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Hypertension. 2004;43:434-444
Published online before print January 5, 2004, doi: 10.1161/01.HYP.0000113044.46326.98
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(Hypertension. 2004;43:434.)
© 2004 American Heart Association, Inc.

Scientific Contribution

Reduced Endothelial NO-cGMP–Mediated Vascular Relaxation and Hypertension in IL-6–Infused Pregnant Rats

Julia M. Orshal; Raouf A. Khalil

From Department of Medicine, Veterans Affairs Medical Center, West Roxbury, Mass and Harvard Medical School, Boston, Mass.

Correspondence to Dr Raouf A. Khalil, Harvard Medical School VA Boston Healthcare Research, 1400 VFW Parkway, 3/2B123, Boston, MA 02132. E-mail raouf_khalil{at}hms.harvard.edu


*    Abstract
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 *Abstract
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 down arrowResults
 down arrowDiscussion
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Placental ischemia during pregnancy is associated with increased plasma cytokines such as interleukin-6 (IL-6), which may contribute to increased vascular resistance and hypertension of pregnancy. We tested the hypothesis that an increase in plasma IL-6 during pregnancy is associated with impaired endothelium-dependent relaxation, enhanced vascular contraction, and hypertension. Systolic blood pressure was measured in virgin and pregnant Sprague-Dawley rats non-treated or infused with IL-6 (200 ng/kg per day for 5 days). Isometric contraction was measured in isolated aortic strips, and endothelial nitric oxide (NO) synthase (eNOS) was measured in aortic homogenate using Western blots. Blood pressure was greater in IL-6–infused (146±3) than in control pregnant rats (117±2 mm Hg). In endothelium-intact vascular strips, phenylephrine (Phe) caused greater increase in active stress in IL-6–infused (maximum: 10.6±0.6) than in control pregnant rats (maximum: 4.1±0.3x104 N/m2). Acetylcholine (ACh)-induced relaxation of Phe contraction and vascular eNOS protein and nitrite/nitrate production were less in IL-6–infused than in control pregnant rats. N{omega}-nitro-L-arginine methyl ester (10-4 mol/L), inhibitor of NOS, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (10-5 mol/L), inhibitor of cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in control but not IL-6–infused pregnant rats. Endothelium removal enhanced Phe-induced stress in control but not in IL-6–infused pregnant rats. The blood pressure and vascular Phe-induced contraction, ACh relaxation, and eNOS protein were not different between control and IL-6–infused virgin rats. Thus, an endothelium-dependent NO-cGMP–mediated relaxation pathway is inhibited in systemic vessels of pregnant rats infused with IL-6. The results support a role for IL-6 as a possible mediator of the increased vascular resistance during hypertension of pregnancy.


Key Words: blood pressure • endothelium • nitric oxide • pregnancy


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
 down arrowDiscussion
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Normal pregnancy is associated with reduced systemic vascular resistance and arterial pressure and decreased vascular contraction to vasoconstrictor agonists.1–3 The hemodynamic and vascular changes observed during normal pregnancy have been attributed, in part, to increased nitric oxide (NO) production by various cells, including vascular endothelial cells.4–8 This is supported by reports that the tissue expression and activity of NO synthase are increased during late gestation9–11 and that the metabolic production and plasma level of cyclic guanosine 3',5'-monophosphate (cGMP), a second messenger of NO and a cellular mediator of vascular smooth muscle relaxation,12 are increased during pregnancy.13

In 3% to 5% of pregnancies, a condition called preeclampsia develops, which is characterized by increased intravascular coagulation, proteinuria, increased systemic vascular resistance, and hypertension.14–16 Although preeclampsia is a major cause of maternal and fetal morbidity and mortality, the mechanisms of this disorder have not yet been clearly identified. Because of the difficulty of performing mechanistic studies in pregnant women, several animal models of hypertension during pregnancy have been developed.17–21

Experimental studies in animal models have suggested that a reduction in the uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia during late pregnancy initiate a cascade of hemodynamic and vascular changes that lead to increased systemic vascular resistance and hypertension.17–21 For example, studies from our laboratory and others’ have demonstrated that reduction in uteroplacental perfusion in pregnant rats results in significant hemodynamic changes and a hypertensive state that closely resembles hypertension of pregnancy in women.18,20,21 We have also found that the reduction in uteroplacental perfusion pressure in pregnant rats is associated with decreased vascular relaxation and enhanced vascular contraction, suggesting that these vascular changes could contribute to the increased vascular resistance and hypertension of pregnancy.20 However, it is not clear how a localized reduction in uteroplacental perfusion pressure could lead to generalized vascular changes in the maternal circulation. For a localized reduction in uterine perfusion pressure to cause generalized vascular changes, one would predict possible release of vasoactive factors from the ischemic/hypoxic placenta into the systemic circulation.

According to the "cytokine" hypothesis, the reduction in uteroplacental perfusion pressure and the ensuing placental ischemia/hypoxia are thought to increase the release of cytokines into the maternal circulation and the increased plasma cytokines would then lead to the generalized vascular changes and hypertension.22–25 In support of the cytokine hypothesis, it has been shown that the plasma levels of tumor necrosis factor-{alpha} (TNF-{alpha}) are elevated in women with preeclampsia.22–25 Studies have also suggested that sources other than the placenta may contribute to the elevated concentrations of TNF-{alpha} found in the circulation of preeclamptic women.26 Additionally, elevation of plasma TNF-{alpha} in rats in late pregnancy results in significant increases in vascular resistance and arterial pressure.27 However, TNF-{alpha} may not be the sole and/or direct cytokine that mediates the vascular changes associated with hypertension of pregnancy.28–30 TNF-{alpha} is known to activate other cytokines such as interleukin-6 (IL-6).31,32 Although some studies suggest that the maternal and cord sera and placental levels of IL-6 may not be different between preeclamptic and normotensive women,33 several other studies have shown that the plasma levels of IL-6 are elevated {approx}2- to 3-fold in women with preeclampsia.23,25,28,34 Additionally, our preliminary observations have shown that chronic elevation of plasma IL-6 {approx}2-fold in late pregnant rats is associated with significant increases in blood pressure. Taken together, these data have suggested a role for IL-6 in the increased vascular resistance associated with hypertension of pregnancy and thus made it important to investigate the vascular mechanisms underlying the IL-6–induced changes in blood pressure during pregnancy.

The present study was designed to test the hypothesis that a 2- to 3-fold elevation in plasma IL-6, produced by infusing the cytokine into chronically instrumented pregnant rats at a rate to mimic the plasma levels observed in preeclamptic women,23,25,28,34 is associated with decreased endothelium-dependent vascular relaxation and enhanced vascular contraction, and thereby increased vascular resistance and hypertension. We used virgin and rats in late pregnancy to investigate: (1) whether the vascular contraction is enhanced in IL-6–infused compared with control pregnant rats; (2) whether endothelium-dependent vascular relaxation is inhibited in IL-6–infused compared with control pregnant rats; and (3) because normal pregnancy is associated with increased vascular NO production,4–8 we investigated whether the reduced vascular relaxation and enhanced vascular contraction in IL-6–infused pregnant rats involve alterations in the endothelium-dependent NO-cGMP pathway.


