F2-isoprostanes affect macrophage migration and CSF-1 signalling.

F2-isoprostanes (F2-IsoP) are formed in vivo via free radical peroxidation of arachidonic acid. Enhanced oxidative stress is implicated in the development of atherosclerosis in humans and F2-IsoP have been detected in atherosclerotic plaque. Colony stimulating factor-1 (CSF-1) is essential to macrophage survival, proliferation and differentiation and has been detected in human atherosclerotic plaques. Accumulation of macrophages within the vascular wall is an important component of atherosclerosis but little is known about the effect of F2-IsoP on the migration of these cells. Our aim was to examine the effect of free and lipid-bound 15-F2t-isoprostane (15-F2t-IsoP) on macrophage migration and investigate the signalling pathways involved. Mouse macrophages (cell line BAC1.2F5) were pre-incubated with 15-F2t-IsoP (free, bound to cholesterol or monoacylglycerol or within oxidized phospholipid) and cell migration was assessed using chemotaxis towards CSF-1 in Boyden chambers. Migration was also measured using the wound healing assay with primary mouse bone marrow derived macrophages. We showed that 15-F2t-IsoP dose-dependently inhibited BAC1.2F5 macrophage spreading and adhesion but stimulated their migration towards CSF-1, with maximum effect at 10 µM. Analysis of CSF-1 stimulated signalling pathways in BAC1.2F5 macrophages showed that phosphorylation of Akt, a key mediator of cell migration, and one of its regulators, the mTORC2 component, Rictor, was significantly decreased. In contrast, phosphorylation of the adhesion kinases, FAK and Pyk2, and the adhesion scaffold protein, paxillin, was enhanced after treatment with 15-F2t-IsoP. Mouse bone marrow macrophages were transfected with FAK or Pyk2 small interfering RNA (siRNA) to examine the role of FAK and Pyk2 in 15-F2t-IsoP signalling. Pyk2 silencing inhibited 15-F2t-IsoP-induced reduction in cell area and phospho-paxillin adhesion numbers. The size distribution of adhesions in the presence of 15-F2t-IsoP was also affected by Pyk2 silencing and there was a trend for Pyk2 silencing to reduce 15-F2t-IsoP-stimulated macrophage migration. These results demonstrate that 15-F2t-IsoP affects macrophage adhesions and migration, which are integral components of macrophage involvement in atherosclerosis.

Effects of postnatal omega-3 fatty acid supplementation on offspring pro-resolving mediators of inflammation at 6 months and 5 years of age: A double blind, randomized controlled clinical trial.

BACKGROUND: Resolution of inflammation is an active process involving specialised pro-resolving mediators (SPMs) generated from the omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Omega-3 fatty acid supplementation during infancy may provide an intervention strategy to modify SPMs and reduce oxidative stress. This study evaluates the effect of omega-3 fatty acid supplementation in infancy on SPMs and F2-isoprostanes from 6 months to 5 years of age. METHODS: In a double-blind, placebo-controlled, parallel-group study design, 420 infants were randomized to a daily supplement of omega-3 fatty acids (280mg DHA and 110mg EPA) or olive oil (control), from birth to age 6 months. Blood was collected at birth (cord blood), 6 months, 12 months and 5 years. Plasma SPMs included 18-HEPE, E-series resolvins, 17-HDHA, D-series resolvins, 14-HDHA, 10S,17S-DiHDoHE, MaR1 and PD1. F2-isoprostanes were measured in plasma and urine, as markers of oxidative stress in vivo. RESULTS: The change in the concentration of 18-HEPE from birth to 6 months was greater in the omega-3 fatty acid group (Ptimepoint*group=0.04) with levels at 6 months significantly higher than controls (P=0.02). Other SPMs were not different between the groups at any time point. Plasma 18-HEPE concentration were associated with erythrocyte EPA concentrations after age and group adjustments (P<0.001), but not with allergic outcomes at 12 months. There were no between-group differences in plasma and urinary F2-isoprostanes at any time point. CONCLUSION: Omega-3 fatty acid supplementation from birth to 6 months of age increased SPM at 6 months but the effects were not sustained after supplementation ceased. Given that 18-HEPE is a biologically active metabolite, future studies should examine how the increase in 18-HEPE relates to potential health benefits of omega-3 fatty acid supplementation in infancy.

Effects of omega-3 and omega-6 fatty acids on human placental cytokine production.

