The role of sirtuin 6 (SIRT6) in atherosclerotic progression of diabetic patients is unknown. We evaluated SIRT6 expression and the effect of incretin-based therapies in carotid plaques of asymptomatic diabetic and nondiabetic patients. Plaques were obtained from 52 type 2 diabetic and 30 nondiabetic patients undergoing carotid endarterectomy. Twenty-two diabetic patients were treated with drugs that work on the incretin system, GLP-1 receptor agonists, and dipeptidyl peptidase-4 inhibitors for 26 ± 8 months before undergoing the endarterectomy. Compared with nondiabetic plaques, diabetic plaques had more inflammation and oxidative stress, along with a lesser SIRT6 expression and collagen content. Compared with non-GLP-1 therapy–treated plaques, GLP-1 therapy–treated plaques presented greater SIRT6 expression and collagen content, and less inflammation and oxidative stress, indicating a more stable plaque phenotype. These results were supported by in vitro observations on endothelial progenitor cells (EPCs) and endothelial cells (ECs). Indeed, both EPCs and ECs treated with high glucose (25 mmol/L) in the presence of GLP-1 (100 nmol/L liraglutide) presented a greater SIRT6 and lower nuclear factor-κB expression compared with cells treated only with high glucose. These findings establish the involvement of SIRT6 in the inflammatory pathways of diabetic atherosclerotic lesions and suggest its possible positive modulation by incretin, the effect of which is associated with morphological and compositional characteristics of a potential stable plaque phenotype.

Cardiovascular disease represents the leading cause of death in patients with type 2 diabetes (1). Diabetes leads to increased vulnerability for plaque disruption, and mediates increased incidence and severity of clinical events (2). Inflammation, particularly in diabetes, plays a central role in the cascade of events that result in plaque erosion and fissuring (2). There is now increasing evidence that a number of transcription factors, including the Sir2 family of enzymes, namely sirtuins (SIRTs), regulate multiple genes whose products are putatively involved in the regulation of inflammation and endothelial cell (EC) function (3). The Sir2 family consists of seven enzymes (SIRT1 to SIRT7) that share a conserved core catalytic domain, but differ in their cellular localization and tissue distribution (4). Among the SIRTs, SIRT6, a chromatin-associated deacetylase, is considered to have a leading role in regulating genomic stability, cellular metabolism, stress response, and aging (58). A recent study (9) in mice suggested a role for SIRT6 in inflammation. Moreover, the knockdown of SIRT6 resulted in the increased expression of proinflammatory cytokines (interleukin [IL]-1β, IL-6, and IL-8), extracellular matrix remodeling enzymes (matrix metalloproteinase [MMP]-2, MMP-9, and plasminogen activator inhibitor 1), and intracellular adhesion molecule-1 (4). In ECs, the loss of SIRT6 was associated with an increased expression of nuclear factor-κB (NF-κB), whereas overexpression of SIRT6 was associated with decreased NF-κB transcriptional activity (4), indicating that SIRT6 may be associated with the upregulation of genes involved in inflammation, vascular remodeling, and angiogenesis. However, the role of SIRT6 in human atherosclerotic plaques has not yet been described. Although it has been demonstrated that diabetes may be implicated in the regulation of SIRT6 expression in diabetes-induced neurodegeneration (10), still no evidence exists about the potential role of SIRT6 in the evolution of atherosclerotic plaques in diabetic patients. We hypothesized that, by acting on SIRT6, diabetes may enhance the inflammatory potential of atherosclerotic plaques, favoring their instability. Thus, this study was designed to identify differences in SIRT6 expression, as well as in inflammatory infiltration, between carotid plaques of asymptomatic diabetic and nondiabetic patients. Experimental studies suggest that in obese mice, GLP-1–based therapies may reduce inflammation (1113) and enhance the protein expression of SIRT1 (14). Moreover, human studies showed that sitagliptin (15) and exenatide (16), even at a single dose, exert a potent anti-inflammatory effect, and that many of these effects were persistent over a period of 12 weeks, thus suggesting that the anti-inflammatory effects of GLP-1–based therapies could help to reduce atherogenesis.

Here, we evaluated the effect of incretin therapy in diabetic patients on SIRT6 expression in carotid plaques and early outgrown circulating endothelial progenitor cells (EPCs). Furthermore, a set of in vitro experiments in ECs and EPCs was designed to evaluate the effect of incretin on SIRT6 and NF-κB during high-glucose treatment.

Patients were recruited from the outpatient Department of Cardiology and Cardiovascular Surgery of the Cardarelli Hospital, Naples, Italy, from January 2009 to June 2013. From among these patients, we selected 52 type 2 diabetic and 30 nondiabetic patients (nondiabetic group) with asymptomatic carotid stenosis (according to North American Symptomatic Carotid Endarterectomy Trial classification), enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid artery stenosis (17). Asymptomatic patients underwent a baseline clinical examination, gave a medical history, and had never developed neurologic symptoms or cerebral lesions assessed by computed tomography. All patients underwent computed tomography scanning or MRI. Diabetes was categorized according to the criteria of the American Association of Clinical Endocrinologists and the American Diabetes Association (18). Furthermore, the diabetic patients answered a specific questionnaire about medicines used for diabetes treatment before the beginning of the study, the dates of the beginning and the end of treatment, the route of administration, and the duration of use. Information from the medicine inventory during the study and this specific questionnaire was used to classify the subjects. The patients with diabetes who never used incretin, such as GLP-1 agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, were classified as “never incretin users.” The patients with diabetes who had already used GLP-1 agonists or DPP-4 inhibitors were classified as “current incretin users.” Among the 52 diabetic patients enrolled in the study, 24 were current incretin users, and 28 were never incretin users. The current incretin users were patients who had been treated with incretin for at least 6 months. Patients treated with incretin for a period of <6 months were excluded from the study. Information on the duration of treatment was available for all current users. The mean (±SD) duration of incretin treatment was 26 ± 8 months. No patients had clinical or laboratory evidence of heart failure, previous stroke, valvular defects, malignant neoplasms, or secondary causes of hypertension. Carotid sonography was performed on a single ultrasound machine (Aloka 5500). The study was approved by the local ethics committee, and informed written consent was obtained for each patient.

Laboratory Analysis

After an overnight fast, plasma glucose, HbA1c, and serum lipid levels were measured by enzymatic assays in the hospital chemistry laboratory. GLP-1 levels (Active GLP-1 [7-36] Specific ELISA Kit; Epitope Diagnostics) were measured after an overnight fast (at 8:00 a.m.) and after breakfast. A standardized breakfast contained 419 kcal (57% carbohydrate, 17% protein, and 26% fat). After breakfast, blood samples for the measurement of GLP-1 were obtained every 30 min over a 2-h period. The mean of the four GLP-1 evaluations was defined as the postprandial GLP-1 value. The standardized meal tolerance test and baseline evaluations were performed 7 days before surgery. Thereafter, the patients were asked to self-monitor their blood glucose level (fasting, postbreakfast, postmeal, and postdinner glucose levels) until the day of surgery. Levels of fasting blood glucose were evaluated before surgery. Fasting and postprandial plasma glucose data were obtained from the average of each assessment.

