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Department of Gynecology and Obstetrics (A.G., A.E.H., L.C.G., N.R.N.), Stanford University, Stanford, California 94305; California National Primate Research Center (L.S.L., B.L.L.), University of California-Davis, Davis, California 95616; and Oregon National Primate Research Center (R.M.B.), Beaverton, Oregon 97006
Address all correspondence and requests for reprints to: Nihar R. Nayak, Ph.D., Department of Gynecology and Obstetrics, Stanford University School of Medicine, 300 Pasteur Drive, HH-333, Stanford, California 94305-5317. E-mail: nayakn{at}stanford.edu.
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Introduction |
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Different growth factors belonging to the vascular endothelial growth factor (VEGF) family, including VEGF, placental growth factor, VEGF-B, VEGF-C, and VEGF-D, act as modulators and inducers of angiogenesis, vasculogenesis, and vascular permeability in different organs, including the female reproductive tract (5, 6). Human VEGF encompasses five different isoforms of 121, 145, 165, 189, and 206 amino acids resulting from alternative splicing from a single gene containing eight exons (6). All VEGF splice variants can bind to their receptors (VEGFRs), VEGFR-1 and VEGFR-2, which transmit VEGF actions through their intrinsic tyrosine kinase activity (7). Numerous studies show cyclic changes and hormonal regulation of VEGF in both human and nonhuman primate endometrium during the menstrual cycle and suggest a role of VEGF in endometrial angiogenesis (5). VEGF165 and VEGF121 are the most abundant isoforms expressed in the human endometrium (8, 9), and a recent study (10) shows transient up-regulation of VEGF189 during the secretory phase.
Because VEGFR-1 and VEGFR-2 are typically expressed in endothelial cells, VEGF is considered as an endothelial cell-specific growth factor. However, several reports show VEGF interactions with nonendothelial cells. We previously suggested a link between VEGF-KDR and the menstrual induction cascade based on our observation that VEGFR-2, normally expressed in the vascular endothelium throughout the menstrual cycle, was expressed in the stromal cells of the upper endometrial zones during the premenstrual phase in both human and macaque endometrium (11). VEGF is known to stimulate migration of monocytes and osteoblasts (12, 13, 14), and a recent study shows that blockade of VEGF action can inhibit estradiol (E2)-dependent luminal epithelial cell proliferation in the mouse endometrium (15). However, these cells do not express VEGFR-1 or VEGFR-2. Cell surface receptor cross-linking studies first suggested the existence of additional VEGFRs that are neither VEGFR-1 nor VEGFR-2. Soker et al. (16) identified these VEGFRs as neuropilins (NPs)-1 and -2, originally described as receptors for the semaphorin family of proteins associated with axonal guidance in the developing nervous system (17). Other members of the VEGF family are also ligands for NPs; placental growth factor binds to both NP-1 and NP-2 (18, 19), and VEGF-B binds to NP-1 (20).
NPs are transmembrane glycoproteins of approximately 140 kDa with a short cytoplasmic domain and an extracellular portion, containing three different structural domains. NP-1 is 47% homologous to NP-2 and has higher affinity for VEGF than NP-2 (16). Interestingly, these receptors act as VEGF isoform-specific receptors in both endothelial and nonendothelial cells, and NP-1 enhances the binding of VEGF165 to VEGFR-2 and facilitates VEGF165-mediated chemotaxis (16, 21). VEGF165 can bind to both NP-1 and NP-2, whereas VEGF145 binds to only NP-2 (18, 22). Gene targeting studies implicate NPs as important modulators of VEGF function in vivo. Overexpression of NP-1 results in excess capillary formation and extensive hemorrhage (23), and conditional overexpression of NP-1 in prostate carcinoma causes increased vascular proliferation and vascular density along with suppression of apoptosis (24). NP-1 or NP-2 single knockouts are embryonically lethal [embryonic d (E) 10–10.5] due to neuronal and cardiovascular defects. However, the double NP-1/NP-2 knockout mice die very early (E8.5) due to more severe abnormal vascular phenotype than the single knockouts (25).
