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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2001;21:6.)
© 2001 American Heart Association, Inc.

Brief Reviews

Estrogen and Angiogenesis

A Review

Douglas W. Losordo; Jeffrey M. Isner

From the Divisions of Cardiovascular Medicine and Research, St. Elizabeth’s Medical Center, Boston, Mass.

Correspondence to Douglas W. Losordo, MD, Divisions of Cardiovascular Medicine and Research, St. Elizabeth’s Medical Center, 736 Cambridge St, Boston, MA 02135. E-mail dlosordo{at}opal.tufts.edu

	Abstract

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Abstract—Multiple lines of evidence suggest that estrogen directly modulates angiogenesis via effects on endothelial cells. Under physiological conditions, angiogenesis is routinely observed in the uterus in association with fluctuations in the levels of circulating estradiol and other sex steroids. In pathological circumstances, such as breast cancer, a clear association between estrogen, estrogen receptor expression by endothelial cells, angiogenic activity, and/or tumor invasiveness has been made. Studies performed in our laboratory have revealed that estradiol accelerates functional endothelial recovery after arterial injury. Despite these consistent observations, the mechanisms by which estrogen regulates angiogenesis under physiological and pathological circumstances have not been defined.

Key Words: estrogen • angiogenesis • vasculogenesis • endothelium • endothelial progenitor cells

	Introduction

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
In adult organisms, angiogenesis is virtually absent under normal conditions, except in the female reproductive tract. This observation suggests the potential for sex steroids to influence neovascularization. Several other associations, including the predilection of certain diseases (such as Takayasu’s arteritis and lupus, both of which involve endothelial cell proliferation) for premenopausal females, also imply the potential role of estrogen in angiogenesis.^{1 2} The role of estradiol in uterine angiogenesis, mediated by at least 1 of the estrogen receptors, has been further suggested by findings in the estrogen receptor- knockout mouse, in which angiogenesis is impaired,³ and by the demonstration that estrogen receptor antagonists can inhibit angiogenesis.⁴ The positive correlation between estrogen receptor expression, angiogenic activity, and breast tumor invasiveness also supports the angiogenic effect of estrogen mediated by the estrogen receptor(s).^{5 6 7 8} Finally, some reports suggest that the antitumor effect of tamoxifen may in fact relate to an antiangiogenic action of this estrogen receptor agonist/antagonist.⁹

Several additional lines of experimental evidence suggest that estrogen and other sex steroids play important roles in physiological and pathological angiogenesis. One of the first observations suggesting a hormonal influence on angiogenesis was an inhibitory effect on tumor vascularization by medroxyprogesterone.¹⁰ Subsequent studies have indicated that the mechanism of this inhibition may relate to the regulation of thrombospondin-1, an angiogenesis inhibitor, by this hormone.¹¹

Angiogenesis has been noted to be a prognostic marker in breast cancer,¹² and inhibition of angiogenesis in these tumors has been effected by antiestrogens.⁴ In addition, the estrogen metabolite 2-methoxyestradiol has been shown to be a potent antiangiogenic agent¹³ mediated by actions on cytoskeletal structure¹⁴ and by increasing endothelial cell apoptosis.^{15 16}

	Vascular Endothelial Cell Proliferation and Migration Are Enhanced by Estradiol

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Estradiol induces endothelial proliferation and migration¹⁷ mediated by the classic estrogen receptor, which is expressed by endothelial cells.^{18 19} This effect provides 1 potential mechanism contributing to the angiogenic effect of estradiol. Moreover, disruption of the anatomic and functional integrity of the endothelium has been postulated as a mechanism for the initiation of atherosclerosis.²⁰ A corollary hypothesis is that restoration of endothelial integrity, which would require endothelial proliferation and migration, should inhibit atherogenesis. Accordingly, the finding that estradiol stimulates endothelial proliferation and migration also lends a potential mechanism for the artery-protecting effect of estrogen, ie, the maintenance of endothelial integrity.

	Role of VEGF in Estrogen-Mediated Angiogenesis

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
The possibility that vascular endothelial growth factor (VEGF) may be partially responsible for the angiogenic action of estradiol has been suggested by several findings. Expression of VEGF by uterine and vascular tissue has been shown to be increased by estradiol,^{21 22 23 24} as has VEGF production by macrophages (MPs) in endometriosis.²⁵ In addition, certain estrogen-induced tumors have also been associated with an increase in the expression of VEGF and its receptors.²⁶ Finally, the acceleration of endothelial recovery by estrogen treatment after arterial injury has been associated with increased VEGF expression by vascular smooth muscle cells (SMCs).²⁷ Interestingly, a possible effect of androgens on VEGF-mediated angiogenesis has also been suggested.²⁸