*    Methods
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Animals
Female virgin (non-pregnant) (12 weeks) and time pregnant (day 10 of gestation) Sprague-Dawley rats were purchased from Charles River Laboratory (Wilmington, Mass). The rats were housed individually in the animal facility and maintained on ad libitum standard rat chow and tap water in 12-hour/12-hour light/dark cycle. After 2-day acclimation period the rats were divided into 4 groups with 12 rats each: control pregnant, IL-6–infused pregnant, control virgin, and IL-6–infused virgin. On day 12 of gestation, or the equivalent in virgin rats, all rats were anesthetized with isoflurane and underwent a surgical procedure for catheter implantation. PE-50 tubing was placed in the external jugular vein. The venous catheter was attached to osmotic mini-pump (Alzet) filled with IL-6 or saline and placed in the back of the neck. After the surgery, the rats were housed individually and allowed to recover for 2 days. The IL-6 treated rats were infused intravenously with IL-6 (Biosource International) at a rate of 200 ng/kg per day for 5 days to increase the plasma levels {approx}2-fold. Control rats were infused with normal 0.9% saline. All procedures were performed in accordance with the guidelines of the institutional Animal Care and Use Committee.

Measurement of Blood Pressure and Heart Rate
The systolic blood pressure and heart rate were measured in conscious rats using an automated tail-cuff electrosphygmomanometer system (ITTC, Woodland Hills, Calif). The rats were first preconditioned to a restraint warming chamber for 2 days. Conditioning was performed daily for 15 minutes between 8:00 AM and 11:00 AM. After the training period, blood pressure was measured on 6 consecutive days (day 14 to 20 in pregnant rats or the equivalent in virgin rats) between 8:00 AM and 11 AM. Rats were allowed to rest quietly for 10 minutes in a plexiglas restrainer placed in a warming chamber preset at 30°C to dilate peripheral blood vessels and stimulate blood flow to the tail. A 17-mm tail-cuff with a pneumatic pulse transducer was applied to the base of the tail. The tail-cuff was programmed to insufflate to a maximum pressure of 250 mm Hg. With the use of this method, the reappearance of pulsations after gradual deflation of the pressure cuff was detected by a photoelectric sensor, amplified, and recorded on a 2-channel polygraph that was calibrated before each run. A rest period of 1 minute was allowed between insufflations. Results from the first 5 inflation cycles were discarded, and the average obtained from the next 5 cycles was taken as the individual systolic blood pressure of each rat at any given day. The total time of the blood pressure measurement on each rat was approximately 15 minutes.

Urinary Protein Levels
Rats were placed in metabolic cages on day 19 of gestation, and 24-hour urine sample was collected and stored at -20°C until use. Protein concentration in urine was determined using a Protein Determination Kit (P5656, Sigma) and Peterson’s modification of Lowry method.

Measurement of Plasma IL-6
On the day of the experiment (day 20 of pregnancy), blood samples (0.5 mL) were collected for measurement of plasma IL-6 in control and IL-6–infused virgin and pregnant rats using a rat IL-6 enzyme-linked immunosorbent assay (ELISA) system (Cytoscreen; Biosource International). This assay is a solid-phase sandwich-type system that uses a specific anti-rat IL-6 antibody coated onto the wells of microtiter plates. Serum samples (50 µL) and standards were pipetted in triplicate into appropriate microtiter wells and the assay was performed according to manufacturer’s instructions. The sensitivity of this IL-6 ELISA system is 0.7 pg/mL and the upper limit of detection is 150 pg/mL. The average recovery of IL-6 in serum pools from normal rats is 97%. No significant cross-reactivity was noted with a battery of other human and murine cytokines. Using this protocol, the plasma level of IL-6 was increased approximately 2-fold in the IL-6 infused compared with control pregnant and virgin rats (Table 1).


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  		TABLE 1. Body Weight, Systolic Blood Pressure, Heart Rate, Protein Excretion, Plasma IL-6, Estradiol, and Progesterone and Litter Size and Pup Weight in Control and IL-6–Infused Pregnant Rats on Day 20 of Gestation or the Equivalent in Virgin Rats

Measurement of Plasma Estradiol 17-ß and Progesterone Levels
Plasma estradiol 17-ß and progesterone concentrations were determined in control and IL-6–infused pregnant and virgin rats using radioimmunoassay kits (ICN Biomedicals). The estrogen assay reactivity with estradiol 17ß is 100%, and cross-reactivity with estrone, estriol, and other steroids is 6, 1.45, and <0.01%, respectively. The progesterone assay reactivity with progesterone is 100%, and cross-reactivity with 17{alpha}-hydroxyprogesterone and other steroids is 2.5 and <0.3%, respectively.

The plasma estrogen and progesterone levels are known to vary during the ovarian cycle. In the present study, the plasma hormone levels were not determined at specific stages of the ovarian cycle in the virgin rats because synchronization of the animals at specific stages of the ovarian cycle would require administering exogenous sex hormones such as estrogen and progesterone and abortifacient drugs such as prostaglandin F2{alpha}, which have been shown to change the vascular reactivity,35 and thus would affect the measurements of the contractile response in the vascular strips. Therefore, the virgin rats were studied using random selection regardless of the stage of the ovarian cycle. Because the ovarian cycle in rats, unlike that in larger mammals, is more frequent (every 4 to 5 days), and because the estrous stage is significantly shorter ({approx}12 hours), the average data from n=12 virgin rats should cancel out any possible peak-like and trough-like fluctuations in sex hormone levels at specific stages of the ovarian cycle and should, approximately, represent the average changes in sex hormone levels during all stages of the ovarian cycle.

Tissue Preparation
On the day of the experiment (day 20 of pregnancy), the rats were anesthetized by inhalation of isoflurane. The thoracic aorta was rapidly excised, placed in oxygenated Krebs solution, and cleaned of connective tissue. The aorta was cut into 3-mm-wide rings. Aortic rings were cut open into strips. For endothelium-intact vascular strips, extreme care was taken throughout the procedure to avoid injury of the endothelium. For endothelium-denuded vascular strips, the endothelium was removed by gently rubbing the vessel interior with wet filter paper.

Isometric Tension
One end of the vascular strip was attached to a glass hook using a thread loop and the other end was connected to a Grass force transducer (FT03; Astro-Med). Vascular strips were stretched to Lmax (1.5 the unloaded initial length, L). Lmax was measured separately in vascular strips of virgin and pregnant rats. Lmax was not significantly different between virgin and pregnant rats. Vascular strips were allowed to equilibrate for 1 hour in a water-jacketed, temperature-controlled tissue bath filled with 50 mL Krebs solution continuously bubbled with 95% O2 5% CO2 at 37°C. The changes in isometric tension were recorded on a Grass polygraph (Model 7D; Astro-Med).

