Thrombosis Research 133 (2014) 418–425

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Regular Article

Simvastatin attenuates the endothelial pro-thrombotic shift in saphenous vein grafts induced by Advanced glycation endproducts Cristiano Spadaccio a,b,⁎,1, Federico De Marco c, Fabio Di Domenico d, Raffaella Coccia d, Mario Lusini a, Raffaele Barbato a, Elvio Covino a, Massimo Chello a a

Department of Cardiovascular Sciences, University Campus Bio Medico of Rome, Italy Department of Cardiac Surgery, University Hospital UZ Leuven, Belgium Laboratory of Virology, Regina Elena Institute for Cancer Research, Rome, Italy d Department of Biochemical Sciences, University La Sapienza, Rome, Italy b c

a r t i c l e

i n f o

Article history: Received 25 September 2013 Received in revised form 30 November 2013 Accepted 17 December 2013 Available online 21 December 2013 Keywords: Graft failure CABG Diabetes Mellitus Advanced Glycation Endproducts Saphenous graft

a b s t r a c t Background: Advanced glycation endproducts (AGEs) and its receptors (RAGEs) are heterogeneous signaling proteins associated to diabetes and responsible of endothelial alterations leading to atherosclerosis progression and graft failure. The aim of this study was to investigate the role of statin in reducing AGEs related endothelial damage. Methods: Endothelial cell(EC) obtained from leftovers of saphenous vein grafts of non-diabetic patients were incubated with AGEs (2 and 20 μM) and subsequently treated with Simvastatin. Neutrophils (PNM) adherence, ROS production and RAGE and peroxisome proliferator-activated receptors-gamma (PPAR-γ) expression were analyzed. As clinical validation of the in vitro findings, ECs of diabetic patients in optimized glycaemic control administered with a 3 weeks Simvastatin regimen were similarly processed. Results: Simvastatin blunted the rise in PMN adhesion and ROS generation following stimulation of saphenous vein EC culture with AGEs in vitro. This effect was time dependent and was associated to an increase in PPAR-γ induction paralleled by a decrease in RAGEs expression. Parallely, data from diabetic patients administered with Simvastatin showed a similar significant reduction in PNM adhesion and ROS generation. Simvastatin treatment significantly decreased RAGEs expression in ECs from diabetic patients and determined a slight increase in PPAR-γ expression but the latter failed to reach statistical significance. Interference in the function of these two crucial pathways might be at the root of the statin antinflammatory and antithrombotic effect in the context of AGEs-associated damage. Conclusions: Despite the recently raised warning on the use of statins in the diabetic population, this study elucidates their cornerstone position in endothelial homeostasis of saphenous grafts in patients with controlled diabetes. © 2013 Elsevier Ltd. All rights reserved.

Introduction Increasing evidences are pointing at advanced glycation endproducts (AGEs) and its receptors (RAGE) as the responsible of a large spectrum of molecular alterations eventually leading to plaque progression and complications [1] but also to graft failure [2] or restenosis after Abbreviations: CABG, Coronary Artery Bypass Grafting; AGEs, Advanced Glycation Endproducts; RAGEs, Receptors of Advanced Glycation Endproducts; PPAR-γ, peroxisome proliferator-activated receptors-gamma; DM, Diabetes Mellitus; ROS, Reactive Oxygen Species; PNM, polymorphonucleates; EC, Endothelial Cells; BSA, Bovine Serum Albumine; DCF, 2′,7′-dichlorofluorescein. ⁎ Corresponding author at: Dept. of Cardiovascular Sciences–University Campus Bio Medico of Rome, Via Alvaro del Portillo 200, Roma 00128, Italy. Tel.: +39 06 225411140; fax: +39 06 22541456. E-mail addresses: [email protected], [email protected] (C. Spadaccio). 1 Alternative address: Department of Cardiac Surgery, University Hospital UZ Leuven, Belgium Herestraat 49, Leuven 3000 Belgium. Tel.: +32 16344260; fax: +32 16344615. 0049-3848/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.thromres.2013.12.023

