Time-Course of the Uterine Response to Estradiol-17ß in Ovariectomized Ewes: Expression of Angiogenic Factors¹

^a Department of Animal & Range Sciences, ^b and Cell Biology Center, Biotechnology Institute, North Dakota State University, Fargo, North Dakota 58105

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Uterine expression of angiogenic factors (vascular endothelial growth factor [VEGF] and basic fibroblast growth factor [bFGF]) was evaluated in ovariectomized ewes at 0, 2, 4, 8, 24, 48, or 72 h after estradiol (E₂) treatment. Endometrial VEGF mRNA increased more than 5-fold from 0 to 4 h, remained elevated at 8 h, and then declined through 72 h after E₂ treatment. In contrast, endometrial bFGF mRNA remained constant from 0 to 4 h, increased 2.2-fold from 4 to 8 h, remained elevated at 24 h, and then declined through 72 h. Immunostaining for VEGF was present in myometrial and endometrial microvessels (arterioles, venules, and/or capillaries) and also in myometrial smooth muscle; the pattern of VEGF immunostaining followed that of mRNA expression, being elevated at 4 and 8 h after E₂ treatment. Immunostaining for bFGF was present exclusively in uterine glands; the pattern of bFGF immunostaining also followed that of its mRNA, being elevated at 8 and 24 h after E₂. On the basis of these observations, we suggest that VEGF and bFGF are probably important factors responsible for the dramatic uterine microvascular response that occurs 8 to 24 h after E₂ treatment in ovariectomized ewes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
In several previous studies, we have shown that the ovine endometrium produces angiogenic factors, and that these factors are heparin-binding [1, 2]. On the basis of these observations as well as their biochemical properties and biological activities, we have suggested that these factors belong to one of the major families of heparin-binding angiogenic factors, namely the fibroblast growth factor (FGF) or vascular endothelial growth factor (VEGF) families of proteins [3–5]. This suggestion also is supported by observations in rodents, pigs, and humans, in which uterine expression of FGF and FGF receptors has been identified during uterine growth and remodeling [6–11]. Similarly, VEGF and VEGF receptors have been identified in the uterus of rodents and humans, and uterine VEGF mRNA expression is stimulated by estrogen treatment in ovariectomized (OVX) rats [12–15].

In OVX ewes, we have shown that treatment with estradiol-17ß (E₂) results in 2- to 3-fold increases in uterine fresh weight, dry weight, and DNA content by 24–48 h after treatment [16, 17]. Endometrial microvascular volume also exhibits a similar dramatic increase by 24 h after E₂ treatment [17]. We hypothesized that basic FGF (bFGF) or VEGF, potent angiogenic factors produced by the endometrium, might play a role in this dramatic microvascular response.

The purpose of the present study, therefore, was to determine the relationship between the dramatic endometrial microvascular response and endometrial expression of bFGF and VEGF after E₂ treatment of OVX ewes.

	MATERIALS AND METHODS

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Animals, Treatments, and Sample Collection

Ewes that had been ovariectomized for 4 wk were slaughtered at 0, 2, 4, 8, 24, 48, or 72 h (n = 4–5 per group) after s.c. insertion of silicone elastomer implants containing 100 mg of E₂, as described previously [16, 17]. These ewes were the same animals as described in the companion manuscript [17]. At slaughter, the uterus was obtained from each ewe and separated from the adnexa, and then cross sections (~1 cm wide) were obtained from the mid-portion of a uterine horn, fixed in Carnoy's solution [16–19], and subsequently used for bFGF and VEGF immunohistochemistry. Additional samples (~1–2 g) of the endometrium (uterine mucosa) were snap-frozen on dry ice and stored at -80°C until analyzed for bFGF and VEGF mRNA by ribonuclease protection assay (RPA).

RPA

Total cellular RNA was isolated from snap-frozen endometrial samples by using the guanidinium-isothiocyanate extraction method followed by CsCl density centrifugation, as we have described previously [19]. The integrity of the RNA samples was assessed visually by ethidium bromide staining of the RNA after electrophoresis on a denaturing 1% agarose gel, and only intact RNA was used for the RPA [20]. In addition, the quantity and purity of the RNA samples were determined spectrophotometrically, as we have reported previously [20].