Atherectomy Specimens

After surgery, the specimens were cut perpendicular to the long axis into two halves. The first half was frozen in liquid nitrogen for the following ELISA analysis. A portion of the other half of the specimen was immediately immersion fixed in 10% buffered formalin. Sections were serially cut at 5 µm, mounted on lysine-coated slides, and stained with hematoxylin-eosin and trichrome stain. Carotid artery specimens were analyzed by light microscopy.

Immunohistochemistry

After the surgical procedure, atherectomy specimens were immediately frozen in isopentane and cooled in liquid nitrogen. Similar regions of the plaque were analyzed (Fig. 1). Serial sections were incubated with the following specific antibodies: anti-SIRT6 (Millipore); anti–HLA-DR; anti-CD68 (cluster of differentiation 68 glycoprotein) and anti-CD3 (cluster of differentiation 3 T-cell coreceptor protein) (Dako), markers of T cells and macrophages, respectively; anti–MMP-9 (Santa Cruz Biotechnology), proteases involved in the degradation of the collagen content in the plaque; and anti–tumor necrosis factor-α (TNF-α) (R&D Systems). Analysis of immunohistochemistry was performed with a personal computer–based, quantitative, 24-bit color image analysis system (IM500; Leica Microsystems AG).

Figure 1

Representative example of the plaques studied. The inset is representative of the plaque region analyzed. The section was stained with hematoxylin-eosin. The main plaque section is shown at low magnification (×5), and the section analyzed is shown at high magnification (inset ×100).

Figure 1

Representative example of the plaques studied. The inset is representative of the plaque region analyzed. The section was stained with hematoxylin-eosin. The main plaque section is shown at low magnification (×5), and the section analyzed is shown at high magnification (inset ×100).

Sirius Red Staining for Collagen Content

Sections were stained as previously described (19). After dehydration, the sections were observed under polarized light after being placed on coverslips. The sections were photographed with identical exposure settings for each section.

Biochemical Assays

Plaques were lysed and centrifuged for 10 min at 10,000g at 4°C. After centrifugation, 20 µg of each sample was loaded, electrophoresed in polyacrylamide gel, and electroblotted onto a nitrocellulose membrane. Each determination was repeated at least three times.

MMP-9, TNF-α, and nitrotyrosine levels were quantified in plaques using specific ELISA kits (from Santa Cruz Biotechnology, R&D Systems, and Imgenex). SIRT6 activity was measured using a fluorimetric SIRT6 assay kit (Abcam, Cambridge, U.K.). NF-κB binding to κB sites was assessed on nuclear extracts from plaque specimens (19) by the TransAM NF-κB p65 Transcription Factor Assay Kit (Active Motif Europe, Rixensart, Belgium).

Isolation and Culture of EPCs

Early outgrown EPCs were isolated from the leukocyte-rich buffy coat of human healthy volunteers, and peripheral blood from diabetic patients (n = 5 current incretin users and n = 5 never incretin users) and nondiabetic patients (n = 10) was isolated and characterized as previously described (20,21).

In Vitro Cell Treatments

ECs, human aortic ECs, were from Lonza (Cologne, Germany and Walkersville, MD). Cells were cultured with an EGM-2MV BulletKit (Lonza) supplemented with 5% FBS, 0.6% HEPES, maintained at 37°C in a 5% CO2 humidified atmosphere, and used in experiments between passage 4 and 7. EPCs (5 × 106 cells/mL medium) and ECs (cultured at 70–80% confluence in six-well plates [Costar, Corning, NY]) were subjected to short-term exposure to high glucose (25 mmol/L) in the presence or absence of a GLP-1 receptor agonist (exenatide [Byetta]) (100 nmol/L) in complete culture media for 3 days. GLP-1 receptor agonists were added 30 min before starting the high-glucose treatment and were left in the culture media throughout the high-glucose treatment. Control cells were cultured for 3 days under basal conditions.

Confocal Laser-Scanning Microscopy

Immunofluorescence detection of SIRT6 in deparaffinized atherosclerotic plaque sections from diabetic and nondiabetic patients and in in vitro–cultured EPCs and ECs was performed by confocal laser-scanning microscope analysis (LSM 510; Zeiss) by using specific antibodies against SIRT6 (1:500) (Cell Signaling Technology, Danvers, MA), vimentin (1:1,000) (Sigma-Aldrich, St. Louis, MO), or antibodies against von Willebrand factor (1:500) (Abcam), as previously described (20,21). Secondary antibodies were conjugated to Alexa Fluor 633 (1:1,000) or Alexa Fluor 488 (1:1,000) (Life Technologies Italia, Monza, Italy) (20,21).

Western Blot Analysis

Western blot analysis of atherosclerotic plaque sections, EPCs, or EC total protein extracts was performed, as previously described (21), using antibodies against SIRT6 (Cell Signaling Technology), NF-κB (Cell Signaling Technology), and γ-tubulin protein (GTU-88) (Sigma-Aldrich). As for plaque section homogenization, 800 μL of 2D lysis buffer (7 mol/L urea, 2 mol/L thiourea, 4% CHAPS [3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate] buffer, 30 mmol/L Tris-HCl, pH 8.8), were added to tissues (400 mg) cut into small pieces. Tissue homogenized with a Precellys 24 system (Bertin Technologies, Montigny-le-Bretonneux, France) was centrifuged at 800g for 10 min at 4°C to collect the supernatant. Proteins were then precipitated by adding 100% cold methanol.

Statistical Analysis

Data are presented as mean ± SD. Continuous variables were compared among the groups of patients with one-way ANOVA for normally distributed data and Kruskal-Wallis test for non–normally distributed data. When differences among the groups were found, Bonferroni correction to make pairwise comparisons was used. P < 0.05 was considered to be statistically significant. All calculations were performed using SPSS 12.

Demographic data for the study population are presented in Table 1 and Supplementary Table 1. The percentage of carotid diameter reduction, risk factors, and concomitant nonhypoglycemic therapy did not differ among the groups (Table 1 and Supplementary Table 1). In diabetic patients, mean, fasting, and postprandial plasma glucose levels at baseline as well as during the week before the surgery, HOMA-IR, and HbA1c levels did not differ among never incretin users and current incretin users (Table 1 and Supplementary Table 1). However, basal and postprandial GLP-1 levels were higher in current incretin users compared with never incretin users (P < 0.01) (Table 1).

