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Main regulators of angiogenesis and their role
in preeclampsia and intrauterine growth restriction
A bibliographic review

Dr. Benedict Ancar, University of Medicine ‘Carol Davila’, Bucharest, Romania
Dr. Didier Chardonnens, University Hospital of Geneva, Switzerland

Summary - The aim of this bibliographic review is to compile the up-to-date knowledge regarding angiogenesis in humans, its main regulators and factors and, also, its relation with certain pathologies, such as preeclampsia and intrauterine growth restriction (IUGR). On doing this, we’ve searched most actual bibliographies, that related to the normal aspects of angiogenesis, as well as to the abnormal aspects of it that can concur to the pathological situations mentioned above. The normal angiogenesis (meaning formation of vessels from already preexisting ones) is regulated by a series of factors, also known as growth factors, such as the vascular endothelial growth factor (VEGF), the placenta growth factor (PlGF) and the basic fibroblastic growth factor (bFGF), as well as the partial pressure of oxygen in the fetoplacental vessels. The presence and action of those factors, as well as many others (some stimulating, others inhibiting factors), concure to a normal angiogenesis, while any imballance in their expression leads to a pathological condition. Angiogenesis can be branching, as in the early stages of placental development, or nonbranching, beginning with the 26th week of pregnancy. In establishing the outcome of an pregnancy, the most important role is attributed to the branching angiogenesis, as the normal development in this stage concurs to an normal outcome of the pregnancy. Any change or abnormality at this stage can lead to an severe outcome of the pregnancy, such as preeclampsia or IUGR.

Keywords – branching angiogenesis, nonbranching angiogenesis, vascular endothelial growth factor, placenta growth factor, basic fibroblastic growth factor, oxygen, hypoxia, preeclampsia, intrauterine growth restriction.

Introduction.

The implanting embryo requires a blood supply in order to grow. This means that there have to be formed blood vessels between the ovum and the decidua, thus making a direct connection between the mother and the embryo.

The cardiovascular system is the first functional organ to develop in the vertebrate embryo, thus also in the human embryo. It forms through several parallel processes. While the primordial vascular system is defined by vasculogenesis, angiogenesis is the result of anticipation of the demands of the growing embryo for oxygen and nutrients1.

Blood vessel differentiation and growth is the result of the action and interaction of different proteins, including cell adhesion molecules, extracellular matrix components, transcription factors, angiogenic growth factors and their receptors.

This bibliographic review tries to emphasize the process of angiogenesis and its main regulators, as well as the changes that lead to preeclampsia and intrauterine growth restriction.

Vessel growth. Angiogenesis.

Two distinct types of vessel growth have been defined :

  • vasculogenesis, meaning new blood vessel formation from angioblast precursor cells, essentially occuring during fetal development, and
  • angiogenesis, meaning new branches from preexisting vessels, which is occuring in the female reproductive tract during the formation of the corpus luteum2, during endometrial development3 and during embryo implantation4,5 and placentation6. This type of vessel growth also occurs during pathologic conditions, such as retinopathies, artropathies, wound healing, tumor growth and metastases.

The process of angiogenesis has three phases: initiation, proliferation-invasion and maturation-differentiation.

The vascularisation of placental villi starts at day 21 postconception, being the result of local de novo formation of capillaries rather than protrusion of embryonic vessels into the placenta. The villous trees at this stage are made up of solid trophoblastic villi (primary villi) and of loose mesenchyme derived from the extraembryonic coelomic cavity in the centre of the villi (secondary villi). In the latter, prior to the formation of the first vessels, mesenchymal derived macrophages (Hofbauer cells) appear in their mesenchyme. Those macrophages will express angiogenic growth factors and, as they appear early, they suggest a paracrine role in the initiation of vasculogenesis7.

Angiogenic growth factors are also expressed by the maternal decidua and macrophages, suggesting that there also exists a paracrine mechanism mediating the trophoblast invasion7,8.

