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.

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.

Basic mechanisms of blood vessel formation

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

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

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

Table 1

Key events of angiogenesis


Phase
 

Key events
 
Endothelial cell and pericyte activation Morphological changes of endothelial cells priming them for proliferation and secretion, local vasodilatation, increased vascular permeability, accumulation of extravascular fibrin 
Degradation of basement membrane Angiogenic stimulus results in proteolytic vascular basement membrane degradation 
Migration of endothelial cells Chemotactic factors produced by fibroblasts, monocytes and platelets induce endothelial cell migration and sprouting 
Proliferation of endothelial cells Locally produced mitogens induce endothelial cells DNA synthesis and mitosis 
Differentiation of endothelial cells Endothelial cell proliferation decreases and cell–cell contact re-establish, sprout develops lumen 
Reconstitution of basement membrane Vessel maturation achieved by reconstitution of basement membrane synthesized by endothelial cells and pericytes 
Vasculature maturation and stabilization
 
Capillary remodelling by stabilization and regression
 

Phase
 

Key events
 
Endothelial cell and pericyte activation Morphological changes of endothelial cells priming them for proliferation and secretion, local vasodilatation, increased vascular permeability, accumulation of extravascular fibrin 
Degradation of basement membrane Angiogenic stimulus results in proteolytic vascular basement membrane degradation 
Migration of endothelial cells Chemotactic factors produced by fibroblasts, monocytes and platelets induce endothelial cell migration and sprouting 
Proliferation of endothelial cells Locally produced mitogens induce endothelial cells DNA synthesis and mitosis 
Differentiation of endothelial cells Endothelial cell proliferation decreases and cell–cell contact re-establish, sprout develops lumen 
Reconstitution of basement membrane Vessel maturation achieved by reconstitution of basement membrane synthesized by endothelial cells and pericytes 
Vasculature maturation and stabilization
 
Capillary remodelling by stabilization and regression
 

From references 6,8,29,30 and elsewhere.

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

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.

Table 2

Phenotypes of transgenic mice with embryonic defects in vascular development


Affected gene
 

Stage of vessel development
 

Detected phenotype
 
VEGF-A Vasculogenesis and angiogenesis Malformation of dorsal aorta, defective heart and vessel sprouting. Delayed EC differentiation 
VEGFR-1 Vasculogenesis Disordered EC assembly causing enlarged blood vessel and impaired vasculogenesis 
VEGFR-2 Vasculogenesis Undifferentiated EC result in anomalous vessel structure and breakdown of vasculogenesis 
VEGFR-3 Vasculogenesis Abnormal vessel sprouting, organization and remodelling 
Angl Angiogenesis Impaired neural tube angiogenesis. Deficient vascular remodelling and endocardial branching 
Ang2 Maturity Deficient vessel integrity leading to haemorrhage and vascular oedema 
Tie-1 Maturity Deficient vessel integrity leading to haemorrhage and vascular oedema 
Tie-2 Angiogenesis Defective vascular remodelling and endocardial branching. Impaired neural tube angiogenesis 
Neu-1
 
Angiogenesis
 
Inadequate development of vascular networks
 

Affected gene
 

Stage of vessel development
 

Detected phenotype
 
VEGF-A Vasculogenesis and angiogenesis Malformation of dorsal aorta, defective heart and vessel sprouting. Delayed EC differentiation 
VEGFR-1 Vasculogenesis Disordered EC assembly causing enlarged blood vessel and impaired vasculogenesis 
VEGFR-2 Vasculogenesis Undifferentiated EC result in anomalous vessel structure and breakdown of vasculogenesis 
VEGFR-3 Vasculogenesis Abnormal vessel sprouting, organization and remodelling 
Angl Angiogenesis Impaired neural tube angiogenesis. Deficient vascular remodelling and endocardial branching 
Ang2 Maturity Deficient vessel integrity leading to haemorrhage and vascular oedema 
Tie-1 Maturity Deficient vessel integrity leading to haemorrhage and vascular oedema 
Tie-2 Angiogenesis Defective vascular remodelling and endocardial branching. Impaired neural tube angiogenesis 
Neu-1
 
Angiogenesis
 
Inadequate development of vascular networks
 

Modified from references 190,191 and elsewhere.

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.

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

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.

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).

Table 3

Properties of the members of the VEGF family


VEGF protein(references insuperscript)
 

Chromosomal location
 

Soluble VEGF isoform
 

Heparin-binding
 

Heparan-sulphate proteoglycan binding isoform
 
VEGF (VEGF-A)70,72 6q21.3 VEGF-A165, VEGF A145, VEGF-A121 VEGF-A189, VEGF-A206, weakly: VEGF-A121, VEGF A145, VEGF-A165 VEGF-A145, VEGF-A189, VEGF-A206 
VEGF-B81,82 1lq13 VEGF-B167 VEGF-B167 VEGF-B189 
VEGF-C66,85 4q34 No  No 
VEGF-D92 Xq22.31 Yes Yes No 
VEGF-E97 Orf Virus genome Yes No No 
PlGF100
 
14q24
 
PlGF-1
 
PlGF-2, PlGF-3
 
PlGF-1
 

VEGF protein(references insuperscript)
 

Chromosomal location
 

Soluble VEGF isoform
 

Heparin-binding
 

Heparan-sulphate proteoglycan binding isoform
 
VEGF (VEGF-A)70,72 6q21.3 VEGF-A165, VEGF A145, VEGF-A121 VEGF-A189, VEGF-A206, weakly: VEGF-A121, VEGF A145, VEGF-A165 VEGF-A145, VEGF-A189, VEGF-A206 
VEGF-B81,82 1lq13 VEGF-B167 VEGF-B167 VEGF-B189 
VEGF-C66,85 4q34 No  No 
VEGF-D92 Xq22.31 Yes Yes No 
VEGF-E97 Orf Virus genome Yes No No 
PlGF100
 
14q24
 
PlGF-1
 
PlGF-2, PlGF-3
 
PlGF-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

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

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

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

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

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

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

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).