cardiovascular surgical procedures in diabetic patients [3]. In the clinical context, the latter achieves a particular significance considering the increasing number of interventions performed on diabetics and the possible therapeutic scenarios that a pharmacologic approach oriented against AGEs-RAGE axis might open. We recently demonstrated in vitro that the presence of a large AGE burden in the vessel wall increases the likelihood of an exaggerated and prolonged inflammatory reaction and determines a pro-thrombotic state, defining a common mechanism potentially explaining the increased rate of vein graft failure after coronary bypass surgery. In vitro stimulation of saphenous vein endothelial cells with AGEs determined i) an upregulation of endothelial RAGE expression, ii)a dose dependent increment of polymorphonucleates (PMN)-endothelium adhesion iii) increased production of reactive oxygen species (ROS) from endothelial cells (ECs) and iv) decreased endothelial PPAR-γ expression [2]. On account of the reported pleiotropic effects of HGMCoA-reductase inhibitors and of their mitigating action on AGE-RAGE-mediated damage [3], we followed our previous set of

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experiments investigating the effect of Simvastatin in AGEs-induced endothelial activation including PMN adhesion, ROS production, RAGEs expression and PPAR-γ induction. Methods Study design This study was conducted on saphenous vein grafts leftovers obtained from 30 non-diabetic patients scheduled for coronary surgery. Patients enrolled have no evidence of systemic inflammatory diseases, malignancies, hematopoietic disorders, renal failure, leg vein insufficiency and were not administered with statin or PPAR-γ agonist (i.e. thiazolidinediones) and/or steroids within the last 6 months. Segments of saphenous vein grafts were ex vivo cultured in presence of AGEs for 24 hours and then treated with Simvastatin for 24 hours to perform leukocyte adhesion assay. Remaining segments were used to isolate and culture ECs which have been further stimulated with AGEs, similarly treated with statin and eventually assayed for ROS production, RAGEs expression and PPAR-gamma induction. In parallel to our previous study, we performed an additional set of experiments with diabetic patients for validation purposes. Thirty type 2 diabetic patients under optimal glycaemic control (HbA1c level b 6.0%), scheduled for coronary surgery were also enrolled (demographics in Table 1). Exclusion criteria were represented by non-optimized glycemic control (HbA1c level N 6.0%), proliferating retinopathy, peripheral neuropathy, renal failure, leg vein insufficiency, any therapy with PPAR-γ agonist (i.e. thiazolidinediones or statin) and/or steroid therapy within the last 6 months. AGEs

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serum levels were measured and patient exhibiting values N 7 μg/ml were included [2]. Accordingly to the literature and our previous report, this value is associated to diabetic state and to diabetes-induced damage to endothelium of cardiac vessels [4,5] and microvasculature [6]. Routine biochemical markers, Hb1Ac, inflammatory parameters and oxidative stress markers, including serum malondialdehyde (MDA) [7] and oxidized low density lipoprotein (oxLDL) [8], were measured and the values reported in Table 1. Fifteen patients received 20 mg/day Simvastatin for 3 weeks before surgery. Venous conduits were harvested and endothelium isolated and further processed similarly to the principal study group. In order to estimate the total number of patients and samples required to demonstrate the study outcomes an inverse power analysis was performed. Data previously generated on the modulation of neutrophil-endothelium interaction on saphenous vein of patients undergoing coronary bypass grafting [9] and on RAGEs and PPAR-γ expression induction [2] were used in the calculation. The study conforms to the Declaration of Helsinki. The Local Ethical Committee approved the protocol, and all individuals provided informed consent. Preparation of AGE – BSA complexes (AGEs) The glycated Bovine Serum Albumine (BSA) was prepared according to the method of Horiuchi et al. [10]. with minor modifications. The AGE-BSA complex was characterized by mobility in sodium dodecyl sulphate-polyacrylamide gel electrophoresis, absorption and fluorescent spectra (370–440 nm). Saphenous vein harvesting, AGE-mediated activation and statin treatment

Table 1 Demographic data and laboratory findings including AGEs and Hb1AC levels, inflammatory parameters and oxidative stress markers of both the control and the diabetic population. Parameter

Control (N 30)

Diabetes (N 30)

