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(Hypertension. 1996;28:758-764.)
© 1996 American Heart Association, Inc.
Articles |
Distinct Factors in Plasma of Preeclamptic Women Increase Endothelial Nitric Oxide or Prostacyclin
the Magee-Womens Research Institute and Department of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh (Pa).
Correspondence to Sandra T. Davidge, Perinatal Research Institute, 220 HMRC, University of Alberta, Edmonton, AB, Canada, T6G 2S2. E-mail sdavidge@gpu.srv.ualberta.ca.
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Abstract |
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The pathogenesis of preeclampsia is proposed to be due to uncharacterized circulating factors that activate endothelial cells. Support for this hypothesis is provided by in vitro activation of endothelial cells by plasma from preeclamptic women, eg, increased nitric oxide and prostacyclin generation. We performed molecular sizing, lipid extraction, and lipoprotein fractionation of plasma from normal pregnant and preeclamptic women and determined the ability of these plasma fractions to increase nitric oxide or prostacyclin generation by endothelial cells. Fractions from plasma of preeclamptic women were consistently more active than fractions from normal pregnant women, although characterization was qualitatively similar. The factors stimulating nitric oxide and prostacyclin were different. The factor (or factors) stimulating nitric oxide generation was extractable by charcoal and present in lipid extracts and lipoprotein isolates with a molecular weight greater that 1.5 million daltons, which is characteristic of a lipoprotein or lipoprotein aggregate. By contrast, activity to stimulate prostacyclin persisted after charcoal stripping or lipoprotein removal, partitioned to the aqueous fraction, and had a molecular weight of approximately 50 000 D. Two distinct factors in the blood of preeclamptic women alter endothelial function in vitro. This information should guide the search for circulating factors contributing to the pathophysiology of preeclampsia.
Key Words: preeclampsia • endothelium • nitric oxide • prostaglandin • lipids • lipoproteins • pregnancy
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Introduction |
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Preeclampsia is a multisystemic complication of pregnancy estimated to affect 7% to 10% of all pregnancies.1 The pathophysiological features of preeclampsia include increased vascular reactivity and activation of the coagulation cascade and have been attributed to altered endothelial cell function.2 Endothelial cells modulate vascular homeostasis through the release of vasodilator agents such as NO and prostacyclin. In women with preeclampsia, there is evidence that intravascular production of prostacyclin is deficient.3 4 The involvement of NO in preeclampsia is uncertain. Urinary and plasma NO metabolites have been reported to be unchanged or decreased in preeclamptic women compared with normal pregnant women.5 6 7 8 Bioassay has shown umbilical vessels from infants of preeclamptic women to produce less NO than vessels from infants of normal pregnant women.9
For many years it has been proposed that materials produced by the poorly perfused placenta of preeclamptic women are released into the circulation and lead to the systemic pathophysiological changes of the disorder.10 Several years ago, this hypothesis was extended to propose that the vascular endothelium was the primary target of these agents.11 Support for a circulating factor (or factors) is provided by studies examining the effect of serum/plasma from preeclamptic women on the behavior of endothelial cells in culture. Serum from preeclamptic women increases platelet-derived growth factor production, increases intracellular triglyceride accumulation, and stimulates cellular fibronectin release from cultured endothelial cells.12 13 14 Paradoxically, although preeclampsia is associated with deficient intravascular production of the vasodilators prostacyclin3 4 and possibly NO,5 8 we and other investigators have reported increased in vitro production of prostacyclin and NO from endothelial cells exposed to the plasma of preeclamptic women compared with plasma of normal pregnant women.15 16 17 18 These data suggest that a factor (or factors) that causes endothelial activation is present in the circulation of preeclamptic women. However, the nature of this factor is not known.
The present study begins to isolate and characterize the factor (or factors) responsible for the in vitro endothelial cell activation of NO and prostacyclin. We report that in vitro endothelial NO and prostacyclin production stimulated by plasma of preeclamptic women is mediated by two distinctly different plasma factors. NO stimulation resulted from a lipid-containing factor with a high molecular weight—likely a lipoprotein. By contrast, prostacyclin was stimulated by an aqueous factor with a lower molecular weight.