A control contraction was elicited by applying phenylephrine (Phe, 10-5 mol/L) to the tissue bath solution. Once the Phe contraction reached a plateau, the tissue was rinsed with Krebs solution 3 times for 10 minutes each. The whole procedure of contraction and washing was repeated 2 times. Increasing concentrations of Phe were applied, the contractile responses were recorded, and concentration–response curves were constructed.

In other tissues, a contraction to submaximal concentration of Phe was elicited. Increasing concentrations of acetylcholine, bradykinin, or sodium nitroprusside were added and the extent of vascular relaxation was measured. In other experiments, the tissues were pretreated for 30 minutes with N{omega}-nitro-L-arginine methyl ester (L-NAME, 10-4 mol/L), to inhibit NO synthase, or with 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10-5 mol/L), to inhibit cGMP production in smooth muscle,36 and the effects on Phe-induced contraction and ACh-induced relaxation of Phe contraction were observed.

Nitrite/Nitrate Production
Endothelium-intact vascular strips were placed in test tubes containing 1.5 mL Krebs aerated with 95% O2 5% CO2 at 37°C and the solution was changed every 10 minutes for 1 hour. Samples for basal accumulation of nitrite (NO2-) formed from released NO were first taken. Krebs solution was replaced, and the strips were stimulated with ACh for 10 minutes. The vascular strips were rapidly removed, dabbed dry with filter paper, and weighed. The incubation solutions were assayed for the stable end product of NO, NO2-. Briefly, samples of incubation solution (50 µL, in triplicate) were mixed in a 96-well microtiter plate with 100 µL of the Griess reagent.37 The chromophore generated by the reaction with nitrite was detected spectrophotometrically (535 nm) using a THERMOmax microplate reader (Molecular Devices). The concentration of nitrite was calculated using a reference calibration curve with known concentrations of NaNO2.

Western Blots
Endothelium-intact aortic strips were transferred to a homogenization buffer containing 20 mmol/L 3-[N-morpholine]propane sulfonic acid, 4% SDS, 10% glycerol, 2.3 mg dithiothreitol, 1.2 mmol/L EDTA, 0.02% BSA, 5.5 µmol/L leupeptin, 5.5 µmol/L pepstatin, 2.15 µmol/L aprotinin, and 20 µmol/L 4-(2-aminoethyl)-benzenesulfonyl fluoride. The tissue was homogenized using a 2-mL tight-fitting homogenizer (Kontes Glass) at 4°C. Protein-matched samples were subjected to electrophoresis on 8% SDS polyacrylamide gels then transferred to nitrocellulose membranes. The membranes were incubated in 5% BSA in phosphate buffered saline (PBS)-Tween at 22°C for 1 hour, then incubated in the antibody solution at 4°C overnight. PBS-Tween contained (in mmol/L): 80 Na2HPO4, 20 NaH2PO4, 100 NaCl, and 0.05% Tween. To quantify NOS III, monoclonal anti-eNOS antibody (1:1000; Transduction Laboratory) was used. To maintain the labeling conditions constant, we used the same antibody titer (1:1000) and protein concentration (10 µg) in all tissue samples. The nitrocellulose membranes were washed 5 times for 15 minutes each time in PBS-Tween then incubated in horseradish peroxidase-conjugated anti-mouse IgG for 90 minutes. The blots were visualized with enhanced chemiluminescence detection system (Amersham). To verify equal loading of sample protein, the immunoblots were stripped in stripping solution (100 mmol/L ß-mercaptoethanol, 2% SDS, 62.5 mmol/L Tris HCl, pH 6.8) at 60°C for 60 minutes and re-probed with monoclonal anti ß-actin antibody (1:5000; Sigma). The reactive bands were analyzed quantitatively by optical densitometry using a GS-700 imaging densitometer (Bio-Rad), and the amount of eNOS was expressed as the ratio of the eNOS/ß-actin signals.

Solutions, Drugs, and Chemicals
Normal Krebs solution contained (in mmol/L): NaCl 120; KCl 5.9; NaHCO3 25; NaH2PO4 1.2; dextrose 11.5; MgCl2 1.2; and CaCl2 2.5 at pH 7.4. Stock solutions of phenylephrine, acetylcholine, bradykinin, sodium nitroprusside, L-NAME (Sigma) were prepared in distilled water. ODQ (Calbiochem) was dissolved in dimethyl sulfoxide (final concentration <0.1%). All other chemicals were of reagent grade or better.

Statistical Analysis
The developed force was corrected for the cross sectional area of each individual strip and expressed as active stress (N/m2) using the equation: stress=force/cross sectional area, where cross-sectional area=wet weight/(tissue densityxlength of the strip) and tissue density=1.055 g/cm3 as previously described.3,37 Data from vascular strips of the same rat were averaged and presented as the data for one rat. The data from different rats were analyzed and expressed as the mean±SEM, with the n values indicating the number of rats. Data were compared using ANOVA with multiple classification criteria: rat type (pregnant versus virgin), condition of endothelium (intact versus denuded), and treatment (control versus long-term infusion with IL-6, or nontreated versus acutely treated with L-NAME, or ODQ). Then Bonferroni posttest was performed to compare selected groups. Differences were considered statistically significant if P<0.05.


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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On the day of the experiment (day 20 of gestation or the equivalent in virgin rats), the systolic blood pressure was 117±2 mm Hg in control pregnant rats and was significantly elevated (P<0.001) in IL-6-infused pregnant rats (Table 1). In contrast, the systolic blood pressure was not significantly different between control and IL-6-infused virgin rats (P=0.555). The heart rate was significantly faster in pregnant than virgin rats (P=0.005), but it was not significantly different between control and IL-6 infused pregnant rats (P=0.257) or virgin rats (P=0.67) (Table 1).

Other whole-animal measurements indicated that the body weight was significantly lower in IL-6 infused than control pregnant rats (P=0.017). Also, a significant increase in urinary protein excretion was observed in IL-6 infused compared with control pregnant rats (P<0.001) (Table 1). The plasma levels of estradiol 17-ß and progesterone were significantly elevated in pregnant compared with virgin rats (P<0001). Although plasma estradiol 17-ß showed a slight decrease and plasma progesterone showed slight increase in IL-6 infused compared with control pregnant rats, these differences did not reach statistical significance. The litter size was significantly reduced in IL-6 infused compared with control pregnant rats (P=0.001) (Table 1). Intrauterine growth restriction was also evident because pup weights were significantly decreased in IL-6 infused compared with control pregnant rats (P=0.014) (Table 1).

