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Research Article |
Department of Reproductive Medicine,3 University of California San Diego, La Jolla, California 92093
Ciphergen Biosystems, Inc.,4 Fremont, California 92054
Perinatal Research Laboratories,5 Departments of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison, Wisconsin 53715
ABSTRACT
Ovariectomized (OVX) ewes were assigned to receive vehicle, progesterone (P4, 0.9-g controlled internal drug release vaginal implants), estradiol-17ß (E2, 5 µg/kg bolus + 6 µg kg–1 day–1), or P4 + E2 for 10 days (n = 3/group). Uterine artery endothelial proteins were mechanically isolated on Day 10. The samples were used for protein expression profiling by the Ciphergen Proteinchip system and immunoblotting analysis of endothelial nitric oxide synthase (NOS3, also termed eNOS) and caveolin 1. Uterine artery rings were cut and analyzed by immunohistochemistry to localize NOS3 and caveolin 1 expression. With the use of the IMAC3 protein chip with loading as little as 2 µg protein/sample, many protein peaks could be detected. Compared to vehicle controls, a
133.1-kDa protein was identified to be upregulated by 2- to 4-fold in OVX ewes receiving E2, P4, and their combination, whereas a 22.6-kDa protein was downregulated by 2- to 4-fold in OVX ewes receiving E2 and E2/P4, but not P4 treatments. Western blot analysis revealed that E2, P4, and their combination all increased NOS3 protein, whereas E2 and its combination with P4, but not P4 alone, downregulated caveolin 1 expression. Immunohistochemical analysis revealed that NOS3 was mainly localized in the endothelium and upregulated by E2, whereas caveolin 1 was localized in both endothelium and smooth muscle and downregulated by E2. Thus, our data demonstrate that uterine artery endothelial NOS3 and caveolin 1 are regulated reciprocally by estrogen replacement therapy. In keeping with the facts that E2, but not P4, causes uterine vasodilatation and that E2 and P4 increase NOS3 expression, but only E2 decrease caveolin 1 expression, our current study suggests that both increased NOS3 expression and decreased caveolin 1 expression are needed to facilitate estrogen-induced uterine vasodilatation.
caveolin 1, endothelium, estradiol, estrogen, gene regulation, nitric oxide, NOS3, progesterone, sheep, uterine artery, uterus
INTRODUCTION
Estrogen is a potent vasoactive hormone that stimulates acute and chronic vasodilatation in various vascular beds and tissue perfusion throughout the body [1, 2]. Historically, physiological studies have identified estrogen as one of the key factors controlling uterine vasodilatation during the follicular phase of the ovarian cycle and during human pregnancy, both conditions that have significantly elevated circulating estrogen levels [3–5]. Of particular importance in perinatal medicine, uterine blood flow is instrumental to fetal development, because a substantial increase (up to 80-fold) in uterine blood flow is required for delivering nutrients and oxygen supplies from mother to fetus and for exhausting respiratory gases and metabolic wastes from fetus to mother [5, 6]. In ovariectomized (OVX) animal models, chronic estrogen administration provokes generalized vasodilatation, i.e., increased cardiac output, mildly decreased blood pressure, and reduction in systemic vascular resistance. Together, these changes result in increases in blood flow in various vascular beds [2]. Typically in the uterine circulation, following estrogen administration a rise in uterine blood flow begins to take place within 30 min, increases up to 10-fold in 90–120 min, plateaus until 8–10 h, then gradually declines but remains elevated just above baseline through a 10-day course of estrogen replacement therapy [4, 7].
Mechanistic studies over the last decade or so have demonstrated that the potent vasodilator nitric oxide (NO) as the main mediator for estrogen-induced uterine vasodilatation because inhibition of NO synthase (NOS) attenuates, to a great extent (60%–70%), estrogen-induced rise in uterine blood flow [8, 9]. NO is produced during the conversion of L-arginine to L-citrulline catalyzed by a family of NO synthesizing isoenzymes including endothelial NOS (NOS3 or eNOS), neuronal NOS (NOS1 or nNOS) and inducible NOS (NOS2 or iNOS). Extensive studies have shown that the constitutively expressed endothelial NOS3 is the primary NOS accounting for the NO-mediated uterine vasodilatation [10, 11]. Compelling data suggest that chronic estrogen treatment increases NOS3 protein expression in uterine as well as in many systemic vascular endothelia [10, 12, 13]. Apart from increased NOS3 expression, rapid activation of NOS3 via nongenomic pathways of estrogen signaling [14, 15] apparently also is involved in blood flow regulation because of the rapid timing of estrogen-induced vasodilatation in uterine as well as in some systemic vascular beds [4, 7]. Inducible NOS (NOS1) and nNOS (NOS2) might also regulate uterine vasodilatation. However, these NOS isozymes seem to participate in endothelium-independent pathways in estrogen-induced uterine vasodilation because none of them are expressed in uterine artery endothelium [16, 17].
