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Increased circulating lipid peroxides in severe preeclampsia activate NF-B and upregulate ICAM-1 in vascular endothelial cells

P. TAKACS^*, S. W. KAUMA^*,, M. M. SHOLLEY^§, S. W. WALSH^*,, M. J. DINSMOOR^* and K. GREEN^*

^* Departments of Obstetrics and Gynecology,
Microbiology and Immunology,
Physiology, and
^§ Anatomy, Virginia Commonwealth University, Medical College of Virginia

²Correspondence: Scott Kauma, M.D., Department of Obstetrics and Gynecology, Virginia Commonwealth University/Medical College of Virginia, 1101 E. Marshal Street, Sanger Hall, Rm. 11-029, Box 980034, Richmond, VA 23298. E-mail: skauma{at}hsc.vcu.edu

SPECIFIC AIMS

In this study we tested the hypothesis that increased circulating lipid peroxides in preeclamptic women activate nuclear transcription factor kappa B (NF-B) in vascular endothelial cells and result in the increased expression of intercellular adhesion molecule-1 (ICAM-1). Furthermore, because antioxidant therapy decreases the incidence of preeclampsia in women at high risk, we also determined if vitamin E or N-acetyl-cysteine could effectively inhibit NF-B activation and ICAM-1 expression in human umbilical endothelial cells (HUVEC) induced by plasma from preeclamptic women.

PRINCIPAL FINDINGS

1. Circulating levels of lipid peroxides are increased in severe preeclampsia
Plasma levels of malondialdehyde were used to estimate circulating lipid peroxides in normal and severe preeclamptic pregnant women. Plasma samples from women with preeclampsia showed a 4.5-fold higher concentration of malondialdehyde than those of normal pregnant patients (6.12 vs. 1.36 µM, P<0.001). The significantly higher malondialdehyde concentration indicates increased circulating levels of lipid peroxides in severe preeclamptic patients.

2. Plasma from women with severe preeclampsia upregulated HUVEC NF-B activity and ICAM-1 expression compared to plasma from normal pregnant women
Lipid peroxides and oxidative stress are known to activate NF-B in a variety of cell types. To measure NF-B activity, HUVEC were transfected with the pNFB-luc plasmid and the pRL-TK plasmid, which served as internal control for transfection efficiency. After transfection, the HUVEC were treated with 5% normal or severe preeclamptic plasma for 48 h. Plasma from women with severe preeclampsia upregulated endothelial NF-B activity by 2.5-fold compared with plasma from normal pregnant women (P<0.001, Fig. 1 ).

View larger version (22K): [in this window] [in a new window]   Figure 1. NF-B activation and ICAM-1 upregulation in HUVEC severe preeclampsia. HUVEC were transfected with pNFB-Luc and pRL-TK luciferase reporter constructs. Transfected HUVEC were treated with 5% normal (N = 12) or preeclamptic plasma (N = 12) for 48 h. NF-B induced luciferase activity was normalized to renilla luciferase activity. Results are expressed as an index of relative light units (RLU). Preeclamptic plasma significantly up-regulated NF-B activation in HUVEC compared with plasma from normal pregnant women (P<0.001). In addition, HUVEC were cultured with 5% plasma from normal (N = 5) or severe preeclamptic women (N = 5) for 48 h, and analytic flow cytometric analysis was then performed for ICAM-1. Severe preeclamptic plasma significantly upregulated ICAM-1 expression by 30% compared with control (P<0.001). The data represent the means ± SE.

ICAM-1 is regulated transcriptionally by NF-B, and activation of NF-B results in increased ICAM-1 expression. To further test our hypothesis that lipid peroxide activation of NF-B upregulates ICAM-1 expression, HUVEC were cultured in media with 5% plasma from normal or severe preeclamptic patients for 48 h. Flow cytometric analysis revealed that severe preeclamptic plasma significantly upregulated ICAM-1 cell surface expression when compared with normal pregnant patients by 30% (P<0.001, Fig. 1 ).

3. Antioxidant treatment with N-acetyl-cysteine and vitamin E reduced NF-B activity and ICAM-1 expression in HUVEC treated with severe preeclamptic plasma
N-acetyl-cysteine and vitamin E, both potent antioxidants, are known inhibitors of NF-B activation. To further test our hypothesis that NF-B activation by lipid peroxides in plasma from women with severe preeclampsia can be inhibited with antioxidants, transfected HUVEC were treated with 5% severe preeclamptic plasma with or without 5 mM N-acetyl-cysteine or 50 µM vitamin E for 48 h. In HUVEC treated with severe preeclamptic plasma, N-acetyl-cysteine reduced NF-B activity by 76% (P<0.01) and vitamin E reduced NF-B activity by 77% (P<0.005, Fig. 2 ) compared with HUVEC treated with severe preeclamptic plasma alone.

