The effect of per os colchicine administration in combination with fenofibrate and N-acetylcysteine on triglyceride levels and the development of atherosclerotic lesions in cholesterol-fed rabbits.

OBJECTIVE: Atherosclerosis is a chronic inflammatory disease promoted by pro-inflammatory cytokines produced by NOD-, LRR- and pyrin domain-containing protein 3 (NLRP 3) inflammasome. Colchicine is an anti-inflammatory agent that inhibits inflammasome's action and stabilizes atherosclerotic lesions. N-acetylcysteine (NAC) reduces low-density lipoprotein (LDL) oxidation, metalloproteinase levels, and foam cell count and volume. Fenofibrate also has antioxidant, anti-inflammatory, and anticoagulant properties while also having a beneficial effect on the vasomotor function of the endothelium. The purpose of this study is to investigate the effect of per os colchicine administration in combination with fenofibrate and NAC on triglyceride levels and the development of atherosclerotic lesions in cholesterol-fed rabbits. MATERIALS AND METHODS: Twenty-eight male, 2 months old New Zealand White rabbits were separated into four groups and were fed with different types of diet for 7 weeks: standard, cholesterol 1% w/w, cholesterol 1% w/w plus colchicine 2 mg/kg body weight plus 250 mg/kg body weight/day fenofibrate, and cholesterol 1% w/w plus colchicine 2 mg/kg body weight plus 15 mg/kg body weight/day NAC. Blood samples were drawn from all animals. Lipid profiles were assessed, and interleukin 6 (IL-6) measurements were performed using an enzyme-linked immunosorbent assay (ELISA) kit. Histologic examination was performed on aorta specimens stained with eosin and hematoxylin. Aortic intimal thickness was evaluated using image analysis. RESULTS: Colchicine administration in combination with fenofibrate or NAC statistically significantly reduced the extent of atherosclerotic lesions in aortic preparations. Co-administration of colchicine with NAC has a stronger anti-atherogenic effect than the colchicine plus fenofibrate regimen. Triglerycide levels were decreased in the colchicine plus fenofibrate group and the colchicine plus NAC group at the end of the experiment (p < 0.05), whereas the Cholesterol group had increased levels. A favorable significant lower concentration of IL-6 was detected in the colchicine plus NAC group vs. the other groups. CONCLUSIONS: In an experimental rabbit model, it appears that colchicine statistically significantly reduces the development of atherosclerosis of the aorta, especially in combination with NAC. Colchicine, as an NLRP3 inflammasome inhibitor, and NAC, as an agent that directly targets IL-6 signaling, can reduce the inflammatory risk. Fenofibrate enhances the attenuating role of colchicine on triglyceride levels. Clinical studies should investigate whether similar effects can be observed in humans.

The complex crosstalk between inflammatory cytokines and ventricular arrhythmias.

The network of cytokines consists one of the most extensively studied signaling systems of human body. Cytokines appear to modulate pathogenesis and progress of many different diseases in the human body, particularly in regards to cardiovascular system. However, their effects on the electrical system of the heart has been neglected. Over the past decade, attemps to understand this relationship led to the uncovering of the direct and indirect effects of cytokines on action potential propagation and cell depolarization. This relationship has been depicted in clinical practice as serum levels of cytokines are increasingly associated with prevalence of ventricular arrhythmias either isolated or secondary to either a heart condition or a systemic auto-immune disease. Thus, they present an appealing potential as a biomarker for prediction of arrhythmia generation, as well as the ourtcome of electrophysiological interventions.

The impact of type 2 diabetes and atorvastatin treatment on serum levels of MMP-7 and MMP-8.