From this stage of development until the end of the first trimester there is a branching angiogenesis, as the villous vasculature increases rather in number than in types of vessels.

There is an increase in the number of fetal red blood cell containing capillaries, while branching angiogenesis from the existing capillaries gives rise to a primitive capillary network, surrounded by an incomplete layer of pericytes9.

From the 6th week of development a basal lamina begins to form around the capillaries. As a result of this, there will be a web like arrangement of capillaries within the stroma of mesenchymal villi, and a superficially location of most of the capillaries in the immature intermediate villi (beneath the trophoblast, covering the villous surface). In the latter, from the 15th week onwards, fibrosal stromal core is being formed by the fusion of the adventicia of large central vessels, thus becoming a stem villus. In these larger villi a few central endothelial tubes (or early villous arteries and veins) have larger diameters, up to 100μm, and become surrounded by cells expressing alpha and gamma smooth muscles actins, vimentin and desmin. These contractile cells concentrate around the lumina, acquiring the full spectrum of cytoskeleton antigens10.

In the stem villi the fibrotic process expands towards the outer layer of the trophoblast and the peripheral capillaries of the villous stroma remain in place, as the paravascular capillary network and new primitive capillary nets of the newly formed side branches of mesenchymal villi branch off. As the fibrous process continues, the capillary networks of the stem villi regress gradually, thus declining the formation of mesenchymal side branches. As an effect, at term the larger stem villi possess very few paravascular capillaries.

The villous trees and their vascular bed expand continuously from the peripheral ends of the newly differentiated villous stems, giving rise to new villous outgrowths with a new capillary network, which in turn differentiates into stem villi10.

From the 26th week until term the villous vascular growth becomes a non-branching angiogenesis, due to the formation of mature intermediate villi, specialized in gas exchange. Those villi contain 1-2 long, poorly branched capillary loops, which coil and bulge through the trophoblastic surface, forming the terminal villi as they grow in excess of the villi9. These structures are the main site of diffusional gas exchange between the maternal and fetal circulations.

As gestation increases, the terminal capillaries focally dilate and form large sinusoids, which counterbalance the effects of the long poorly branched capillaries on total fetoplacental vascular impedance. Increasing fetal blood pressure aids this dilation and fetoplacental blood flow rises throughout gestation to 40% of fetal cardiac output at term11.

Implantation and angiogenic regulators.

Human endometrium undergoes specific phases of proliferation and secretion. If implantation does not occur, the endometrium is shed. The processes that drive the endometrium through these phases and prepare it for implantation are under the control of specific control mechanisms12.

Implantation is a complex process that initially requires the interaction of the blastocyst and, further on, the developing embryo and placenta with the endometrium. In implantation, both endometrial and embryonic factors are involved.

It is postulated that in humans, like in other mammals, an implantation window exists during which the endometrium becomes receptive to the implantation of the blastocyst. This phase is followed by a nonreceptive phase, when the endometrium becomes refractory to the implantation process13.

The initiation of embryonic implantation is associated with the acquisition of an invasive cellular phenotype, which comprises a host of cellular processes that include expression or repression of specific cell adhesion molecules, elaboration of matrix-digesting enzymes, and acquisition of a blood supply14.

The most common mediators of angiogenesis are the vascular endothelial growth factor (VEGF), the basic fibroblast growth factor (bFGF) and the placenta growth factor (PlGF), but there also are many more factors that influence the process (table 1).

Table 1. Regulators of angiogenesis.

Stimulators Inhibitors

  • Vascular endothelial growth factor Thrombospondin (Tsp-1)
  • Fibroblast growth factor (acidic and basic) bFGF soluble receptor
  • Placenta growth factor Fibronectin (29-kd fragment)
  • Transforming growth factor-α Tissue inhibitor of metalloproteins
  • Interleukin-8 Interferon-α
  • Angiogenin Angiostatin (plasminogen fragment)
  • Transforming growth factor (α and β) Prolactin (16 kd)
  • Platelet-derived endothelial cell growth factor Platelet factor 4
  • Granulocyte colony growth factor
  • Hepatocyte growth factor
  • Proliferin

From Murray M, Lessey BA – Embryo implantation and tumor metastasis.