Table 4

VEGF receptors


Receptor
 

Ligand
 

Function
 
VEGFR-1 (Flt-1) VEGF-A121 Promotion of cell migration 
 VEGF-A165 Organization of blood vessels 
 VEGF-B Gene expression of monocytes and macrophages 
 PlGF-1  
 PlGF-2  
VEGFR-2 (KDR, Flk-1) VEGF-A121 Mitogenesis, differentiation of endothelial cells 
 VEGF-A145 Promotion of cell migration 
 VEGF-A165 Enhancement of vascular permeability 
 VEGF-C  
 VEGF-D  
VEGFR-3 (Flt-4) VEGF-A145 Remodelling of primary capillary vasculature 
 VEGF-A165 Embryonic cardiovascular development 
 VEGF-A189 Regulation of growth and maintenance of lymphatic system 
 PlGF-2  
 VEGF-B167  
Neu-1 VEGF-A165 Development of cardiovascular system 
 PlGF-2  
Neu-2 VEGF-A165 Organization of peripheral nerve fibres 

 

 
Development of vascular networks
 

Receptor
 

Ligand
 

Function
 
VEGFR-1 (Flt-1) VEGF-A121 Promotion of cell migration 
 VEGF-A165 Organization of blood vessels 
 VEGF-B Gene expression of monocytes and macrophages 
 PlGF-1  
 PlGF-2  
VEGFR-2 (KDR, Flk-1) VEGF-A121 Mitogenesis, differentiation of endothelial cells 
 VEGF-A145 Promotion of cell migration 
 VEGF-A165 Enhancement of vascular permeability 
 VEGF-C  
 VEGF-D  
VEGFR-3 (Flt-4) VEGF-A145 Remodelling of primary capillary vasculature 
 VEGF-A165 Embryonic cardiovascular development 
 VEGF-A189 Regulation of growth and maintenance of lymphatic system 
 PlGF-2  
 VEGF-B167  
Neu-1 VEGF-A165 Development of cardiovascular system 
 PlGF-2  
Neu-2 VEGF-A165 Organization of peripheral nerve fibres 

 

 
Development of vascular networks
 

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.

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.

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

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.

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.

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

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.

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.

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.

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.106,110Table 2 details the phenotypic mutations observed with targeted gene mutation of VEGF-A, the angiopoietins and their respective receptors.

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

Table 5

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


References
 

Study sample(s)
 

Observations
 

Comments
 
Couffinhal et al., 1997223 Normal arteries, veins and atherosclerotic coronary arteries VEGF was immunolocalized predominantly to SMC in normal and atherosclerotic vascular tissue Localization of VEGF to normal and atherosclerotic vascular tissue implicates VEGF in vascular physiology 
Ruef et al., 1997218 Umbilical vein endothelial cells and vascular smooth muscle cells VEGF expression of vascular endothelial cells increased after oxidative stress and balloon-injuries VEGF may enhance neovascularization of atherosclerotic and restenotic arteries 
Inoue et al., 1998221 Coronary artery segments stained for VEGF, VEGFR-1 and VEGFR-2 VEGF activity, VEGFR-1 and VEGFR-2 were detected in atherosclerotic but not normal arteries Small autopsy study, role for VEGF in progression of CAD as well as recanalization 
Chen et al., 1999222 Coronary artery Number of VEGF positive cells correlates with number of intimal blood vessels More advanced type of atherosclerotic lesion contain more VEGF positive cells 
Lee et al., 2000220 Biopsied myocardial tissue Expression of VEGF mRNA was more pronounced in tissue with acute ischemia compared with normal ventricle or those with past episodes of infarction Acute myocardial injury may result in increased production of VEGF transcripts in humans 
Bobryshev et al., 2001224
 
Aortocoronary saphenous vein grafts
 
Areas of intimal neovascularization and neovascular endothelial cells were VEGF positive
 
VEGF local regulator of intimal neovascularization in saphenous vein grafts
 

References
 

Study sample(s)
 

Observations
 

Comments
 
Couffinhal et al., 1997223 Normal arteries, veins and atherosclerotic coronary arteries VEGF was immunolocalized predominantly to SMC in normal and atherosclerotic vascular tissue Localization of VEGF to normal and atherosclerotic vascular tissue implicates VEGF in vascular physiology 
Ruef et al., 1997218 Umbilical vein endothelial cells and vascular smooth muscle cells VEGF expression of vascular endothelial cells increased after oxidative stress and balloon-injuries VEGF may enhance neovascularization of atherosclerotic and restenotic arteries 
Inoue et al., 1998221 Coronary artery segments stained for VEGF, VEGFR-1 and VEGFR-2 VEGF activity, VEGFR-1 and VEGFR-2 were detected in atherosclerotic but not normal arteries Small autopsy study, role for VEGF in progression of CAD as well as recanalization 
Chen et al., 1999222 Coronary artery Number of VEGF positive cells correlates with number of intimal blood vessels More advanced type of atherosclerotic lesion contain more VEGF positive cells 
Lee et al., 2000220 Biopsied myocardial tissue Expression of VEGF mRNA was more pronounced in tissue with acute ischemia compared with normal ventricle or those with past episodes of infarction Acute myocardial injury may result in increased production of VEGF transcripts in humans 
Bobryshev et al., 2001224
 