Table 1

Characteristics of study patients

Control subjects
(n = 30)Never incretin users
(n = 28)Current incretin users
(n = 24)
Age (years) 71 ± 4 69 ± 7 70 ± 5 
Sex    
 Female 17 17 15 
 Male 13 11 
Patient characteristics    
 Family history of IHD 14 (47) 17 (60) 13 (54) 
 Hypertension 12 (40) 13 (46) 12 (50) 
 Hypercholesterolemia 12 (40) 13 (46) 10 (41.6) 
 Coronary artery disease 16 (53) 17 (61) 15 (63) 
 BMI (kg/m227.3 ± 3 29.8 ± 4 28.7 ± 2 
 HbA1c (%) 4.8 ± 1.0* 8.0 ± 1.4 7.9 ± 1.3 
 Blood glucose (mmol/L) 6 ± 0.6* 9.5 ± 1.4 9.4 ± 1.6 
 Insulin (µU/mL) 8.09 ± 3.6* 10.9 ± 2.9 10.4 ± 2.7 
 Basal GLP-1 (pmol/L) 7.9 ± 1.1* 5.1 ± 1.1 6.6 ± 1.4* 
 Postprandial GLP-1 (pmol/L) 22.6 ± 3.2* 11.4 ± 2.8 20.1 ± 2.8* 
 HOMA-IR 2.14 ± 0.8* 4.60 ± 1.5 4.59 ± 1.4 
 CRP (mg/dL) 0.84 ± 0.07* 1.20 ± 0.07 1.24 ± 0.09 
 Total cholesterol (mmol/L) 5.66 ± 0.08 5.68 ± 0.10 5.62 ± 0.05 
 HDL cholesterol (mmol/L) 1.24 ± 0.11 1.20 ± 0.09 1.21 ± 0.09 
 LDL cholesterol (mmol/L) 3.58 ± 0.10 3.59 ± 0.11 3.49 ± 0.09 
 Triglycerides (mmol/L) 1.84 ± 0.36 1.98 ± 0.31 1.97 ± 0.39 
Plaque characteristics    
 Stenosis severity (%) 77.1 ± 6.6 79.2 ± 5.9 78.1 ± 6.4 
 Macrophage-rich areas (%) 6 ± 2* 24 ± 4 17 ± 3* 
 HLA-DR–rich areas (%) 8 ± 2* 28 ± 9 14 ± 8* 
 T cells/mm2 section area 19.7 ± 9.3* 79.1 ± 16.2 28.9 ± 11.7* 
 Collagen content (%) 28.2 ± 3.8* 9.7 ± 3.9 19.1 ± 3.2* 
 p50 (ng/mg) 20.6 ± 11.2* 34.8 ± 15.4* 71.1 ± 16.4 
 p65 (ng/mg) 13.5 ± 7.7* 31.3 ± 15.1* 59.9 ± 16.3 
 MMP-9 (μg/mg) 4.3 ± 2.5* 17.6 ± 2.8 8.8 ± 2.1* 
 Nitrotyrosine (nmol/pg) 1.3 ± 0.7* 6.1 ± 0.9 3.5 ± 1.2* 
 TNF-α (pg/mg) 23.9 ± 4.5* 94.6 ± 6.5 61.4 ± 7.3* 
Incretin therapy    
 GLP-1 agonists   4 (17) 
  Exenatide   3 (12) 
  Liraglutide   1 (4) 
 DPP-4 inhibitors   20 (83) 
Control subjects
(n = 30)Never incretin users
(n = 28)Current incretin users
(n = 24)
Age (years) 71 ± 4 69 ± 7 70 ± 5 
Sex    
 Female 17 17 15 
 Male 13 11 
Patient characteristics    
 Family history of IHD 14 (47) 17 (60) 13 (54) 
 Hypertension 12 (40) 13 (46) 12 (50) 
 Hypercholesterolemia 12 (40) 13 (46) 10 (41.6) 
 Coronary artery disease 16 (53) 17 (61) 15 (63) 
 BMI (kg/m227.3 ± 3 29.8 ± 4 28.7 ± 2 
 HbA1c (%) 4.8 ± 1.0* 8.0 ± 1.4 7.9 ± 1.3 
 Blood glucose (mmol/L) 6 ± 0.6* 9.5 ± 1.4 9.4 ± 1.6 
 Insulin (µU/mL) 8.09 ± 3.6* 10.9 ± 2.9 10.4 ± 2.7 
 Basal GLP-1 (pmol/L) 7.9 ± 1.1* 5.1 ± 1.1 6.6 ± 1.4* 
 Postprandial GLP-1 (pmol/L) 22.6 ± 3.2* 11.4 ± 2.8 20.1 ± 2.8* 
 HOMA-IR 2.14 ± 0.8* 4.60 ± 1.5 4.59 ± 1.4 
 CRP (mg/dL) 0.84 ± 0.07* 1.20 ± 0.07 1.24 ± 0.09 
 Total cholesterol (mmol/L) 5.66 ± 0.08 5.68 ± 0.10 5.62 ± 0.05 
 HDL cholesterol (mmol/L) 1.24 ± 0.11 1.20 ± 0.09 1.21 ± 0.09 
 LDL cholesterol (mmol/L) 3.58 ± 0.10 3.59 ± 0.11 3.49 ± 0.09 
 Triglycerides (mmol/L) 1.84 ± 0.36 1.98 ± 0.31 1.97 ± 0.39 
Plaque characteristics    
 Stenosis severity (%) 77.1 ± 6.6 79.2 ± 5.9 78.1 ± 6.4 
 Macrophage-rich areas (%) 6 ± 2* 24 ± 4 17 ± 3* 
 HLA-DR–rich areas (%) 8 ± 2* 28 ± 9 14 ± 8* 
 T cells/mm2 section area 19.7 ± 9.3* 79.1 ± 16.2 28.9 ± 11.7* 
 Collagen content (%) 28.2 ± 3.8* 9.7 ± 3.9 19.1 ± 3.2* 
 p50 (ng/mg) 20.6 ± 11.2* 34.8 ± 15.4* 71.1 ± 16.4 
 p65 (ng/mg) 13.5 ± 7.7* 31.3 ± 15.1* 59.9 ± 16.3 
 MMP-9 (μg/mg) 4.3 ± 2.5* 17.6 ± 2.8 8.8 ± 2.1* 
 Nitrotyrosine (nmol/pg) 1.3 ± 0.7* 6.1 ± 0.9 3.5 ± 1.2* 
 TNF-α (pg/mg) 23.9 ± 4.5* 94.6 ± 6.5 61.4 ± 7.3* 
Incretin therapy    
 GLP-1 agonists   4 (17) 
  Exenatide   3 (12) 
  Liraglutide   1 (4) 
 DPP-4 inhibitors   20 (83) 

Data are presented as mean ± SD or n (%). CRP, C-reactive protein; IHD, ischemic heart disease; IR, insulin resistance.

*P < 0.05 compared with never users group.

P < 0.05 compared with current users group.

Plaque Composition

Compared with nondiabetic patients, diabetic patients (n = 52) had a significantly greater portion of plaque area occupied by macrophages and T cells, as well as greater expression of HLA-DR antigen (Table 1 and Fig. 2). Compared with the never incretin users group, the current incretin users group presented with a significantly smaller portion of plaque area occupied by macrophages (P < 0.01) and T cells (P < 0.01), as well as lower expression of HLA-DR (P < 0.01) (Table 1 and Fig. 2). Both immunohistochemistry and ELISA revealed markedly higher staining and levels of TNF-α in all diabetic versus nondiabetic lesions (P < 0.001). In diabetic patients, staining and levels of TNF-α were significantly more abundant in lesions from never incretin users than in lesions from current incretin users (P < 0.001) (Table 1 and Fig. 2). Moreover, TNF-α levels were inversely correlated with GLP-1 levels (r = −0.67, P < 0.001). MMP-9 levels were more abundant in diabetic than in nondiabetic lesions (P < 0.001). Specifically, in diabetic patients, MMP-9 levels were more abundant in lesions from never incretin users than in lesions from current incretin users (P < 0.001). As for the content of interstitial collagen, a lower amount of interstitial collagen was found in plaques of all diabetic patients compared with nondiabetic patients (P < 0.001). The amount of interstitial collagen in plaques from never incretin users was lower than in lesions from current incretin users (P < 0.001) (Table 1 and Fig. 3). Higher nitrotyrosine levels were found in diabetic plaques than in nondiabetic plaques (P < 0.001). Among diabetic plaques, nitrotyrosine levels were significantly higher in never incretin users than in current incretin users (P < 0.01) (Table 1 and Fig. 3).

