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Circulating Endothelial Progenitor Cells during Human Pregnancy

Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, Sendai, 980-8574, Japan

Address all correspondence and requests for reprints to: Junichi Sugawara, M.D., Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, Tohoku University Graduate School of Medicine, 1-1, Seiryomachi, Aobaku, Sendai, 980-8574, Japan. E-mail: sugawara{at}mail.tains.tohoku.ac.jp.

	Abstract

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The precise molecular and cellular mechanisms that regulate maternal vascular development during gestation are largely unknown. Endothelial progenitor cells (EPCs), which play an important role in vascular homeostasis, have been discovered in the circulation. We examined the level of circulating EPCs throughout uncomplicated pregnancies (n = 20) and assessed the correlation between serum estradiol levels and the number of EPCs. The number of circulating EPCs increased gradually and paralleled the progression of gestational age. In addition, the number of EPCs correlated significantly with the level of serum estradiol. The present study suggests that EPCs may play an important role in the regulation and maintenance of the placental development and vascular integrity during pregnancy.

	Introduction

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DURING PREGNANCY, MATERNAL vascular adaptation is necessary to supply nutrients effectively to meet the increasing demands of the growing fetus. The precise molecular and cellular mechanisms that regulate maternal vascular changes during gestation are largely unknown. Endothelial function has been reported to be up-regulated in pregnancy, producing vasodilatation by either an increased release of vasodilators (e.g. nitric oxide) or a decreased release of vasoconstrictors. Recently, endothelial progenitor cells (EPCs) have been detected among circulating mononuclear cells (MNCs) (1) and in cord blood (2). They have been reported to play an important role in vascular homeostasis. It is currently thought that bone marrow-derived EPCs contribute to neovascularization by vasculogenesis, the formation of blood vessels de novo from precursors, where no blood vessels previously existed. The recruitment, mobilization, and incorporation of bone marrow-derived EPCs have been shown to restore an intact endothelial lining (3).

Estrogens play an important vasoprotective role by causing an increase in the production of nitric oxide and a decrease in reactive oxygen species (4). More recently, it has been reported that estrogens lead to increased numbers of circulating EPCs via antiapoptotic effects (5, 6). They suggest that estrogens exert their vasoprotective effects by leading to an increase in the number of these progenitor cells. In the present study, we investigated the number of EPCs in the maternal circulation according to gestational week. Additionally, we assessed the correlation between serum estradiol levels and the number of circulating EPCs.

	Subjects and Methods

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Twenty women with uncomplicated pregnancies were enrolled in this study (gestational age, 12–41 wk). At study entry, all women had singleton pregnancies, were free of any medications, had no personal or family history of cardiovascular disease, and had no uterine contractions. Study subjects were between the ages of 18 and 39 yr. All women had appropriately grown fetuses. Ethical approval was obtained from the Ethical Commission on Research on Humans (School of Medicine, Tohoku University, Sendai, Japan). Informed consent was obtained from each patient.

Peripheral blood (20 ml) was diluted with the same volume of PBS, and MNCs were isolated by Ficoll density gradient centrifugation. MNCs were suspended in medium 199 supplemented with 20% fetal bovine serum, bovine pituitary extract as an endothelial cell growth supplement, heparin, and antibiotic-antimycotic reagent (Standard Medium, Life Technologies, Grand Island, NY). Aliquots of 1.5 x 10⁶ MNCs were plated on 48-well plastic dishes coated with human fibronectin (BIO-COAT, Becton Dickinson, San Jose, CA). On d 7, three randomly selected microscopic fields in a minimum of three wells were evaluated, and mean numbers of cell clusters were calculated by three independent investigators (J.S., M.M.-S., and T.H.). Samples of 4 x 10⁶ MNCs were plated on chamber slides coated with human fibronectin (BIO-COAT, Becton Dickinson), and adherent cells underwent cytochemical analysis. To assess the ability of cells to take up acetylated low-density lipoprotein (ac-LDL), attached cells were incubated in medium containing 15 µg/ml DiI-labeled ac-LDL (DiI-ac-LDL, Molecular Probes, Eugene, OR) for 24 h at 37 C. Afterward, cells were fixed with 2% paraformaldehyde for 10 min and stained with fluorescein isothiocyanate-labeled Ulex europaeus agglutinin I (Lectin, 10 µg/ml; Sigma, St. Louis, MO). Confirmation of endothelial-cell lineage was performed in samples from 10 subjects. Cells at d 7 of culture were subjected to immunocytochemistry to analyze the expression of von Willebrand factor, KDR/Flk-1, CD31, and ecNOS. The specificity of the immunocytochemical staining was confirmed by the deletion of the primary antibody.

We took 5-ml samples of blood at the time of EPC studies to measure plasma concentrations of estradiol. Enzyme immunoassay was performed with commercially available enzyme immunoassay kits according to the manufacturer’s instructions. All assays were done in duplicate, and the protein levels were calculated using a standard curve derived from known concentrations of standard. Data are expressed as means ± SE. Univariate correlations were performed with the use of Spearman’s rank-correlation coefficient.

