Immunohistochemistry Antibody Validation Report for Anti-GSK3β Antibody (STJ93447)
Sirtuin 6 Expression and Inflammatory Activity in Diabetic ... · Carotid artery specimens were...
Transcript of Sirtuin 6 Expression and Inflammatory Activity in Diabetic ... · Carotid artery specimens were...
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Revised 1
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Sirtuin 6 Expression and Inflammatory 7
Activity in Diabetic Atherosclerotic 8
Plaques: Effects of Incretin Treatment 9
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Maria Luisa Balestrieri1, BiolD, PhD; Maria Rosaria Rizzo2, MD, PhD; Michelangela Barbieri2, 11
MD, PhD; Pasquale Paolisso2, Student; Nunzia D’Onofrio1, BiolD; Alfonso Giovane1, BiolD; 12
Mario Siniscalchi3, MD, PhD; Fabio Minicucci3, MD; Celestino Sardu2, MD; Davide D’Andrea3, 13
MD; Ciro Mauro3, MD; Franca Ferraraccio4, MD; Luigi Servillo1, PhD; Fabio Chirico5, MD; 14
Pasquale Caiazzo5, MD; Giuseppe Paolisso2 MD; Raffaele Marfella2, MD, PhD. 15
1Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, 16
Italy; 2Department of Medical, Surgical, Neurological, Aging and Metabolic Sciences, Second 17
University of Naples, Italy; 3Department of Cardiology, Cardarelli Hospital, Naples, Italy; 18
4Department of Clinical, Public and Preventive Medicine Second University of Naples, Italy; 19
5Department of Neurosurgery, Cardarelli Hospital, Naples, Italy. 20
Short title: atherosclerosis in diabetics and sirtuin-6 21
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Address correspondence to: 24
Prof. Raffaele Marfella, 25
Piazza Miraglia, 2. 80138 Napoli, Italy 26
Telephone ++39 081 5665110 27
Fax ++39 081 5665303 28
Email [email protected] 29
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Page 1 of 34 Diabetes
Diabetes Publish Ahead of Print, published online October 16, 2014
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ABSTRACT 1
The role of sirtuin-6 (SIRT6) in atherosclerotic progression of diabetic patients is unknown. We 2
evaluated SIRT6 expression and the effect of incretin-based therapies in carotid plaques of 3
asymptomatic diabetic and non-diabetic patients. Plaques were obtained from 52 type 2 diabetic and 4
30 non-diabetic patients undergoing carotid endarterectomy. Twenty-two diabetic patients were 5
treated with the drugs that work on the incretin system, glucagon-like-peptide-1 (GLP-1) receptor 6
agonists and dipeptidyl-peptidase-4 (DPP-4)-inhibitors, for 26±8 months before endarterectomy. 7
Compared to non-diabetic plaques, diabetic plaques had more inflammation and oxidative stress, 8
along with a lesser SIRT6 expression and collagen content. Compared with no-GLP-1 therapies-9
treated plaques, GLP-1 therapies-treated plaques presented greater SIRT6 expression and collagen 10
content, less inflammation and oxidative stress, indicating a more stable plaque phenotype. These 11
results were supported by in vitro observations on endothelial progenitor cells (EPCs) and 12
endothelial cells (EC). Indeed, both EPCs and EC treated with high-glucose (25mM) in the presence 13
of GLP-1 (100 nM liraglutide) presented a greater SIRT6 and lower nuclear factor-kappa B (NF-14
ĸB) expression compared to cells treated only with high-glucose. These findings establish the 15
involvement of SIRT6 in the inflammatory pathway(s) of diabetic atherosclerotic lesions and 16
suggest its possible positive modulation by incretin, whose effect is associated with morphological 17
and compositional characteristics of a potential stable plaque phenotype. 18
Key Words: Atherosclerosis; Type 2 diabetes mellitus; Sirtuin-6; Inflammation. 19
Page 2 of 34Diabetes
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INTRODUCTION 1
Cardiovascular disease represents the leading cause of death in patients with type 2 diabetes 2
(1). Diabetes leads to increased vulnerability for plaque disruption and mediates increased incidence 3
and severity of clinical events (2). Inflammation, particularly in diabetes, plays a central role in the 4
cascade of events that result in plaque erosion and fissuring (2). There is now increasing evidence 5
that a number of transcription factors, including the Sir2 family of enzymes, namely sirtuins 6
(SIRT), regulate multiple genes whose products are putatively involved in the regulation of 7
inflammation and endothelial cell function (3). The Sir2 family consists of seven enzymes (SIRT1-8
SIRT7) that share a conserved core catalytic domain but differ in their cellular localization and 9
tissue distribution (4). Among the sirtuins, SIRT6, a chromatin-associated deacetylase, is 10
considered to have a leading role in regulating genomic stability, cellular metabolism, stress 11
response and aging (5-8). A recent study in mice suggested a role for SIRT6 in inflammation (9). 12
Moreover, the knockdown of SIRT6 resulted in the increased expression of proinflammatory 13
cytokines (IL-1β, IL-6, IL-8), extracellular matrix remodelling enzymes (MMP-2, MMP-9 and PAI-14
1), and the adhesion molecule ICAM-1 (4). In endothelial cells (EC), the loss of SIRT6 was 15
associated with an increased expression of nuclear factor-kappa B (NF-ĸB), whereas overexpression 16
of SIRT6 was associated with decreased NF-ĸB transcriptional activity (4), indicating that SIRT6 17
may be associated with the upregulation of genes involved in inflammation, vascular remodelling, 18
and angiogenesis. However, the role of SIRT6 in human atherosclerotic plaques has not been yet 19
described. Although it has been demonstrated that diabetes may be implicated in the regulation of 20
SIRT6 expression in diabetes-induced neurodegeneration (10), still no evidence exists about the 21
