Prevalence of cardiovascular disease risk factors in Tallinn, Estonia.

BACKGROUND AND OBJECTIVE: Cardiovascular diseases are still a major public health concern in Estonia despite the decline in the mortality rate during the past decade. For better preventive strategies we aimed to investigate the prevalence of cardiovascular disease risk factors and their relations with age, gender and ethnicity. MATERIALS AND METHODS: The cross-sectional study was carried out in Tallinn, Estonia. Two hundred individuals from each of the sex and 10-year age group (range 20-65 years of age) were randomly selected and invited to participate. Final study sample consisted of 511 men and 600 women (mean age of 46 years). Physiological measurements were taken and blood samples were drawn for standard measurements of the following markers: total cholesterol, high- and low-density lipoprotein cholesterol, apolipoproteins, triglycerides, glucose and inflammatory markers. RESULTS: Overall, 31% of the study subjects had high blood pressure, 23% had metabolic syndrome, and 55% were overweight/obese. The prevalence of all risk factors increased with age amongst both genders. The proportion of individuals having increased cholesterol, apolipoprotein B-100, and homocysteine levels was very high amongst both genders (60-80%). More Russians and other ethnic minorities compared to ethnic Estonians had calculated 10-year CHD risk≥10%. CONCLUSIONS: The study established a high prevalence of cardiovascular disease risk factors in Estonian adults (20-65 years of age). Younger portion of the population and some extent ethnic considerations should be taken into account when designing future studies, health prevention activities and interventions.

Amyloid ß25-35 induced ROS-burst through NADPH oxidase is sensitive to iron chelation in microglial Bv2 cells.

Iron chelation therapy and inhibition of glial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can both represent possible routes for Alzheimer's disease modifying therapies. The metal hypothesis is largely focused on direct binding of metals to the N-terminal hydrophilic 1-16 domain peptides of Amyloid beta (Aß) and how they jointly give rise to reactive oxygen species (ROS) production. The cytotoxic effects of Aß through ROS and metals are mainly studied in neuronal cells using full-length Aß1-40/42 peptides. Here we study cellularly-derived ROS during 2-60min in response to non-metal associated mid domain Aß25-35 in microglial Bv2 cells by fluorescence based spectroscopy. We analyze if Aß25-35 induce ROS production through NADPH oxidase and if the production is sensitive to iron chelation. NADPH oxidase inhibitor diphenyliodonium (DPI) is used to confirm the production of ROS through NADPH oxidase. We modulate cellular iron homeostasis by applying cell permeable iron chelators desferrioxamine (DFO) and deferiprone (DFP). NADPH oxidase subunit gp91-phox level was analyzed by Western blotting. Our results show that Aß25-35 induces strong ROS production through NADPH oxidase in Bv2 microglial cells. Intracellular iron depletion resulted in restrained Aß25-35 induced ROS.

Pro-survival effects of JNK and p38 MAPK pathways in LPS-induced activation of BV-2 cells.

Identifying MAPK pathways and understanding their role in microglial cells may be crucial for understanding the pathogenesis of neurodegenerative diseases since activated microglia could contribute to the progressive nature of neurodegeneration. In this study we show that the JNK pathway plays an important role in the survival of resting microglia BV-2 cells, as evidenced by Annexin-V positive staining and caspase-3 activation in cells treated with the specific JNK inhibitor SP600125. During LPS-induced activation of BV-2 cells inhibition of the p38 and JNK pathways with SB203580 and SP600125, respectively, results in apoptosis as detected by apoptotic markers. In the presence SP600125 the phosphorylation of p38 was significantly increased both in control and LPS-activated BV-2 cells. This suggests that the pro-survival role of JNK is possible due to its abrogation of a potentially apoptotic signal mediated by p38 MAPK pathway. Furthermore, inhibition of the p38 MAPK pathway during LPS-induced activation of BV-2 cells resulted in an increased phosphorylation of c-Jun, suggesting that the pro-survival effect of p38 MAPK during inflammatory conditions involves the JNK pathway. In conclusion, the results of this study demonstrate that both the JNK and p38 MAPK pathways possess anti-apoptotic functions in the microglial cell line BV-2 during LPS-induced activation.

New capillary coatings in open tubular CEC as models for biological membranes.

