Regulation of Angiogenic Growth Factors in the Female Reproductive Tract by Estrogens and Progestins

Salman M. Hyder and George M. Stancel

Department of Integrative Biology, Pharmacology, and Physiology The University of Texas Medical School at Houston Houston, Texas 77225

BACKGROUND AND INTRODUCTION

Since time immemorial, women and men alike have undoubtedly recognized that pronounced changes periodically occur in the vasculature of the female reproductive tract. However, it was not until the beginning of the 20th century that it became clear these changes were under endocrine control by ovarian hormones. Within the last two decades, specific genes that regulate vascular growth and function have been identified (1), and we can now investigate their hormonal regulation. This is required to elucidate the physiological mechanisms that regulate changes in the endometrium throughout the menstrual cycle, the pronounced vascular changes that occur during implantation and placentation, and the pathophysiology of conditions such as dysfunctional uterine bleeding, uterine cancers, and endometriosis, which are major health problems in women.

A key function of the vasculature is to provide a nutrient supply to the reproductive tract, and in most species three major types of regulation occur: vasodilation; changes in capillary permeability; and growth and development of new vessels. In virtually all species studied, estrogens produce dramatic increases in uterine blood flow caused by vasodilation, and in general uterine hyperemia (i.e. an increase in blood in the tissue) increases as the ratio of circulating estrogen/progesterone increases. Despite the fact that these dramatic changes in uterine hyperemia have been recognized for many years, the molecular mechanism(s) for this effect remain to be established and will not be further considered here.

In addition to hyperemia, estrogens increase the permeability of uterine capillaries to water, small molecules, and proteins (2). While slightly slower in onset, changes in permeability are also quite rapid and are typically seen within several hours after hormone administration to experimental animals. These changes are thought to occur as the result of fenestrations that develop in the endothelial surface of capillaries after treatment with hormones (see Ref. 2 and references therein). Changes in blood flow and vascular permeability provide increased oxygen, nutrients, and plasma-borne growth factors, i.e. a perfusion effect, and also cause stromal edema. This edema and increased blood flow could also facilitate uterine growth by diluting or removing inhibitory factors. While admittedly speculative this possibility has not been well explored, and we believe that it merits consideration.

In addition to hyperemia and changes in permeability, marked changes also occur in the growth of vessels. In primates there is an increase in the length, branching, and coiling of the spiral arteries that supply the functionalis layer of the endometrium during each menstrual cycle as the endometrium is regenerated. The sole ovarian hormones required to produce these changes are estrogens and progesterone (3). In nonprimates this cyclical regeneration does not occur in the estrous cycle, but the growth of new vessels takes place if implantation and placentation occur. In either case, the production of new vessels adds to the perfusion effect as noted above, but it is now recognized that endothelial cells secrete a large number of cytokines and growth factors (4). Increasing the number of endothelial cells and/or altering their production and release of growth factors thus represents a potential paracrine effect that could contribute to the growth and proliferation of uterine cells, the infiltration of migratory cells, and/or autocrine effects on the endothelial cells themselves. This is another possibility that has not yet been widely studied in the female reproductive tract, but which we believe also merits consideration.

ANGIOGENESIS AND ANGIOGENIC FACTORS

Angiogenesis is the process of generating new capillaries from existing vessels. It occurs extensively during development, but in the adult organism angiogenesis is limited to relatively few tissues, including the ovary and uterus, but the formation of new vessels commonly occurs during tumor growth (1, 5) and in several other diseases (6), including endometriosis (7). During angiogenesis new capillaries originate from a preexisting microvasculature, mainly capillaries and venules, rather than from larger vessels with a smooth muscle layer. One of the first things that occurs in response to an angiogenic stimulus is an increase in capillary permeability (8). This allows fibrinogen and other serum proteins to exit the capillary space and form an extravascular fibrin gel. This matrix then supports the ingrowth of endothelial cells and other elements to generate new vascularized stroma. The generation of new capillaries involves: 1) local degradation of the basement membrane by matrix metalloproteinases; 2) movement of endothelial cells through the basement membrane and their ingrowth into the provisional perivascular matrix noted above; 3) proliferation of the endothelial cells; and 4) organization of the cells to form new vessels and the subsequent production of basement membrane along their surface. For additional information, interested readers are referred to several recent reviews about the basic process of angiogenesis (1, 5), and earlier reviews of angiogenesis in the female reproductive tract (9, 10). Despite this complexity, two factors emerging as key regulators of angiogenesis in most systems are basic fibroblast growth factor (FGF) or bFGF (11) and vascular endothelial growth factor or VEGF (12, 13), and the remainder of this article will thus focus on these specific proteins.

