Paraoxonase-1 (PON1) activity, but not PON1(Q192R) phenotype, is a predictor of coronary artery disease in a middle-aged Serbian population.

BACKGROUND: Paraoxonase-1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that protects lipoproteins from oxidative modifications. Polymorphisms in the gene, including PON1Q192R, have been studied. However, inconsistencies regarding the above-mentioned polymorphism obscure its association with vascular disease. METHODS: Using a two-substrate (paraoxon/diazoxon) activity method, we investigated the frequencies of PON1Q192R phenotypes in 261 middle-aged subjects: 156 patients with angiographically assessed coronary heart disease (CHD) and 105 CHD-free subjects as the control group. The PON1(192) phenotype was predicted from examination of the two-dimensional plot of hydrolysis rates of diazoxon vs. paraoxon and by using the antimode of the histogram of the ratio of diazoxonase/paraoxonase activity. RESULTS: The PON1Q192R phenotype frequencies in 113 patients with occlusion >50% (coronary artery disease-positive, CAD+ group) vs. control population were as follows: QQ (0.552 vs. 0.510), QR (0.382 vs. 0.408) and RR (0.066 vs. 0.082); chi2=0.414, p=0.813. We found lower paraoxonase (POase) and diazoxonase (DZOase) activities in the CAD+ patients when compared to the control population. According to logistic regression analysis, POase activity was a better predictor of coronary disease onset compared with DZOase activity measurements and PON1Q192R phenotyping. CONCLUSIONS: We conclude that enzyme activity (within a particular phenotypic group) is more important than phenotype alone in predicting susceptibility to coronary artery disease.     

Role of stem cells in kidney repair

End-stage renal disease and acute renal failure are the most important issues of practical and clinical nephrology, bearing in mind their high mortality rate, solely symptomatic treatment, and overall economic impact on society. The advances in stem cell biology opened the door for the new era in treatment of many disorders, including renal, offering new therapeutical solutions. Findings suggesting that the adult kidney contains stem cells and that stem cells from bone marrow have potential to differentiate into renal cells focused research on the possible application of these cells in therapy of kidney disorders. The other promising candidates for stem cell therapy for the kidney are embryonic stem cells and amniotic fluid-derived stem cells. This article focuses on the characteristics and possible application of these types of stem cells.     

Possible interactions of genetic and immuno-neuro-endocrine regulatory mechanisms in pathogenesis of congenital anomalies

The process of organogenesis depends on genetic and environmental factors. Besides genetic background, congenital anomalies can also be influenced by micro environmental changes, which are related to maternal-foetal interactions followed by the production of cytokines, hormones, neurotransmitters, growth factors and biochemical mediators, and stress proteins. Pre-natal maternal stress, including infections, psychological stress and other teratogens, can influence a disregulation of maternal immune, endocrine and nervous systems, during pregnancy. This is a crucial condition for the abnormal growth and development of the foetus. Activated maternal immune system can alter the cytokine network and make it inadequate for normal embryogenesis and organogenesis. Heat-shock proteins play an important role in stress physiology repairing DNA errors or activating pro-inflammatory response. Regarded from this aspect, the altered cytokine network suggests aetiopathogenetic basis of congenital anomalies in neonates. It is our wish to point out our potentially harmful conditions in the development of congenital anomalies, as well as their control by using pre-natal and pre-conceptional diagnostics and treatment.     

Antiatherosclerotic and Renoprotective Effects of Ebselen in the Diabetic Apolipoprotein E/GPx1-Double Knockout Mouse

OBJECTIVE

To investigate the effect of the GPx1-mimetic ebselen on diabetes-associated atherosclerosis and renal injury in a model of increased oxidative stress.

RESEARCH DESIGN AND METHODS

The study was performed using diabetic apolipoprotein E/GPx1 (ApoE^−/−GPx1^−/−)-double knockout (dKO) mice, a model combining hyperlipidemia and hyperglycemia with increased oxidative stress. Mice were randomized into two groups, one injected with streptozotocin, the other with vehicle, at 8 weeks of age. Groups were further randomized to receive either ebselen or no treatment for 20 weeks.

RESULTS

Ebselen reduced diabetes-associated atherosclerosis in most aortic regions, with the exception of the aortic sinus, and protected dKO mice from renal structural and functional injury. The protective effects of ebselen were associated with a reduction in oxidative stress (hydroperoxides in plasma, 8-isoprostane in urine, nitrotyrosine in the kidney, and 4-hydroxynonenal in the aorta) as well as a reduction in VEGF, CTGF, VCAM-1, MCP-1, and Nox2 after 10 weeks of diabetes in the dKO aorta. Ebselen also significantly reduced the expression of proteins implicated in fibrosis and inflammation in the kidney as well as reducing related key intracellular signaling pathways.

