Apoptosis is a form of cell death which is important in many physiological processes. Four apoptotic mechanisms have been identified but two have been well examined: the intrinsic and the extrinsic mechanism. Due to many pro/antiapoptotic factors, these processes take place on a physiologically useful level. In cases of apoptosis dysregu lation, illnesses occur such as neurodegenerative diseases combined with an increased level of cell death or cancerogenesis associated with uncontrolled cell proliferation. Apoptosis can be triggered by the activation of the first caspase in a series and stopped by its deactivation, which represents a new challenge: determining the »point of no return«. Besides the antiapoptotic proteins (Bcl 2, Bcl XL), a family of proteins called the Inhibitors of Apoptosis Proteins (IAPs) play a key role in the regulation of apoptosis. Members of the IAP family are: cIAP1, cIAP2, XIAP, Survivin, Livin and TsIAP. Domain BIR is the most important in the IAP structure since it determines their specificity for caspases. The interaction of IAPs with caspases is complex and not completely understood, however, IAPs are considered to be important target proteins in the therapy of tumor and autoimmune diseases.
Dušan Lazarević, Vladimir Đorđević, Vladan Ćosić, Predrag Vlahović, Suzana Tošić-Golubović, Tatjana Ristić and Vidosava Đorđević
Increased Lymphocyte Caspase-3 Activity in Patients with Schizophrenia
A growing body of evidence indicates that cortical brain cells of schizophrenic patients are vulnerable to apoptosis. As apoptosis is an important mechanism in organism modeling during development, active since the early phase of intrauterine life, it could be involved in the pathogenesis of schizophrenia. To test this hypothesis, caspase-3 activity was determined in peripheral blood mono nuclear cells from 30 patients with schizophrenia and from 30 age and gender matched healthy subjects by a colorimetric commercially available kit. Consistent with increased susceptibility to apoptosis, caspase-3 activity in lymphocytes of patients with schizophrenia was significantly increased (0.111±0.055 μmol/mg protein, p<0.05) in comparison with those in the matched control group (0.086±0.030 μmol/mg protein). The highest activity was obtained in the group showing almost equally positive and negative symptoms (0.159±0.096 μmol/mg protein) and it was significantly higher (p<0.05) compared to the group with a relative predomination of positive symptoms (0.100±0.029 μmol/mg protein). Caspase-3 activity in patients receiving typical antipsychotic drugs (0.124± 0.071 μmol/mg protein) was not significantly different from that in patients treated with atypical antipsychotics (0.104±0.039 μmol/mg protein). To our knowledge to date, this has been the first demonstration that there is a significant increase in caspase-3 activity, determined in native cells, in patients with schizophrenia, indicating a dysregulated apoptotic mechanism in this disease.
Vidosava Đorđević, Tatjana Ristić, Vladan Ćosić, Predrag Vlahović, Lilika Zvezdanović and Gordana Đorđević
Inflammatory and Apoptotic Markers in Ischemic Heart Disease Patients
Ischemic heart disease is the most frequent cause of cardiovascular morbidity and mortality. It is developed on the basis of atherosclerosis which is today considered a chronic inflammatory disease. It is documented by an increase in inflammatory and immune biomarkers, such as C-reactive protein, fibrinogen, neopterin, leukocytes, lymphocytes and others, that are significantly changed in patients with unstable angina or acute myocardial infarction. CRP is mostly studied. Increased concentrations of CRP are associated with a series of risk factors. CRP may predict recurrent events and mortality independently of cardiac troponin levels, and it is also an independent predictor of a cardiovascular event after adjustment for traditional risk factors. Although CRP currently appears to be the most promising biological marker, there is still controversy regarding its use in clinical practice. Both necrotic and apoptotic cell death are documented during atherogenesis, however, limited data are available about apoptotic markers in ischemic heart disease patients. Increasing evidence supports the existence of apoptotic death initiated by ligation of membrane-bound death receptors or by release of cytochrome c from mitochondria, as well as their regulators in the heart. The studies of serum markers show that the apoptotic process is disregulated in ischemic heart disease patients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is present in stable atherosclerotic lesions, is increased in vulnerable plaques, but its serum levels are reduced significantly in patients with unstable angina. Serum Fas concentrations are increased and FasL are decreased in subjects at high cardiovascular risk. The results of our study show significant changes in serum Fas, FasL, and Bcl-2 concentrations, and lymphocyte caspase-3 activity in different stages of ischemic heart disease. For now, there is evidence that statins are effective in the regulation of some apoptotic markers. The better understanding of the pathways of apoptosis and their regulation is promissing in yielding novel therapeutic targets for cardiovascular disease.