P value

Age (yrs) Male gender (%) Hypertension (%) Dislipidemia (%) Smoke (%) Family History of CVD Peripheral vascular disease Recent AMI Medications • ACE-inhibitors assumption • B-Blockers • Calcium antagonists • Diuretics WBC (cells/μl) Hb (g/dL) AST (IU/L) ALT (IU/L) γ-GTP (IU/L) Total bilirubin (mg/dL) BUN (mg/dL) Creatinine (mg/dL) Uric acid (mg/dL) TC (mg/dL) LDL-C (mg/dL) TG (mg/dL) HDL-C (mg/dL) hs-CRP(mg/L) HbA1c (%) AGEs (μg/ml) Malondialdheide MDA (μmol/L) Ox-LDL (ng/ml)

68,75 ± 6,54 23 (76,7%) 28 (93,3%) 25 (83,3%) 14 (46,6%) 21 (70%) 18 (60%) 11(36,6%)

66,91 ± 10,1 22 (73,3%) 26(86,6%) 28 (93,3%) 11 (36,6%) 23 (76,6%) 20 (66,6%) 12 (40%)

0,78 0,45 0,57 0,48 0,37 0,87 0,52 0,47

26 (86,6%) 23 (76,6%) 16 (53,3%) 8 (26,6%) 6,546 ± 4,372 12.3 ± 2,1 72.4 ± 21,48 30.2 ± 11,38 31.9 ± 13.7 0.9 ± 0.2 12.6 ± 3,2 0.94 ± 0.33 4,1 ± 1.9 243.5 ± 28.2 147.8 ± 27.4 112.6 ± 49.3 43.7 ± 11.8 23.7 ± 26,8 4.3 ± 1.7 2.3 ± 1,2 2,82 ± 0,7 91.82 ± 5,98

24 (80%) 21 (70%) 19 (63,3%) 9 (30%) 7,289 ± 2,897 11.4 ± 1.4 75,98 ± 37,45 31,8 ± 18,79 34.5 ± 12,32 1,02 ± 0.7 13,8 ± 2.8 0.92 ± 0.49 4.9 ± 2.3 241.9 ± 32.7 143.1 ± 31.7 114.8 ± 51.6 44.1 ± 11.2 30.1 ± 19.7 5.1 ± 0,8 9.1 ± 3.9 3,27 ± 1,4 93.31 ± 4.12

0,46 0,86 0,27 0,64 0.47 0.42 0.59 0.65 0.97 0.47 0.84 0.61 0.28 0.46 0.79 0.47 0.81 0.78 0.023 0.04 0.87 0.42

Abbreviations WBC, white blood cell counts; Hb, hemoglobin; AST, aspartate aminotransferase; ALT, alanine aminotransferase; γ-GTP, γ-guanosine 5’-triphosphate; BUN, blood urea nitrogen; TC, total cholesterol; LDL-C, low density lipoprotein-cholesterol; TG, triglyceride; HDL-C, high density lipoprotein cholesterol; hs-CRP, high-sensitivity C-reactive protein; HbA1c, haemoglobin A1c (glycated haemoglobin); AGEs, Advanced glycation end products AGE; Ox-LDL, oxidized LDL.

Great saphenous vein was harvested by a no touch technique, stored in heparinized blood and immediately employed for grafting. Discarded vein segments were immediately stored in sterile M 199 culture medium in humidified incubator at 37 °C and 5% CO2. Segments were then incubated with AGE at concentrations of both 2 μM and 20 μM and control media represented by no-glycated BSA in order to avoid biases concerning non-specific effect of BSA [11]. After 24 hours of culturing 5μM Simvastatin (Merck Sharp&Dohme, Whitehouse Station, NJ) was added and segments collected after 24 hours for further adhesion assay. Neutrophil isolation and adhesion assay Blood sample were collected by venipuncture from patients and neutrophils were isolated by Ficoll-Hypaque density gradient centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes. Adhesion assay following fluorescent labeling of isolated neutrophils was performed as previously described [9]. Number of neutrophils adhering to the endothelial surface in five separate microscopic fields were counted manually on a microscope equipped for fluorescence, using the filter IF355–550. Endothelial cell cultures, AGE-mediated activation and statin treatment Human ECs were isolated from segments of saphenous veins and cultured as previously described [2]. All the experiments were performed on ECs at passages 2 to 5. Similarly to vein segments, ECs were cultured in presence of AGE at concentrations of 2 μM and 20 μM or control medium containing non-glycated BSA for 24 hours and subsequently cultures were added with 5μM Simvastatin (Merck Sharp&Dohme, Whitehouse Station, NJ). Endothelial cell RNA Extraction and Reverse Transcription (RT)-PCR Total RNA was extracted from cultured endothelial cells using TRI Reagent (Sigma Aldrich), as described by Chomczynski and cDNA obtained [12]. Amplification of PPARγ and RAGE cDNA was performed as