AIM: Novel members of matrix metalloproteinases (MMPs), MMP-7 and MMP-8, have emerged as predictors of cardiovascular events. Our study aimed to evaluate serum MMP-7 and MMP-8 concentrations in patients with type 2 diabetes mellitus (T2DM) and the effects of atorvastatin on them. METHODS: We enrolled 85 statin-free subjects with concomitant T2DM and hypercholesterolemia, but without overt micro-/macro-vascular complications (diabetic group - DG). 42 age- and gender-matched healthy subjects without chronic diseases or therapy served as healthy group (HG). All diabetic patients received fix dose of atorvastatin (20 mg/day). Clinical and anthropometrical parameters, lipids, fasting plasma glucose (FPG), serum MMP-7, MMP-8, their inhibitor (TIMP-1), IL-18, hsCRP and insulin resistance (HOMA-IR) were assayed at baseline in all participants and after 3 months in the DG. RESULTS: At baseline, DG showed higher levels of BMI, systolic blood pressure, insulin resistance and FPG compared to HG (p<0.05). Similarly, DG appeared with elevated concentrations of MMP-7 (4.28±1.01 ng/ml vs 2.63±1.11 ng/ml, p<0.001), MMP-8 (73.07±21.96 ng/ml vs. 21.27±10.49 ng/ml, p<0.001) and inflammatory markers (WBC, hsCRP, IL-18, p<0.010). Importantly, atorvastatin treatment improved lipid profile, significantly reduced the concentrations of MMP-7, MMP-8 and inflammatory markers (p<0.01). Moreover, there was considerable suppression of both MMP-7/TIMP-1 and MMP-8/TIMP-1 ratios (p<0.01). In standard multiple regression analysis, the atorvastatin-induced reduction in MMP-7 was independently associated with LDL and IL-18 downregulation (R(2)=0.648, p=0.017). Similarly, IL-18 changes emerged as an independent determinant of MMP-8 alterations (R(2)=0.678, p=0.007). CONCLUSIONS: Hypercholesterolemic patients with T2DM showed elevated MMP-7 and MMP-8 serum concentrations. Atorvastatin reduced the latter concentrations and their ratio with TIMP-1. Those effects seemed mediated by the atorvastatin-induced suppression of inflammatory mediators. ClinicalTrials.gov Identifier: NCT00636766.

The anti-inflammatory effects of exercise training promote atherosclerotic plaque stabilization in apolipoprotein E knockout mice with diabetic atherosclerosis.

Physical exercise is the cornerstone of cardiovascular disease treatment. The present study investigated whether exercise training affects atherosclerotic plaque composition through the modification of inflammatory-related pathways in apolipoprotein E knockout (apoE(-/-)) mice with diabetic atherosclerosis. Forty-five male apoE(-/-) mice were randomized into three equivalent (n=15) groups: control (CO), sedentary (SED), and exercise (EX). Diabetes was induced by streptozotocin administration. High-fat diet was administered to all groups for 12 weeks. Afterwards, CO mice were euthanatized, while the sedentary and exercise groups continued high-fat diet for 6 additional weeks. Exercising mice followed an exercise program on motorized-treadmill (5 times/week, 60 min/session). Then, blood samples and atherosclerotic plaques in the aortic root were examined. A considerable (P<0.001) regression of the atherosclerotic lesions was observed in the exercise group (180.339 ± 75.613 x10(3)µm(2)) compared to the control (325.485 ± 72.302 x10(3)µm(2)) and sedentary (340.188 ± 159.108 x 10(3)µm(2)) groups. We found decreased macrophages, matrix metalloproteinase-2 (MMP-2), MMP-3, MMP-8 and interleukin-6 (IL-6) concentrations (P<0.05) in the atherosclerotic plaques of the exercise group. Compared to both control and sedentary groups, exercise training significantly increased collagen (P<0.05), elastin (P<0.001), and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) (P<0.001) content in the atherosclerotic plaques. Those effects paralleled with increased fibrous cap thickness and less internal elastic lamina ruptures after exercise training (P<0.05), while body-weight and lipid parameters did not significantly change. Plasma MMP-2 and MMP-3 concentrations in atherosclerotic tissues followed a similar trend. From our study we can conclude that exercise training reduces and stabilizes atherosclerotic lesions in apoE-/- mice with diabetic atherosclerosis. A favorable modification of the inflammatory regulators seems to explain those beneficial effects.

The differential anti-inflammatory effects of exercise modalities and their association with early carotid atherosclerosis progression in patients with type 2 diabetes.