Tatjana Cvetković, Radmila Pavlović, Vidosava Đorđević, Ivana Stojanović, Radmila Veličković-Radovanović, Aleksandra Ignjatović, Nikola Stefanović, Slavoljub Živanović and Vidojko Đorđević
Decreased nitric oxide (NO) production and/or impaired NO bioavailability may occur in patients with the chronic kidney disease (CKD), and could contribute to elevation of blood pressure, cardiovascular disease (CVD) and progression of renal injury in these patients. Free guanidinomethylated arginine residues occur endogenously as a result of proteolysis of post-translational methylated tissue proteins. The asymmetric dimethyl arginine (ADMA) is a competitive inhibitor of the nitric oxide synthase (NOS) enzymes. The kidney has a predominant role in ADMA elimination by combining two mechanisms; urinary excretion and metabolization of ADMA The degradation of ADMA is accomplished intracellularly by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). ADMA is not only a uremic toxin, but also a strong marker of the endothelial dysfunction and atherosclerosis and a stronger independent predictor of all-cause mortality and cardiovascular outcome in patients with the chronic renal failure. There are at least four mechanisms that may explain the accumulation of ADMA in CKD: increased methylation of proteins, increased protein turnover, decreased metabolism by DDAH and impaired renal excretion. A strong positive correlation between symmetric dimethyl arginine (SDMA) and creatinine suggests that SDMA might be of value as a marker of the renal function. Reduced NO elaboration secondary to accumulation of ADMA and elevated inflammation may be important pathogenic factors for endothelial dysfunction in patients with the renal disease. Elevation of ADMA may be a missing link between CVD and CKD.
Lilika Zvezdanović, Vidosava Đorđević, Vladan Ćosić, Tatjana Cvetković, Slavica Kundalić and Aleksandra Stanković
The Investigation of Cytokines and Oxidative Stress in Patients with Systemic Lupus Erythematosus
Numerous factors can influence the onset of SLE and development of some clinical disease manifestations with various organ involvements and occurrence of characteristic symptoms and disease signs. This paper studies the balance between proinflammatory and antiinflammatory cytokines, investigates the presence of oxidative stress measuring certain prooxidative factors and determines the activation of antioxidative protection pathways aiming to establish possible correlations between the studied parameters. ELISA, enzymatic spectrophotometry and colorimetric methods were used to determine the above-mentioned parameters. The results obtained indicate that disturbed pro/antioxidative status is associated with the change of antioxidative factors, with the fall od SOD activity and increase of GPx and CAT activity in the erythrocytes of all studied groups of patients. At the same time, the cytokine production was altered, not only compared to the healthy control samples, but also in various clinical disease manifestations. Altered relationships of pro and antiinflammatory cytokines and the consequential disorders of other studied systems provide us with useful strategic targets for diagnostic monitoring and possible therapeutic interventions in SLE patients.
Tatjana Ristić, Vladan Ćosić, Predrag Vlahović, Marina Deljanin-Ilić and Vidosava Đorđević
Could Lymphocyte Caspase-3 Activity Predict Atherosclerotic Plaque Vulnerability?