potential role of SIRT6 in the evolution of atherosclerotic plaques of diabetic patients. We 22
hypothesized that, by acting on SIRT6, diabetes may enhance the inflammatory potential of 23
atherosclerotic plaques favoring their instability. Thus, this study was designed to identify 24
differences in SIRT6 expression, as well as in inflammatory infiltration, between carotid plaques of 25
asymptomatic diabetic and non-diabetic patients. Experimental studies suggest that in obese mice 26
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glucagon-like peptide-1 (GLP-1) based therapies may reduce inflammation (11-13) and enhance 1
protein expression of SIRT1 (14). Moreover, human studies showed that sitagliptin (15) and 2
exenatide (16), even at a single dose, exert a potent anti-inflammatory effect and many of these 3
effects were persistent over a period of 12 weeks, thus suggesting that the anti-inflammatory effects 4
of GLP-1 based therapies could contribute to reduce atherogenesis 5
Here, we evaluated the effect of incretin therapy of diabetic patients on SIRT6 expression in carotid 6
plaques and early outgrown circulating endothelial progenitor cells (EPCs). Furthermore, a set of 7
in-vitro experiments on EC and EPCs was designed to evaluate the effect of incretin on SIRT6 and 8
NF-ĸB during high-glucose treatment. 9
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RESEARCH DESIGN AND METHODS 1
Patients were recruited from the outpatient Department of Cardiology and Cardiovascular Surgery 2
of the Cardarelli Hospital, Naples, Italy, from January 2009 to June 2013. Among them we selected 3
52 type 2 diabetic and 30 non-diabetic patients (non-diabetic group) with asymptomatic carotid 4
stenosis (according to North American Symptomatic Carotid Endarterectomy Trial classification), 5
enlisted to undergo carotid endarterectomy for extracranial high-grade (>70%) internal carotid 6
artery stenosis (17). Asymptomatic patients underwent a baseline clinical examination, as well as 7
medical history, and had never developed neurologic symptoms or cerebral lesions assessed by 8
computed tomography. All patients received computed tomography or MRI. Diabetes was 9
categorized according to American Diabetes Association criteria (18). Furthermore, the diabetic 10
patients answered a specific questionnaire about medicines used for diabetes treatment before the 11
beginning of the study, the date of the beginning and end of treatment, route of administration, and 12
duration of use. Information from medicine inventory during the study and this specific 13
questionnaire were used to classify the subjects. The diabetics who never used incretin, such as 14
GLP-1 agonists and DPP-4 inhibitors, were classified as “never incretin-users.” The diabetics who 15
had already used GLP-1 agonists or DPP-4 inhibitors were classified as “current incretin-users.” 16
Among the 52 diabetic patients, enrolled for the study, 24 were current incretin-users, and 28 were 17
never incretin-users. The current incretin-users were patients with incretin for at least 6 months. 18
Patients treated with incretin for a period of less than 6 months were excluded from the study 19
Information on duration of treatment was available for all current users. A duration of incretin 20
treatment was 26±8 months (mean±SD). All patients had no clinical or laboratory evidence of heart 21
failure, previous stroke, valvular defects, malignant neoplasms, or secondary causes of 22
hypertension. Carotid sonography was performed on a single ultrasound machine (Aloka 5500). The 23
study was approved by the ethics committee, and informed written consent was obtained for each 24
patient. The study was approved by the local Ethics Review Committee. 25
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Laboratory analysis. After an overnight fast, plasma glucose, HbA1c and serum lipids were 1
measured by enzymatic assays in the hospital’s chemistry laboratory. GLP-1 levels (Active GLP-1 2
7-36, Epitope ) were measured after an overnight fast (8:00 am) and after breakfast. Standardized 3
breakfast contained 419 kcal (57% carbohydrate, 17% protein, and 26% fat). After breakfast, blood 4
samples for measurement of GLP-1 were obtained every 30 min along 2 h. The mean of the four 5
GLP-1 evaluations were defined as post-prandial GLP-1 value. The standardized meal tolerance test 6
and baseline evaluations were performed 7 days before surgery. Thereafter, the patients were asked 7
to perform self-monitoring blood glucose (fasting, post-breakfast, post-meal and post-dinner 8
glucose levels) until the day of surgery. Levels of fasting blood glucose were evaluated before 9
surgery. Fasting and post-prandial plasma glucose data were obtained from the average of each 10
assessment. 11
Atherectomy specimens. After surgery, the specimens were cut perpendicular to the long axis into 12
two halves. The first half was frozen in liquid nitrogen for the following ELISA analysis. A portion 13
of the other half specimen was immediately immersion-fixed in 10% buffered formalin. Sections 14
were serially cut at 5 µm, mounted on lysine-coated slides, and stained with hematoxylin and eosin 15
and with the trichrome method. Carotid artery specimens were analyzed by light microscopy. 16
Immunohistochemistry. After the surgical procedure, atherectomy specimens were immediately 17
frozen in isopentane and cooled in liquid nitrogen. Similar regions of the plaque were analyzed 18
(Figure 1). Serial sections were incubated with following specific antibodies: anti-SIRT6 19
(Millipore); anti–HLA-DR; anti-CD68 (cluster of differentiation 68 glycoprotein) and anti-CD3 20
