Adult Tissue Angiogenesis: Evidence for Negative Regulation by Estrogen in the Uterus

W. Ma1, J. Tan1, H. Matsumoto, B. Robert, D. R. Abrahamson, S. K. Das and S. K. Dey

Departments of Molecular and Integrative Physiology (W.M., H.M., S.K.Dey), Anatomy and Cell Biology (B.R., D.R.A.), and Obstetrics and Gynecology (J.T., S.K.Das), Ralph L. Smith Research Center, University of Kansas Medical Center, Kansas City, Kansas 66160

Address all correspondence and requests for reprints to: S. K. Dey, Ph.D., Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, Kansas 66160-7338. E-mail: sdey{at}kumc.edu


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Increased uterine vascular permeability and angiogenesis are two major events of embryo implantation and placentation during pregnancy. These latter processes require coordinated, uterine-specific interactions between progesterone (P4) and estrogen (E) signaling. Although roles of these steroids have long been suspected, definitive functions of E and/or P4 in uterine angiogenesis still remain elusive. We have therefore exploited the availability of reporter and mutant mice to explore the regulation of angiogenesis in response to steroid hormonal changes in vivo. We present here molecular, genetic, physiological, and pharmacological evidence that E and P4 have different effects in vivo: E promotes uterine vascular permeability but profoundly inhibits angiogenesis, whereas P4 stimulates angiogenesis with little effect on vascular permeability. These effects of E and P4 are mediated by differential spatiotemporal expression of proangiogenic factors in the uterus.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
UNDER PHYSIOLOGICAL CONDITIONS, angiogenesis, the process by which new blood vessels originate from preexisting vessels, primarily occurs in the uterus and ovary in the adult during the reproductive cycle and pregnancy (1). Indeed, increased vascular permeability and angiogenesis are essential to successful implantation and placentation. Several studies have provided indirect and descriptive evidence for the potential roles of estrogen (E) and progesterone (P4) in these processes in various species (reviewed in Refs. 2, 3, 4). As described below, these studies primarily examined the changes in the whole uterus and the expression of a number of gene products known to control vascular permeability and angiogenesis, including vascular endothelial growth factor (VEGF) and its receptors, without investigating its angiogenic status. Thus, in vivo roles for E and P4 in uterine angiogenesis are still unclear. In this study, we have used a combination of molecular, genetic, physiological, and pharmacological approaches to address this question.

VEGF, originally discovered as a vascular permeability factor (reviewed in Ref. 5), is also a potent mitogen for endothelial cells and a key regulatory growth factor for vasculogenesis and angiogenesis (6). Targeted disruption of even one allele of the Vegf gene results in embryonic death in utero on d 10.5 with aberrant blood vessel formation (7, 8). Differential splicing of the Vegf gene generates several VEGF isoforms in both humans and mice; VEGF121 and VEGF165 are the predominant isoforms in humans, whereas isoforms VEGF120 and VEGF164, which are shorter by one amino acid, are most abundant in mice (4, 9).

VEGF effects are primarily mediated by two tyrosine kinase receptors: FLT1 (VEGF receptor 1) and FLK1/KDR (VEGF receptor 2) (10, 11, 12, 13). Although FLT1 activation does not stimulate endothelial cell mitosis, targeted disruption of the Flt1 gene produces impaired endothelial cell assembly into blood vessels and embryonic lethality (14). FLK1 is the major transducer of VEGF signals that induce chemotaxis, actin reorganization, and proliferation of endothelial cells (6, 15, 16). Targeted deletion of the Flk1 gene in mice produces defects in hematopoietic and endothelial cell development leading to embryonic death by d 9.5 (17).

Recently, another multifunctional VEGF receptor has been identified as neuropilin-1 (NRP1). NRP1 was originally described as a neuronal transmembrane receptor that participates in axonal guidance in the developing nervous system (18, 19) and is a receptor for the collapsin/semaphorin family of proteins (20, 21). It is now known that NRP1 functions as a receptor for at least five different ligands, collapsin-1/semaphorin-IIII/D, semaphorin-E, semaphorin-IV, VEGF165, and placental growth factor, which are involved in different biological processes such as nervous system development, vasculogenesis, and angiogenesis (21, 22). NRP1 is expressed in human endothelial cells as a VEGF165-specific receptor. When coexpressed in endothelial cells with FLK1, NRP1 enhances the binding of VEGF165 to FLK1 and VEGF165-mediated chemotaxis severalfolds higher than that of FLK1 alone (23). Conversely, inhibition of VEGF165 binding to NRP1 inhibits its binding to FLK1 and its mitogenic activity for endothelial cells. Nrp1-deficient mice show peripheral nervous system abnormalities and die in midgestation due to yolk sac vascular insufficiency and developmental anomalies of the cardiovascular system (24). Mice overexpressing NRP1 also show cardiovascular abnormalities including increased number of blood vessels and abnormal hearts (25).

