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Biology of Reproduction 61, 1393-1401 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Specialized Patterns of Vascular Differentiation Antigens in the Pregnant Mouse Uterus and the Placenta1

Andrea Kruse2,a,b, Rupert Hallmannc, and Eugene C. Butchera,b

a Laboratory of Immunology and Vascular Biology, Department of Pathology and the Digestive Disease Center, Department of Medicine, Stanford University Medical School, Stanford, California, 94305 b Center for Molecular Biology and Medicine, Foothill Research Center, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304 c Institut für Experimentelle Medizin, Universität Erlangen-Nürnberg, 91054 Erlangen, Germany


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The success of pregnancy depends on the ability of trophoblast cells to infiltrate the maternal decidua and breach uterine vessels. To ask whether the antigenic phenotype of maternal endothelial cells (EC) in the vascular zone and central decidua basalis may reflect a specialized programming of these vessels for interaction with the trophoblast, we did a survey of several mouse EC differentiation antigens, including MECA-32, MECA-99, and endoglin. Our results revealed striking differences in the phenotype of endothelial lining of vessels in the distinct compartments of the pregnant uterus during Day 9 of pregnancy and at midgestation. Vessels in the central decidua basalis and the vascular zone showed strong expression of MECA-99 but only weak expression of MECA-32, contrasting with the MECA-99lo, MECA-32hi vessels in the capsularis. The vascular zone in addition stained brightly with anti-endoglin. Importantly, invading trophoblast as well as trophoblast cells lining maternal blood spaces were MECA-99+, MECA-32-, and endoglin-, suggesting that the expression of MECA-99 may reflect a specialized co-programming of these trophoblast and EC for future interaction, but also that trophoblast cells may mimic selected antigenic characteristics of endothelium in association with their role in lining maternal blood spaces. In the term pregnant uterus the expression of all differentiation antigens decreased dramatically, suggesting that trophoblast cells as well as maternal EC lose their selected antigenic characteristics when the process of placentation is complete.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The development of the mammalian embryo depends critically upon the formation of a functional placenta. During the process of hemochorial placenta formation (as in rodents and primates), the fetally derived trophoblast is invading the decidua and breaching uterine vessels; as a result, maternal blood is in direct contact with the trophoblast [1]. Failure of trophoblast invasion of the uterine vessels is associated with intrauterine growth retardation and fetal morbidity and mortality [2]. The antigenic phenotype of endothelial cells (EC) lining vessels of the pregnant mouse uterus has received little attention in the past but may represent a critical factor regarding trophoblast-EC recognition and interaction.

Our laboratory has previously produced and utilized a panel of antibodies against mouse EC differentiation antigens, including MECA-32, MECA-99, and anti-endoglin (MJ7/18). The MECA-32 antigen is expressed on most EC in the embryonic and the adult mouse, except in the brain and in skeletal and cardiac muscle, where it has a more restricted distribution. In skeletal and cardiac muscle, the MECA-32 antigen is confined to arterioles and venules; in the brain, MECA-32 antigen expression occurs only in the circumventricular organs and the neurohemal tissue [3, 4]. MECA-99 is a previously unpublished EC-specific rat monoclonal antibody (mAb), which in most settings selectively stains capillary EC in adult mice. Endoglin is a homodimeric membrane glycoprotein that can bind several isoforms of transforming growth factor-ß (TGF-ß) and is characterized by its distribution on human vascular EC in situ and by its expression on human syncytiotrophoblast and the multinucleated placental layer [5, 6]. Murine endoglin is a pan-endothelial antigen that is variably expressed on EC in murine tissues [7]. The presence of endoglin in endothelium of the mouse, human, and other species suggests that it is an important regulator of the effects of TGF-ß on angiogenesis [8].

E-selectin, a member of the smallest and most recently identified CAM gene family, is expressed by endothelium and can be induced in vitro and in vivo by proinflammatory cytokines and endotoxin [9, 10]. It binds neutrophils, monocytes, and lymphocytes and plays an important role in leukocyte contact and rolling in certain inflammatory models [11, 12]. In addition to its function in leukocyte adhesion, E-selectin may also be involved in angiogenesis [13, 14].