Angiogenesis can be induced by VEGF infusion and effectively inhibited by neutralising antibodies to VEGF or its receptor3,15,16.

VEGF is unique as an angiogenic factor, because the two known human receptors, VEGFreceptor - 1 (VEGFR-1) and VEGFreceptor - 2 (VEGFR-2), are expressed only on endothelial cells and, as an exception, on human trophoblast cells17. Both of these tyrosine kinase receptors are present in the first trimester placental tissue7.

VEGF was identified as an endothelium-specific mitogen and inducer of angiogenesis. It also turned out to be identical to the previously described vascular permeability factor (VPF). But its main function was discovered only recently : the stimulation of endothelial cell survival in newly formed blood vessels18.

VEGF (also called VEGF-A) occurs in different isoforms that are encoded by mRNA splice variants derived from a single gene. In human there have been described at last five isoforms : VEGF121, VEGF145, VEGF165, VEGF189 and VEGF206. These isoforms differ primarily in heparin binding, which may affect their diffusion rates in the extracellular space19.

VEGF is the ‘founding member’ of a growing family of growth factors, comprising at least six different proteins that bind to different members of the VEGF receptors family. In the human placenta, VEGF has been demonstrated to be localized in the villous trophoblast and macrophages of both fetal and maternal origin11.

The function of the VEGF/VEGF receptor system is not restricted to early stage vascular development, as they are expressed in all developing organs. The high affinity VEGF-A receptors are localized primarily in developing blood vessels, whereas VEGF-A is secreted from cells in the vicinity. VEGF-A has been demonstrated to be a potent stimulator of endothelial cell proliferation, migration and production of the plasminogen activators required for proteolytic degradation of the extracellular matrix, all of these being markers of angiogenic activity. This indicates that VEGF-A stimulates the growth and survival of blood vessels in developing organs in a paracrine way18.

Another property of VEGF-A is its inductibility to hypoxia. The upregulation of VEGF-A can therefore be considered as a major mechanism by which the compensatory formation of blood vessels is initiated in hypoxic tissue. The increased VEGF-A mRNA transcription in response to hypoxia is largely attributed to the activity of hypoxia-inducible factors (HIF), such as the HIF-1 and HIF-220. The mechanisms of VEGF-A upregulation in response to hypoxia were initially implicated in the control of pathological angiogenesis15, but they may also be active both in developmental and physiological adult angiogenesis.

Aside from VEGF, the only other angiogenic protein that directly influences endothelial cells is the placenta growth factor (PlGF). It shares a 50% homology with VEGF and binds to VEGFR-1. In the human placenta, PlGF is expressed in both villous syncytiotrophoblast and in the media of larger stem vessels6,21.

Being a close relative of VEGF-A, which is strongly expressed in the placenta, PlGF has been suggested to play a definite role in placental angiogenesis. Even if not, it might act primarily on trophoblast cells that express VEGFR-1, that seems to regulate negatively the commitment of angioblasts during vasculogenesis. PlGF has been demonstrated to be a very weak stimulator of endothelial cell chemotaxis and proliferation8,22.

This different effects may be explained by the fact that VEGF binds both VEGFR-1 and VEGFR-2, resulting branching angiogenesis, while PlGF binds VEGFR-1, but not VEGFR-2, resulting non-branching angiogenesis.

Both VEGF and PlGF are thought to be important for the embryonic development and placental growth.

Correlations of these growth factors, their effects and their expression patterns throughout gestation with the development of the villous angioarchitecture suggest that VEGF and VEGFR-2 are involved in the first two trimesters of pregnancy in the establishment of the richly branched capillary beds of the mesenchymal and immature intermediate villi, while PlGF and VEGFR-1 are more likely to be involved in the formation of the long, poorly branched, terminal capillary loops in the third trimester. As VEGF declines throughout gestation, it suggest a possible mechanism for the regression of the capillary nets during stem villus formation14.