In endothelium-intact vascular strips of control pregnant rats, Phe caused concentration-dependent increases in contraction (Figure 1 A). The Phe-induced contraction appeared to be greater in IL-6–infused pregnant rats (Figure 1B) compared with control pregnant rats (Figure 1A), but did not appear to be different between control virgin (Figure 1C) and IL-6–infused virgin rats (Figure 1D). To correct for the difference in the size of the vascular strips, the Phe contraction was normalized for the cross-sectional area of the vascular strip and presented as active stress as described in the Methods. The Phe concentration-active stress curve in IL-6–infused pregnant rats was enhanced compared with that in control pregnant rats (Figure 2 A). The maximal Phe-induced active stress was significantly greater in IL-6–infused than control pregnant rats (P<0.001) (Table 2). Removal of the endothelium significantly enhanced the Phe-induced stress in control pregnant but caused slight and insignificant increase in Phe-induced stress in IL-6–infused pregnant rats (Figure 2A, Table 2). In contrast, the Phe-induced active stress was not significantly different between control virgin and IL-6-infused virgin rats (Figure 2B, Table 2). When the Phe response was presented as percent of the maximum Phe contraction and the Phe ED50 was calculated, Phe was significantly more potent in causing contraction in endothelium-denuded than endothelium-intact vascular strips of control pregnant rats (P=0.003) (Figure 2C, Table 2). In contrast, Phe was not significantly different (P=0.491) in causing contraction in endothelium-denuded compared with endothelium-intact vascular strips of IL-6–infused pregnant rats (Figure 2C, Table 2). The Phe ED50 was not significantly different between control virgin and IL-6–infused virgin rats (P=0.077) (Figure 2D, Table 2).



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  		Figure 1. Representative traces of Phe-induced contraction in vascular strips isolated from control pregnant (A), IL-6–infused pregnant (B), control virgin (C), and IL-6–infused virgin rats (D). Endothelium-intact strips were incubated in normal Krebs solution, then stimulated with increasing concentrations of Phe. Unlabeled arrow represents a 3-fold greater concentration than the preceding arrow.



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  		Figure 2. Phe-induced contraction in vascular strips isolated from control and IL-6 infused pregnant (A and C) and virgin rats (B and D). Endothelium-intact (+Endo) and endothelium-denuded (-Endo) strips were incubated in normal Krebs then stimulated with increasing concentrations of Phe. The Phe contraction was measured and presented as active stress (A and B) or as percent of maximum Phe contraction (C and D). Data points represent the mean±SEM of measurements in 18 to 24 vascular strips from 6 to 8 rats of each group. *Measurements in -Endo strips are significantly greater (P<0.05) than corresponding measurements in +Endo strips.


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  		TABLE 2. Maximal Phe (10-5 mol/L)-Induced Active Stress, Phe ED50, Maximal ACh (10-5 mol/L)-Induced Relaxation, and Nitrite/Nitrate (NOx) Production in Vascular Strips of Control and IL-6–Infused Pregnant and Virgin Rats

In endothelium-intact vascular strips, pretreatment with L-NAME (10-4 mol/L) for 30 minutes to inhibit NO synthase significantly enhanced the Phe-induced stress in control pregnant rats (Figure 3 A, Table 2). Also, calculation of the Phe ED50 showed that Phe was significantly more potent in causing contraction in L-NAME pretreated than nontreated vascular strips of control pregnant rats (P=0.003) (Figure 3C, Table 2). In contrast, the maximal Phe-induced stress and the Phe ED50 were not significantly different between L-NAME pretreated and nontreated vascular strips of IL-6-infused pregnant rats (Figure 3B and 3D, Table 2).



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  		Figure 3. Effect of L-NAME and ODQ on Phe-induced contraction in endothelium-intact vascular strips of control pregnant (A and C) and IL-6 infused pregnant rats (B and D). Endothelium-intact vascular strips were either nontreated or pretreated with L-NAME (10-4 mol/L) or ODQ (10-5 mol/L) for 30 minutes, then stimulated with increasing concentrations of Phe. The Phe contraction was measured and presented as active stress (A and B) or as percent of maximum Phe contraction (C and D). Data points represent the mean±SEM of measurements in 18 to 24 vascular strips from 6 to 8 rats of each group. *Measurements in the presence of L-NAME or ODQ are significantly greater (P<0.05) than corresponding measurements in the absence of L-NAME or ODQ.

Similarly, in endothelium-intact strips, pretreatment with ODQ (10-5 mol/L) for 30 minutes to inhibit cGMP production in smooth muscle36,37 enhanced Phe-induced stress in control pregnant rats (Figure 3A, Table 2). Also, the Phe ED50 indicated that Phe was significantly more potent in causing contraction in ODQ-pretreated than nontreated vascular strips of control pregnant rats (P=0.018) (Figure 3C, Table 2). In contrast, the maximal Phe-induced stress and the Phe ED50 were not significantly different between ODQ pretreated and nontreated vascular strips of IL-6–infused pregnant rats (Figure 3B and D, Table 2).

In endothelium-intact vascular strips of control pregnant rats, ACh caused concentration-dependent relaxation of submaximal Phe (ED50=6x10-7 mol/L)-induced contraction (Figure 4 A and Figure 5 A). Because the Phe contraction in other groups of rats was greater than that in control pregnant rats, the Phe concentration was adjusted in the IL-6–infused pregnant (3x10-8 mol/L), control virgin (3x10-7 mol/L), and IL-6–infused virgin rats (3x10-7 mol/L) to produce a submaximal contraction that is approximately equal in magnitude to that in control pregnant rats. The ACh-induced relaxation of Phe contraction was significantly reduced in IL-6–infused compared with control pregnant rats (Figure 4B, 5DownA, Table 2). In contrast, the ACh-induced relaxation was not significantly different between control virgin and IL-6-infused virgin rats (Figure 4C, 4D, 5 DownA, Table 2).



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  		Figure 4. Representative traces of ACh-induced relaxation of Phe contraction in vascular strips isolated from control pregnant (A), IL-6 infused pregnant (B), control virgin (C), and IL-6 infused virgin rats (D). Endothelium-intact strips were incubated in normal Krebs then stimulated with a submaximal concentration of Phe to produce a contraction approximately equal in magnitude in the different groups of rats. Increasing concentrations of ACh were added and the relaxation of Phe contraction was measured. At the end of the experiment, sodium nitroprusside (SNP) (10-6 mol/L) was added to ensure the ability of the smooth muscle to relax.



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  		Figure 5. ACh-induced relaxation of Phe contraction in endothelium-intact vascular strips isolated from control and IL-6–infused pregnant and virgin rats (A), and in vascular strips of control pregnant (B) and IL-6–infused pregnant rats (C) nontreated or pretreated with L-NAME (10-4 mol/L) or ODQ (10-5 mol/L) for 30 minutes. A submaximal Phe contraction was elicited then increasing concentrations of ACh were added and the percent relaxation of Phe contraction was measured. Data points represent the mean±SEM of measurements in 18 to 24 vascular strips from 6 to 8 rats of each group. *Measurements in IL-6–infused pregnant rats are significantly different (P<0.05) from corresponding measurements in control pregnant rats. {dagger}Measurements in the presence of L-NAME or ODQ are significantly different (P<0.05) from corresponding measurements in the absence of L-NAME or ODQ.