Vascular cell adhesion molecule-1 (VCAM-1) is a transmembrane glycoprotein member of the immunoglobulin gene superfamily [15]. It is induced on endothelium in nonmucosal sites of inflammation, where it supports adhesion of lymphocytes and monocytes through both {alpha}4ß1 and activated {alpha}4ß7 integrins [16, 17], although {alpha}4ß1 appears to dominate VCAM-1 binding in most situations involving normal leukocytes [18]. In addition, VCAM-1 has also been implicated in events during development. Through its interactions with {alpha}4 integrin, it has been shown to participate in secondary myogenesis [19] and in chorioallantoic fusion and placentation [20].

All the evidence taken together suggests that during the process of hemochorial placenta development, trophoblast invade the maternal decidua basalis and its vascular network and promote the formation of sinusoidal spaces in which blood can have direct contact with trophoblast cells [21, 22]. The mechanisms involved, however, are not known. In this study we investigated at several stages of pregnancy the expression of EC differentiation antigens and E-selectin, which may play a role in trophoblast-endothelial recognition. Our results revealed striking differences in the phenotype of endothelial lining of vessels in distinct compartments of the pregnant uterus, including distinct expression of MECA-32 and MECA-99, and endoglin as well as E-selectin. Moreover, the distinct antigenic phenotype of EC in the decidua basalis may reflect specialized programming of these vessels for future interaction with the trophoblast.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice

Female and male BALB/c mice were bred at the VA Medical Center Research Animal Facilities, Palo Alto, CA. Male C57BL/6J mice were obtained from the Charles River Breeding Laboratories, Wilmington, MA. BALB/c females were paired to either C57BL/6J or BALB/c males for one night. The first day of gestation was determined by the detection of a vaginal plug on the morning following nocturnal mating. Pregnant uteri were collected on Day 9, Day 12, and Day 20 of pregnancy. Six animals were evaluated for each of the 3 days of pregnancy.

All procedures involving animals were approved by the Stanford University Institutional Animal Care and Use Committee and were conducted in accordance with the Guide for Care and Use of Agricultural Animals in Teaching and Research.

Antibodies

The primary rat anti-mouse mAbs used were anti-mouse EC antigen mAb MECA-32 [3], anti-endoglin mAb MJ7/18 [7], and anti-MECA-99 mAb. MECA-99 is a rat IgG2c mAb made by immunizing rats with mouse lymph node stroma and screening by immunohistology for EC-specific staining. It recognizes a 140-kDa antigen on a major subset of capillary EC in all tissues, few arterioles, and syncytiotrophoblast. It also stains cultured mouse endothelium (bEND3 cells) (unpublished results). Further rat anti-mouse mAbs used were anti-cytokeratin mAb K8 (TROMA I; from the National Institute of Child Health and Human Development, Bethesda, MD; a gift of M.B. Omary); anti-E-selectin mAb 10E.9 (a gift of D. Vestweber, Max-Planck-Institute, Freiburg, Germany, and of PharMingen, San Diego, CA) [23]; and anti-VCAM-1 mAb MK2.7 [24], anti-ß7 mAbs FIB21, FIB30, FIB504 [25], and anti-{alpha}4 mAb PS/2 [26]. Anti-human CD44 mAb Hermes-1 [27] was used as a rat IgG2a isotype-control mAb. The mAbs were purified and concentrated from serum-free culture supernatant by ammonium sulfate precipitation. Rat anti-mouse cytokeratin mAb was conjugated with fluorescein isothiocyanate (FITC; Sigma Chemical Co., St. Louis, MO) according to the method of Goding [28]. R-Phycoerythrin (PE)-conjugated affinity-purified mouse anti-rat IgG Fab2 polyclonal (Chromaprobe, Mountain View, CA) was used as second stage.

All antibodies were titered and used at saturation for staining.