Figure 2

Inflammation in atherosclerotic plaques. Immunochemistry for macrophages (CD68) (×400), lymphocytes (CD3) (×400), inflammatory cells (HLA-DR) (×400), and TNF-α (×400) in asymptomatic plaques of a control subject, a never incretin user, and a current incretin user. Similar regions of plaque are shown. These results are typical of the asymptomatic plaques of control subjects, never incretin users, and current incretin users.

Figure 2

Inflammation in atherosclerotic plaques. Immunochemistry for macrophages (CD68) (×400), lymphocytes (CD3) (×400), inflammatory cells (HLA-DR) (×400), and TNF-α (×400) in asymptomatic plaques of a control subject, a never incretin user, and a current incretin user. Similar regions of plaque are shown. These results are typical of the asymptomatic plaques of control subjects, never incretin users, and current incretin users.

Figure 3

Atherosclerotic plaque phenotypes. Immunochemistry for MMP-9 (×400), and sirius red staining for collagen (×400), nitrotyrosine (×400), and SIRT6 (×400) content in control subjects, never incretin users, and current incretin users with asymptomatic plaques. Similar regions of plaque are shown. These results are typical of control subjects, never incretin users, and current incretin users with asymptomatic plaques.

Figure 3

Atherosclerotic plaque phenotypes. Immunochemistry for MMP-9 (×400), and sirius red staining for collagen (×400), nitrotyrosine (×400), and SIRT6 (×400) content in control subjects, never incretin users, and current incretin users with asymptomatic plaques. Similar regions of plaque are shown. These results are typical of control subjects, never incretin users, and current incretin users with asymptomatic plaques.

Interestingly, both immunohistochemistry (Fig. 3) and confocal laser-scanning microscopy analyses (Fig. 4A and B) revealed that levels of SIRT6 are consistently lower in plaques from diabetic patients compared with plaques from nondiabetic patients and, specifically, in plaques from diabetic never incretin users (P < 0.01). Indeed, levels of SIRT6 in plaques from diabetic current incretin users were significantly higher than those observed in plaques from never incretin users (P < 0.05), and were near the values observed in nondiabetic patients (Figs. 3 and 4A and B). In addition, in order to define the cellular type expressing SIRT6 within diabetic and nondiabetic plaques, sections were incubated with antibodies against SIRT6 and von Willebrand factor, a specific EC marker. Results showed the coexpression of SIRT6 and von Willebrand factor, thus suggesting that SIRT6 is expressed by ECs (Fig. 4B). Consistent with immunofluorescence findings, Western blot analysis of SIRT6 protein levels in atherosclerotic plaques from nondiabetic and diabetic patients (current and never incretin users) showed a similar trend (Fig. 4C). Moreover, SIRT6 expression levels in plaques were inversely correlated with GLP-1 levels (r = −0.58, P < 0.001). NF-κB activation, as reflected by the selective analysis of the activated form of both p50 and p65, was significantly higher in plaques from both current incretin users (P < 0.01) and never incretin users (P < 0.01) than in those of nondiabetic patients. In plaques from diabetic patients, the NF-κB level was significantly higher in never incretin users than in current incretin users (Table 1).

Figure 4

Detection of SIRT6 expression in atherosclerotic plaques. Immunofluorescence detection of SIRT6 was performed on deparaffinized atherosclerotic plaque sections from diabetic and nondiabetic patients. A: Bar graph of SIRT6 arbitrary fluorescence units (AFU) determined by confocal laser-scanning microscopy. B: Representative confocal images of SIRT6 (red) and von Willebrand factor (green). C: Representative bar graph of SIRT6 protein levels determined by Western blot analysis of atherosclerotic plaque homogenates from nondiabetic patients (P1 and P2), diabetic never incretin user patients (P3 and P4), and diabetic current incretin user patients (P5 and P6). Inset, representative image of Western blot analysis. Lanes 1 and 2, nondiabetic patients (P1 and P2). Lanes 3 and 4, diabetic never incretin user patients (P3 and P4). Lanes 5 and 6, diabetic current incretin user patients (P5 and P6). Data are mean ± SD. *P < 0.01 vs. nondiabetic patients; §P < 0.05 vs. never incretin users.

Figure 4

Detection of SIRT6 expression in atherosclerotic plaques. Immunofluorescence detection of SIRT6 was performed on deparaffinized atherosclerotic plaque sections from diabetic and nondiabetic patients. A: Bar graph of SIRT6 arbitrary fluorescence units (AFU) determined by confocal laser-scanning microscopy. B: Representative confocal images of SIRT6 (red) and von Willebrand factor (green). C: Representative bar graph of SIRT6 protein levels determined by Western blot analysis of atherosclerotic plaque homogenates from nondiabetic patients (P1 and P2), diabetic never incretin user patients (P3 and P4), and diabetic current incretin user patients (P5 and P6). Inset, representative image of Western blot analysis. Lanes 1 and 2, nondiabetic patients (P1 and P2). Lanes 3 and 4, diabetic never incretin user patients (P3 and P4). Lanes 5 and 6, diabetic current incretin user patients (P5 and P6). Data are mean ± SD. *P < 0.01 vs. nondiabetic patients; §P < 0.05 vs. never incretin users.

SIRT6 Protein Levels in EPCs From Diabetic and Nondiabetic Patients

It is already known from experimental and clinical studies (22) that atherosclerosis is associated with a reduced number and dysfunction of EPCs. Here, for a better understanding of the molecular mechanisms underlying EPC impairment in atherosclerosis, we looked at the possible involvement of SIRT6 by evaluating its expression levels in EPCs isolated from the peripheral blood of 10 nondiabetic patients and 10 diabetic patients (5 current incretin users and 5 never incretin users). Western blot analysis revealed that EPCs from diabetic never incretin users had lower values of SIRT6 protein arbitrary units and that diabetic current incretin users had levels of SIRT6 higher than those observed in EPCs from never incretin users (P < 0.05) (Fig. 5).

Figure 5

SIRT6 expression in EPCs from diabetic and nondiabetic patients. Western blot analysis of SIRT6 protein levels on EPCs from nondiabetic and diabetic patients (never incretin users and current incretin users) was performed as described in the 2research design and methods section. Representative bar graph and image of the Western blot analysis of SIRT6 expression in EPCs from nondiabetic patients (P1, lane 1), never incretin users (P2, lane 2), and current incretin users (P3, lane 3). Data are expressed as mean ± SD arbitrary units. *P < 0.05 vs. nondiabetic patients; §P < 0.05 vs. never incretin users.

Figure 5

SIRT6 expression in EPCs from diabetic and nondiabetic patients. Western blot analysis of SIRT6 protein levels on EPCs from nondiabetic and diabetic patients (never incretin users and current incretin users) was performed as described in the 2research design and methods section. Representative bar graph and image of the Western blot analysis of SIRT6 expression in EPCs from nondiabetic patients (P1, lane 1), never incretin users (P2, lane 2), and current incretin users (P3, lane 3). Data are expressed as mean ± SD arbitrary units. *P < 0.05 vs. nondiabetic patients; §P < 0.05 vs. never incretin users.