Similarly, bradykinin caused concentration-dependent relaxation of submaximal Phe contraction. The maximum bradykinin (10-5 mol/L)-induced relaxation of Phe contraction was significantly reduced (P<0.001) in IL-6-infused pregnant (31.6%±2.3%) compared with control pregnant rats (73.7%±4.8%). In contrast, the maximum bradykinin-induced relaxation was not significantly different (P=0.46) between IL-6-infused virgin (56.8%±3.6%) and control virgin rats (61.3%±4.7%).

Pretreatment of endothelium-intact strips with L-NAME (10-4 mol/L) or ODQ (10-5 mol/L) significantly inhibited the ACh-induced relaxation of Phe contraction in control pregnant (Figure 5B) rats but not IL-6–infused pregnant rats (Figure 5C). Removal of the endothelium completely inhibited the ACh-induced relaxation of Phe contraction in all groups of rats.

Western blot analysis using tissue homogenates of endothelium-intact vascular strips and anti-eNOS antibody showed a prominent band at {approx}140 kDa, with the optical density significantly greater in control pregnant than virgin rats (P=0.01). The optical density of eNOS was significantly reduced in IL-6–infused compared with control pregnant rats (P=0.042) but was not significantly different between IL-6-infused and control virgin rats (P=0.874) (Figure 6 A).



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  		Figure 6. Western blot analysis of eNOS using anti eNOS antibody (1:1000; Transduction Laboratory) as normalized to ß-actin (A) and the basal and ACh-induced nitrite/nitrate production (B) in endothelium-intact vascular strips of control and IL-6–infused pregnant and virgin rats. Data points represent the mean±SEM of measurements in 6 tissue samples/vascular strips from 6 rats of each group. *Measurements in IL-6–infused pregnant rats are significantly different (P<0.05) from corresponding measurements in control pregnant rats.

In endothelium-intact vascular strips, the basal nitrite/nitrate production was 51.2±6.5 pmol/mg tissue weight in pregnant rats and was significantly reduced in IL-6–infused pregnant rats (13.9±5.8 pmol/mg tissue weight, P<0.05). ACh caused concentration-dependent increases in nitrite/nitrate production that was significantly reduced in IL-6–infused pregnant compared with control pregnant rats (Figure 6B), but not significantly different between control virgin and IL-6–infused virgin rats (Figure 6B).

In endothelium-denuded vascular strips of all groups of rats, sodium nitroprusside, an exogenous NO donor and a standard guanylate cyclase activator,12 caused concentration-dependent relaxation of Phe contraction. However, no significant differences in the magnitude of sodium nitroprusside-induced relaxation of Phe contraction were observed between control and IL-6–infused pregnant or virgin rats.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The main findings of the present study are: (1) the blood pressure in late pregnant rats with 2-fold elevation of plasma IL-6 is greater than that in control pregnant rats; (2) vascular contraction is greater in IL-6–infused pregnant compared with control pregnant rats; (3) endothelium-dependent vascular relaxation is less in IL-6–infused pregnant than control pregnant rats; (4) the activity of the endothelium-dependent NO-cGMP pathway is reduced in IL-6–infused pregnant compared with control pregnant rats; and (5) the blood pressure, vascular contraction, and vascular relaxation are not significantly different between control virgin and IL-6–infused virgin rats.

The present study showed that 2-fold elevation of plasma IL-6 in pregnant rats was associated with proteinuria and intrauterine growth retardation. Also, the blood pressure was reduced in control pregnant compared with virgin rats but was significantly increased in pregnant rats when their plasma IL-6 was elevated 2-fold. Previous studies in pregnant rats have shown that intravenous infusion of a very low dose (1 µg/kg body weight) of the endotoxin lipopolysaccharide (LPS), which is known to increase IL-6,38 results in increased urinary albumin excretion and significant increase in blood pressure.39 The observed increases in protein excretion and blood pressure in IL-6–infused pregnant rats are consistent with those previously reported in the endotoxin-induced model of preeclampsia.39 The data in the IL-6–infused pregnant rats are also consistent with previous reports that long-term infusion of pregnant rats with the cytokine TNF-{alpha}, which is known to activate IL-6,31,32 is associated with significant increases in blood pressure.27

Consistent with previous reports, the vascular contraction to Phe was significantly reduced (P<0.05) in pregnant rats compared with virgin rats.3,19 The vascular contraction to Phe was enhanced in IL-6–infused compared with control pregnant rats. The present findings in IL-6–infused pregnant rats are in agreement with other studies that have shown that the vascular contraction is enhanced in other animal models of hypertension during pregnancy.19,20,27 In search for the possible mechanisms involved in the enhanced vascular contraction in IL-6–infused pregnant rats, we found that removal of the endothelium enhanced Phe contraction in control pregnant but had minimal effects in IL-6–infused pregnant rats. Also, the ACh-induced relaxation was reduced in IL-6–infused pregnant compared with control pregnant rats. The decreased ACh relaxation in IL-6–infused compared with control pregnant rats did not appear to be caused by blockade of endothelial cholinergic receptor, because similar inhibition of bradykinin-induced relaxation was also observed in IL-6–infused pregnant rats. These results suggest that an endothelium-dependent relaxation pathway is intact in control pregnant rats but is possibly impaired during elevation of plasma IL-6 in pregnant rats.

One important vasodilator released from endothelial cells is NO.40,41 The reduced ACh-induced relaxation in IL-6–infused pregnant rats could be due to either a decrease in the synthesis/release of NO from endothelial cells, increased consumption of NO by reactive oxygen species, or a change in the sensitivity of vascular smooth muscle to relaxation by NO. The observation that the relaxation of endothelium-denuded vascular strips by the exogenous NO donor sodium nitroprusside was not significantly different between control and IL-6–infused pregnant rats provided evidence that the endothelium-independent mechanisms of vascular relaxation and the sensitivity of vascular smooth muscle to relaxation by NO are not impaired in IL-6–infused pregnant rats, thereby suggesting that the impaired ACh-induced relaxation in IL-6–infused pregnant rats is more likely caused by a decrease in the synthesis/release of NO from endothelial cells.

To further investigate the role of NO synthesis/release in the impaired endothelium-dependent relaxation pathway in IL-6–infused pregnant rats we found that pretreatment of the vascular strips with L-NAME, which blocks NO synthesis, significantly inhibited ACh-induced vascular relaxation, and enhanced Phe-induced vascular contraction in control pregnant but had minimal effects in IL-6–infused pregnant rats. These results suggest that NO synthesis by endothelial cells is intact in control pregnant rats but impaired during elevation of plasma IL-6 in late pregnant rats. This is further supported by the observation that the basal and the ACh-induced nitrite/nitrate production were reduced in vascular strips of IL-6–infused compared with control pregnant rats.