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described [2] and PCR products were then analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Internal triplicate for each experiment have been performed. ROS production analysis 2′,7′-dichlorofluorescein (DCF) diacetate assay on cultured saphenous vein ECs was performed after 24 hour stimulation with 2 μM and 20 μM AGEs and Simvastatin according to the above mentioned protocol. ROS accumulated in the cells was estimated by spectrofluorimetric analysis of DCF fluorescence using a Shimadzu F-5000 spectrophotofluorimeter. Excitation and emission wavelengths were set at 498 and 522 nm, respectively[13]. Internal triplicate for each experiment have been performed. Statistic analysis The data are expressed as mean ± SD. Differences between groups were tested using the ANOVA test or unpaired Student’s t test when appropriate. P values less than 0.05 (two-tailed) were taken to indicate statistical significance. Analysis was performed with the SPSS version 20.0 software for Mac. Results Neutrophil-endothelium adhesion Values of five replicates from each plate were averaged, and the coefficient of variations between replicates was b10%. Incubation with AGE at 2 μM and 20 μM increased neutrophil-endothelial adhesion in a dose dependent fashion (2 μM: 7.5 ± 1.2%, P = 0.01 vs. control; 20 μM: 16.8 ± 2,1, P b 0.001 vs. control). Simvastatin treatment significantly reduced the percentage of adhered neutrophils in both the subsets incubated with 2 μM AGEs (4.3 ± 1.1%, P b 0.001 vs AGEs 2 μM) and 20 μM AGEs (8.3 ± 1.6%, P b 0.001 vs AGEs 20 μM). Interestingly, under basal conditions, Simvastatin reduced neutrophils adherence to the vein endothelium of non-diabetic patients compared with nontreated controls (4.7 ± 1.3% vs. 2.9 ± 1.2%, P b 0.001) (Fig. 1). Conduits from diabetic patients behaved similarly to cultures treated with AGEs 2 μM (P = NS) and patients treated with Simvastatin showed

Fig. 1. Adherence of neutrophils to saphenous vein. All treated groups showed statistical significant difference in respect to both the control non treated and the control treated with Simvastatin(P b 0.001)*:significant vs relative AGEs treated; **:significant vs 2 μM AGEs; ***:significant vs all treated groups.

values comparable to in vitro cultures stimulated with AGEs 2 μM plus Simvastatin (P = NS). Interestingly, Simvastatin treatment in diabetic patients was able to reduce PNM adhesion at levels similar to those obtained in vitro in control cultures from non-diabetic patients (P = NS) (Fig. 6A).

ROS production Fig. 2 and 3 show the effect of Simvastatin treatment on ROS production from ECs cultures following 24 and 48 hours incubation with AGE at both 2 μM and 20 μM respectively, compared with baseline conditions. Under normal conditions, cell cultures from diabetic patients showed a higher ROS production compared with cells from control group after 24 hours culture, even if the difference failed to reach statistical significance (Diabetic 0,074 ± 0.005 vs Control 0.063 ± 0.009 A.U. P = 0.13). ECs isolated from diabetic patients and treated with Simvastatin showed a significant reduction in ROS production in comparison to diabetic control (Fig. 6B). After incubation with AGEs, the ROS production increased in a time and dose depended fashion while Simvastatin was able to significantly blunt AGE-induced elevation of ROS at each time point. Interestingly the greater mitigating effect of statin could be observed in the group incubated for 48 hours with AGEs rather than in the acute exposition group and also a higher percentage degree of reduction in the ROS production could be detected in the 48 hours treated group (Fig. 3).

