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. Author manuscript; available in PMC: 2011 Jul 5.
Published in final edited form as: Am J Hypertens. 2010 Aug 19;24(1):110–113. doi: 10.1038/ajh.2010.180

ROLE OF REACTIVE OXYGEN SPECIES DURING HYPERTENSION IN RESPONSE TO CHRONIC ANTI-ANGIOGENIC FACTOR (sFlt-1) EXCESS IN PREGNANT RATS

Kiran B Tam Tam 1, Babbette LaMarca 1, Marietta Arany 1, Kathy Cockrell 1, Lillian Fournier 1, Sydney Murphy 1, James N Martin Jr 1, JOEY P GRANGER 1
PMCID: PMC3129783  NIHMSID: NIHMS289768  PMID: 20725052

Abstract

Background

Preeclampsia is associated with increased levels of reactive oxygen species (ROS) and the anti-angiogenic factor, soluble fms-like tyrosine kinase (sFlt-1). Moreover, recent studies have indicated that chronic sFlt-1 excess causes hypertension in pregnant animals. The purpose of this study was to evaluate the role of ROS in mediating sFlt-1 induced hypertension in the pregnant rat.

Methods

Mean arterial pressure (MAP), plasma sFlt-1 and tissue ROS levels were measured in the following groups: (i) pregnant controls; (ii) sFlt-1 treated pregnant rats; (iii) Tempol treated pregnant rats; (iv) sFlt-1 and Tempol treated pregnant rats.

Results

Mean arterial pressure increased from 104 +/−2 mmHg in pregnant control rats to 118 +/−3 mmHg (P = 0.002) in sFlt-1 infused rats. Basal and NADPH stimulated levels of tissue ROS were increased in response to excess sFlt-1 during pregnancy. Pre-treatment with Tempol attenuated oxidative stress and hypertension in response to sFlt-1.

Conclusion

ROS play an important role in mediating hypertension in response to chronic sFlt-1 excess during pregnancy.

Keywords: sFlt-1, Tempol, reactive oxygen species, hypertension, preeclampsia

BACKGROUND

While preeclampsia is a major cause of poor maternal and fetal outcomes complicating 5–8% of pregnancies in the United States, the pathophysiology of preeclampsia has not been fully elucidated1–5. Systemic endothelial dysfunction is a well recognized hallmark of preeclampsia and is thought to be responsible for many of its clinical features such as hypertension2–3. The endothelial dysfunction in preeclampsia is also associated with an increase in the anti-angiogenic factor, soluble fms-like tyrosine kinase-1 (sFlt-1) which is hypothesized to antagonize vascular endothelial growth factor and placental growth factor during pregnancy6. Although recent studies have shown that chronic sFlt-1 excess causes hypertension in pregnant animals, the mechanisms underlying this effect of sFlt-1 on blood pressure regulation is unknown7–8.

There is growing evidence that suggests an increase in pro-oxidant forces in women with preeclampsia9. Placentas from women with preeclampsia contain more lipid peroxides and malondialdehydethan women with normal pregnancies 9. Lipid peroxides and oxygen radicals can damage endothelial cells as they are highly reactive compounds. 8-isoPGF2a, the free form of isoprostane, is also increased in women with preeclampsia9. In addition, we have recently reported enhanced production of sFlt-1 and reactive oxygen species in a placental ischemic model of preeclampsia. Moreover, we found that chronic administration of the superoxide dismutase (SOD) mimetic, Tempol, markedly attenuates the hypertension induced by reductions in uterine perfusion pressure in pregnant rats10. While these data suggest that ROS are important mediators in hypertension in response to placental ischemia, in which sFlt-1 is also increased, the importance of reactive oxygen species in mediating the hypertension in response to chronic sFlt- 1 excess during pregnancy is unknown. Therefore, we tested the hypothesis that generation of ROS was elevated in sFlt-1 infused hypertensive pregnant rats. Furthermore, we tested the hypothesis that the increase in ROS plays an important role in mediating sFlt-1 induced hypertension in the pregnant rat.