	Role of NO in Estrogen-Mediated Angiogenesis

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Estradiol has been shown to regulate NO in a variety of settings. NO synthase has been shown to be upregulated in human aortic endothelial cells by estradiol,²⁹ and disruption of estrogen receptors in patients has been associated with endothelial dysfunction.³⁰ Estradiol regulates uterine NO synthase expression,³¹ and short-term estradiol has been shown to augment NO-mediated vasodilation in multiple arterial beds^{32 33} by mechanisms that may involve increased basal NO release^{34 35} and increased NO synthase activity.^{36 37} Recently, the activation of plasmalemmal caveolae, where membrane estrogen receptors reside, has been shown to be impaired by oxidized LDL, a finding that may have important implications for the pathogenesis of atherosclerosis and the inhibitory effect of estradiol on that process.³⁸

The role of NO in angiogenesis has been controversial. Previous reports have suggested that NO inhibits the migration of endothelial cells,³⁹ which is an essential step in angiogenesis.⁴⁰ In addition, NO donors have been shown to inhibit angiogenesis in vitro⁴¹ and in vivo.⁴² More recently, however, NO has been shown to be critical for the angiogenic properties of VEGF.⁴³ Finally, and most compelling, the angiogenic response to ischemia has been shown to be severely impaired in a mouse model with targeted disruption of endothelial NO synthase,⁴⁴ and these mice have also displayed delayed endothelial recovery after vascular injury.⁴⁵

	Regulation of Adhesion Molecule Expression by Estrogen

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Estrogen-mediated alteration in the expression of certain adhesion molecules has been shown in endothelial cells^{46 47} and other cell types.^{48 49 50 51} The potential mechanistic importance of these effects is reflected in human and animal studies noting the association between alterations in sex hormones, changes in the expression patterns of adhesion molecules and matrix proteins, and modification of angiogenic activity.^{11 12 17 52}

	Vascular Cells Express Estrogen Receptors

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
The expression of estrogen receptor- has been demonstrated in endothelial cells^{18 19} and vascular SMCs.^{53 54} In addition to the form of the receptor, the ß isoform has also been demonstrated in vascular tissue with increased expression after vascular injury,⁵⁵ although the function of the ß-receptor in vascular tissue has not yet been fully defined.⁵⁶

	Effect of Estrogen on Bone Marrow Precursors

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Recent evidence (see below) suggests a significant role for circulating, bone marrow–derived, endothelial precursor cells in postnatal vasculogenesis. An effect of estrogen on hematopoietic stem cells and granuloid progenitors has been noted for >2 decades.^{57 58 59 60} Estrogen deficiency has been shown to stimulate ß-lymphopoiesis in bone marrow⁶¹ and to increase osteoblastogenesis.⁶² Peripheral blood monocytes as well as T and B cells have been shown to express intact estrogen receptor-,⁶³ and an intact estrogen receptor transactivation domain has been shown to be required for estradiol to inhibit the differentiation of avian erythroid progenitors.⁶⁴ Interestingly, estradiol has also been shown to regulate erythropoietin production locally in the uterus in association with angiogenic activity in that organ.⁶⁵ Together, these prior studies provide evidence of direct actions of estradiol on the bone marrow and the regulation of bone marrow–derived precursor cells.

	Steroid Hormone–Mediated Recruitment of Inflammatory Cells to the Uterus

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
Inflammatory cells, including MPs, leukocytes,^{66 67 68 69 70} and mast cells,⁷¹ have been shown to be constituents of the normal uterus. Among these cells, the MPs have been most extensively studied. Initially considered for their potential role in local immune suppression,^{72 73} these ubiquitous cells have been shown to exert a variety of potential influences on the normal physiology of the cycling and pregnant uterus.^{74 75} It is clear that sex steroids influence the recruitment of MPs, their function,⁷⁶ and their distribution in the uterus.⁷⁷ The relative roles of estradiol and progesterone have recently been clarified by studies revealing estradiol-mediated recruitment of MP, which is inhibited by progesterone⁷⁸ via its receptor.⁷⁹ Importantly, it has been noted that uterine inflammatory cells are the result of recruitment from the bloodstream rather than the result of local proliferation.⁸⁰

	Role of MPs in the Uterus

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
The abundance of MPs in the uterus and the regulation of cell number and distribution during normal menstrual cycling and pregnancy are findings that imply significant function for these cells. MPs have been shown to express a variety of growth factors and cytokines, including tumor necrosis factor⁷⁵ and NO,^{81 82} both of which are pertinent as possible mediators of angiogenesis.