(cluster of differentiation 3 T-cell co-receptor protein) (Dako), markers of T cell and macrophages, 21
respectively; anti-matrix metalloproteinase-9 (MMP-9) (Santa Cruz), proteases involved in the 22
degradation of the collagen content in the plaque; and anti–tumor necrosis factor (TNF)-α (R&D). 23
Analysis of immunohistochemistry was performed with a personal computer-based quantitative 24-24
bit color image analysis system (IM500; Leica Microsystem AG). 25
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Sirius red staining for collagen content. Sections were stained as previously described (19). After 1
dehydration, the sections were observed under polarized light after being placed on coverslips. The 2
sections were photographed with identical exposure settings for each section. 3
Biochemical assays. Plaques were lysed and centrifuged for 10 min at 10,000 g at 4°C. After 4
centrifugation, 20 µg of each sample were loaded, electrophoresed in polyacrylamide gel, and 5
electroblotted onto a nitrocellulose membrane. Each determination was repeated at least three times. 6
MMP-9, TNF-α, and nitrotyrosine levels were quantified in plaques using a specific ELISA kits 7
(from Santa Cruz, R&D Systems, and Imgenex). SIRT6 activity was measured using fluorimetric 8
SIRT6 assay kit (Abcam, Cambridge, UK). NF-ĸB binding to kB sites was assessed on nuclear 9
extracts from plaque specimens (19) by the Trans-AM NF-ĸB p65 transcription factor assay kit 10
(Active Motif Europe; Rixensart). 11
Isolation and culture of EPCs. Early outgrown EPCs were isolated from leukocyte-rich buffy coat 12
of human healthy volunteers and peripheral blood from diabetic (n=5 current incretin-users and n=5 13
never incretin-users) and non-diabetic (n=10) patients were isolated and characterized as previously 14
described (20, 21). 15
In vitro cell treatments. EC, human aortic endothelial cells (HAECs), were from Lonza (Cologne, 16
Walkersville, MD, USA). Cells were cultured with EGM-2MV BulletKit (Lonza) supplemented 17
with 5 % FBS, 0.6 % HEPES, maintained at 37 °C in a 5 % CO2 humidified atmosphere, and used 18
in experiments between passage 4 and 7. EPCs (5 x 106 cells/ml medium) and EC (cultured at 70-19
80% confluence, , cultured in six-well plates (Costar, Corning, NY, USA), were subjected to short-20
term exposure to high-glucose (25 mM) in the presence or absence of GLP-1 receptor agonist 21
(Exenatide, Byetta) (100 nM) in complete culture media for 3 days. GLP-1 receptor agonists was 22
added 30 min before starting the high-glucose treatment and was left in the culture media 23
throughout the high-glucose treatment. Control cells were cultured for 3 days under basal 24
conditions. 25
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Confocal laser-scanning microscopy. Immunofluorescence detection of SIRT6 in deparaffinized 1
atherosclerotic plaque sections from diabetic and non-diabetic patients and in in vitro-cultured EPCs 2
and EC was performed by confocal laser-scanning microscope analysis (Zeiss LSM 510 ) by using 3
specific antibodies against SIRT6 (Signaling Technology, Danvers, MA, USA) (1:500), Vimentin 4
(1:1000) (Sigma, St. Louis, MO, USA), or antibodies against von Willebrand Factor (1:500) 5
(Abcam, Cambridge, UK), as previously described (20, 21). Secondary antibodies were conjugated 6
to Alexafluor 633 (1:1000) or Alexafluor 488 (1:1000) (Invitrogen, Life Technologies Italia, 7
Monza, Italy) (20, 21). 8
Western blot analysis. Western blot analysis of atheroscherotic plaque sections , EPCs, or EC total 9
protein extracts was performed as previously described (21) using antibody against SIRT6 (Cell 10
Signaling Technology, Danvers, MA, USA), NF-kB (Signaling Technology, Danvers, MA, USA) 11
and γ -tubulin protein (GTU-88) (Sigma, St. Louis, MO, USAAs for plaque section 12
homogenization, 800 µl of 2D lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 30 mM Tris-HCl, 13
pH 8.8) were added to tissues (400 mg) cut into small pieces. Tissue homogenized with Precellys 24 14
system (Bertin Technologies, Bertin Pharma, Montigny-le-Bretonneux, France) were centrifuged at 15
800 x g for 10 min at 4°C to collect the supernatant. Proteins were then precipitated by adding 16
100% cold methanol. 17
Statistical analysis. Data are presented as mean±SD. Continuous variables were compared among 18
the groups of patients with one-way ANOVA for normally distributed data and Kruskal-Wallis for 19
non–normally distributed data. When differences among the groups were found, Bonferroni 20
correction to make pairwise comparisons was used. P < 0.05 was considered statistically 21
significant. All calculations were performed using SPSS 12. 22
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RESULTS 1
Demographic data for the study population are presented in Table 1 and supplementary Table. 2
Percentage of carotid diameter reduction, risk factors, and concomitant non-hypoglycemic therapy 3
did not differ among the groups (Table 1 and supplementary Table). In diabetic patients, mean, 4
fasting and post-prandial plasma glucose levels at baseline as well as during the week before the 5
surgery, HOMA-IR and HbA1c (A1C) levels did not differ among never incretin-users and current 6
incretin-users (Table 1 and supplementary Table). However, basal and post-prandial GLP-1 7
levels were higher in current incretin-users compared to never incretin-users (P<0.01) (Table 1). 8
Plaque composition. Compared with non-diabetic patients, diabetic patients (n=52) had a 9
significantly greater portion of plaque area occupied by macrophages and T-cells, as well as greater 10
expression of HLA-DR antigen (Table 1 and Fig. 2). Compared with the never incretin-users 11