Tissue Preparation and Immunofluorescence Histology

Pregnant uteri were collected and rapidly frozen in Tissue-Tek OCT (Miles, Naperville, IL). Cryostat sections (6 µm) were air dried overnight, acetone fixed, and stained using a two-step immunofluorescence technique. To assist with orientation and selection, every 10th tissue section was stained with hematoxylin and eosin. To facilitate confident identification of maternal blood spaces versus fetal vessels, in most experiments mice were injected with luconyl blue [29] i.v. 5 min before they were killed. Luconyl blue is retained in the lumen of maternal blood spaces and is readily visualized in frozen and routine histologic sections as bright blue particles.

For immunofluorescence staining, tissue sections were incubated with 10% normal mouse serum in PBS, pH 7.1, for 15 min and then exposed for 30 min to individual primary mAbs diluted in PBS. Samples were washed with several changes of PBS for 10 min, incubated for 30 min with PE-conjugated secondary mouse anti-rat IgG (50 µg/ml), and rinsed in PBS. For two-color staining, tissue sections were then incubated with 10% normal rat serum in PBS, pH 7.1, for 15 min and exposed for 30 min to FITC-conjugated rat anti-mouse cytokeratin mAbs. Either rat anti-human CD44 mAb Hermes-1 or mouse IgG was substituted for primary antibodies as controls for nonspecific staining. The staining patterns of antibodies were tested on at least 10 different tissue samples. Sections were analyzed on the day of staining. Samples were examined with a Nikon (Garden City, NY) phase/epifluorescence microscope UFX-II equipped with the appropriate filters. A permanent record of immunofluorescence staining was obtained by a sensitive silicon-intensified target video camera allowing rapid capture of immunofluorescence images. In some instances, the same field was captured under transillumination to allow visualization of luconyl blue as marker of maternal vessels or maternal blood spaces. The images were then processed and overlaid (Photoshop; Adobe, Mountain View, CA) as appropriate for presentation in the figures.

To confirm luminal expression of E-selectin in some experiments, we examined staining of trophoblast cells as well as decidual EC by circulating mAbs. Briefly, 0.5 mg of rat mAb against E-selectin was injected i.v. and allowed to circulate for 1–2 h; pregnant animals were then killed and perfused to remove free antibody. The perfusion was performed as described by Rannie and Donald [30]. Frozen sections were then stained with PE-conjugated mouse anti-rat IgG to determine whether mAb had reacted with the luminal face of vascular endothelium or trophoblast cells. This approach has been used previously to distinguish luminal from adluminal and cytoplasmic reactivity of high endothelial venules for vascular addressins [31].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Differences in the Phenotype of the Vascular Endothelium Associated with Distinct Compartments of the Pregnant Uterus at Day 9 of Gestation

The Day 9 pregnant mouse uterus comprises three histologically defined zones: the central decidua basalis, the vascular zone (a region of sinusoidal vessels within the decidua basalis), and the decidua capsularis. Schematic diagrams of the pregnant uterus at various stages of gestation are shown in Figure 1. The invasive epithelial trophoblast, which intercalates in a complex fashion with other cell types, can be marked effectively and identified with anti-mouse cytokeratin mAb (Fig. 2, d and f). To ask whether these distinctive uterine zones could be correlated with features of vascular specialization, we did a survey of the EC differentiation antigens MECA-32 [4], MECA-99, and anti-endoglin (MJ7/18) [7].



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  		FIG. 1. Schematic structure of the pregnant uterus or the placenta from gestational Days 9 (a), 12 (b), and 20 (c). The regions illustrated in Figures 2–4 are indicated for reference. MV, Maternal vessel; LAB, labyrinthine zone; SP, spongy zone



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  		FIG. 2. Immunohistologic staining for mouse EC differentiation antigens revealed specialization of vascular phenotypes in different decidual zones. a and b) At Day 9 of pregnancy, EC lining most vessels in the central decidua basalis (DB) were MECA-99hi (a) and MECA-32lo (b). c–f) Sections including decidua capsularis (DC), vascular zone (VZ), and trophoblast (T) were double stained for MECA-32 (c) and cytokeratin (d), or endoglin (MJ7/18) (e) and cytokeratin (f). Vascular zone EC were MECA-32lo (c) and endoglinhi (e). In contrast, most vessels in the decidua capsularis were MECA-32hi and endoglinlo (upper left corner, c and e). Invasive cytokeratin-positive epithelial trophoblast (d, f) failed to stain for MECA-32 (c) or anti-endoglin mAbs (e). In the term pregnant uterus (Day 20), the vascular lining EC of the decidua basalis (DB) (g), as well as the invading trophoblast cells (g, h), had a MECA-99lo phenotype. a–h) x100 (published at 67%).

