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Angiogenesis: basic pathophysiology and implications for disease

D.C Felmeden, A.D Blann, G.Y.H Lip
DOI: http://dx.doi.org/10.1016/S0195-668X(02)00635-8 586-603 First published online: 1 April 2003
  • Angiogenesis
  • Vasculogenesis
  • Vascular endothelial growth factor
  • Fibroblast growth factor
  • Angiopoietin

1 Introduction

The development of new blood vessels is essential to embryonic growth and throughout life for physiological repair processes such as wound healing, post-ischaemic tissue restoration, and the endometrial changes of the menstrual cycle. However, abnormal development of new blood vessels has been implicated in numerous pathophysiological processes. For example, inhibited growth of blood vessels is associated with bowel atresia and peptic ulcers.1–3 Furthermore, although generally focussing on tumour growth, increased vascular growth has been demonstrated in many other non-malignant diseases such rheumatoid arthritis, systemic lupus erythematosus, psoriasis, proliferative retinopathy and atherosclerosis.3–5 It is therefore clear that the subject is currently attracting considerable research energies as tools are becoming available to assess possible therapeutic options.

The formation of the vascular system is fashioned by three processes. During embryogenesis, there is differentiation of embryonic mesenchymal cells (the endothelial precursor cells or angioblasts) into endothelial cells resulting in de novo development of blood vessels (vasculogenesis).6 Secondly, angiogenesis refers to the formation of new blood vessels by sprouting from pre-existing small vessels in adult and embryonic tissue (sprouting angiogenesis) or by intravascular subdivision (intussusception). The existing vasculature can betransformed into a mature network by processes of pruning and remodelling. Thirdly, arteriogenesisis defined as rapid proliferation of pre-existingcollateral vessels.7 Angiogenesis also seems to bean organ-specific process reliant on the stage of microvascular network.8

Since angiogenesis seems to play a key role inthe pathophysiology of various disease processes,recent attempts have been made to utilize this knowledge in the development of new therapeutic approaches. For example, inhibition of angiogenesis has been used in the restriction of tumour growth and the seeding of metastases, as well asin rheumatoid arthritis, where an aim is to reduce the infiltration of inflammatory cells and soluble mediators.9–11

Angiogenesis related research in cardiovascular medicine has initially been linked to ischaemic heart disease and atherosclerosis. The observed raised angiogenic markers resulted in a theory of impaired angiogenesis in cardiovascular disease.12 One therapeutic direction in ischaemic vascular disease has been to use various angiogenic growth factors in an effort to improve vascularization,12–14 and more recently the role of angiogenesis in hypertension has also been investigated.15 However, in order to discuss the potential implications of angiogenesis in disease states, the mechanisms of vascular growth need to be fully understood.

2 Search strategy

In order to achieve our objective of summarizing current literature on angiogenesis, fibroblast growth factor (FGF) and vascular endothelial cell growth factor, we entered these and other key words into online literature search engines such as PubMed and EMBASE, as well as obtaining data and copy from other current reviews, reference listsof current literature, information from expert colleagues and abstracts from meetings of relevant societies.

3 Basic mechanisms of blood vessel formation

3.1 Vasculogenesis

In embryogenesis, vasculogenesis is a complexbut ordered process involving the differentiationof endothelial precursor cells (angioblasts) from primitive mesoderm commencing with gastrulation.16,17 This process is probably induced by FGF.18 The angioblasts can be distinguished adjacent to primitive blood cells, and are located in distinct zones that when merged together are the first indication of a primitive vasculature. In the next step, these mesoderm-derived angioblasts differentiate into endothelial cells and form de novo vessels.19 The process of vasculogenesis occurs predominantly during embryonic development. These initial blood vessels consist purely of endothelial cells and are referred to as capillary plexus.8 The succeeding development of various diverse blood vessels is a complex process. The ultimate vessel structure is determined by the derivation of the endothelial cells and smooth muscle cells comprising the vessel wall.

The process of subendothelial smooth muscle cell layer development incorporates migration, and proliferation of different cell types such as pericytes, smooth muscle cells and fibroblasts. The precise mechanisms involved in early vessel formation have yet to be elucidated but observations indicate that the primordial endothelium can recruit undifferentiated locally derived mesenchymal cells and direct their differentiation into pericytes in microvessels, and smooth muscle cells in large vessels.20 In comparison to the rather uniformendothelial cells, vascular smooth muscle cells are much more diverse. They can develop from endothelial cells as well as fibroblasts.21,22 In additionto endothelial and splanchnic mesodermal origin, there is also evidence of derivation from themesectoderm of the neural crest.23,24 The diverse origin of the vascular smooth muscle cell is an important factor in the tissue specific make-up of the final blood vessel.