In Vitro Effect of High Glucose and GLP-1 Receptor Agonist on SIRT6 and NF-κB Expression in EPCs and ECs

In light of the observational data that diabetic never incretin users showed decreased levels of SIRT6 protein in either atherosclerotic plaque sections or EPCs, and that incretin therapies seem to prevent SIRT6 downregulation (Figs. 3, 4, and 5), we next evaluated whether the detrimental effect of high-glucose concentration on EPCs and ECs (20) is exerted via a SIRT6/NF-κB pathway. Western blot and confocal laser-scanning microscopy analysis revealed that short-term exposure of early EPCs (Fig. 6) and ECs (Fig. 7) to high-glucose concentrations induces the downregulation of SIRT6 protein (P < 0.01) with a concomitant upregulation of NF-κB protein expression (P < 0.01) (Figs. 6 and 7).

Figure 6

Effect of short-term exposure to high glucose and GLP-1 on SIRT6 and NF-κB protein levels in EPCs. Peripheral blood mononuclear cells (5 × 106 cells/mL in medium), isolated from the leukocyte-rich buffy coat of healthy donor subjects, were subjected to short-term exposure to high glucose (25 mmol/L) in the presence or absence of the GLP-1 receptor agonist exenatide (100 nmol/L) for 3 days. Representative bar graph and image of Western blot analysis of SIRT6 (A and B) and NF-κB (A and C) protein levels in control (Ctr), high glucose–treated cells (hGluc), GLP-1–treated cells (GLP-1), and cells treated with high glucose in the presence of GLP-1 (hGluc + GLP-1). D: Representative confocal images of SIRT6 (red) and vimentin (green). E: Bar graph of SIRT6 fluorescence intensity (in arbitrary fluorescence units [AFU]) determined by confocal laser-scanning microscopy. Data are mean ± SD (n = 6). *P < 0.01 vs. Ctr; §P < 0.05 vs. hGluc; ‡P < 0.01 vs. hGluc.

Figure 6

Effect of short-term exposure to high glucose and GLP-1 on SIRT6 and NF-κB protein levels in EPCs. Peripheral blood mononuclear cells (5 × 106 cells/mL in medium), isolated from the leukocyte-rich buffy coat of healthy donor subjects, were subjected to short-term exposure to high glucose (25 mmol/L) in the presence or absence of the GLP-1 receptor agonist exenatide (100 nmol/L) for 3 days. Representative bar graph and image of Western blot analysis of SIRT6 (A and B) and NF-κB (A and C) protein levels in control (Ctr), high glucose–treated cells (hGluc), GLP-1–treated cells (GLP-1), and cells treated with high glucose in the presence of GLP-1 (hGluc + GLP-1). D: Representative confocal images of SIRT6 (red) and vimentin (green). E: Bar graph of SIRT6 fluorescence intensity (in arbitrary fluorescence units [AFU]) determined by confocal laser-scanning microscopy. Data are mean ± SD (n = 6). *P < 0.01 vs. Ctr; §P < 0.05 vs. hGluc; ‡P < 0.01 vs. hGluc.

Figure 7

Effect of short-term exposure to high glucose and GLP-1 on SIRT6 and NF-κB protein levels in ECs. ECs were seeded in six-well plates at 70–80% confluence 12 h before treatments with high glucose (25 mmol/L) in the presence or absence of the GLP-1 receptor agonist exenatide (100 nmol/L). ECs were cultured for 3 days in complete media alone (control [Ctr]), with high glucose (hGluc), GLP-1, or high glucose in the presence of GLP-1 (hGluc + GLP-1). Representative bar graph and image of Western blot analysis of SIRT6 (A and B) and NF-κB (A and C) protein levels. D: Representative confocal images of SIRT6 (red) and vimentin (green). E: Bar graph of SIRT6 fluorescence intensity (in arbitrary fluorescence units [AFU]). Data are mean ± SD (n = 6). *P < 0.05 vs. Ctr; **P < 0.01 vs. Ctr; §P < 0.05 vs. hGluc; ‡P < 0.01 vs. hGluc.

Figure 7

Effect of short-term exposure to high glucose and GLP-1 on SIRT6 and NF-κB protein levels in ECs. ECs were seeded in six-well plates at 70–80% confluence 12 h before treatments with high glucose (25 mmol/L) in the presence or absence of the GLP-1 receptor agonist exenatide (100 nmol/L). ECs were cultured for 3 days in complete media alone (control [Ctr]), with high glucose (hGluc), GLP-1, or high glucose in the presence of GLP-1 (hGluc + GLP-1). Representative bar graph and image of Western blot analysis of SIRT6 (A and B) and NF-κB (A and C) protein levels. D: Representative confocal images of SIRT6 (red) and vimentin (green). E: Bar graph of SIRT6 fluorescence intensity (in arbitrary fluorescence units [AFU]). Data are mean ± SD (n = 6). *P < 0.05 vs. Ctr; **P < 0.01 vs. Ctr; §P < 0.05 vs. hGluc; ‡P < 0.01 vs. hGluc.

The dose-dependent response (from 1 to 1,000 nmol/L) effect of GLP-1 on SIRT6 activity during short-term exposure to high-glucose levels showed a significant effect starting at a concentration of 100 nmol/L (810 ± 51 vs. 398 ± 25 relative fluorescence units in high glucose–treated cells) (P < 0.05) and with no further increase at 1,000 nmol/L. GLP-1 alone (from 1 to 1,000 nmol/L) showed no significant effect on SIRT6 activity. Thus, a 100 nmol/L concentration was used to test the in vitro effect of GLP-1 on SIRT6 and NF-κB protein levels in EPCs and ECs exposed to high glucose. Notably, in EPCs, GLP-1 receptor agonist (100 nmol/L) significantly counteracted the effect of high-glucose concentrations on both SIRT6 and NF-κB protein levels (P < 0.05) (Fig. 6A–C).

Similar results were obtained when ECs were subjected to high-glucose treatment in the presence or absence of GLP-1 (100 nmol/L) (Fig. 7A–C). Indeed, the presence of GLP-1 during high-glucose treatment prevented SIRT6 downregulation and NF-κB upregulation (P < 0.05) (Fig. 7A–C). According to Western blot analysis, measurements of the SIRT6 fluorescence intensity units (Figs. 6D and E, and 7D and E) showed similar results.

This study provides novel insights into the relationship between the SIRT6 pathway and the inflammatory process of atherosclerotic plaques of type 2 diabetic patients. In particular, SIRT6 protein expression was downregulated in diabetic atherosclerotic lesions, compared with nondiabetic lesions, and the impaired SIRT6 expression was associated with higher oxidative stress and higher NF-κB, proinflammatory cytokine, and MMP-9 levels along with less interstitial collagen content. On the whole, all these factors might increase the risk of future acute ischemic events precipitated by inflammation-dependent rupture of atherosclerotic plaques. Moreover, we provide evidence that in diabetic patients the drugs that work on the incretin system, such as GLP-1 receptor agonists and DPP-4 inhibitors, may prevent plaque progression to an unstable phenotype and the downregulation of SIRT6 expression.