Our immunohistological results revealed striking differences in the phenotype of the endothelial lining of vessels, which underscore the formation of the distinct uterine zones at this period. The EC lining of most vessels in the central decidua basalis was MECA-99hi (Fig. 2a), MECA-32lo (Fig. 2b), and endoglinlo (not shown), contrasting with the MECA-99lo, MECA-32hi, and endoglinlo vessels in the capsularis (Fig. 2, c and e; and Fig. 3a). The vascular zone EC had the MECA-99hi (Fig. 3a), MECA-32lo (Fig. 2c) phenotype of basalis vessels but in addition stained brightly with anti-endoglin mAb (Fig. 2e). The trophoblast (Fig. 2, d and f) also expressed the EC differentiation antigen MECA-99 (Fig. 3a) but failed to stain for MECA-32 (Fig. 2c) or endoglin (Fig. 2e).



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  		FIG. 3. a) Immunohistology of the maternal-trophoblast interface at different stages of pregnancy. a, b) Day 9 pregnant uterus. a) The invasive epithelial trophoblast (T) was identified with FITC-conjugated mAb to cytokeratin (green). Double staining with anti-MECA-99 mAb (red) revealed that MECA-99 was expressed on vascular zone (VZ) EC, as well as in the invading trophoblast (double staining indicated by yellow). In contrast, most vessels in the decidua capsularis (DC) were MECA-99lo (upper left corner). Luconyl blue (L) in maternal vessels confirmed that MECA-99+ EC faced the maternal bloodstream. b) Staining with anti-E-selectin mAb (E; red) revealed an almost linear array of maternal blood spaces in the outer spongy zone next to the decidua basalis. Double staining with FITC-conjugated anti-cytokeratin mAb (ck; green) confirmed that these blood spaces were lined by trophoblast, and suggested that these trophoblastic cells were themselves E-selectin positive (double staining indicated by yellow). c, d) At midgestation (Day 12), double staining with anti-cytokeratin (ck; green) vs. anti-MECA-99 mAbs (red) revealed MECA-99 expression on trophoblast of the outer spongy zone (SP) lining maternal blood spaces (c; double staining indicated by yellow). MECA-99 was further expressed on trophoblast cells lining the blood sinuses in the deeper spongy zone (SP) (c; yellow). The large trophoblast cells of the spongy zone outside the blood sinuses were MECA-99- (c). Double staining of cytokeratin (ck; green) vs. E-selectin (E; red) revealed that E-selectin expression, which was only weak at Day 9 of pregnancy, increased dramatically but remained associated with the trophoblast-lined maternal blood spaces of the outer spongy zone (d; double staining indicated by yellow). e and f) Double staining of cytokeratin (ck; green) vs. MECA-99 (red) in the term placenta (Day 20) revealed a decreased MECA-99 expression on trophoblast cells lining maternal blood sinuses in the spongy zone (SP) (e; double staining indicated by yellow). E-selectin (E; red) was near background levels on trophoblast cells (ck; green) of the outer spongy zone (as indicated by yellow; f). a, c, e, f) x100; b and d) x200. M99: MECA-99.

Staining with anti-E-selectin mAb revealed an almost linear array of E-selectin-positive maternal blood spaces in the outer spongiotrophoblast next to the decidua basalis (Fig. 3b). Double staining with FITC-conjugated anti-cytokeratin mAb revealed that maternal EC as well as trophoblast cells lining these blood spaces were E-selectin positive. Negative control staining with class-matched rat IgG confirmed that the E-selectin reactivity was specific. To evaluate the localized expression of E-selectin further, we injected mice with 0.5 mg anti-E-selectin mAb i.v. Sections of the perfused uterus were stained with PE-anti-rat IgG. Double staining with FITC-conjugated anti-cytokeratin mAb confirmed that the trophoblastic cells lining the maternal blood spaces were themselves E-selectin positive (Fig. 3b).