Novel stationary phases in open tubular CEC were investigated. The coating procedure was fast and simple. The coating material contained membrane suspension of different neuronal cell lines. The performance and stability of three cell lines: human neuroblastoma SH-SY5Y, murine microglia Bv-2 and human glioma U87-MG cells were studied. The coating solution was expected to contain both membrane proteins and membrane lipids. The presence of membrane proteins was tested by Western blotting and the presence of phospholipids by the analysis of phosphorus content. The stability of the coating was estimated by monitoring the mobility of EOF over successive runs. The effects of pH, storage time and temperature on the coating stability were also studied. The results showed that the cell membrane-based coating was stable over pH range of 6.5-8.5. Coatings derived from different cells yielded similar stability and EOF mobility. Capillary coated with a membrane solution was stable over 3-day period. The same coating solution could be used for 3 weeks.

LPS-induced iNOS expression in Bv-2 cells is suppressed by an oxidative mechanism acting on the JNK pathway--a potential role for neuroprotection.

Activated microglia cells, observed during chronic inflammation, produce and secrete compounds that at high concentrations or during sustained production might cause neuronal cell death. Inducible nitric oxide synthase (iNOS) is expressed in response to various immunological stimuli and catalyses the formation of the free radical nitric oxide (NO), that at low and regulated levels participate in cell signaling and cytoprotective events, whereas its higher and unregulated production can promote neurotoxicity in cells or in tissues. Regulation of NO production is therefore central for maintaining NO-levels within a safe window. We have analyzed iNOS protein expression and NO production, in murine microglial Bv-2 cells after 16h treatment with the bacterial endotoxin lipopolysaccharide (LPS). We have further analyzed three MAPK pathways, by co-treating the cells with LPS and the inhibitors of ERK1/2, p38 or JNK MAPK activities. To investigate participation of an oxidative regulatory mechanism, cells were also treated with the antioxidant N-acetyl-L-cysteine (NAC). Our results show that LPS-induced iNOS expression in Bv-2 cells is mainly mediated through JNK MAPK. In addition, co-treatment of the Bv-2 cells with LPS and NAC surprisingly further increased the iNOS expression, an effect also found to be mediated through the JNK MAPK pathway. The level of phosphorylated JNK MAPK (p46) was strongly increased by LPS alone and was further increased when combined with NAC. Our data indicate that iNOS and NO production are suppressed by an oxidative mechanism acting on the JNK MAPK pathway and we speculate that it might constitute a potential regulatory mechanism controlling the NO level.

Sulfide increases labile iron pool in RD4 cells.

A linkage between sulfur and iron metabolism has been suggested since sulfide has the ability to release iron from ferritin in the presence of iron acceptors in vitro. Nevertheless, this linkage is still lacking evidence in vivo as well as in cellular models. In this study we have treated human RD4 skeletal muscle cells with sodium sulfide and measured the level of the labile iron pool (LIP) as well as the intracellular sulfide concentration. We have also detected the amounts of L-ferritin protein as well as the iron regulatory protein 2 (IRP2). The sulfide treatment resulted in a 100% increase in the amount of LIP after 1 and 2 h. We also found that the raise of the LIP levels was coupled to an elevation of the amounts of intracellular sulfide that increased by 60%. The bioavailability of the released iron was confirmed by a 100% increase in L-ferritin protein as well as a 60% decrease of the IRP2 protein levels. These results suggest that there is a linkage between sulfur metabolism and intracellular iron regulation in mammalian cells.

NADPH oxidase inhibitor diphenyliodonium abolishes lipopolysaccharide-induced down-regulation of transferrin receptor expression in N2a and BV-2 cells.

The activation of cellular inflammatory response is tightly linked to induced production of reactive oxygen species (ROS) and nitric oxide (NO), which in turn have been identified as important regulators of cellular iron metabolism. In the present study, we have used the microglia cell line BV-2 and the neuroblastoma cell line N2a to study the regulatory effects of the microbial agent lipopolysaccharide (LPS) on the expression of the transferrin receptor (TfR) and ferritin in cell lines with different characteristics. The receptor mainly responsible for LPS recognition is the Toll-like receptor 4 (TLR4) that triggers a variety of intracellular signalling cascades leading to the induction of transcription of target genes involved in the innate immune response. Among the pathways to be activated is the MAPK cascade leading to the activation of nuclear factor-kappaB that induces transcription of a variety of genes, e.g., inducible nitric oxide synthase (iNOS). The TLR4-mediated LPS response also induces the production of ROS through a mechanism(s) suggested to involve the activation of NADPH oxidase(s). This study shows that exposure of BV-2 and N2a cells to LPS results in decreased TfR protein levels and increased H-ferritin mRNA levels. The LPS down-regulatory effect on TfR protein expression is abolished by the NADPH oxidase inhibitor diphenyliodonium (DPI) but is not affected by the free radical scavenger N-acetyl-L-cysteine (NAC) or the iNOS inhibitor aminoguanidine (AG). The increased H-ferritin mRNA levels in response to LPS are not affected by DPI, NAC, or AG.