During vasculogenesis, mesodermal precursor cells form a primitive vascular plexus. Vascular structures such as the dorsal aorta and the heart are also formed. This process involves the differentiation and organization of endothelial cells into capillary tubes and the interplay between growth factors and cytokines. The subsequent process of remodelling of the primary capillary plexus is termed angiogenesis.25

3.2 Embryonic angiogenesis

The primary step of angiogenesis is thought to be initiated by activation of endothelial cells of pre-existing vessels in response to increasing levelsof local angiogenic stimuli. This results in local vasodilatation, increased vascular permeability and the disruption of the basement membrane encompassing endothelial cells of the existing capillaries via proteolytic degradation.26 These enzymes may be activated by growth regulatory molecules.27 The disturbance of the basement membrane allowscytoplasmatic processes to extend from the activated endothelial cells, directing their migration and sprouting into the extravascular space toward the angiogenic stimulus. After the proliferation, elongation and alignment of the endothelial cells follows the formation of capillary sprouts. The growing sprout eventually develops a lumen and consequently these tubular structures anastomose with neighbouring vessels. The resulting capillary loop then permits blood flow.8,25 In the final stage these vessels are again remodelled by stabilization and regression. The development of establishing and remodelling of blood vessels is believed to be mediated by paracrine signals, and the formation of the basement membrane completes the maturation process.28–30

3.3 Post-embryonic angiogenesis

In post-embryonic development the main form of vasculature expansion is angiogenesis, also referred to as neovascularization. Post-embryonicangiogenesis follows the pattern of embryonicangiogenesis, and as tissue grows expansion ofthe vasculature is essential. This process includes growth and disappearance of capillaries and formation of arterioles and venules6,8,28 (Table 1). Angiogenesis also involves the differentiation and organization of endothelial cells into capillary tubes and the interplay between growth factors and cytokines. Cell adhesion molecules generally mediate innumerable cell–cell and cell–matrix interactions. These, in conjunction with the recruitment of supporting pre-endothelial cells that encasethe endothelial tubes, provide maintenance and modulatory functions to the vessel. Supporting cells usually include pericytes in small capillaries and smooth muscle cells in larger vessels.29,30

View this table:
Table 1

Key events of angiogenesis

In a healthy mature organism endothelial cell turnover is, with the exception of angiogenesis, very low. Angiogenesis is essential during vessel growth in most organs particularly in pathophysiological processes occurring in response to injury such as gastrointestinal ulcers, strokes, myocardial infarction and left ventricular hypertrophy.31–34Female reproductive organs demonstrate ongoing physiological angiogenesis to ensure the proper biological functioning of these organs during their lifespan.35–37 The expression of numerous angiogenic growth factors is required in the development of ovarian follicles and corpus luteum.38,39

4 Angiogenic growth factors

The existence of angiogenic factors was first observed with the isolation of a tumour factor that generated mitogenic activities in endothelial cells and later found to be a member of the FGF family.40 Angiogenetic growth factors are produced by a variety of different cells, and their functions include close involvement in developmental as well as tumour angiogenesis.41 Indeed, angiogenic growth factors such as vascular endothelial growth factor (VEGF), FGF and angiopoietin are essential to angiogenesis.19,40–42 Further to the initiation of angiogenesis these growth regulators establish the rate and extent of angiogenesis. However, little data are available about the resolution phase of angiogenesis. It is still unclear if this process results from exhaustion of the growth factors or if negative regulators predominate in this phase.

Angiogenic growth factors are so-called because of their varying ability to induce the proliferationof various cells in vitro, which contribute to the process of angiogenesis in vivo, as demonstrated by studies of animal models (Table 2). These growth factors are produced by various cell types and include a diverse range of proteins in addition to VEGF and FGF: platelet derived growth factor, tumour necrosis factor, insulin like growth factor-1, transforming growth factor, angiogenin, hepatocyte growth factor, placental growth factor and several others.43,44 Of the vast number of angiogenetic growth factors described, the FGF and VEGF families have been most extensivelyresearched and will be described in more detail.

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Table 2

Phenotypes of transgenic mice with embryonic defects in vascular development

4.1 Fibroblast growth factor

The first angiogenic growth factor to be discovered,40 this family currently comprises at least20 molecules with extensive mitogenic potentials representing some of the most potent angiogenic peptides. They are produced by vascular endothelial and smooth muscle cells, hence their almost omnipresent distribution. With numerous biological activities, including induction of proliferation ofa wide range of cells, the FGFs are closely involved in several developmental and pathophysiological processes.44,45 They stimulate fibroblast as well as endothelial cell growth and are therefore of vital importance in the process of angiogenesis,41 and also play a significant part in at least three ofthe four phases of wound healing: inflammation, repair and regeneration.42,46,47 Further importantfunctions of FGFs include tumour development and progression.

One characteristic of the FGF family is the ability to interact with heparan-like glycosaminoglycansof the extra-cellular matrix.48 The biological responses of FGF are mediated through the activation of four specific receptors, membrane-spanningtyrosine kinases resulting in an increase of multiple isoforms of FGF due to alternative mRNA splicing.45,49 The two most widely researched isoforms are FGF-1 and FGF-2.