group, the current incretin-users group presented a significantly smaller portion of plaque area 12
occupied by macrophages (P< 0.01) and T-cells (P<0.01), as well as lower expression of HLA-DR 13
(P< 0.01) (Table 1 and Fig. 2). Both immunohistochemistry and ELISA revealed markedly higher 14
staining and levels of TNF-α in all diabetic vs. non-diabetic lesions (P< 0.001). In diabetic patients, 15
staining and levels of TNF-α were significantly more abundant in lesions from never incretin-users 16
than lesions from the current incretin-users group (P<0.001) (Table 1 and Fig. 2). Moreover, TNF-17
α levels were inversely correlated with GLP-1 levels (r=-0.67, P<0.001). MMP-9 levels were more 18
abundant in diabetic than in non-diabetic lesions (P< 0.001). Specifically, in diabetic patients, 19
MMP-9 levels were more abundant in lesions from never incretin-users than lesions from the 20
current incretin-users group (P<0.001). As for the content of interstitial collagen, lower content of 21
interstitial collagen was found in plaques of all diabetic patients compared with non-diabetic 22
patients (P<0.001). Content of interstitial collagen of plaques from never incretin-users was lower 23
than lesions from the current incretin-users group (P<0.001) (Table 1 and Fig. 3). Higher 24
nitrotyrosine levels were found in diabetic than in non-diabetic plaques (P< 0.001). Among diabetic 25
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plaques, nitrotyrosine levels were significantly higher in never incretin-users than in current 1
incretin-users (P<0.01) (Table 1 and Fig. 3). 2
Interestingly, both immunohistochemistry (Fig. 3) and confocal laser-scanning microscopy analyses 3
(Fig. 4, panel A and B) revealed that levels of SIRT6 are consistently lower in plaques from 4
diabetic patients compared to plaques from non-diabetic patients and, specifically, in plaques from 5
diabetic never incretin-users (P<0.01). Indeed, levels of SIRT6 in plaques from diabetic current 6
incretin-users were significantly higher than those observed in patients never incretin-users 7
(P<0.05), and near to values observed in non-diabetic patients (Fig. 3 and 4 Fig. 4, panel A and 8
B). In addition, in order to define the cellular type expressing SIRT6 within diabetic and non-9
diabetic plaques, sections were incubated with antibody against SIRT6 and against von Willebrand 10
Factor, a specific EC marker. Results showed the co-expression of SIRT6 and von Willebrand 11
Factor, thus, suggesting that SIRT6 is expressed by EC (Fig. 4, panel B). Consistently with 12
immunofluorescence findings, Western blot analysis of SIRT6 protein levels in atherosclerotic 13
plaques from non-diabetic and diabetic patients (current and never incretin-users) showed a similar 14
trend (Fig. 4, panel C). Moreover, SIRT6 expression levels in plaques were inversely correlated 15
with GLP-1 levels (r=-0.58, P<0.001). NF-ĸB activation, as reflected by the selective analysis of the 16
activated form of both p50 and p65, was significantly higher higher in both current incretin-users 17
(P<0.01) and never incretin-users (P< 0.01) than in non-diabetic plaques. In plaques from diabetic 18
patients, NF-ĸB were significantly higher in never incretin-users than in current incretin-users 19
(Table 1). 20
SIRT6 protein levels in EPCs from diabetic and non-diabetic patients. 21
It is already known from experimental and clinical studies that atherosclerosis is associated with 22
reduced number and dysfunction of EPCs (22). Here, for a better understanding of the molecular 23
mechanisms underlying EPC impairment in atherosclerosis, we looked at the possible involvement 24
of SIRT6 by evaluating its expression levels in EPCs isolated from peripheral blood of 10 non-25
diabetic patients and 10 diabetic patients (5 current incretin-users and 5 never incretin-users). 26
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Western blot analysis revealed that EPCs from diabetic never incretin-users had lower values of 1
SIRT6 protein arbitrary units (AU) and that diabetic patients current incretin-users had levels of 2
SIRT6 higher than those observed in EPCs from never incretin-users (P<0.05) (Fig. 5). 3
In vitro effect of high-glucose and GLP-1 receptor agonist on SIRT6 and NF-ĸB expression in 4
EPCs and EC. 5
In light of the observational data that diabetic patients never incretin-users showed decreased levels 6
of SIRT6 protein in either atherosclerotic plaque sections and EPCs, and that incretin therapies 7
seems to prevent SIRT6 downregulation (Fig. 3, 4, 5), we next evaluated whether the detrimental 8
effect high-glucose concentration on EPCs and EC (20) is exerted via SIRT6/NF-ĸB pathway. 9
Western blot and confocal-laser scanning microscopy analysis revealed that short-term exposure of 10
early EPCs (Fig. 6) and EC (Fig. 7) to high-glucose concentration induces downregulation of 11
SIRT6 protein (P<0.01) with a concomitant upregulation of NF-ĸB protein expression (P<0.01) 12
(Fig. 6, 7). 13
The dose-dependent response (from 1 to 1000 nM) effect of GLP-1 on SIRT6 activity during short-14
term exposure to high-glucose showed a significant effect starting at 100 nM concentration (810±51 15
vs. 398±25 relative fluorescence units in high-glucose treated cells) (P<0.05) and with no further 16
increase at 1000 nM. GLP-1 alone (from 1, to 1000 nM) showed no significant effect on SIRT6 17
activity. Thus, 100 nM concentration was used to test the in-vitro effect of GLP-1 on SIRT6 and 18
NF-ĸB protein levels in EPCs and EC exposed to high-glucose. Noticeably, in EPCs, GLP-1 19
receptor agonist (100 nM) significantly counteracted the effect of high-glucose concentration on 20