OBJECTIVE: Adipokines, visfatin, apelin, vaspin and ghrelin have emerged as novel cardiovascular risk factors. We aimed to evaluate the effects of different exercise modalities on the aforementioned novel adipokines and carotid intima-media thickness in patients with Type 2 diabetes mellitus. METHODS: One hundred patients with Type 2 diabetes were equivalently (n = 25) randomized into four groups: (1) a control group with patients encouraged to perform self-controlled exercise; (2) a supervised aerobic exercise group (exercise four times/week, 60 min/session, 60-75% of maximum heart rate); (3) a resistance training group (60-80% baseline maximum load achieved in one repetition); and (4) a combined aerobic exercise plus resistance training group, as in groups 2 and 3. All participants had HbA(1c) levels ≥ 48 mmol/mol (≥ 6.5%), without overt diabetic vascular complications. Blood samples, clinical characteristics, peak oxygen uptake and carotid intima-media thickness measurements were obtained at baseline and at the end of the study, after 6 months. RESULTS: At baseline, there were non-significant differences between groups. All active groups significantly ameliorated glycaemic profile, insulin sensitivity and triglycerides levels compared with the control group (P < 0.05). Aerobic training further improved lipids, systolic blood pressure and exercise capacity compared with the resistance training and the control groups (P < 0.05). Moreover, high-sensitivity C-reactive protein and visfatin decreased, while vaspin and apelin circulating levels increased within the aerobic exercise group and the aerobic exercise plus resistance training group, and compared with the other groups (P < 0.05). Within- and between-group comparisons showed negligible alterations in ghrelin serum levels and body weight after all exercise modalities. Finally, aerobic training attenuated the carotid intima-media thickness progression (0.017 ± 0.006 mm) compared with the control subjects (0.129 ± 0.042 mm, P < 0.001). That effect was independently associated with visfatin and amelioration of peak oxygen uptake. CONCLUSIONS: In subjects with Type 2 diabetes, all exercise training modalities improved metabolic profile. Importantly, aerobic training predominantly ameliorated adipokines concentrations and carotid intima-media thickness progression.

Effects of rosiglitazone/metformin fixed-dose combination therapy and metformin monotherapy on serum vaspin, adiponectin and IL-6 levels in drug-naïve patients with type 2 diabetes.

OBJECTIVE: Vaspin, adiponectin and interleukin-6 (IL-6) constitute novel adipose-tissue derivatives, known as adipokines, which mediate insulin resistance. The aim of the present study was to evaluate the effects of metformin and rosiglitazone on serum levels of those novel adipokines in drug-naïve patients with type 2 diabetes mellitus (T2DM). METHODS: 140 patients with T2DM, already treated with diet, but without adequate glycemic control (HbA1c > 7%), were randomly assigned to: RSG+MET group, (n = 70): Combination therapy with fixed dose of 4 mg rosiglitazone plus 500 mg metformin. MET group, (n = 70): Half-maximum dose of metformin monotherapy (1 700 mg/day). Before and after 6-month treatment, body-mass index (BMI), blood pressure (BP), fat-mass, fasting plasma glucose (FPG), HbA1c, insulin resistance indexes (HOMA-IR, insulin), lipids, high-sensitivity CRP (hsCRP), vaspin, adiponectin, and interleukin-6 (IL-6) were measured. RESULTS: Glucose regulation and insulin resistance were equivalently improved from baseline within both groups (p < 0.05). There was a considerable amelioration of hsCRP, WBC, adiponectin, IL-6, systolic and diastolic BP with rosiglitazone/metformin combined treatment as compared to baseline (p < 0.05) and MET group (p < 0.05). In contrast, metformin monotherapy significantly reduced BMI (p < 0.001), total-cholesterol (p = 0.012) and LDL (p = 0.020) levels compared to RSG+MET group. Importantly, serum vaspin concentration was equivalently decreased from baseline in both RSG+MET (-0.96 ± 0.75 ng/ml, p < 0.001) and MET (-0.92 ± 0.57 ng/ml, p=0.001) group. The aforementioned vaspin changes correlated with changes in WHR, HbA1c, FPG, HOMA-IR, insulin, IL-6 (only in the RSG+MET group) and fat-mass. In standard multiple regression analysis, FPG, HbA1c, HOMA-IR and insulin remained independent determinants of serum vaspin levels changes (R² = 0.836, p = 0.004). CONCLUSIONS: Both rosiglitazone/metformin combination therapy and metformin monotherapy decreased serum vaspin levels through glucose and insulin sensitivity regulation, while they exerted differential effects on adiponectin, IL-6 and other cardiovascular risk factors in drug-naïve patients with T2DM.

The role of ex-vivo gene therapy of vein grafts with Egr-1 decoy in the suppression of intimal hyperplasia.