4.1.1 Fibroblastic growth factor-1

Also known as the acidic FGF, in its mature form itis a 16kD peptide. FGF-1 (as well as FGF-2) doesnot have a signal peptide for channelling throughthe classical secretory pathway, but possesses a nuclear localization motif.50,51 FGF-1 has also been shown to stimulate DNA synthesis without signalling through a cell surface receptor, suggestive ofan intracrine mechanism transmitting a nuclear localization signal.52

Like other members of the family, FGF-1 has mitogenic and chemotactic effects especially on fibroblasts, endothelial cells and smooth muscle cells. It also contributes to the control of capillary progression, wound healing and tumour progression. Not surprisingly, FGF-1 expression is increased during regeneration of endothelial cells, hypoxia and collateral formation.44,53–55 However, so far in vivo studies looking into its potential therapeutic use have been disappointing.56

4.1.2 Fibroblastic growth factor-2

This single chain 18kDa polypeptide is also referred to as basic FGF and has a 55% sequence identity with FGF-1.57 Hypoxia, in addition to a number of other growth factors, increases its activity.55 FGF-2 is one of the most potent mitogens and chemotactic factors of the vascular endothelial cell. Recently, it has been demonstrated that basic FGF and VEGF have synergistic effects on angiogenesis in vivo.58 Numerous studies are currently investigating the potential role of FGF-2 and VEGF in the treatment of coronary artery disease.

4.2 Vascular endothelial growth factor

Initially purified as vascular permeability factor (VPF) from tumour cell ascites,59 its biologicaleffects were subsequently shown to extend toendothelial cell mitogenesis, prompting the name change to VEGF.60–62

VEGF is now known to be a multifunctional peptide capable of inducing receptor-mediated endothelial cell proliferation and angiogenesis both in vivo and in vitro.60–63 In addition to its crucial role in embryonic vascular development, VEGF has been implicated in the process of neovascularization in adult pathophysiology.63–65 VEGF is a basic, 45kDa disulfide-linked dimeric glycoprotein, that binds heparin and is structurally related to platelet derived growth factors.63 VEGF loses all biological activities following reduction and dissociates into monomeric units between 17 and 23kDa.60 The various VEGF iso-proteins have been described which have a circulating half-life of between 10min and 6h, depending upon the isoform, and the exogenous stimulus.66–69 The whole VEGFfamily currently consists of at least five members whose effects are mediated via three VEGF receptors (VEGFR), (Table 3). These receptors communicate with the cell interior via transmembrane receptor tyrosine kinases (RTKs).

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Table 3

Properties of the members of the VEGF family

4.2.1 VEGF-A (VEGF)

Interestingly, human chromosome 6p21.3, that encodes for the VEGF-A gene, the first VEGF protein identified, is also a location giving origin to several human disorders with unidentified genetic defects.70,71 The VEGF gene sequence extends over approximately 14kb, encoding eight exons that are separated by seven introns.72,73 Through alternate exon splicing of this gene different mRNA areencoded producing five biologically active proteins (VEGF121, VEGF145, VEGF165, VEGF189and VEGF206).62,72–74 All VEGF-A transcriptions have the amino terminal 141 amino acids in common. This consists of a signal peptide enabling its identification by VEGFR Flt-1 and KDR. Exons six and seven code for peptides determining the capability of binding to the extra-cellular matrix and/or heparan sulphate proteoglycan. All VEGF isoforms aresecreted glycoproteins. They are able to homodimerize and bind to heparin (except VEGF121).75,76

VEGF165, often referred to as VEGF-A or simply VEGF, is the predominant human isoform secreted by a variety of normal and transformed cells.Although all human VEGF-A isoforms are able to induce in vivo angiogenesis,73 there are, however, differences in their capability to bind heparansulphate and VEGFR (Flt-1). The soluble glycoproteins VEGF121, VEGF145and VEGF165can bedetected by biochemical assays (e.g. ELISA) of fluid samples such as human serum and plasma.77–80 VEGF121is a weakly acidic polypeptide failing to bind to heparan sulphate, whereas the VEGF isoforms VEGF189and VEGF206are more basic and exhibit higher affinity to heparin than VEGF165.72 The differences in the affinity for heparan sulphate and in the isoelectric point have a profound effect on the bioavailability of VEGF, leaving larger VEGF isoforms almost completely cell associated and bound to extra-cellular matrix.74,75 Only the isoform VEGF165is freely diffusible and able to bind to heparin, which is an indicator of its mitotic activity for vascular endothelial cells. There is also evidence to suggest that the stability of the VEGF–heparan sulphate receptor complex may contribute to effective signal transduction and therefore proliferation of the vascular endothelial cells. In contrast, VEGF206is the rarest isoform and has so far only been discovered in human foetal liver cDNA library.74–76