FGF

The FGF family consists of nine distinct FGFs and a roughly equal number of other factors with a high degree of homology and many similar activities. The two prototype factors are FGF-1, or acidic FGF (aFGF), which is usually isolated as a form with a molecular mass of 16–18 kDa, depending upon proteolysis, and FGF-2 or bFGF, which generally has a molecular mass of 18 kDa but may exist as larger forms up to 24 kDa. This family of growth factors interacts with at least four plasma membrane receptors, which are receptor tyrosine kinases, with the FGF-receptor 1 (FGF-R-1) having the widest pattern of cellular expression. The FGF receptors (FGFRs) are prototypical membrane receptors that dimerize upon FGF binding to activate a tyrosine kinase activity and autophosphorylate the receptor in trans. The activated receptors are in turn coupled to a number of signal transduction pathways in various cells, including phospholipase C{gamma}; the src family of tyrosine kinases; the Grb2-ras pathway; phosphatidyl-inositol 3'-kinase; as well as a number of other specific FGF adaptor proteins and kinase substrates. The FGFR system is complex in that it requires proteoglycans containing heparan sulfate for activity. These proteoglycans are cell membrane or extracellular matrix-bound proteins with carbohydrate side chains containing heparan sulfate that are essential for FGFR activation. The heparan sulfate is thought to either help bind and position the FGF correctly for presentation to its cognate receptor tyrosine kinase, or because FGF, its receptor, and heparan sulfate form a ternary complex necessary for activity. The expression of FGF is highly ubiquitous, and this growth factor produces mitogenesis, migration, and differentiation of a large number of cells studied in vitro. A very high percentage of cultured cells contain FGFRs, but it is not clear whether this widespread expression of FGFRs reflects the in vivo expression of this receptor tyrosine kinase or whether it results from explantation of cells and their growth in culture (see Refs. 11, 14 , and references therein for recent reviews of FGFs and their cognate receptors).

Of the various factors known to exhibit angiogenic activity in vitro, FGF is one of the prime candidates to regulate angiogenesis in vivo. Basic FGF was actually the first purified growth factor shown to have angiogenic activity (15), and it has been shown to increase the proliferation, migration, and differentiation of endothelial cells, smooth muscle cells, and fibroblasts, all of which express FGFRs (11). FGF acts synergistically with VEGF (16), and FGF has been reported to stimulate the synthesis of VEGF in a number of tumor cell lines (17). FGF may thus regulate angiogenesis by a combination of actions (11, 14).

Basic FGF is the most potent of the peptide angiogenic growth factors described to date (16). In vivo, its angiogenic actions may be initiated by its release from heparan sulfate of cell membranes or the extracellular matrix, or by increased production and release from cells. Historically a major question has been the mechanism by which FGF is secreted from cells because, despite the fact that this protein lacks a classical signal sequence, it is clearly found in the extracellular matrix as well as intracellularly. This has led to a variety of suggestions including the release of FGF from cells after their death or wounding or some type of export mechanism that does not require a prototypical signal sequence. In addition, it has also been suggested that some actions of FGF may be intracrine, i.e. the growth factor may exert an action in the cell in which it is produced without first being exported to act via an autocrine mechanism. The expression of FGF is observed in most cell types examined, including fibroblasts, endothelial cells, and smooth muscle cells, and its angiogenic actions may thus be due to both paracrine, autocrine, or intracrine actions (see Refs. 11, 14 for further details on mechanisms of FGF action).

bFGF AND THE REPRODUCTIVE TRACT

bFGF has been identified in the normal, nonpregnant uterus of a number of species including human (18, 19), monkey (20), sheep (21), pig (22), mouse (23), and rat (24), and the growth factor has also been identified in uterine secretions (25, 26, 27) from several species. Acidic FGF has also been identified in the nongravid human (18), monkey (20), and pig (22) uterus. There are some differences in the patterns of localization of bFGF observed by immunohistochemical analysis in these 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 (19), raising the possibility that this growth factor exerts numerous intracrine, autocrine, and/or paracrine interactions in the uterus. Several studies have shown that bFGF stimulates endometrial-derived fibroblasts to proliferate (19, 28, 29) and differentiate (28). bFGF transcripts have been observed in human fetal uterus as early as the 10th week of gestation (30) and in the atrophic postmenopausal human uterus (31). These observations suggest that the growth factor could exert effects in the uterus throughout the entire lifespan of mammals.