Another major positive regulator of angiogenesis is the basic fibroblastic growth factor (bFGF), first isolated from tumors23. bFGF is also secreted by the trophoblast24. It's synthesis, like that of VEGF, appears to be modulated by estradiol25. bFGF activates a variety of cell types, and as endothelial cells lack receptors for this growth factor, it seems that it influences the vascular endothelium indirectly26.

During the invasive phase of the angiogenesis, VEGF increases vascular membrane permeability, bathing the angiogenic network in extracellular matrix and cell growth modulating factors. Also, both VEGF and bFGF are associated with the expression of cell adhesion molecules that are necessary for endothelial cell invasion through the extracellular matrix.

Oxygen and angiogenesis.

Another important factor is the effect of the local oxygen environment during gestation. Thus, in placental and related chorioallantoic tissues VEGF is upregulated by hypoxia and downregulated by hyperoxia15.

Successful placentation means development of a low-impedance uteroplacental circulation, following trophoblast invasion and transformation of the maternal intramyometrial portion of the spiral arterioles9.

Placental development in the first trimester occurs in a relative hypoxia, stimulating cytotrophoblast proliferation and inhibiting trophoblast invasion. Only at about 12 weeks of gestation and towards the second trimester maternal blood flow starts and partial pressure of oxygen (PO2) increases27.

The rise of PO2 may be the trigger for the trophoblast to change to an invasive extravillous trophoblast, thus the secondary wave of trophoblast invasion of the maternal spiral arterioles, establishing a high flow, low impedance uteroplacental circulation.

Haemochorial placentation is also dependent upon the establishment and maintenance of a competent fetoplacental vascular network through the process of vasculogenesis. Consequently, a careful coordination of trophoblast and endothelial cell development, proliferation, invasion and differentiation must occur during the first stages of placental development. This is considered to be mediated by locally acting growth factors, as the ones already described8, that are themselves likely to be regulated by the partial pressure of oxygen and mechanical stimuli28.

Preeclampsia and intrauterine growth restriction.

In preeclampsia and intrauterine growth restriction (IUGR) the uterine blood vessels do not undergo adequate vascular transformation, so that the rate of delivery of oxygenated blood to the fetus falls. It comes to an ‘uterine insufficiency’, that led to the term of placental hypoxia29.

Placentae from IUGR conditions display a failure of elongation, branching and dilation of the capillary loops and failure of terminal villi formation. Thus, fetoplacental blood flow is severely impaired and transplacental gas exchange is poor, placing the fetus at risk of hypoxia and acidosis30.

In growth restricted pregnancies with reduced or absent end-diastolic flow velocity there appears to be a poor placental blood vessel development, with straight and unbranched capillaries, along with reduced cytotrophoblast proliferation, increased syncytial nuclei and erythrocyte congestion. All these suggest an increased rate of trophoblast proliferation, thus an aged syncytium. This situation has been interpreted as placental hyperoxia9,30,31.

Since hypoxia promotes angiogenesis, it seems that the relatively high oxygen levels in the intervillous space, in relation with placental villi of IUGR gestations will limit angiogenesis, thus increasing the negative effects on the gestation28.

PlGF is significantly increased in severe IUGR placentae, whereas VEGF expression is decreased. Being merely a consequence of this, the action of PlGF may have deleterious effects on placental growth and development, as seen in IUGR.

There have been three types of hypoxia described, that may occur in the fetoplacental unit and may influence the fetoplacental angiogenesis31.

1. Pre-placental hypoxia, when the mother, the placenta and the fetus are hypoxic (such as gestation at high altitude, maternal anaemia, cyanotic maternal heart diseases). The peripheral placental villi show increased branching angiogenesis, which means formation of richly branched but shorter terminal capillary loops32.