Increased susceptibility to oxidative stress in scrapie-infected neuroblastoma cells is associated with intracellular iron status.

The molecular mechanism of neurodegeneration in prion diseases remains largely uncertain, but one of the features of infected cells is higher sensitivity to induced oxidative stress. In this study, we have investigated the role of iron in hydrogen peroxide (H(2)O(2))-induced toxicity in scrapie-infected mouse neuroblastoma N2a (ScN 2 a) cells. ScN 2 a cells were significantly more susceptible to H(2)O(2) toxicity than N2a cells as revealed by cell viability (MTT) assay. After 2h exposure, significant decrease in cell viability in ScN 2 a cells was observed at low concentrations of extracellular H(2)O(2) (5-10 microM), whereas N2a cells were not affected. The increased H(2)O(2) toxicity in ScN 2 a cells may be related to intracellular iron status since ferrous iron (Fe(2+)) chelator 2,2'-bipyridyl (BIP) prevented H(2)O(2)-induced decrease in cell viability. Further, the level of calcein-sensitive labile iron pool (LIP) was significantly increased in ScN 2 a cells after H(2)O(2) treatment. Finally, the production of reactive oxygen species (ROS) was inhibited by 30% by iron chelators desferrioxamine (DFO) and BIP in ScN 2 a cells, whereas no significant effect of iron chelators on basal ROS production was observed in N2a cells. This study indicates that cellular resistance to oxidative stress in ScN 2 a cells is associated with intracellular status of reactive iron.

Increased iron-induced oxidative stress and toxicity in scrapie-infected neuroblastoma cells.

The mechanisms behind the pathology of prion diseases are still unknown, but accumulating evidence suggests oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we have investigated iron-induced oxidative stress in scrapie-infected mouse neuroblastoma N2a (ScN2a) cells. Uninfected N2a and ScN2a cells were treated with ferric ammonium citrate (FAC) for 1-16 h, and the levels of labile iron pool (LIP), the formation of reactive oxygen species (ROS), cell viability and ferritin protein levels were measured. The increase in LIP in N2a cells was transient with a quick recovery to normal levels within 4h accompanied by a moderate increase of formation of ROS after 3h followed by the decrease to the basal level. In ScN2a cells, the increase in LIP was lower, but the process of recovery was prolonged and accompanied by high ROS formation and decreased cell viability. Ferritin protein levels were significantly lower in ScN2a cells than in wild-type cells in all iron treatments. These results suggest that ScN2a cells are more sensitive to iron treatment as compared to wild-type cells with respect to ROS formation and cell viability, and that ferritin deficiency in infected cells may contribute to iron-induced oxidative stress in scrapie-infected cells.

Changed iron regulation in scrapie-infected neuroblastoma cells.

Prion diseases are characterized by the conversion of the normal cellular prion protein PrP(C) into a pathogenic isoform, PrP(Sc). The mechanisms involved in neuronal cell death in prion diseases are largely unknown, but accumulating evidence has demonstrated oxidative impairment along with metal imbalances in scrapie-infected brains. In this study, we report changes in cellular iron metabolism in scrapie-infected mouse neuroblastoma N2a cells (ScN2a). We detected twofold lower total cellular iron and calcein-chelatable cytosolic labile iron pool (LIP) in ScN2a cells as compared to the N2a cells. We also measured in ScN2a cells significantly lower activities of iron regulatory proteins 1 and 2 (IRP1 and IRP2, respectively), regulators of cellular iron by sensing cytosolic free iron levels and controlling posttranscriptionally the expression of the major iron transport protein transferrin receptor 1 (TfR1) and the iron sequestration protein ferritin. IRP1 and IRP2 protein levels were decreased by 40% and 50%, respectively, in ScN2a cells. TfR1 protein levels were fourfold reduced and ferritin levels were threefold reduced in ScN2a cells. TfR1 and ferritin mRNA levels were significantly reduced in ScN2a cells. ScN2a cells responded normally to iron and iron chelator treatment with respect to the activities of IRP1 and IRP2, and biosynthesis of TfR1 and ferritin. However, the activities of IRP1 and IRP2, and protein levels of TfR1 and ferritin, were still significantly lower in iron-depleted ScN2a cells as compared to the N2a cells, suggesting lower need for iron in ScN2a cells. Our results demonstrate that scrapie infection leads to changes in cellular iron metabolism, affecting both total cellular and cytosolic free iron, and the activities and expression of major regulators of cellular iron homeostasis.

Stimulation of G-proteins in human control and Alzheimer's disease brain by FAD mutants of APP(714-723): implication of oxidative mechanisms.