View larger version (29K): [in this window] [in a new window]   Figure 2. Vitamin E inhibition of NF-B activation and ICAM-1 upregulation in HUVEC in response to preeclamptic plasma. HUVEC were transfected with pNFB-Luc and pRL-TK luciferase reporter constructs. Transfected HUVEC were treated with 5% preeclamptic plasma (N = 12) with or without 50 µM vitamin E for 48 h. NF-B–induced luciferase activity was normalized to renilla luciferase activity. Results are expressed as an index of relative light units (RLU). Vitamin E reduced NF-B activation by 77% (P<0.005) compared with HUVEC treated with severe preeclamptic plasma without vitamin E. In addition, HUVEC were cultured with 5% plasma from severe preeclamptic patients (N = 5) for 48 h with or without vitamin E. Analytic flow cytometric analysis was then performed for ICAM-1. Vitamin E significantly reduced ICAM-1 expression by 39% compared with HUVEC treated with severe preeclamptic plasma without vitamin E (P<0.005). The data represent the means ± SE.

HUVEC were also cultured with 5% plasma from severe preeclamptic patients in the presence of 5 mM N-acetyl-cysteine or 50 µM vitamin E to determine the effect of antioxidant treatment on ICAM-1 upregulation. Both N-acetyl-cysteine and vitamin E significantly reduced HUVEC ICAM-1 expression in response to severe preeclamptic plasma. N-acetyl-cysteine reduced ICAM-1 expression by 62% (P<0.0001) and vitamin E by 39% (P<0.005, Fig. 2 ) compared with HUVEC treated with severe preeclamptic plasma without antioxidants.

CONCLUSIONS

Preeclampsia is a systemic disease characterized by diffuse endothelial dysfunction, vasospasm, increased oxidative stress and antioxidant deficiencies, hyperlipidemia, and activation of the coagulation system. Over the years, various theories have been proposed to explain the pathophysiology of preeclampsia, including vascular endothelial damage, cardiovascular maladaptation, immunologic phenomena, abnormal trophoblast invasion, coagulation abnormalities, genetic predisposition, and dietary deficiencies or excesses. Despite ongoing research into the etiology of preeclampsia, the cause remains unknown. However, most investigators would agree that the pathogenic mechanisms of preeclampsia converge on the vascular endothelium and result in endothelial activation and dysfunction. Consequently, most of the clinical pathological findings in preeclampsia, including hypertension, edema, proteinuria, and activation of the coagulation system, can be attributed to abnormalities in endothelial function.

A major factor leading to endothelial cell dysfunction in preeclampsia is increased circulating levels of lipid peroxides. Preeclampsia is associated with increased levels of triglycerides and free fatty acids. In a case control study, women with preeclampsia exhibited 65% higher mean triglyceride levels. The importance of the increased free fatty acids and hypertriglyceridemia is that these compounds can undergo either spontaneous or induced oxidation, which leads to an increase in lipid peroxides and oxidative stress. During normal pregnancy, the placenta produces significant quantities of reactive oxygen species and lipid peroxides. In women with dyslipidemia and increased lipid peroxides, the ability of the antioxidant systems to neutralize placental lipid peroxides is exceeded and a pathologic state of oxidative stress develops. The placental contribution to increased circulating levels of lipid peroxides may serve as the ‘catalyst’ for increased oxidative stress in preeclampsia which, when eliminated at the time of delivery, results in disease regression. Evidence has accumulated that women with preeclampsia have increased circulating levels of oxygen free radicals and lipid peroxides. Conversely, women with preeclampsia have decreased levels of circulating antioxidants such as vitamin E.

This study is the first to report that plasma from women with severe preeclampsia activates NF-B in vascular endothelial cells. Lipid peroxides, oxidative stress, and reactive oxygen intermediates previously have been shown to activate NF-B in a variety of cell types. Our findings suggest that increased circulating lipid peroxides in preeclamptic women are responsible for endothelial cell NF-B activation, as plasma samples from preeclamptic women used in these studies had 4.5-fold higher levels of lipid peroxides than those of normal pregnant women. This hypothesis is further supported by the fact that vitamin E and N-acetyl-cysteine, both antioxidants, significantly inhibited endothelial cell NF-B activation by preeclamptic plasma. Although there was a small decrease in NF-kB activity by N-acetyl-cysteine in the normal group without preeclampsia, this was probably due to the presence of low levels of lipid peroxides known to be present in the plasma of normal pregnant women.