While it is well established that FGF and its receptor are expressed in the uterus, the regulation of these proteins by ovarian steroids is not yet well understood. In cultured human endometrial fibroblasts (32), several endometrial cancer cell lines (33), and rodents (24) and sheep in vivo (21), estrogens increase bFGF mRNA or protein, and progesterone decreases estradiol-stimulated bFGF production in some cell lines (32). Depending on the age of the animal and the treatment protocol used, progesterone can inhibit or augment the increase in uterine bFGF mRNA caused by estrogens, and progesterone alone can induce growth factor expression in some conditions (24). These studies indicate that estrogens stimulate bFGF production, and this is consistent with several studies showing that expression of the growth factor and its receptor generally correlate with expected estrogen levels throughout the menstrual and estrous cycles (19, 22, 34). In contrast, other studies have shown no change in bFGF expression throughout the estrous cycle in rats (35) or the menstrual cycle in humans (31). Others reported that estrogen treatment of monkeys did not change bFGF expression (20) and that hormone replacement therapy actually decreased bFGF expression in the postmenopausal human endometrium (36). At the molecular level, it is not yet established whether the regulation of FGF expression by steroids is a primary or secondary effect. The 5'-flanking region of the human gene contains a potential progesterone response element as well as an estrogen response element half-site (24) based upon sequence homologies, but functional studies have not yet been performed.

Given the importance of angiogenesis in implantation and decidualization, a number of studies have also investigated FGF expression during the peri-implantation period and during pregnancy. In rodents, expression of bFGF and its receptor increase (23, 26, 37, 38), and the pattern of expression is spatially and temporally related to the pattern of angiogenesis that occurs at this time. Progesterone (39) and PRL (37) appear to up-regulate bFGF expression, and several of these studies suggest that signals from the embryo also increase growth factor expression in the periimplantation region. bFGF expression also increases in the sheep (27) and pig endometrium (22) during pregnancy. FGF may also play a role in regulation of embryonic development since the growth factor is present in uterine secretions during pregnancy (22, 25, 26).

VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF)/VASCULAR PERMEABILITY FACTOR (VPF)

VEGF is a multifunctional cytokine that was originally identified as a protein produced by tumor cells that increased the permeability of capillaries to proteins (see Ref. 8). It was subsequently discovered to be selectively mitogenic for endothelial cells and to stimulate angiogenesis (see Refs. 12, 13 and references therein). This factor has been referred to as VPF, 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 (see Refs. 1, 12, 13, 40 and references therein).

Both the human and murine VEGF genes contain eight exons and seven introns, and differential exon splicing generates predominant forms containing 121, 165, 189, and 206 amino acids in the human, and corresponding forms with 1 amino acid less in the mouse (13). In most systems VEGF-121 and -165 are the major species expressed. A major difference between the different forms of VEGF is their heparin-binding capability. VEGF 121 is freely 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 (see Ref. 12). Unlike bFGF, VEGF has a prototypical signal sequence and appears to be freely secreted from a variety of cells that would enable it to exert both autocrine and paracrine actions on angiogenesis and endothelial cell function. Quite recently, a number of VEGF-related genes referred to as VEGF B, C, D, E, and placental growth factor have been identified (13, 40, 41). 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. In addition, since 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.

The angiogenic actions of VEGF are thought to be mediated primarily by two cell surface receptors termed VEGF-R1 (also referred to in the literature as flt) and VEGF-R2 (flk or KDR), which are tyrosine protein kinases expressed primarily, but not exclusively, on endothelial cells. At present, 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 (see Refs. 12, 13, 40 for recent reviews). Occupancy of VEGF receptors leads to autophosphorylation as well as phosphorylation of other effectors including phospholipase C{gamma}, the GTPase-activating protein Ras-GAP, and the adaptor proteins Nck and Shc. After receptor activation in endothelial cells, a number of cellular responses occur that are likely to play a role in angiogenesis and tissue remodeling, e.g.induction of urokinase and urokinase receptors, tissue plasminogen activators (PA), PA inhibitors, metalloproteinase activity, VCAM-1 (vascular cell adhesion molecule 1) and ICAM-1 (intercellular adhesion molecule 1) expression, and hexose transport (see Ref. 13). Special note should be made of the ability of VEGF to induce fenestrations in capillaries in vivo (42) that are reminiscent of estrogenic effects in the rodent reproductive tract (see Ref. 2 and references therein). In contrast to bFGF, which affects multiple cell types including endothelial cells, smooth muscle cells, and fibroblasts, VEGF is much more selective and exerts its actions primarily on endothelial cells.