CONCLUSIONS

Ebselen has an antiatherosclerotic and renoprotective effect in a model of accelerated diabetic complications in the setting of enhanced oxidative stress. Our data suggest that ebselen effectively repletes the lack of GPx1, and indicate that ebselen may be an effective therapeutic for the treatment of diabetes-related atherosclerosis and nephropathy. Furthermore, this study highlights the feasibility of addressing two diabetic complications with one treatment regimen through the unifying approach of targeted antioxidant therapy.Chronic kidney disease is associated with enhanced morbidity and mortality, particularly caused by accelerated cardiovascular disease (1). Diabetes is emerging as an independent risk factor for both chronic kidney disease and atherosclerosis (2). Diabetic renal and cardiovascular complications are known to share common underlying pathogenic mechanisms, with oxidative stress and systemic inflammation contributing to both (3,4). Limiting oxidative stress through the removal of reactive oxygen species (ROS) is one strategy postulated to limit ROS-mediated chronic kidney disease and atherosclerosis in diabetic patients (5). However, antioxidant therapies such as vitamins E and C have shown limited benefits in clinical trials targeted at reducing cardiovascular outcomes (6,7). This has led to intensive efforts to define alternative antioxidant strategies for clinical applications (8).Recent studies by our group have suggested that targeting antioxidant defenses, which are reduced in organs susceptible to injury in the diabetic setting (9), may be an appropriate strategy (10). Our studies have shown an involvement of the key antioxidant enzyme, glutathione peroxidase-1 (GPx1), in diabetes-associated proatherogenic pathways that included proinflammatory and profibrotic mediators, suggesting that this antioxidant holds promise as a therapeutic target (10). Our data are also supported by clinical findings where a reduction in GPx1 activity has been linked to an increased risk of cardiovascular disease, both within a diabetic setting and associated with coronary artery disease (11–13).Modulation of GPx1 activity can be achieved through administration of selenium, the essential trace element found within the active site of GPx1, (14) although this holds less pharmacologic appeal because of selenium toxicity (15). In addition, lack of specificity caused by incorporation of selenium into several key enzymes makes this approach less attractive (14). Thus, compounds that mimic GPx1 activity offer an alternate way to increase GPx1-like activity (16).Ebselen, a lipid-soluble low molecular weight seleno-organic compound and a known GPx1-mimetic, (17) has shown potential in reducing pathogenesis in various experimental models (18–20). Of significance in the Zucker diabetic fat rat, a model of type 2 diabetes associated with the metabolic syndrome, ebselen improved endothelial dysfunction and renal insufficiency (21,22). Furthermore, data from our laboratory have shown that ebselen reduces diabetes-associated lesions and proinflammatory mediators in the ApoE-deficient mouse (23). However, no study has investigated the effects of ebselen on atherosclerosis and nephropathy, two clinically relevant and often linked comorbidities, in one animal model. Such a study would strengthen the notion that oxidative stress underpins these diabetic complications, and would highlight the effectiveness of a targeted antioxidant approach against both conditions.Consequently, in this study our objectives were as follows: First, our study investigated whether ebselen protects against diabetic nephropathy and diabetes-associated atherosclerosis in the diabetic ApoE/GPx1-deficient mouse. Second, we specifically chose the ApoE-deficient mouse additionally lacking Gpx1 to establish whether ebselen replenishes the lack of GPx1 activity. This has been performed to further clarify the mechanism of action of ebselen in an in vivo context, as well as strengthen the idea that replenishing GPx1-like activity may be of therapeutic benefit (11). Finally, our study also addressed potential pathways affected by ebselen in a rat kidney cell line.     

Quantification of blood flow and volume in arterioles and venules of the rat cerebral cortex using functional micro-ultrasound

Relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), and blood flow speed are key parameters that characterize cerebral hemodynamics. We used contrast-enhanced functional micro-ultrasound (fMUS) imaging employing a disruption-replenishment imaging sequence to quantify these hemodynamic parameters in the anesthetized rat brain. The method has a spatial resolution of about 100μm in-plane and around 600μm through-plane, which is comparable to fMRI, and it has a superior temporal resolution of 40ms per frame. We found no significant difference in rCBV of cortical and subcortical gray matter (0.89±0.08 and 0.61±0.09 times the brain-average value, respectively). The rCBV was significantly higher in the vascular regions on the pial surface (3.89±0.71) and in the area of major vessels in the subcortical gray matter (2.02±0.31). Parametric images of rCBV, rCBF, and blood flow speed demonstrate spatial heterogeneity of these parameters on the 100μm scale. Segmentation of the cortex in arteriolar and venular-dominated regions identified through color Doppler imaging showed that rCBV is higher and flow speed is lower in venules than in arterioles. Finally, we show that the dependence of rCBV on rCBF was significantly different in cortical versus subcortical gray matter: the exponent α in the power law relation rCBV=s·rCBF(α) was 0.37±0.13 in cortical and 0.75±0.16 in subcortical gray matter. This work demonstrates that functional micro-ultrasound imaging affords quantification of hemodynamic parameters in the anesthetized rodent brain. This modality is a promising tool for neuroscientists studying these parameters in rodent models of diseases with a cerebrovascular component, such as stroke, neurodegeneration, and venous collagenosis. It is of particular import for studying conditions that selectively affect arteriolar versus venular compartments.     