We have recently shown that the genes encoding murine VEGF isoforms and their receptors, FLT1, FLK1, and NRP1, are differentially expressed in the mouse uterus in a spatiotemporal manner during implantation, and that the predominant VEGF164 isoform interacts with FLK1 and NRP1 (2, 4). These results provide evidence that the VEGF system is important for uterine vascular permeability and angiogenesis during implantation. Others have also shown the expression of VEGF and its receptors in the uterus as a whole during pregnancy and in response to steroid hormones (reviewed in Ref. 3). For example, E rapidly induces uterine vascular permeability and Vegf expression transcriptionally via nuclear ER (reviewed in Refs. 3 and 26). In addition, the Vegf gene contains E response elements (26). P4 also up-regulates uterine Vegf expression, but at a slower rate, via activation of its nuclear receptor, PR (26). Because E rapidly stimulates uterine vascular permeability and Vegf expression, and because vascular permeability is considered a prerequisite for angiogenesis, it is widely believed that E is a potent stimulator of uterine angiogenesis during normal reproductive processes in vivo. However, there is no experimental evidence to support this conclusion.

In this paper, we have used a combination of approaches to address in detail the roles of ovarian steroids on uterine angiogenesis in mice. Unexpectedly, we found that although E promotes uterine vascular permeability, it profoundly attenuates angiogenesis. In contrast, P4 stimulates uterine angiogenesis with little effect on vascular permeability.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
E and P4 Differentially Regulate the Spatiotemporal Expression of Vegf and Flk1 in the Uterus
In the mouse uterus, E elicits an early response (phase I) and a late response (phase II) (27, 28). The phase I response is characterized by increased uterine vascular permeability and edema that reach a maximum after 6 h of E administration. The phase II uterine response occurs between 12 and 24 h of E treatment and represents the "true growth" phase, i.e. cellular proliferation and hypertrophy (28, 29). Furthermore, P4 attenuates the phase I estrogenic responses (reviewed in Ref. 30). Because VEGF signaling is a strong stimulator of vascular permeability, we therefore examined the temporal and cell-specific expression of Vegf and Flk1 in the mouse uterus in response to estradiol-17ß (E2) and/or P4 by Northern and in situ hybridization. Ovariectomized mice were given an injection of oil (control), E2, P4, or E2 plus P4, and uterine RNA was extracted at various times. As previously reported (2), we detected multiple transcripts of Vegf in uterine poly(A)+ RNA samples with 4.2 kb as a major transcript (Fig. 1Go). When normalized to a gene encoding ribosomal protein L7 (rpL7), the levels of Vegf mRNA showed an early, but transient, increase after E2 injection, with peak levels between 2 and 4 h (260% increase over the oil-treated controls) followed by a decline reaching a nadir by 24 h (Fig. 1Go). In contrast, Vegf mRNA levels after a P4 injection were modest and remained more or less steady, except an increase (125% increase compared with controls) at 12 h. The pattern of uterine levels of Vegf mRNA after a combined injection of E2 and P4 was similar to those of E2 treatment alone, except the levels were a little lower (170% increase at 4 h). These results corroborate those of a recently published report (26).



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Figure 1. Northern Blot Detection of Vegf and Flk1 mRNAs in the Mouse Uterus

Poly(A)+ RNA samples (2 µg) of uteri after steroid hormone treatments were separated by formaldehyde-agarose gel electrophoresis, transferred to nylon membranes, UV cross-linked, and hybridized to specific 32P-cRNA probes. The same blots were stripped and rehybridized to a rpL7 (a house-keeping gene) probe to confirm integrity of RNA samples.