Distribution of Vascular Differentiation Antigens in the Decidua Basalis and Placenta at Midgestation

At Day 12 the basic decidual zones defined at Day 9 remained almost intact with their local patterns of vascular differentiation antigens. Double staining of cytokeratin versus E-selectin revealed that E-selectin expression, which was only weak at Day 9 of pregnancy, had increased dramatically over the last few days (Fig. 3d) but remained restricted to the trophoblast-lined maternal blood spaces of the outer spongiotrophoblast next to the decidua basalis, which also expressed the MECA-99 antigen (Fig. 3, c and d), but not MECA-32 or endoglin (not shown).

The murine placenta can be subdivided at midgestation into several zones: the innermost labyrinthine zone containing the most extensive area of blood sinuses, the central spongiotrophoblast or spongy zone of large trophoblast cells with less numerous and smaller blood sinuses, and the giant cell layer of very large trophoblasts with occasional sinuses that remains embedded in the decidual tissue [32–34]. Double staining with FITC-conjugated anti-cytokeratin versus PE-conjugated anti-MECA-99 mAbs revealed MECA-99 expression on trophoblast of the outer spongy zone lining maternal blood spaces (Fig. 3c), which also expressed E-selectin (as mentioned above; Fig. 3d). MECA-99 was further expressed on trophoblast cells lining the blood sinuses in the deeper spongy zone (Fig. 3c). Interestingly, the large trophoblast cells of the spongy zone outside the blood sinuses were MECA-99- (Fig. 3c). The trophoblast cells again failed to stain with anti-MECA-32 or anti-endoglin mAbs (not shown).

In the labyrinthine zone, MECA-99 was expressed on MECA-32-, endoglin- trophoblast cells lining the maternal blood sinuses (Fig. 4, a and b; and not shown). These maternal blood sinuses are interlaced with but not connected to fetal vessels, which were MECA-99+ (Fig. 4a), MECA-32+ (not shown), and endoglin+ (Fig. 4b). To facilitate confident identification of maternal blood sinuses versus fetal vessels, mice were injected with luconyl blue. Fetal and maternal circulation never mix; thus luconyl blue was retained in the lumen of maternal blood spaces. Sensitive immunofluorescence microscopy revealed, surprisingly, that some fetal vessels expressed {alpha}4 integrin (Fig. 4c), but not ß7 integrin (not shown), and also VCAM-1 (Fig. 4d).



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  		FIG. 4. Immunohistologic staining of fetal vessels and maternal blood sinuses in the labyrinthine zone at midgestation (Day 12). Double staining of trophoblast-lined maternal blood sinuses of the labyrinthine zone with anti-cytokeratin mAb (green) vs. anti-MECA 99 (red; a) or anti-endoglin (red; b) mAbs revealed that these trophoblast cells expressed MECA-99 (a; double staining indicated by yellow), but not endoglin (b). The EC of fetal blood vessels were MECA-99+ (red; a) and endoglin+ (red; b). c, d) Fetal vessels expressed significant levels of {alpha}4 (red; c) (but not ß7, not shown) and VCAM-1 (red; d). x200 (published at 96%). L, Luconyl blue

Altered Vascular Differentiation Antigens and Decreased E-Selectin Expression in the Term Pregnant Uterus

