Regulation of Vascular Endothelial Growth Factor Expression by Estrogens and Progestins

This article is based on a presentation at the conference on Women's Health and the Environment: The Next Century--Advances in Uterine Leiomyoma Research held 7-8 October 1999 in Research Triangle Park, North Carolina, USA.
Address correspondence to S.M. Hyder, Dept. of Integrative Biology and Pharmacology, University of Texas-Houston Medical School, PO Box 20708, Houston, TX 77225 USA. Telephone: (713) 500-7459. Fax: (713) 500-7344. E-mail: salman.hyder@uth.tmc.edu
This work was supported by National Institutes of Health grants HD-08615 and ES-06995, the Texas Affiliate of the American Heart Association, and a Fleming/Davenport Award from the Texas Medical Center.
Received 23 February 2000; accepted 9 May 2000.

Angiogenesis is the growth of new blood vessels from an existing capillary network, and there are a number of important reasons to consider the regulation of angiogenic factors by sex steroids in the setting of uterine fibroids. a) Leiomyomas are vascularized tumors, and the nutrient supply they require for growth must be provided via an increased capillary network in order for these smooth muscle tumors to expand. These tumors can be very large in some cases and clearly contain a well-developed blood supply that must involve expansion of the myometrial vasculature. b) Although the precise etiology of fibroids is not known, there is a general association of the disease with ovarian steroids. The disease is most prevalent during childbearing years and regresses during menopause. Medical management most commonly focuses on reducing ovarian steroid levels, e.g., by the continuous administration of gonadotropin-releasing hormone (GnRH) agonists. This historical information may now be coupled with more recent studies showing that estrogens and progestins regulate the expression of several potent angiogenic factors, including vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF). c) Common symptoms of leiomyomas include prolonged and/or heavy menstrual bleeding (menorrhagia) as well as bleeding between menstrual periods or after intercourse. d) In addition to medical castration via GnRH administration, antiprogestins and antiestrogens are potential therapeutic options for the treatment of fibroids. Because sex steroids affect expression of factors such as VEGF and FGF, systemic use of these agents has the potential to disrupt angiogenesis and vascular function in normal uterine tissue and other target sites. Studies in our laboratories have focused on the regulation of VEGF by sex steroids, and we present several of our findings in this area in this article.

VEGF is a multifunctional cytokine, originally identified as a protein produced by tumor cells, that increased the permeability of capillaries to proteins (1). It was subsequently discovered to be mitogenic for endothelial cells and to stimulate angiogenesis (2,3). Although numerous factors are now known to regulate angiogenesis and capillary function, VEGF remains the most selective angiogenic factor discovered to date and appears to regulate these processes in many normal tissues and tumors. This factor has been referred to as vascular permeability factor, VEGF, or vasculotropin in the literature. VEGF is expressed in both epithelial and mesenchymal cells in a wide variety of tissues, and it is highly expressed in many tumors [(2,4-6), and references therein]. Both the human and murine VEGF genes contain eight exons and seven introns, and differential exon splicing generates four predominant forms containing 121, 165, 189, and 206 amino acids in the human, and corresponding forms with 1 amino acid less in the mouse (2). In most systems VEGF-121 and VEGF-165 are the major species expressed. A major difference between these different forms of VEGF is their heparin-binding capability. VEGF-121 is readily diffusible, but the other major forms contain heparin-binding regions that can mediate binding to cell surfaces and the extracellular matrix and thus provide a potential reservoir for locally controlled release by heparinases or plasmin (3). This is a potentially important consideration, as leiomyomas often contain large amounts of collagen and other components of the extracellular matrix that have the potential to serve as reservoirs for VEGF and other heparin-binding growth factors.

The first form of VEGF identified is now referred to as VEGF A, but a number of VEGF-related genes referred to as VEGF B, C, D, E, and placental growth factor have subsequently been identified (7,8). Some of these forms may have selectivity for certain functions, e.g., VEGF-C may be the factor that regulates capillary growth in the lymphatic system (9,10). In addition, as VEGF binds to its receptor as a dimer (see below), the various VEGFs can form heterodimers, either with variants of the same family or with members of another family, that may have different activities. A reproductive tract-specific VEGF 145 has also been reported in the literature (11), but this variant is not consistently observed. VEGFs act by binding to plasma membrane receptors that have tyrosine kinase activity. There are three members of the VEGF receptor family: VEGFR-1 (Flt), VEGFR-2 (flk/KDR), and VEGFR-3 (Flt4). The first two bind the classical VEGF (VEGF A) and the latter interacts with VEGF C and D. These receptors are expressed primarily, but not exclusively, on endothelial cells, and the mitogenic actions of VEGF are thought to be mediated primarily by VEGF-R2. Several other forms of VEGF receptors have been identified, but their physiological functions are not clearly established (6,12). A thorough analysis of the types of VEGF and VEGF receptors that may be present in leiomyomas has not yet been reported.