4.2.2 VEGF-B

This member of the VEGF gene family is composed of 188 amino acids and can be expressedas homodimer or heterodimer with VEGF-A..81–83 Alternate splicing of the VEGF-B gene, situatedon chromosome 11q13, results in two isoforms. VEGF-B167is a soluble peptide and VEGF-B189is bound to the cell and extra-cellular matrix82and has been shown to stimulate vascular endothelial cell proliferation. These findings resulted in the hypothesis that VEGF-B may contributeto the regulation of angiogenesis in muscletissue.81

4.2.3 VEGF-C

VEGF-C is a protein composed of 419 amino acids, with a predicted molecular mass of 47kDa whose gene is located on chromosome 4q34.83,84 VEGF-C shares 30% of the VEGF homology domain and can be found in small quantities in myocardium, placental tissue, skeletal muscle, ovaries, in certain tumour cell lines and is present in platelets.66,85,86 It is involved in the formation and maintenance ofthe venous and lymphatic systems and promotes lymphatic endothelial cell proliferation and vessel enlargement.87–89 Nonetheless, there is also data to suggest that VEGF-C may possess angiogenic properties relating to capillaries.90 The actions of both VEGF-C and VEGF-B are mediated via their receptors Flt-1 and Flt-4 resulting in a paracrine pathway.85,91

4.2.4 VEGF-D

The latest member of the human VEGF family to be described in detail, VEGF-D, shares 61% homology with VEGF-C and its gene is located on chromosome Xp22.31.92 Human VEGF-D seems to be generated by proteolytic processing of precursor polypeptides.93,94 VEGF-D is recognized by VEGFR-2 and VEGFR-3, which are present on endothelial cells,93 and appears to be capable of stimulating lymphangiogenesis.95 There is further evidence to suggest that VEGF-D may promote the spread of tumour cells via the lymphatic system.96

4.2.5 VEGF-E

Based on the sequence of VEGF-A121, a further VEGF variant, VEGF-E, was discovered in the genome of Orf virus.97 The Orf virus is an epitheliotropic parapoxvirus which induces proliferative skin lesionsin goats, sheep and humans (seen as ‘milker's nodules’).98 In addition to the characteristic cysteine residue present in all mammalian VEGF proteins, VEGF-E possesses a conserved threonine and proline rich region at the carboxyl terminus.97 VEGF-E binds with high affinity to VEGFR-2 resulting in stimulation of angiogenesis and vascular permeability, therefore enhancing viral infection.99

4.3 Placenta growth factor

The first VEGF-related protein, placenta growth factor (PlGF), discovered in 1991, owes its name to the predominance in placental tissue. It waslater identified as a member of the VEGF family asthe molecule shares 53% of a homologous domain with the platelet derived growth factor-like region of VEGF.100 Three isoforms arise by means ofalternate splicing, PlGF-1/PlGF131, PlGF-2/PlGF152 and PlGF-3.101 These molecules are, like VEGF, dimeric glycoproteins. However, the PlGF expression pattern is limited to the placenta and some forms of tumours such as brain tumours and renal cell carcinoma.102,103 PlGF homodimers bind VEGFR-1 (Flt-1), but have little effect on angiogenesis in vitro.101 On the other hand, naturally occurring VEGF/PlGF heterodimers, identified in rat glioma cells, are mitogenic; their potency is approximately sevenfold lower than that of the VEGF homodimer. Taking into consideration differential binding affinity and reports of hypoxia-induced up-regulation of VEGF/PlGF in vitro, it seems possible that PlGF and VEGF may becoexpressed in vivo.102–104

4.4 Angiopoietin

A further family of growth factors involved in the early processes of angiogenesis and vasculogenesis are the angiopoietins. One isotype, angiopoietin 1 (Ang1) is present in tissues adjacent to blood vessels suggesting a paracrine mode of action, whilst another, angiopoietin 2 (Ang2) is only found at sites of tissue remodeling.105,106 Both angiopoietins, including the two recently discovered angiopoietin-3 (in mouse) and angiopoietin-4 (in humans), have been identified as ligands for the Tie-2/Tek receptor.105,107 In vitro neither Ang1 nor Ang2 havemitogenic effects mediated via Tie-2.105 However, Ang1 facilitates endothelial cell sprouting and vascular network maturation.58,108 Ang2 antagonises Ang1 by blocking Ang1-induced phosphorylization of Tie-2.106 On the other hand Ang2, in combination with VEGF, promotes neovascularization.58 Knock-out mice for either Tie-2 or Ang1 genes demonstrate an embryonic lethal phenotype caused by defective embryonic development of the vasculature resulting in immature vessels and lack of branch network.109,110 The findings indicate a contribution of the angiopoietin/Tie-2 system at later stages in the vascular development. This system appears to be particularly involved in the determination of the subdivision of the initially homogeneous capillary network into larger arterioles and venules.110 A mutation of the RTK Tie-2 in mice leads to vascular dysmorphogenesis, possibly instigated by a lack of peri-endothelial support cell recruitment resulting in underdevelopment of smooth muscle cell layers.111