In resting endothelial cells, NOS3 is rendered less active or inactive by direct association with caveolin 1, i.e., a major residual protein of the flask-shaped plasma membrane caveolae [18, 19]. Upon stimulation with estrogen [20] or shear stress [21], NOS3 dissociates from caveolin 1, which facilitates an increase in NOS3 activity in endothelial cells both in vitro and in vivo. These data suggest that caveolin 1 functions as an endogenous inhibitor of NOS3 activity. We therefore hypothesize that, in addition to increased NOS3 expression, downregulation of caveolin 1 may also be required for the full development of estrogen-induced uterine vasodilatation. In this study, the well-established OVX sheep model receiving hormone (estrogen/progesterone) replacement therapy was used to investigate the hypothesis that the expressions of NOS3 and caveolin 1 proteins are regulated reciprocally in uterine artery endothelium by estrogen replacement therapy. Furthermore, available data show that blockade of protein synthesis by cycloheximide [22], but not blockade of RNA synthesis by actinomycin-D [23], inhibits estrogen-induced uterine vasodilatation. Thus, it is possible that the global protein expression profile in uterine artery endothelium might be altered upon hormone replacement therapy. However, this has never been studied. By using the Ciphergen ProteinChip system developed according to the surface-enhanced laser desorption-ionization-time-of-flight (SELDI-TOF) mass spectrometry (MS), we also investigated the effects of chronic hormone replacement therapy treatment on the global protein expression profile in OVX sheep uterine artery endothelium in vivo.
MATERIALS AND METHODS
Ovariectomized Ewes and Hormone Replacement Therapy
Procedures for animal handling and protocols for experimental procedures were approved by the University of Wisconsin–Madison Research Animal Care Committees of both the Medical School and the College of Agriculture and Life Sciences and followed the recommended American Veterinary Medicine Association guidelines for euthanasia of laboratory farm animals. Mixed Western breed ewes (n = 12, purchased from the farm of the Department of Animal Sciences, University of Wisconsin-Madison) were ovariectomized via a midventral laparotomy as previously described [24]. After at least 10 days of recovery, an indwelling 19-gauge polyvinyl catheter was placed in the right ventricle via the jugular vein. The ewes were then divided into four groups (n = 3/group) receiving either vehicle control, 17ß-estradiol (E2), progesterone (P4), or E2/P4 combination. For the hormone replacement therapy groups, ewes received a 5-µg/kg bolus infusion of E2 (Sigma; dissolved in 3 ml of saline containing 10–11% ethanol) via the catheter. The catheter was flushed with 5 ml of saline immediately followed by 6 µg E2 kg body weight–1 day–1 continuous infusion in 9.5% ethanol isotonic saline (0.0123 ml/min) for 10 days. The E2 dose and timed tissue collection were based on NOS3 expression and hemodynamic responses as well as on blood levels of E2 and P4 achieved in our previous studies [13, 24]. For administration of P4, three controlled internal drug release (CIDR) implants with 0.9 g P4 (Carter Holt Harvey) were placed in the vagina of OVX ewes for 10 days [24]. For combined treatment (P4 + E2), both E2 infusion and CIDR P4 implants were used as described above. Control ewes received vehicle (ethanol in saline) infusion and/or blank CIDR implants. The ewes were killed with intravenous injection (50 mg/kg) of pentobarbital sodium and uterine artery samples were collected as described below.