Two recent reports demonstrate expression of NPs in both endothelial and nonendothelial cells in the mouse (26) and rat (27) endometrium and suggest a role of VEGF-NP in induction of the vascular permeability and angiogenesis in mouse uterus (26). Despite the dramatic vascular changes that occur in the primate endometrium during each menstrual cycle, the mechanisms of VEGF action in this organ remain unknown. The aim of this study was to examine the temporal and spatial expression of NP-1 and NP-2 in the rhesus macaque endometrium during hormonally controlled menstrual cycles and in the human endometrium during natural menstrual cycles. In rhesus macaques the menstrual cycle is essentially identical with the human cycle (28), and sequential hormonal treatments can be used after ovariectomy to mimic the natural endometrial cycle (2).
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Materials and Methods |
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All animal studies were conducted at the Oregon National Primate Research Center and the California National Primate Research Center, according to the National Institutes of Health guidelines for use of nonhuman primates under the approved protocol by the respective Primate Center Institutional Animal Care and Use Committee. A total of 20 adult female rhesus macaques (Macaca mulatta) were ovariectomized and artificially cycled as described previously (2, 29). All macaques received sc crystalline E2 (Sigma, St. Louis, MO) implants in the form of 3-cm SILASTIC capsules (Dow Corning Corp., Midland, MI) to induce a proliferative-phase endometrium. After 14 d a 6-cm crystalline progesterone (P) SILASTIC implant was placed sc and remained in place for 14 d to induce a secretory-phase endometrium. Menstruation followed removal of the P implant, whereas the E2 implant was kept in place. Endometrium from all animals was collected at five different time points: early proliferative phase, i.e. endometrium harvested after 5–6 d of P withdrawal (E5/6P; n = 4); midproliferative phase, 8–10 d after P withdrawal (E8/10P; n = 4); late-proliferative phase, 14 d after P withdrawal (E14P; n = 3); midsecretory phase, 7–8 d after P treatment (E+7/8P; n = 3); and late-secretory phase, 14 d after P treatment (E+14P; n = 3). In a second set of animals, both E2 and P implants were removed [hormone deprived (HD)] at the end of the cycle to induce menstruation, and the endometria were collected 10–14 d later (HD 10–14; n = 3). Endometrial tissue samples were collected by hysterectomy or during necropsy, and serum E2 and P concentrations were measured by radioactive immunoassay, as described previously (29, 30). Tissue samples within normal ranges of serum E2 and P levels for the rhesus menstrual cycle, as previously reported, were included in this study (11, 31).
Human subjects
Endometrial samples were also obtained from human subjects undergoing hysterectomy for benign indications during the mid- to late-proliferative (n = 4) and mid- to late-secretory phases (n = 5) of the cycle as described previously (32). All samples were obtained from normally cycling women in the age group between 28 and 39 yr, after informed consent under an approved protocol by the Stanford University Committee on the Use of Human Subjects in Medical Research. Full-thickness endometrial blocks (2–3 mm thickness) were embedded in optimal cutting temperature (OCT) compound and frozen in liquid nitrogen, and 10-µm cryosections were used for NP-1 and -2 in situ hybridization (ISH) as described below. The stage of the cycle was based on last menstrual period and histological evaluation by pathologists (32). Women under any treatment including hormonal preparations in the preceding 3 months were excluded from this study.
Immunocytochemistry (ICC)
A double-immunostaining method that detected both Ki-67 and von Willebrand factor (vWF) in the same sections was used to differentiate proliferating endothelial cells from other proliferating cells in the endometrium (31). Tissues embedded in OCT were cryosectioned at 7 µm, fixed in 0.2% picric acid-2% paraformaldehyde in phosphate buffer saline at pH 7.3 for 10 min at room temperature, rinsed twice for 2 min each in 85% ethanol and 1.5% polyvinylpyrollidone (PVP) at 4 C, and washed in PBS followed by 0.37% glycine in PBS and PVP and then in 0.1% gelatin in PBS and PVP at 4 C. The sections were then incubated 45 min in glucose oxidase (1 U/ml) with Na-Azide (1 mM) and glucose (10 mM) in PBS to block endogenous peroxidase activity. After washing and incubation with blocking serum, the sections were incubated with a mouse monoclonal primary antibody for Ki-67 (1:300, BioGenex Laboratories, Inc., San Ramon, CA) overnight at 4 C, followed by blocking and incubation with a biotinylated second antibody at room temperature. Nuclei of Ki-67-positive cells were stained brown using the ABC kit (Vector Laboratories, Inc., Burlingame, CA) following the manufacturer’s instructions. The sections were again incubated with the primary rabbit polyclonal antibody for vWF (1:4000, Dako Corp., Carpinteria, CA) and the secondary antirabbit antibody. The cytoplasm of endothelial cells was stained blue-gray with a peroxidase substrate kit (Vector Laboratories). The slides were lightly counterstained with hematoxylin, and the number of Ki-67-positive and -negative nuclei in each field were counted under a x40 objective of a microscope (Carl Zeiss, New York, NY) (31). At least 10 different fields in each section were evaluated, and the percentage of proliferating luminal epithelial, glandular, stromal, and endothelial cells was calculated.