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Subjects
Nulliparous subjects were recruited at the time of admission to the labor and delivery unit at Magee-Womens Hospital, Pittsburgh, with the use of protocols approved by the hospital Ethics Committee. Preeclampsia was defined by the criteria of hypertension, proteinuria, hyperuricemia, and reversal of hypertension and proteinuria after delivery.1 Hypertension was defined as an increase of 30 mm Hg systolic or 15 mm Hg diastolic pressure compared with values obtained before 20 weeks of gestation, or an absolute blood pressure greater than 140/90 mm Hg after 20 weeks of gestation if earlier blood pressures were not known. Proteinuria was defined as greater than 500 mg/24 h collection or greater than 2+ on a voided or greater than 1+ on a catheterized random urine specimen. Hyperuricemia was defined as more than 1 SD above usual values (at term, >5.5 mmol/L). Control subjects had uncomplicated pregnancies, were normotensive throughout gestation, and had no proteinuria. No subject was known to have chronic hypertension or renal or metabolic disease. To verify that observed effects were not due to hemoconcentration, we determined that hematocrits did not differ between the two groups of women.
Blood was collected by routine forearm venipuncture before delivery. Plasma was prepared from blood anticoagulated with EDTA to avoid the confounding effects of cellular products released into serum during blood coagulation. Samples were aliquoted under sterile conditions and stored at -80°C.
Isolation and characterization studies were conducted on plasma pooled from four preeclamptic women and four normal pregnant women. Pooled plasma from other women was used for repeating procedures to assure reproducibility.
Reagents
Horse serum, L-glutamine, gentamicin, kanamycin, nystatin, and trypsin-EDTA were obtained from GIBCO Laboratories. Heparin, sulfanilamide, naphthylethylenediamine dihydrochloride, phosphoric acid, fat red 7B, myoglobin, carbonic anhydrase, ovalbumin, bovine serum albumin, 
-globulin, lysozyme, and thyroglobulin were from Sigma Chemical Co. Dialysis cassettes were purchased from Pierce Chemical Co. Alpha-modified minimum essential medium, Dulbecco's phosphate-buffered saline, butanol, isopropyl ether, charcoal, imido black, and agarose gels were purchased from Fisher Scientific.
Endothelial Cell Culture
A bovine coronary microvascular endothelial cell line was obtained from Gensia, Inc. Since the establishment of this cell line, the phenotype of these endothelial cells has been maintained for more than 180 passages. Cellular characteristics include growth as a monolayer; a cobblestone morphology at confluence; positive immunostaining for von Willebrand factor–related antigen; the presence of receptors for acetylated LDL; and secretion of prostaglandins, NO, and tissue-type plasminogen activator.
Cells were grown at 37°C in a humidified atmosphere of 5% CO2/95% air with alpha-modified minimum essential medium (0.6 mmol/L L-arginine) containing 10% horse serum, 2 mmol/L L-glutamine, gentamicin (5 µg/mL), kanamycin (20 µg/mL), and nystatin (10 U/mL). Cell cultures were dispersed with 0.05% trypsin/0.53 mmol/L EDTA, plated at 105 cells in 24-well plates, and grown to confluent monolayers. The consistency of the cell number in each well was confirmed by measurement of protein content by the Bradford method.19
Experimental Design
Cells were made quiescent in serum-free medium containing 0.05% bovine serum albumin for 24 hours before experimental stimulation. Basal release of NO and prostacyclin by the cells during quiescence was subtracted from the observed stimulation by plasma or plasma fractions. The medium was supplemented with heparin (final concentration, 10 U/mL) to prevent clotting of the dilute EDTA plasma when exposed to the endothelial cells. Plasma or plasma fractions were then added to the cells at a final concentration of 2%. We chose this concentration on the basis of our previous work indicating that 2% plasma gave reproducible stimulation without affecting cellular viability.17 All experiments were performed with triplicate wells. Medium was removed after 24 hours for measurement of nitrite, a stable end product of NO, and 6-keto-PGF1
, a stable end product of prostacyclin.