Circulating levels of soluble ICAM-1 are increased in women with preeclampsia and have been thought to be a marker of endothelial activation in this disease. In addition, circulating soluble ICAM-1 levels are significantly increased at 18 wks’ gestation in women who eventually develop preeclampsia. The current studies demonstrate that plasma from preeclamptic women significantly upregulates the expression of cell surface ICAM-1 in vascular endothelial cells. Oxidized LDL is known to cause endothelial cell activation and up-regulation of ICAM-1. In addition, NF-B activation plays an important role in the up-regulation of ICAM-1 expression by binding to corresponding cis-regulatory elements in the promotor region of the ICAM-1 gene. Given our findings that preeclamptic plasma activates NF-B in endothelial cells, it is likely that increased lipid peroxides in preeclamptic plasma stimulate ICAM-1 expression through a NF-B mediated mechanism. This hypothesis is supported by the findings that vitamin E and N-acetyl cystine not only inhibited endothelial cell NF-B activation by preeclamptic plasma but also ICAM-1 expression. Conclusions about early pathogenic mechanisms in preeclampsia based on the results from this current study are limited by the fact that the plasma samples were obtained from patients who had preeclampsia. Consequently, our findings may be the result of other underlying pathogenic mechanisms leading to the development of this disease, and not the cause of preeclampsia.

A recent study revealed that supplementation with vitamin E and C of pregnant women at high risk for preeclampsia was beneficial in the prevention of preeclampsia. In this prospective, randomized, placebo-controlled study, women at increased risk for preeclampsia received daily supplementation with 400 IU of vitamin E and 1000 mg of vitamin C beginning at 18–20 wks’ gestation. They subsequently found a significantly decreased incidence of preeclampsia, from 18% in the placebo group to 8% in the treatment group. The rationale for using vitamin E and vitamin C in this study and the likely explanation of the observed beneficial effect was to decrease oxidative stress. That oxidative stress was decreased was demonstrated by decreased circulating 8-isoprostane levels in the treatment group. The present study provides a potential molecular mechanism for the beneficial effects of antioxidant treatment for the prevention of preeclampsia. According to our data, plasma from women with preeclampsia activates NF-B and upregulates ICAM-1 expression in endothelial cells in vitro. Furthermore, the addition of 50 µM vitamin E, a dose comparable with serum levels that result from taking 400 IU of vitamin E orally, inhibited NF-B activation and significantly decreased ICAM-1 expression in endothelial cells cultured with preeclamptic plasma. Taken together, these studies support the hypothesis that, in part, the beneficial effect of antioxidant therapy in the prevention of preeclampsia is through the inhibition of lipid peroxide activation of vascular endothelial cells.

Elevated lipid peroxides in preeclampsia may mediate increased leukocyte adhesion to vascular endothelial cells, thus contributing to the pathophysiology of this disease. Oxidized low-density lipoprotein has been shown to increase endothelial cell expression of ICAM-1, vascular cell adhesion molecule 1, and E-selectin in vitro. Increased circulating levels of ICAM-1 are seen in patients with elevated triglycerides, atherosclerotic coronary artery disease, obesity, and hypertension, as well as preeclampsia. Monocyte and neutrophil adhesion to vascular endothelial cells is modulated by cell surface adhesion molecules, such as ICAM-1. Up-regulation of endothelial cell ICAM-1 expression in preeclampsia may increase monocyte and neutrophil adherence to vascular endothelial cells. Because both monocytes and neutrophils have increased superoxide production in preeclamptic women, the increased adherence and interaction of these activated leukocytes with the vascular endothelium could lead to further endothelial dysfunction and damage. Similar mechanisms for leukocyte-mediated endothelial dysfunction and damage have been proposed in the pathogenesis of atherosclerosis. The presence of ‘acute atherosis’ in the spiral arteries of women with preeclampsia further supports the commonality between endothelial dysfunction in preeclampsia and atherosclerosis. These findings suggest that models of lipid peroxide-induced endothelial dysfunction in atherosclerosis may be useful in the study of endothelial dysfunction in preeclampsia.

In summary, severe preeclampsia is associated with elevated levels of circulating lipid peroxides. Plasma from preeclamptic women activates NF-B and increases ICAM-1 expression in HUVEC. In addition, antioxidant treatment with vitamin E or N-acetyl-cysteine of HUVEC exposed to preeclamptic plasma inhibits NF-B activation and ICAM-1 expression (Fig. 3 ). These findings suggest that, in preeclampsia, increased lipid peroxides upregulate ICAM-1 expression in vascular endothelial cells through a NF-B mediated mechanism.

View larger version (16K): [in this window] [in a new window]   Figure 3. In women who develop preeclampsia, elevated levels of circulating lipid peroxides activate vascular endothelial cell NF-B, which results in increased ICAM-1 expression. NF-B activation by lipid peroxides with subsequently increased ICAM-1 expression can be inhibited by antioxidants such as vitamin E and N-acetyl-cysteine (NAC).

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0549fje To cite this article, use (December 8, 2000) FASEB J. 10.1096/fj.00-0549fje

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