Apoptotic cell death may play a critical role in a variety of cardiovascular diseases, especially in those developing on the basis of atherosclerosis. The goal of this study was to compare the activity of caspase-3 in different forms of ischemic heart disease and to correlate caspase-3 activity with inflammatory and lipid markers as well as risk factors. This enzyme activity was determined in peripheral blood mononuclear cells (PBMC) of 30 patients with stable angina pectoris (SAP), 27 with unstable angina (USAP), 39 with acute ST-elevation myocardial infarction (STEMI) and 27 healthy volunteers by a colorimetric commercially available ELISA method. In the SAP group caspase-3 activity was 0.093±0.036 μmol/mg protein, in patients with STEMI it was 0.110±0.062 μmol/mg protein, and both values were insignificantly higher in comparison with controls (0.092±0.022 μmol/mg protein). In PBMC of USAP patients caspase-3 activity (0.122±0.062 μmol/mg protein) was significantly higher (p<0.05) compared to the control group. In SAP patients caspase-3 activity showed a significant positive correlation with triglicerydes (p<0.05). Caspase-3 activity may be a valid parameter for assessing the atherosclerotic plaque activity, and a new target for therapeutic intervention.
Vidosava B. Đorđević, Ivana Stojanović, Slavica Kundalić, Tatjana Ristić, Radmila Pavlović, Vladan Ćosić and Tatjana Cvetković
Nitric oxide (NO) is produced by many cells in the body; however, its production by vascular endothelium is particularly important in the regulation of blood flow. Vascular actions of NO include the following: direct vasodilation, indirect vasodilation by inhibiting the vasoconstrictor influences, anti-thrombotic, anti-inflammatory and anti-proliferative effects. Due to its importance in vascular function, abnormal production of NO, occurring in different diseases can adversely affect blood flow and other vascular functions. It has been suggested that alterations in NO generation are a critical cause of injury in the ischemic heart. A biologic link between the endothelial damage and atherosclerotic coronary arterial disease has been presumably related to de - creased arterial bioavailability of NO through the increased leucocyte and platelet adhesions, vasoconstriction and smooth muscle cell proliferation. However, the precise me - chanism of the impaired NO generation is not known, and there is a considerable controversy regarding whether myo - cardial ischemia results in increased or decreased NO for - mation. Asymmetric dimethylarginine (ADMA) is a natural, com petitive inhibitor, and one of the primary factors controlling the nitric oxide production. ADMA was found to be elevated and closely correlated with the impaired vasodilator function in conditions associated with the endothelial dysfunction, such as hypercholesterolemia, hypertension, insulin resistance and type 2 diabetes, and renal failure. But ADMA also seems to be involved in myocardial ischemia, since its plasma levels predict future coronary events in patients with the elevated cardiovascular risk. It has been recently reported that the elevated plasma ADMA concentrations in the acute coronary events are an independent cardiovascular risk factor.
Tatjana Stanković, Vidosava Đorđević, Borislav Kamenov, Hristina Stamenković, Vladan Ćosić, Radovan Milićević and Vjeroslava Slavić
Antioxidative Enzyme Activities and Lipid Peroxidation in Children with Inflammatory Endothelial Injury
During the inflammatory process endothelial cells are activated and a proadherent ability is assumed. The synthesis of reactive oxygen metabolites, which follows the immunological processes, can cause oxidative damage to endothelial cells leading to the clinical expression of disease including a variety of skin manifestations. In this study the activity of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) and the malondialdehyde concentration were examined in 36 children with inflammation-mediated damage to microvascular endothelial cells. On the basis of clinical manifestations the studied children were divided into 4 groups (1st group-macular skin manifestations, 2nd group-maculo-papular skin manifestations, 3rd group-papular skin manifestations, 4th group- erythematous skin manifestations). All the examined children showed symptoms of inflammation (mainly respiratory tract infections) with leukocytosis and monocytosis before actual skin manifestations took place. Superoxide dismutase activity was significantly decreased in three groups of patients, except in the group with erythematous skin manifestations. Catalase activity was significantly increased in all the groups compared to the control group. The values of malondialdehyde were significantly increased in the groups of children with maculo-papular and erythematous skin manifestations. The results have confirmed the presence of a changed antioxidant enzyme pattern indicating oxidative stress during inflammatory endothelial cells injury. Malondialdehyde was not an adequate parameter in its evaluation.