METHODS

Pregnant Sprague-Dawley rats purchased from Harlan Sprague Dawley Inc (Indianapolis, IN) were used in the study. Animals were housed in a temperature-controlled room (23 °C) with a 12:12-hour light/dark cycle. All experimental procedures executed in this study were in accordance with the National Institute of Health guidelines for use and care of animals. All protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Mississippi Medical Center.

Experimental Design

sFlt-1 induced hypertension during pregnancy

Normal pregnant Sprague Dawley rats were used as controls (n=7). Mini osmotic pump (Alzet 2001) infusing sFlt-1(R&D Systems: recombinant mouse NSO-derived; 1ng/kg/hr) were surgically implanted intraperitoneally on day 13 into normal pregnant rats (n=6). Carotid catheters were inserted on day 18 of gestation. On day 19 of gestation mean arterialpressures were recorded and blood, kidneys, heart, aorta, placentas andpups were collected. Tissues were flash frozen in liquid nitrogen and stored at −80°C. Serum and plasma from the rats was stored at −20 °C.

The effect of Tempol on sFlt-1 induced hypertension

On day 12 of gestation Tempol (Sigma-Aldrich; 97% 4-Hydroxy-Tempo) was administered in the drinking water of pregnant rats at a dose of 30mg/kg/day. Normal pregnant rats treated with Tempol only were used ascontrols (n=10). Chronic sFlt-1 infusion (1ng/kg/hr) using intrapreitoneal osmotic pumps previously described was started on gestation day 13 in normal pregnant Tempol treated rats (n=7). Carotid catheters were inserted on day 18 of gestation. On day 19 of gestation mean arterial pressureswere recorded and blood, kidneys, heart, aorta, placentas and pups were collected. Tissues were flash frozen in liquid nitrogen and stored at −80°C. Serum and plasma from the rats was stored at −20 °C.

Measurement of arterial pressure in chronically instrumented conscious rats

Arterial pressure was determined in all groups of pregnant rats at day 19 of gestation as previously described11–14. Briefly, on day 18 of gestation, pregnant rats were catheterized under anesthesia using isoflurane(Webster, Sterling MA) delivered by an anesthesia apparatus (Vaporizer for Forane Anesthetic, Ohio Medical Products, Madison, WI). A catheter of V-3 tubing (SCI, Lake HavasuCity, AZ) was inserted into the carotid artery for blood pressure monitoring. Thecatheter was tunneled to the back of the neck and exteriorized after implantation. On day 19 of gestation, pregnant rats were placed in individual restraining cages for arterial pressure measurements. Arterial pressure was monitored with a pressure transducer (Cobe III Transducer CDX Sema, Birmingham, AL) and was recorded continuously for a 2 hour period following a 1 hour stabilization.

Determination of tissue ROS

Superoxide production in the placenta and thoracic aorta was measured by using the lucigenin Technique15,16. Briefly, placentae and thoracic aorta were removed, cleansed of extraneous tissues and homogenized (1:8 wt/vol) in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS, and a protease inhibitor cocktail; Sigma, St. Louis, MO) as described previously15,16. The samples were centrifuged at 12,000 g for 20 min, the supernatant aspirated and the remaining cellular debris discarded. Basal tissue oxidative stress was determined as follows. The supernatant (100 μl) was incubated with PBS (350 μl) and lucigenin (50μl) at a final concentration of 5 μM. The samples were allowed to equilibrate for 3 min in the dark, and luminescence was measured every second for 5–15 minuteswith a luminometer (Berthold, Oak Ridge, TN). Luminescence was recorded as relative light units per minute (RLU/min). This process was slightly modified to determine NADPH stimulated tissue oxidative stress (RLU/min). The supernatant (100 μl), PBS (300 μl) and NADPH (Sigma-Aldrich; 100 μl of 1mM solution) was incubated with lucigenin (50 μl) at a final concentration of 5 μM. An assay of a blank with no homogenate(PBS 450 μl)but containing lucigenin (50μl) was subtracted from the reading before transformation of the data. The protein concentration was measured using a Pierce (Rockford, IL)protein assay with BSA standards. The data are expressed as RLU per min per milligram protein.

Determination of plasma sFlt-1

Murine sVEGF R1 colorimetric ELISA (R&D Systems, Minneapolis, MN) was used for quantification of plasma sFlt-1 levels. Procedures were carried out following instructions outlined by the manufacturer. This assay displayed a sensitivity level of less than 10 pg/ml.