 
Because signaling of VEGF via FLK1 is an essential component of angiogenesis, we also examined the uterine expression of the gene encoding FLK1 in response to steroid hormones (Fig. 1Go). An E2 injection resulted in an early increase in uterine Flk1 mRNA (~7.0 kb) levels that peaked at 6 h (205% over the controls). This was followed by a decline reaching a level similar to that of the control (oil) at 24 h. After an injection of P4, however, an increase in the level of Flk1 mRNA was observed at 6 h, reaching a peak at 12 h (165% increase over the controls), and sustained through 24 h. The pattern of levels in E2 plus P4-treated uteri was similar to those of E2 treatment alone. These results suggest that whereas E2 regulates the uterine expression of Vegf and its receptor Flk1 in an early and transient manner, P4 has more sustained effects on the expression of these genes.

The early but transient rise in uterine levels of Vegf and Flk1 mRNAs seen within 6 h of an E2 injection (Fig. 1Go) is consistent with a role for VEGF in uterine vascular permeability. However, Northern analysis gives no indication of the uterine cell types that are responding by increased gene expression, and levels of whole uterine mRNAs by Northern hybridization may have limited meaning because of the dilution effects resulting from heterogeneous uterine cell types in which myometrial cells constitute the major cell population. We therefore examined the cell-specific expression of Vegf and Flk1 mRNAs by in situ hybridization in the ovariectomized uterus at different hours after an E2 injection. Indeed, differential cell-specific expression of Vegf and Flk1 mRNAs were observed with changing time. As shown in Fig. 2AGo, Vegf mRNA accumulation was markedly up-regulated, primarily in stromal cells, within 2 h of an E2 injection and persisted through 6 h. In contrast, whereas the levels of Flk1 mRNA were very low at 2 h, a marked increase was noted at 6 h of E2 treatment. The prompt increase in Vegf expression at 2 and 6 h was accompanied by an up-regulation of Flk1 expression in the stromal bed at 6 h after an injection of E2 (Fig. 2AGo). This result suggests that VEGF is important for uterine vascular permeability changes that are induced by E during the phase I response.



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Figure 2. In Situ Hybridization of Vegf and Flk1 mRNA in Mouse Uteri After Steroid Hormone Treatments

A, Two and 6 h after oil (control) or E2 treatment. B, Twenty-four hours after oil, E2, P4, or E2 plus P4 treatment. Representative dark-field photomicrographs of longitudinal sections of uteri are shown (bar, 60 µm). le, luminal epithelium; s, stroma; myo, myometrium.

 
Because vascular permeability is normally followed by angiogenesis, we next examined the expression of Vegf at 24 h after an injection of oil (control), E2, P4, or E2 plus P4 (Fig. 2BGo). Low to modest levels of Vegf mRNA accumulation were noted in both the epithelial and stromal cells of ovariectomized mice receiving an injection of oil (control). Surprisingly, Vegf expression was primarily limited to the luminal epithelium at 24 h of E2 treatment; the stromal expression was very low. In contrast, Vegf mRNA accumulation was prominent and primarily stromal in response to P4 alone. The expression pattern in response to P4 plus E2 was similar to that of P4 alone, but at lower levels (Fig. 2BGo). The differential cell-specific expression of Vegf in response to E2 and/or P4 prompted us to examine the cell-specific expression of Flk1 in steroid-exposed ovariectomized uteri. As shown in Fig. 2BGo, the accumulation of Flk1 mRNA in stromal endothelial cells was very low in oil-treated control uteri and was not altered 24 h after an E2 injection. In contrast, the accumulation of Flk1 mRNA was remarkably up-regulated in endothelial cells of the stromal bed 24 h after an injection of P4. The combined injection of P4 with E2 resulted in an accumulation pattern similar to that of P4 alone, albeit at lower levels. The late induction of stromal Vegf and Flk1 by P4 alone suggests that P4 positively regulates uterine angiogenesis. E, on the other hand, appears to be a negative regulator of uterine angiogenesis, because Vegf and Flk1 expression in the stromal bed is very low in response to this steroid during the late phase, and P4-induced changes are attenuated by E.