both SIRT6 and NF-ĸB protein levels (P<0.05) (Fig. 6, panel A, B, C). 21
Similar results were obtained when EC were subjected to high-glucose treatment in the presence or 22
absence of GLP-1 (100 nM) (Fig. 7, panel A, B, C). Indeed, the presence of GLP-1 during high-23
glucose treatment prevented SIRT6 downregulation and NF-ĸB upregulation (P<0.05) (Fig. 7, 24
panel A, B, C. According to Western blot analysis, measurements of the SIRT6 fluorescence 25
intensity units (AFU) (Fig 6 panel D, E and Fig. 7, panel D, E) showed similar results. 26
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DISCUSSION 1
This study provides novel insights into the relationship between the SIRT6 pathway and the 2
inflammatory process of atherosclerotic plaques of type-2 diabetic patients. In particular, SIRT6 3
protein expression was down-regulated in diabetic atherosclerotic lesions, as compared with non-4
diabetic lesions, and the impaired SIRT6 expression was associated with higher oxidative stress, 5
and higher NF-ĸB, pro-inflammatory cytokines, and MMP-9 levels along with less interstitial 6
collagen content. On the whole, all these factors might increase the risk of future acute ischemic 7
events precipitated by inflammatory-dependent rupture of atherosclerotic plaques. Moreover, we 8
provide evidence that in diabetic patients the drugs that work on the incretin system, such as GLP-1 9
receptor agonists and DPP-4 inhibitors, may prevent plaque progression to an unstable phenotype 10
and the downregulation of SIRT6 expression. 11
A previous study in mice has suggested a role for SIRT6 in inflammation (9), as well as a 12
recent experimental study on endothelial cells which showed that the loss of SIRT6 is associated 13
with up-regulation of genes involved in inflammation, vascular remodelling, and angiogenesis (4). 14
Up to date, there are no evidence about the possible role of SIRT6 in subgroups of high-risk 15
plaques, such as those found in diabetic patients, and about the specific pathway(s) transducing 16
environmental stimuli in the modulation of SIRT6 levels in atherosclerotic plaques. In our study, 17
the novelty is represented by the evidence that the endothelial cell-associated SIRT6 expression is 18
markedly down-regulated, and is associated with more macrophages, T-cells, and HDLA-DR+ 19
inflammatory cells in the diabetic plaques compared to non-diabetic plaques. These results suggest 20
that the presence of an active inflammatory reaction in diabetic plaques may be associated to a 21
lower production of SIRT6 in endothelial cells. In line with such evidence, lower expression of 22
SIRT6 and concomitantly higher levels of oxidative stress (nitrotyrosine levels) and inflammatory 23
cytokine were found in plaques obtained from the asymptomatic patients with type 2 diabetes 24
compared with non-diabetic patients. In agreement with the difference in SIRT6 staining pattern, 25
the histological milieu of the lesions appears different with regard to cellularity, but not in the 26
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degree of vessel stenosis, thus suggesting that diabetic and non-diabetic lesions are different only as 1
regards to SIRT6 protein expression and inflammatory burden. These data are consistent with our 2
previous findings that the inflammatory response, as well as oxidative stress levels, were higher in 3
diabetic than in non-diabetic plaques (23). Therefore, in this study, the observed decreased 4
expression SIRT6 in carotid plaques of diabetic patients might be related to the expansion of 5
oxidative and inflammatory processes, thinning of the fibrous cap, and plaque instability. These 6
results are in agreement with studies showing that in endothelial cells the loss of SIRT6 is paralleled 7
by the increased expression of the proinflammatory transcription factor NF-ĸB and, conversely, the 8
overexpression of SIRT6 is associated with a downregulation of NF-ĸB activity as well as the 9
expression of its target genes (4). These findings suggest that SIRT6, a critical enzyme in the 10
maintenance of genomic stability, could also play a key role in reducing oxidative stress and 11
cellular damage associated to plaque instability by acting in the pathway(s) controlling for vascular 12
oxidative stress and inflammation (24). Collectively, this suggests that SIRT6 is a negative 13
regulator of vascular oxidative stress and inflammation. In addition, our observational results on the 14
negative modulation of SIRT6 levels in EPCs from diabetic patients open a new scenario in the 15
signaling pathways underlying the EPC functional impairment during atherosclerosis and diabetes 16
(22, 20). However, although our findings suggest that diabetes determines an increase of oxidative 17
stress, NF-ĸB activation, and SIRT6 expression, the mechanism by which SIRT6 acts in the 18
regulation of type-2 diabetic plaques phenotype is unknown. 19
In this context, previous reports evidenced the involvement of the incretin system in the 20
regulation of the sirtuins, particularly, under conditions of metabolic diseases such as diabetes and 21
high-fat diet induced-obese mice (14). Incretin system deregulation plays a pivotal role in the 22
pathogenesis of type-2 diabetes and patients with type 2 diabetes show a significant reduction in 23
meal-stimulated levels of GLP-1 (25). However, no evidence exists about the potential role of GLP-24
1in the regulation of SIRT6 on atherosclerotic plaques of diabetic patients. In this study, we 25
observed that plaque SIRT6 expression levels were inversely correlated with GLP-1 levels. SIRT6 26
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antagonizes NF-ĸB induced gene expression programs by associating with chromatin-bound NF-1
