Mohsen Kerkeni

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.

Hyperhomocysteinaemia, methylenetetrahydrofolate reductase polymorphism and risk of coronary artery disease.

Background: Hyperhomocysteinaemia is an independent, graded risk factor for coronary artery disease (CAD). The methylenetetrahydrofolate reductase (MTHFR) polymorphism is associated with hyperhomcysteinaemia and may therefore influence individual susceptibility to CAD. We have investigated this risk factor in a Tunisian Arab population. Methods: Polymerase chain reaction-restriction fragment length polymorphism analysis was used to detect the C677T and A1298C variants of the MTHFR gene in 100 patients with CAD and 120 healthy controls. The severity of CAD was expressed as the number of affected vessels. Plasma total homocysteine (tHcy) concentration was determined using a direct chemiluminescence assay. Results: MTHFR CC, CT and TT genotype frequencies in the CAD group were significantly different from those observed in the control group (49%, 35% and 16% versus 48.3%, 45.8% and 5.8%, respectively; P=0.031). However, MTHFR AA, AC and CC genotypes frequencies in the CAD group were not significantly different from the control group ( P = 0.568). Patients with CAD showed higher plasma tHcy concentrations than patients without CAD (15.86±8.63 μmol/L versus 11.90 ± 3.25 μmol/L, P<0.001). There was no association between the MTHFR polymorphisms and the number of stenosed vessels. Patients with the MTHFR TT genotype had higher plasma tHcy, serum creatinine, cholesterol and triglyceride concentrations than patients with the MTHFR CC genotype. Conclusions: The C677T polymorphism of the MTHFR gene is associated with hyperhomocysteinaemia, lipid dysregulation and the presence of CAD in this Tunisian Arab population.

Pentosidine as a biomarker for microvascular complications in type 2 diabetic patients

Serum soluble receptor for advanced glycation end product (sRAGE) may reflect the activity of the advanced glycation end product (AGE)–receptor for advanced glycation end product (RAGE) axis, which has been proposed as a potential mechanism linking hyperglycaemia to vascular complications in diabetes. We have investigated whether serum AGEs, sRAGE and pentosidine levels were increased and correlated with microvascular complications in type 2 diabetes mellitus (DM). We included 30 healthy control subjects, and 200 diabetic patients were divided into two subgroups: 100 patients with diabetic retinopathy and 100 patients with diabetic nephropathy. AGEs, sRAGE and pentosidine were measured in serum by enzyme-linked immunosorbent assay (ELISA). Serum AGEs, sRAGE and pentosidine levels were significantly increased in diabetic patients with retinopathy and in diabetic patients with nephropathy compared to control subjects (p < 0.001). Serum AGEs, sRAGE and pentosidine levels are positively associated with microvascular complications in type 2 DM. Multiple regression analysis reveals serum pentosidine as an independent determinant of the presence of diabetic retinopathy (p = 0.004) and the presence of hypertension (p = 0.018) and hyperlipidaemia (p = 0.036). Pentosidine levels may be a biomarker for microvascular complications in type 2 diabetic patients.

Elevated serum levels of AGEs, sRAGE, and pentosidine in Tunisian patients with severity of diabetic retinopathy

OBJECTIVES The advanced glycation end products (AGEs)-receptor for AGE (RAGE) axis activity are implicated in diabetic vascular complications. We measured serum AGE, sRAGE and pentosidine levels in Tunisian patients with diabetic retinopathy (DR) and examined whether these biomarkers are related to the severity of DR. DESIGN AND METHODS We included 30 healthy control subjects and 100 diabetic patients were divided into 2 subgroups: 40 patients with nonproliferative diabetic retinopathy (NPDR), and 60 patients with proliferative diabetic retinopathy (PRD). AGEs, sRAGE and pentosidine were measured in serum by ELISA. RESULTS Serum levels of AGEs, sRAGE and pentosidine were significantly increased in patients with diabetes mellitus compared to nondiabetic controls (P<.01, P<.001, P<.001 respectively). In diabetic patients, serum AGEs, sRAGE and pentosidine levels were significantly higher in patients who had PDR than in those with NPDR (P=.001, P=.01, P=.005 respectively). Furthermore, in stepwise multivariate regression analysis, the levels of pentosidine and duration of diabetes were independently associated with severity of DR. CONCLUSION Serum AGEs, sRAGE, and pentosidine levels are related with the presence of DR. Duration of diabetes and pentosidine were independently correlated with the severity of DR.