Tissue Fixation and Isolation of Uterine Artery Endothelium Protein Samples
Uterine arteries were excised immediately after animals were killed, placed in PBS (8 mM sodium phosphate, 2 mM potassium phosphate, and 0.15M NaCl, pH7.4), dissected free of connective tissue, and rinsed free of blood. Intact artery segments were cut immediately and fixed in 4% formaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 (EM Science) for 24 h and then stored at 4°C in sodium cacodylate buffer containing 0.01% sodium azide until dehydration and placement in paraffin blocks. The rest of the uterine arteries from each ewe were used to collect uterine artery endothelial protein samples according to a rapid mechanical isolation procedure as previously described and validated [25]. Briefly, the artery was opened longitudinally and the endothelium/tunica intima was gently scraped (3–6 times) from the artery. The "scraping" uterine artery endothelial protein samples were placed in a lysis buffer (50 mM Tris, pH 7.4, 0.15 M NaCl, 10 mM EDTA, 0.1% Tween-20, 0.1% ß-mercaptoethanol, 0.1 M phenylmethylsulfonyl fluoride, and 5 µg/ml each of leupeptin and aprotinin; all chemicals are from Sigma). The samples were snap-frozen in liquid nitrogen immediately and were stored at –20°C.
SELDI-TOF MS
The primary uterine artery endothelial "scraping" proteins isolated mechanically were homogenized in lysis buffer and then sonicated. After centrifugation (250 x g for 10 min) to remove particulate materials from the protein extracts, the protein concentrations were determined by the Bio-Rad procedure using BSA as a standard. Protein extracts (20 µg/sheep) were mixed with ice-cold PBS containing urea to adjust the final urea concentration to 8 M and protein concentration to 1 µg/ul. Protein samples were diluted 20x in a binding buffer (PBS, 0.5 M NaCl, and 0.1% Triton X-100) for the immobilized metal affinity capture (IMAC3) protein chip. The samples (2 µg/sample) were spotted onto IMAC3 Protein Chips (Ciphergen). The chips were equilibrated in 2x binding buffers, and protein binding was allowed for 2 h at room temperature with shaking. After washing 3x with corresponding binding buffer and 3x with HPLC grade water, 0.8 µl of saturated sinapinic acid was applied onto the chip as a matrix solution twice. The chip arrays were analyzed in a Ciphergen Biosystems PBS-IIC ProteinChip Reader according to an automated protocol for data collection. The instrument using a nitrogen laser was used in positive ion mode. Laser intensity was set at approximately 200–230 µJ, emitting at 337 nm. The digitizer was operated at 250 MHz. The SELDI-TOF MS spectra data were obtained and analyzed with the Ciphergen ProteinChip analysis software 3.2.1.
Western Blot Analysis
Proteins (5–10 µg/sheep) mixed and boiled in SDS sample buffer were separated on Bio-Rad precasted 10% polyacrylamide gels and transferred to Immobilon P membranes by using a Fisher semidry blotter (0.15 mA/100 cm2, 1.5 h). Western blot analysis of NOS3 and caveolin proteins was performed as previously described [26]. Briefly, the membranes were incubated with 20 mM Tris, pH7.5, 500 mM NaCl, and 0.1% Tween-20 (TBST) containing 5% skim milk to block nonspecific binding, followed by incubation with TBST containing 1% BSA anti-NOS3 monoclonal antibody (1:750, N30020) for 2h, or anti-caveolin 1 polyclonal antibody (1:20,000, N20) and ß-actin monoclonal antibody (1:5000, Qiagen) for 1 h at room temperature. After washing four times (5 min each) with TBST, the membranes were incubated with TBST containing 0.5% skim milk and anti-mouse (1:2000) or anti-rabbit IgG (1:3000, Amersham) for 1 h at room temperature. After washing four times (5 min each) with TBST, the membranes were incubated with Chemi-Glow Chemiluminescent substrate (Alpha Innotech Corp.) to visualize bound antibodies. Digital images of bound antibodies were captured with the ChemiImager Imaging System (Alpha Innotech Corp.) with a high-resolution charge-coupled device camera and quantified by Alpha Innotech ChemiImager 4400 software.
Immunohistochemical Analysis of NOS3 and Caveolin Proteins
Uterine artery segments were collected from OVX ewes receiving vehicle and estrogen replacement therapy as described above. Immediately after the ewes were killed, the uterine artery rings were fixed with 4% paraformaldehyde and embedded in paraffin, and 6-µm sections were cut and mounted onto slides. Immunohistochemical analysis using a monoclonal antibody against human NOS3 (N30020, 5 µg/ml) from Transduction Laboratories was performed using the PicTure-Plus kit from Zymed Laboratories, Inc., following a protocol as described previously [27]. Polyclonal antibodies against caveolin 1, caveolin 2, and caveolin 3 (2–5 µg/ml, all from Santa Cruz Biotechnolgies Inc.) were used to localize these caveolins in paraffin-embeded uterine artery sections immunohistochemically by using the Vectastain ABC Elite kit (Vector Laboratories) as previously described [25]. The same concentration of anti-mouse IgG or anti-rabbit IgG, respectively, was used as negative control.
