Phosphoprotein enriched in astrocytes (PEA)-15 is a novel regulator of adipose tissue expansion.

Excessive expansion of adipose tissue in obesity typically leads to overflow and accumulation of lipids in other tissues, causing fatty liver disease and atherosclerosis. The intracellular protein, phosphoprotein enriched in astrocytes (PEA)-15 has been linked to metabolic disease but its role in lipid storage has not been examined. To delineate the role of PEA-15 in adipose tissue, we placed PEA-15-/- mice on a high fat diet. These mice developed increased body weight and greater white adipose tissue expansion compared to high fat diet-fed wild type mice. This was due to increased adipocyte cell size in PEA-15-/- mice consistent with greater lipid storage capacity. Surprisingly, PEA-15-/- mice exhibited improvements in whole body insulin sensitivity, lower hepatic weight and decreased serum triglycerides indicating a protective phenotype. To determine effects on atherosclerosis, PEA-15-/- mice were crossed with the ApoE-/- mice on a high fat diet. Strikingly, these mice were protected from atherosclerosis and had less hepatic lipid accumulation despite increased adiposity. Therefore, we reveal for the first time that PEA-15 plays a novel role in regulating the expansion of adipose tissue. Decreasing PEA-15 expression increases the sequestering of lipids in adipose tissue, protecting other tissues in obesity, thereby improving metabolic health.

Requirement for sphingosine kinase 1 in mediating phase 1 of the hypotensive response to anandamide in the anaesthetised mouse.

In the isolated rat carotid artery, the endocannabinoid anandamide induces endothelium-dependent relaxation via activation of the enzyme sphingosine kinase (SK). This generates sphingosine-1-phosphate (S1P) which can be released from the cell and activates S1P receptors on the endothelium. In anaesthetised mice, anandamide has a well-characterised triphasic effect on blood pressure but the contribution of SK and S1P receptors in mediating changes in blood pressure has never been studied. Therefore, we assessed this in the current study. The peak hypotensive response to 1 and 10 mg/kg anandamide was measured in control C57BL/6 mice and in mice pretreated with selective inhibitors of SK1 (BML-258, also known as SK1-I) or SK2 ((R)-FTY720 methylether (ROMe), a dual SK1/2 inhibitor (SKi) or an S1P1 receptor antagonist (W146). Vasodilator responses to S1P were also studied in isolated mouse aortic rings. The hypotensive response to anandamide was significantly attenuated by BML-258 but not by ROMe. Antagonising S1P1 receptors with W146 completely blocked the fall in systolic but not diastolic blood pressure in response to anandamide. S1P induced vasodilation in denuded aortic rings was blocked by W146 but caused no vasodilation in endothelium-intact rings. This study provides evidence that the SK1/S1P regulatory-axis is necessary for the rapid hypotension induced by anandamide. Generation of S1P in response to anandamide likely activates S1P1 to reduce total peripheral resistance and lower mean arterial pressure. These findings have important implications in our understanding of the hypotensive and cardiovascular actions of cannabinoids.

PEA-15 (Phosphoprotein Enriched in Astrocytes 15) Is a Protective Mediator in the Vasculature and Is Regulated During Neointimal Hyperplasia.

BACKGROUND: Neointimal hyperplasia following angioplasty occurs via vascular smooth muscle cell proliferation. The mechanisms involved are not fully understood but include mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2). We recently identified the intracellular mediator PEA-15 (phosphoprotein enriched in astrocytes 15) in vascular smooth muscle cells as a regulator of ERK1/2-dependent proliferation in vitro. PEA-15 acts as a cytoplasmic anchor for ERK1/2, preventing nuclear localization and thereby reducing ERK1/2-dependent gene expression. The aim of the current study was to examine the role of PEA-15 in neointimal hyperplasia in vivo. METHOD AND RESULTS: Mice deficient in PEA-15 or wild-type mice were subjected to wire injury of the carotid artery. In uninjured arteries from PEA-15-deficient mice, ERK1/2 had increased nuclear translocation and increased basal ERK1/2-dependent transcription. Following wire injury, arteries from PEA-15-deficient mice developed neointimal hyperplasia at an increased rate compared with wild-type mice. This occurred in parallel with an increase in a proliferative marker and vascular smooth muscle cell proliferation. In wild-type mice, PEA-15 expression was decreased in vascular smooth muscle cells at an early stage before any increase in intima:media ratio. This regulation of PEA-15 expression following injury was also observed in an ex vivo human model of hyperplasia. CONCLUSIONS: These results indicate, for the first time, a novel protective role for PEA-15 against inappropriate vascular proliferation. PEA-15 expression may also be repressed during vascular injury, suggesting that maintenance of PEA-15 expression is a novel therapeutic target in vascular disease.