Endometrial bFGF and VEGF mRNA were quantified with the RPA by using a protocol similar to that which we have validated and reported previously [20], except that nonradioactive, biotin-labeled riboprobes were used. The nonradioactive in vitro transcription reactions and nonradioactive detection were performed by using kits from Ambion (Austin, TX) as described in their technical manuals. For the RPA, 25 µg of total cellular (tc)RNA was co-precipitated with excess bFGF, VEGF, and actin riboprobes, and then resuspended in hybridization buffer. For bFGF, a 292-base biotin-labeled riboprobe (bases 91–382 from a partial cDNA sequence of ovine bFGF; a gift from Dr. Russel V. Anthony, Dept. of Physiology, Colorado State University, Fort Collins, CO) was used for hybridization. For VEGF, a 464-base biotin-labeled riboprobe (bases 1–464 from the entire coding sequence of ovine VEGF₁₂₀ [20]) was used for hybridization. Similarly, for actin, a 266-base biotin-labeled riboprobe (202 bases transcribed from a partial cDNA sequence for ovine ß-actin [20]) was used. After hybridization and RNase digestion, the protected hybrids were separated on a 6% polyacrylamide denaturing gel, electroblotted to a nylon membrane, and detected by using BrightStar BioDetect chemiluminescence reagents (Ambion). Protected fragments corresponding in size to their respective riboprobes were quantified by densitometry as we have described [19, 20]. For each protected fragment, an autoradiographic exposure was used that was within the linear range of detection based on a validation conducted with differing amounts (3.12, 6.25, 12.5, 25.0, and 50 µg) of tcRNA from ovine liver. Densitometric data for VEGF and bFGF mRNA were expressed relative to that of actin mRNA.

Immunohistochemistry

Basic FGF and VEGF proteins were immunolocalized in paraffin-embedded, Carnoy's-fixed uterine tissue sections (6 µm) by using specific primary antibodies (bFGF: SC-79 rabbit polyclonal anti-peptide [amino acids 40–63 of human bFGF] antibody, Santa Cruz Biotech., Santa Cruz, CA; VEGF: Red1 rabbit polyclonal anti-peptide [amino acids 27–41 of ovine VEGF] antibody [20]) and the avidin-biotinylated peroxidase complex (ABC) system (Vector Lab., Burlingame, CA) with Vector SG (Vector Lab.) as the peroxidase substrate, as we have described before [16, 19, 21]. For controls, the primary antibodies were preadsorbed with their respective peptides (bFGF: AG-1, Santa Cruz Biotech.; VEGF: Red1 peptide [20]), and no immunohistochemical staining was detected when the preadsorbed antibodies were used (data not shown).

Statistical Analysis

Data from the densitometric analysis of the RPA were analyzed by using General Linear Models procedures, with time after E₂ implant as the main effect [22]. Differences between specific means were determined by using Bonferroni's t-test [18]. Unless otherwise indicated, data are reported as means with their respective pooled SE.

	RESULTS

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RPA

Protected RNA fragments 464, 420, 292, and 202 nucleotides in length were observed, which, on the basis of previous [20] as well as preliminary studies, represented VEGF₁₂₀, VEGF_164+188, bFGF, and actin, respectively (Fig. 1). Because the expression of VEGF₁₂₀ and VEGF_164+188 mRNA followed similar patterns (Fig. 1), only densitometric evaluation of VEGF₁₂₀ mRNA is presented in Figure 2. Whether or not it was expressed relative to that of actin mRNA, endometrial expression of VEGF₁₂₀ mRNA increased (p < 0.01) more than 5-fold from 0 to 4 h, remained elevated at 8 h, and then declined (p < 0.01) through 72 h (Fig. 2). Although they followed similar patterns, the ratio of VEGF₁₂₀ to VEGF_164+188 mRNA increased (p < 0.01) slightly (1.3-fold; SE = 0.1) from 0 to 24 h and then remained elevated through 72 h. In contrast with the pattern of VEGF mRNA expression, endometrial expression of bFGF mRNA remained relatively constant from 0 to 4 h, increased (p < 0.01) 2.2-fold from 4 to 8 h, remained elevated at 24 h, and then declined (p < 0.01) through 72 h (Fig. 2).

View larger version (68K): [in this window] [in a new window]   FIG. 1. RPA of endometrial mRNA for VEGF, bFGF, and actin in OVX, E2-treated ewes. Lanes represent individual samples obtained at the indicated hours post-implantation.