Statistical Analysis

All data are expressed as mean standard error of the mean. Difference between control and experimental groups were analyzed using ANOVA with Tukey-Kramer multiple comparison tests. The t-test was used when comparing two groups of dams. Data was considered statistically different at P values < 0.05.

RESULTS

Elevated sFlt-1 increases blood pressure during pregnancy

Figure 1 illustrates the plasma sFlt-1 levels and mean arterial pressure in pregnant controls and sFlt-1 treated pregnant rats with and without Tempol treatment. Infusion of sFlt-1 achieved comparable elevations of plasma sFlt-1 levels in the sFlt-1 treated pregnant rats andsFlt -1 and Tempol treated pregnant rats (Figure 1a). The mean plasma sFlt-1 level for the pregnant controls; sFlt-1 treated pregnant rats; Tempol treated pregnant rats; sFlt-1 and Tempol treated pregnant rats was 301 +/−93, 947 +/− 268, 177 +/− 44 and 942 +/− 210 pg/ml respectively (P = 0.026). Administration of sFlt-1 produced a significant increase in blood pressure (Figure 1b). The MAP for the pregnant controls and sFlt-1 treated pregnant rats was 104 +/− 2 and 118 +/− 3 mm Hg, respectively. Administration of Tempol attenuated sFlt -1 induced hypertension. The MAP for the Tempol treated pregnant rats and sFlt-1 and Tempol treated pregnant rats was 104 +/− 2 and 107 +/− 2 mm Hg, respectively.

Figure 1.

Figure 1

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Effect of chronic infusion of soluble fms-like tyrosine kinase (sFlt-1) on plasma sFlt-1 levels and meanarterial pressure (MAP) in pregnant rats in the presence and absence of Tempol treatment. Data are expressed as mean ± SEM, * indicates P< 0.05.

sFlt-1 induced hypertension is associated with increases in reactive oxygen species

In response to sFlt-1 infusion, basal superoxide levels in the placenta, renal cortex and aorta increased from 9 +/− 4 to 27 +/− 8 RLU/mg/min (P = 0.445), 147 +/− 36 to 333 +/− 154 RLU/mg/min (P = 0.423), and 161 +/− 35 to 905 +/− 155 RLU/mg/min (P < 0.05), respectively. NADPH stimulated superoxide production in the placenta, renal cortex and aorta increased from 129 +/− 29 to 179 +/− 35 RLU/mg/min (P = 0.444), 266 +/− 114 to 970 +/− 402 RLU/mg/min (P = 0.303), and 364 +/− 77 to 1008 +/− 194 RLU/mg/min (P <0.05), respectively. In contrast basal superoxide production in the placenta, renal cortex and aorta in Tempol treated dams did not increase (21 +/−5 to 32 +/− 12 RLU/mg/min (P = 0.355)), 243 +/− 46 to 197 +/− 61 RLU/mg/min (P = 0.572), 270 +/−47 to 475 +/− 118 RLU/mg/min (P = 0.1), respectively. In addition, Tempol administration attenuated the increase in NADPH stimulated superoxide production in the placenta, renal cortex and aorta from 128 +/−40 to 130 +/− 56 RLU/mg/min (P= 0.504), 594 +/− 85 to 574 +/ 116 RLU/mg/min (P=0.897), 507 +/− 54 to 712 +/− 201 RLU/mg/min (P=0.277), respectively, with chronic sFlt-1. Figure 2 illustrates the effects of chronic sFlt-1 excess and Tempol pre-treatment on vascular oxidative stress.

Figure 2.

Figure 2

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Effect of chronic infusion of soluble fms-like tyrosine kinase (sFlt-1) on basal and NADPH stimulated vascular superoxide levels in pregnant rats in the presence and absence of Tempol treatment. Data are expressed as mean ± SEM, * indicates P < 0.05.