The precise mechanism by which IL-6 could inhibit endothelial NO production/release is not clear at the present time but could be related to changes in eNOS protein expression or activity. Although high levels of IL-6, as observed during septic shock,38 may activate gene expression of iNOS and promote vasodilation,42,43 modest levels of LPS, which activate the cytokines IL-6 and TNF-{alpha} may downregulate eNOS.44 This is supported by reports that modest levels of cytokines such as TNF-{alpha} downregulate eNOS.45,46 It has also been shown that small-dose infusion of TNF-{alpha} in pregnant rats is associated with a significant decrease in the expression of renal nNOS isoform.47 The present study suggest that the amount of vascular eNOS is reduced in IL-6–infused compared with control pregnant rats. However, IL-6 infusion in pregnant rats may also cause posttranscriptional and/or posttranslational modifications of eNOS. For example, IL-6 has been localized in the plasma membrane caveolae,48 which may promote tighter association of eNOS with the inhibitory protein caveolin-1 and thereby prevent the initial activation of eNOS and its dissociation from the plasma membrane.49 IL-6 may also affect the activity of mitogen-activated protein kinase or the phosphatidylinositol-3-kinase (PI3-kinase)-dependent Akt/protein kinase B pathway and thereby prevent phosphorylation of eNOS and its relocation to the plasma membrane, a process required for its full activation.49 Furthermore, IL-6 could alter eNOS activity via a protein kinase C (PKC)-dependent mechanism. This is supported by reports that IL-6 activates endothelial PKC50 and that PKC causes eNOS phosphorylation and inhibition of eNOS activity.51

The NO produced by endothelial cells is known to promote vascular relaxation by activating guanylate cyclase and increasing cGMP production in vascular smooth muscle.12,41 We found that ODQ, which inhibits guanylate cyclase and decreases cGMP production in smooth muscle,36,37 significantly inhibited ACh-induced vascular relaxation and enhanced Phe-induced contraction in endothelium-intact vascular strips of control pregnant, but not IL-6–infused pregnant rats. These results further support the contention that NO production/release by endothelial cells and thereby the activity of the NO-cGMP pathway in vascular smooth muscle is reduced in IL-6-infused compared with control pregnant rats.

It is important to emphasize the following cautionary remarks regarding the aforementioned interpretations. First, although the observed decrease in endothelial cell function and increase in vascular contraction in the IL-6–infused pregnant rats could contribute to the observed increase in blood pressure, the changes in endothelial cell function and vascular contraction may be secondary to blood pressure elevation. Analysis of the time course of the changes in vascular contraction and the increase in blood pressure should help determine whether the relationship between these two parameters is causal or associative in nature. Second, the long-term effects of IL-6 in vivo could be caused by direct effects on the vascular endothelium or perhaps indirect effects through the release of other factor(s). Although IL-6 has been shown to directly affect vascular relaxation in isolated arterial segments of normal male rats,52 whether any direct effects of IL-6 on the mechanisms of vascular relaxation are altered in females, particularly during pregnancy, remain to be investigated. Third, the vascular endothelium is known to release other vasodilator substances, in addition to NO, such as prostacyclin and endothelium-derived hyperpolarizing factor. This may explain why in the vascular strips of IL-6–infused pregnant rats some relaxation to ACh was still observed and was not completely inhibited by L-NAME or ODQ. However, the complete absence of ACh-induced relaxation in endothelium-denuded strips of IL-6–infused pregnant rats still supports the contention that the ACh-induced relaxation is endothelium-dependent. Fourth, although the present results provided evidence that the enhanced vascular contraction in the IL-6–infused pregnant rats may involve inhibition of the endothelium-dependent NO-cGMP pathway, we cannot rule out the possibility that an increase in the release of contracting factors from the endothelium or an increase in the sensitivity of vascular smooth muscle to endothelium-derived contracting factors also occurs. This is supported by reports that IL-6 stimulates endothelin-1 mRNA expression and endothelin-1 release from endothelial cells.53 This is also supported by the present observation that removal of the endothelium or pretreatment of vascular strips of control pregnant rats with L-NAME or ODQ caused an enhancement of Phe-induced vascular contraction to levels that were still less than that observed in the IL-6 infused pregnant rats. These findings suggest that elevation of plasma IL-6 during pregnancy in rats may be associated with additional alterations in the cellular mechanisms of vascular smooth muscle contraction and should be further examined in future investigations.

The causes of the lack of vascular effects of IL-6 in virgin rats and its dramatic effects in pregnant rats are unclear at the present time but could be related, in part, to the plasma levels of estrogen and progesterone and possible synergistic actions of the sex hormones on the vascular effects of IL-6. The plasma estrogen and progesterone levels are elevated during pregnancy and have been shown to be higher in pregnant than virgin rats.21 Also, estrogen and progesterone have been shown to modulate eNOS expression/activity and NO production via genomic and non-genomic mechanisms,35,54 which in turn may promote feedback regulation of the effects of IL-6 on eNOS expression/activity. Studying the effects of acute and long-term exposure to estrogen and progesterone on the vascular effects of IL-6 should help further identify the mechanisms underlying the possible synergistic interactions between sex hormones and the cytokine and should represent important areas for future investigations.

Perspectives
The search of the mechanisms underlying the increased vascular resistance in animal models of hypertension during pregnancy should help to understand better the pathophysiological basis of preeclampsia in pregnant women. The present results suggest that an endothelium-dependent relaxation pathway involving the production and release of NO from endothelial cells and increased cGMP production in smooth muscle is inhibited in systemic vessels of pregnant rats with long-term infusion of IL-6. The results suggest a role for IL-6 as one possible mediator of the increased vascular resistance and blood pressure associated with hypertension of pregnancy. However, limitations of studying the relation between vascular reactivity in the aorta and the changes in blood pressure should be considered and should highlight the importance of studying the pregnancy-associated vascular effects in the more relevant resistance vessels. Also, the present data represent the changes in endothelium-dependent vascular relaxation at one specific point in time, namely day 20 of pregnancy in rats and after 5 days infusion of IL-6. Time course studies should be useful to identify the time of onset and the time to peak changes in endothelium-dependent vascular relaxation in IL-6–infused pregnant rats and to determine whether the vascular changes precede or coincide with the changes in arterial pressure. Additionally, it is not clear whether the long-term effects of IL-6 represent direct vascular effects of the cytokine or are mediated by other factors. Studying the acute vascular effects of IL-6 should help further delineate the role of cytokines as possible mediators of preeclampsia.


*    Acknowledgments
 
This work was supported by grants from the National Heart, Lung and Blood Institute (HL-52696, HL-65998, and HL-70659). R.A. Khalil is an Established Investigator of the American Heart Association.

Received September 30, 2003; first decision November 3, 2003; accepted December 4, 2003.