RT-PCR and cloning of cDNA
NP-1 and NP-2 cDNAs specific to the rhesus macaque were generated by RT-PCR and cloning as described previously (11). Total RNA was isolated using TRIZOL (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. RNA was treated with DNase (Qiagen, Santa Clarita, CA) and purified thereafter, using RNeasy spin columns (Qiagen). Two micrograms of the sample were reverse transcribed with the Omniscript kit (Qiagen) using oligo (dT)16–18 and random hexamers (Invitrogen) in equal amounts according to the manufacturer’s instructions. The product was amplified with respective primer pairs in a standard PCR, and the amplified product was isolated in agarose gel. The NP-1 cDNA was subcloned into pGEM-T (Promega Corp., Madison, WI) and NP-2 into pGEM-T easy (Promega) vector. A clone with the insert was maxiprepped (Qiagen) and sequenced. NP-1 (forward: 3'-ACCCGCACCTCATTCCTACAT-5', reverse: 3'-TGCTGGGCTGTGAAGTGGAA-5') and NP-2 (forward: 5'-TGTACCGGCATGGCAAAAAC-3', reverse 5'-GATGATGACACCTTTCACTG-3') primers were designed based on the homologous human GenBank sequences BC007533.1 (1682–2018 bp) and AF022860 (1121–1509 bp), respectively. The partial cDNA sequence of both NP-1 and NP-2 were 97% homologous to the corresponding human sequences.
In situ hybridization
35S-UTP-labeled (Amersham Biosciences, Piscataway, NJ) sense and antisense riboprobes from NP-1 and NP-2 cDNA templates were prepared with the MAXIscript in vitro transcription kit from Ambion, Inc. (Austin, TX), purified by mini-Quick Spin RNA columns (Roche Diagnostics Corp., Indianapolis, IN) according to the manufacturer’s instructions, and ISH was performed as described previously (11, 31). Frozen sections (10 µm) of endometrium were mounted on SuperFrost Plus slides (Fisher Scientific, Fair Lawn, NJ) and fixed in 4% paraformaldehyde in PBS for 20 min at 4 C. The tissue sections were rinsed in 2x standard saline citrate (SSC), and acetylated with 0.25% acetic anhydride in 0.1 mol/liter triethanolamine (pH 8.0) for 10 min. Slides were then rinsed in 2x SSC, dehydrated through an ascending series of alcohols, and air dried. Appropriate concentration of the labeled probe was empirically determined by serial dilution, and the sections were incubated at 55 C overnight with the hybridization solutions [10 mmol/liter dithiothreitol, 0.3 mol/liter NaCl, 20 mmol/liter Tris (pH 8.0), 5 mmol/liter EDTA, 1x Denhardt’s solution, 10% dextran sulfate, 50% formamide] containing the respective sense and antisense probes. The slides were then washed in 5x SSC with 10 mM dithiothreitol at 50 C for 45–60 min, followed by 50% formamide and 2x SSC containing 10–15 mM dithiothreitol at 65 C for 20–30 min. Then the slides were incubated in solution A [0.5 M NaCl, 10 mM Tris (pH 7.5), and 5 mM EDTA (pH 8.0)] containing 20 µg/ml RNase A at 37 C for 30 min, rinsed in solution A, and incubated in 50% formamide at 65 C for 20–30 min. The sections were then washed in SSC in descending order (2x SSC, 1x, 0.5x, 0.1x), followed by dehydration in alcohol solution containing 300 mM NH4Ac, vacuum dried, coated with NTB-2 autoradiographic emulsion (Eastman Kodak Co., Rochester, NY), stored at 4 C for 2–3 wk, developed in D-19 (Eastman Kodak), lightly counterstained with hematoxylin, dehydrated in an ascending series of alcohol dilutions, cleared with xylene, and then coverslipped with Permount (Fisher Scientific).