Tatjana Cvetković, Predrag Vlahović, Vidosava đorđević, Lilika Zvezdanović, Dušica Pavlović, Gordana Kocić and Dušan Sokolović
The Significance of Urinary Markers in the Evaluation of Diabetic Nephropathy
Oxidative stress is considered to be a unifying link between diabetes mellitus (DM) and its complications, including nephropathy (DN). The aim of this study was to determine the parameters of oxidative injury of lipids and proteins as well as the activity of ectoenzymes in the urine of DN patients. The study included 40 individuals: 10 patients with type 2 diabetes mellitus and microalbuminuria (DMT2-MIA), 10 type 2 diabetic patients with macroalbuminuria (DMT2-MAA), 10 patients with type 1 diabetes and microalbuminuria (DMT1-MIA) and 10 age- and sex-matched healthy subjects (control). In the urine we determined TBA reactive substances (TBARS), reactive carbonyl groups (RCG), and the activity of ectoenzymes N-acetyl-β-d-glucosaminidase (NAG), plasma cell differentiation antigen (PC-1), aminopeptidase N (APN) and dipeptidyl peptidase IV (DPP IV). A higher concentration of TBARS in the urine was found in DMT2-MIA and DMT1-MIA, compared to the control group (p<0.001 and P<0.05). The urine concentration of RCD shows similar results with a significant elevation in the groups with DMT2-MAA and DMT1-MIA, compared to the DMT2-MIA (p<0.001) and control group (p<0.001). Activities of NAG, APN and DPPIV were significantly higher in the urine of DMT2-MAA, compared to the control (p<0.01). The activity of PC-1 was slightly increased in that group, but not significantly. In conclusion, the level of oxidative stress markers and activities of brush border ectoenzymes in the urine may be a useful non-invasive and easily repeatable test in DN.
Vladimir V. Đorđević, Tatjana Jevtović-Stoimenov, Dušan Lazarević, Ivana Stojanović, Ljiljana Trajanović, Olivera Žikić and Vidosava Đorđević
Background: Single nucleotide polymorphisms (SNP) of many genes, including the gene for neuronal nitric oxide syn-thase (NOS1), were found significantly associated with schizo-phrenia. According to our previously published results of increased plasma nitric oxide concentration in patients with schizophrenia, we hypothesized that the NOS1 gene polymorphism might be a cause of increased nitric oxide production in patients with schizophrenia and tested the interdependence between plasma nitrite/nitrate concentrations and SNP (a CT transition located in exon 29) of the human NOS1 gene.
Methods: Nitrite/nitrate concentration was measured in blood plasma of 38 patients with schizophrenia and of 39 age and gender matched healthy persons by the colorimet-ric test. The NOS1 gene polymorphism was determined by polymerase chain reaction analysis.
Results: A significantly higher plasma nitrite/nitrate concentration was found in patients with schizophrenia (97.5±33.3 μmol/L, p<0.001) in comparison with controls (61.4±18.9 μmol/L). No T/T genotype was found in healthy individuals and there was a significant difference in the genotype distribution between patients and controls (χ2=24.54, p=0.0000047). Furthermore, a significant difference in the allele frequencies between patients and controls (χ2=19.00, p<0.000013, OR=4.45, 95% CI=2.12–9.39) was noted. Also, a significant difference in plasma nitrite/nitrate concentration was observed between patients having the C/T genotype (99.97±33.83 μmol/L) and the corresponding control (C/T) subgroup (63.88±10.26 μmol/L, p<0.01). However, there were no significant differences in nitrite/nitrate concentration between the patient subgroups with different genotypes (C/C, C/T, T/T).
Conclusions: CT transition located in exon 29 of the human NOS1 gene may be responsible for the increased plasma nitrite/nitrate levels.