VEGF IN THE REPRODUCTIVE TRACT

In 1993 VEGF expression was initially reported in the uterus of the mouse (43), human (44), and rat (45) and has since been identified in the ewe (21), the rabbit (46), and the monkey (47). These and other studies have found that expression of this growth factor and its mRNA occurs primarily in the epithelial and stromal layers of the uterus, but expression of VEGF (44, 48) and its receptor (49) has also been observed in the myometrium. As in other tissues, multiple forms of VEGF are produced in the uterus, including VEGF-189, -165, -145, and -121 in the human (44), VEGF-188, -164, and -120 in the rat (45, 50), and VEGF-189, -165, and -121 in the monkey (47). In all these systems VEGF-121(120) and -165(164) appear to be the predominant forms produced, and immunological measurements have demonstrated that VEGF protein is also produced in monkeys (47), mice (43, 51), rabbits (46), and humans (52, 53). VEGF expression has also been reported in human endometrial carcinoma cells (44), leiomyoma (48), endometriosis (7, 52), and various gynecological malignancies (54).

Estrogens regulate VEGF mRNA expression in human endometrial adenocarcinoma cells (44), primary cultures of human uterine stromal cells (52), and the rat uterus in vivo (45, 50). These are likely to be primary estrogen receptor- mediated effects because induction is very rapid, is blocked by pure antiestrogens (55), and is inhibited by actinomycin D but not puromycin or cycloheximide (45, 50), although an estrogen response element has not yet been identified in the VEGF gene. Partial estrogen agonists such as tamoxifen (50) also induce VEGF expression in the uterus, which is consistent with their agonist activity in this tissue. This regulation by estrogens is consistent with most studies on the expression of VEGF throughout the estrous cycle in rodents (43, 51) and the menstrual cycle in humans (52). In contrast, others have reported that VEGF expression is highest in hypoestrogenic monkeys (47). One possible explanation of this apparent discrepancy is that the endometrium of hypoestrogenic animals is relatively hypoxic, since hypoxia is known to be a strong inducer of VEGF transcription (12, 13).

There are several reports that progestins can also induce VEGF expression in human stromal cells (47, 52) and the rodent uterus (45). The effects of progestins on VEGF expression have not been as well studied as those of estrogens, and little information is available concerning the possible molecular mechanism of progesterone regulation of VEGF. However, progesterone increases VEGF protein expression in a human breast cancer cell line, and this effect is blocked by RU-486, suggesting it is mediated by the progesterone receptor (56). The overall picture that emerges from these studies is that VEGF expression is likely to be under the control of both estrogens and progestins, although very little is known about the molecular mechanisms that regulate expression in the reproductive tract at present, and substantial differences may exist between cell types and in different species.

A number of studies have also investigated expression of VEGF and its receptor in the uterus and placenta during pregnancy in the rat (37), mouse (57), rabbit (46), and human (58, 59, 60). VEGF expression correlates spatially and temporally with changes in angiogenesis and vascular reactivity at implantation sites and in decidua and placental tissue in most cases, except in the rat where these changes correlate primarily with bFGF, but not VEGF, expression (37). On balance, however, these studies support an important role for VEGF in implantation and placentation.

SUMMARY AND CONCLUSIONS

Proper regulation of angiogenesis and vascular permeability is essential for the physiological functioning of the female reproductive tract, and major health problems in women, such as dysfunctional uterine bleeding, endometriosis, and uterine cancer, involve a vascular component. There is a large body of literature that describes the effects of sex steroids on the vasculature of the reproductive tract, but far less is known about the molecular mechanisms that regulate these important actions. We hope that this minireview will help emphasize the need for mechanistic studies in this area to improve treatment and prevention of these major health problems in women. Specifically, we believe it will be important to 1) define the exact roles of FGF, VEGF, and other factors in physiological and pathological events in the reproductive tract and the cell types and receptors involved; 2) identify estrogen and progesterone receptor subtypes, the DNA elements, nuclear protein factors, and signaling pathways that mediate regulation of these genes by sex steroids; 3) elucidate any mechanisms of cross-talk between sex steroids and other regulatory factors in the overall regulation of FGF, VEGF, and other angiogenic/permeability factors; and 4) eventually understand how genetic polymorphisms of key regulatory elements affect angiogenesis and the regulation of vascular function in the female reproductive tract.

FOOTNOTES

Address requests for reprints to: Dr. George Stancel, Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Medical School, 6431 Fannin Street, Room 5.036, P.O. Box 20708. Houston, Texas 77225. E-mail: gstancel{at}farmr1.med.uth.tmc.edu

This work was supported by NIH Grant HD-08615, the Texas Affiliate of the American Heart Association, and a Fleming/Davenport Award from the Texas Medical Center.

Received for publication March 2, 1999. Revision received March 23, 1999. Accepted for publication March 23, 1999.

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