Sirtuin 6 Expression and Inflammatory Activity in Diabetic Atherosclerotic Plaques: Effects of Incretin Treatment

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 (5–8). 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 (11–13) 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.

After an overnight fast, plasma glucose, HbA_1c, 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.

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.

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).

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.

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).

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).

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.

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 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.

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 HbA_1c 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).

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).

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).

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).

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).

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.