A previous study (9) in mice has suggested a role for SIRT6 in inflammation, and a recent experimental study (4) on ECs has shown that the loss of SIRT6 is associated with the upregulation of genes involved in inflammation, vascular remodeling, and angiogenesis. To date, there has been no evidence concerning the possible role of SIRT6 in subgroups of high-risk plaques, such as those found in diabetic patients, and about the specific pathways transducing environmental stimuli in the modulation of SIRT6 levels in atherosclerotic plaques. In our study, the novelty is represented by the evidence that EC-associated SIRT6 expression is markedly downregulated, and is associated with more macrophages, T cells, and HLA-DR+ inflammatory cells in the diabetic plaques compared with nondiabetic plaques. These results suggest that the presence of an active inflammatory reaction in diabetic plaques may be associated with a lower production of SIRT6 in ECs. In line with such evidence, lower expression of SIRT6 and concomitantly higher levels of oxidative stress (nitrotyrosine levels) and inflammatory cytokines were found in plaques obtained from the asymptomatic patients with type 2 diabetes compared with nondiabetic patients. In agreement with the difference in the SIRT6 staining pattern, the histological milieu of the lesions appears different with regard to cellularity, but not in the degree of vessel stenosis, thus suggesting that diabetic and nondiabetic lesions are different only with regard to SIRT6 protein expression and inflammatory burden. These data are consistent with our previous findings that the inflammatory response, as well as the oxidative stress level, were higher in diabetic than in nondiabetic plaques (23). Therefore, in this study, the observed decreased expression of SIRT6 in carotid plaques of diabetic patients might be related to the expansion of oxidative and inflammatory processes, thinning of the fibrous cap, and plaque instability. These results are in agreement with studies showing that in ECs the loss of SIRT6 is paralleled by the increased expression of the proinflammatory transcription factor NF-κB, and, conversely, the overexpression of SIRT6 is associated with a downregulation of NF-κB activity as well as the expression of its target genes (4). These findings suggest that SIRT6, a critical enzyme in the maintenance of genomic stability, could also play a key role in reducing oxidative stress and cellular damage associated with plaque instability by acting in the pathways controlling for vascular oxidative stress and inflammation (24). Collectively, this suggests that SIRT6 is a negative regulator of vascular oxidative stress and inflammation. In addition, our observational results on the negative modulation of SIRT6 levels in EPCs from diabetic patients open a new scenario in the signaling pathways underlying the EPC functional impairment during atherosclerosis and diabetes (20,22). However, although our findings suggest that diabetes determines an increase of oxidative stress, NF-κB activation, and SIRT6 expression, the mechanism by which SIRT6 acts in the regulation of the type 2 diabetic plaque phenotype is unknown.

In this context, previous reports (14) evidenced the involvement of the incretin system in the regulation of the SIRTs, particularly under conditions of metabolic disease such as in diabetic and high-fat diet–induced obese mice. Incretin system deregulation plays a pivotal role in the pathogenesis of type 2 diabetes, and patients with type 2 diabetes show a significant reduction in meal-stimulated levels of GLP-1 (25). However, no evidence exists on the potential role of GLP-1 in the regulation of SIRT6 in atherosclerotic plaques of diabetic patients. In this study, we observed that plaque SIRT6 expression levels were inversely correlated with GLP-1 levels. SIRT6 antagonizes NF-κB–induced gene expression programs by associating with chromatin-bound NF-κB, directing deacetylation of histone H3 lysine 9 (H3K9), and destabilizing the binding of NF-κB to chromatin (26). Thus, we hypothesized that the decreased expression of SIRT6 in plaques, as a consequence of GLP-1 reduction, may enhance NF-κB activity, and that this could represent a crucial step in the pathophysiology of diabetic plaque instability. In this context, our data suggest the possibility of a novel pathway to be unveiled through which the incretin system impairment, by reducing SIRT6 expression, could mediate inflammatory activity in diabetic atherosclerotic plaques. Thus, the modulation of SIRT6 through the incretin system could be beneficial for many inflammatory diseases associated with endothelial dysfunction (27) and could play a pivotal role in the stabilization of diabetic atherosclerotic plaques. In this context, the present findings also show a stimulatory effect of the drugs that work on the incretin system, such as GLP-1 receptor agonists and DPP-4 inhibitors, in the SIRT6 pathway in diabetic lesions. Indeed, at the same blood glucose levels, diabetic patients treated with both GLP-1 receptor agonists and DPP-4 inhibitors had the lowest levels of plaque inflammatory cells, cytokines, oxidative stress, and MMP-9 associated with the highest expression of SIRT6 and amount of plaque interstitial collagen. Thus, patients assigned to incretin-based therapy had less plaque progression to an unstable phenotype than patients treated without incretin-based therapy. In particular, the increased SIRT6 expression observed in diabetic plaques from the current incretin users suggests a low inflammatory activity linked to decreased NF-κB activation. This hypothesis is supported by the results of in vitro experiments on EPCs and ECs showing that the loss of SIRT6 during short-term exposure with high glucose is paralleled by the increased expression of NF-κB, whereas overexpression of SIRT6 in the presence of cotreatment with GLP-1 relates to a decreased NF-κB expression. In the process of vascular inflammation, EPCs and activated ECs are critically involved in the formation of new blood vessels, which plays an important role in several pathologies, including atherosclerosis and diabetes (22). A strong correlation between the number of circulating EPCs and the combined Framingham risk factor score for atherosclerosis exists, suggesting that EPCs can be used as a predictive biomarker for cardiovascular risk and vascular function (22). Our data, according to reports showing that the loss of SIRT6 in ECs is associated with increased expression of intracellular adhesion molecule-1, NF-κB level, and senescence (4), shed light on the role of SIRT6 as the regulator of endothelial function during altered glucose homeostasis within a pathway that links inflammation, metabolic diseases, and atherosclerosis.