Aortocoronary saphenous vein grafts
 
Areas of intimal neovascularization and neovascular endothelial cells were VEGF positive
 
VEGF local regulator of intimal neovascularization in saphenous vein grafts
 
Table 6

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


References
 

n
 

Observations
 

Comments
 
Seko et al., 199778 19 Serum VEGF levels were significantly elevated in patients with AMI compared to controls. After reperfusion, levels were normalized Small observational study, illustrating the acute induction of circulating VEGF and also reflecting the relatively short half-life of this growth factor 
Fleisch et al., 1999225 76 Levels of intra-coronary VEGF in patients undergoing angioplasty correlated with collateral flow and proximal VEGF levels were higher in patients with more stenotic lesions Large observational study suggesting that serum VEGF levels may be dependent on the degree of coronary atherosclerosis and/or disease severity 
Hojo et al., 2000227 30 Levels of VEGF in serum increased gradually after the onset of AMI and peaked on day 14 Medium sized progressive study, also showing acute induction of VEGF in humans with CVD 
Burton et al., 2000226 32 Post-operative serum VEGF levels were significantly greater than pre-operative levels following coronary artery bypass surgery Medium sized comparative study showing acute induction of VEGF following cardiovascular surgery 
Belgore et al., 200115 21 Levels of plasma VEGF and sFlt-1 were significantly raised in hypertensives compared with controls and these were normalized after successful therapy Medium sized intervention study highlighting the possible involvement of VEGF in hypertension and the effect of therapy on levels of VEGF and sFlt-1 
Blann et al., 200277
 
140
 
Levels of plasma VEGF were significantly raised while levels of sFlt-1 were lower in the patients with PAD or CAD compared with controls. Also, diabetic data.
 
Large comparative study of two patient groups with variant atherosclerosis. First study to examine the levels of sFlt-1 in plasma in atherosclerosis.
 

References
 

n
 

Observations
 

Comments
 
Seko et al., 199778 19 Serum VEGF levels were significantly elevated in patients with AMI compared to controls. After reperfusion, levels were normalized Small observational study, illustrating the acute induction of circulating VEGF and also reflecting the relatively short half-life of this growth factor 
Fleisch et al., 1999225 76 Levels of intra-coronary VEGF in patients undergoing angioplasty correlated with collateral flow and proximal VEGF levels were higher in patients with more stenotic lesions Large observational study suggesting that serum VEGF levels may be dependent on the degree of coronary atherosclerosis and/or disease severity 
Hojo et al., 2000227 30 Levels of VEGF in serum increased gradually after the onset of AMI and peaked on day 14 Medium sized progressive study, also showing acute induction of VEGF in humans with CVD 
Burton et al., 2000226 32 Post-operative serum VEGF levels were significantly greater than pre-operative levels following coronary artery bypass surgery Medium sized comparative study showing acute induction of VEGF following cardiovascular surgery 
Belgore et al., 200115 21 Levels of plasma VEGF and sFlt-1 were significantly raised in hypertensives compared with controls and these were normalized after successful therapy Medium sized intervention study highlighting the possible involvement of VEGF in hypertension and the effect of therapy on levels of VEGF and sFlt-1 
Blann et al., 200277
 
140
 
Levels of plasma VEGF were significantly raised while levels of sFlt-1 were lower in the patients with PAD or CAD compared with controls. Also, diabetic data.
 
Large comparative study of two patient groups with variant atherosclerosis. First study to examine the levels of sFlt-1 in plasma in atherosclerosis.
 

n, number of patients.

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.