4.5 VEGF receptors

In humans, the effects of VEGF on endothelial cells is mediated via two high-affinity membrane-spanning receptors, VEGFR-1 and VEGFR-2. They are also referred to as RTK. Both receptors have a high affinity for VEGF and possess seven characteristic immunoglobulin-like domains that form the extra-cellular section. Additionally, a kinase-insert domain links a single transmembrane region and a consensus tyrosine kinase.112–115 VEGFR-1 and VEGFR-2 are 33% identical in their extra-cellular domain and 80% in their kinase domains. Bothreceptors are predominantly expressed on endothelial cells, but have also been detected on human uterine, colonic and aortic smooth muscle cells, trophoblasts and in foetal kidney.116,117 VEGFR-3 is a further RTK with seven immunoglobulin-likedomains. This receptor is mainly expressed in lymphatic vessels and binds only VEGF-C and -D118 (Table 4).

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Table 4

VEGF receptors

4.5.1 VEGFR-1

Vascular endothelial growth factor receptor-1 (VEGFR-1) also known as fms-like tyrosine kinase-1 (Flt-1), is a 180kDa surface associated RTK.115 The human gene is located on chromosome 13q12.119 Flt-1 and VEGFR-2 are predominantly expressed on the vascular endothelium, but traces of mRNA have been located in monocytes, renal mesangial cells and stroma of human placenta.120–122 PlGF, VEGF-A121, VEGF-A165, and VEGF-B, associate with this receptor with varying affinity.123,124 VEGF-A165binds to VEGFR-1 with high affinity than VEGF-A121.125,126 The ability of the receptor to attach heparan-sulphate proteoglycan is eluded after the removal of the second immunoglobulin-like domain of VEGFR-1.127

In addition to the full-length receptor, the VEGFR-1 gene encodes for a soluble form carrying only six immunoglobulin domains. This form results from differential splicing of the Flt-1 mRNA and was first discovered in human umbilical vein endothelial cells.128,129 This soluble receptor, referred to as soluble Flt-1 (sFlt-1), attaches itself to VEGF121with a high affinity, and is present in human plasma15,77 and amniotic fluids from pregnant women.130,131 Currently, the biological implications of sFlt-1 remain unknown although in vitro studies have demonstrated that it is capable of reducing VEGF-induced mitogenesis.128,129 Therefore, sFlt-1 may correspond to a physiologicalregulatory mechanism for reducing VEGF action.

4.5.2 VEGFR-2

The gene of the second VEGF tyrosine-kinase receptor, VEGFR-2, is located on chromosome 4q12.132 VEGFR-2 is also known as kinase-insert-domain containing receptor (KDR), and is homologous to the foetal liver kinase-1 (flk-1) receptor in mice. KDR is predominantly expressed in endothelial cells and was cloned from a human endothelial cell cDNA library.133–135 However, the mRNA for this receptor can also be detected in haematopoietic stem cells, megakaryocytes and retinal progenitor cells.136–140 VEGFR-1 and VEGFR-2 transduce signals for endothelial cells in response to ligands of the VEGF family. Their individual reaction is distinctivelydifferent. Unlike Flt-1, the final glycosylatedform of KDR undergoes VEGF-triggered auto-phosphorylation, which may explain the much weaker response to VEGFR-1 activation.141 KDR binds VEGF121, VEGF145, VEGF165VEGF-C and VEGF-D.126,142 Despite numerous similarities between VEGFR-1 and VEGFR-2, a naturally occurring soluble form of KDR comparable to sFlt-1 has not been described.

4.5.3 VEGFR-3

The VEGFR-3 gene is encoded in the chromosomal region 5q34–q35.143 VEGFR-3 is also known as fms insert-like tyrosine kinase 4 (Flt-4) and its extra-cellular domain is 80% homologue to the other VEGFR.118 Only VEGF-C and VEGF-D of the VEGF family are associated with Flt-4.83,93 Unlike VEGFR-1 and VEGFR-2, Flt-4 is predominantlyexpressed in lymphatic endothelium in adult tissue.85,95,144 However, in most vascular endothelial cells low levels of VEGFR-3 are detectable. Its presence, particularly on lymphatic endothelial cells and on developing vessels of several organs suggests that Flt-4 together with its ligandsmay have a role in the regulation of growth and differentiation of the lymphatic system.96

4.5.4 Neuropilins

In addition to VEGFR-1 and VEGFR-2, endothelial cells express neuropilin-1 (Neu-1) and neuropilin-2 (Neu-2), which selectively bind (but with low affinity) VEGF-A165. Due to a short intracellular domain of these receptors they are not likely to operate as an independent receptor. This is further supported by lack of cellular response when stimulating only the neuropilins.145 However, during the embryonic stages of angiogenesis neuropilin-1 seems toregulate blood vessel development, suggesting a role as coreceptor for VEGFR-2.146 The geneticencoding and exact biological purpose has yet to be discovered.