In addition, uterine epithelial cells and endometrial stromal cells are also the source of a number of cytokines, such as VEGF,^{22 23} which may influence angiogenesis by directly inducing endothelial cell proliferation⁸³ or by recruiting endothelial progenitor cells.⁸⁴

	Mechanisms of Angiogenesis

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
The full paradigm for angiogenesis⁸⁵ has been suggested to begin with "activation" of endothelial cells within a parent vessel, followed by disruption of the basement membrane and the subsequent migration of endothelial cells into the interstitial space, possibly in the direction of an ischemic stimulus. Concomitant and/or subsequent endothelial cell proliferation, intracellular-vacuolar lumen formation, pericyte "capping," and production of a basement membrane complete the developmental sequence.

During angiogenesis, migration precedes proliferation by 24 hours.⁸⁶ This principle was best demonstrated in classic experiments performed by Sholley et al,⁴⁰ who used a model of inflammation-induced angiogenesis of the rat cornea, in which initiation of vascular sprouting was shown to occur in the absence of endothelial cell proliferation, which was suppressed by x-irradiation before application of the inflammatory stimulus. In irradiated corneas displaying no cellular proliferation, vascular sprouting at 2 days was similar to that seen in contralateral shielded corneas. Although neovascular growth was subsequently blunted and ultimately ceased by 4 to 7 days, these experiments documented the critical if not exclusive roles of migration and redistribution of preexisting endothelial cells in the commencement of neovascularization. Similar implications resulted from work by Nicosia et al⁸⁷ : Fibronectin was shown to promote, in a dose-dependent fashion, the elongation of microvessels that sprout from explants of rat aorta placed in serum-free collagen gel, despite the fact that neither DNA synthesis nor mitotic activity was increased in serum-free collagen gels compared with fibronectin-negative gels. Therefore, fibronectin was inferred to promote angiogenesis in vitro by migratory recruitment of preexisting endothelial cells. Subsequent studies have established the critical role played by plasmin and other proteases in promoting migration through preexisting matrix.^{88 89}

In contrast to angiogenesis in vivo inflammatory and in vitro organ culture models, angiogenesis that develops in response to experimental vascular obstruction, ie, collateral vessel development, has been shown by several previous investigators to involve proliferation of not only endothelial cells but also SMCs. Several important principles have been elucidated by these studies.

First, evidence of endothelial cell proliferation is nearly absent in normal arteries,^{90 91} a finding that is consistent with an estimated endothelial cell turnover time of "thousands of days" in quiescent microvasculature.⁹² Even a relatively low percentage of endothelial cell proliferation observed in response to arterial occlusion or exogenous growth factors may therefore represent considerable enhancement of endothelial cell proliferative activity and, when considered in relation to a denominator of thousands of endothelial cells, is clearly sufficient to provide the basis for new blood vessel formation. Second, endothelial cell proliferation that contributes to naturally occurring collateral development in the setting of vascular occlusion varies from 2.6% to 3.5% in the canine coronary^{90 93} and from 5% to 6% in the rodent renal vasculature⁹⁴ and is <1% in swine coronary arteries.⁹⁵ The contrasting rates of endothelial cell proliferation between the canine and swine coronary circulations are in parallel with the relative propensity for natural collateral artery development in these 2 species. Third, proliferation of SMCs, the additional requisite cell type for the formation of larger blood vessels, is an implicit component of angiogenesis, regardless of animal species or circulatory site.⁹⁶ Fourth, proliferative activity (for SMCs as well as endothelial cells) is highest at the level of the smallest-diameter collateral vessels, the so-called midzone collateral segments.^{90 93 97 98} Fifth, although evidence of endothelial cell and SMC proliferation alone does not necessarily distinguish new vessel development from an increase in the size of preexisting vessels, adjunctive data regarding increased capillary density^{83 99} support the notion that proliferative activity does in fact reflect true angiogenesis.

	Postnatal Vasculogenesis

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
In the embryonic yolk sac, vasculogenesis involves growth and fusion of multiple blood islands, which ultimately give rise to the yolk sac capillary network¹⁰⁰ ; after the onset of blood circulation, this network differentiates into an arteriovenous vascular system.¹⁰¹ The integral relationship between the elements that circulate in the vascular system (ie, the blood cells) and the cells that are principally responsible for the vessels themselves (ie, the endothelial cells) is implied by the composition of the embryonic blood islands. The cells destined to generate hematopoietic cells are situated in the center of the blood island and are termed hematopoietic stem cells. Endothelial progenitor cells (EPCs), or angioblasts, are located at the periphery of the blood islands. In addition to this spatial association, hematopoietic stem cells and EPCs share certain antigenic determinants, including Flk-1, Tie-2, Sca-1, and CD-34. These progenitor cells have consequently been considered to derive from a common precursor, putatively termed a hemangioblast.^{102 103 104}

Postnatal neovascularization has been previously considered to result exclusively from the proliferation, migration, and remodeling of fully differentiated endothelial cells derived from preexisting blood vessels, ie, angiogenesis (see Figure).^{90 92 100} The formation of blood vessels from EPCs (ie, vasculogenesis) has been considered to be restricted to embryogenesis.^{101 105}

View larger version (128K): [in this window] [in a new window]   Figure 1. Estradiol influences angiogenesis via activation of endothelial cells within a parent vessel and vasculogenesis by mobilization and recruitment of EPCs.