2. Uteroplacental hypoxia. Maternal oxygenation is normal, whereas the placenta and the fetus are hypoxic. This is due to impaired uteroplacental circulation and occurs in preeclampsia with preserved end diastolic flow. The peripheral placental villi show formation of richly branching nests. There is a increased expression of VEGF and a reduced expression of PlGF, suggesting that placental hypoxia upregulates VEGF and causes the changes in angiogenesis8.

3. Postplacental hypoxia or placental hyperoxia. In this case the mother is normoxic, the placenta hyperoxic and the fetus hypoxic. That is all because the capillaries are poorly developed, capillary branching being virtually absent. The result is an increase in fetoplacental flow impedance. Thus, VEGF is decreased, while PlGF is increased, suggesting that an early onset of placental hypoxia leads to dominating PlGF effects too early in gestation. This has as final result the decrease of branching angiogenesis and the failure of terminal villi formation30,31,33.

Conclusions.

The up-to-date bibliography states that in the development of placental vasculature there is a balance between VEGF, PlGF and PO2. This balance regulates the switch between branching and non-branching angiogenesis, thus the development of vessels and the moment when they come to a stop in their branching.

A main role in this process have VEGF, PlGF and bFGF, as well as PO2. The up- and downregulation of various growth factors, thus of various predominant structures, is in a close relation with the PO2 of the fetoplacental blood.

Pathologic conditions can be the result of :

- placental hypoxia and increased expression of VEGF, associated with decreased expression of PlGF, thus an increased branching angiogenesis, such as in preeclampsia with preserved end diastolic flow, or

- an early onset of placental hyperoxia with decreased expression of VEGF, that causes a switch to premature PlGF dominance, thus the observed decreased branching angiogenesis, the failure of non-branching angiogenesis within the terminal villi and the disturbances in the rate of trophoblast growth, which all lead to an increase in fetoplacental flow impedance, such as in IUGR.

References.