INTRODUCTION: Dietary supplementation with omega-3 long chain polyunsaturated fatty acids (n-3 PUFAs) may exert benefits in pregnancy through inhibition of placental inflammation. However, studies on the anti-inflammatory effects of n-3 PUFAs in the placenta are lacking. We compared the cytokine responses of human placental explants in vitro after 4 days pre-incubation with either: a) individual n-3 or n-6 PUFAs (20 µM), or b) physiologically relevant combinations of low, medium or high n-3 or n-6 PUFA concentrations. METHODS: Placental cytokine (IL-6 and TNF-α) mRNA expression and protein production were assessed at 4 h and 12 h, respectively. Cytokine and fatty acid concentrations were also measured in placentas delivered at term by women who ingested either low (n = 12) or high (n = 10) amounts of fish/fish oil in the month prior to delivery. RESULTS: Pre-exposure to n-3 PUFAs as individual fatty acids results in reduced placental IL-6 production (P < 0.05), whereas exposure to complex fatty acid mixtures enriched in n-3 PUFAs (high n-3:n-6 ratios) results in a significant stimulation of IL-6 production (P < 0.05). There were no differences in placental n-3 or n-6 PUFA levels between women with either high or low dietary fish oil intake and no differences in cytokine expression. DISCUSSION: In summary, data from our complex lipid explant model and an observational cohort study do not support a role for n3 PUFAs in the suppression of pro-inflammatory cytokine expression in the human placenta. Results from studies of placental tissues exposed to single n-3 and n-6 PUFAs should be interpreted with considerable caution.

Minimizing artifactual elevation of lipid peroxidation products (F2-isoprostanes) in plasma during collection and storage.

F2-isoprostanes (F2-IsoP's) are reliable measures of in vivo lipid oxidation, but care is required to prevent artifactual elevation. We examined the effects of blood collection and storage on plasma F2-IsoP's. Blood was collected into EDTA/butylated hydroxytoluene/reduced glutathione (EDTA/BHT/GSH) or EDTA, at 4 °C or room temperature. Plasma was stored at -20 or -80 °C for 1 or 6 months before F2-IsoP's were assayed by GC-MS. The temperature of blood collection did not affect F2-IsoP's. However, storage at -20 °C or collection into EDTA resulted in significant increases in F2-IsoP's. Blood collection into EDTA/BHT/GSH and storage at -80 °C minimizes artifactual elevation of plasma F2-IsoP's.

Association between the endothelin-1 gene Lys198Asn polymorphism blood pressure and plasma endothelin-1 levels in normal and pre-eclamptic pregnancy.

OBJECTIVE: This study examined the frequency of the Lys198Asn polymorphism in the endothelin-1 (ET-1) gene in women with pre-eclampsia and normal pregnancy; and its contribution to levels of plasma ET-1 and blood pressure. DESIGN AND METHODS: This was a retrospective study examining the frequency of the ET-1 Lys198Asn polymorphism in 72 proteinuric pre-eclamptics and 81 normal pregnant women. Height, weight, blood pressure and plasma ET-1 were measured antenatally and at 6 weeks post-partum. Using specific mutagenic primers, the frequency of the G/G (normal), G/T heterozygote and T/T (mutant) genotypes of the Lys198Asn polymorphism were examined. RESULTS: The polymorphism was not associated with pre-eclampsia. However, in the combined pregnant groups after correction for BMI and group, a significant effect of the T-allele (T/T,G/T) on systolic blood pressure was found (121 +/- 1.5 mmHg compared with 116 +/- 1.3 mmHg in the G/G homozygotes). A significant interaction was found between the T-allele and pregnancy in determining systolic blood pressure, so that the effect was no longer seen post-partum. Pregnant women with the T/T genotype had significantly elevated plasma ET-1 levels 5.8 pg/ml [confidence interval (CI) 3.7-9.1] compared with 3.1 pg/ml (CI 2.6-3.8) in the G/T heterozygotes and 3.6 pg/ml (CI 3.0-4.1) in the normal G/G homozygotes. CONCLUSION: The Lys198Asn polymorphism does not directly contribute to the incidence of pre-eclampsia. However, the association of the T-allele with raised blood pressure and the T/T genotype with increased plasma ET-1 levels suggest that this polymorphism may interact with other genes or environmental factors to sensitize pregnant women to develop pre-eclampsia.

Is proteinuric pre-eclampsia a different disease in primigravida and multigravida?