In the term pregnant uterus, the decidua capsularis is no longer identified. At this stage of pregnancy, the trophoblast cells line most maternal blood spaces. Cytokeratin reactivity was used as a positive marker. Scanning of luconyl blue-defined maternal vessels revealed very few MECA-32+ EC-lined segments. MECA-32 expression at this stage was less intense than in the Day 9 or Day 12 pregnant uterus (not shown). Endoglin expression was restricted to dilated maternal blood vessels (not shown). Surprisingly, EC lining maternal vessels in the decidua basalis mainly had a MECA-99lo phenotype; only scattered single EC lining maternal blood vessels still stained brightly with anti-MECA-99 (Fig. 2g). Double staining of cytokeratin and MECA-99 in the term placenta revealed that invading trophoblast cells (Fig. 2, g and h) were MECA-32-, endoglin-, and MECA-99lo. In the spongy zone, expression of MECA-99 on trophoblast lining maternal blood spaces (Fig. 3e) was quantitatively less intense than on Day 12, although the patterns and distribution were the same. E-selectin decreased dramatically on trophoblast cells lining maternal blood spaces of the outer spongy zone (Fig. 3f). In the labyrinthine zone, MECA-32, endoglin, {alpha}4, and VCAM-1 (not shown) expression on fetal vessels was quantitatively less intense than on Day 12. Staining patterns of vascular differentiation antigens and E-selectin at the maternal/fetal interface from different regions at different days of gestation are summarized in Table 1.


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  		TABLE 1. Staining patterns of vascular differentiation antigens and E-selectin at the maternal/fetal interface at different days of gestation.a


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The establishment of a functional maternal-fetal interface is essential for the subsequent development of the fetus [21, 35]. Many species, including primates and rodents, form a hemochorial placenta in which the fetal trophoblast is directly exposed to the maternal blood. To reach the maternal circulation, the trophoblast must invade the maternal decidua and its vascular network and promote the formation of sinusoidal spaces in which blood can have direct contact with the trophoblast cells engaged in nutrient and gas exchange [21, 22]. The mechanisms of these complex activities, which are sensitive to disruption as shown by the high incidence of intrauterine growth retardation and fetal mortality [2], remain to be determined.

To analyze whether the EC in the decidua basalis have a unique phenotype that might reflect specialized programming of these EC for interaction with the trophoblast, we did a survey of several EC differentiation antigens, including MECA-32, MECA-99, and endoglin, at various stages of pregnancy. We also investigated the expression of E-selectin, which is exclusively expressed by endothelium [9, 10].

At Day 9 of pregnancy the uterus comprises three histologically defined zones: the central decidua basalis, the vascular zone (a region of sinusoidal vessels within the decidua basalis), and the decidua capsularis. Our data showed striking differences in the phenotype of endothelial lining of vessels in the distinct compartments of the pregnant uterus. Vessels in the central decidua basalis, as well as in the vascular zone, showed strong expression of MECA-99, but only weak expression of MECA-32. This contrasts with observations in the vessels in the capsularis, which were MECA-32hi and MECA-99lo. The EC lining the vessels in the vascular zone in addition stained brightly with anti-endoglin. The epithelial trophoblast, invading the vascular zone and central decidua basalis during the process of placentation but not the decidua capsularis, is subdivided at this stage in the spongiotrophoblast layer and trophoblast giant cells lying on the outside of the placenta and forming the interface with maternal cells in the decidua. At this stage of pregnancy, the formation of the labyrinthine zone begins by the establishment of contact between the chorionic trophoblast and the allantoic mesoderm [1, 34, 36]. Importantly, our data revealed that the trophoblast also expressed the EC differentiation antigen MECA-99 but failed to stain with anti-MECA-32 or anti-endoglin mAbs; this suggested that the expression of MECA-99 by trophoblast cells, as well as EC in the vascular zone and central decidua basalis, may reflect a specialized co-programming of these trophoblast and EC for future interaction. Interestingly, Zhou et al. [37] reported that human cytotrophoblasts that colonize spiral arterioles transform their adhesion receptor phenotype so as to resemble EC, suggesting that this adhesion phenotype switch is required for successful endovascular invasion and normal placentation. It should be mentioned, however, that the area of trophoblast interaction with the uterine wall and vasculature is distinct among primates and rodents. A relatively sharp demarcation is maintained between trophoblast and decidua in the mouse, whereas intermingling of trophoblast and maternal cells in the decidua and myometrial compartments in the human is extensive [22]. In addition, human cytotrophoblasts are much more invasive, since they migrate to maternal uterine vessels where they replace EC [1, 22]. There is little evidence that EC replacement occurs in the mouse, although we observed trophoblast cell migration into the uterus beyond the giant cell layer as well as into the mesometrial arteries from Day 12 of pregnancy.

