Prenatal vitamin C and E supplementation in smokers is associated with reduced placental abruption and preterm birth: a secondary analysis.

OBJECTIVE: Smoking and pre-eclampsia (PE) are associated with increases in preterm birth, placental abruption and low birthweight. We evaluated the relationship between prenatal vitamin C and E (C/E) supplementation and perinatal outcomes by maternal self-reported smoking status focusing on outcomes known to be impacted by maternal smoking. DESIGN/SETTING/POPULATION: A secondary analysis of a multi-centre trial of vitamin C/E supplementation starting at 9-16 weeks in low-risk nulliparous women with singleton gestations. METHODS: We examined the effect of vitamin C/E by smoking status at randomisation using the Breslow-Day test for interaction. MAIN OUTCOME MEASURES: The trial's primary outcomes were PE and a composite outcome of pregnancy-associated hypertension (PAH) with serious adverse outcomes. Perinatal outcomes included preterm birth and abruption. RESULTS: There were no differences in baseline characteristics within subgroups (smokers versus nonsmokers) by vitamin supplementation status. The effect of prenatal vitamin C/E on the risk of PE (P = 0.66) or PAH composite outcome (P = 0.86) did not differ by smoking status. Vitamin C/E was protective for placental abruption in smokers (relative risk [RR] 0.09; 95% CI 0.00-0.87], but not in nonsmokers (RR 0.92; 95% CI 0.52-1.62) (P = 0.01), and for preterm birth in smokers (RR 0.76; 95% CI 0.58-0.99) but not in nonsmokers (RR 1.03; 95% CI 0.90-1.17) (P = 0.046). CONCLUSION: In this cohort of women, smoking was not associated with a reduction in PE or the composite outcome of PAH. Vitamin C/E supplementation appears to be associated with a reduction in placental abruption and preterm birth among smokers.

Haptoglobin phenotype, pre-eclampsia, and response to supplementation with vitamins C and E in pregnant women with type-1 diabetes.

OBJECTIVE: The phenotype of the antioxidant and pro-angiogenic protein haptoglobin (Hp) predicts cardiovascular disease risk and treatment response to antioxidant vitamins in individuals with diabetes. Our objective was to determine whether Hp phenotype influences pre-eclampsia risk, or the efficacy of vitamins C and E in preventing pre-eclampsia, in women with type-1 diabetes. DESIGN: This is a secondary analysis of a randomised controlled trial in which women with diabetes received daily vitamins C and E, or placebo, from 8 to 22 weeks of gestation until delivery. SETTING: Twenty-five antenatal metabolic clinics across the UK (in north-west England, Scotland, and Northern Ireland). POPULATION: Pregnant women with type-1 diabetes. METHODS: Hp phenotype was determined in white women who completed the study and had plasma samples available (n = 685). MAIN OUTCOME MEASURE: Pre-eclampsia. RESULTS: Compared with Hp 2-1, Hp 1-1 (OR 0.59, 95% CI 0.30-1.16) and Hp 2-2 (OR 0.93, 95% CI 0.60-1.45) were not associated with significantly decreased pre-eclampsia risk after adjusting for treatment group and HbA1c at randomisation. Our study was not powered to detect an interaction between Hp phenotype and treatment response; however, our preliminary analysis suggests that vitamins C and E did not prevent pre-eclampsia in women of any Hp phenotype (Hp 1-1, OR 0.77, 95% CI 0.22-2.71; Hp 2-1, OR 0.81, 95% CI 0.46-1.43; Hp 2-2, 0.67, 95% CI 0.34-1.33), after adjusting for HbA1c at randomisation. CONCLUSIONS: The Hp phenotype did not significantly affect pre-eclampsia risk in women with type-1 diabetes.

OS098. Increased adiposity decreases the intensity myeloperoxidase in neutrophils from pregnant women at delivery.