Effects of E and P4 on Uterine Expression of Angiogenic Factors are Mediated by Their Nuclear Receptors
E and P4 effects in the uterus are primarily mediated via activation of nuclear ER{alpha} and PR, respectively. Because these steroids differentially regulate uterine expression of Vegf and Flk1 in a spatiotemporal manner, we sought to examine whether the effects of E or P4 on the expression of these genes are direct and mediated via their nuclear receptors. Thus, we examined the expression of Vegf and Flk1 in uteri of mice with a null mutation for the ER{alpha} or PR gene by in situ hybridization. Our results show distinct expression of stromal Vegf and endothelial Flk1 in the endometrial bed in ER{alpha}(-/-) mice (Fig. 3AGo). In contrast, uterine expression of Vegf and Flk1 in PR(-/-) mice was very scanty (Fig. 3BGo), and the response was not altered in ovariectomized PR(-/-) mice by treatment with E2 or P4 (data not shown). The level and pattern of expression of Flk1 mRNA in ER{alpha}(-/-) and PR(-/-) uteri correlate well with lacZ-stained endometrial blood vessels under a Flk1 promoter in ER{alpha}(-/-) x Flk1(+/-)lacZ and PR(-/-) x Flk1(+/-)lacZ double mutant female mice, respectively (compare Fig. 3Go vs. Fig. 6AGo). Uteri of ER{alpha}(-/-) mice contain PR and respond to P4 with respect to gene expression and decidualization (31, 32, 33). The results provide genetic evidence that higher Flk1 expression in ER{alpha}(-/-) uteri is primarily due to P4 effects, whereas the attenuated expression in PR(-/-) uteri is the result of predominant E action. We next sought to determine the effects of E and/or P4 on uterine angiogenesis.



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Figure 3. In Situ Hybridization of Vegf and Flk1 mRNAs in Uteri of Intact ER{alpha}(-/-) (A) and PR(-/-) (B) Mice

Representative bright- and dark-field photomicrographs of longitudinal uterine sections are shown (bar, 60 µm). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.

 


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Figure 6. lacZ-Stained Endometrial Blood Vessels in ER{alpha}(-/-) x Flk1(+/-)lacZ or PR(-/-) x Flk1(+/-)lacZ Double Mutant Mice

A, Photomicrographs of lacZ-stained endometrial blood vessels in representative longitudinal uterine sections and cross-sections of the ovary, brain, and skeletal muscle from intact ER{alpha}(-/-) x Flk1(+/-)lacZ or PR(-/-) x Flk1(+/-)lacZ double mutant mice are shown (bar, 60 µm). B, lacZ-stained endometrial blood vessels in representative cross-sections of ovariectomized PR(-/-) x Flk1(+/-)lacZ double mutant mice in response to steroid hormones are shown (bar, 60 µm). Ovariectomized mice were injected with oil or steroids once daily for 2 d and killed 24 h after the last injection. Arrowheads indicate the location of lacZ-stained blood vessels. le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.

 
E and P4 Differentially Regulate Uterine Angiogenesis
FLK1 (VEGF receptor 2) is a well established marker of endothelial cell development and angiogenesis (34, 35). Mice in which the Flk1 gene has been disrupted with an insertion of the Escherichia coli ß-galactosidase (lacZ) gene (17) were used to study uterine angiogenesis. Although the Flk1(-/-)lacZ embryos die in utero, Flk1(+/-)lacZ embryos are viable, and Flk1(+/-)lacZ females have normal fertility. Thus, Flk1(+/-)lacZ mice serve as a powerful genetic model, with ß-galactosidase expression as a read-out for Flk1 promoter activity and as an endothelial cell marker to examine uterine angiogenesis under different physiological and experimental conditions.