5 Regulation of VEGF production

As a key regulator, it is essential that the expression of VEGF is itself correctly controlled in order to prevent uncontrolled angiogenesis. There are a plethora of cytokines, growths factors and physiological parameters modulating the production of VEGF, depending on the current status quo. In the mature organism, VEGF expression is limited and a balance between angiogenic and anti-angiogenic stimuli is maintained.41 However, in response to tissue damage, a wide array of growth factors, cytokines and other molecules is released stimulating angiogenesis directly or indirectly via VEGF which is essential for the repair process.

In pathophysiological situations such as cancer and diabetes mellitus, stimulated VEGF expression might result in increased pathological angiogenesis. This hypothesis is further supported by datademonstrating a suppression of neovascularization by inhibition of VEGF or its effects.147,148 However, in other circumstances, such as atherosclerosis and diabetes, the increased plasma VEGF concentration77 might be an attempt to compensate for tissue damage or hypoxia, or may simply reflect endothelial cell damage apparent in these conditions.

5.1 The interaction of VEGF with cytokines and other growth factors

Factors that can alter VEGF production include platelet derived growth factor, tumour necrosis factor-α (TNF-α), fibroblast growth factor 4 (FGF 4), bFGF, transforming growth factor-β (TGF-β), PDGF, angiotensin-2, insulin-like growth factor I, keratinocyte growth factor, interleukin 1 (IL-1) and IL-6.69,149–160 A few substances, such as the cytokines IL-10 and IL-13, decrease VEGF production.161

The angiopoietins also influence VEGF release.85,105 Ang-1 stimulates vessel sprouting whereas Ang-2 inhibits this effect, but also mediates destabilization of vessel integrity, which in turn facilities vessel sprouting in response to VEGF.106,110,162 These effects are mediated via the Tie-2 receptor. The combination of VEGF, Ang-1 and Ang-2 is essential for successful angiogenesis as established in vivo experiments.58

5.2 Effect of oxygen on VEGF expression

Apart from growth factors there is a variety of chemical stimuli affecting the release of VEGF. Hypoxia, which occurs in pathophysiological processes such as atherosclerosis, solid tumours and proliferative retinopathy, is a major stimulator of VEGF expression resulting in neovascularization.163 Hypoxia induces a protein called hypoxia inducible protein complex (HIPC) or hypoxia-inducible factor (HIF).

This heteromeric basic helix–loop–helix transcriptional regulator is activated by reduced oxygen tension and up-regulates the transcription of VEGF mRNA. HIF increases production of VEGF mRNAwith enhanced stability by directly attaching to a HIF-1 binding-site located in the VEGF promoterregion.67,164,165 Furthermore VEGFR-1 seems to be up-regulated through hypoxia induced HIF.166

Hypoxia not only increases VEGF production but it also seems to increase the stability of some VEGFisoforms.149,167–169 With regard to stability, VEGF-A isoforms are hypoxia sensitive whereas hypoxia has little or no effect on VEGF-B and VEGF-C mRNA.66 This variation in the behaviour of VEGF isoforms may be another regulatory mechanism, that ensures that the different VEGF species are tissue and/or functionally specific.

Further mechanisms leading to hypoxia-induced increase of VEGF production may be related to often associated features of hypoxia such as tissue damage, necrosis and apoptosis. These events may therefore trigger the release of cytokines and other chemical mediators from cells of the surrounding tissue, initiating a cascade of events leading tothe production of VEGF.65,170 These events arediscussed below.

The importance of oxygen as a regulator of VEGF production is further emphasized by demonstrating inhibitory properties of the normoxic or even hyperoxic environment. Hypoxia-induced VEGF increase returns to baseline levels within 24h of the return of the cells to normoxia.171 VEGF expression is decreased in in vitro and in vivo studies following hyperoxia.172,173 Additionally, hyperoxia-induced retinopathy in prematurely born mice can be prevented by intraoccular VEGF injection.174 These data clearly demonstrate the importance of oxygen as a regulatory mechanism of VEGF expression.