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

1
Folkman
J
, Szabo S, Stovroff M et al. Duodenal ulcer: discovery of a new mechanism and development of angiogenic therapy which accelerates healing.
Ann Surg
 .
1991
;
214
:
414
–427.
2
Folkman
J
. Clinical applications of research on angiogenesis.
N Engl J Med
 .
1995
;
333
:
1757
–1763.
3
Folkman
J
. Angiogenesis in cancer, vascular, rheumatoid and other disease.
Nat Med
 .
1995
;
1
:
27
–31.
4
Adamis
AP
, Miller JW, Bernal MT et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy.
Am J Ophthalmol
 .
1994
;
118
:
445
–450.
5
Couffinhal
T
, Kearney M, Witzenbichler B et al. Vascular endothelial growth factor (VEGF/VPF) in normal and atherosclerotic human arteries.
Am J Pathol
 .
1997
;
150
:
1673
–1685.
6
Risau
W
, Flamme I. Vasculogenesis.
Annu Rev Cell Dev Biol
 .
1995
;
11
:
73
–91.
7
Buschmann
I
, Schaper W. Arteriogenesis versus angiogenesis: two mechanisms of vessel growth.
News Physiol Sci
 .
1999
;
14
:
121
–125.
8
Risau
W
. Mechanisms of angiogenesis.
Nature
 .
1997
;
386
:
671
–674.
9
Ferrara
N
, Alitalo K. Clinical applications of angiogenic growth factors and their inhibitors.
Nat Med
 .
1999
;
5
:
1359
–1364.
10
Timar
J
, Dome B, Fazekas K et al. Angiogenesis-dependent diseases and angiogenesis therapy.
Pathol Oncol Res
 .
2001
;
7
:
85
–94.
11
Peacock
DJ
, Banquerigo ML, Brahn E. A novel angiogenesis inhibitor suppresses rat adjuvant arthritis.
Cell Immunol
 .
1995
;
160
:
178
–184.
12
Marti
HH
, Risau W. Angiogenesis in ischemic disease.
Thromb Haemost
 .
1999
;
82
(Suppl 1):
44
–52.
13
Losordo
DW
, Vale PR, Isner JM. Gene therapy for myocardial angiogenesis.
Am Heart J
 .
1999
;
138
(2 Pt 2):
S132
–S141.
14
Tabibiazar
R
, Rockson SG. Angiogenesis and the ischaemic heart.
Eur Heart J
 .
2001
;
22
:
903
–918.
15
Belgore
F
, Blann AD, Li-Saw-Hee FL et al. Plasma levels of vascular endothelial growth factor and its soluble receptor (sFlt-1) in essential hypertension.
Am J Cardiol
 .
2001
;
87
:
805
–807.
16
Gonzalez Crussi
F
. Vasculogenesis in the chick embryo. An ultrastructural study.
Am J Anat
 .
1972
;
130
:
441
–460.
17
Kessel
J
, Fabian B. Graded morphogenetic patterns during the development of the extraembryonic blood system and coelom of chick blastoderm: a scanning electron microscope and light microscope study.
Am J Anat
 .
1985
;
173
:
99
–112.
18
Flamme
I
, Risau W. Induction of vasculogenesis and hematopoiesis in vitro.
Development
 .
1992
;
116
:
435
–439.
19
Shalably
F
, Rossant J, Yamaguchi TP et al. Failure of blood-island formation and vasculogenesis in FLK-1 deficient mice.
Nature
 .
1995
;
376
:
62
–66.
20
Hirschi
KK
, D'Amore PA. Pericytes in the microvasculature.
Cardiovasc Res
 .
1996
;
32
:
687
–698.
21
DeRuiter
MC
, Poelmann RE, VanMunsteren JC et al. Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro.
Circ Res
 .
1997
;
80
:
444
–451.
22
Campbell
GR
. Development of the vessel wall: an overview. Schwartz SM, Mecham RP. The vascular smooth muscle cell. San Diego: Academic Press; 1995. p. 1–17.
23
Rosenquist
TH
, Beall AC. Elastogenic cells in the developing cardiovascular system: smooth muscle, nonmuscle, andcardiac neural crest.
Ann NY Acad Sci
 .
1990
;
558
:
106
–109.
24
Gittenberger-de Groot
AC
, Slomp J, DeRuiter MC et al. Smooth muscle cell differentiation during early development and during intimal thickening formation in the ductus arteriosus. Schwartz SM, Mecham R. The vascular smooth muscle cell. San Diego: Academic Press; 1995. p. 17–36.
25
Risau
W
. Differentiation of endothelium.
FASEB J
 .
1995
;
9
:
926
–933.
26
Pepper
MS
. Manipulating angiogenesis. From basic science to bedside.
Arterioscler Thromb Vasc Biol
 .
1997
;
17
:
605
–619.
27
Patan
S
, Munn LL, Jain RK. Intussusceptive microvascular growth in a human colon adenocarcinoma xenograft: a novel mechanism of tumor angiogenesis.
Microvasc Res
 .
1996
;
51
:
260
–272.
28
Hudlicka
O
, Wright AJ, Ziada AM. Angiogenesis in the heart and skeletal muscle.
Can J Cardiol
 .
1986
;
2
:
120
–123.
29
Folkman
J
, D'Amore PA. Blood vessel formation: what is its molecular basis?
Cell
 .
1996
;
87
:
1153
–1155.
30
Hanahan
D
. Signalling vascular morphogenesis and maintenance.
Science
 .
1997
;:
48
–50.
31
Kovacs
EJ
, DiPietro LA. Fibrogenic cytokines and connective tissue production.
FASEB J
 .
1994
;
8
:
854
–861.
32
Krupinski
J
, Kaluza J, Kumar P et al. Role of angiogenesis in patients with cerebral ischaemic stroke.
Stroke
 .
1994
;
25
:
1794
–1798.
33
Sabri
MN
, DiSciascio G, Cowley MJ et al. Coronary recruitment: functional significance and relation to rate of vessel closure.
Am Heart J
 .
1991
;
121
(3Pt1):
876
–880.
34
Tomanek
RJ