ĸB, directing deacetylation of histone H3 lysine 9 (H3K9), and destabilizing binding of NF-ĸB to 2
chromatin (26). Thus, we hypothesized that the decreased expression of SIRT6 in plaques, as a 3
consequence of GLP-1 reduction, may enhance NF-ĸB activity and that this could represent a 4
crucial step in the pathophysiology of diabetic plaque instability. In this context, our data suggest 5
the possibility of a novel pathway to be unveiled through which the incretin system impairment, by 6
reducing SIRT6 expression, could mediate inflammatory activity in diabetic atherosclerotic plaques. 7
Thus, modulation of SIRT6 through incretin system could be beneficial for many inflammatory 8
diseases associated with endothelial dysfunction (27) and could play a pivotal role in the 9
stabilization of diabetic atherosclerotic plaques. In this context, the present findings also show a 10
stimulatory effect of the drugs that work on the incretin system, such as GLP-1 receptor agonists 11
and DPP-4 inhibitors, on SIRT6 pathway in diabetic lesions. Indeed, at the same level of blood 12
glucose levels, diabetic patients treated with both GLP-1 receptor agonists and DPP-4 inhibitors had 13
the lowest level of plaque inflammatory cells, cytokines, oxidative stress, and MMP-9 associated 14
with the highest expression of SIRT6 and content of plaque interstitial collagen. Thus, patients 15
assigned to incretin based therapy had lesser plaque progression to an unstable phenotype than 16
patients treated without incretin based therapy. In particular, the increased SIRT6 expression 17
observed in diabetic plaques from the current incretin-users suggests a low inflammatory activity 18
linked to decreased NF-ĸB activation. This hypothesis is supported by the results of in vitro 19
experiments on EPCs and EC showing that the loss of SIRT6 during short-term exposure with high-20
glucose is paralleled by the increased expression of NF-ĸB whereas overexpression of SIRT6 in the 21
presence of co-treatment with GLP-1 relates to a decreased NF-ĸB expression. In the process of 22
vascular inflammation, EPCs and activated EC and are critically involved in the formation of new 23
blood vessels which plays an important role in several pathologies, including atherosclerosis and 24
diabetes (22). A strong correlation between the number of circulating EPCs and the combined 25
Framingham risk factor score for atherosclerosis exists suggesting that EPCs can be used as a 26
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predictive biomarker for cardiovascular risk and vascular function (22). Our data, accordingly to 1
reports showing that the loss of SIRT6 in EC associated with increased expression of the cell 2
adhesion molecule ICAM-1, NF-ĸB, and senescence (4), shed light on the role of SIRT6 as 3
regulator of endothelial function during altered glucose homeostasis within a pathway that links 4
inflammation, metabolic diseases, and atherosclerosis. 5
As whole, our data might have strong clinical implications because in a large series of 6
carotid endarterectomy specimens it has been shown that plaque inflammation is one of the major 7
determinants of ischemic events in patients affected by carotid atherosclerotic disease (28). 8
Therefore, a direct anti-inflammatory effects of GLP-1 analogues and DPP-IV inhibitors, which 9
increase plasma concentrations of GLP-1, and go above and beyond the glycemic control, should 10
also be considered. In addition to their metabolic actions, various beneficial cardiovascular effects 11
have been described for GLP-1 based therapies (29). It has previously been reported that GLP-1 12
reduce in vitro monocyte migration while the DPP-IV inhibitor, sitagliptin, reduced plaque 13
inflammation and enhanced plaque stability in ApoE-/- mice (11). Similar effects of DPP-IV 14
inhibitors on plaque inflammation were observed (30). Finally, sitagliptin, vildagliptin (15, 31), and 15
exenatide (16), a GLP1 agonist, have recently been shown to exert a rapid and significant anti-16
inflammatory effect which is evident within 2h of the first dose of each of these drugs. Such results 17
support the hypothesis that the anti-atherosclerotic effect we observed may be also due to a direct 18
anti-inflammatory effect independently of the effect on glycemic control. However, the full 19
mechanism by which GLP-1 acts on of SIRT6, still uncovered by this study, requires further 20
investigations. Indeed, we did not identified the complete mechanism by which GLP-1 can regulate 21
the inflammatory status of atherosclerotic plaque through the modulation of SIRT6 levels. This 22
study highlights the relevance of modulating SIRT6/NF-ĸB signaling with incretin based-therapy to 23
reduce the inflammatory burden of diabetic plaques. At the moment, we do not exclude the 24
possibility that GLP-1 could inhibit NF-ĸB activation through additional mechanisms 25
independently of SIRT6. Indeed, it has previously been reported that exendin-4, a GLP-1R agonist, 26
Page 15 of 34 Diabetes
16
attenuates atherosclerosis through PKA–PI3K/Akt–eNOS–p38 MAPK–JNK- dependent pathways 1
via a GLP-1R-dependent mechanism, without affecting metabolic parameters (32). Furthermore, 2
recent evidences have shown a promising role for DPP-4 inhibitors in the attenuation of 3
atherosclerosis involving vascular dysfunction and endothelial inflammation by upregulating the 4
Akt/eNOS signaling pathway and suppressing the activation of ERK1/2 in vascular tissues (33). 5
However, despite the incretin-mimetic drugs have shown favorable effects on pathogenic 6
mechanisms of atherosclerosis, the recently published large outcome trials have not shown any 7