OBJECTIVES: To test the hypothesis that vein graft intimal hyperplasia can be significantly suppressed by a single intra-operative transfection of the graft with a decoy oligonucleotide (ODN) binding the transcription factor Egr-1. DESIGN: Experimental study. MATERIALS AND METHODS: Jugular vein to carotid artery interposition grafts in rabbits were treated with Egr-1 decoy, mutant decoy ODN, vehicle alone, using a non-distending pressure of 300 mm Hg for 20 min, or were left untreated. All animals were fed a 2% cholesterol diet. The animals were sacrificed after 48h, 6 weeks and 12 weeks. Paraffin-embedded vein sections were subjected to angiometric analysis. RESULTS: Successful delivery of the ODN was confirmed by DAPI staining. Quantitative real-time PCR revealed a 60% decrease of the Egr-1 gene expression in the animals in which the Egr-1 decoy ODN was delivered. Cellular proliferation was also significantly decreased as indicated by the Ki-67 labelling index. An increase in intimal and medial thickness was found in all vein grafts. However, intimal thickness was significantly reduced in the grafts treated with Egr-1 decoy ODN, whereas luminal area was significantly increased. CONCLUSION: A single intra-operative pressure-mediated transfection of vein grafts with Egr-1 decoy ODN significantly suppresses intimal hyperplasia in a rabbit hypercholesterolaemic model.

Exercise ameliorates serum MMP-9 and TIMP-2 levels in patients with type 2 diabetes.

AIM: This study assessed the impact of regular exercise on inflammatory markers (high-sensitivity C-reactive protein [hsCRP], fibrinogen), and matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), in patients with type 2 diabetes mellitus (T2DM). PATIENTS: Fifty overweight patients with T2DM were randomly assigned to two groups: (A) an exercise group (EXG, n=25), with self-controlled exercise for at least 150 min/week and one additional supervised exercise session/week; and (B) a control group (COG, n=25), with no exercise instructions. All participants were taking oral antidiabetic drugs, and none had diabetic complications. Clinical parameters, exercise capacity (VO(2 peak)), ventilatory threshold (VT), insulin resistance indices (fasting insulin, HOMA-IR, HOMA%S), hsCRP, fibrinogen, MMP-2, MMP-9, TIMP-1 and TIMP-2 were assessed at baseline and after 16 weeks. RESULTS: No significant changes were found in body mass index, waist/hip ratio, insulin-resistance indices, MMP-2 and TIMP-1 throughout the study in either group (P>0.05). Compared with controls, the EXG showed a significant decrease in systolic and mean blood pressure, total and LDL cholesterol, and HbA(1c) (P<0.05). Also, exercise significantly suppressed levels of fibrinogen (P=0.047), hsCRP (P=0.041) and MMP-9 (P=0.028), and the MMP-9-to-TIMP-1 ratio (P=0.038), whereas VO(2 peak) (P=0.011), VT (P=0.008) and plasma TIMP-2 levels (P=0.022) were considerably upregulated in the EXG vs. COG. Standard multiple-regression analyses revealed that MMP-9 changes were independently associated with fibrinogen and HbA(1c) changes, while fibrinogen changes independently predicted TIMP-2 alterations with exercise. CONCLUSION: Mostly self-controlled exercise of moderate intensity ameliorated serum levels of pro- and anti-atherogenic markers in patients with T2DM, with no effects on body weight. These data offer further insight into the cardioprotective mechanisms of exercise in patients with T2DM.

Visfatin (nampt) and ghrelin as novel markers of carotid atherosclerosis in patients with type 2 diabetes.

OBJECTIVE: Visfatin (nampt) and ghrelin are the most recently identified adipocytokines, but their role in atherosclerosis is poorly clarified. In our study we investigated their association with advanced carotid atherosclerosis and carotid intima-media thickness (CIMT) in patients with type 2 diabetes mellitus (T2DM). METHODS: 122 patients (50 males) with T2DM, aged 55-70 were enrolled. Sixty-four age- and sex-matched healthy individuals served as controls (group A). CIMT was assayed in all participants by ultrasound. Among diabetic patients, 47 appeared with carotid plaques (group B), while 75 without plaques (group C). Anthropometric parameters, blood pressure, glycemic and lipid profile, high-sensitivity CRP (hsCRP), insulin resistance (HOMA-IR), fibrinogen, nampt and ghrelin were measured. RESULTS: Diabetic patients had a higher mean-CIMT, increased body-mass index, worse lipid profile, elevated blood pressure and higher levels of white blood cells count, nampt and hsCRP with respect to controls (p<0.01). Among diabetic patients, groups B and C were comparable in anthropometric, glycemic and lipid parameters. Serum nampt was significantly higher in group B rather than in groups A and C (p<0.05). On the other hand, ghrelin levels were considerably lower only in diabetic patients with carotid atherosclerosis compared with healthy individuals. In univariate analysis, mean-CIMT correlated with age (r=0.312; p=0.003), nampt (r=0.341; p<0.001) and ghrelin (r=-0.421; p=0.002) and the latter associations remained significant in multiple regression analysis. CONCLUSIONS: High nampt and low ghrelin serum levels are significantly associated with advanced carotid atherosclerosis in patients with T2DM. Moreover these adipocytokines are independently associated with CIMT, implicating their role as novel atherosclerotic biomarkers and providing another important link between adiposity and atherosclerosis.