Statistical Analysis
Data are presented as means ± SEM, and analyzed by one-way ANOVA using Ciphergen Biomarker Wizard Software for the SELDI/TOF MS data or Sigma Stat/Plot software (Jandel Scientific) for Western blot analysis. When the F-test was significant (P < 0.05), treatment responses were compared with vehicle controls by Fisher multiple comparisons. P < 0.05 was considered significantly different.
RESULTS
SELDI-TOF MS Analysis
Using the IMAC3 protein chip, we initially tried to spot 1- to 10-µg proteins to normalize the protein profiling procedure. Many protein peaks could be detected on this chip with loading as low as 2-µg proteins. Specifically, two peaks were detectable in all the samples (Fig. 1). These two proteins are approximately 22.6 kDa and 133.1 kDa in molecular weight, which are similar to weights of a monomer of caveolin 1 or NOS3 protein, respectively. Statistical analysis revealed that compared to controls, the 22.6-kDa peak was downregulated nearly 4-fold in E2- and 2-fold in E2/P4-treated, whereas unchanged in P4-treated OVX ewes. On the other hand, the 133.1-kDa peak was upregulated 4-fold in E2- and E2/P4- and 2-fold in P4-treated OVX ewes in comparison to controls. According to the spectra data shown in Figure 1, it is apparent that the signal intensities of the peaks detected by SELDI-TOF MS were reduced significantly from low- to high-molecular-weight proteins.
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Western Blot Analysis
As illustrated in Figure 2, uterine artery endothelial caveolin 1 protein levels were dramatically downregulated by 3-fold in OVX ewes receiving chronic estrogen replacement therapy compared to vehicle controls. In OVX ewes receiving P4 alone, uterine artery endothelial caveolin 1 protein levels were not changed significantly in compared to controls. However, in OVX ewes receiving E2 and P4 combination replacement therapy, uterine artery endothelial caveolin 1 protein levels were also downregulated significantly (P < 0.05). In contrast, uterine artery endothelial NOS3 protein levels were upregulated by 3-fold in OVX ewes receiving E2 or E2 plus P4 combination replacement therapy in comparison to controls. Progesterone alone also upregulated uterine artery endothelial NOS3 protein expression, but to a lesser, although still significant (P < 0.05) extent, compared to controls. Western blot analysis with the anti-ß-actin antibody revealed that chronic hormone replacement therapy did not alter the protein expression of this "housekeeping" gene in uterine artery endothelium, which shows equal protein loading in the Western blot analysis of caveolin 1 and NOS3 proteins.
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Immunohistochemical Analysis
To further confirm above findings in the reciprocal regulation of uterine artery endothelial caveolin 1 and NOS3 expression in OVX ewes receiving estrogen replacement therapy alone, immunohistochemical analysis of paraffin-embedded sections of intact uterine artery rings was performed. As shown in Figure 3, high levels of caveolin 1 protein expression was detected in both the endothelial and smooth muscle cells of uterine arteries in vehicle-treated control OVX ewes, whereas NOS3 protein was also detectable in the endothelial, but not the smooth muscle cells (Fig. 3, a and b, top right panel images). In comparison to vehicle-treated controls, OVX ewes receiving estrogen replacement therapy displayed a dramatic reduction in caveolin 1 protein expression in uterine artery endothelial and smooth muscle cells, whereas by contrast an induction of NOS3 protein expression in uterine artery endothelial but not smooth muscle cells (Fig. 3, a and b, lower right panel images). Omitting (data not shown) or replacing (Fig. 3, a and b, lower right panel images) the primary antibodies against caveolin 1 or NOS3 with the corresponding rabbit or mouse IgG showed weak nonspecific background signals (Fig. 3, left panels), indicating the specificity of the antibodies for immunohistochemical analysis. Caveolin 2 and caveolin 3 are both undetectable by immunohistochemical analysis with the commercially available antibodies (data not shown).