INTRODUCTION: The cardiovascular risk factor myeloperoxidase (MPO) is a lysozomal enzyme found within azurophillic granules of inflammatory cells. Upon activation, inflammatory cells degranulate and release MPO. In the pregnancy disorder preeclampsia, MPO is elevated in the circulation and placenta and greater numbers of neutrophils are positive for hydrogen peroxide. OBJECTIVES: To determine if during preeclampsia, increases in circulating MPO result in losses of intracellular MPO within neutrophils. We also sought to determine if the degree by which neutrophil functionand intracellular MPO levels varies by BMI differsin women with preeclampsia compared to normal control pregnancies. METHODS: Reactive oxygen species (nitric oxide, superoxide and hydrogen preoxide) weredetermined in fresh whole blood samples from non-smoking women with normal pregnancies (n=8) and preeclampsia (n=9) by flow cytometry using intracellular fluorescent probes. Granulocytes were permeabilized (Dako, Glostrup, Denmark) and stained for MPO (anti-MPO, BD, San Jose, CA), and the number and percentage of CD15 and MPO positive cells and the intensity of MPO staining (MFI) was determined by flow cytometry. Student t-test or one-way ANOVA was used to test for significance. Dataare reported as mean±sem. RESULTS: There wasno significant difference in the number (percentage) orintensity of neutrophils staining for MPO between normal pregnancies and preeclampsia (MPO positive cells: 2816±981(39% positive) vs 2714±901(28% positive), Intensity: 3337±434 vs 3178±367 MFI units). However, there was a dramatic reduction in intensity of MPO staining in neutrophils from overweight women (BMI>27) compared to normal weight women regardless of pregnancy outcome (2508±160 vs 4091±376 MFI units, p=0.001). Among normal pregnancies, lean women had significantly more MFI of MPO than overweight women (4500±750 vs 2639±178 MFI units, p=0.04). The same trend was seen among women with preeclampsia (lean; 3845±428 vs overweight; 2344±292MFI units, p=0.07). CONCLUSION: The increased circulating levels of MPO in preeclampsia did not result in decreased intensity of MPO staining in circulating neutrophils. Neutrophils from normal weight women have greater amounts of intracellular MPO available upon activation than overweight and obese women. ACKNOWLEDGEMENT: This project was supported byNational Institutes of Health grant PO1-HD30367.

Binding of fatty acids facilitates oxidation of cysteine-34 and converts copper-albumin complexes from antioxidants to prooxidants.

As a transition metal capable of undergoing one-electron oxidation-reduction conversions, copper (Cu) is essential for life and fulfills important catalytic functions. Paradoxically, the same redox properties of copper can make it extremely dangerous because it can catalyze production of free radical intermediates from molecular oxygen. Factors involved in regulation of redox activity of albumin-bound copper have not been well characterized. In the present study, effects of modification of the albumin cysteine-34 (Cys-34) and binding of nonesterified fatty acids on the redox-cycling activity of the complex of copper with human serum albumin (Cu/HSA) were studied. Because ascorbate is the most abundant natural reductant/scavenger of free radicals in blood plasma, the electron paramagnetic resonance assay of ascorbate radical formation was used as a method to monitor Cu/HSA redox-cycling activity. At Cu/HSA ratios below 1:1, the bound Cu was virtually redox inactive, as long as Cys-34 was in reduced state (Cu/HSA-SH). Alkylation, nitrosylation, or oxidation of Cu/HSA resulted in the appearance of redox-cycling activity. Experiments with ultrafiltration of Cu/HSA alkylated with N-ethylmaleimide (Cu/HSA-NEM) showed that at Cu/HSA-NEM ratios below 1:1, the ascorbate radicals were produced by Cu tightly bound to HSA rather than by Cu released in solution. The rate of ascorbate radical production in HSA-NEM and S-nitrosylated HSA (HSA-NO) was, however, more than one order of magnitude lower than that in a solution containing equivalent concentration of free copper ions. While Cu/HSA-SH was redox inactive, binding of oleic or linoleic acids induced Cu-dependent redox-cycling with maximal activity reached at a fatty acid to protein molar ratio of 3:1 for oleic acid and 2:1 for linoleic acid. Binding of fatty acids caused profound conformational changes and facilitated oxidation of the Cys-34 SH-group at essentially the same ratios as those that caused redox-cycling activity of Cu/HSA. We conclude that fatty acids regulate anti-/prooxidant properties of Cu-albumin via controlling redox status of Cys-34.

Endothelial function and myogenic reactivity in small mesenteric arteries of hyperlipidemic pregnant rats.

Supraphysiological increases in serum triglycerides and cholesterol often occur during pregnancy, but their effects on vascular function are poorly understood. Intraperitoneal injection of the nontoxic surfactant poloxamer 407 (P-407) results in sustained elevation of triglycerides and cholesterol. We asked if P-407-induced hyperlipidemia during late pregnancy adversely affects mesenteric resistance artery vasodilator function. On days 13-15 of pregnancy, rats were given a single intraperitoneal injection of P-407, sterile water vehicle, or non-lipid-altering pluronic F-88 (P-88). Four days postinjection, serum triglycerides, cholesterol, free fatty acids, and the lipid peroxidation product malondialdehyde were significantly increased in P-407-treated rats. Mesenteric arteries from P-407-treated rats displayed significant increases in myogenic reactivity (constrictor responses to step increases in intraluminal pressure). The nitric oxide (NO) blocker N(alpha)-methyl-L-arginine increased the myogenic response in control but not in P-407 arteries, normalizing group differences. Endothelial removal increased myogenic reactivity beyond that of prior NO synthase inhibition in controls and potentiated myogenic reactivity in P-407 arteries such that responses again converged. Relaxation responses to the endothelium-dependent vasodilator methacholine did not differ. We conclude that that P-407-induced hyperlipidemia during pregnancy increases myogenic reactivity due to selective attenuation of an NO-mediated vasodilator component of the myogenic response.