Hypothalamic noradrenaline synthesis, uptake and storage in rats during adaptation to long-term individual housing

The brain monoamine systems play a crucial role in mediating social behaviors. In order to assess the effect of socially isolated rats on neurochemical substrates in hypothalamus we measured levels of tyrosine hydroxylase (TH), noradrenaline transporter (NET) and vesicular monoamine transporter 2 (VMAT 2). Our results show that 12 weeks of social isolation produced no significant changes in noradrenaline content in hypothalamus. The levels of TH and NET were elevated in these animals, whereas level of VMAT 2 was unchanged. The data suggests that both enhanced synthesis and reuptake may aid in the recovery of noradrenaline content in the hypothalamus. The adaptation of the hypothalamic noradrenaline to long period of stress may have great physiological importance and may represent mechanism designed to help the organism adapt to the deleterious effects of chronic indiviual housing.     

Survivin expression in odontogenic keratocysts and correlation with cytomegalovirus infection

Oral Diseases (2010) 16 , 156–159 Objectives: The aim of this study was to investigate the expression of survivin, an inhibitor of apoptosis, in odontogenic keratocysts and to compare it to the findings in non‐neoplastic jaw cysts – periapical cysts, as well as to establish a possible relationship between survivin expression and human cytomegalovirus presence within these cysts. Materials and methods: Samples of 10 odontogenic keratocysts (five positive and five negative for the presence of cytomegalovirus, as determined by polymerase chain reaction) and 10 periapical cysts (five positive and five negative for the cytomegalovirus presence) were analysed. The expression of survivin was assessed by immunohistochemical methods, using monoclonal antibody that selectively recognizes the cytoplasmic form of survivin. Results: All 10 odontogenic keratocysts showed immunostaining for survivin, while all 10 periapical cysts were negative for its presence. There was no correlation between cytomegalovirus presence and expression of survivin within odontogenic keratocysts. Conclusion: Survivin may contribute to the aggressive behavior of odontogenic keratocysts, and thus support the emerging opinion of their neoplastic nature.     

PON1 status is influenced by oxidative stress and inflammation in coronary heart disease patients

Objectives

High-density lipoprotein (HDL) associated paraoxonase 1 (PON1) is an essential component of HDLs' capability to protect low-density lipoproteins (LDL) from oxidative modification and thus to limit the atherosclerotic process. The aim of the current study was to investigate the association between oxidative stress status, indices of inflammation and PON1 status parameters.

Design and methods

We determined the relationship between the oxidative stress status, inflammatory markers and PON1 status parameters in 261 middle-aged subjects: 156 coronary heart disease (CHD) patients and 105 CHD-free subjects (as the control group). The PON1 status involved PON1 activity measurements towards two substrates: paraoxon (POase activity) and diazoxon (DZOase activity) and subsequent PON1_Q192R activity phenotype determination.

Results

A statistically significant greater malondialdehyde (MDA) concentration in the RR phenotype subjects compared to QQ subjects within the CHD group was apparent (P < 0.05). Multiple linear regression analysis revealed an independent influence of plasmatic SOD activity (P < 0.05) on POase values and MDA (P < 0.01) and O₂⁻ (P < 0.05) on DZOase values. Involvement of inflammatory markers (fibrinogen and hsCRP) in the regression model did not hinder the influence of SOD and MDA on POase and DZOase activities, respectively.

Conclusions

Our CHD patients were in a state of oxidative stress, which was most evident in the RR phenotype group. The QQ phenotype group is associated with the lowest oxidative stress status level and also with a better capacity for anti-oxidative protection. Oxidative stress in CHD patients is maintained by systemic low-grade inflammation, which results in PON1 enzymatic activity exhaustion. Therefore, deeper investigation of an effective anti-oxidative and anti-inflammatory therapy should be necessary in order to increase anti-oxidative potency and improve PON1 status of CHD patients.