*    References
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 up arrowDiscussion
 *References
 

  1. Molnar M, Hertelendy F. N{omega}-nitro-L-arginine, an inhibitor of nitric oxide synthesis, increases blood pressure in rats and reverses the pregnancy-induced refractoriness to vasopressor agents. Am J Obstet Gynecol. 1992; 166: 1560–1567.[Medline] [Order article via Infotrieve]
  2. Nathan L, Cuevas J, Chaudhuri G. The role of nitric oxide in the altered vascular reactivity of pregnancy in the rat. Br J Pharmacol. 1995; 114: 955–960.[Medline] [Order article via Infotrieve]
  3. Khalil RA, Crews JK, Novak J, Kassab S, Granger JP. Enhanced vascular reactivity during inhibition of nitric oxide synthesis in pregnant rats. Hypertension. 1998; 31: 1065–1069.[Abstract/Free Full Text]
  4. Ahokas RA, Mercer BM, Sibai BM. Enhanced endothelium-derived relaxing factor activity in pregnant, spontaneously hypertensive rats. Am J Obstet Gynecol. 1991; 165: 801–807.[Medline] [Order article via Infotrieve]
  5. Bird IM, Zhang L, Magness RR. Possible mechanisms underlying pregnancy-induced changes in uterine artery endothelial function. Am J Physiol Regul Integr Comp Physiol. 2003; 284: R245–R258.[Abstract/Free Full Text]
  6. Davidge ST, McLaughlin MK. Endogenous modulation of the blunted adrenergic response in resistance-sized mesenteric arteries from the pregnant rat. Am J Obstet Gynecol. 1992; 167: 1691–1698.[Medline] [Order article via Infotrieve]
  7. Sladek SM, Magness RR, Conrad KP. Nitric oxide and pregnancy. Am J Physiol. 1997; 272: R441–R463.[Medline] [Order article via Infotrieve]
  8. Williams DJ, Vallance PJ, Neild GH, Spencer JA, Imms FJ. Nitric oxide-mediated vasodilation in human pregnancy. Am J Physiol. 1997; 272: H748–H752.[Medline] [Order article via Infotrieve]
  9. Conrad KP, Joffe GM, Kruszyna H, Kruszyna R, Rochelle LG, Smith RP, Chavez JE, Mosher MD. Identification of increased nitric oxide biosynthesis during pregnancy in rats. FASEB J. 1993; 7: 566–571.[Abstract]
  10. Weiner CP, Knowles RG, Moncada S. Induction of nitric oxide synthases early in pregnancy. Am J Obstet Gynecol. 1994; 171: 838–843.[Medline] [Order article via Infotrieve]
  11. Alexander BT, Miller MT, Kassab S, Novak J, Reckelhoff JF, Kruckeberg WC, Granger JP. Differential expression of renal nitric oxide synthase isoforms during pregnancy in rats. Hypertension. 1999; 33: 435–439.[Abstract/Free Full Text]
  12. Ignarro LJ, Kadowitz PJ. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol. 1985; 25: 171–191.[CrossRef][Medline] [Order article via Infotrieve]
  13. Conrad KP, Vernier KA. Plasma level, urinary excretion, and metabolic production of cGMP during gestation in rats. Am J Physiol. 1989; 257: R847–R853.[Medline] [Order article via Infotrieve]
  14. Friedman SA, Lubarsky SL, Ahokas RA, Nova A, Sibai BM. Preeclampsia and related disorders. Clinical aspects and relevance of endothelin and nitric oxide. Clin Perinatol. 1995; 22: 343–355.[Medline] [Order article via Infotrieve]
  15. Morris NH, Eaton BM, Dekker G. Nitric oxide, the endothelium, pregnancy and pre-eclampsia. Br J Obstet Gynaecol. 1996; 103: 4–15.[Medline] [Order article via Infotrieve]
  16. Roberts JM, Pearson G, Cutler J, Lindheimer M. Summary of the NHLBI working group on research on hypertension during pregnancy. Hypertension. 2003; 41: 437–445.[Abstract/Free Full Text]
  17. Losonczy G, Brown G, Venuto RC. Increased peripheral resistance during reduced uterine perfusion pressure hypertension in pregnant rabbits. Am J Med Sci. 1992; 303: 233–240.[Medline] [Order article via Infotrieve]
  18. Abitbol MM. Simplified technique to produce toxemia in the rat: considerations on cause of toxemia. Clin Exp Hypertens B. 1982; 1: 93–103.[Medline] [Order article via Infotrieve]
  19. Crews JK, Novak J, Granger JP, Khalil RA. Stimulated mechanisms of Ca2+ entry into vascular smooth muscle during NO synthesis inhibition in pregnant rats. Am J Physiol. 1999; 276: R530–R538.[Medline] [Order article via Infotrieve]
  20. Crews JK, Herrington JN, Granger JP, Khalil RA. Decreased endothelium-dependent vascular relaxation during reduction of uterine perfusion pressure in pregnant rat. Hypertension. 2000; 35: 367–372.[Abstract/Free Full Text]
  21. Alexander BT, Kassab SE, Miller MT, Abram SR, Reckelhoff JF, Bennett WA, Granger JP. Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide. Hypertension. 2001; 37: 1191–1195.[Abstract/Free Full Text]
  22. Kupferminc MJ, Peaceman AM, Wigton TR, Rehnberg KA, Socol ML. Tumor necrosis factor-alpha is elevated in plasma and amniotic fluid of patients with severe preeclampsia. Am J Obstet Gynecol. 1994; 170: 1752–1759.[Medline] [Order article via Infotrieve]
  23. Vince GS, Starkey PM, Austgulen R, Kwiatkowski D, Redman CW. Interleukin-6, tumour necrosis factor and soluble tumour necrosis factor receptors in women with pre-eclampsia. Br J Obstet Gynaecol. 1995; 102: 20–25.[Medline] [Order article via Infotrieve]
  24. Conrad KP, Benyo DF. Placental cytokines and the pathogenesis of preeclampsia. Am J Reprod Immunol. 1997; 37: 240–249.[Medline] [Order article via Infotrieve]
  25. Williams MA, Mahomed K, Farrand A, Woelk GB, Mudzamiri S, Madzime S, King IB, McDonald GB. Plasma tumor necrosis factor-alpha soluble receptor p55 (sTNFp55) concentrations in eclamptic, preeclamptic and normotensive pregnant Zimbabwean women. J Reprod Immunol. 1998; 40: 159–173.[CrossRef][Medline] [Order article via Infotrieve]
  26. Benyo DF, Smarason A, Redman CW, Sims C, Conrad KP. Expression of inflammatory cytokines in placentas from women with preeclampsia. J Clin Endocrinol Metab. 2001; 86: 2505–2512.[Abstract/Free Full Text]