  1. Breier G. Angiogenesis in embryonic development – a review. Troph Res 2000 ; 14 : 11-15
  2. Folkman J. Clinical application of research on angiogenesis. N Engl J Med 1995 ; 333 : 1757-1763
  3. Ferrara N, Chen H, Davis-Smyth T, et al. Vascular endothelial growth factor is essential for corpus luteum angiogenesis. Nat Med 1998 ; 4 : 336-340
  4. Smith SK. Vascular endometrial growth factor and the endometrium. Hum Reprod 1996 ; 11 (Suppl 2) : 56-61
  5. Torry DS, Holt VJ, Keenan JA, Harris G, Caudle MR, Torry RJ. Vascular endothelial growth factor expression in cycling human endometrium. Fertil Steril 1996 ; 66 : 72-80
  6. Zhou Y, Fisher SJ, Janatpour M, et al. Human cytotrophoblasts adopt a vascular phenotype as they differentiate-A strategy for successful endovascular invasion ? J Clin Invest 1997 ; 99 : 2139-2151
  7. Ahmed AS, Li XF, Dunk CE, Whittle MJ, Rollason T. Colocalisation of vascular endothelial growth factor and its flt-1 receptor in human placenta. Growth Factors 1995 ; 12 : 235-243
  8. Ahmed AS, Whittle MJ, Khaliq A. Differential expression of placenta growth factor (PlGF) and vascular endothelial growth factor (VEGF) in abnormal placentation. J Soc Gynaecol Invest 1997 ; 4 : A663
  9. Benirschke K, Kaufmann P (Eds). Pathology of Human Placenta. London : Springer-Verlag 1995
  10. Kohen G. Anatomy of the stem villus. IUGR. (Eds) Baker P, Kingdom JCP. London : Springer-Verlag 1999
  11. Ahmed A, Dunk C, Ahmad S, Khaliq A. Regulation of placental vascular endothelial growth factor (VEGF) and placenta growth factor (PlGF) and soluble flt-1 by oxygen. A Review. Troph Res 2000 ; 14 : 16-24
  12. Tabibzadeh S, Babaknia A. The signals and molecular pathways involved in implantation, a symbiotic interaction between blastocyst and endometrium involving adhesion and tissue invasion. Mol Hum Reprod 1995 ; 10 : 1579-1602
  13. Tabizadeh S, Shea W, Lessey BA, Broome J. From endometrial receptivity to infertility. Reprod Endocrin 1999 ; 17 : 197-203
  14. Murray M, Lessey BA. Embryo implantation and tumor metastasis : Common pathways of invasion and angiogenesis. Reprod Endocrin 1999 ; 17 : 275-290
  15. Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia-initiated angiogenesis. Nature 1992 ; 359 : 843-845
  16. Kim KJ, Li B, Winer J, et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 1993 ; 362 : 841-844
  17. Charnock-Jones DS, Sharkey AM, Boocock CA, et al. Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells. Biol Reprod 1994 ; 51 : 524-530
  18. Ferrara N. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidn Int 1999 ; 56 : 794-814
  19. Alon T, Hemo I, Itin A, Pe’er J, Stone J, Keshet E. Vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity. Nature Med 1995 ; 1 : 1024-1028
  20. Semenza GL, Agani F, Iyer N, Kotch L, Laughner E, Leung S, Yu A. Regulation of cardiovascular development and physiology by hypoxia-inducible factor 1. Ann N Y Acad Sci 1999 ; 874 : 262-268
  21. Khaliq A, Li XF, Shams M, et al. Localisation of placenta growth factor in human term placenta. Growth Factors 1996 ; 13 : 243-250
  22. Athanassiades A, Hamilton GS, Lala PK. Vascular endothelial growth factor stimulates proliferation but not migration or invasiveness in human extravillous trophoblast. Biol Reprod 1995 ; 59 : 643-654
  23. Folkman J, Merler E, Abernathy C, Williams G. Isolation of a tumor factor responsible of angiogenesis. J Exp Med 1971 ; 133 : 275-288
  24. Hamai Y, Fujii T, Yamashita T, Kozuma S, Okai T, Taketani Y. Evidence for basic fibroblast growth factor as a crucial angiogenic growth factor, released from human trophoblasts during early gestation. Placenta 1998 ; 19 : 149-155
  25. Huang J-C, Liu D-Y, Dawood MY. The expression of vascular endothelial growth factor isoforms in cultured human endometrial stroma cells and its regulation by 17β-oestradiol. Hum Reprod 1998 ; 4 : 603-607
  26. Risau W. What, if anything, is an angiogenic factor ? Cancer Metastast Rev 1996 ; 15 : 149-151
  27. Genbacev O, Joslin R, Damsky CH, Polliotti BM, Fisher SJ. Hypoxia alerts early gestation human cytotrophoblast differentiation / invasion in vitro and models placentals defects that occurs in preeclampsia. J Clin Invest 1996 ; 97 : 540-550
  28. Ahmed A, Kilby MD. Placental insufficiency : hypoxia or hyperoxia ? Lancet 1997 ; 350 : 826-827
  29. Brosens I, Dixon HG, Robertson WB. Fetal growth retardation and the arteries of the placental bed. Br J Obstet Gynaecol 1997 ; 84 : 656-664
  30. Macara LM, Kingdom JCP, Kaufmann P, Kohen G, Hair J, More IRA, Lyall F, Greer IA. Structural analysis of placental terminal villi from growth-restricted pregnancies with abnormal umbilical artery Doppler waveforms. Placenta 1996 ; 17 : 37-48
  31. Kingdom JCP, Kaufmann P. Oxygen and placental villous development : Origins of fetal hypoxia. Placenta 1997 ; 18 : 613-621
  32. Krebs C, Longo LD, Leiser D. Term ovine placental vasculature : comparison of sea level and high altitude conditions by corrosion cast and histomorphometry. Placenta 1997 ; 18 : 43-51
  33. Carter AM. Placental oxygen consumption. Part I : In vivo studies a review. Troph Res 2000 ; 14 : 31-37