superiority of treatment with a DPP-4 inhibitor (alogliptin or saxagliptin) on the outcomes of a 8
composite of atherosclerosis related events (34, 35). However, it should be reminded that these 9
trials were Food and Drug Administration (FDA)-mandated to simply rule out cardiovascular harm. 10
Furthermore, it should be noted that in both studies patients were randomized to DPP-4 inhibitors or 11
placebo on top of standard therapy, usually metformin (alone or in combination with other agents), 12
which was taken by about two-thirds of the patients. Moreover, almost half of the patients were 13
taking sulfonylureas, that may be associated with increased mortality (36). Finally, both studies 14
were conducted in patients with cardiovascular disease and, therefore, the assessment of prevention 15
of events with incretins may not be accurate. However, the effects of incretin-mimetic drugs on 16
cardiovascular events and mortality cannot be considered established unless it is evaluated in long-17
term cardiovascular outcomes trials in patients without cardiovascular diseases. 18
In conclusion, we demonstrated that GLP-1 reduced plaque macrophage infiltration and MMP-9 19
expression, which resulted in an increased plaque collagen content and a thickened fibrous cap in 20
diabetic plaques from humans. As these plaque characteristics are features of plaque stability, the 21
results of this study suggest that GLP-1 reduce plaque vulnerability. These findings may be of 22
clinical importance for patients with type 2 diabetes mellitus, who are known to exhibit a higher 23
burden of inflamed, rupture prone atherosclerotic plaques in comparison to non-diabetic subjects 24
(37). Overall, it is tempting to speculate that activators of SIRT6 might be an effective strategy for 25
inflammatory vascular diseases such as diabetes and atherosclerosis. Future studies are needed to 26
Page 16 of 34Diabetes
17
determine whether the beneficial effects of GLP-1 receptor agonists and DPP-4 inhibitors on 1
features of plaque vulnerability though SIRT6 pathway translate in a reduction of cardiovascular 2
events in patients with type-2 diabetes treated with GLP-1 based regimes. Moreover, this study also 3
raises an important question whether asymptomatic diabetic patients with >70 % carotid artery 4
stenosis on GlP-1 based therapies really need carotid endarterectomy as they are likely to have 5
stable plaques. Prospectively randomized large scale clinical trials are required to further clarify this 6
relationship. Finally, although the number are small for incretin users, it would be of interest to 7
know if GLP-1 agonists showed more SIRT6 expression compared to DPP-4 inhibitors. Moreover, 8
further investigations are required to determine whether GLP-1 agonists are more effective than 9
DPP4 inhibitors in modulating SIRT6 expression and activity. Also, within the class of GLP-1 10
agonists, evaluation of the effect of GLP-1 agonist administration rules (once or twice daily and 11
weekly) on SIRT 6 expression need to be determined. 12
Acknowledgments 13
Guarantor: Raffaele Marfella. 14
Funding: Ricerca Ateneo 2006-2010 Second University Naples 15
The manuscript, or part of it, neither has been published nor is currently under consideration for 16
publication by any other journal. All authors have approved its submission to Diabetes. Each author 17
contributed significantly to the submitted work. Balestrieri and Marfella have contributed to: 1) 18
conception, design, analysis and interpretation of data; 2) drafting and revision of the manuscript; 19
and 3) final approval of the submitted manuscript. Rizzo, Siniscalchi, D’Onofrio, Servillo, Giovane, 20
Barbieri and Ferraraccio have contributed to the analysis and interpretation of data. Minicucci, 21
D’Andrea, Pasquale Paolisso, Chirico and Mauro have contributed to drafting and revision of the 22
manuscript. Giuseppe Paolisso and Caiazzo have contributed to conception and design. All authors 23
do not have any conflict of interest in connection with the submitted article. 24
25
26
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Investigators. Alogliptin after acute coronary syndrome in patients with type 2 diabetes. N 15
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Figure legends 1
FIGURE 1. Representative example of plaques studied. This section is representative of the 2
plaque region analyzed. The section was stained with hematoxylin-eosin. Plaque section at low 3
magnification (X5). High magnification (inset; X100) indicated the section analyzed. 4
FIGURE 2. Inflammation in atherosclerotic plaques. Immunochemistry for macrophages 5
(CD68) (X400), lymphocytes (CD3) (X400), inflammatory cells (HLA-DR) (X400), and TNF-α 6
(X400) in control, current incretin-user, and never incretin-user asymptomatic plaques. Similar 7
regions of plaque are shown. These results are typical of control, current incretin-user, and never 8
incretin-user asymptomatic plaques. 9
FIGURE 3. Atherosclerotic plaque phenotypes. Immunochemistry for matrix metalloproteinase-10
9 (MMP-9) (X400), sirius red staining for collagen content (X400), nitrotyrosine (X400) and sirtuin 11
6 (SIRT6) (X400) in control, current incretin-user, and never incretin-user asymptomatic plaques. 12
Similar regions of plaque are shown. These results are typical of control, current incretin-user, and 13
never-incretin user asymptomatic plaques. 14
FIGURE 4. Detection of SIRT6 expression in atherosclerotic plaques. Immunofluorescence 15
detection of SIRT6 was carried out on deparaffinized atherosclerotic plaque sections from diabetic 16
and non-diabetic patients. (A) Bar graph of SIRT6 arbitrary fluorescence units (AFU) determined 17