Conditioned medium from hypoxic cytotrophoblasts alters arterial function.

OBJECTIVE: Our goal was to test the hypothesis that cytotrophoblasts, under low oxygen tension, release substances that affect vascular behavior. STUDY DESIGN: We studied the vascular response to the vasoconstrictors phenylephrine (receptor dependent) and potassium (receptor independent), the relaxation response to methacholine, and the vasomotor behavior of isolated resistance (mesenteric) arteries from early pregnant rats after incubation in conditioned medium from first-trimester cytotrophoblasts, maintained in standard or hypoxic (2%; 14 mm Hg) culture conditions. RESULTS: After incubation in medium from hypoxic cytotrophoblasts, arterial segments were more responsive to phenylephrine and to potassium-induced constriction but were less responsive to methacholine, and the vasomotor activity was increased compared with that found in vessels incubated in control medium. CONCLUSIONS: These changes in vascular behavior are similar to those reported in isolated arteries from women with preeclampsia. These studies provide evidence which suggests that the link between abnormal placentation and maternal vascular abnormality in preeclampsia is the elaboration of vasoactive factors by cytotrophoblasts in response to hypoxia.

Endothelin and nitric oxide mediate reduced myogenic reactivity of small renal arteries from pregnant rats.

We tested the hypothesis that endothelin acting through the endothelial ET(B) receptor subtype and the nitric oxide (NO) pathway accounts for reduced myogenic reactivity of the renal resistance vasculature during pregnancy. Small renal arteries (100-200 microm) were isolated from virgin and midterm pregnant rats when gestational renal hyperfiltration and vasodilation are maximal in this species. Myogenic reactivity (the adjustment of arterial diameter in response to a change in transmural pressure) was assessed with a pressurized myograph system. A rapid increase in transmural pressure from 60 to 80 mmHg resulted in a 2.4% diameter increase in vessels from virgin compared with an 8.1% increase in arteries from midgestation rats (n = 8 each, P < 0.05). Thus myogenic reactivity is markedly reduced during pregnancy. Incubation with the NO synthase inhibitors, an ET(B) receptor subtype antagonist (RES-701-1), the nonselective ET(A/B) receptor blocker (SB-209670), or endothelial removal abrogated the reduced myogenic reactivity of vessels from gravid rats without affecting myogenic reactivity in arteries from virgin animals. Thus the endothelium mediates the reduced myogenic reactivity of small renal arteries of midgestation rats most likely through the ET(B) receptor subtype and NO pathway.

Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein.

Although the function of metallothionein (MT), a 6- to 7-kDa cysteine-rich metal binding protein, remains unclear, it has been suggested from in vitro studies that MT is an important component of intracellular redox signaling, including being a target for nitric oxide (NO). To directly study the interaction between MT and NO in live cells, we generated a fusion protein consisting of MT sandwiched between two mutant green fluorescent proteins (GFPs). In vitro studies with this chimera (FRET-MT) demonstrate that fluorescent resonance energy transfer (FRET) can be used to follow conformational changes indicative of metal release from MT. Imaging experiments with live endothelial cells show that agents that increase cytoplasmic Ca(2+) act via endogenously generated NO to rapidly and persistently release metal from MT. A role for this interaction in intact tissue is supported by the finding that the myogenic reflex of mesenteric arteries is absent in MT knockout mice (MT(-/-)) unless endogenous NO synthesis is blocked. These results are the first application of intramolecular green fluorescent protein (GFP)-based FRET in a native protein and demonstrate the utility of FRET-MT as an intracellular surrogate indicator of NO production. In addition, an important role of metal thiolate clusters of MT in NO signaling in vascular tissue is revealed.

Endothelin mediates renal vasodilation and hyperfiltration during pregnancy in chronically instrumented conscious rats.