Sections hybridized with the sense probe or treated with RNase showed no specific signals. Grain counts were performed as described previously (31). Silver grains over the stroma and luminal epithelium were expressed as the number of grains per cell, and grains over endothelial cells were expressed as the number of grains per unit area of vascular endothelium.
Statistical analysis
Data were analyzed by one-way ANOVA and Fisher’s protected least significant difference test (31). P < 0.05 was considered significant. Correlations between different attributes and coefficients of simple determination (R2) were calculated using the StatView software (SAS Institute, Cary, NC), as described previously (31).
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NP-1 mRNA cellular localization.
NP-1 mRNA was expressed by three cell types: stromal cells, vascular endothelial cells (arteries and veins), and luminal epithelial cells, but not by glandular epithelial cells (Fig. 1
). Dark-field microscopy of the in situ preparations showed clearly that in the stroma, the NP-1 mRNA signal was expressed at a moderate level and did not change under different hormonal conditions (Fig. 1, C, G, K, and O). Similarly, in the vascular endothelium, NP-1 mRNA expression was not different under different hormonal conditions (Fig. 2). However, in the luminal epithelium, the NP-1 mRNA signal was very high in the mid- to late-proliferative phase and was almost nondetectable in the secretory phase, early proliferative phase, and hormone-deprived animals (in Fig. 1, compare E with A, I, and M). Control sections incubated with sense probe or RNase-treated sections incubated with the antisense probe showed signals similar to background and on the glass slide away from the sections (data not shown). Grain counts of the ISH preparations (Fig. 3, A–C) confirmed these observations. NP-1 expression in the stroma (Fig. 3A) and vascular endothelium (Fig. 3B) showed no statistically significant changes during the menstrual cycle or in hormone-deprived animals. However, in the luminal epithelium, the NP-1 mRNA signal was significantly (P < 0.01) up-regulated during the mid- to late-proliferative phase, suppressed to low or nondetectable levels by P treatment during the mid- to late-secretory phases, and also very low in hormone-deprived animals and the newly formed epithelium during the early-proliferative phase (Fig. 3C).
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Combination immunohistochemistry with Ki-67 and vWF antibodies clearly marked proliferating blood vessels and distinguished them from proliferating stromal and luminal epithelial cells (Fig. 4). Cell counts on such preparations showed a significant increase in luminal epithelial cell proliferation in the mid- to late-proliferative stage (Fig. 3D
). This increase in proliferation was E2 dependent because similar changes did not occur in the absence of E2 in hormone-deprived animals (Fig. 3D, HD 10–14). After P treatment, the percentage of proliferating luminal epithelial cells was dramatically reduced during the midsecretory phase (P < 0.01) and was nondetectable during the late-secretory phase (Figs. 3D and 4C). In the luminal epithelium, the pattern of NP-1 mRNA expression paralleled the pattern of proliferation throughout the cycle. For example, in the luminal epithelium, the Ki-67 labeling index and the NP-1 mRNA grain counts were highly correlated (R2 = 0.895, P < 0.001). However, there were no significant correlations between Ki-67 and NP-1 expression in either the vascular endothelium (R2 = 0.010, P < 0.692) or stroma (R2 = 0.168, P < 0.090) (data not shown).
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During the menstrual cycle, NP-2 mRNA expression was confined to the endothelium of small and large veins (Fig. 2H) in the endometrium. No specific signal was evident in the endothelium of spiral arteries (Fig. 2, E and F), stroma, glands, or luminal epithelium (data not shown). This venous signal was very low or nondetectable during the proliferative phase (Fig. 2G) and hormone-deprived animals (data not shown) and was significantly (P < 0.001) up-regulated by P during the secretory phase (Fig. 2, panel III).