The hypotensive effect of acute and chronic AMP-activated protein kinase activation in normal and hyperlipidemic mice.

AMP-activated protein kinase (AMPK) is present in the arterial wall and is activated in response to cellular stressors that raise AMP relative to ADP/ATP. Activation of AMPK in vivo lowers blood pressure but the influence of hyperlipidemia on this response has not been studied. ApoE(-/-) mice on high fat diet for 6weeks and age-matched controls were treated with the AMPK activator, AICAR daily for two weeks. Under anesthesia, the carotid artery was cannulated for blood pressure measurements. Aortic tissue was removed for in vitro functional experiments and AMPK activity was measured in artery homogenates by Western blotting. ApoE(-/-) mice had significantly raised mean arterial pressure; chronic AICAR treatment normalized this but had no effect in normolipidemic mice, whereas acute administration of AICAR lowered mean arterial pressure in both groups. Chronic AICAR treatment increased phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase in normolipidemic but not ApoE(-/-) mice. In aortic rings, AMPK activation induced vasodilation and an anticontractile effect, which was attenuated in ApoE(-/-) mice. This study demonstrates that hyperlipidemia dysregulates the AMPK pathway in the arterial wall but this effect can be reversed by AMPK activation, possibly through improving vessel compliance.

Differential effects of chlorinated and oxidized phospholipids in vascular tissue: implications for neointima formation.

The presence of inflammatory cells and MPO (myeloperoxidase) in the arterial wall after vascular injury could increase neointima formation by modification of phospholipids. The present study investigates how these phospholipids, in particular oxidized and chlorinated species, are altered within injured vessels and how they affect VSMC (vascular smooth muscle cell) remodelling processes. Vascular injury was induced in C57BL/6 mice and high fat-fed ApoE-/- (apolipoprotein E) mice by wire denudation and ligation of the left carotid artery (LCA). Neointimal and medial composition was assessed using immunohistochemistry and ESI-MS. Primary rabbit aortic SMCs (smooth muscle cells) were utilized to examine the effects of modified lipids on VSMC proliferation, viability and migration at a cellular level. Neointimal area, measured as intima-to-media ratio, was significantly larger in wire-injured ApoE-/- mice (3.62±0.49 compared with 0.83±0.25 in C57BL/6 mice, n=3) and there was increased oxidized low-density lipoprotein (oxLDL) infiltration and elevated plasma MPO levels. Relative increases in lysophosphatidylcholines and unsaturated phosphatidylcholines (PCs) were also observed in wire-injured ApoE-/- carotid arteries. Chlorinated lipids had no effect on VSMC proliferation, viability or migration whereas chronic incubation with oxidized phospholipids stimulated proliferation in the presence of fetal calf serum [154.8±14.2% of viable cells at 1 µM PGPC (1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine) compared with control, n=6]. In conclusion, ApoE-/- mice with an inflammatory phenotype develop more neointima in wire-injured arteries and accumulation of oxidized lipids in the vessel wall may propagate this effect.

Phosphoprotein enriched in astrocytes (PEA)-15: a potential therapeutic target in multiple disease states.