Nitrite and 6-Keto-PGF1 Assays
Nitrite production was determined with the spectrophotometric Greiss reaction.20 Preliminary studies with nitrate reductase indicated that nitrate levels were less than 10% of total nitrite and nitrate levels. Therefore, we measured only nitrite levels for these studies. An aliquot of medium (180 µL) from each culture well was mixed with 20 µL Greiss reagent (1% sulfanilamide and 0.1% naphthylethylenediamine dihydrochloride in 2% phosphoric acid). The mixture was incubated for 10 minutes at room temperature, and the absorbance (550 nm) was read in a Vmax kinetic microplate reader (Molecular Devices). Concentrations were determined by comparison with a standard solution of sodium nitrite in plasma-free medium. The reaction was linear from 0.25 to 64 nmol/mL.
Prostacyclin was measured as its stable metabolite 6-keto-PGF1 by an enzyme immunoassay (Cayman Co). The lower detection limit was 3.9 pg/mL.
Molecular Weight Estimation
Size-exclusion chromatography was conducted with either Sephacryl or polyacrylamide beads as the gel filtration medium. Sephacryl S-300 HR (Pharmacia Biotech) packing was used in 1.5x50-cm columns with a fractionation range of 1.0x104 to 1.5x106 D. Each Sephacryl-packed column was calibrated with six protein standards ranging in molecular weight from 19 000 to 670 000 D (myoglobulin [19 kD], carbonic anhydrase [30 kD], ovalbumin [43 kD], bovine serum albumin [67 kD], -globulin [158 kD], and thyroglobulin [670 kD]). Bio-Gel P100 (Bio-Rad) packing was used in 1.5x50-cm columns with a fractionation range of 5x103 to 1x105 D. Each polyacrylamide-packed column was calibrated with four protein standards ranging in molecular weight from 19 000 to 67 000 D (myoglobulin [19 kD], carbonic anhydrase [30 kD], ovalbumin [43 kD], and bovine serum albumin [67 kD]).
Pooled plasma (1 mL) was placed on the columns and eluted with Dulbecco's phosphate-buffered saline. The flow rate was 0.47 mL/min. Fractions of 1.0 mL were collected into 13x100-mm tubes and read at an absorbance of 280 nm. Fractions were then filter sterilized and placed on cells (200 µL sample plus 300 µL medium). Experiments included a phosphate-buffered saline control.
Lipid Removal
We used charcoal treatment (modified method of Chen21 ) to remove lipophilic, hydrophobic factors. Charcoal was first prepared by washing with 0.9% saline and 4% bovine serum albumin (10% suspension) overnight. The saline wash was removed by centrifugation at 1500 rpm for 10 minutes. Plasma was then resuspended with the charcoal (10% suspension) and stirred for 6 hours. For charcoal removal, the plasma/charcoal mixture was centrifuged at 1500 rpm for 10 minutes. Plasma was retained, and further charcoal was removed by centrifugation at 16 000 rpm for 5 minutes. Samples were then filter sterilized and placed on endothelial cells at a 2% concentration.
To further evaluate lipid and aqueous fractions, we extracted plasma with butanol/isopropyl ether. Pooled plasma (250 µL) was added to 0.5 mL butanol/isopropyl ether (40:60, vol/vol) and centrifuged for 2 minutes at 1000g. An additional 1 mL of butanol/isopropyl ether was added to the supernatant and centrifuged for 2 minutes at 1000g. The organic phases from the two steps were pooled. Both aqueous and organic phases were placed in a SpeedVac for 1 hour and reconstituted to 0.5 mL phosphate-buffered saline. Samples were then filter sterilized and placed on endothelial cells at a 2% concentration.