	Results

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Isolated peripheral-blood MNCs formed cell clusters, and spindle-shaped attached cells sprouted from the clusters as reported previously (1, 7) (Fig. 1). By immunocytochemistry with endothelial cell markers, more than 80% of attaching cells expressed those markers (data not shown). We further confirmed that isolated cells indicated endothelial lineage by being positive for both DiI-ac-LDL incorporation and lectin binding (data not shown). We next assessed whether the level of endothelial progenitors correlated with gestational weeks. As shown in Fig. 2, the number of circulating EPCs increased gradually with gestational age (P = 0.03; r = 0.48). We next examined the relation between the number of cell clusters and plasma estrogen levels. Simple regression analysis revealed that circulating levels of EPCs correlated significantly with plasma estradiol levels (P = 0.002; r = 0.72; Fig. 3). We also examined the relation between age and EPC levels. Circulating EPC levels were slightly decreased with increasing age (P = 0.53; r = 0.15); however, no significant correlation was observed (data not shown).

View larger version (186K): [in this window] [in a new window]   FIG. 1. Phase-contrast micrograph of a purified EPC cell cluster (magnification, x200). Isolated peripheral-blood MNCs formed cell clusters, and spindle-shaped attached cells sprouted from the clusters.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Relation between the number of circulating EPCs and gestational weeks. On d 7 of culture, mean numbers of cell clusters were calculated according to the gestational weeks.

View larger version (15K): [in this window] [in a new window]   FIG. 3. The number of circulating EPCs and serum estradiol levels. Significant correlation was seen with a correlation coefficient of 0.72.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Currently, little is known about the precise mechanisms regulating maternal vascular changes during pregnancy. However, increasing evidence suggests that maternal vascular development is explained by three adaptive changes: vasodilation, increased permeability, and neovascularization. Up-regulated endothelial cells would contribute to these dynamic changes. In human pregnancy, flow-mediated vasodilation, an established noninvasive method for the assessment of endothelium-mediated vascular function (8), has been shown to increase with gestational weeks (9). Strikingly, a recent study revealed that the number of circulating EPCs correlated significantly with the reactivity of flow-mediated vasodilation (7). Taken altogether, the present study indicates that EPCs in the maternal circulation may contribute to the maintenance of endothelium-mediated vascular function during pregnancy.

Estrogens exert multiple cellular and molecular effects on the endothelium by increasing nitric oxide production and reducing reactive oxygen species (4). Estrogens have recently been reported to increase the number of circulating EPCs by antiapoptotic effects (5). Moreover, estradiol stimulated mobilization, proliferation, and incorporation of EPCs into the recovering endothelium (6). These studies provide evidence that estrogens exert their vasoprotective effects by increasing the number of circulating EPCs. To determine whether the levels of serum estrogen would correlate with the number of circulating EPCs, we examined the relation between the number of circulating EPCs and the serum estradiol levels. Interestingly, serum estradiol levels and the number of circulating EPCs showed a significant positive correlation. These results indicate that estrogens may exert their vasoprotective effects via regulating the number of circulating EPCs during human pregnancy.

The kinetics of EPC mobilization from bone marrow is stimulated by vascular endothelial growth factors (VEGF) (10), granulocyte-macrophage colony-stimulating factor (11), angiopoietin-1, fibroblast growth factor, and stromal cell-derived growth factor-1 (12, 13). VEGFR-1, also termed soluble fms-like tyrosine kinase-1 (sFlt-1), acts as a potent VEGF antagonist (14), and it is an important factor in the development of the placental vasculature (15). Therefore, we measured sFlt-1 levels according to gestational week and assessed the correlation with circulating EPC levels. As reported by others (16), we also demonstrated that serum sFlt-1 levels increased progressively with gestational weeks (data not shown). However, there was no significant correlation between EPC and sFlt-1 levels (data not shown). Together with these results, one may speculate that estradiol overrides the inhibitory effects of the VEGF antagonist, sFlt-1, on EPC proliferation and migration, and thereby up-regulates the number of circulating EPCs. The present study was a cross-sectional study using samples from each subject once during pregnancy. Longitudinal studies would be more informative in investigating the relationship between EPC levels and gestational weeks.

Although there is convincing evidence for the improvement of neovascularization by EPC transplantation (2, 17, 18, 19), the lineage and exact phenotype of functional EPCs still have been unclear (20, 21, 22). A recent study demonstrated that primitive endothelial precursor cells were present only in samples from maternal blood and not in nonpregnant controls (23). With these results, it might be possible that their observations indicate the lineage heterogeneity in the population of peripheral MNCs capable of assuming an endothelial phenotype. Interestingly, a recent study revealed that ac-LDL(+)ulex-lectin(+) cells, commonly referred to as EPCs, did not proliferate and actually showed angiogenic effects by secreting several growth factors in a paracrine manner (24).

Whether circulating EPCs were of maternal or fetal origin was not confirmed in this study. However, Gussin et al. (23) clearly demonstrated that late-outgrowth endothelial cells from peripheral blood MNCs in pregnant women were of maternal origin.

In summary, we have demonstrated that EPCs can be isolated from the maternal circulation and increase gradually with gestational weeks. Serum estradiol levels correlated significantly with the level of circulating EPCs. These results not only give potential insight into the cellular mechanism of maternal vascular adaptation and remodeling, but may additionally provide new approaches to clarify the pathophysiology of endothelial disorders such as preeclampsia.

	Acknowledgments

 
We thank Dr. Linda Giudice for critically reviewing the manuscript and for helpful suggestions.

	Footnotes

 
First Published Online December 7, 2004

Abbreviations: ac-LDL, Acetylated low-density lipoprotein; EPC, endothelial progenitor cell; MNC, mononuclear cell; sFlt-1, soluble fms-like tyrosine kinase-1; VEGF, vascular endothelial growth factor(s).

Received March 30, 2004.

Accepted November 23, 2004.

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