Beneficial changes of serum calcification markers and contralateral carotid plaques echogenicity after combined carotid artery stenting plus intensive lipid-lowering therapy in patients with bilateral carotid stenosis.

OBJECTIVES/DESIGN: In symptomatic patients treated with ipsilateral carotid artery stenting (CAS) plus intensive lipid lowering, we assessed the changes of osteopontin (OPN), osteoprotegerin (OPG) and the Gray-Scale Median (GSM) score contralateral to symptomatic carotid stenosis. MATERIALS/METHODS: Forty-six symptomatic patients (group A) with significant carotid stenosis (North American Symptomatic Carotid Endarterectomy Trial (NASCET): >70%) underwent ipsilateral CAS. Those patients had simultaneously contralateral low-grade carotid stenosis (NASCET: 30-69%). Group B included 67 symptomatic patients with low-grade bilateral carotid stenosis (NASCET: 30-69%), but without indications for revascularisation. All patients were treated with atorvastatin (10-80mg) to target low-density lipoprotein (LDL)<100mgdl(-1). Blood samples and plaques' GSM score contralateral to brain infarct were assayed at baseline and after 6 months. RESULTS: At baseline, there were no significant differences between groups (p>0.05). Six-month atorvastatin treatment equivalently improved lipid profile in both groups (p<0.05). The parameters hsCRP, OPN and OPG were significantly down-regulated within both groups, but to a greater extent in group A (p<0.05). Besides this, contralateral GSM score was significantly improved from baseline in both groups (p<0.01), but that increment was more pronounced in group A (vs. group B; p=0.041). These changes were inversely correlated with changes in OPN (p=0.014), OPG (p=0.011) and LDL (p=0.041). CONCLUSION: Ipsilateral CAS plus intensive lipid-lowering therapy was associated with enhanced contralateral carotid plaque stability and attenuated inflammatory burden and calcification inhibitors to a greater extent than atorvastatin therapy alone in patients with bilateral carotid stenosis.

Intensive lipid-lowering therapy ameliorates novel calcification markers and GSM score in patients with carotid stenosis.

OBJECTIVES/DESIGN: Carotid plaque echogenicity quantified by the Gray-Scale Median (GSM) score has been associated with plaque vulnerability. The aim of this study was to assess whether intensive lipid-lowering treatment with atorvastatin in patients with carotid artery stenosis ameliorates novel vascular calcification inhibitors, such as osteopontin (OPN) and osteoprotegerin (OPG), and improves GSM score. METHODS: Ninety-seven patients with carotid stenosis (>40%), but without indication for intervention, were treated for 6 months with atorvastatin (10mg-80mg) to target LDL<100mg/dl. Fifty-two age-and sex-matched healthy individuals served as the control group. Blood samples and GSM were obtained at the beginning and after 6 months. RESULTS: Systolic blood pressure, hsCRP, fibrinogen, OPN and OPG levels differed significantly between patients with carotid stenosis and healthy controls at baseline (p<0.05). Atorvastatin treatment improved lipid profile and significantly reduced hsCRP (p=0.002), WBC count (p=0.041), OPN (p<0.001) and OPG levels (p<0.001). GSM score increased considerably after atorvastatin therapy (from 58.33+/-24.38 to 79.33+/-22.3; p<0.001) and that effect appeared related to OPN (p=0.001), OPG (p=0.013) and LDL (p=0.01) reduction. CONCLUSIONS: In patients with carotid stenosis, intensive lipid-lowering therapy with statins attenuates serum OPN and OPG levels and enhances carotid plaque echogenicity, outlining their beneficial effects on plaque stability.