This study aimed to identify if the clinical features of proteinuric pre-eclampsia or the biochemical markers of endothelial dysfunction associated with this syndrome are altered according to parity in a direction that would suggest a different pathophysiology. Groups of 27 primigravid and 35 multigravid women with pre-eclampsia (defined as blood pressure >140/90 mmHg and 2+ proteinuria) were studied ante-partum, and at 6 weeks and 6 months post-partum. Clinical markers of severity of pre-eclampsia, including blood pressure, markers of renal, hepatic and coagulatory function, and biochemical markers of endothelial dysfunction were measured. Fetal outcome was assessed by birthweight and birthweight percentile. Ante-partum systolic blood pressure was 10 mmHg higher in the primigravida, and this difference was independent of age and anti-hypertensive medication. Analysis of systolic blood pressure before and after delivery showed the primigravid women to have elevated systolic blood pressure over the whole time period (P<0.01). The primigravid women had more severe hepatic dysfunction, with elevated aspartate aminotransferase levels, but plasma creatinine, proteinuria, platelet counts and haematocrit were similar, indicating that renal and coagulatory function and plasma volume were affected to the same extent in the two groups and were independent of parity. Birthweight was similar in the two groups, and the percentage of infants weighing less than the 10th centile for gestation was also similar. Biochemical markers of endothelial dysfunction, assessed by measuring the urinary prostacyclin metabolite 2, 3-dinor-6-oxo-prostaglandin F(1alpha) and plasma endothelin 1, did not differ according to parity. There were no differences in a number of other biochemical markers of pre-eclampsia, including plasma albumin, uric acid, triacylglycerol, and total, low-density lipoprotein and high-density lipoprotein cholesterol. Basophil, monocyte and lymphocyte counts were elevated before delivery in primigravid women with pre-eclampsia. The differences in lymphocyte counts persisted post-partum. Further studies are required to clarify the role, if any, of monocytes, basophils and lymphocytes in the pathophysiology of pre-eclampsia. In conclusion, the elevated systolic blood pressure and raised aspartate aminotransferase levels observed in primigravida suggest a more severe form of pre-eclampsia. The lack of differences in birthweight and other biochemical and endothelial markers of severity of pre-eclampsia do not suggest a different pathophysiology; however, the persistently higher white cell counts in the primigravid pre-eclamptics are of interest, and might reflect differences in immune responses in the two groups. We suggest that studies of the pathophysiology of pre-eclampsia should include multigravida, as long as there is adequate post-partum follow-up to exclude underlying disease.

Does a predisposition to the metabolic syndrome sensitize women to develop pre-eclampsia?

OBJECTIVE: This study aimed to identify those factors in the non-pregnant state that distinguished women who developed pre-eclampsia from those who had normotensive pregnancies. DESIGN AND SETTING: This was a retrospective analysis of anthropometry, blood pressure, biochemical and haematological variables in 62 women with pre-eclampsia and 84 normotensive pregnant women who took part in studies of the pathophysiology of pre-eclampsia. Pregnant volunteers were seen, after admission to hospital or in the outpatient clinic, and followed-up at 6 weeks and 6 months post-partum in the outpatient clinic or their home. Participants Proteinuric pre-eclampsia was defined as blood pressure > or = 140/90 mmHg with proteinuria of at least 300 mg/24 h after 20 weeks gestation, in women with no history of hypertension and whose blood pressure returned to normal levels by 6 months post-partum. Normotensive pregnancy was defined as blood pressure < 130/90 mmHg without proteinuria. MAIN OUTCOME MEASURES: The primary outcome measures were blood pressure, body mass index (BMI), triglycerides, total cholesterol, low density lipoprotein (LDL) and high density lipoprotein cholesterol and markers of severity of pre-eclampsia. RESULTS: Regardless of parity, women with pre-eclampsia had elevated BMI before, during and after pregnancy compared with women who had normotensive pregnancies. Triglycerides were significantly elevated in women who had pre-eclampsia both before and after delivery, while total and LDL cholesterol were elevated significantly at both visits after delivery. Systolic and diastolic blood pressure, which by definition were elevated antepartum in women with pre-eclampsia, remained higher at post-partum visits compared with women who had normotensive pregnancies. Women with pre-eclampsia reported a greatly increased frequency of both maternal hypertension and pre-eclampsia. Markers of severity of pre-eclampsia, which normalized by 6 months postpartum, included plasma creatinine, uric acid, albumin, endothelin 1 and urinary protein, 2,3, dinor-6-keto-PGF1alpha, blood platelet and neutrophil counts. CONCLUSION: The relative elevation of blood pressure, BMI and lipids in the non-pregnant state are features of the metabolic syndrome and may be important sensitizing factors contributing to the pathogenesis of pre-eclampsia. A familial predisposition to pre-eclampsia may operate partly through these mechanisms.

Potassium supplementation does not lower blood pressure in normotensive women.

Forty-four normotensive females, who were selected on the basis of lower prevailing potassium intake, participated in a two-period crossover study to assess the effects of potassium supplementation on blood pressure. They were randomly allocated to one of two groups who took either 80 mmol/day of KCl (Slow-K, Ciba Geigy), or matching placebo, for the first of two 4-week treatment periods. The treatments were reversed during the second 4-week period. Despite significant increases in both urinary and plasma potassium no consistent fall in blood pressure was seen during 80 mmol/day potassium intake.

Frusemide releases renin in the rat kidney when prostacyclin synthesis is suppressed.