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DISCUSSION
Nitric oxide produced locally by the uterine artery endothelium via NOS3 contributes to the majority of the endothelium/NO-dependent mechanism(s) for estrogen-induced rises in blood flows in the uterine and placental circulations essential for providing the nutrients and oxygen supplies for the growing fetus during pregnancy [11]. This is supported directly by a recent report showing abnormal uterine artery remodeling thus reduced pregnancy outcomes (reduced litter size) in NOS3-null compared to wild-type mice [28]. In the present study, we used the well-established OVX sheep model to investigate the effects of hormone (estrogen/progesterone) replacement therapy on uterine arterial caveolin 1 and NOS3 protein expression. Our data, obtained by using three different approaches—proteomics, immunoblotting and immunohistochemistry—consistently rectified our hypothesis that estrogen regulates uterine artery endothelial caveolin 1 and NOS3 expression in a reciprocal fashion. Increased NOS3 expression is expected to play a role in the control of uterine blood flow, because estrogen increases endothelial NOS3 expression in uterine artery in vivo, as shown by previous studies [13, 17, 25, 29, 30] and confirmed by this study as well as by the fact that blockade of NO biosynthesis by the NOS inhibitor L-NAME attenuates estrogen-induced uterine vasodilatation [18, 19]. Furthermore, upon estrogen or shear stress stimulation, increased NO production via NOS3 activation is associated with NOS3 dissociation from caveolin 1 [18, 19], because binding to the latter tonically renders NOS3 less active or completely inactive [18, 19]. We were the first to hypothesize downregulation of uterine artery caveolin 1 expression as an additional mechanism for estrogen-induced rise in uterine blood flow [26]. Thus, the observation that caveolin 1 is downregulated by estrogen in uterine artery highlights a novel additional mechanism for estrogen-induced uterine vasodilatation. This conclusion was also supported by the phenotypes of caveolin 1-null mice, i.e., increased NOS3 activity and NO production as well as increased vascular permeability [31]
We have successfully developed a proteomic approach using SELDI-TOF MS technology to profile the protein expression patterns in uterine artery endothelium of OVX ewes receiving hormone replacement therapy. With this method, we are able to conduct proteomic analysis of protein expression profiling by using as little as 2 µg protein/assay. This method has made the proteomic profiling of endothelial protein expression in vivo feasible because only very limited endothelial protein samples can be obtained from any blood vessels in situ. For example, even using large animals like sheep, we normally are able to isolate approximately <50 µg "scraping" protein samples from both uterine arteries of an OVX ewe. Thus, this approach would be useful for in vivo protein expression profiling in other systemic arterial endothelial or any tissues with sampling limited input materials. In the current study, we also used other protein chips, i.e., weak cation exchanger WCX2 and strong anion exchanger SAX2, to profile the protein expression patterns in all the samples (data not shown). Similarly to the IMAC3 chip, with the use of these chips many protein peaks were detected, but their identifications awaits further investigation. However, once combined with other protein analysis tools, such as antibody arrays and on-chip peptide mapping, the SELDI-TOF MS will be a powerful tool for data mining in protein expression studies.
The vasodilatory effects of estrogen have been of intense interest for many decades, because it is reasoned from numerous epidemiological studies that estrogen apparently contributes to the major cause of the low incidence of the cardiovascular diseases in premenopausal women compared to age-matched men or postmenopausal women [32]. Although recent clinical data from the Heart and Estrogen/Progestin Replacement Study [33] and the Women's Health Initiative Investigations [34] have led to reevaluation of the cardiovascular protective role of estrogen deduced from the epidemiological studies, it is becoming clear that the beneficial effects of estrogen on the cardiovascular systems, if any, are mediated, to a great extent, by endothelial NO synthesized via NOS3 [35]. Extensive studies in the last few years have unraveled vascular endothelial cells as direct targets for estrogen actions, because specific estrogen receptors, i.e., ESR1 (also referred to ER
) and ESR2 (also referred to ERß), are expressed in various endothelial cells including uterine artery endothelial cells [27, 36]. More recently, we have demonstrated that estrogen-induced uterine vasodilatation is mediated by a ER-dependent mechanism [37]. It is also clear now that estrogen regulation of NOS3-NO production takes place both rapidly by increasing NOS3 activity through nongenomic pathways via interacting with plasma membrane ER and chronically by upregulating NOS3 protein expression through classical genomic pathways via binding to nuclear ER [15]. We have recently reported that in uterine artery endothelial cells estrogen rapidly stimulates NO production via rapid NOS3 activation through membrane ER-mediated mitogen-activated protein kinase pathway [14]. The nongenomic pathways are initiated within seconds and minutes upon estrogen stimulation and may be a mechanism by which estrogen initiates rapid NO-mediated vasodilatation [14, 15]. Additionally, the nuclear actions of estrogen in the endothelial cells are exemplified by the fact that in association with chronic vasodilatation, endothelial NOS3 expression is increased in the uterine and some systemic artery endothelia [30, 36]. Obviously, nuclear pathways apparently play a critical role in the regulation of uterine artery endothelial gene expression, as shown by the current study and others that indicate that chronic estrogen treatment increases the expression of enzymes for synthesizing vasodilators such as NO and prostacyclin in the vascular bed [13, 24]. Our current study also has raised a critical question as to by what mechanism(s) estrogen regulates NOS3 and caveolin 1 expression in the nucleus in the uterine artery endothelium. Apparently, this is a very interesting and complex area that deserves further investigation because the expression of these two NO regulatory genes is controlled reciprocally in the same uterine artery endothelial cells expressing both ER and ERß [27, 36]. This is even more complex when the fact that membrane ER-mediated rapid signaling pathways may also participate in the regulation of nuclear transcription activities in estrogen-responsive tissues [38] is taken into consideration.