  27. Davis JR, Giardina JB, Green GM, Alexander BT, Granger JP, Khalil RA. Reduced endothelial NO-cGMP vascular relaxation pathway during TNF-{alpha}-induced hypertension in pregnant rats. Am J Physiol Regul Integr Comp Physiol. 2002; 282: R390–R399.[Abstract/Free Full Text]
  28. Greer IA, Lyall F, Perera T, Boswell F, Macara LM. Increased concentrations of cytokines interleukin-6 and interleukin-1 receptor antagonist in plasma of women with preeclampsia: a mechanism for endothelial dysfunction? Obstet Gynecol. 1994; 84: 937–940.[Abstract/Free Full Text]
  29. Meekins JW, McLaughlin PJ, West DC, McFadyen IR, Johnson PM. Endothelial cell activation by tumour necrosis factor-alpha (TNF-alpha) and the development of pre-eclampsia. Clin Exp Immunol. 1994; 98: 110–114.[Medline] [Order article via Infotrieve]
  30. Ellis J, Wennerholm UB, Bengtsson A, Lilja H, Pettersson A, Sultan B, Wennergren M, Hagberg H. Levels of dimethylarginines and cytokines in mild and severe preeclampsia. Acta Obstet Gynecol Scand. 2001; 80: 602–608.[CrossRef][Medline] [Order article via Infotrieve]
  31. Riccioli A, Filippini A, De Cesaris P, Barbacci E, Stefanini M, Starace G, Ziparo E. Inflammatory mediators increase surface expression of integrin ligands, adhesion to lymphocytes, and secretion of interleukin 6 in mouse Sertoli cells. Proc Natl Acad Sci U S A. 1995; 92: 5808–5812.[Abstract/Free Full Text]
  32. De Cesaris P, Starace D, Riccioli A, Padula F, Filippini A, Ziparo E. Tumor necrosis factor-alpha induces interleukin-6 production and integrin ligand expression by distinct transduction pathways. J Biol Chem. 1998; 273: 7566–7571.[Abstract/Free Full Text]
  33. Al-Othman S, Omu AE, Diejomaoh FM, Al-Yatama M, Al-Qattan F. Differential levels of interleukin 6 in maternal and cord sera and placenta in women with pre-eclampsia. Gynecol Obstet Invest. 2001; 52: 60–65.[CrossRef][Medline] [Order article via Infotrieve]
  34. Madazli R, Aydin S, Uludag S, Vildan O, Tolun N. Maternal plasma levels of cytokines in normal and preeclamptic pregnancies and their relationship with diastolic blood pressure and fibronectin levels. Acta Obstet Gynecol Scand. 2003; 82: 797–802.[CrossRef][Medline] [Order article via Infotrieve]
  35. Thompson J, Khalil RA. Gender differences in the regulation of vascular tone. Clin Exp Pharmacol Physiol. 30: 1–15, 2003.[CrossRef][Medline] [Order article via Infotrieve]
  36. Hussain AS, Marks GS, Brien JF, Nakatsu K. The soluble guanylyl cyclase inhibitor 1H-[1, 2, 4]oxadiazolo[4, 3-alpha]quinoxalin-1-one (ODQ) inhibits relaxation of rabbit aortic rings induced by carbon monoxide, nitric oxide, and glyceryl trinitrate. Can J Physiol Pharmacol. 1997; 75: 1034–1037.[CrossRef][Medline] [Order article via Infotrieve]
  37. Giardina JB, Green GM, Rinewalt AN, Granger JP, Khalil RA. Role of endothelin B receptors in enhancing endothelium-dependent nitric oxide-mediated vascular relaxation during high salt diet. Hypertension. 2001; 37: 516–523.[Abstract/Free Full Text]
  38. Forfia PR, Zhang X, Ochoa F, Ochoa M, Xu X, Bernstein R, Sehgal PB, Ferreri NR, Hintze TH. Relationship between plasma NOx and cardiac and vascular dysfunction after LPS injection in anesthetized dogs. Am J Physiol. 1998; 274: H193–H201.[Medline] [Order article via Infotrieve]
  39. Faas MM, Schuiling GA, Baller JF, Visscher CA, Bakker WW. A new animal model for human preeclampsia: ultra-low-dose endotoxin infusion in pregnant rats. Am J Obstet Gynecol. 1994; 171: 158–164.[Medline] [Order article via Infotrieve]
  40. Furchgott RF, Zawadzki JV. The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature. 1980; 288: 373–376.[CrossRef][Medline] [Order article via Infotrieve]
  41. Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res. 1989; 65: 1–21.[Free Full Text]
  42. Bhagat K, Hingorani AD, Palacios M, Charles IG, Vallance P. Cytokine-induced venodilation in humans in vivo: eNOS masquerading as iNOS. Cardiovasc Res. 1999; 41: 754–764.[Abstract/Free Full Text]
  43. Yu X, Kennedy RH, Liu SJ. JAK2/STAT3, not ERK1/2, mediates interleukin-6-induced activation of inducible nitric-oxide synthase and decrease in contractility of adult ventricular myocytes. J Biol Chem. 2003; 278: 16304–16309.[Abstract/Free Full Text]
  44. Hallemeesch MM, Janssen BJ, De Jonge WJ, Soeters PB, Lamers WH, Deutz NE. Increased iNOS-mediated and decreased cNOS-mediated NO production reflect blood pressure changes in LPS-challenged mice. Am J Physiol Endocrinol Metab. 2003; 285: E871–E875.[Abstract/Free Full Text]
  45. Alonso J, Sanchez de Miguel L, Monton M, Casado S, Lopez-Farre A. Endothelial cytosolic proteins bind to the 3' untranslated region of endothelial nitric oxide synthase mRNA: regulation by tumor necrosis factor alpha. Mol Cell Biol. 1997; 17: 5719–5726.[Abstract]
  46. Yoshizumi M, Perrella MA, Burnett JC Jr., Lee ME. Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. Circ Res. 1993; 73: 205–209.[Abstract]
  47. Alexander BT, Cockrell KL, Massey MB, Bennett WA, Granger JP. Tumor necrosis factor-alpha-induced hypertension in pregnant rats results in decreased renal neuronal nitric oxide synthase expression. Am J Hypertens. 2002; 15: 170–175.[CrossRef][Medline] [Order article via Infotrieve]
  48. Podar K, Tai YT, Cole CE, Hideshima T, Sattler M, Hamblin A, Mitsiades N, Schlossman RL, Davies FE, Morgan GJ, Munshi NC, Chauhan D, Anderson KC. Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells. J Biol Chem. 2003; 278: 5794–5801.[Abstract/Free Full Text]
  49. Michel T. Targeting and translocation of endothelial nitric oxide synthase. Braz J Med Biol Res. 1999; 32: 1361–1366.[Medline] [Order article via Infotrieve]
  50. Desai TR, Leeper NJ, Hynes KL, Gewertz BL. Interleukin-6 causes endothelial barrier dysfunction via the protein kinase C pathway. J Surg Res. 2002; 104: 118–123.