The human endometrium
Overall the pattern of expression and cellular localization of both NP-1 and NP-2 in the human endometrium during the menstrual cycle was essentially identical with that seen in the rhesus macaque endometrium. In the luminal epithelium, NP-1 expression was elevated during the proliferative phase and undetectable during the secretory phase (compare Fig. 5A vs. 5C). In the stroma and vessels, NP-1 mRNA was abundantly and constantly expressed without change during the cycle (Fig. 5, E–L), and in the glands, NP-1 mRNA was nondetectable at all times (Fig. 5, E–H). As in the macaque, NP-2 expression was evident only in large and small veins of three samples during the secretory phase and was undetectable in any samples from the proliferative phase (Fig. 5, M–P).
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This is the first report on the cellular localization and hormonal regulation of NP-1 and NP-2 mRNA in the primate endometrium. The expression of these NPs is cell type specific. For example, NP-1 mRNA is expressed by the luminal, but not the glandular, epithelium. It is present in the endothelium of both arteries and veins and is expressed by endometrial stromal cells. NP-2 mRNA was expressed only by the endothelium of veins. In agreement with our findings, Gluzman-Poltorak et al. (18) noted that both NP-1 and NP-2 were expressed in human endothelial cells derived from the umbilical vein; and NP-2 is expressed preferentially in veins in chick embryos (33). Furthermore, in the mouse uterus, VEGFR-2 mRNA is exclusively expressed in endothelial cells, whereas NP-1 mRNA is expressed in both endothelial and stromal cells (26). However, there appear to be species differences because Pavelock et al. (27), who studied the rat uterus, reported that NP-1 protein (by ICC) was expressed in glands and vessels, and NP-2 was seen only in glandular epithelium, not vessels. It is not clear whether NP-1 is expressed in the mouse and rat uterine luminal epithelium because the latter two studies did not comment on luminal epithelial expression. Although we tried several commercially available NP-1 and -2 antibodies to examine protein localization by ICC, most of these antibodies are indicated for use in Western blots, and they were not satisfactory as ICC reagents in either human or rhesus monkey endometrium. We plan future studies to evaluate NP-1 and -2 protein expression in different endometrial cell types by laser capture microdissection and Western blotting.
NP-1 mRNA expression in the luminal epithelium is estrogen dependent
NP-1 mRNA expression in luminal epithelium was high during the mid- to late-proliferative phase, down-regulated in the secretory phase, and absent during hormone deprivation. Because only E2 was present during the induced proliferative phase and because P is known to block many estrogen-dependent processes, these data indicate that the increased expression of NP-1 mRNA was estrogen driven. Also, the low expression of NP-1 mRNA in the luminal epithelium of the hormone-deprived group is consistent with estrogen dependence because these animals lacked both E2 and P. VEGF, which is also estrogen dependent in the primate endometrium (31, 34), can mediate estrogen-dependent proliferation in the luminal epithelium in mice (15) and can induce epithelial cell proliferation in human fetal lung tissue (35). However, classical VEGFRs (VEGFR-1 and VEGFR-2) are lacking in the luminal epithelium of the primate endometrium (31). Because our data reveal that NP-1 mRNA expression is highly correlated with the proliferation marker Ki-67 in the luminal epithelium, we hypothesize that NP-1 mediates the proliferative effects of VEGF on these cells in the primate endometrium. Studies with antagonists of VEGF and NP-1 in the primate endometrium are needed to test this hypothesis.
However, during the postmenstrual repair (early proliferative) phase, NP-1 mRNA signal was very low in the newly formed luminal epithelium in the presence of E2, indicating that E2 has no regulatory role on NP-1 expression during this period. We previously reported that expression of VEGF, VEGFR-1, and VEGFR-2 is also independent of E2 action during the postmenstrual repair phase (31). In fact, postmenstrual healing and reepitheliazation of primate endometrium can occur normally after P withdrawal in the absence of E2 (2).