Phosphoprotein enriched in astrocytes-15 (PEA-15) is a cytoplasmic protein that sits at an important junction in intracellular signalling and can regulate diverse cellular processes, such as proliferation and apoptosis, dependent upon stimulation. Regulation of these processes occurs by virtue of the unique interaction of PEA-15 with other signalling proteins. PEA-15 acts as a cytoplasmic tether for the mitogen-activated protein kinases, extracellular signal-regulated kinase 1/2 (ERK1/2) preventing nuclear localisation. In order to release ERK1/2, PEA-15 requires to be phosphorylated via several potential pathways. PEA-15 (and its phosphorylation state) therefore regulates many ERK1/2-dependent processes, including proliferation, via regulating ERK1/2 nuclear translocation. In addition, PEA-15 contains a death effector domain (DED) which allows interaction with other DED-containing proteins. PEA-15 can bind the DED-containing apoptotic adaptor molecule, Fas-associated death domain protein (FADD) which is also dependent on the phosphorylation status of PEA-15. PEA-15 binding of FADD can inhibit apoptosis as bound FADD cannot participate in the assembly of apoptotic signalling complexes. Through these protein-protein interactions, PEA-15-regulated cellular effects have now been investigated in a number of disease-related studies. Changes in PEA-15 expression and regulation have been observed in diabetes mellitus, cancer, neurological disorders and the cardiovascular system. These changes have been suggested to contribute to the pathology related to each of these disease states. As such, new therapeutic targets based around PEA-15 and its associated interactions are now being uncovered and could provide novel avenues for treatment strategies in multiple diseases.

The effect of reactive oxygen species on whole blood aggregation and the endothelial cell-platelet interaction in patients with coronary heart disease.

BACKGROUND: The effect of reactive oxygen species (ROS) on platelet function in coronary heart disease (CHD) is complex and poorly defined. Platelet aggregation studies in healthy volunteers have demonstrated contrasting results when platelets are exposed to ROS. We investigated the effect of ROS on whole blood aggregation (WBA) and the endothelial cell-platelet interaction in patients with CHD. METHODS AND RESULTS: ROS generated by xanthine and xanthine oxidase caused a concentration-dependent inhibition of WBA in blood from healthy donors and patients with CHD. In patients with CHD, 100 µM xanthine and 100 mU/ml xanthine oxidase inhibited WBA in response to 3 µg/ml collagen by 28.9% (95% CI 15.9%-41.8%, p<0.001) and in response to 5 µM ADP by 36.0% (95% CI 9.6%-62.4%, p=0.005). Using nitrotyrosine expression, platelets isolated from patients with CHD were found to be susceptible to peroxynitrite damage. The addition of 1 × 10(5) cultured endothelial cells inhibited WBA in response to 3 µg/ml collagen by 31.2% (95% CI 12.2%-50.2%, p<0.05) and in response to 5 µM ADP by 31.6% (95% CI 2.5-60.7%, p<0.05). Addition of xanthine and xanthine oxidase did not alter this effect, however pre-treatment of endothelial cells with a nitric oxide synthase inhibitor (L-NAME) partly reversed the inhibition. CONCLUSION: ROS inhibit WBA in blood from patients with CHD. Whilst endothelial cells also inhibit WBA, the effect is attenuated by L-NAME, suggesting that nitric oxide is likely to remain an important protective mechanism against thrombosis in CHD.

Physiological effects of oxidized phospholipids and their cellular signaling mechanisms in inflammation.

Oxidized phospholipids, such as the products of the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine by nonenzymatic radical attack, are known to be formed in a number of inflammatory diseases. Interest in the bioactivity and signaling functions of these compounds has increased enormously, with many studies using cultured immortalized and primary cells, tissues, and animals to understand their roles in disease pathology. Initially, oxidized phospholipids were viewed largely as culprits, in line with observations that they have proinflammatory effects, enhancing inflammatory cytokine production, cell adhesion and migration, proliferation, apoptosis, and necrosis, especially in vascular endothelial cells, macrophages, and smooth muscle cells. However, evidence has emerged that these compounds also have protective effects in some situations and cell types; a notable example is their ability to interfere with signaling by certain Toll-like receptors (TLRs) induced by microbial products that normally leads to inflammation. They also have protective effects via the stimulation of small GTPases and induce up-regulation of antioxidant enzymes and cytoskeletal rearrangements that improve endothelial barrier function. Oxidized phospholipids interact with several cellular receptors, including scavenger receptors, platelet-activating factor receptors, peroxisome proliferator-activated receptors, and TLRs. The various and sometimes contradictory effects that have been observed for oxidized phospholipids depend on their concentration, their specific structure, and the cell type investigated. Nevertheless, the underlying molecular mechanisms by which oxidized phospholipids exert their effects in various pathologies are similar. Although our understanding of the actions and mechanisms of these mediators has advanced substantially, many questions do remain about their precise interactions with components of cell signaling pathways.