View larger version (19K): [in this window] [in a new window]   FIG. 2. Densitometric quantification of endometrial expression of mRNA for VEGF120 and bFGF in OVX, E2-treated ewes. Data are means for n = 4–5 ewes per group and are expressed as arbitrary optical density units relative to actin mRNA expression. SE = pooled SE.

Immunohistochemistry

Immunostaining for VEGF was observed throughout the uterus at all times (Figs. 3 and 4). In the myometrium, VEGF immunostaining was present in the walls of the microvessels, including both larger microvessels and capillaries, and also was present in the myometrial smooth muscle (e.g., Fig. 3D). In the endometrium, however, VEGF immunostaining was localized exclusively to the walls of the microvessels (Figs. 3 and 4). In both the myometrium and endometrium, the intensity of immunostaining for VEGF increased from 0 to 4 h after E₂ implant, remained relatively intense at 8 h, and then decreased again by 24 h (Figs. 3 and 4). In the walls of the larger endometrial microvessels, VEGF immunostaining was present primarily in the vascular smooth muscle cells near the vascular lumen at 0 (Fig. 4, A and B) and at 24 to 72 h (Fig. 3, E and F; Fig. 4F), but was observed throughout the vascular wall at 4–8 h (Fig. 3, C and D; Fig. 4D). In addition, at 0 and at 24–72 h, VEGF immunostaining was present primarily in deep endometrial microvessels, but at 4 and 8 h VEGF immunostaining was present in deep endometrial microvessels and also in those near the uterine lumen (Fig. 4). Thus, the pattern of VEGF immunostaining followed the pattern of VEGF mRNA expression in that it was increased dramatically at 4 and 8 h after E₂ implant.

View larger version (120K): [in this window] [in a new window]   FIG. 3. Immunohistochemical localization of VEGF in sections of deep endometrium of OVX, E2-treated ewes. Sections represent caruncular (A) and intercaruncular (B) endometrium at 0 h; caruncular (C) and intercaruncular (D) endometrium at 4–8 h; and caruncular (E) and intercaruncular (F) endometrium at 24 h after E2 treatment. *Lumens of some of the microvessels (red blood cell ghosts are clearly visible in several of these); m, myometrium; s, stroma; g, uterine gland(s). Bar = 50 µm.

View larger version (120K): [in this window] [in a new window]   FIG. 4. Immunohistochemical localization of VEGF in sections of luminal endometrium of OVX, E2-treated ewes. Sections represent caruncular (A) and intercaruncular (B) endometrium at 0 h; caruncular (C) and intercaruncular (D) endometrium at 4–8 h; and caruncular (E) and intercaruncular (F) endometrium at 24 h after E2 treatment. *Lumens of some of the microvessels (red blood cell ghosts are clearly visible in several of these); e, luminal epithelium; s, stroma; g, uterine gland(s). Bar = 50 µm.

In contrast with that of VEGF, immunostaining for bFGF was observed exclusively in the luminal epithelium and glands of the endometrium at all times (Fig. 5) but was not detected in the myometrium. At 0 h, immunostaining for bFGF was relatively strong in deep glands (Fig. 5A), whereas the intensity of staining was relatively weak in luminal epithelium and luminal glands (Fig. 5B). By 8 h, the intensity of the immunostaining for bFGF had increased dramatically in the deep glands (Fig. 5C) and also in the luminal epithelium and luminal glands (Fig. 5D). In addition, at 8 h, immunoreactive bFGF also seemed to be present in the lumen of some of the deep glands (Fig. 5C). By 24 h, the intensity of bFGF immunostaining was reduced in the epithelial cells of the deep glands, but abundant immunoreactive bFGF was present in their lumina (Fig. 5E). At 24 h, immunostaining also was reduced in the luminal epithelium and luminal glands (Fig. 5F). At 72 h, the intensity of bFGF immunostaining was reduced in the deep glands, where it was present primarily in the apical regions of the cells (Fig. 5G), and also remained low in the luminal epithelium and luminal glands (Fig. 5H). Thus, the time-course of bFGF immunostaining appeared to follow that of bFGF mRNA expression, with a transient increase at 8–24 h. In contrast, a relatively weak immunostaining for bFGF was present in the luminal stroma at all times (Fig. 5, B, D, F, and H).