In 1993, VEGF expression was reported in the mouse (13), human (11), and rat uterus (14) and has since been identified in the uteri of other species (15). Most studies have observed VEGF expression in the epithelial and stromal layers of the uterus (15), but expression of the angiogenic factor (11,16) and its receptor (17) has also been observed in the myometrium. In 1993, multiple reports (11,13,14) indicated that female sex steroids regulated expression of VEGF, and in the remainder of this chapter we will summarize our studies on the nature and mechanism of this regulation.

Results

Regulation of Uterine VEGF Expression by Estrogens

For our initial studies we employed the immature rat uterus as a model system, as it has been used extensively to study the regulation of gene expression by estrogens and progestins. As seen in Figure 1, administration of either the endogenous hormone 17ß-estradiol or the widely used synthethic estrogen 17-ethinyl estradiol causes a massive (> 10-fold) increase in expression in the level of VEGF mRNA. The data in Figure 1 were obtained by densitometric analysis of the density of VEGF transcripts observed by Northern blot analysis. Of the many genes we have studied in this animal model, such as immediate early genes, growth factors, and protooncogenes [(18) and references therein], VEGF is the most rapidly induced after estrogen treatment. In addition, the dose-response curve for induction of this gene by either estrogen used in Figure 1 is shifted slightly to the left of the dose-response curves for induction of other genes in the uterus (18), suggesting that VEGF is more sensitive to induction by estrogens than other estrogen-responsive genes studied in this experimental system. In the immature animal, this induction of VEGF occurs primarily in the stromal cell layer, as previously reported (18,19), and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that VEGFs 164 and 120 are the major forms of the growth factor induced (19). This induction is blocked by inhibitors of RNA but not protein synthesis (19), suggesting that the hormone increases de novo synthesis of the transcript and that this is a primary estrogen receptor (ER)-mediated effect.

Figure 1. Induction of VEGF mRNA by estrogens. Immature rats were treated with estradiol (E₂) or ethinyl estradiol (EE) for the indicated times prior to sacrifice and preparation of uterine RNA. VEGF mRNA levels in total uterine RNA were measured by Northern blot analysis, as described by Hyder et al. (18). Values are in arbitrary units based upon densitometric scans of the resultant films.

Another indication that VEGF induction occurs via the classical ER pathway is that the pure antiestrogen ICI 182,780 blocks increased production of the growth factor transcript by estrogen (20). Interestingly, however, the blockade of VEGF induction by the ICI compound displays a dose-response curve shifted to the right relative to that for inhibition of other responses such as the estrogenic induction of the protooncogene c-fos (20). This indicates that the antiestrogen selectively blocks the induction of other uterine genes relative to VEGF, suggesting differences in the exact mechanism by which estrogens induce this growth factor and other genes. At present we do not understand the molecular basis for this selectivity, but it could be because of differences in hormone response elements, co-activators, or corepressors involved in ER induction of different genes.

Estradiol also regulates VEGF expression in cultured human endometrial stromal cells, as shown in Figure 2. This illustrates that estradiol (10 nM) increases the level of VEGF protein in the media of stromal cells within 2 hr after hormone addition to the medium and that VEGF secretion continues to increase for at least 40 hr. Other studies in this experimental system showed that estrogen also increased the level of VEGF mRNA in the cells; RT-PCR analysis showed that the predominant forms of the transcript in these cells code for VEGF 121 and 165 as in the rat system (21). As in the rat, the antiestrogen ICI 182,780 was able to partially blunt, although not completely abolish, the increase in VEGF (21). Thus, estrogens are able to regulate VEGF expression in both rodent and human stromal cells.

Figure 2. Secretion of VEGF protein by human endometrial stromal cells. Primary cultures of human endometrial stromal cells were prepared as described by Huang et al. (21), and 10 nM estradiol was added. VEGF protein levels in the media at various times after hormone addition were measured with a specific enzyme immunoassay, as described by Huang et al. (21).