As recently shown, endoglin, which is expressed on vessels in the vascular zone, interacts specifically with the ß1 and ß3 isoforms of TGF-ß; and it has been suggested that the presence of endoglin in endothelium of the mouse and other species is an important regulatory and/or mediator of the effects of TGF-ß on angiogenesis [8, 38]. The female reproductive organs (ovary, uterus, placenta) exhibit periodic growth and regression, which are extremely rapid and are accompanied by equally rapid changes in rates of blood flow [39–41]. Therefore, it is not surprising that female reproductive tissues are some of the few tissues in which angiogenesis occurs as a normal process [41, 42]. In the pregnant uterus we observed a high expression of endoglin on EC of maternal blood vessels in the vascular zone. We therefore speculate that the endoglin expression is important for this angiogenic process.

During the process of hemochorial placenta development, dilated maternal vessels become eroded by the trophoblast; as a result, trophoblast cells are in direct contact with the maternal blood [43]. Particularly surprising was the apparent display of E-selectin not only by EC lining maternal vessels in this site but also by trophoblast cells. Staining with anti-E-selectin revealed an almost linear array of E-selectin+ maternal blood spaces in the outer spongy zone in direct contact with the decidua basalis. Double staining with FITC-conjugated anti-cytokeratin mAb revealed that the maternal EC, as well as the trophoblastic cells lining the blood spaces, were themselves E-selectin+. Staining of serial sections for E-selectin and endoglin confirmed that most E-selectin+ lining cells lack the EC antigen endoglin, again consistent with invading trophoblast. Our immunohistologic studies, however, do not determine whether the E-selectin on maternal blood spaces lining trophoblast is expressed by the trophoblast cells or is bound by the trophoblast from circulating E-selectin following production elsewhere. E-selectin plays an important role in leukocyte contact and rolling in certain inflammatory models [11, 12]. In the pregnant uterus, E-selectin activity was often associated with vessels containing luminally bound neutrophils, and it is likely to be important in neutrophil recruitment to areas of necrosis in the ectoplacental zone [44]. Furthermore, expression of E-selectin on maternal EC, as well as on trophoblast in the outer spongy zone, may also be involved in trophoblast-EC recognition and interaction. Interestingly, several studies demonstrated the expression of E-selectin in proliferating EC of hemangiomas, neonatal foreskin, and human placenta [14, 45]. Weak E-selectin expression has also been reported in the human decidua during early pregnancy [45]. Vascular EC of reproductive tissues exhibit a mitotic rate equal to or greater than that observed for tumor EC, and it has been suggested that E-selectin may also function in angiogenesis [14]. This is underscored by the results of Koch et al. [13] suggesting that soluble E-selectin may exert a novel function as mediator of angiogenesis.

At midgestation, the basic decidual zones defined at Day 9 remain almost intact with their local patterns of vascular differentiation antigens. E-selectin, however, which was only poorly expressed at Day 9 of pregnancy, increased dramatically at midgestation, but remained restricted to the trophoblast-lined maternal blood spaces of the outer spongy zone, which also expressed the MECA-99 antigen.

In the placenta from Day 10 on, fetal vessels intermingle closely with maternal blood sinuses, creating the loose network of vascular cells that comprises the mature labyrinth layer [34]. The murine placenta can now be subdivided into several zones: the innermost labyrinthine zone containing the most extensive area of blood sinuses, the central spongy zone of large trophoblast cells with less numerous and smaller blood sinuses, and the giant cell layer of very large trophoblasts with occasional sinuses that remains embedded in the decidual tissue [32–34]. The blood sinuses of the labyrinthine zone and the spongy zone, containing maternal blood, were lined by MECA-99+, MECA-32- and endoglin- trophoblast cells. Interestingly, the large trophoblast cells of the spongy zone outside the blood sinuses were MECA-99-. These observations suggest that trophoblast cells mimic selected antigenic as well as functional characteristics of endothelium in association with their role as lining in maternal blood spaces.