View larger version (115K): [in this window] [in a new window]   FIG. 5. Immunohistochemical localization of bFGF in uterine tissue sections of OVX, E2-treated ewes. Sections represent intercaruncular tissues at 0 h (A, B), 8 h (C, D), 24 h (E, F), and 72 h (G, H) after E2 treatment. Sections are from deep (A, C, E, and G) or luminal (B, D, F, and H) regions of the endometrium. e, Luminal epithelium; s, stroma; g, uterine gland(s). Bar = 25 µm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
In the present study, we have shown that uterine expression of VEGF and bFGF are dramatically increased within 4–8 h after E₂ treatment of OVX ewes. In addition, for both VEGF and bFGF, the expression of mRNA and protein followed similar patterns. In the endometrium, immunodetectable VEGF protein was low or undetectable at 0 h after E₂, increased dramatically at 4–8 h, and then declined. Surprisingly, although endometrial bFGF protein increased dramatically by 8 h after E₂ in association with increased bFGF mRNA, substantial bFGF protein was detectable at 0 h, indicating that even after long-term withdrawal of ovarian steroids, angiogenic factors—and especially bFGF—persist in the endometrium. In a companion study, we found that endometrial vascularity also was surprisingly high after long-term ovariectomy [17]. In addition, the timing of increased expression of angiogenic factors VEGF and bFGF preceded the dramatic uterine growth and microvascular responses to estrogen, which occur at 8–24 h after treatment [17]. Thus, the present data provide further evidence that VEGF and bFGF are important uterine angiogenic factors. Moreover, maintenance of both angiogenic factors and a relatively healthy vascular bed even after long-term ovariectomy may be keys to enabling the uterus to grow so rapidly and dramatically upon exposure to E₂.

The present data also indicate that estrogen is a major regulator of VEGF and bFGF expression in the uterus. Previous studies have shown that E₂ stimulates expression of bFGF in endometrial adenocarcinoma cell lines in culture [23]. In addition, Rider and Psychoyos [10] demonstrated that inhibition of progesterone receptors with RU-486 results in the loss of bFGF expression and reduced stromal cell proliferation during early pregnancy in rats. Since estrogen appears to be an important regulator of uterine responsiveness to progesterone [24], probably via maintenance of uterine progesterone receptors [25], it seems likely that one mechanism by which estrogen regulates uterine levels of bFGF is through maintenance of progesterone receptors.

In primates including humans, endometrial VEGF expression varies throughout the menstrual cycle, suggesting that ovarian steroids also regulate endometrial VEGF expression in these species [26]. In mice, endometrial expression of VEGF mRNA changed throughout the estrous cycle, shifting from the luminal epithelium during the proliferative phase to the luminal stroma during the secretory phase, again indicating that ovarian steroids modulate endometrial VEGF expression [27]. Similar to our observations in the present study, E₂ or estriol induced rapid (within 1 h, and maximal at 2 h) and large (8-fold) increases in uterine VEGF expression in OVX rats [13].

E₂ also has been shown to stimulate VEGF expression in endometrial adenocarcinoma cell lines as well as primary cultures of human endometrial cells [12, 15]. As observed for the in vivo models, the VEGF response to E₂ also was rapid in primary endometrial cell cultures, reaching maximal levels by 1 h after estrogen treatment [15]. In addition, concentrations of VEGF in peritoneal fluid were greater in women with moderate to severe endometriosis compared with those with minimal to mild endometriosis, suggesting that VEGF may be important not only in normal but also in pathological angiogenic processes in the endometrium [15].

Vascular endothelial growth factors are unique among growth factors that influence angiogenic processes, in that their effects appear to be exclusively on vascular endothelial cells [28, 29]. Interestingly, VEGF and VEGF receptors are present not only in growing but also in quiescent endothelial cells, for example in adult tissues such as kidney, adrenal, liver, pituitary, heart, lung, and brain [29, 30], suggesting their role not only in vascular growth and development but also in vascular maintenance. Recently, abnormal cardiovascular development and organization, resulting in fetal death by midgestation, was reported for mouse embryos in which the genes for VEGF or VEGF receptors were inactivated, demonstrating the critical requirement for VEGF in vascular development and organization in the mouse fetus and placenta [31–34]. Thus, a role for VEGF as a major regulator of endometrial angiogenesis, as suggested in the present study, seems not only plausible but highly likely. An additional role for VEGF is in stimulating increased vascular permeability, leading to the increased uterine tissue water content observed in sheep as described in our companion manuscript [17] and suggested by previous studies in rats [13, 35, 36].