As a whole, our data might have strong clinical implications because in a large series of carotid endarterectomy specimens it has been shown that plaque inflammation is one of the major determinants of ischemic events in patients affected by carotid atherosclerotic disease (28). Therefore, the direct anti-inflammatory effects of GLP-1 analogs and DPP-4 inhibitors, which increase plasma concentrations of GLP-1, and go above and beyond glycemic control, should also be considered. In addition to their metabolic actions, various beneficial cardiovascular effects have been described for GLP-1–based therapies (29). It has previously been reported (11) that GLP-1 reduces in vitro monocyte migration, while the DPP-4 inhibitor sitagliptin reduced plaque inflammation and enhanced plaque stability in ApoE−/− mice. Similar effects of DPP-4 inhibitors on plaque inflammation were observed (30). Finally, sitagliptin, vildagliptin (15,31), and exenatide (16), a GLP-1 agonist, have recently been shown to exert a rapid and significant anti-inflammatory effect that is evident within 2 h of administration of the first dose of each of these drugs. Such results support the hypothesis that the anti-atherosclerotic effect we observed may also be due to a direct anti-inflammatory effect that is independent of the effect on glycemic control. However, the full mechanism by which GLP-1 acts on SIRT6, which was not uncovered by this study, requires further investigation. Indeed, we did not identify the complete mechanism by which GLP-1 can regulate the inflammatory status of atherosclerotic plaque through the modulation of SIRT6 levels. This study highlights the relevance of modulating SIRT6/NF-κB signaling with incretin-based therapy to reduce the inflammatory burden of diabetic plaques. At the moment, we do not exclude the possibility that GLP-1 could inhibit NF-κB activation through additional mechanisms independently of SIRT6. Indeed, it has previously been reported (32) that exendin-4, a GLP-1 receptor agonist, attenuates atherosclerosis through cAMP-dependent protein kinase-phosphatidylinositol 3-kinase/Akt-endothelial nitric oxide synthase-p38 mitogen-activated protein kinase-Jun NH2-terminal kinase–dependent pathways via a GLP-1 receptor–dependent mechanism, without affecting metabolic parameters. Furthermore, recent evidence has shown a promising role for DPP-4 inhibitors in the attenuation of atherosclerosis involving vascular dysfunction and endothelial inflammation by upregulating the Akt/endothelial nitric oxide synthase signaling pathway and suppressing the activation of extracellular signal–related kinase 1/2 in vascular tissues (33). However, despite the fact that incretin-mimetic drugs have shown favorable effects on pathogenic mechanisms of atherosclerosis, the recently published large outcome trials (34,35) have not shown any superiority of treatment with a DPP-4 inhibitor (alogliptin or saxagliptin) for the outcomes of a composite of atherosclerosis-related events. However, it should be pointed out that these trials were mandated by the U.S. Food and Drug Administration simply to rule out cardiovascular harm. Furthermore, it should be noted that in both studies patients were randomized to receive DPP-4 inhibitors or placebo on top of standard therapy, usually metformin (alone or in combination with other agents), which was taken by about two-thirds of the patients. Moreover, almost half of the patients were taking sulfonylureas, which may be associated with increased mortality (36). Finally, both studies were conducted in patients with cardiovascular disease, and, therefore, the assessment of the prevention of events with incretin therapy may not be accurate. However, the effects of incretin-mimetic drugs on cardiovascular events and mortality cannot be considered to be established unless they are evaluated in long-term cardiovascular outcomes trials in patients without cardiovascular diseases.

In conclusion, we demonstrated that GLP-1 reduced plaque macrophage infiltration and MMP-9 expression, which resulted in increased plaque collagen content and a thickened fibrous cap in diabetic plaques from humans. As these plaque characteristics are features of plaque stability, the results of this study suggest that GLP-1 reduces plaque vulnerability. These findings may be of clinical importance for patients with type 2 diabetes, who are known to exhibit a higher burden of inflamed, rupture-prone atherosclerotic plaques in comparison with nondiabetic subjects (37). Overall, it is tempting to speculate that the use of activators of SIRT6 might be an effective strategy for treating inflammatory vascular diseases such as diabetes and atherosclerosis. Future studies are needed to determine whether the beneficial effects of GLP-1 receptor agonists and DPP-4 inhibitors on features of plaque vulnerability through the SIRT6 pathway translate into a reduction of cardiovascular events in patients with type 2 diabetes treated with GLP-1–based regimens. Moreover, this study also raises an important question about whether asymptomatic diabetic patients with >70% carotid artery stenosis who are receiving GLP-1–based therapies really need to undergo carotid endarterectomy, because they are likely to have stable plaques. Prospectively randomized, large-scale clinical trials are required to further clarify this relationship. Finally, although the numbers of incretin users are small, it would be of interest to know whether administration of GLP-1 agonists showed more SIRT6 expression compared with administration of DPP-4 inhibitors. Moreover, further investigations are required to determine whether GLP-1 agonists are more effective than DPP-4 inhibitors in modulating SIRT6 expression and activity. Also, within the class of GLP-1 agonists, an evaluation of the effect of the rules for GLP-1 agonist administration (once or twice daily and weekly) on SIRT6 expression needs to be undertaken.

Funding. This research was supported by Ricerca Ateneo 2006–2010 and the Second University of Naples.