Contrasting to the endothelial localization of NOS3 protein expression demonstrated by previous studies [10] and confirmed by the current study is the fact that uterine artery caveolin 1 protein is expressed in both endothelial and smooth muscle cells. Upon hormone (e.g., estrogen and progesterone) replacement therapy, although estrogen, progesterone, and their combination were all capable of increasing uterine artery endothelial NOS3 expression, it is intriguing that estrogen and estrogen plus progesterone, but not progesterone alone, could downregulate uterine artery endothelial and smooth muscle cell caveolin 1 protein expression. We were unable to detect caveolin 2 and caveolin 3 in uterine artery ex vivo by immunohistochemistry. The data agree with our previous report that these caveolins are undetectable in cultured uterine artery endothelial cells [26]. Upregulation of uterine artery endothelial NOS3 by estrogen and its combination with progesterone is not a surprise because these have been reported previously [13], and these treatment regimes are accompanied by the well-established chronic uterine vasodilatation [2, 39]. The scenario of the effects of progesterone on uterine vasodilatation is much less distinct. It has been previously reported that progesterone treatment alone does not stimulate uterine blood flow [40] and that during prolonged estrogen infusion, progesterone treatment does not have any major effects on estrogen-induced uterine vasodilatation [41], even though both treatments are capable of stimulating uterine artery endothelial NOS3 expression [13, present study]. Thus, our finding that progesterone does not inhibit uterine artery caveolin 1 expression seems to have provided a reasonable, direct explanation as to why progesterone does not increase uterine blood flow in various animal models. Additionally, concomitant progesterone treatment partially attenuates the estrogen-induced acute (120-min) increase in uterine blood flow in OVX sheep [40, 42, 43]. These previous data may have implied that progesterone might be able to inhibit the observed stimulatory effects of estrogen on NOS3 dissociation from caveolin 1 [20], thereby antagonizing the stimulatory effects of estrogen on NOS3 activation, although this theory needs to be tested. Regardless, our current study has demonstrated that both upregulation of NOS3 and downregulation of caveolin 1 are required for estrogen-induced uterine vasodilatation.
FOOTNOTES
1 Supported in part by National Institutes of Health (NIH) grants RO1 HL70562 and HL74947 to D.B.C. and HD33325 and HL49210 to R.R.M. and HL64703 to J.Z. A Postdoctoral Fellowship from the Lalor Foundation partially supports S.M.L., and E.M-G. is partially supported by a Minority Supplemental Postdoctoral Fellowship from the National Heart, Lung and Blood Institute. 
2 Correspondence: Dong-bao Chen, Division of Maternal-Fetal Medicine (MC0802), Department of Reproductive Medicine, University of California San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0802. FAX: 619 543 2919; dochen{at}ucsd.edu
Received: 16 December 2005.
First decision: 9 January 2006.
Accepted: 23 January 2006.
REFERENCES
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  K. Chang and Lubo Zhang Review Article: Steroid Hormones and Uterine Vascular Adaptation to Pregnancy Reproductive Sciences, April 1, 2008; 15(4): 336 - 348. [Abstract] [PDF]
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