5.3 Regulation of VEGF by nitric oxide

VEGF is known to induce the release of nitric oxide (NO) from endothelial cells, and vascular endothelium and inducible NO synthase (iNOS) production is amplified during VEGF-induced angiogenesis. Therefore the physiological effects of VEGF may, at least in part, be mediated by endothelium derived NO.175,176 The vital role of NO in VEGF-induced angiogenesis has also been demonstrated in NOS knock-out mice as well as after NOS inhibition, both resulting in reduction of angiogenesis.175,177 NO, on the other hand, also has regulatory effects on VEGF production. Protein kinase C mediated binding of the transcription activator protein-1 (AP-1) is decreased by NO.178 This results in reduced stimulation of the promoter region of the VEGF gene, hence lower VEGF expression. Pathological circumstances coupled with impaired NO availability, such as atherosclerosis, are associated with increased VEGF levels consistent with the presence of a negative feedback loop.178,179 Increased levels of plasma VEGF have been demonstrated in patients with various risk factors for atherosclerosis such as diabetes mellitus and hypertension,15,77 further supporting this theory although, as discussed, raised VEGF may also be related to tissue hypoxia or may simply reflect endothelial damage. The same rationale may also partly explain raised plasma VEGF in certain cancers180,181 as the demands of the growing tumour may create a local hypoxia.

5.4 Effect of glucose on VEGF expression

Hypoglycaemia increases VEGF expression, which was initially thought to be an indirect consequence mediated via associated hypoxia. However, up-regulation and increased production of VEGF have been described in cells exposed to hypoglycaemia independently of HIF (hypoxia).169,182–184 After equilibration of the glucose concentrations VEGF production returned to pre-experimental levels184 suggesting that acute hypoglycaemia may trigger VEGF mediated angiogenesis.

Furthermore,185 increased intracellular Ca2+levels in a glucose-deprived environment leads to activation of protein kinase C. This process induces the activation of AP-1 resulting in increase ofVEGF expression, thus not only confirming previous studies but exposing its underlying mechanism.

Remarkably, not only lack of glucose but also high glucose levels result in an upsurge of VEGF mRNA,150,186,187 as well as production of VEGF and VEGFR-2.180 Recent studies have demonstrated that hyperglycaemia can directly increase VEGF expression via a protein kinase C dependent mechanism, and this effect can be abolished by a protein kinase C inhibitor.186–188 Hyperglycaemia induced VEGF up-regulation is also reversible by normalizing the extra-cellular glucose concentration in SMC.150 Therefore, and possibly difficult to explain simply, and type of non-euglycaemia seems a strong up-regulatory factor for VEGF expression. Hence the apparent relationship between angiogenesis, VEGF and diabetes4,77,137,189 requires clarification.

6 Pathophysiological consequences of the interactions between growth factors and their receptors

The importance of the specific angiogenic activities of VEGF and its receptor interactions in the process of endothelial cell proliferation, differentiation, migration and growth has been considerably enhanced by analysis of knock-out mice.190,191 The pattern of abnormalities observed provides some evidence for the role of VEGF and its receptors Flt-1, KDR and Flt-4, along with Tie-2/Tek and its ligands angiopoietins 1 and 2. Certainly, all four receptors are essential for vasculogenesis as mutations in the loci of any of the gene coding for these receptors leads to embryonic lethality due to imperfections in the haemotopoietic and endothelial cell lineage. Mutations in different genes encoding VEGF or its receptors become evident as different phenotypic defects.192,193 Homozygous VEGF receptor deficiency resulting in embryonic death varies from heterozygous VEGF gene mutation, which generates an embryonic lethal phenotype.192,193

There are also different patterns arising from receptor mutants. Unlike KDR, Flt-1 not only affects endothelial cell proliferation and differentiation, but also blood vessel construction as demonstrated by certain mutations in Flt-1 loci causing embryonic lethality due to inadequate vessel assembly.19,42 After targeted inactivation of the Flt-4 gene,vasculogenesis and angiogenesis occur but thelarge blood vessel development is disorganized with irregular sized vessels and defective lumens leading to cardiovascular failure.162 However, mutation in the genes for angiopoietin or its receptors results in disrupted vessel structure and impaired capillary functions leading to haemorrhage.106,110 Findings from these studies suggest that in embryonic vasculogenesis, KDR-mediated processes precede those of Flt-1. KDR is involved in endothelial cell formation, proliferation and migration in the early stages of vasculogenesis, whilst Flt-1 plays a role in embryonic vascular assembly following differentiation of endothelial cells. At an even later stage Flt-4 is involved in organizing large vessels and the emergence of lymphatic vessel formation but preceding the angiopoietins and their receptors.106,110 Table 2 details the phenotypic mutations observed with targeted gene mutation of VEGF-A, the angiopoietins and their respective receptors.