Because the widely used antiestrogen tamoxifen has estrogen-agonist activity in the uterus, we also examined the ability of the drug and related triphenylethylene antiestrogens to induce VEGF in this experimental system. These studies revealed that tamoxifen (19) and other antiestrogens in this class, including nafoxidine, 4-hydroxytamoxifen, and clomiphene, all induce VEGF expression (22). The induction of VEGF gene expression by these partial agonists is blocked by pure antiestrogens and inhibitors of RNA, but not protein synthesis, suggesting that the triphenylethylene compounds are also acting via a classical ER-mediated pathway. As for 17ß-estradiol and 17-ethinyl estradiol, tamoxifen induction of VEGF mRNA levels also occurs primarily in the stromal layer of the immature rat uterus (19).

To further explore the mechanism by which estrogens regulate expression of VEGF, we next searched for functional estrogen response elements (EREs) in the growth factor gene. Our initial efforts focused on the upstream 5´-flanking region of the gene, as most steroid response elements identified to date are found in this region of target genes. We were unable to identify any sequences in this region, however, that specifically bound the ER or appeared to confer hormone inducibility to reporter constructs. We thus broadened our search and examined the entire VEGF gene for sequences with homology to the consensus ERE, GGTCAnnnTGACC, present in the vitellogenin gene. In a somewhat surprising series of experiments, we identified two regions of the VEGF gene that selectively bound the ER in band shift assays. One of these elements is located in the 5´ untranslated region (UTR) of the gene and the other in the 3´ UTR, as shown schematically in Figure 3. The sequence of these elements is also indicated in the figure, and it is seen that both have several base changes (shown in lowercase letters) relative to the consensus ERE. To our knowledge this is the first example of a gene that contains EREs in both 5´ and 3´ UTRs.

Figure 3. Location and sequence of putative EREs in the rat VEGF gene. The rat VEGF gene contains eight exons and seven introns, as schematically illustrated. The sequence GGgCAnnnTGACt is located in the 5´ untranslated region of exon 1 between the start site of transcription and ATG that initiates translation, and the sequence GagCAnnnTGcCC in the 3´ untranslated region, as illustrated. (The bases shown in lowercase letters indicate those that differ at the indicated positions from the consensus element, GGTCAnnnTGACC.) Both VEGF sequences bind the ER and confer hormone inducibility to reporter constructs (see text for details).

Both of these sequences bind to human recombinant ER-, which is the predominant form of the ER present in the uterus. This binding is specific, as it is inhibited by excess unlabeled DNA, the complex is supershifted by antibodies to ER-, and binding is destroyed by several mutations in either half-site of the elements. Other experiments revealed that both of these elements also bind human recombinant ER-ß in band shift experiments, although we have not yet directly compared the affinities of the wo forms of the ER for these sequences. These studies have been completed recently and submitted for publication (23).

In addition to binding the ER, both the 5´ and 3´ elements illustrated in Figure 3 confer estrogen inducibility to reporter constructs. Co-transfection experiments with expression plasmids for ER- or ER-ß indicate that both forms of the receptor can affect expression from reporters containing these EREs. These transcription studies are complicated, however, because the nature (activation or repression) and the magnitude of these transcriptional effects depends upon both the receptor type present and the orientation of the element in the constructs (23). These studies support our previous pharmacological studies that estrogens regulate VEGF expression at the transcriptional level but indicate that the nature and exact mechanism of this regulation may be more complex than that previously observed for other genes.

Regulation of Uterine VEGF Expression by Progestins

In addition to studies on estrogenic regulation of VEGF in the uterus, we and others have also examined the regulation of this angiogenic factor by progestins. Cullinan-Bove and Koos (14) originally showed that progestins increase VEGF expression in the immature rat uterus, and we have confirmed their observations. The magnitude of induction by progestins is less than that observed by estrogens, however, and the onset of induction is also slower. More recently we have observed, by in situ hybridization studies, that progestins also induce VEGF transcription primarily in the stromal layer of the rat uterus (24). Interestingly, however, progestins seem to elevate mRNA levels uniformly throughout the stromal layer, whereas estrogens appear to affect expression primarily in the stromal layer nearest the uterine lumen. Although this preliminary observation needs to be firmly established by quantitative morphometry, it raises the possibility that the two hormones may affect VEGF expression in different stromal cell populations. In other recent studies we have also observed that the induction of VEGF expression in the uterus is blocked by the antiprogestin mifepristone (RU-486), and that the 5´-flanking region of the VEGF gene contains sequences that confer progestin inducibility to reporter constructs in transfection studies. However, this region of the gene does not appear to contain a classical progesterone-response element, and we have not yet extended these studies to include deletion experiments (25). Although it remains to be firmly established, these studies suggest that progestins also regulate uterine VEGF expression by a classical nuclear receptor-mediated pathway.