Surprisingly, the NP-1 gene in endometrial stroma and blood vessels showed no responsiveness to changes in hormonal state. Rather, expression in these cells was similar throughout the cycle and during hormone deprivation. The specific functions of NP-1 in these cells are currently not clear. However, in the vessels, NP-1 may function as an enhancer of tyrosine-kinase receptor (VEGFR-1 and -2) signaling because these receptors are also expressed in the vascular endothelium throughout the cycle (31), and NP-1 is known to form complexes with both VEGFR-1 and -2 and stimulate VEGF165 activity (22). In the stroma, NP-1 may also play a similar role to enhance VEGF activity when other tyrosine kinase receptors are expressed in the stromal cells. For example, in an earlier study, we have shown transient expression of VEGFR-2 in the stromal cells during the premenstrual/menstrual phase and a potential role of VEGF in induction of matrix metalloproteinases in these cells (11). Nevertheless, the difference in NP-1 regulation implies that cell-specific regulatory factors, rather than gene promoter differences, are responsible for these differences in hormonal responsiveness. Consequently, studies aimed at discovering which cell-specific factors regulate NP-1 gene expression in the different cell types of the primate endometrium should be of high priority.
We recently observed expression of semaphorin E, another ligand for NP-1, in the human endometrium (32). Its expression was down-regulated in the secretory phase during the window of implantation. But semaphorin E level was elevated in the endometrium of patients with endometriosis during the window of implantation (36). Thus, semaphorin actions may be mediated through NP-1 in the primate endometrium. Additional studies are underway in our laboratory to examine cellular localization and hormonal regulation of different members of this family of ligands and possible interactions with NPs in regulating endometrial functions.
NP-2 mRNA expression in the venous endothelium is P dependent
NP-2 mRNA provides a further example of cellular and regulatory specificity because it is expressed only in the venous endothelium and is up-regulated by P, not E2. Pavelock et al. (27) reported no change in NP-2 expression during the estrous cycle in the rat uterus and suppression of NP-2 by E2. However, in our study, NP-2 was not up-regulated in hormone-deprived macaques.
Of the various receptors expressed in vascular endothelium, including VEGFR-1, VEGFR-2, and NP-1, only NP-2 is up-regulated in the venous endothelium during the secretory phase. Because VEGF acts in the primate endometrium during the secretory phase to enhance permeability, venous NP-2 could mediate this effect. VEGF is a potent permeability factor (5, 6), and the edema it induces immediately subjacent to the luminal epithelium of the primate endometrium is essential for proper implantation (1, 3, 4, 5). Also, NP-1 and -2 can form heteromultimer complexes (37), and NP-1/NP-2 double-knockout mice show severe abnormal vascular phenotype, compared with single knockouts (25). Thus, expression of NP-2 in addition to NP-1 in the secretory phase venous endothelium may further potentiate VEGF action in this endothelium.
In summary, our ISH of NP-1 and NP-2 revealed that these VEGFRs are expressed in a cell-specific and hormonally responsive manner in the endometrium of both cycling women and nonhuman primates. NP-1 is expressed by several cell types, but hormonal regulation of this receptor occurs only in the luminal epithelium. In these cells, NP-1 expression is highly correlated with Ki-67 expression, an indication that NP-1 could mediate VEGF-dependent luminal epithelial proliferation. NP-2 mRNA exists at significant levels only in veins and only during the secretory phase of the menstrual cycle, in which it could mediate the inductive effects of VEGF on physiological edema. Because there is no shift in expression of the major VEGF isoform (VEGF165) during the menstrual cycle, it appears that hormonally induced, cell-specific expression of these isoform-specific receptors may be an important factor in mediating VEGF functions in the primate endometrium. Additional research is essential to discover the cell-specific factors that regulate NP expression and function in the primate endometrium.
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Acknowledgments |
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Footnotes |
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Present address for A.G.: Department of Reproductive Endocrinology and Infertility, University of Heidelberg, Heidelberg, Germany.
First Published Online December 21, 2004
Abbreviations: E, Embryonic day; E2, estradiol; HD, hormone deprived; ICC, immunocytochemistry; ISH, in situ hybridization; NP, neuropilin; P, progesterone; PVP, polyvinylpyrollidone; SSC, standard saline citrate; VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; vWF, von Willebrand factor.
Received September 7, 2004.
Accepted December 12, 2004.
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