7 Summary and clinical perspectives

The majority of our knowledge of VEGF originates from work done as part of studies in cancer research, as the ability of a tumour to metastasize seems be related to the quantity of VEGF produced.134 VEGF has been detected in numerous tumour cells and in the plasma of patients with various cancers,101,180,181,194–202 and hypoxia appears to play an important part as the expressionof VEGF mRNA and production of the growth factor is intensified in regions neighbouring the necrotic area.197,203 Furthermore, surgical excision of alocalized tumour resulted in a prompted reduction in circulating VEGF.204 In addition, VEGF may also have a role in the regulation of inflammatory repair processes as VEGF increases vascular permeability and acts as chemotactic agent for phagocytic cells, both processes of eminent importance duringinflammation.205 VEGF expression is dramatically up-regulated in chronic wounds such as venous leg ulceration particularly in the hyperplastic epithelial region of the wound margin.206 Similar findings have been observed in resected liver where higher levels of VEGF have been demonstrated when compared to normal liver.207 Again hypoxia, a common feature in damaged tissue, seems to be the underlying mechanism.208

In chronic inflammatory disorders such as rheumatoid arthritis and systemic lupus erythematosus, raised levels of VEGF have been noted in plasma, serum and synovial fluid.209,210 Regrettably, however, in some of these cases (and, indeed, in any clinical study), VEGF data derived from serum isof limited value in the study of pure vascular responses as VEGF may also arise from platelets.80,211 However, the existence of VEGF in the sub-synovial macrophages, leukocytes, fibroblasts and synovial lining cells implies some participation in the inflammatory process.212,213 Indeed, it has been suggested that the amount of VEGF in rheumatoid synovium may be a marker for joint destruction.214 Overall, therefore, it appears plausible thatVEGF-induced angiogenesis and increased vascular permeability may promote these chronic inflammatory processes. More recently, possible roles for VEGFs C and D and their receptors in the development of arthritic synovia have been proposed.215

Recently, a link between VEGF and cardiovascular disease has been established. Atherosclerosis eventually results in progressive arterial occlusion which leads to ischaemia, hypoxia and subsequently to necrosis. These processes trigger the expression of a variety of vasoactive substances, matrix proteins and growth factors, which mediate neovascularization, remodelling of the vasculature and surrounding tissue.203 Animal studies of VEGF in various aspects of cardiovascular disease216–220 have provided pilot data for studies in man. For example, histological studies of coronary atherosclerotic plaques, saphenous vein bypass grafts, and areas of recent myocardial infarction that demonstrated increased VEGF expression221–224 have given way to observational clinical studies.225,226

Pathophysiological, possibilities include the suggestion that acute myocardial ischaemia rapidly induced up-regulation of VEGF and its receptors VEGFR-1 and VEGFR-2, whereas areas of healed myocardial infarction failed to demonstrate that effect.216,220 These data would suggest that VEGF plays a role in neovascularization in connection with myocardial ischaemia and atheroscleroticarteries. Atherosclerotic lesions in human coronary arteries demonstrate distinct expression of VEGF, VEGFR-1 and VEGFR-2 on endothelial cells, macrophages and partially differentiated smooth muscle cells.221,222 Moreover, in patients with coronary artery disease there is a correlation between the directly measured index of collateral blood flow and intracoronary levels of VEGF, suggesting that VEGF is influenced by degree of coronary atherosclerosis.225 However, generally, the precise role(s) of large amounts of circulating VEGF in the plasma of subjects with long-standing peripheral or coronary atherosclerosis, or in acute myocardial infarction compared to asymptomatic controls77,78,227,228 is unclear. As histological data confirms amplified angiogenicity in atherosclerotic lesions by demonstrating a plethora of blood vessels within the atheromatous plaques itself and in the surrounding vessel walls,221–223,229,230 VEGF-mediated neovascularization of the media and adventitia ofdiseased vessels may be relevant in enhancing the supply of oxygen and nutrients to the affected tissue.231

View this table:
Table 5

Human tissue/cell studies of VEGF and angiogenesis in cardiovascular disease

View this table:
Table 6

Summary of studies measuring VEGF in the plasma or serum of patients with cardiovascular disease

Against this background is the presumption by many commentators that exogenous VEGF supplied as a therapy may provide a benefit in cardiovascular disease by enhancing collateral development232–235 and preliminary methodological work has been published236–238 with some success.239 However, recent animal data suggest that exogenously-supplied VEGF may actually enhance atherosclerotic plaque progression,240 implying that raised plasma VEGF in man77,78,227,228 may not be advantageous. Indeed, rheumatologists, studying a different disease where there is raised plasma VEGF209,210 and evidence of involvementin pathogenesis,212–215 seek to reduce angiogenesis,241 as do oncologists.40,181,194,196,204,242

The involvement of VEGF in atherosclerosis therefore seems undoubted, as summarized inTables 5 and 6 although its precise effects (and the value of interventions) are subject of an ongoing debate. It is heartening to note from recent data that therapeutic angiogenesis (e.g. with recombinant fibroblastic growth factor-2) in intermittent claudication does provide some clinical benefit, at least in phase II trials.243 Nonetheless, the variable results in clinical trials could at least in part reflect the inadequacy of preclinical in vitro and animal models. Only time will tell whether this approach would bring the potential morbidity and mortality benefits that we hope would arise.

Acknowledgments

We acknowledge the support of the City Hospital Research and Development programme for theHaemostasis Thrombosis and Vascular Biology Unit. We thank Dr F. Belgore for expert technical advice.

References