It thus appears that the overall regulation of VEGF expression in the uterus will represent an interplay of estrogen and progestin effects, but little specific information is available at present about the precise interaction between these 2 steroids. An understanding of the physiological interactions between estrogens and progestins in the regulation of uterine VEGF expression will probably require studies in mature animals, however, as the interactions in that setting may be quite different than those in the immature rat model that we have studied to date. For example, it has been known for some time (13) that uterine VEGF expression varies throughout the estrus cycle of rodents, and the expression of both VEGF and its receptors appears to vary throughout the human menstrual cycle (26-28).

Regulation of VEGF Expression in Breast Cancer Cells

Uterine leiomyoma occurs with such a high incidence that many women with fibroids undoubtedly have other diseases affected by steroid hormones, including cancers such as those of the endometrium and breast. Because the etiology of all these diseases may involve exposure to endogenous or exogenous steroids and because various hormonal therapies are employed in their treatment, we and others have been interested in studying the regulation of VEGF by sex steroids in hormone-responsive cancers as well as normal target tissues. In particular, we are interested in control of VEGF expression in breast cancer.

It is increasingly clear that angiogenesis plays an important role in the growth and metastasis of many types of cancer, including breast cancer (29,30). For example, tumors may not grow past a certain size unless the density of microvessels in their vicinity increases, and the density of blood vessels near a tumor can influence its ability to metastasize. It is also known that the production of angiogenic factors such as VEGF is high in many human breast tumors and that there is, in general, an inverse relationship between the level of VEGF expression in a patient's tumor and her overall prognosis (31-33). In addition at least one recent experimental study demonstrated that estrogens increase VEGF expression in rat dimethylbenz[a]anthracene (DMBA)-induced mammary tumors (34). Given these observations and the clear effect of steroid hormones on breast cancer, we decided to extend our studies on estrogen and progestin regulation of VEGF to human breast cancer cells.

To initially investigate the effects of progestins, we used human T47-D breast cancer cells, which express high levels of the progesterone receptor (PR). These studies illustrated that progesterone causes a 3- to 4-fold induction of VEGF protein secretion into the cell growth medium (Figure 4). This effect is specific for progesterone, and interestingly, neither estradiol nor the synthetic estrogen diethylstilbestrol increases VEGF secretion in these cells that do contain ER. Other experiments revealed that medroxyprogesterone acetate and a variety of synthetic progestins used for contraception and hormone replacement therapy cause similar increases in VEGF secretion. Thus, this appears to be a generalized effect of progestins, both endogenous and synthetic. This effect occurs with physiological levels of progesterone, with increases in VEGF secretion observed in the dose range of 0.1 to 10 nM progesterone (35). Not all PR-containing cell lines display increased VEGF production in response to progestins, however, as we have not observed this effect in MCF-7 cells, ZR-75 cells, MDA-MB-231 cells, or the Ishikawa endometrial carcinoma cell line (35). This clearly indicates that regulation of VEGF expression by estrogens and progestins is tissue and cell-type specific.

Figure 4. Induction of VEGF secretion by progesterone (Prog) in T47-D human breast cancer cells. Cells were incubated overnight with 10 nM progesterone alone (Prog), progesterone plus a 100-fold excess of the antiprogestin RU-486 (Prog + RU), the antiprogestin alone (RU), or the vehicle (control). VEGF was then measured in the medium using an enzyme-linked immunosorbent assay as described by Hyder et al. (35).

The mechanism of progestin regulation of VEGF expression is not unequivocally established but appears most likely to represent a direct PR-mediated transcriptional event. This is suggested by the blockade of progesterone induction by the antagonist RU-486 (Figure 4) and the dose-response relationship for VEGF induction (35). More importantly, As noted in a previous section, we have shown in transfection studies that the 5´-flanking region of the rat VEGF gene confers progestin inducibility to reporters in HeLa cells co-transfected with human PR, and preliminary studies have also shown that progestins increase the level of VEGF mRNA in T-47D cells (25).