In the labyrinthine zone, maternal blood sinuses are interlaced with but not connected to fetal vessels, which are MECA-99+, MECA-32+, and endoglin+. Surprisingly, fetal vessels expressed significant levels of {alpha}4 (but not ß7 and hence presumably {alpha}4ß1) and also VCAM-1. Expression was not associated with dramatic leukocyte traffic (not shown). The significance of expression of this receptor ligand pair by fetal EC is uncertain. However, coordinate {alpha}4 and VCAM-1 expression has been reported previously in inflamed brain [46, 47]. In addition, VCAM-1 has been implicated in events during development. Through its interaction with {alpha}4 integrins, it has been shown to participate in secondary myogenesis [19] and in chorioallantoic fusion and placentation [20]. One possibility is that on EC, this receptor ligand pair is used to enhance EC-EC adhesion.

In the term placenta, the trophoblast lines most maternal blood spaces as indicated by staining with anti-cytokeratin mAbs. Invading trophoblast cells, as well as trophoblast cells lining maternal blood sinuses in the spongy zone, showed a decreased expression of MECA-99. In addition, the EC lining maternal vessels in the decidua basalis also had a MECA-99lo phenotype; only single EC stained brightly with anti-MECA-99. MECA-32 expression on these vessels was weak. Endoglin expression was restricted to dilated maternal blood vessels. Importantly, also E-selectin expression decreased dramatically on trophoblast cells lining maternal blood spaces of the outer spongy zone next to the decidua basalis, suggesting that trophoblast cells and maternal EC lose their selective antigenic characteristics when the process of placentation and angiogenesis is complete and as the placenta dies. However, the concept that the placenta may have a finite life span and that it ages is controversial [1]. Nevertheless, it has been shown that as pregnancy proceeds, human cytotrophoblast stem cells rapidly lose the ability to differentiate and to undergo integrin switching [1, 48–50]. Whether the changes in MECA-99 as well as E-selectin expression are indicative of trophoblast aging remains to be determined.

Although not illustrated, most of the findings discussed here in detail are not restricted to BALB/c x C57BL/6J F1 pregnancies but were also replicated faithfully in Day 9 BALB/c syngeneic pregnancies. A minor difference included quantitatively less intense staining for E-selectin (although the patterns and distribution were the same).

In conclusion, our results revealed striking differences in the phenotype of endothelial lining of vessels in distinct compartments of the pregnant uterus between Day 9 of pregnancy and midgestation, including distinct expression of MECA-32, MECA-99, endoglin, and E-selectin. The expression of MECA-99 by trophoblast, as well as by EC in the vascular zone and decidua basalis, may reflect a specialized co-programming of these trophoblast and EC for future interaction. In the term pregnant uterus, the expression of all vascular differentiation antigens as well as E-selectin decreased dramatically, suggesting that trophoblast cells as well as maternal EC lose their selective antigenic characteristics when the process of placentation is complete.


   ACKNOWLEDGMENTS
 
We would like to thank Evelyn Resurreccion and June Twelves for excellent technical assistance and M. Bishr Omary, Dietmar Vestweber, and PharMingen, San Diego, CA, for antibodies.


   FOOTNOTES
 
1 This work was supported in part by NIH grants GM37734, AI37832, and by the FACS Core and by the Molecular Biology and Cell Imaging Core Facilities of the Stanford Digestive Disease Center under DK38707, and by an Award from the Department of Veterans Affairs. A.K. was a recipient of a fellowship from the Deutsche Forschungsgemeinschaft. Back

2 Correspondence and current address: Andrea Kruse, Institute of Immunology, University of Lübeck, School of Medicine, Ratzeburger Allee 160, 23538 Lübeck, Germany. FAX: 49 451 5003069; kruse{at}immu.mu-luebeck.de Back

Accepted: July 7, 1999.

Received: April 19, 1999.


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 DISCUSSION
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