Lipoprotein Fractionation
Lipoprotein isolation was conducted by density gradient ultracentrifugation from pooled plasma as well as individual samples from two preeclamptic women and two normal pregnant women. Isolation of CM/VLDL (density range, 1.006 to 1.019 g/mL) and LDL (density range, 1.019 to 1.063 g/mL) fractions was achieved with the density gradient ultracentrifugation method of Havel et al22 adapted for the rapid, sequential isolation of low-volume lipoprotein fractions. Lipoprotein separations were made following the TL-100 Tabletop Ultracentrifuge Application Note (Beckman Instruments, Inc). Briefly, 0.5 mL saline (0.9% NaCl containing 0.1 g/L EDTA) was mixed with 0.5 mL plasma in polycarbonate centrifuge tubes and centrifuged at 436 000g for 1 hour and 45 minutes at 10°C using a TLA-100.2 fixed-angle rotor (Beckman). The top, CM/VLDL-containing layer (0.5 mL) was obtained by careful aspiration; the remaining fraction was adjusted to a density of 1.019 g/mL (with 0.5 mL of 16.7% NaCl, 0.1 g/L EDTA) and centrifuged as previously described. The top, LDL-containing layer (0.5 mL) was adjusted to a density of 1.10 g/mL using solid potassium bromide and recentrifuged so that LDL was purified of albumin contamination. The remaining fraction (devoid of lipoproteins, with density <1.063 g/mL) was filter sterilized (Amicon, 0.22 µm) and stored under nitrogen at 4°C in the dark for less than 24 hours before use. LDL and CM/VLDL fractions were dialyzed in the dark against two changes of deoxygenated phosphate-buffered saline containing 10 µmol/L EDTA at 4°C for 12 hours with the use of dialysis cassettes. Fractions were subsequently sterile filtered and stored at 4°C under nitrogen before use. The purity of fractions was confirmed by agarose gel electrophoresis23 with both lipid (fat red 7B) and protein (imido black) staining. Total protein was estimated by Peterson's modification of Lowry.24 Fractions were filter sterilized and placed on endothelial cells at a 2% concentration.
CM/VLDL and LDL fractions were subsequently treated with magnesium (240 U/mL) and heparin (0.1012 mol/L) for precipitation of apoprotein B followed by centrifugation for removal of lipoproteins from each fraction.25 The status of intact and treated lipoproteins was confirmed with agarose gel electrophoresis and lipid staining. Intact and treated lipoproteins were placed on endothelial cells at a 2% concentration.
Data Analysis
Data are presented as mean±SE of triplicate wells of endothelial cells stimulated with pooled plasma and their fractions. Lipoprotein fractionation was performed with plasma pooled from preeclamptic and normal pregnant women as well as with plasma from two preeclamptic and two normal pregnant women and is presented as mean±SE. ANOVA was conducted with post hoc analysis by Fisher's protected least significant difference test. Differences among means were considered significant at a value of P<.05.
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Results |
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Molecular Weight Estimation
Fractions that stimulated either nitrite or 6-keto-PGF1
generation were qualitatively similar in preparations from the plasma of normal and preeclamptic women. However, the area under the peak of activity was greater in fractions from preeclamptic women (P<.05). Fig 1
depicts the activity of fractions from S-300 columns (fractionation range, 1.0x104 to 1.5x106 D). Fractions with a molecular weight greater than 670 000 D stimulated nitrite generation (Fig 1, top). An extrapolated estimate of molecular weight was greater than 1.5 million daltons. By contrast, most 6-keto-PGF1 stimulation (Fig 1, bottom) was in response to fractions with a molecular weight of approximately 50 000 D (peak activity occurred in fractions with molecular weights between 43 000 and 67 000 D). These findings were confirmed in P100 columns (fractionation range, 5x103 to 1x105 D; Fig 2). Nitrite stimulation (Fig 2, top) was by fractions of plasma from preeclamptic women in the void volume (>1x105 D), whereas 6-keto-PGF1 stimulation (Fig 2, bottom) was by fractions of approximately 50 000 D.
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The concentration and isolation of the activating factor enhanced production of 6-keto-PGF1 but not of nitrite. However, it is important to point out that differences in 6-keto-PGF1 concentration in cells stimulated with the isolated fractions compared with the native plasma were only about doubled when the total activity in the volume of plasma added to the column and recovered as the area under the curve of the peak of prostacyclin activation was considered.
Lipid Removal
Similar to our previous report,18 charcoal stripping significantly (P<.05) reduced nitrite generation by endothelial cells exposed to plasma from preeclamptic women (Fig 3, top). In contrast, charcoal stripping did not reduce 6-keto-PGF1 generation by endothelial cells exposed to plasma from either preeclamptic or normal pregnant women (Fig 3, bottom). Nitrites and 6-keto-PGF1 could not be detected in media containing 2% plasma that were not exposed to cells.