RAGE expression mRNA for RAGE was analyzed by real-time PCR. RAGE mRNA was only lightly expressed in unstimulated ECs from non-diabetic patients. Incubation of unstimulated ECs with AGE (2 and 20 μM) for 24 hours led to a sharp increase of RAGE expression in a concentrationdependent manner (Fig. 4). In parallel, Simvastatin treatment was able to decrease RAGEs induction in statistically significant fashion. Interestingly, the level of expression observed in EC from diabetic patients was comparable with that observed in control EC after incubation with AGE at 2 μM and Simvastatin treatment blunted the rise in RAGEs mRNA expression to a value comparable to the one observed in vitro after treatment with the same concentration of AGEs and statin (Fig. 6C).

Fig. 2. Reactive oxygen species generation in saphenous ECs following 24 hours stimulation. All treatment groups showed statistical significant difference in respect to both the control non treated and the control treated with Simvastatin (P b 0.001) *:significant vs relative AGEs treated; **:significant vs 2 μM AGEs; ***:significant vs all treated groups.

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Fig. 3. ROS generation in saphenous ECs following 48 hours stimulation. P b0.001*:significant vs relative AGEs treated; **:significant vs 2 μM AGEs; ***: significant vs all treated groups. Significance of the difference between AGEs 2 μM and AGEs 2 μM + Simvastatin treated group was at level of P b 0.05. Note the greater benefit of statin treatment for higher doses of AGEs.

Endothelial PPAR- γ expression The expression of the PPAR γ receptor in cultures of EC was evaluated by RT-PCR. Incubation of unstimulated ECs with AGE (2 and 20 μM) for 24 hours determined a significant reduction of PPAR-γ in a dose dependent manner, with decrease of 31% and 47% after incubation with 2 μM and 20 μM, respectively. Simvastatin treatment induced a significant increase in PPAR-γ in all groups (fig. 5). At baseline PPAR-γ expression was significantly reduced in EC cultures from diabetic patients compared with those from non-diabetic patients, while Simvastatin induced a slight increase in PPAR-γ expression in ECs from diabetic patients, which failed to reach statistical significance (Fig. 6D). Comment The non-enzymatic glycation and oxidation of proteins, lipids, and nucleic acids results in the formation of an heterogeneous class of irreversible adducts collectively named AGEs[14]. The interaction of AGEs with their receptor (RAGEs) triggers a molecular cascade converging to the activation of NF-κB[15] and leading in turn to the up-regulation of adhesion molecules, cytokines, pro-inflammatory genes, tissue factor expression[16] and generation of reactive oxygen species[17]. The complex interplay among these factors has been shown to regulate atherosclerotic lesion generation, progression and instability[18]. The direct effect of AGEs and RAGE axis on these molecular mechanisms justifies its recognized involvement in atherosclerosis in diabetes[19], but a large piece of evidence is underpinning its role in the restenosis phenomenon after interventional vascular procedures[20] and in pathogenesis of graft failure after coronary bypass surgery[2]. Indeed, beside the cited direct effect on inflammation, thrombosis, adhesion molecules upregulation and free radicals generation, AGEs have been shown to affect vascular smooth muscle cells proliferation and migration and extracellular matrix protein turnover, other crucial factors in restenosis pathogenesis. Also, AGEs are claimed to be one of the responsible of neointimal hyperplasia induction after balloon-induced endothelial injury[21]. Several studies underpinned the significance of AGEsRAGEs axis in thrombogenesis in both micro and macrovascular districts[22]. AGEs have been claimed to initiate thrombotic phenomena inducing the expression of monocyte chemoattractant protein-1 (MCP-1) and ICAM-1 in microvascular endothelial cells throughout mechanisms mediated by intracellular ROS generation and eventually determining T-cell adhesion to endothelium[23]. Oxidative stress