Profound vasodilation of the kidneys and other nonreproductive organs transpires during early pregnancy. Because nitric oxide (NO) was found to mediate renal vasodilation and hyperfiltration in conscious pregnant rats, and endogenous endothelin (ET) was suggested to be vasodilatory in the renal circulation of nonpregnant rats, we tested whether endothelin mediates the NO-dependent changes in the renal circulation during pregnancy. Glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) were measured in conscious pregnant and virgin rats before and during infusion of 30 micrograms/min RES-701-1 (a selective ETB receptor subtype antagonist). Baseline GFR and ERPF were significantly increased by 35% in gravid rats relative to virgin controls. During infusion of RES-701-1, the pregnant rats responded more robustly, showing a greater decline in both GFR and ERPF such that renal function converged in the two groups of rats. ERPF also converged in pregnant and virgin rats during infusion of SB-209760, a nonselective ETA/B receptor subtype antagonist. Combined infusion of Nomega-nitro-L-arginine methyl ester [L-NAME, an NO synthase (NOS) inhibitor] and RES-701-1 reduced GFR and ERPF to levels comparable to those reached with either agent given alone, suggesting inhibition of a common vasodilatory pathway. RES-701-1 and SB-209670 significantly lowered the cGMP content of small renal arteries from gravid and virgin rats in vitro, strengthening the link between the renal endothelial ETB receptor subtype and NO. Importantly, we showed that RES-701-1 is not a direct inhibitor of NOS. We conclude that endothelin mediates the NO-dependent changes in the renal circulation of conscious rats during pregnancy.

Contribution of vasomotion to vascular resistance: a comparison of arteries from virgin and pregnant rats.

Intrinsic oscillatory activity, or vasomotion, within the microcirculation has many potential functions, including modulation of vascular resistance. Alterations in oscillatory activity during pregnancy may contribute to the marked reduction in vascular resistance. The purpose of this study was 1) to mathematically model the oscillatory changes in vessel diameter and determine the effect on vascular resistance and 2) to characterize the vasomotion in resistance arteries of pregnant and nonpregnant (virgin) rats. Mesenteric arteries were isolated from Sprague-Dawley rats and studied in a pressurized arteriograph. Mathematical modeling demonstrated that the resistance in a vessel with vasomotion was greater than that in a static vessel with the same mean radius. During constriction with the alpha1-adrenergic agonist phenylephrine, the amplitude of oscillation was less in the arteries from pregnant rats. We conclude that vasomotor activity may provide a mechanism to regulate vascular resistance and blood flow independent of static changes in arterial diameter. During pregnancy the decrease in vasomotor activity in resistance arteries may contribute to the reduction in peripheral vascular resistance.

Small low-density lipoproteins and vascular cell adhesion molecule-1 are increased in association with hyperlipidemia in preeclampsia.

The pregnancy disorder preeclampsia is characterized by endothelial cell dysfunction that may be promoted by abnormal increases in circulating lipids, particularly triglycerides and free fatty acids. Serum triglyceride concentration is a major regulatory determinant of low-density lipoprotein (LDL) size and density distribution. Smaller, denser LDL particles have several intrinsic properties capable of inducing endothelial dysfunction. The present nested, case-control study of gestationally matched preeclamptic and normal pregnant women tested the hypothesis that hypertriglyceridemia in preeclampsia is accompanied by decreases in LDL peak particle diameter (predominant LDL size). Plasma LDL peak particle diameter was determined by nondenaturing 2% to 16% polyacrylamide gel electrophoresis. Correlations of LDL diameter with the concentration of serum triglycerides, free fatty acids, total cholesterol, LDL-cholesterol, and apolipoprotein B (apo B) were determined. In the same individuals, we measured serum concentrations of a marker of vascular dysfunction previously reported to be increased in preeclampsia, soluble vascular cell adhesion molecule-1 (VCAM-1), and examined the association of VCAM-1 with LDL diameter and serum lipids. LDL peak particle diameter was decreased in preeclampsia relative to normal pregnancy (P < .01). The LDL-cholesterol:apo B ratio, which frequently decreases with decreasing LDL diameter, was also decreased (P < .04). Triglyceride concentrations were increased in preeclampsia (P < .0002), and there was a significant inverse relationship between LDL peak particle diameter and triglycerides (r = -.55, P < .02). Serum soluble VCAM-1 concentrations were markedly increased in preeclampsia (P < .0003). Apo B (P < .004), free fatty acids (P < .01), total cholesterol (P < .01), and LDL-cholesterol (P < .02) were also increased. VCAM-1 correlated with apo B (r = .50, P < .03) and LDL-cholesterol (r = .50, P < .03), but showed no relationship with the LDL diameter, LDL-cholesterol:apo B ratio, or other lipids. We conclude that the predominance of smaller, denser LDL, a potential contributor to endothelial cell dysfunction, is a feature of preeclampsia. However, the serum VCAM-1 level, one indicator of endothelial involvement, may be influenced more by quantitative lipoprotein changes (serum apo B or LDL-cholesterol) than by LDL particle size.