However, we reasoned that the use of hematopoietic stem cells derived from peripheral blood in lieu of bone marrow to provide sustained hematopoietic recovery constituted inferential evidence for circulating stem cells. Circulating CD34 antigen–positive EPCs were recently isolated from adult species; once adherent, these cells were shown to differentiate along an endothelial cell lineage in vitro.¹⁰⁶ Heterologous, homologous, or autologous EPCs administered systemically to animals with operatively induced hindlimb ischemia were found to incorporate themselves into foci of neovascularization in ischemic muscles of the affected hindlimb. These findings, together with those of other recent studies,^{107 108 109 110} have been interpreted as evidence of postnatal "vasculogenesis."^{101 105}

	Hormone-Regulated Neovascularization

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
In the female reproductive system, neovascularization is imposed recurrently by cyclic development of transient structure and cyclical repair of damaged tissues.^{28 92 111 112} The ovarian sex steroid hormones, estrogen and progesterone, are primarily uterotropic and control the cyclical patterns of uterine cell proliferation and vascular growth that occur throughout the nonpregnant menstrual cycle. Given the synchronized nature of neovascularization in this cyclical process, it is assumed that angiogenic growth factor expression is induced by steroid hormones and regulates blood vessel formation in reproductive organogenesis.^{113 114 115 116} Indeed, physiological changes in the reproductive tract during the normal menstrual cycle²⁸ as well as physiological and pathological symptoms, including telangiectasia of pregnancy¹¹⁷ and telangiectasia in postsclerotherapy patients receiving gonadal hormones,¹¹⁸ are all associated with elevated levels of serum estradiol.

Despite clinical evidence of the significant role of steroid hormones in endometrial neovascularization, the results of previous experiments have yielded inconclusive results in terms of defining specific elements of pathophysiological mechanisms, including experiments involving endothelial cells in vitro and in vivo.^{3 4 10 17 119 120 121} Moreover, estrogen has been shown to exhibit an inhibitory effect on certain hematopoietic kinetics, including lymphocytes and monocytes, numerically^{60 122 123} and functionally.^{124 125}

Conventionally, endometrial vascularization has been considered to develop as the result of angiogenesis, ie, proliferation and migration of fully differentiated endothelial cells (endothelial cells) from preexisting "parent" vessels.¹²⁶ However, normal monthly physiological endometrial proliferation would require that endothelial cells in the uterus replicate >1000 times during the reproductive life span of the average human female. Therefore, it is unlikely that differentiated endothelial cells in situ could accomplish this mission without the occurrence of replicative senescence.¹²⁷

As outlined above, a large body of previous literature has documented the recruitment of MPs and other nucleated cells to the uterus under the influence of sex steroids. Our data¹²⁸ indicate that a subpopulation of these cells is capable of differentiating into endothelial cells (ie, EPCs) and thereby constitutes a previously unidentified source of vascular cells for cyclical neovascularization. In future studies, we will explore the influence of estradiol on EPC recruitment, differentiation, and incorporation in several models of postnatal angiogenesis/vasculogenesis.

	Conclusions

Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 
A potent angiogenic effect of estradiol is suggested by the normal physiology of the female reproductive tract, which requires recurrent neovascularization. In every 1 of >300 estrous cycles, an elaborate capillary network develops and regresses in association with fluctuations in estradiol levels. This unique system of blood vessel development involves hormone-mediated in situ differentiation of bone marrow–derived precursor cells and constitutes a natural model for postnatal vasculogenesis. Further study of the precise mechanisms for hormone-induced neovascularization will also yield a better understanding of the regulation of blood vessel development under physiological and pathological circumstances.

	Acknowledgments

 
This work was supported in part by National Institutes of Health grants AG-16332, HL-63414, and HL-63695 (D.W.L) and HL-40518, HL-02824, and HL-57516 (J.M.I).

Received April 3, 2000; accepted August 15, 2000.

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Top Abstract Introduction Vascular Endothelial Cell... Role of VEGF in... Role of NO in... Regulation of Adhesion Molecule... Vascular Cells Express Estrogen... Effect of Estrogen on... Steroid Hormone-Mediated... Role of MPs in... Mechanisms of Angiogenesis Postnatal Vasculogenesis Hormone-Regulated... Conclusions References

 

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