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generation and prostanoid secretion are also at the root of AGEsinduced platelet aggregation[24]. However, a direct in vivo effect of AGEs on platelet activation has also been reported[25]. The glycated products present on erythrocyte membranes have been showed to interact with RAGEs on endothelial cells and to trigger expression of ICAM-4 on the endothelium, which is responsible for binding to integrins present on leukocytes and on platelets inducing adhesion aggregation and favoring thrombosis[26]. In our previous study on AGEs-induced saphenous vein endothelial damage, we demonstrated their deleterious effect on endothelium resulting in increased PMN adhesion and ROS generation, and, more interestingly, in a downregulation of endothelial peroxisome proliferator-activated receptors-γ (PPAR-γ) expression[2]. PPAR-γ has been reported to have protective effects on endothelial cells by inhibiting endothelin-1 release[27] and, more widely, by mitigating or preventing endothelial inflammatory response[28]. PPAR-γ might therefore constitute an attractive target for medical therapy of atherosclerosis. In this extent, beside the known lipid lowering function, statins have been widely reported to exert pleiotropic effects and have been numbered among PPAR-γ agonists [29]. Thus we sought to demonstrate a possible protective role of statin in the AGEs-mediated damage to endothelium of saphenous grafts and in the molecular mechanisms underlying AGEs effects. With an eye on the clinical side, a consistent number of evidences showed the correlation between low levels of s-RAGEs and post percutaneous coronary intervention restenosis[20]. Our group recently published the results of the ARMYDA-AGE study which demonstrated the predictive role of serum AGEs levels in the determinism of restenosis after drug-eluting stent implantation also in a population of diabetics in optimal glycaemic control (Hb1Ac b 6%)[4]. It has been demonstrated by Jinnouchi and colleagues that Atorvastatin decreases the serum levels of AGEs in diabetic patients[30]. Additionally statins are able to influence the balance in the AGE-RAGE axis increasing circulating levels of esRAGE and decreasing the expression of RAGE[31] and therefore mitigating the effect of its activation. We demonstrated that Simvastatin blunts the rise in PMN adhesion and ROS generation following stimulation of saphenous vein endothelial cell culture with AGEs in vitro. Statin might therefore interfere with the aforementioned molecular cascade triggered by AGEs-RAGEs interaction, which is leading to thrombotic phenomena. Indeed, the direct effects resulting from AGE-RAGEs activation include PMN chemotaxis, endothelial adhesion and migration, ROS generation and nitric oxide depletion, and, eventually, NF-κB mediated transcription of inflammatory mediators with consequent increase in endothelial permeability and vasoconstriction [32]. Clearly, as statins has here been shown to reduce RAGEs expression, activation of these multiple pathways would be deeply impaired and therefore the majority of the effects induced by the AGEs-RAGEs axis would become dramatically inhibited [33]. Parallely, the positive induction of PPAR-γ has been shown to exert a further down-regulating activity on RAGEs expression[34] and pharmacological activation of PPAR-γ is widely accepted to have an inhibitory effect on both AGEs formation, throughout antioxidant actions and blockade of glyoxalase-I and –II enzymes, and on RAGEs downstream activation pathway, resulting in reduction of PAI1 and expression of adhesive integrin on the endothelium [35]. Taken together these molecular effects would blunt the well-known selfperpetuating circle converging towards thrombosis. The effect of statins unveiled in the current study was time dependent and was associated to an increase in PPAR-γ induction paralleled by a decrease in RAGEs expression. Whether the final common effect of RAGEs blunting was due to a direct effect of Simvastatin on AGE-RAGEs system or mediated by PPAR-γ induction or by a combination of these activities deserve further studies. Interestingly and similarly to our previous report, in this study we used an internal control constituted by ex vivo explants from diabetic patients treated or not with 20 mg Simvastatin per day for 3 weeks. This regimen was previously demonstrated to be effective and safe in diabetics[36]. This additional control allowed to validate the simulation of diabetic conditions achieved in the in vitro experiments through the

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Fig. 4. Effect of AGEs and Simvastatin on ECs expression of RAGE mRNA. Labels per each sample are reported on the top of the gel. Lower panel shows quantified data of independent determinations. Each column represents the mean + SD of the relative 30 experiments performed for each group. Values are expressed as % relative to internal control house keeping gene glyceraldehyde-3-phosphate dehydrogenase. Figure shows data from one representative experiment. All treatment groups showed statistical significant difference in respect to both the control non treated and the control treated with Simvastatin(P b 0.001) *:significant vs AGEs treated or control.