We report the effects of amyloid precursor protein (APP) fragment 714-723 (APP(714-723); peptide P1) and its V717F and V717G mutants (peptides P2 and P3, respectively) on G-protein activity ([35S]GTPgammaS binding) in membranes from postmortem human control and Alzheimer's disease (AD) brains. The peptides P1, P2, and P3 revealed a significant stimulatory effect on [35S]GTPgammaS binding in control temporal cortex. The most potent stimulator, P3, at 10 microM concentration enhanced [35S]GTPgammaS binding by 500%. The effect was threefold stronger than that for wild-type P1 and twofold stronger than that for P2. In sporadic AD, the stimulatory effect of P1, P2, and P3 on G-proteins was reduced significantly whereas in Swedish familial AD (SFAD), only P1 elicited marked stimulation (at 10 microM by 50%). In control sensory postcentral cortex, the stimulation of G-proteins by P3 was 1.5-fold lower than that in control temporal cortex, whereas in AD and SFAD the effect showed no remarkable regional difference. Treatment of membranes with H2O2 produced 1.5-fold higher stimulation in [35S]GTPgammaS binding to temporal cortex than that in binding to sensory postcentral cortex. In AD and SFAD, the stimulation by H2O2 revealed no significant regional difference. Glutathione, desferrioxamine (DFO), and 17beta-estradiol markedly decreased the strong stimulatory effect by P3 on [35S]GTPgammaS binding to control temporal cortex, with the protective effect by DFO being most potent. The G(alphaO)-protein levels were not changed in AD or SFAD brain membranes as compared to levels in control membranes. We suggest that strong G-protein stimulation by P3 in the human brain implies the specific (per)oxidation mechanism that might be affected by regional content of peroxidizing substrates and antioxidants.

Targeting of antisense PNA oligomers to human galanin receptor type 1 mRNA.

In this work, we have targeted positions 18-38 of the human galanin receptor type 1 (GalR1) mRNA coding sequence with different peptide nucleic acid (PNA) oligomers. This region has previously been shown to be a good antisense region and therefore we aimed to identify the subregions and/or thermodynamic parameters determining the antisense efficacy. Nine different PNA oligomers were conjugated to a cell-penetrating peptide, transportan, to enhance their cellular uptake. Concentration-dependent down-regulation of GalR1 protein expression in human melanoma cell line Bowes was measured by radioligand binding assay. No reduction of GalR1 mRNA level was observed upon PNA treatment, thus, the effect was concluded to be translational arrest. Judging from the EC50 values, antisense PNA oligomers targeting regions 24-38 (EC50=70 nM) or 27-38 (EC50=80 nM) were the most potent suppressors of protein expression. No parameter predicted by M-fold algorithm was found to correlate with the measured antisense activities. Presence of some subregions was found not to increase antisense efficiency of PNA. Presence of a short unpaired triplet between nucleotides 33 and 35 in the target region was, on the other hand, found to be the most critical for efficient GalR1 down-regulation. Thus, the results are of high impact in designing antisense oligomers. Specific results of this study demonstrate 20-fold more efficient antisense down-regulation of GalR1 as achieved before.

Amyloid precursor protein carboxy-terminal fragments modulate G-proteins and adenylate cyclase activity in Alzheimer's disease brain.

The influence of three C-terminal sequences and of transmembrane domain from amyloid precursor protein (APP) on the activity of G-proteins and of the coupled cAMP-signalling system in the postmortem Alzheimer's disease (AD) and age-matched control brains was compared. 10 microM APP(639-648)-APP(657-676) (PEP1) causes a fivefold stimulation in the [35S]GTPgammaS-binding to control hippocampal G-proteins. APP(657-676) (PEP2) and APP(639-648) (PEP4) showed less pronounced stimulation whereas cytosolic APP(649-669) (PEP3) showed no regulatory activity in the [35S]GTPgammaS-binding. PEP1 also showed 1.4-fold stimulatory effect of on the high-affinity GTPase and adenylate cyclase activity in control membranes, whereas in AD hippocampal membranes the stimulatory effect of PEP1 was substantially weaker. The PEP1 stimulation of the [35S]GTPgammaS-binding to the control membranes was significantly reduced by 1.5 mM glutathione, 0.5 mM antioxidant N-acetylcysteine and, in the greatest extent, by 0.01 mM of desferrioxamine. In AD hippocampus these antioxidants revealed no remarkable reducing effect on PEP1-induced stimulation. Our results suggest that C-terminal and transmembrane APP sequences possess receptor-like G-protein activating function in human hippocampus and that abnormalities of this function contribute to AD progression. The stimulatory action of these sequences on G-protein mediated signalling suggests the region-specific formation of reactive species.