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

Author Contributions. M.L.B. and R.M. contributed to the conception, design, analysis, and interpretation of the data, and the drafting and revision of the manuscript; and gave final approval of the submitted manuscript. M.R.R., M.B., N.D.O., A.G., M.S., F.F., and L.S. contributed to the analysis and interpretation of the data, and approved the submitted manuscript. P.P., F.M., D.D.A., C.M., and F.C. contributed to the drafting and revision of the manuscript, and approved the submitted manuscript. C.S. contributed to the analysis of the data. P.C. and G.P. contributed to the conception and design of the study, and approved the submitted manuscript. R.M. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Grundy
SM
,
Benjamin
IJ
,
Burke
GL
, et al
.
Diabetes and cardiovascular disease: a statement for healthcare professionals from the American Heart Association
.
Circulation
1999
;
100
:
1134
1146
[PubMed]
2.
Creager
MA
,
Lüscher
TF
,
Cosentino
F
,
Beckman
JA
.
Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I
.
Circulation
2003
;
108
:
1527
1532
[PubMed]
3.
Michishita
E
,
Park
JY
,
Burneskis
JM
,
Barrett
JC
,
Horikawa
I
.
Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins
.
Mol Biol Cell
2005
;
16
:
4623
4635
[PubMed]
4.
Lappas
M.
Anti-inflammatory properties of sirtuin 6 in human umbilical vein endothelial cells
.
Mediators Inflamm
2012
;
2012
:
597514
5.
Kanfi
Y
,
Peshti
V
,
Gil
R
, et al
.
SIRT6 protects against pathological damage caused by diet-induced obesity
.
Aging Cell
2010
;
9
:
162
173
[PubMed]
6.
Grimley
R
,
Polyakova
O
,
Vamathevan
J
, et al
.
Over expression of wild type or a catalytically dead mutant of Sirtuin 6 does not influence NFκB responses
.
PLoS One
2012
;
7
:
e39847
[PubMed]
7.
Kim
HS
,
Xiao
C
,
Wang
RH
, et al
.
Hepatic-specific disruption of SIRT6 in mice results in fatty liver formation due to enhanced glycolysis and triglyceride synthesis
.
Cell Metab
2010
;
12
:
224
236
[PubMed]
8.
Michishita
E
,
McCord
RA
,
Berber
E
, et al
.
SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin
.
Nature
2008
;
452
:
492
496
[PubMed]
9.
Kawahara
TLA
,
Michishita
E
,
Adler
AS
, et al
.
SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span
.
Cell
2009
;
136
:
62
74
[PubMed]
10.
Favero
G
,
Rezzani
R
,
Rodella
LF
.
Sirtuin 6 nuclear localization at cortical brain level of young diabetic mice: an immunohistochemical study
.
Acta Histochem
2014
;
116
:
272
277
[PubMed]
11.
Vittone
F
,
Liberman
A
,
Vasic
D
, et al
.
Sitagliptin reduces plaque macrophage content and stabilises arteriosclerotic lesions in Apoe (-/-) mice
.
Diabetologia
2012
;
55
:
2267
2275
[PubMed]
12.
Arakawa
M
,
Mita
T
,
Azuma
K
, et al
.
Inhibition of monocyte adhesion to endothelial cells and attenuation of atherosclerotic lesion by a glucagon-like peptide-1 receptor agonist, exendin-4
.
Diabetes
2010
;
59
:
1030
1037
[PubMed]
13.
Shah
Z
,
Kampfrath
T
,
Deiuliis
JA
, et al
.
Long-term dipeptidyl-peptidase 4 inhibition reduces atherosclerosis and inflammation via effects on monocyte recruitment and chemotaxis
.
Circulation
2011
;
124
:
2338
2349
[PubMed]
14.
Lee
J
,
Hong
SW
,
Chae
SW
, et al
.
Exendin-4 improves steatohepatitis by increasing Sirt1 expression in high-fat diet-induced obese C57BL/6J mice
.
PLoS One
2012
;
7
:
e31394
[PubMed]
15.
Makdissi
A
,
Ghanim
H
,
Vora
M
, et al
.
Sitagliptin exerts an antinflammatory action
.
J Clin Endocrinol Metab
2012
;
97
:
3333
3341
[PubMed]
16.
Chaudhuri
A
,
Ghanim
H
,
Vora
M
, et al
.
Exenatide exerts a potent antiinflammatory effect
.
J Clin Endocrinol Metab
2012
;
97
:
198
207
[PubMed]
17.
Young
B
,
Moore
WS
,
Robertson
JT
, et al
.
An analysis of perioperative surgical mortality and morbility in the asymptomatic carotid atherosclerosis study. ACAS Investigators. Asymptomatic Carotid Atherosclerosis Study
.
Stroke
1996
;
27
:
2216
2224
[PubMed]
18.
Moghissi
ES
,
Korytkowski
MT
,
DiNardo
M
, et al.;
American Association of Clinical Endocrinologists
;
American Diabetes Association
.
American Association of Clinical Endocrinologists and American Diabetes Association consensus statement on inpatient glycemic control
.
Diabetes Care
2009
;
32
:
1119
1131
[PubMed]
19.
Marfella
R
,
D’Amico
M
,
Di Filippo
C
, et al
.
Increased activity of the ubiquitin-proteasome system in patients with symptomatic carotid disease is associated with enhanced inflammation and may destabilize the atherosclerotic plaque: effects of rosiglitazone treatment
.
J Am Coll Cardiol
2006
;
47
:
2444
2455
[PubMed]
20.
Balestrieri
ML
,
Servillo
L
,
Esposito
A
, et al
.
Poor glycaemic control in type 2 diabetes patients reduces endothelial progenitor cell number by influencing SIRT1 signalling via platelet-activating factor receptor activation
.
Diabetologia
2013
;
56
:
162
172
[PubMed]
21.
Balestrieri
ML
,
Giovane
A
,
Milone
L
,
Servillo
L
.
Endothelial progenitor cells express PAF receptor and respond to PAF via Ca(2+)-dependent signaling
.
Biochim Biophys Acta
2010
;
1801
:
1123
1132
22.
Du
F
,
Zhou
J
,
Gong
R
, et al
.
Endothelial progenitor cells in atherosclerosis
.
Front Biosci (Landmark Ed)
2012
;
17
:
2327
2349
[PubMed]
23.
Marfella
R
,
D’Amico
M
,
Esposito
K
, et al
.
The ubiquitin-proteasome system and inflammatory activity in diabetic atherosclerotic plaques: effects of rosiglitazone treatment
.
Diabetes
2006
;
55
:
622
632
[PubMed]
24.
Etchegaray
JP
,
Zhong
L
,
Mostoslavsky
R
.
The histone deacetylase SIRT6: at the crossroads between epigenetics, metabolism and disease
.
Curr Top Med Chem
2013
;
13
:
2991
3000
[PubMed]
25.
Drucker
DJ
,
Nauck
MA
.
The incretin system: glucagon-like peptide-1 receptor agonists and dipeptidyl peptidase-4 inhibitors in type 2 diabetes
.
Lancet
2006
;
368
:
1696
1705
[PubMed]
26.
Beauharnois
JM
,
Bolívar
BE
,
Welch
JT
.
Sirtuin 6: a review of biological effects and potential therapeutic properties
.
Mol Biosyst
2013
;
9
:
1789
1806
[PubMed]
27.
Sundaresan
NR
,
Vasudevan
P
,
Zhong
L
, et al
.
The sirtuin SIRT6 blocks IGF-Akt signaling and development of cardiac hypertrophy by targeting c-Jun
.
Nat Med
2012
;
18
:
1643
1650
[PubMed]
28.
Spagnoli
LG
,
Mauriello
A
,
Sangiorgi
G
, et al
.
Extracranial thrombotically active carotid plaque as a risk factor for ischemic stroke
.
JAMA
2004
;
292
:
1845
1852
[PubMed]
29.
Burgmaier
M
,
Heinrich
C
,
Marx
N
.
Cardiovascular effects of GLP-1 and GLP-1-based therapies: implications for the cardiovascular continuum in diabetes
?
Diabet Med
2013
;
30
:
289
299
[PubMed]
30.
Matsubara
J
,
Sugiyama
S
,
Sugamura
K
, et al
.
A dipeptidyl peptidase-4 inhibitor, des-fluoro-sitagliptin, improves endothelial function and reduces atherosclerotic lesion formation in apolipoprotein E-deficient mice
.
J Am Coll Cardiol
2012
;
59
:
265
276
[PubMed]
31.
Barbieri
M
,
Rizzo
MR
,
Marfella
R
, et al
.
Decreased carotid atherosclerotic process by control of daily acute glucose fluctuations in diabetic patients treated by DPP-IV inhibitors
.
Atherosclerosis
2013
;
227
:
349
354
[PubMed]
32.
Erdogdu
O
,
Eriksson
L
,
Xu
H
,
Sjöholm
A
,
Zhang
Q
,
Nyström
T
.
Exendin-4 protects endothelial cells from lipoapoptosis by PKA, PI3K, eNOS, p38 MAPK, and JNK pathways
.
J Mol Endocrinol
2013
;
50
:
229
241
[PubMed]
33.
Zeng
Y
,
Li
C
,
Guan
M
, et al
.
The DPP-4 inhibitor sitagliptin attenuates the progress of atherosclerosis in apolipoprotein-E-knockout mice via AMPK- and MAPK-dependent mechanisms
.
Cardiovasc Diabetol
2014
;
13
:
32
36
[PubMed]
34.
White
WB
,
Cannon
CP
,
Heller
SR
, et al.;
EXAMINE Investigators
.
Alogliptin after acute coronary syndrome in patients with type 2 diabetes
.
N Engl J Med
2013
;
369
:
1327
1335
[PubMed]
35.
Scirica
BM
,
Bhatt
DL
,
Braunwald
E
, et al.;
SAVOR-TIMI 53 Steering Committee and Investigators
.
Saxagliptin and cardiovascular outcomes in patients with type 2 diabetes mellitus
.
N Engl J Med
2013
;
369
:
1317
1326
[PubMed]
36.
Monami
M
,
Genovese
S
,
Mannucci
E
.
Cardiovascular safety of sulfonylureas: a meta-analysis of randomized clinical trials
.
Diabetes Obes Metab
2013
;
15
:
938
953
[PubMed]
37.
Moreno
PR
,
Murcia
AM
,
Palacios
IF
, et al
.
Coronary composition and macrophage infiltration in atherectomy specimens from patients with diabetes mellitus
.
Circulation
2000
;
102
:
2180
2184
[PubMed]

Supplementary data