use of different AGEs concentrations and further demonstrated the effect of statins in AGEs-mediated endothelial damage. Demographic data and laboratory findings including AGEs and Hb1AC levels, inflammatory parameters and oxidative stress markers of both the control and the diabetic population are reported in Table 1. Noteworthy, we could not detect statistical significance differences in hsCRP, MDA and ox-LDL between control patients and diabetics in optimized glycaemic control, thus indicating a similar baseline oxidative and inflammatory status. This finding on a side shelters from potential biases due to unequal inflammatory or oxidative conditions in the interpretation of the results. On the other, is in line with several studies demonstrating that a durable glycaemic control, as testified by Hb1AC b 6%, induces a normalization of both oxidative levels and inflammation parameters in diabetics[7,8]. This phenomenon has been suggested to be due to the antioxidant effect of sulfonylureas, metformin and other hypoglycemic agents that are chronically assumed in long-lasting diabetic disease

[8] as for the patients enrolled in this study. Diabetic controls showed results comparable to the 2 μM AGEs treated groups in all the assays performed. Treatment with Simvastatin decreased the intensity of these inflammatory phenomena with final values similar to the group treated with 2 μM AGEs and 5 mM Simvastatin. AGEs concentrations in the study design were similar to those of our previous report and corresponded to the quantity shown to induce a biological effect comparable to the in vivo situation in diabetic patients [5,11,37]. Also, considering the reported AGEs serum levels in diabetic patients in our and other reports, 2 μM AGEs is a concentration that well matches in in vitro settings the findings described in vivo in the normal blood volume[2,4,5]. Beside the caveat of the limited number of observations, the evidence of a statistical significant dose dependent effect demonstrated in the in vitro set of experiments, allow us to speculate that a more intensive treatment with statin in terms of both dosage and time of exposure might result in enhanced anti-inflammatory effect.

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Fig. 5. Effect of AGEs and Simvastatin ECs expression of PPARγ mRNA. Sample from each treatment group has been run in duplicate. Labels per each sample are reported on the top of the gel. Lower panel shows quantified data of independent determinations. Each column represents the mean + SD of the relative 30 experiments performed for each group. Values are expressed as % relative to internal control house keeping gene glyceraldehyde-3-phosphate dehydrogenase. Figure shows data from one representative experiment. All treatment groups showed statistical significant difference in respect to both the control non treated and the control treated with Simvastatin(P b 0.001). *:significant vs AGEs treated or control.

The observed protective role of Simvastatin on saphenous graft endothelial stability is in accord with previous reports and a beneficial effect of statin in the prevention of saphenous vein graft disease and in the progression of atherosclerosis in native coronary arteries has also been demonstrated [38]. The Post-Coronary Artery Bypass Graft trial showed that aggressive lowering lipid treatment with 80 mg Atorvastatin improved CABG outcomes in terms of saphenous vein graft patency and need for repeat post-surgery revascularization in a average follow up time of 4.3 years. Being based on angiographic study, this trial provides anatomic evidence of a role of statins in the attenuation of saphenous vein graft disease onset and progression, with a reduction in the incidence of new vein graft lesions or occlusions and need for revascularization procedures [39]. Unfortunately, even if in this and other studies the population analyzed included diabetic patients[40], no specific ad hoc evaluation on this particular subgroup is available at the moment. In this context, the set of experiments here performed on ex vivo explants from patients treated with Simvastatin validated the in vitro findings of the study and constituted at the same time a clinical correlate of the molecular mechanisms occurring at the cellular level. Well away from any reductionist view pretending to mimic the complexity of the conditions occurring in vivo by single cell experiments, the concordant results obtained in patients after Simvastatin treatment further remark the potential effectiveness of statins in determining endothelial stability in diabetic patients. The results on RAGEs expression find a correlate in the work of Cuccurullo et al. that reported reduced RAGE expression