by confocal laser-scanning microscopy. (B) Representative confocal images of SIRT6 (red) and 18
Willebrand Factor (green). (C) Representative bar graph of SIRT6 protein levels determined by 19
Western blot analysis of atherosclerotic plaque homogenates from non-diabetic patients (P1, P2), 20
diabetic never incretin-user patients (P3, P4), and diabetic current incretin-user patients (P5, P6). 21
(D) Inset, representative image of Western blot analysis. Lane 1 and 2, non-diabetic patients (P1, 22
P2). Lane 3 and 4, diabetic never incretin-user patients (P3, P4). Lane 5 and 6, diabetic current 23
incretin-user patients (P5, P6). Data are mean±SD with *P< 0.01 vs. non-diabetic; § P<0.05 vs. 24
never incretin-users. 25
Page 23 of 34 Diabetes
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FIGURE 5. SIRT6 expression in EPCs from diabetic and non-diabetic patients. Western blot 1
analysis of SIRT6 protein levels on EPCs from non-diabetic and diabetic patients (never incretin-2
users and current incretin-users) was performed as described under Methods section. Representative 3
bar graph and image of the Western blot analysis of SIRT6 expression in EPCs from non-diabetic 4
patients (P1, lane 1), never incretin-users (P2, lane 2), and current incretin-users (P3, lane 4). Data 5
were expressed as arbitrary units (AU) ±SD with *P< 0.05 vs. non-diabetic; § P<0.05 vs. never 6
incretin-users. 7
FIGURE 6. Effect of short-term exposure to high-glucose and GLP-1 on SIRT6 and NF-ĸB 8
protein levels in EPCs. PBMCs (5×106 cells/ml medium), isolated from of leukocyte-rich buffy 9
coat of healthy donor subjects, were subjected to short-term exposure to high-glucose (25 mM) in 10
the presence or absence of GLP-1 receptor agonist, exenatide (100 nM) for 3 days. Representative 11
bar graph and image of Western blot analysis of SIRT6 (A, B) and NF-ĸB (A, C) protein levels in 12
control (ctr), high-glucose treated cells (hGluc), GLP-1 treated cells (GLP-1), and cells treated with 13
high-glucose in the presence of GLP-1 (hGluc + GLP1). (D) Representative confocal images of 14
SIRT6 (red) and vimentin (green).(E) Bar graph of SIRT6 arbitrary fluorescence units (AFU) 15
determined by confocal laser-scanning microscopy. Data are mean±SD (n= 6) with * P< 0.01 vs. 16
ctr; § P<0.05 vs. hGluc, ǂ P<0.01 vs. hGluc. 17
FIGURE 7. Effect of short-term exposure to high-glucose and GLP-1 on SIRT6 and NF-ĸB 18
protein levels in EPCs. EC were seeded in six-well plates at 70-80 % confluence 12 h before 19
treatments with high-glucose (25 mM) in the presence or absence of GLP-1 receptor agonist, 20
exenatide (100 nM). EC were cultured for 3 days in complete media alone (control, ctr), with high-21
glucose (hGluc), with GLP-1 (GLP-1), or with high-glucose in the presence of GLP-1 (hGluc + 22
GLP1). Representative bar graph and image of Western blot analysis of SIRT6 (A, B) and NF-ĸB 23
(A, C) protein levels. (D) Representative confocal images of SIRT6 (red) and vimentin (green).(E) 24
Bar graph of SIRT6 arbitrary fluorescence units (AFU). Data are mean±SD (n= 6) with *P< 0.05 25
vs. ctr; **P< 0.01 vs. ctr; § P<0.05 vs. hGluc, ǂ P<0.01 vs. hGluc. 26
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Table 1. Characteristics of study patients. 1
2
Patients characteristics Control
(N= 30)
Never incretin users
(N=28)
Current incretin
users (N=24)
Age, years 71 ± 4 69 ± 7 70 ± 5
Sex (female/male) 17/13 17/11 15/9
Patients 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/m2) 27.3 ± 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*
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T-cells per 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*
P 50, ng/mg 20.6±11.2*† 34.8±15.4* 71.1±16.4
P 65, 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*
Nytrotyrosine, 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-1agonists — — 4 (17)
Exenatide 3 (12)
Liraglutide 1 (4)
DPP-4 inhibitors — — 20 (83)
Data are presented as mean±SD, or number (%).*P<0.05 compared with never current user group. †P<0.05 1
compared with current user group. MMP-9= metalloproteinases-9; TNF-α= tumor necrosis factor- α; CRP= 2
C-reactive protein IHD = ischemic heart disease; BMI = body mass index; HDL = high-density lipoprotein; 3
GLP-1=glucagon-like peptide-1; DPP-4= dipeptidyl-peptidase-4. 4
5
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Supplementary Table. Characteristics of study patients.
Patients characteristics Control
(N= 30)
Never incretin users
(N=28)
Current incretin users
(N=24)
Cigarette smoking 9 (30) 8 (29) 6 (25)
Systolic blood pressure (mmHg) 130 ± 14 132 ± 14 131 ± 13
Diastolic blood pressure (mmHg) 80 ± 5 80 ± 5 82 ± 3
Glycemic control
Baseline fasting blood glucose (mmol/l) 6 ± 0.6*† 9.5 ± 1.4 9.4 ± 1.6
Baseline postprandial blood glucose (mmol/l) 6.39±0.3*† 11.06±0.7 11.28±0.8
Follow-up fasting blood glucose (mmol/l) 5.3±0.2*† 8.78±0.5 8.94±0.6
Follow-up postprandial blood glucose (mmol/l) 6.15±0.3*† 9.8±0.5 9.9±0.4
Blood glucose before surgery (mmol/l) 5.2±0.2*† 7.5±0.1 7.67±0.2
Active therapy (%)
Aspirin 25 (83.3) 25 (89) 22 (92)
Warfarin 2 (6.67) 1 (4) 1 (4)
β-Blocker 8 (26.67) 6 (21.4) 6 (25)
Calcium-channel blocker 3 (10) 4 (14) 3 (12.5)
Statin 20 (66,67) 21 (75) 18 (75)
ACE inhibitor 15 (50) 17 (61) 15 (62)
Diuretic agent 9 (30) 7 (25) 6 (25)
AT-2 antagonist 11 (36.67) 9 (32) 7 (29)
Insulin — 4 (14) 2 (8)
Sulfonylureas — 4 (14) 3 (12)
Metformin — 20 (71) 14 (58)
PPAR-γ agonists — 5 (18) 3 (12)
Acarbose — 4 (14) 3 (12)
Data are presented as mean±SD, or number (%).*P<0.05 compared with never current user group.
†P<0.05 compared with current user group.
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