serum or plasma.