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After lipid extraction, the activity for stimulation of nitrite production was greater (P<.05) in the organic phase than in the aqueous phase of plasma fractions from both preeclamptic and normal pregnant women (Fig 4, top). In contrast to nitrite production, the activity for stimulation of 6-keto-PGF1 production was greatest (P<.05) in the aqueous phase (Fig 4, bottom). After extraction, the activity for stimulation of nitrite and 6-keto-PGF1 production did not differ in fractions of plasma from preeclamptic women compared with fractions from normal pregnant women.
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Lipoprotein Fractionation
To further characterize the lipid fraction that stimulated nitrite generation by endothelial cells, we isolated lipoprotein fractions of the plasma by density gradient ultracentrifugation. Both CM/VLDL and LDL fractions stimulated (P<.01) nitrite generation by endothelial cells, although nitrite production by these individual fractions was less than that by the native plasma (Fig 5, top). However, the sum of nitrite accumulation by endothelial cells stimulated by CM/VLDL and LDL accounted for greater than 90% of the activity of native plasma. The plasma fractions devoid of these lipoproteins minimally (<5%) stimulated nitrite production from the cells. The lipoprotein fractions from preeclamptic women stimulated greater (P<.05) nitrite release than fractions from normal pregnant women.
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In contrast to nitrite production, 6-keto-PGF1 production from endothelial cells was stimulated by plasma fractions devoid of lipoproteins (density <1.063 g/mL); CM/VLDL and LDL fractions did not stimulate 6-keto-PGF1 production (Fig 5
, bottom). The differences between the two groups of women did not achieve significance.
To further confirm that VLDL and LDL were the entities responsible for nitrite stimulation by endothelial cells, we treated VLDL and LDL fractions of plasma from preeclamptic women with heparin/manganese to precipitate apoprotein B and followed this by centrifugation to remove the lipoproteins (confirmed by agarose gel electrophoresis). Removal of VLDL and LDL reduced (P<.05) the ability of these plasma fractions to stimulate nitrite production by endothelial cells (Fig 6). These data further support the observation that lipoproteins stimulated nitrite release from endothelial cells.
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Discussion |
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The vascular pathophysiology of preeclampsia has been attributed to activation of the maternal vascular endothelium.11 Endothelial cells have complex functions, such as modifying vascular tone and preventing intravascular coagulation, which are relevant to preeclampsia. Endothelial cells are strategically interposed between the circulating blood and vascular smooth muscle. We have proposed that the uteroplacental unit secretes factors into the maternal circulation that activate the endothelium. We report in this study that two distinct factors in the plasma of pregnant women that stimulate NO or prostacyclin production by endothelial cells in vitro are increased in the plasma of preeclamptic women. NO stimulation was by a lipid-containing, high molecular weight factor—likely a lipoprotein. By contrast, prostacyclin stimulation was by a non–lipid-containing, smaller molecular weight factor.
The plasma factor that stimulated endothelial NO production is a lipid-containing molecule (determined by lipid extraction and charcoal stripping) with a molecular weight of greater than 1.5 million daltons. Subsequent lipoprotein isolation determined that this activity resides in the CM/VLDL and LDL fractions. This finding is especially intriguing in light of increasing attention to the striking abnormalities of lipid metabolism that accompany preeclampsia.26 The normal hypertriglyceridemia of pregnancy is accentuated in preeclampsia.26 Furthermore, Endresen and coworkers27 have found that sera from preeclamptic women induce excessive accumulation of triglycerides into endothelial cells in culture. In general, hypertriglyceridemia is associated with evidence of increased free radical reactions.28 29 Furthermore, in other disorders associated with hypertriglyceridemia, there is an increased prevalence of smaller, more dense LDL30 that are more susceptible to oxidative modification and are known to adversely affect vascular function.31 32
The formation of oxidized lipid products and oxidative stress in general are proposed as components of the pathophysiology of preeclampsia.33 34 35 Although we do not know whether the changes we have identified are due to such modified lipids, there are interesting parallels. We previously reported that increased NO generation in response to plasma from preeclamptic women is associated with increased endothelial NO synthase activity and mass.18 Low concentrations of oxidized LDL upregulate endothelial NO synthase mRNA expression in bovine aortic endothelial cells.36 Oxidized LDL also increases intracellular free calcium,37 which could activate the calcium-sensitive endothelial NO synthase. In contrast, however, oxidized LDL has also been shown to impair endothelium-dependent relaxation of arteries38 39 and reduce NO synthase activity in human neutrophils.40 These diverse results indicate the complexity of the system. In the present study, we have identified plasma lipoproteins as stimulators of NO production from endothelial cells, and the ability to stimulate cells is greater in plasma from preeclamptic women than in that from normal pregnant women. The difference in the lipoproteins of the plasma of preeclamptic women that accounted for the greater biological activity remains to be determined.