To examine the status of uterine angiogenesis in response to E2 and/or P4, ovariectomized Flk1(+/-)lacZ mice received injections of oil, P4, and/or E2 once daily for 2 d and were killed 24 h after the last injection. When compared with oil-treated (control) mice, endometria of mice treated with P4 showed an increased density of lacZ-stained blood vessels (Fig. 4AGo). In contrast, mice treated with E2 exhibited a remarkably reduced number of such endometrial blood vessels, even lower than those of the oil-treated mice. Mice that received both P4 and E2 injections displayed a number of lacZ-stained endometrial blood vessels intermediate between P4- and E2-treated mice (Fig. 4AGo). To quantify the extent of endometrial angiogenesis, we measured the area of uterine sections occupied by lacZ-stained blood vessels. The mean percentage of endometrial area occupied by lacZ-stained blood vessels is shown in Fig. 4BGo. Furthermore, the inhibitory response of E2 or stimulatory effects of P4 on uterine angiogenesis were reversed by IC1–182,780, an ER antagonist, or RU-486, a PR antagonist (Fig. 4CGo). These results suggest that whereas E2 is profoundly inhibitory, P4 is stimulatory to uterine angiogenesis. Furthermore, E2 is capable of counteracting the stimulatory effects of P4 on uterine angiogenesis. This observation prompted us to examine the long-term effects of sustained stimulation of the uterus with P4 or E2 using SILASTIC brand (Dow Corning Corp, Midland, MI) implants (36). In ovariectomized Flk1(+/-)lacZ mice with SILASTIC brand implants containing P4 or E2 for 4 d, a similar pattern of lacZ staining was noted as observed for steroid treatments of shorter duration (data not shown). We also examined by in situ hybridization the expression of Vegf and Nrp1 in uteri of these mice (Fig. 5AGo). In ovariectomized uteri without the steroid treatment, the expression of Vegf and Nrp1 was modest throughout the endometrium. E2 treatment again suppressed stromal expression of Vegf; the expression was primarily restricted to epithelial cells, as observed at 24 h after an E2 injection (compare Fig. 2BGo vs. Fig. 5AGo). In contrast, the expression was distinct and mostly stromal in P4-treated mice. The expression of Nrp1 in E2-treated uteri was observed in both epithelial and stromal cells at very low levels. In contrast, Nrp1 was abundantly expressed in stromal cells of P4-treated mice (Fig. 5AGo). Our observation of up-regulated expression of Nrp1 in the stroma by P4 is consistent with a recent report of up-regulated Nrp1 expression in the rat uterus by P4, but not by E2 (37). Although the observation of heightened expression of Vegf, Flk1, and Nrp1 in the stromal bed in response to P4 suggests its stimulatory role, their attenuated expression in the stromal bed by E2 suggests its inhibitory role in uterine angiogenesis. Immunolocalization of the platelet endothelial cell adhesion molecule (PECAM), another endothelial cell marker (35), showed similar distribution patterns as lacZ staining in steroid-treated uteri (Fig. 5BGo).



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Figure 4. lacZ-Stained Endometrial Blood Vessels in Flk1(+/-)lacZ Mice After Steroid Hormone Treatments

Ovariectomized mice were treated with oil, E2, P4, or E2 plus P4 once daily for 2 d and killed 24 h after the last injection. A, Photomicrographs of lacZ staining of representative uterine cross-sections are shown. B, Quantification of lacZ staining is shown as mean percentage of stained endometrial area. Four to six sections from two to three mice in each group were evaluated for quantification. Results are presented as mean ± SEM. Values were statistically significant from each other (P < 0.05, ANOVA). C, Ovariectomized mice were treated for 2 d with either P4, E2, P4 plus RU-486, or E2 plus ICI-182,780 (ICI). RU-486 and ICI were injected 30 min prior to steroid injections. Mice were killed 24 h after the last injection. Photomicrographs of lacZ staining of representative uterine longitudinal sections are shown (bar, 60 µm). Arrowheads indicate the location of lacZ-stained blood vessels. le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.

 


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Figure 5. In Situ Hybridization of Vegf and Nrp1 mRNAs and Immunolocalization of PECAM in Mouse Uteri After Long-Term E2 or P4 Treatment

Ovariectomized mice carrying SILASTIC brand implants containing oil, E2, or P4 were killed after 4 d, and uteri were processed for in situ hybridization and immunohistochemistry. A, Representative dark-field autoradiographic signals of in situ hybridization in longitudinal uterine sections are shown (bar, 75 µm). B, Frozen longitudinal uterine sections (10 µm) were subjected to immunostaining using a rat-antimouse monoclonal antibody to PECAM (PECAM-1). Red color indicates the sites of immunoreactive PECAM (bar, 60 µm). le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium.