We have not yet observed an induction of VEGF protein or mRNA expression by estrogens in any of the human breast cancer cell lines we have studied in vitro (T-47D, MCF-7, ZR-75, or MDA-MB-231). This is in contrast to the more recent work of Ruohola et al. (36), who have reported that estrogens can induce expression of certain forms of VEGF in cultured MCF-7 cells. This may indicate that different clonal cell lines of these cells respond differently, or it may be due to other differences in experimental protocols. Brodie and colleagues have also observed that estrogens increase VEGF expression in rat DMBA-induced tumors in vivo (34). This indicates that estrogen can control expression of the gene in DMBA-induced rodent tumors, which provides another experimental system for studies. This in vivo system may be especially useful to determine if estrogen effects involve paracrine interactions between different cell types, which might not be observed in cell culture studies. Although many specific questions remain to be answered, the data available at present strongly indicate that estrogens and progestins may regulate VEGF expression in breast cancer cells as well as normal uterine stromal cells.

Regulation of VEGF Expression by Cross-Talk between Steroids and Other Signaling Pathways

VEGF expression is regulated in other systems by a number of signaling pathways, including those involving activation of protein kinase A and protein kinase C activities [(5) and references therein]. It is becoming increasingly clear that cross-talk between these pathways and steroid receptor-mediated pathways regulates expression of many genes. We have thus initiated studies to determine if regulation occurs via these pathways in the uterus and/or breast cancer cells. In preliminary studies we have found that the tumor promoter 12-O-tetradecanoyl-13-acetate increases expression of VEGF in T47-D cells at both the mRNA and protein levels. We are now in the process of investigating if cross-talk occurs between steroid and protein kinase C-mediated pathways. Although the regulation of VEGF expression by nonsteroidal factors is beyond the scope of this manuscript, this is a key area for future studies if we are to understand the overall regulation of VEGF expression in steroid-responsive tissues and tumors, including fibroids.

Discussion

In 1993 VEGF expression was initially reported in the uterus of the mouse (13), human (11), and rat (14), and has since been identified in the ewe, the rabbit, and the monkey [reviewed in (15)]. As in other tissues, multiple forms of VEGF are produced in the uterus, including VEGF-189, -165, -145, and -121 in the human, VEGF-188, -164, and -120 in the rat, and VEGF-189, -165, and -121 in the monkey. In all these systems VEGF-121(120) and -165(164) appear to be the predominant forms produced; immunological measurements have demonstrated that VEGF protein is also produced in monkeys, mice, rabbits, and humans (15,37). It thus seems clearly established that VEGF is ubiquitously expressed in uterine tissue of various species. It is especially noteworthy that VEGF has been identified in human uterine leiomyomas (16) and that VEGF receptors are also present in myometrial cells (17).

Estrogens regulate VEGF mRNA expression in human endometrial adenocarcinoma cells (11), primary cultures of human uterine stromal cells (21,26), and the rat uterus in vivo (14,18,19). These are likely to be primary ER-mediated effects because induction is very rapid, blocked by pure antiestrogens (18-20), and inhibited by actinomycin D but not puromycin or cycloheximide (14,19). In addition we have recently identified functional EREs in the VEGF gene (23). Partial estrogen agonists such as tamoxifen (19) and related triphenylethylene antiestrogens (22) also induce VEGF expression in the uterus, which is consistent with their agonist activity in that tissue. This regulation by estrogens is consistent with most studies on the expression of VEGF throughout the estrus cycle in rodents (13,38) and the menstrual cycle in humans (26). There are several reports that progestins can also induce VEGF expression in human stromal cells (39,26) and the rodent uterus (14). The effects of progestins on VEGF expression have not been as well studied as those of estrogens, and less information is available about the possible molecular mechanism of progesterone regulation of VEGF. However, progesterone increases VEGF protein expression in a human breast cancer cell line; this effect is blocked by RU-486, suggesting it is mediated by the PR (35). Thus, the overall picture that emerges from these studies is that VEGF expression is likely to be under the control of both estrogens and progestins. However, at present very little is known about the molecular mechanisms that regulate expression in the reproductive tract and substantial differences may exist between cell types and in different species.