In contrast to the plasma factor that stimulated NO, endothelial prostacyclin stimulation was by an aqueous, smaller molecular weight factor. The plasma factor or factors that stimulated prostacyclin production remained in the fractions devoid of lipid or lipoproteins (density <1.063 g/mL). It is intriguing that unlike NO production, prostacyclin production was enhanced by the concentrated, isolated activating factor. The concentration of 6-keto-PGF1 from cells stimulated with column fractions appears much greater than that in cells stimulated with 2% plasma. The cells may have reached a maximum capacity to produce NO, while the concentration of the factor enhanced prostacyclin stimulation. Furthermore, the column separation (ie, isolation of the factor) may have removed inhibitors or activated additional factors. Nonetheless, the results of the fractionation indicate that compared with NO stimulation, a separate, smaller factor stimulated prostacyclin production.
We had previously predicted that the activity that increases prostacyclin generation would be due to a lipid, more specifically, a lipid peroxidation product, because endothelial cells exposed to oxidized LDL or lipid peroxidation in vitro alters the function of several enzymes of arachidonate metabolism.41 42 43 In fact, one biochemical action of lipid peroxides is the modulation of prostaglandin synthesis. The cyclooxygenase component of prostaglandin endoperoxide synthase requires low levels of lipid peroxides for activation and continued catalysis.41 Furthermore, oxidized glycated LDL enhances prostacyclin production in cultured endothelial cells.42 In endothelial cells exposed to plasma from preeclamptic women in vitro, we have found evidence of enhanced cyclooxygenase activation and augmented prostacyclin stimulation after 24 hours,16 consistent with low levels of lipid peroxidation and/or oxidized glycated LDL. Nonetheless, three lines of evidence—removal of activity by charcoal stripping or lipid extraction or removal of lipoproteins (with density <1.063 g/mL)—indicate that the ability of plasma to increase prostacyclin is not due to a lipid component. Indeed, the prostacyclin stimulant has an estimated molecular weight of 50 000 D. Obviously, numerous plasma proteins exist at this molecular weight. Further isolation and characterization of the factor responsible for prostacyclin stimulation in plasma from preeclamptic women is under way.
This study provides evidence that two distinct factors stimulate NO and prostacyclin. We have previously reported that plasma from preeclamptic women stimulated endothelial NO and prostacyclin production more than plasma from normal pregnant women.16 17 18 In the present study, plasma fractionated with the molecular weight exclusion column showed greater NO stimulation and prostacyclin production from women with preeclampsia compared with normal pregnant women. However, after organic extraction, there was no difference between fractions from the two groups of women. These data were generated with a pooled sample. Our previous findings16 17 18 indicate that it is likely that a greater number of individual samples would be needed for determination of whether the extraction procedure affected the stimulating capacity of the plasma from preeclamptic women. Similarly, with three samples, prostacyclin stimulation by native plasma from preeclamptic women was not significantly different than stimulation by plasma from normal pregnant women. Nonetheless, this study indicates that the characteristics of the stimulating factors are similar in plasma from preeclamptic and normal pregnant women.
In summary, two distinct factors are present in the plasma of pregnant women that stimulate NO or prostacyclin production by endothelial cells in vitro, and these factors are increased in the plasma of preeclamptic women. NO stimulation resulted from a lipid-containing, high molecular weight factor that was likely a lipoprotein. By contrast, an aqueous, smaller molecular weight fraction was responsible for the prostacyclin stimulation. This information should provide direction in the search for circulating factors contributing to the pathophysiology of preeclampsia.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health Grant 1PO1HD30367-01. We thank the Clinical Data Core and the nursing staff of the Magee-Womens Hospital in Pittsburgh for invaluable help in sample collection. We also thank Marcia Gallaher and Leslie Minich for their excellent technical support.
Received March 18, 1996; first decision April 24, 1996; accepted June 20, 1996.
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References |
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