Altered active but not passive properties of mesenteric resistance arteries from the vitamin E-deprived rat.

1 We tested the hypothesis that lowering antioxidant protection through dietary vitamin E deprivation would alter active and passive mechanical properties in resistance arteries of the rat. Specifically, we hypothesized that vascular tone in isolated mesenteric arteries of the vitamin E-deprived rats would be altered due to impaired endothelial influences of nitric oxide and/or prostaglandins. 2 Lumen diameter and wall thickness were measured in pressurized arteries (approximately 250 microm diameter) from control (n=9) and vitamin E deprived (n=9) Sprague-Dawley female rats by use of a dimension analysing system. 3 Treatment with a cyclo-oxygenase inhibitor (meclofenamate) did not affect the basal vascular tone in either group. Treatment with a nitric oxide synthase inhibitor (NG-methyl-L-arginine) caused a significant increase in basal tone only in the vitamin E-deprived rats (% tone: 6.2+/-1.1 vs 1.2+/-0.3%; P<0.05). When tone was induced to 25% of the initial diameter with phenylephrine, treatment with the nitric oxide synthase inhibitor resulted in a greater potentiated tone in the vitamin E-deprived rats compared to the controls (26.5+/-2.7 vs 16.4+/-3.4%; P<0.05); suggesting a greater nitric oxide affect in the vessels from the vitamin E-deprived rats. Meclofenamate treatment in the induced tone arteries significantly relaxed (-17.4+/-4.0%; P<0.05) only the arteries from the vitamin E-deprived rats, indicating that a vasoconstrictor was modifying tone. The passive characteristics of distensibility and stress-strain relationship were not different between the two groups of rats. 4 In summary, vitamin E deprivation in the rat enhanced the modulation of vascular tone by both the nitric oxide and cyclo-oxygenase pathways but did not alter passive characteristics of mesenteric arteries.

Contribution of chondroitin-dermatan sulfate-containing proteoglycans to the function of rat mesenteric arteries.

Proteoglycans are an important nonfibrous matrix component of the arterial wall. Direct evidence for their role in resistance-sized arteries is lacking, although they likely have an important role in coordinating and regulating vessel behavior, presumably via interactions of their glycosaminoglycan chains or core proteins with other matrix molecules and/or the smooth muscle cell surface. The purpose of this study was to determine whether the removal of specific glycosaminoglycan chains from proteoglycans in resistance-sized mesenteric arteries would change the mechanical properties of the arterial wall, thereby affecting their functional behavior. The major finding of the study was that 65% removal of chondroitin-dermatan sulfate-containing glycosaminoglycans from the arterial wall increased vascular wall stiffness and altered the myogenic behavior of the artery. The significant alterations in myogenic behavior associated with changes in passive mechanics following partial glycosaminoglycan chain removal support our hypothesis that chondroitin-dermatan sulfate-containing proteoglycans contribute significantly to the functional behavior of resistance arteries. We speculate that these alterations are the result of changes in stress transfer between collagen fibrils and/or stress transfer between cells and collagen fibrils under applied pressure.

Intrinsic tone and passive mechanics of isolated renal arteries from virgin and late-pregnant rats.

The purpose of this study was to investigate whether there are alterations in the intrinsic properties of renal interlobar arteries during pregnancy. Renal interlobar arteries (internal diameter approximately 250 microns) from virgin and late-pregnant rats were mounted in a pressurized arteriograph system. Intrinsic tone was quantified as the percent difference in luminal diameter of each artery in the presence of physiological saline solution and while pharmacologically relaxed with papaverine. At pressures between 75 and 125 mmHg, tone was 35-50% less in arteries from pregnant rats (P < 0.05). Endothelial removal reduced tone in arteries from virgin rats but had no effect on arteries from pregnant rats. Analysis of stress-strain curves (rate constants: pregnant, 6.31 +/- 0.38; virgin, 7.81 +/- 0.78; P < 0.05) indicate that there is a decrease in arterial stiffness in gestation. Thus pregnancy is associated with a reduced intrinsic tone, possibly because of a reduction in an endothelial constrictor influence on the vascular smooth muscle in isolated rat renal interlobar arteries. This effect, coupled with the decreased arterial stiffness, demonstrates the significant arterial adaptation occurring during pregnancy.