 
Heightened Angiogenesis with the Loss of ER{alpha} Function Is Consistent with Reduced Angiogenesis with the Loss of PR Function in the Uterus
Although uterine estrogenic effects are virtually absent in ER{alpha}-deficient mice, uteri in PR-deficient mice are highly estrogenized due to the absence of P4 effects. These findings led us to examine the status of uterine angiogenesis in mice homozygous for ER{alpha} or PR null mutations but heterozygous for the Flk1lacZ mutation. ER{alpha}(+/-) or PR(-/-) males were mated with Flk1(+/-)lacZ females to generate ER{alpha}(-/-) x Flk1(+/-)lacZ or PR(-/-) x Flk1(+/-)lacZ double mutant female mice. Our results of lacZ staining show that the density of endometrial blood vessels in ER{alpha}(-/-) x Flk1(+/-)lacZ mice was remarkably higher than that observed in PR(-/-) x Flk1(+/-)lacZ mice (Fig. 6AGo). In fact, the lower density of endometrial vessels in the latter group of mice was similar to that observed in ovariectomized Flk1(+/-)lacZ mice treated with E2 (Fig. 4AGo vs. Fig. 6AGo). The density of these endometrial blood vessels was even higher in ovariectomized PR(-/-) x Flk1(+/-)lacZ mice, and P4 did not alter this response. However, treatment of such ovariectomized mice with E2 reduced the number of lacZ-stained endometrial blood vessels, and as expected, treatment with P4 did not alter the E2 response (Fig. 6BGo). However, the angiogenic status in the ovarian theca was similar in both ER{alpha}(-/-) x Flk1(+/-)lacZ and PR(-/-) x Flk1(+/-)lacZ mice. Likewise, there was no significant alteration in lacZ staining in adult brains or skeletal muscles of these double mutant mice (Fig. 6AGo).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The present investigation provides molecular, genetic, physiological, and pharmacological evidence that E is a potent inhibitor of uterine angiogenesis in mice, and surprisingly, that P4 is a stimulator of uterine angiogenesis. Furthermore, these effects of E and P4 are mediated via activation of their cognate nuclear receptors and are specific to the uterus, because angiogenesis in the ovary, brain, and skeletal muscle were similar in mice with null mutations for the ER{alpha} or PR gene. These findings challenge the prevailing notion of E-induced stimulation of uterine angiogenesis (reviewed in Ref. 3). This notion is built on the correlative findings that vascular permeability is followed by increased angiogenesis and that E rapidly induces uterine vascular permeability coincident with increased VEGF expression. Indeed, our observation of rapid, but transient, induction of Vegf and Flk1 in the uterine stromal bed by E supports the idea that E increases uterine vascular permeability during the phase I response. However, our findings of reduced uterine vascular density and decreased stromal endothelial Flk1 expression during the late estrogenic response suggest that this steroid has an inhibitory role in uterine angiogenesis. This is further confirmed by genetic evidence in ER{alpha}(-/-) mice that display increased density of lacZ-stained endometrial blood vessels and increased stromal expression of Vegf and Flk1 in the absence of ER{alpha} functions. The role of ERß is questionable, because uterine expression of ERß is very low in ER{alpha}(-/-) mice. Moreover, an ER antagonist (ICI 182,780), which negates both ER{alpha} and ERß functions (38), reversed the inhibitory effects of E2 on uterine angiogenesis. Thus, our findings demonstrate that uterine vascular permeability is not always followed by angiogenesis. This is consistent with the recent observation that although mice deficient in individual Src family kinases show normal angiogenesis, mice lacking in pp60c-src or pp62c-yes, but not fyn, failed to exhibit VEGF-mediated vascular permeability (39).

The restricted expression of Vegf in the uterine epithelium with a very low or undetectable level of expression in the stroma during the late phase of estrogenic stimulation suggests that VEGF is not readily available in the stroma for increased angiogenesis. In contrast, our observation of increased vessel density in P4-treated Flk1(+/-)lacZ mice, as well as increased accumulation of Vegf mRNA in stromal cells and Flk1 mRNA in stromal endothelia of wild-type mice after P4 treatment, suggests that this steroid stimulates uterine angiogenesis. This steroid participates in this process via its nuclear receptor, PR, because the vessel density is severely compromised in uteri of PR(-/-) x Flk1(+/-)lacZ double mutant mice with manifestation of predominantly E actions, and reversal of P4 effects by a PR antagonist RU-486 can be observed in wild-type mice.