The development of research tools used in the STORK Study (the Scottish Trial of Refer or Keep) to explore midwives' intrapartum decision making.

OBJECTIVES: to develop appropriate tools to assess midwives' attitudes and behaviour in relation to decision making involving risk. DESIGN: a questionnaire and series of vignettes were developed and testes to explore midwives' intrapartum decision making in relation to their attitudes towards risk. An innovative online computer package was developed specifically for use in the STORK Study which enabled the programme to be very tightly controlled with limited functions accessible to participants. TESTING: a pilot study was conducted with over 50 midwives and nurses to ensure face and content validity of the vignettes and questionnaire. Initially designed to be a paper-based study, rigorous piloting highlighted the many difficulties in presenting it in that particular format. The solution to this problem was to develop the study as a secure online package. FINDINGS: online data collection provided the researchers with a greater degree of control of the data collection process, not achievable using traditional paper survey methods. Another example of this control is the immediate entry of data from participants' responses to a background database which automatically stores and backs up data this means that no additional time is required for data entry. The cost of employing an information technology professional was easily offset by the financial savings made through the limited use of stationery and postage. KEY CONCLUSIONS: although the development and testing of the research tools for the STORK Study was labour and time intensive, ultimately a questionnaire and vignette package was produced that had been rigorously tested by over 50 midwives and nurses. The researchers are confident in the reliability of the questionnaire and vignettes, as well as the validity of the data collected. The use of an online survey is clearly indicated when the population has readily available internet access, and where controlling the process of data collection is required, as such control cannot be achieved in traditional survey and questionnaire implementation.

The Scottish Trial of Refer or Keep (the STORK study): midwives' intrapartum decision making.

OBJECTIVES: to explore midwives' intrapartum referral decisions in relation to their dispositional attitude towards risk. DESIGN: a web-based correlation study examined the association between midwife's personality (personal risk tendency), place of work (location), years of experience and the timing of their decisions to make referrals (referral score) in a series of fictitious case scenarios (vignettes). PARTICIPANTS: 102 midwives providing labour care in both consultant-led units (CLU) and community maternity units (CMU) from four Scottish health board areas. ANALYSIS: a correlational analysis was carried out to test the association between total risk scores and midwives' referral scores. Between-group comparisons were also conducted for experienced vs inexperienced midwives, midwives practising in CLU vs CMU settings and between the four health board areas. FINDINGS: despite being presented with identical information in the vignettes, midwives made a wide range of referral decisions. There was no association between referral scores and measures of risk, personality or years of experience. No statistically significant difference between the referral scores of midwives working in CLUs or CMUs was observed. However, a statistically significant difference did emerge between the four health board areas, with midwives from one area making referrals at a significantly earlier stage. The maternity services in this area had experienced several high profile adverse events prior to this study; this may have influenced their referral behaviour (the availability heuristic), in terms of making more cautious decisions. KEY CONCLUSIONS: there was no evidence that variability in the range of referral decisions was due to personality factors, risk propensity, experience or whether the midwife worked in a CLU or CMU. Local factors such as recent adverse events may significantly influence subsequent referral behaviour. Further research is required to identify why the midwives showed so much unexplained variability in their responses to the vignettes.

Effect of phosphatidylcholine chlorohydrins on human erythrocytes.

Hypochlorite generated in vivo under pathological conditions is a known oxidant and chlorinating agent, able to react with proteins and lipids, which affects the stability of biological membranes. Reaction with unsaturated fatty acyl chains in glycerophospholipids such as phosphatidylcholine results in the formation of chlorohydrins. The aim of this study was to determine the effects of chlorohydrins formed by the reaction of hypochlorite with 1-stearoyl-2-oleoyl-, 1-stearoyl-2-linoleoyl-, and 1-stearoyl-2-arachidonylphosphatidylcholine on biophysical properties of bilayers and their effects on human erythrocytes. Using electrospray mass spectrometry we observed complete conversion of the lipids into chlorohydrins, which resulted in a decrease in the rotational correlation time and an increase in the order parameter of liposomes. Unilamellar chlorohydrin liposomes had a lower permeation coefficient for calcein than liposomes made of parent lipids. Flow cytometry demonstrated fast incorporation of uni and multilamellar chlorohydrin liposomes labeled with NBD-phosphatidylethanolamine into erythrocytes. This effect was accompanied by changes in erythrocyte shape (echinocyte formation) and aggregation. Similar but less pronounced effects were noticed for parent lipids only after longer incubation. Chlorohydrins showed also a stronger hemolytic action, proportional to the lipid:erythrocyte ratio. These results are important for understanding the effects of HOCl on mammalian cells, such as might occur in inflammatory pathology.