, Doty MK, Sandra A. Early coronary angiogenesis in response to Thyroxine: growth characteristics and upregulation of basic fibroblastic growth factor.
Circ Res
 .
1998
;
82
:
587
–593.
35
Jackson
MR
, Carney EW, Lye SJ et al. Localisation of two angiogenic growth factors (PDECGF and VEGF) in human placenta throughout gestation.
Placenta
 .
1994
;
15
:
341
–353.
36
Koos
RD
, Oslon CE. Expression of basic fibroblast growth factor in the rat ovary: detection of mRNA using reverse transcription-polymerase chain reaction amplification.
Mol Endocrinol
 .
1989
;
3
:
2041
–2048.
37
Redmer
DA
, Reynolds LP.
Rev Reprod
 .
1996
;
1
:
182
–192.
38
Mattioli
M
, Barboni B, Turriani M et al. Follicle activation involves vascular endothelial growth factor productionand increased blood vessel extension.
Biol Reprod
 .
2001
;
65
:
1014
–1019.
39
Stouffer
RL
, Martinez-Chequer JC, Molskness TA et al. Regulation and action of angiogenic factors in the primate ovary.
Arch Med Res
 .
2001
;
32
:
567
–575.
40
Folkman
J
. Tumour angiogenesis: therapeutic implications.
N Engl J Med
 .
1971
;
285
:
1182
–1186.
41
Folkman
J
, Shing Y. Angiogenesis.
J Biol Chem
 .
1992
;
267
:
10931
–10934.
42
Fong
G
, Rossant J, Gartsenstein M et al. Role of the Flt-1 receptor tyrosine kinase in regulating the assembly ofvascular endothelium.
Nature
 .
1995
;
376
:
67
–70.
43
Folkman
J
. Tumour angiogenesis. Mendelsohn J, Howley P, Israel M, Liotta L. The molecular basis of cancer. Philadelphia: W. B. Saunders; 1995. p. 206–232.
44
Folkman
J
, Klagsbrun M. Angiogenic factors.
Science
 .
1987
;
235
:
442
–447.
45
Friesel
RE
, Maciag T. Molecular mechanism of angiogenesis: fibroblast growth factor signal transducing.
FASEB J
 .
1995
;
9
:
919
–925.
46
Besser
D
, Presta M, Nagamine Y. Elucidation of a signalling pathway induced by FGF-2 leading to uPA gene expression in NIH 3T3 fibroblasts.
Cell Growth Differ
 .
1995
;
6
:
1009
–1017.
47
Ichimura
T
, Finch PW, Zhang G et al. Induction of FF-7 after kidney damage—a possible paracrine mechanism for tubule repair.
Am J Physiol
 .
1996
;
271
:
F967
–F976.
48
Wadzinski
MG
, Folkman J, Sasse J et al. Heparin-binding angiogenesis factor: detection by immunological methods.
Clin Physiol Biochem
 .
1987
;
5
:
200
–209.
49
Xu
X
, Weinstein M, Li C et al. Fibroblast growth factor receptor (FGFRs) and their role in limb development.
Cell Tissue Res
 .
1999
;
296
:
33
–43.
50
Jaye
M
, Howk R, Burgess SJ et al. Human endothelial cell growth factor: cloning, nucleotide, sequence, and chromosome location.
Science
 .
1986
;
233
:
541
–545.
51
Imamura
T
, Engleka K, Zhan X et al. Recovery of mitogenic activity of a growth factor mutant with a nuclear translocation sequence.
Science
 .
1990
;
249
:
1567
–1570.
52
Wiedlocha
A
, Falnes PO, Madshus IH et al. Dual mode of signal transducing by externally added acidic fibroblast growth factor.
Cell
 .
1994
;
76
:
1039
–1051.
53
Linder
V
, Reidy MA, Fingerle J. Regrowth of arterialendothelium. Denudation with minimal trauma leads to complete cell regrowth.
Lab Invest
 .
1989
;
61
:
556
–563.
54
Engelman
GL
, Dionne CA, Jaye MC. Acidic fibroblast growth factor and heart development. Role in myocyte proliferation and capillary angiogenesis.
Circ Res
 .
1993
;
72
:
7
–19.
55
Kuwabara
K
, Ogawa S, Matsumoto M et al. Hypoxiamediated induction of acidic/basic fibroblast growth factor and platelet-derived growth factor in mononuclear phagocytes stimulates growth of hypoxic endothelial cells.
Proc Natl Acad Sci USA
 .
1995
;
92
:
4606
–4610.
56
Banai
S
, Jaklischt MT, Casscells W et al. Effects of acidic fibroblast growth factor on normal and ischaemic myocardium.
Circ Res
 .
1991
;
69
:
76
–85.
57
Bohlen
P
, Esch F, Baird A et al. Acidic fibroblast growth factor (FGF) from bovine brain: amino-terminal sequence and comparison with basic FGF.
EMBO J
 .
1985
;
4
:
1951
–1956.
58
Asahara
T
, Chen D, Takahashi T et al. Tie2 receptor ligands, angiopoietin-1 and angiopoietin-2, modulate VEGF-induced postnatal neovascularisation.
Circ Res
 .
1998
;
83
:
233
–240.
59
Senger
DR
, Galli SJ, Dvoral AM et al. Tumour cells secrete a vascular permeability factor that promotes accumulation of ascitic fluid.
Science
 .
1983
;
219
:
983
–985.
60
Connolly
DT
, Heuvelman DM, Nelson R et al. Tumor vascular permeability factor stimulates endothelial cell growth and angiogenesis.
J Clin Invest
 .
1989
;
84
:
1470
–1478.
61
Leung
DW
, Cachianes G, Kuang WJ et al. Vascular endothelial growth factor is a sectered angiogenic mitogen.
Science
 .
1989
;
246
:
1306
–1309.
62
Tischer
E
, Gospodarowicz D, Mitchell R et al. Vascular endothelial growth factor: a new member of the platelet derived growth factor gene family.