Hyperhomocysteinaemia and parameters of antioxidative defence in Tunisian patients with coronary heart disease

Abstract Background An imbalance between oxidative damage and antioxidative protection in association with the pathophysiology of atherosclerosis has been suggested. The aim of this study was to test the parameters of antioxidative defence and to assess their association with hyperhomocysteinaemia and the severity of coronary heart disease (CHD) in Tunisian patients. Methods The study population included 100 patients with CHD and 120 healthy controls. The severity of CHD was expressed as the number of affected vessels. Superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity and total antioxidant status (TAS) concentrations were measured using commercially available methods. Plasma total homocysteine (tHcy) concentration was determined by direct chemiluminescence assay. Serum zinc (Zn) was measured by a colorimetric method. Results Compared with healthy control subjects, patients with CHD had significantly lower activities of SOD (P < 0.01), GPx (P < 0.001), and serum Zn concentrations (P < 0.001) and significantly higher tHcy concentration (P < 0.001). However TAS concentrations were not significantly different between the groups. SOD and GPx activities were negatively correlated with tHcy concentration (P < 0.05, P < 0.001, respectively). Patients with hyperhomocysteinaemia showed a lower GPx and SOD activities than patients with normohomocysteinaemia. Antioxidant enzyme activities tended to be decreased in CHD patients presenting with 0- to 3-vessel stenosis. Conclusions This study indicates that low activity of GPx, SOD and Zn concentration are associated with CHD patients. We hypothesize that hyperhomocysteinaemia and low antioxidant enzyme activities may increase the extent of CHD.

Increased serum concentrations of pentosidine are related to presence and severity of coronary artery disease

BACKGROUND There are limited data regarding the contribution of advanced glycation end products (AGEs) in the presence of coronary artery disease (CAD). We investigated whether serum pentosidine and Nε-carboxymethyllysine (CML) were related to the presence and the severity of CAD. METHODS 69 Tunisian patients with CAD (≥ 50% obstruction in ≥ 1 coronary artery), 32 Tunisian patients without CAD but with potential cardiovascular risk factors and 60 Tunisian control subjects were included in a cross-sectional study. Patients were classified as CAD and non CAD patients according to angiographic results. The severity of CAD was assessed using the Gensini score. Serum pentosidine and CML were measured by LC-MS/MS. RESULTS Serum pentosidine and CML concentrations were significantly higher in non-CAD patients vs control subjects (P<0.001). Serum pentosidine concentrations were significantly higher in CAD patients vs non-CAD patients (P<0.001). A multiple logistic regression analysis demonstrated that pentosidine was independently associated with the presence of CAD (OR=1.52, 95% CI: 1.12-2.07, P=0.007). The area under curve (AUC) determined by ROC analysis was 0.74 (95% CI: 0.64-0.84, P<0.001) and the optimal cut-off value of pentosidine to predict the presence of CAD was 3.2 μmol/mol Lys, with 64% sensitivity and 78% specificity. Furthermore, in a multivariate stepwise regression analysis, pentosidine was independently correlated with Gensini score (standardized β= 0.46, 95% CI: 0.70-1.99, P<0.001). CONCLUSIONS High concentrations of pentosidine show the presence and the severity of CAD with high sensitivity.

Endothelial nitric oxide synthetase, methylenetetrahydrofolate reductase polymorphisms, and cardiovascular complications in Tunisian patients with nondiabetic renal disease.

OBJECTIVES The relationship between endothelial nitric oxide synthase (eNOS), methylenetetrahydrofolate reductase (MTHFR) gene polymorphisms and cardiovascular disease (CVD) in Tunisian patients with chronic renal disease (CRD) has not been examined. We investigated (a) the relationship of these gene polymorphisms with the presence and the severity of renal disease, and (b) their relationships with CVD in these patients. DESIGN AND METHODS We used PCR-RFLP analysis to detect the eNOS G894T, MTHFR C677T and A1298C variants in 100 patients with CRD and in 120 healthy controls. RESULTS MTHFR C677T and A1298C polymorphisms were not associated with the presence of renal disease. However, we found that eNOS G894T polymorphism was associated with the presence and severity of renal disease and with CVD in CRD patients (P=0.028, P=0.018, P=0.016 respectively). We showed that 894T allele was an independent risk factor of severity of renal disease and the incidence of CVD (P=0.01 and P<0.01 respectively). CONCLUSION The G894T polymorphism of the eNOS gene is associated with severity of renal disease. Presence of the 894T allele aggravated renal damage and increased the incidence of CVD in Tunisian CRD patients.