DISCUSSION

The present study reveals that the increase in blood pressure in response to chronic sFlt-1 excessin pregnant rats is associated with increases in the production of reactive oxygen species in a variety of tissues including the placenta, kidneys, and the aorta. Moreover, we found that chronic administration of the superoxide dismutase (SOD) mimetic, Tempol, markedly attenuated the hypertension induced by chronic sFlt-1 excess in pregnant rats. These novel findings suggest that reactive oxygen species play an important role in mediating hypertension in response to chronic sFlt-1 excess during pregnancy.

There is compelling evidence that preeclampsia is strongly linked to an imbalance between pro-angiogenic and anti-angiogenic factors in the maternal circulation5–7. For example, recent studies have reported that increased sFlt-1 may have a predictive valuein diagnosing preeclampsia17. Moreover, recent experimental studies have shown that chronic sFlt-1 excess in mouse and rat models of pregnancy reduces free VEGF levels and causes endothelial dysfunction and hypertension7,8,18. In the present study, our infusion protocol achieved plasma concentrations of sFlt-1 that were increased three-fold compared to the vehicle control group. This increase in sFlt-1 is similar to what we have reported in our reduced uterine perfusion pressure model of placental ischemia13. In addition, we recently reported that this increase in sFlt-1 resulted in decreased circulating free VEGF concentrations8,13. While physiological elevations in sFlt-1 levels in pregnant rats increases blood pressure, the mechanisms by which sFlt-1 mayexert its effects have remained unclear.

Preeclamptic women and rats with placental ischemia have demonstrated the presence of vascular endothelial dysfunction and oxidative stress 5,6,9. We also recently reported that chronic in vivo exposure to 3-fold increased concentrations of sFlt-1 impairs Acetylcholine and Sodium Nitroprusside mediated vasodilation8. We also found that this effect was ameliorated by addition of Tiron, a superoxidescavenger, to the organ chamber bath suggesting t hat oxidative stress plays a role in the observed vascular dysfunction8. Although our recent data showed that increased oxidative stress contributes to the observed endothelial dysfunction induced by sFlt-1, the study did not discern the relative importance of ROS in mediating the hypertension associated with chronic sFlt-1 excess. In the current study we found that infusion of sFlt-1 in normal pregnant rats increased blood pressure by approximately 15 mmHg whereas blood pressure in Tempolpretreated pregnant rats only increased by 5 mmHg in response to sFlt-1. These findings clearly demonstrate that reactive oxygen species play an important role in mediating hypertension in response to chronic sFlt-1 excess during pregnancy.

Human studies have not yielded consistent results when the ability of various anti-oxidants to prevent Pre-eclampia was investigated19–24. These studies primarily evaluated the efficacyof Vitamins C and E to prevent Preeclampsia following their use in the second and third trimesters of pregnancy. It is possible that either Vitamins C and E or their dosage schedule failed to generate an antioxidant effect potent enough to realize the desired clinical benefit. Furthermore, the pro-vasodilator effect of anti-oxidants usually requires an intact endothelium capable of Nitric Oxide production25. Perhaps the beneficial effect of these anti-oxidants in pregnant women is not realized if they are administered later in pregnancy after the onset of systemic endothelium dysfunction despite the absence of clinical disease. This study’s design allowed for timely pre-treatment with Tempol prior to the onset of excess sFlt-1 induced endothelial dysfunction, a feat that may not be easy to achieve in clinical practice to realize the beneficial effects of anti-oxidants.

This study supports the association between anti-angiogenic factors and hypertension. This association may not be unique to pregnancy and has been noted in other contexts such as cancer therapy26. Although the present data demonstrate a novel pathway by which sFlt-1 may contributeto hypertension during pregnancy, it remains unknown by which mechanisms oxidative stress is increased in this model. It appears that the increased tissue oxidative stressproduced by sFlt-1 infusion may be mediated through NADPH oxidases since we found that basal and NADPHstimulated levels of ROS in placenta, renal cortex and aorta are increased in response to sFlt-1 excess in pregnant rats. Further studies are planned to evaluate the role of theNADPH oxidase pathway as well as other mechanisms that may lead to increased oxidative stress in response to chronicsFlt-1 excess during pregnancy.

Acknowledgments

This work was supported by National Institutes of Health grants HL51971 and AHA 0835472N.

Footnotes

Conflict of interest statement: All authors do not have any conflict of interest to declare.

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