The effect of inhibiting prostaglandin (PG) synthesis on basal and frusemide-stimulated renin secretion was examined in the rat isolated perfused kidney. The stable PGI2 derivative, 6-keto PGF1 alpha, was measured by radioimmunoassay in urine collected from the kidney. Treatment of rats with indomethacin (3.0 mg kg-1) reduced 6-keto PGF1 alpha excretion from 121.3 +/- 39.1 (n = 9) to 15.5 +/- 6.6 (n = 9) pg min-1 (P less than 0.02) but had no effect on basal renin secretion. Renal perfusion pressure, flow rate and vascular resistance were similar in treated and control rats. Mean urine flow was lower after treatment. Infusion of frusemide (250 micrograms min-1) did not alter 6-keto PGF1 alpha excretion in control or indomethacin-treated (P greater than 0.05) rats. Although renin secretion was increased during frusemide infusion, there was no significant difference between control (1,806 +/- 384 ng angiotensin I (AI) min-1) and treated (2,310 +/- 554 ng AI min-1) rats (P greater than 0.05). Propranolol, at a dose (8 micrograms min-1) which suppressed renin secretion after isoprenaline stimulation, had no effect on the response to frusemide in indomethacin-treated rats. These results demonstrate that frusemide-stimulated renin secretion in the rat kidney does not require intact renal PGI2 synthesis and is independent of beta-adrenergic mechanisms.

Diurnal rhythms of blood pressure, plasma renin activity, angiotensin II and catecholamines in normotensive and hypertensive pregnancies.

Diurnal patterns of blood pressure and pressor hormones after 26 weeks gestation were compared in 10 normotensive women, 13 subjects with uncomplicated hypertension, and 8 with biochemical evidence of pre-eclampsia. 4 of the pre-eclamptics showed nocturnal hypertension. Levels of plasma renin activity fell progressively from 9 a.m. to midnight in all three groups, and were significantly lower in pre-eclampsia. Plasma angiotensin II levels fell during the day in normotensives and uncomplicated hypertensives, whereas pre-eclamptics showed loss of this pattern and significantly lower levels than the other groups. Free plasma norepinephrine levels fell during the day and to a similar extent in all three groups. A diurnal pattern for free plasma norepinephrine levels, with lower levels at midnight than during the day, was seen in normotensives and uncomplicated hypertensives but not in pre-eclamptics. Plasma norepinephrine sulphate levels rose from 9.00 a.m. to midnight in normotensives and uncomplicated hypertensives. This pattern was reversed in pre-eclamptics, including 3 of the 4 subjects with nocturnal hypertension. Plasma epinephrine sulphate levels rose progressively through the day, with no significant differences between groups. Failure of plasma angiotensin II or epinephrine levels to fall at night in pre-eclampsia may contribute to nocturnal hypertension in subjects with increased vascular reactivity. Suppression of plasma renin activity and angiotensin II levels suggests that perhaps as yet unidentified pressor mechanisms are involved in pre-eclampsia.

Circadian rhythms of blood pressure and pressor hormones in normal and hypertensive pregnancy.

1. Circadian patterns of blood pressure and pressor hormones were studied in late pregnancy in ten normotensive women and twenty-one hypertensives. 2. Plasma renin activity, angiotensin II and free adrenaline and noradrenaline leves were highest at 0900 h in normotensives and uncomplicated hypertensives. Catecholamine sulphate levels were lowest at 0900 h. 3. In eight pre-eclamptic hypertensives, plasma renin activity, and angiotensin II were suppressed. 4. Adrenaline sulphate levels were raised in hypertensives. 5. Four pre-eclamptics with noctural hypertension had the highest blood pressures; their diurnal rhythm for noradrenaline sulphate was reversed but they showed no consistent pattern of pressor hormones. However failure of angiotensin II, adrenaline or noradrenaline to fall at night in subjects with increased vascular reactivity could contribute to their nocturnal hypertension.

Effect of beta-adrenergic receptor blockade with propranolol on the response of plasma catecholamines and renin activity to upright tilting in normal subjects.

1 Relationship between plasma catecholamines (measured as noradrenaline and adrenaline) and plasma renin activity (PRA) were examined at rest and during passive head-up tilting for 30 min in nine normal subjects, before and after treatment with propranolol 160 mg daily for 7 days. 2 Noradrenaline (NA) and adrenaline (A) increased substantially after tilting for 15 min. There were no changes in PRA. After 30 min tilting, NA remained elevated, whereas A had returned to resting levels. A significant increase in PRA was apparent at 30 min. Pulse rate and diastolic blood pressure increased progressively during tilting. Systolic pressure did not change. 3 Treatment with propranolol reduced pulse rate and systolic blood pressure at rest and during tilting. Resting catecholamine concentrations and the response of NA to tilting were unaffected. In contrast, treatment prolonged the A response leading to significantly higher levels after 30 min tilting. Propranolol reduced PRA in six of the nine subjects and prevented the increase with tilting observed before treatment.