It seems important to investigate in further studies the degree to which VEGF expression in normal uterine tissue and leiomyomas is regulated by estrogens and progestins, and to determine if endocrine therapies such as medical castration with GnRH-releasing factors, antiestrogens, and/or antiprogestins may act in part by decreasing VEGF levels in this disease. In this regard, it is important to note that VEGF has other activities besides stimulating the proliferation of capillary endothelial cells. In particular, VEGF is the most potent agent known for affecting capillary permeability (1). Thus, one can consider the possibility that VEGF might have multiple actions in normal tissues and fibroids. One would be an increase in angiogenesis, but a second factor conceivably quite important is the increase in capillary permeability for growth factors, proteins, and other nutrients present in plasma. Even in the absence of frank angiogenesis, such an effect could enhance the growth of fibroids by increasing their nutrient supply and proliferation signals. Finally, it is also possible that VEGF could have indirect actions in leiomyomas and other diseases. It is now recognized that endothelial cells themselves produce a number of growth factors (40). Thus, VEGF or other angiogenic factors might affect the local production of such growth factors by increasing the number of endothelial cells and/or their synthesis and secretion of growth signals for either normal smooth muscle or leiomyomas.

Although VEGF is a very potent and highly selective angiogenic factor, we also wish to note that several other factors are important regulators of angiogenesis and should be considered candidates to regulate this process in leiomyomas, alone or in conjunction with VEGF. In particular, FGF is likely to be an important regulator of angiogenesis in a variety of systems (41-44) including the uterus (15). Basic FGF (bFGF) was actually the first purified growth factor shown to have angiogenic activity (41). It increases the proliferation, migration, and differentiation of endothelial cells, smooth muscle cells, and fibroblasts, all of which express FGF receptors (42,43). FGF acts synergistically with VEGF (44), and FGF has been reported to stimulate the synthesis of VEGF in a number of tumor cell lines (45). FGF may thus regulate angiogenesis by a combination of actions (42,43,46,47).

Importantly for the context of this discussion, bFGF has been identified in the normal, nonpregnant uterus of a number of species, including human, monkey, sheep, pig, mouse, rat, and guinea pig, and the growth factor has also been identified in uterine secretions from several species. Acidic FGF has also been identified in the nongravid human, monkey, and pig uterus. There are some differences in the patterns of localization of bFGF observed by immunohistochemical analysis in various studies, but bFGF staining has been reported in the basal lamina of uterine blood vessels in the endometrium and myometrium, in uterine stromal cells and extracellular matrix, in myometrial cells, and in the basal lamina of both glandular and surface epithelial cells. Immunoreactive FGF receptor 1 has also been identified in glandular epithelial cells, stromal fibroblasts, and endothelial cells in the human endometrium (33), raising the possibility that this growth factor exerts numerous intracrine, autocrine, and/or paracrine interactions in the uterus. There are also a number of reports suggesting that estrogens and progestins may regulate expression of FGF and its receptor in the uterus, although there are conflicting reports in some cases and the exact regulatory patterns remain to be established. See Hyder and Stancel [(15) and references therein] for a recent discussion of FGF in the reproductive tract.

Although much work remains for us to understand the regulation of FGF expression and its resultant effects in the uterus, this growth factor is another prime candidate to play a role in leiomyomas, as a) the growth factor (48) and its receptor (49) are both present in normal myometrium and leiomyomas; b) FGF is mitogenic for both cultured human myometrial and leiomyoma cells (50); c) some studies have reported that bFGF is overexpressed in leiomyomas relative to normal myometrium (48); and d) at least one form of FGF receptor (FGFR1) may be overexpressed in women with leiomyoma-related uterine bleeding (51).

In summary, abnormal uterine bleeding is a common symptom in women with leiomyomas and the growth of fibroids undoubtedly requires some increase in angiogenesis and possibly capillary permeability. This suggests that the disease may involve derangements in the factors that regulate angiogenesis and capillary function. These processes are now known to be regulated by a number of factors; two of these key regulators, VEGF and FGF, as well as their receptors, are expressed in the myometrial and stromal cell layers of the uterus. Furthermore, the level of expression of these proteins in the uterus appears to be regulated in large part by estrogens and progestins, which are clearly associated with the etiology of fibroids. This suggests that the expression of VEGF, FGF, their cognate receptors, and/or other angiogenic factors may represent potential targets for prevention and treatment of this disease, which is such a major public health problem.

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Last Updated: October 3, 2000