Biochem Biophys Res Commun
 .
1989
;
165
:
1198
–1206.
63
Ferrara
N
, Houck K, Jakeman L et al. Molecular and biological properties of vascular endothelial growth factor family of protein.
Endocr Rev
 .
1992
;
13
:
18
–32.
64
Carmeliet
P
, Collen D. Genetic analysis of blood vessel formation. Role of endothelial versus smooth muscle cells.
Trends Cardiovasc Med
 .
1997
;
7
:
271
–281.
65
Neufeld
G
, Cohen T, Gengrinovitch S et al. Vascular endothelial growth factor (VEGF) and its receptors.
FASEB J
 .
1999
;
13
:
9
–22.
66
Enholm
B
, Jussila L, Karkkainen M et al. Vascular endothelial growth factor-C: a growth factor for lymphatic and blood vascular endothelial cells.
Trends Cardiovasc Med
 .
1998
;
8
:
292
–297.
67
Levy
AP
, Levy NS, Goldberg MA. Identification of 5 hypoxia-inducible RNA-protein binding sites conserved in rat and human vascular endothelial growth factor mRNA.
JACC
 .
1997
;
90
:
1
–32.
68
Levy
NS
, Goldberg MA, Levy AP. Sequencing of the human vascular endothelial growth factor (VEGF) 3′ untranslated region (UTR): conservation of five hypoxia-inducible RNA-protein binding sites.
Biochim Biophys Acta
 .
1997
;
1352
:
167
–173.
69
Li
J
, Perrella MA, Tsai JC et al. Induction of vascular endothelial growth factor gene expression by interleukin-1 beta in rat aortic smooth muscle cells.
J Biol Chem
 .
1995
;
270
:
308
–312.
70
Vincenti
V
, Cassano C, Rocchi M et al. Assignment of the vascular endothelial growth factor gene to human chromosome 6p21.3.
Circulation
 .
1996
;
93
:
1493
–1495.
71
Volz
A
, Boyle JM, Cann HM et al. Report of the Second International Workshop on Human Chromosome 6.
Genomics
 .
1994
;
21
:
464
–472.
72
Houck
KA
, Ferrara N, Winer J et al. The vascular endothelial growth factor family: identification of a fourth molecular species and characterisation of alternative splicing of RNA.
Mol Endocrinol
 .
1991
;
5
:
1806
–1814.
73
Tischer
E
, Mitchell R, Hartmann T et al. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing.
J Biol Chem
 .
1991
;
266
:
11947
–11954.
74
Park
JE
, Keller GA, Ferrara N. The vascular endothelial growth factor (VEGF) isoforms: differential deposition into the sub-epithelial extracellular matrix and bioactivity of extracellular matrix-bound VEGF.
Mol Biol Cell
 .
1993
;
4
:
1317
–1326.
75
Houck
KA
, Leung DW, Rowland AM et al. Dual regulationof vascular endothelial growth factor bioavailability bygenetic and proteolytic mechanisms.
J Biol Chem
 .
1992
;
267
:
26031
–26037.
76
Keyt
BA
, Berleau LT, Nguyen HV et al. The carboxyl-terminal domain (111–165) of vascular endothelial growth factoris critical for its mitogenic potency.
J Biol Chem
 .
1996
;
271
:
7788
–7795.
77
Blann
AD
, Belgore FM, McCollum CN et al. Vascular endothelial growth factor and its receptor Flt-1 in the plasma of patients with coronary and peripheral atherosclerosis and type II diabetes.
Clin Sci
 .
2002
;
102
:
187
–194.
78
Seko
Y
, Imai Y, Suzuki S et al. Serum levels of vascular endothelial growth factor in patients with acute myocardial infarction undergoing reperfusion therapy.
Clin Sci
 .
1997
;
92
:
453
–454.
79
Lip
PL
, Belgore FM, Blann AD. Plasma vascular endothelial growth factor and soluble VEGF receptor Flt-1 in prolifer-ative retinopathy: a pilot study of the relationship toendothelial dysfunction and laser treatment.
Invest Ophthalmol Vis Sci
 .
2000
;
41
:
2115
–2119.
80
Webb
NJ
, Bottomley MJ, Watson CJ et al. Vascular endothelial growth factor (VEGF) is released from plateletsduring blood clotting: implications for the measurement of circulating VEGF levels in clinical disease.
Clin Sci
 .
1998
;
94
:
395
–404.
81
Olofsson
B
, Pajusola K, Kaipainen A et al. Vascular endothelial growth factor B, a novel growth factor for endothelial cells.
Proc Natl Acad Sci USA
 .
1996
;
93
:
2576
–2581.
82
Olofsson
B
, Pajusola K, von Euler G et al. Genomic organization of the mouse and human genes for vascular endothelial growth factor B (VEGF-B) and characterization of a second splice isoform.
J Biol Chem
 .
1996
;
271
:
19310
–19317.
83
Joukov
V
, Kaipainen A, Jeltsch M et al. Vascular endothelial growth factors VEGF-B and VEGF-C.
J Cell Physiol
 .
1997
;
173
:
211
–215.
84
Chilov
D
, Kukk E, Taira S et al. Genomic organisation of human and mouse genes for vascular endothelial growth factor C.
J Biol Chem
 .
1997
;
272
:
25176
–25183.
85
Kukk
E
, Lymboussaki ST, Kaipaninen A et al. VEGF-C receptor binding pattern of expression with VEGFR-3 suggestsa role in lymphatic development.
Development
 .
1996
;
122
:
3837
–3839.
86
Wartiovaara
U
, Salven P, Mikkola H et al. Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.
Thromb Haemost
 .
1998
;
80
:
171
–175.
87
Fitz
LJ
, Morris JC, Towler P et al. Characterization of murine Flt4 ligand/VEGF-C.
Oncogene
 .
1997
;
15
:
613
–618.
88
Kaipainen
A
, Korhonen J, Mustonen T et al. Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development.
Proc Natl Acad Sci USA
 .
1995
;
92
:
3566
–3570.
89
Kaipainen
A
, Korhonen J, Pajusola K et al. The related Flt-4, Flt-1, and KDR receptor tyrosine kinases show distinct expression patterns in human foetal endothelial cells.
J Exp Med
 .
1993
;
178
:
2077
–2088.
90
Witzenbichler
B
, Asahara T, Murohara T et al. Vascular endothelial growth factor-C (VEGF-C/VEGF-2) promotesangiogenesis in the setting of tissue ischemia.
Am J Pathol
 .
1998
;
153
:
381
–394.
91
Aase
K
, Lymboussaki A, Kaipainen A et al. Localisation of VEGF-B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature.
Dev Dyn
 .
1999
;
215
:
12
–25.
92
Yamada
Y
, Nezu J, Shimane M et al. Molecular cloning of a novel vascular endothelial growth factor, VEGF-D.
Genomics
 .
1997
;
42
:
483
–488.
93
Achen
MG
, Jeltsch M, Kukk E et al. Vascular endothelial growth factor D (VEGF-D) is a ligand for tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt-4).
Proc Natl Acad Sci USA
 .
1998
;
95
:
548
–553.
94
Orlandini
M
, Marconcini L, Ferruzzi R et al. Identification of a c-fos-induced gene that related to the platelet derived growth factor/vascular endothelial growth factor family.
Proc Natl Acad Sci USA
 .
1996
;
93
:
11675
–11680.
95
Karkkainen
MJ
, Petrova TV. Vascular endothelial growth factor receptors in the regulation of angiogenesis and lymphangiogenesis.
Oncogene
 .
2000
;
19
:
5598
–5605.
96
Staker
SA
, Caesar C, Baldwin ME et al. VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.
Nat Med
 .
2001
;
7
:
186
–191.
97
Meyer
M
, Clauss M, Lepple-Wienhues A et al. A novel vascular endothelial growth factor encoded by Orf virus, VEGF-E mediates angiogenesis via signalling through VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) receptor kinases.
EMBO J
 .
1999
;
18
:
363
–374.
98
Lyttle
DJ
, Fraser KM, Flemings SB et al. Homologs of vascular endothelial growth factor are encoded by the poxvirus Orf virus.
J Virol
 .
1994
;
68
:
84
–92.
99
Savory
LJ
, Stacker SA, Flemming SB et al. Viral vascular endothelial growth factor plays a critical role in Orf virus infection.
J Virol
 .
2000
;
74
:
10699
–10706.
100
Maglione
D
, Guerriero V, Viglietto G et al. Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor.
Proc Natl Acad Sci USA
 .
1991
;
88
:
9271
–9276.
101
Cao
Y
, Ji WR, Qi P et al. Placenta growth factor: identification and characterization of a novel isoform generated by RNA alternative splicing.
Biochem Biophys Res Commun
 .
1997
;
235
:
493
–498.
102
Weindel
K
, Moringlane JR, Marme D et al. Detection and quantification of vascular endothelial growth factor/vascular permeability factor in brain tumour tissue and cyst fluid: the key to angiogenesis.
Neurosurgery
 .
1994
;
35
:
439
–448.
103
Takahashi
A
, Sasaki H, Kim SJ et al. Marked increased amounts of messenger RNAs for vascular endothelial growth factor and placenta growth factor in renal cell carcinoma associated angiogenesis.
Cancer Res
 .
1994
;
54
:
4233
–4237.
104
Cao
Y
, Linden P, Shima D et al. In vivo angiogenesis and hypoxia induction of heterodimer of placenta growth factor/vascular endothelial growth factor.
J Clin Invest
 .
1996
;
98
:
2507
–2511.
105
Davis
S
, Aldrich T, Jones PF et al. Isolation of angiopoietin-1, a ligand for the angiogenic TIE-2 receptor, by secretion-trap expression cloning.
Cell
 .
1996
;
87
:
1161
–1169.
106
Maisonpierre
PC
, Suri C, Jones PF et al. Angiopoietin-2, a natural antagonist for TIE2 that disrupts in vivo angiogenesis.
Science
 .
1997
;
277
:
55
–60.
107
Valenzuela
DM
, Griffith JA, Rojass J et al. Angiopoietin 3 and 4: diverging gene counterparts in mice and humans.
Proc Natl Acad Sci USA
 .
1999
;
96
:
1904
–1909.
108
Koblizek
TI
, Weiss C, Yancopoulos GD et al. Angiopoietin-1 induces sprouting angiogenesis in vitro.
Curr Biol
 .
1998
;
8
:
529
–532.
109
Sato
TN
, Tozawa Y, Deutsch U et al. Distinct roles of the receptor tyrosine kinases Tie-1 and Tie-2 in blood vessel formation.
Nature
 .
1995
;
376
:
70
–74.
110
Suri
C
, Jones PF, Patan S et al. Requisite role of angiopoietin-1, a ligand for the TIE-2 receptor, during embryonic angiogenesis.
Cell
 .
1996
;
87
:
1171
–1180.
111
Vikkula
M
, Boon LM, Carraway KL et al. Vascular dysmorphogenesis caused by an activating mutation in the receptor tyrosine kinase TIE2.
Cell
 .
1996
;
87
:
1181
–1190.
112
Matthews
W
, Jordan CT, Gavin M et al. A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close geneticlinkage to c-kit.