P4 has recently been shown to attenuate in vitro proliferation of human endothelial cells derived from various tissues (40). This P4 effect appears to be mediated via PR, because these cells express PR, and a PR antagonist compromises this P4 effect. Furthermore, using PR(-/-) mice, these investigators provided evidence that P4 also interferes with re-endothelialization of injured aortae, again suggesting the inhibitory role of P4 in endothelial cell proliferation (40). The discrepancy between this study and our present investigation is most likely due to the differences in experimental designs and parameters used and cell types studied. Vázquez et al. (40) studied the influence of P4 on proliferation of human dermal and coronary endothelial cells and of mouse brain endothelial cells in culture, whereas our study examined the effects of P4 and/or E2 on endometrial angiogenesis in mice in vivo. They did not examine the effects of P4 on proliferation of PR-expressing human endometrial endothelial cells. We speculate that ovarian steroids influence endothelial cell functions with respect to angiogenesis in a tissue-specific manner. Because the uterus is a major target for E and P4, and because heterogeneous cell types of the uterus respond differently to these hormones in a dynamic manner, we suspect that uterine endothelial cells respond to these steroids differently from cells in extrauterine sites. It is also highly possible that angiogenic factors generated by the action of steroid hormones on uterine cell types act on endothelial cells for angiogenesis in a paracrine manner. However, such paracrine effects are absent in endothelial cells in culture.

P4-induced angiogenesis in the uterus is physiologically meaningful, because this steroid is an essential hormone for the initiation and maintenance of pregnancy in all mammals examined, and uterine angiogenesis is an essential component during pregnancy. Although E and P4 are critical to implantation and pregnancy maintenance in mice and rats, P4 alone is sufficient for these events in several species including hamsters, guinea pigs, rabbits, and pigs (reviewed in Ref. 41). This suggests that P4 is a prime regulator of uterine angiogenesis during early pregnancy in these species. Furthermore, uterine expression of proangiogenic factors is very low on d 1 of pregnancy in mice when the uterus is under the dominance of preovulatory ovarian E secretion. However, dramatic increases in the expression of these factors are observed from d 4 onward, with rising P4 levels by the newly formed corpora lutea (2, 4). Thus, perhaps a balance between negative and positive influences resulting from coordinated interactions between E and P4 during early pregnancy determines the normal angiogenic status of the uterus during early pregnancy in mice.

Our results suggest that E-induced early endometrial vascular permeability and stromal edema are mediated by an early induction of VEGF and FLK1 in the stromal bed, whereas the attenuated uterine angiogenesis during the late estrogenic growth phase is the result of suppressed Vegf, Nrp1, and Flk1 expression in the stromal bed. In contrast, P4-induced uterine angiogenesis is executed by late and sustained induction of Vegf and Flk1 in conjunction with Nrp1 (Fig. 7Go). Our results also suggest that an increased vascular permeability is not always a prerequisite condition for increased uterine angiogenesis. In conclusion, we provide here the first evidence for the regulation of angiogenesis in a physiologically relevant adult organ system in vivo. This information will be important for comparing normal physiological angiogenesis with the process during pathological conditions such as uterine adenocarcinoma, endometriosis, and dysfunctional uterine bleeding. In this respect, it is interesting to note that xenografts resulting from a human endometrial cancer cell line overexpressing ER{alpha} showed reduced tumor growth and angiogenesis (42).



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Figure 7. A Scheme Depicting Uterine Vascular Permeability and Angiogenesis in Response to Steroids

This scheme suggests that in the stromal bed, E2 rapidly, but transiently, induces Vegf and Flk1, whereas P4 induces these genes in the stroma in a more delayed and sustained manner.

 

    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Animals and Treatments
Adult CD-1 mice were purchased from the Charles River Laboratories, Inc. (Raleigh, NC). Flk1-deficient mice were generated by disruption of the Flk1 gene using homologous recombination in embryonic stem cells (17). A targeting vector was constructed in which the translated portion of the first coding exon and the proximal part of the first intron of the Flk1 gene were replaced by a promoterless ß-galactosidase gene from E. coli, leaving the Flk1 promoter intact. Therefore, ß-galactosidase expression is used as a read-out for Flk1 promoter activity. ER{alpha}-deficient mice (129/J/C57BL/6J) and PR-deficient mice (129SvEv/C57BL/6) were generated as previously described (31, 43) and were kindly provided by Dennis Lubahn (University of Missouri, Columbia, MO) and Bert O’Malley (Baylor College of Medicine, Houston, TX), respectively, for establishing our colonies. ER{alpha}(-/-) x Flk1(+/-)lacZ double mutant mice were generated by crossing Flk1(+/-)lacZ females with ER{alpha}(+/-) males, whereas PR(-/-)/Flk1(+/-)lacZ double mutants were generated by crossing Flk1(+/-)lacZ females with PR(-/-) males. PCR analysis of the genomic DNA determined the genotypes. All mice were housed in the Animal Care Facility at the University of Kansas Medical Center (Kansas City, KS) according to NIH and institutional guidelines for laboratory animals.