in carotid plaques of diabetic patients randomized to simvastatin for 4 months before surgical endoarterectomy[41]. Conversely, we failed to demonstrate a statistical significant increase in PPAR-γ expression in venous tissues from diabetic patients treated with Simvastatin. In attempt to give an explanation of this finding, authors acknowledge that the numerosity of the sample size might have underpowered the experiments to produce a detectable changes in PPAR-γ expression. On the other hand, the dose dependent fashion of the findings obtained in this study might allow to speculate that the dosage of 20 mg/day and the time of exposure (3 weeks) might have been insufficient to produce any detectable effect and that progressive increase in dose and time might induce in in vivo conditions a similarly significant biological change. Alternatively, more potent statins, such as atorvastatin and rosuvastatin, might have a more favorable effect on endothelial function[42] and especially on the induction of PPAR-γ expression[43]. Additional experimental efforts are therefore required in this direction. However, from recent reports and clinical trials, the modulation of PPARs has been more likely attributed to another class of compounds, namely fibrates, pan-PPAR agonists which have been demonstrated to be even superior than statin in reducing cardiovascular events in diabetic patients[44]. This data, together with our findings of a non significant induction of PPAR-γ by statin, might suggest the presence of a more complicated network underneath the PPARs biology and its influence on endothelial homeostasis and corroborate the hypothesis of Tenebaum and colleagues on the need for a more balanced and holistic

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Fig. 6. Exemplificative panel of comparison of data obtained in the in vitro set of experiments with the analogous assays performed on samples from diabetic patients treated or not with Simvastatin 20 mg/day for 3 weeks. NS: non significant. *:P b 0.001.

approach to the activation of PPARs family through the use of combined pharmachological strategy[45]. However, the present study underpins the importance of AGEs plasma levels in surgical management of diabetic patients and remarks the positive effect of Simvastatin on AGEsinduced endothelial damage. Through a hypothetical mechanism defined by an in vitro decrease of RAGE endothelial expression and an induction of PPAR-γ, Simvastatin was able to reduce in saphenous vein grafts from diabetic patients the adhesion of PNMs and the ROS production, which are considered at the basis of graft failure pathogenesis. Despite the safety concerns issued by the FDA on the detrimental effects exerted by statin on tissutal insulin resistance and on disease worsening [46] recent systematic analysis of the literature demonstrated the benefit of statins for cardiovascular events outweighs the small absolute clinical risk[47]. The results of this study remark the biological importance of the pleiotropic effects of statins on diabetesrelated angiopathy and allow to strengthen the possible relevance of their use in this clinical context. A number of different pieces in the complex mosaic of the endothelial pathobiology and homeostasis in diabetes still need to be taken in account and uncovered to individuate the optimal pharmacologic approach, however the results of this study confirm the cornerstone position of statin in this context and in the perioperative management of these patients.

Conflict of Interest Statement Authors declare no conflicts of interest. References [1] Harja E, Bu DX, Hudson BI, Chang JS, Shen X, Hallam K, et al. Vascular and inflammatory stresses mediate atherosclerosis via RAGE and its ligands in apoE−/− mice. J Clin Invest 2008;118:183–94. [2] Chello M, Spadaccio C, Lusini M, Covino E, Blarzino C, De Marco F, et al. Advanced glycation end products in diabetic patients with optimized glycaemic control and their effects on endothelial reactivity: possible implications in venous graft failure. Diabetes Metab Res Rev 2009;25:420–6. [3] Prasad K. Do statins have a role in reduction/prevention of post-PCI restenosis? Cardiovasc Ther 2013;31:12–26. [4] Spadaccio C, Patti G, De Marco F, Coccia R, Di Domenico F, Pollari F, et al. Usefulness of Preprocedural Levels of Advanced Glycation End Products to Predict Restenosis in Patients With Controlled Diabetes Mellitus Undergoing Drug-Eluting Stent Implantation for Stable Angina Pectoris (From the Prospective ARMYDA-AGEs Study). Am J Cardiol 2013 Jul 1;112(1):21–6. http://dx.doi.org/10.1016/j.amjcard.2013.02.046 [Electronic publication ahead of print 2013 Apr 2]. [5] Fukushima Y, Daida H, Morimoto T, Kasai T, Miyauchi K, Yamagishi S, et al. Relationship between advanced glycation end products and plaque progression in patients with acute coronary syndrome: the JAPAN-ACS Sub-study. Cardiovasc Diabetol 2013;12:5. [6] Choudhuri S, Dutta D, Sen A, Chowdhury IH, Mitra B, Mondal LK, et al. Role of Nepsilon- carboxy methyl lysine, advanced glycation end products and reactive

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