Setting priorities for safe motherhood interventions in resource-scarce settings.

OBJECTIVE: Guide policy-makers in prioritizing safe motherhood interventions. METHODS: Three models (LOW, MED, HIGH) were constructed based on 34 sub-Saharan African countries to assess the relative cost-effectiveness of available safe motherhood interventions. Cost and effectiveness data were compiled and inserted into the WHO Mother Baby Package Costing Spreadsheet. For each model we assessed the percentage in maternal mortality reduction after implementing all interventions, and optimal combinations of interventions given restricted budgets of US$ 0.50, US$ 1.00, US$ 1.50 per capital maternal health expenditures respectively for LOW, MED, and HIGH models. RESULTS: The most cost-effective interventions were family planning and safe abortion (fpsa), antenatal care including misoprostol distribution for postpartum hemorrhage prevention at home deliveries (anc-miso), followed by sepsis treatment (sepsis) and facility-based postpartum hemorrhage management (pph). CONCLUSIONS: The combination of interventions that avert the greatest number of maternal deaths should be prioritized and expanded to cover the greatest number of women at risk. Those which save the most number of lives in each model are 'fpsa, anc-miso' and 'fpsa, sepsis, safe delivery' for LOW; 'fpsa, anc-miso' and 'fpsa, sepsis, safe delivery' for MED; and 'fpsa, anc-miso, sepsis, eclampsia treatment, safe delivery' for HIGH settings. Safe motherhood interventions save a significant number of newborn lives.

Ability to pay for maternal health services: what will it take to meet who standards?

High maternal morbidity and mortality in many developing countries are highly associated with poor access to and quality of health care. Here we review the economic feasibility of the WHO's mother-baby package as a means of reducing maternal and neonatal mortality and morbidity in Tanzania. This paper examines the costs of maternal health care in Tanzania, and how much can we expect households to contribute to these expenses, if the MBP were implemented. Using data from the Tanzanian 1993 Living Standard Measurement Survey (LSMS), we analyze responses from 757 women of reproductive age who have had a birth in the 12 months preceding the survey. We estimate current spending on maternal health care by different socio-economic groups and its share in relation to total household expenditures. Using logistic regression analyses, we examine the effect of the prices paid for maternal health care on the likelihood of using antenatal and safe delivery services, controlling for relevant socio-economic and demographic factors. Results show that if the MBP recovered 100% of its costs, most of the households would have to allocate more than half of their annual consumption on maternal health care. Poor socio-economic groups would experience the greatest increase in service utilization if MBP care were subsidized. In the face of scarce resources, subsidies should be targeted according to socio-economic group, in order to attain equitable and sustainable maternal health services.

Multilevel analysis of women's empowerment and HIV prevention: quantitative survey Results from a preliminary study in Botswana.

This preliminary study explored relationships between women's empowerment and HIV prevention on the national and individual level with a focus on Botswana. Among sub-Saharan Africa countries, HIV prevalence was positively correlated with indirect indicators of women's empowerment relating to their education (female enrollment in secondary education and ratio of female to male secondary school enrollment), but not to their economic status (female share of paid employment in industry and services) or political status (women's share of seats in national parliament), while controlling for gross national income, percentage of births attended, and percentage of roads paved. Condom use at last sexual encounter was positively and significantly correlated with both indicators of women's educational empowerment, but was not significantly related to the other two indices. Empowerment at the individual level was explored through a preliminary quantitative survey of 71 sexually active women in Gaborone, Botswana, that was conducted in February 2001. Regression analyses showed that women's negotiating power and economic independence were the factors most strongly related to condom use, and did not show that education was a crucial factor. Economic independence was the factor most strongly related to negotiating power. These results suggest that in Botswana, HIV prevention efforts may need to improve women's negotiating skills and access to income-generating activities.