To examine the effects of E and/or P4 on uterine expression of proangiogenic genes or uterine angiogenesis (lacZ staining), ovariectomized mice were injected with sesame oil (0.1 ml/mouse), estradiol-17ß (E2) (100 ng/mouse), P4 (2 mg/mouse), E2 and P4, E2 and ICI 182,780 (an ER antagonist), or P4 and RU486 (a PR antagonist). ICI 182,780 and RU486 were injected at a dose of 500 µg and 1 mg per mouse per day, respectively. In another set of experiments, ovariectomized mice were implanted sc with SILASTIC brand implants (0.1 x 1 cm) filled with E2, or implants (0.1 x 2 cm) filled with P4, for 4 d. At termination of the treatments, uteri were processed for subsequent analysis. For systemic injections, steroids and antagonists were dissolved in sesame oil and injected sc.

Probes
The cDNA clones for Vegf, Flk1, Nrp1, and rpL7 have been previously described (2, 4). For Northern hybridization, antisense 32P-cRNA probes were generated, whereas for in situ hybridization, sense or antisense 35S-cRNA probes were generated using appropriate polymerases. Probes had specific activities of about 2 x 109 dpm/µg.

Northern Hybridization
For Northern hybridization, total RNA (6.0 µg) or poly(A)+ RNA (2.0 µg) was denatured and separated by formaldehyde/agarose gel electrophoresis, transferred to nylon membranes, and UV cross-linked. Northern blots were prehybridized, hybridized, and washed as previously described (2, 4). Quantification of hybridized bands was analyzed by densitometric scanning.

In Situ Hybridization
In situ hybridization was performed as previously described (2, 4). In brief, frozen sections (10 µm) were mounted onto (poly)L-lysine–coated slides and fixed in cold 4% paraformaldehyde in PBS. The sections were prehybridized and hybridized at 45 C for 4 h in 50% formamide hybridization buffer containing the 35S-labeled antisense cRNA probes. RNase A-resistant hybrids were detected by autoradiography. Sections were post-stained with eosin and hematoxylin. Sections hybridized with the sense probes did not result in any positive hybridization.

lacZ Staining and Quantification
The expression of ß-galactosidase was assessed by lacZ staining as previously described (44). In brief, small pieces of tissues were fixed in 0.2% paraformaldehyde solution followed by infusion in 30% sucrose at 4 C overnight. Tissues were embedded in OCT and snap-frozen. Frozen sections were mounted onto glass slides and stained overnight at 37 C using 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside as a substrate. Sections were counterstained with eosin. Uterine area occupied by lacZ-stained blood vessels was quantitated. Random sections of uteri were used for lacZ staining; digital images were obtained, and measurements were made using the Scion Image program (Scion Corp., Frederick, MD). For consistency of measurements, the total uterine stromal area was defined by subtracting the section area occupied by the uterine luminal and myometrial layers, and the percentage of uterine stromal area occupied by lacZ-positive vascular structures was measured for each section.

Immunohistochemical Localization of PECAM
Frozen longitudinal uterine sections (10 µm) were subjected to immunostaining using a rat-anti-mouse monoclonal antibody to PECAM (PECAM-1, BD PharMingen, San Diego, CA) at a dilution of 1:50 using a Histostain-SP kit (Zymed Laboratories, Inc., San Francisco, CA) as previously described (45). Red color indicates the sites of immunoreactive PECAM.


    ACKNOWLEDGMENTS
 
We thank Brigid Hogan for her valuable advice.


    FOOTNOTES
 
This work was supported by NIH Grants HD-12304, HD-29968, HD-33994-06, Mellon Foundation (to S.K.Dey), ES-07814 (to S.K.Das), and DK-52483 (to D.R.A.), and an American Heart Association grant (to B.R.). A center grant in Mental Retardation provided core facilities. S.K.Dey is the recipient of an NIH MERIT award.

1 These authors contributed equally to this work. Back

Abbreviations: E, Estrogen; E2, estradiol-17ß; NRP1, neurophilin 1; P4, progesterone; PECAM, platelet endothelial cell adhesion molecule; rpL7, ribosomal protein L7; VEGF, vascular endothelial growth factor.

Received for publication June 28, 2001. Accepted for publication August 8, 2001.


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