Plasma Levels of Pentosidine, Carboxymethyl-Lysine, Soluble Receptor for Advanced Glycation End Products, and Metabolic Syndrome: The Metformin Effect

Metabolic syndrome (MetS) is considered one of the most important public health problems. Several and controversial studies showed that the role of advanced glycation end products (AGEs) and their receptor in the development of metabolic syndrome and therapeutic pathways is still unsolved. We have investigated whether plasma pentosidine, carboxymethyl-lysine (CML), and soluble receptor for advanced glycation end products (sRAGE) levels were increased in patients with MetS and the effect of metformin in plasma levels of pentosidine, CML, and sRAGE. 80 control subjects and 86 patients were included in this study. Pentosidine, CML, and sRAGE were measured in plasma by enzyme-linked immunosorbent assay (ELISA). Plasma pentosidine, CML, and sRAGE levels were significantly increased in patients compared to control subjects (P < 0.001, P < 0.001, and P = 0.014, resp.). Plasma levels of pentosidine were significantly decreased in patients who received metformin compared to untreated patients (P = 0.01). However, there was no significant difference between patients treated with metformin and untreated patients in plasma CML levels. Plasma levels of sRAGE were significantly increased in patients who received metformin and ACE inhibitors (P < 0.001 and P = 0.002, resp.). However, in a multiple stepwise regression analysis, pentosidine, sRAGE, and drugs treatments were not independently associated. Patients with metabolic syndrome showed increased levels of AGEs such as pentosidine and CML. Metformin treatment showed a decreased level of pentosidine but not of CML. Therapeutic pathways of AGEs development should be taken into account and further experimental and in vitro studies merit for advanced research.

Increased serum homocitrulline concentrations are associated with the severity of coronary artery disease.

Abstract Background: Carbamylation is a non-enzymatic post-translational modification of proteins that has been recently identified as a non-traditional risk factor for atherosclerosis. The aim of this study was to determine whether serum homocitrulline (HCit), a characteristic carbamylation-derived product, was related to the presence and the severity of coronary artery disease (CAD). Methods: Forty-five control subjects and 109 patients were included in this cross-sectional study. After coronary angiography, the patients were classified as non-CAD patients (patients with normal arteries, n=33) and CAD patients (n=76). The severity of CAD was then evaluated using the Gensini scoring system. Serum total HCit concentrations were determined by LC-MS/MS. Results: Serum HCit concentrations were significantly (p<0.001) higher in CAD patients than in control or non-CAD subjects. The receiver operating characteristic curve analysis showed an area under the curve equal to 0.908 (95% confidence interval, 0.853–0.964, p<0.001) and a threshold HCit concentration of 0.16 mmol/mol Lys for predicting the presence of CAD (78.9% sensitivity and 78.8% specificity). HCit concentrations significantly (p<0.001) increased concomitantly with the severity of CAD and were positively correlated with Gensini scores (r=0.725, p<0.001) as well as with the number of stenotic coronary arteries (p<0.001). Furthermore, in a multiple stepwise regression analysis, HCit was significantly (p<0.001) and independently associated with the presence of CAD, the Gensini score, and the number of stenotic arteries (standardized β values of 0.525, 0.722, and 0.642, respectively). Conclusions: Our results demonstrate that serum HCit concentrations are increased during CAD and are positively associated with the severity of the disease.

Hyperhomocysteinemia, paraoxonase concentration and cardiovascular complications in Tunisian patients with nondiabetic renal disease

OBJECTIVES Hyperhomocysteinemia is associated with an increased risk of cardiovascular diseases. We determine homocysteine levels (Hcy), paraoxonase (PON1) concentration and their relationship on cardiovascular complications in patients with chronic renal disease (CRD). DESIGN AND METHODS The study population included 100 CRD patients and 120 healthy controls. Renal function was assessed using the eGFR by the MDRD study equation. Patients were considered to have CRD when the eGFR was <60 mL/min/1.73 m(2). Hcy concentrations were determined by direct chemiluminescence assay. PON1 concentration was measured spectrophotometrically using phenylacetate as a substrate. RESULTS We found an increased Hcy levels and a decreased eGFR and PON1 concentration in CRD patients compared to the control group (P<0.001, P<0.001, P<0.01 respectively). Patients with cardiovascular complications showed an increased Hcy levels and a lower PON1 concentration than patients without cardiovascular complications (P<0.001, P<0.01 respectively). CONCLUSION We showed that hyperhomocysteinemia and low PON1 concentration are associated with CRD and markedly associated in patients with cardiovascular complications. Additional effects contribute to the severity of renal disease and increase the incidence of cardiovascular disease.