In those species studied, various isoforms of VEGF exist, and they result from alternative splicing of eight exons contained in a single gene [28, 29]. In sheep, the VEGF isoforms contain 120, 164, or 188 amino acids based on the cDNA sequences, which are designated VEGF₁₂₀, VEGF₁₆₄, and VEGF₁₈₈, respectively [20]. The smaller isoform, VEGF₁₂₀, probably does not bind to heparin or heparan sulfates, and therefore is freely soluble in the extracellular compartment [28, 29]. In contrast, VEGF₁₆₄ and VEGF₁₈₈ are thought to bind strongly to heparin and therefore are probably sequestered in the extracellular matrix [28, 29]. Additionally, the VEGF isoforms may differ in their potencies for stimulating various endothelial functions [28, 29]. These differences are interesting in light of the observations from the present study, in which the ratio of VEGF₁₂₀ to VEGF_164+188 increased by 24 h after E₂ treatment and remained elevated through 72 h. Thus, by 24-h, expression of the smaller, more soluble, and presumably more biologically available isoform was stimulated by E₂ to a greater extent than were the larger isoforms.

FGFs, although not exclusively acting on vascular endothelial cells, also are potent angiogenic factors that have been identified in the endometrium of several mammalian species [6–10, 37, 38]. In addition, the FGFs have numerous other actions, including promotion of mesodermal differentiation and cardiovascular development in the embryo/fetus [39, 40]. The role of FGF in embryonic development is intriguing in light of the potential that bFGF is a secretory product of the uterus, as suggested by the present data, as well as by previous reports in which bFGF was identified in uterine flushings of pigs and rats [6, 8]. Thus, FGF may play additional roles in uterine function including regulation of embryonic differentiation.

Moreover, synergism between VEGF and bFGF has been demonstrated with in vitro models of angiogenesis [41, 42]. In the present study, we found VEGF localization to the microvascular smooth muscle of the endometrium, whereas bFGF was localized to the luminal and glandular epithelium as well as the stroma. Because bFGF has been shown to up-regulate the expression of VEGF in vascular smooth muscle cells [43], endometrial bFGF may regulate VEGF expression in endometrial microvascular smooth muscle. However, in the present study, the increase in endometrial VEGF expression preceded that of bFGF expression, indicating that bFGF may not be the primary regulator of VEGF expression in the sheep uterus. Another potential paracrine interaction involves stimulation of endothelial cell growth and permeability during vascular remodeling associated with the dramatic growth response of the uterus to E₂ treatment [17], since VEGF receptors have been shown to be present exclusively on endothelial cells in numerous tissues including the endometrium [28, 29], whereas the present study clearly demonstrates the presence of VEGF in vascular smooth muscle.

In summary, we have shown a dramatic up-regulation of endometrial expression of the angiogenic factors VEGF and bFGF in response to E₂ treatment of OVX ewes. This response preceded by several hours the E₂-induced growth and microvascular responses of the endometrium in the same animals [17]. On the basis of these data as well as previous observations by us and others, we suggest that VEGF and bFGF are major regulators of the profound microvascular response of the endometrium to E₂. This experimental paradigm will enable us to further investigate the role of these factors as well as their mechanisms of action, not only during microvascular growth and development in response to ovarian steroids but also during the dramatic endometrial microvascular response to the conceptus [18, 44].

	NOTE ADDED IN PROOF

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION NOTE ADDED IN PROOF REFERENCES

 
Recently, Rider et al. (J. Endocrinol. 1997; 154: 75–84) have shown dramatic up-regulation of uterine bFGF mRNA within 3 h after estrogen treatment of OVX rats: this response was augmented by progesterone.

	FOOTNOTES

 
¹ A publication of the North Dakota Agric. Exp. Sta., Project 1795.

² Correspondence. FAX: (701) 231–7590; lreynold{at}prairie.nodak.edu

Accepted: April 21, 1998.

Received: January 7, 1998.

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