Proc Natl Acad Sci
 .
1991
;
88
:
9026
–9030.
113
Park
M
, Lee ST. The fourth immunoglobulin-like loop in the extracellular domain of FLT-1, a VEGF receptor, includes a major heparin-binding site.
Biochem Biophys Res Commun
 .
1999
;
264
:
730
–734.
114
De-Vries
C
, Escobedo JA, Ueno H et al. The fms-like tyrosine kinase, a receptor for vascular endothelial growth factor.
Science
 .
1992
;
255
:
989
–991.
115
Shibuya
M
, Yamaguchi S, Yamame A et al. Nucleotide sequence and expression of novel human receptors-type tyrosine kinase gene (flt-1) closely related to fms family.
Oncogene
 .
1990
;
5
:
519
–524.
116
Charnock-Jones
D
, Sharkey A, Boocock C et al. Vascular endothelial growth factor receptor localization and activation in human trophoblast and choriocarcinoma cells.
Biol Reprod
 .
1994
;
51
:
524
–530.
117
Simon
M
, Grone HJ, Johren O et al. Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and adult kidney.
Am J Physiol
 .
1995
;
268
:
F240
–F250.
118
Pajusola
K
, Aprelikova O, Korhonen J et al. Flt-4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines.
Cancer Res
 .
1992
;
52
:
5738
–5743.
119
Rosnet
O
, Mattei MG, Marchetto S et al. Isolation and chromosomal localisation of a novel FMS-like tyrosinekinase gene.
Genomics
 .
1991
;
9
:
380
–385.
120
Barleon
B
, Sozzani S, Zhou D et al. Migration of human monocytes in response to vascular endothelial growthfactor (VEGF) is mediated via the VEGF receptor flt-1.
Blood
 .
1996
;
87
:
3336
–3343.
121
Takahashi
T
, Shiraswa T, Miyake K et al. Protein tyrosine kinase expressed in glomeruli and cultured glomerular cells: FLT-1 and VEGF expression in renal mesangial cells.
Biochem Biophys Res Commun
 .
1995
;
209
:
218
–226.
122
Ahmed
A
, Li XF, Dunk C et al. Co-localisation of vascular endothelial growth factor and its receptor in human placenta.
Growth Factors
 .
1995
;
12
:
235
–243.
123
Keyt
BA
, Nguyen HV, Berleau LT et al. Identification of vascular endothelial growth factor determinants for binding KDR and FLT-1 receptors. Generation of receptor-selective VEGF variants by site-directed mutagenesis.
J Biol Chem
 .
1996b
;
271
:
5638
–5643.
124
Sawano
A
, Takahashi T, Yamaguchi S et al. Flt-1 but not KDR/Flk-1 tyrosine kinase is a receptor for placenta growth factors, which is related to vascular endothelial growth factor.
Cell Growth Differ
 .
1996
;
7
:
213
–221.
125
Gitay-Goren
H
, Cohen T, Tessler S et al. Selective binding of VEGF 121 to one of the three vascular endothelial growth factor receptors of vascular endothelial cells.
J Biol Chem
 .
1996
;
271
:
5519
–5523.
126
Wilting
J
, Birkenhager R, Eichmann A et al. VEGF 121 induces proliferation of vascular endothelial cells and expression of flk-1 without affecting lymphatic vessels of chorioallantoic membrane.
Dev Biol
 .
1996
;
176
:
76
–85.
127
Davis-Smyth
TH
, Park J, Presta LG et al. The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.
EMBO J
 .
1996
;
15
:
4919
–4927.
128
Kendall
RL
, Thomas KA. Inhibition of vascular endothelial growth factor activity by an endogenously encoded soluble receptor.
Proc Natl Acad Sci USA
 .
1993
;
90
:
10705
–10709.
129
Kendall
RL
, Wang G, Thomas KA. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR.
Biochem Biophys Res Commun
 .
1996
;
226
:
324
–328.
130
Hornig
C
, Barleon B, Ahmad S et al. Release and complex formation of soluble VEGFR-1 from endothelial cells and biological fluids.
Lab Invest
 .
2000
;
80
:
443
–454.
131
Vuorela
P
, Helske S, Hornig C et al. Amniotic fluid-soluble vascular endothelial growth factor receptor-1 in preeclampsia.
Obstet Gynecol
 .
2000
;
95
:
353
–357.
132
Terman
BI
, Jani-Sait S, Carrion ME et al. The KDR gene maps to human chromosome 4q31.2Q32, a locus which is distinct from locations for the other type III growth factor receptor tyrosine kinases.
Cytogenet Cell Genet
 .
1992
;
60
:
214
–215.
133
Terman
BI
, Carrion ME, Rasmussen BA et al. Identification of a new endothelial growth factor receptor tyrosine kinase.
Oncogene
 .
1991
;
6
:
1677
–1683.
134
Millauer
B
, Wizigmann-Voos S, Schnurch H et al. High affinity VEGF binding and developmental expression flk-1 as a major regulator of vasculogenesis and angiogenesis.
Cell
 .
1993
;
72
:
835
–846.
135
Terman
BI
, Dougher-Vermazen M, Carrion ME et al. Identification of the KDR tyrosine kinase as a receptor for vascular endothelial cell growth factor.
Biochem Biophys Res Commun
 .
1992
;
187
:
1579
–1586.
136
Khaliq
A
, Li XF, Shams M et al. Localisation of placenta growth factor (PIGF) in human term placenta.
Growth Factors
 .
1996
;
13
:
243
–250.
137
Thieme
H
, Aiello LP, Takagi H et al. Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells.
Diabetes
 .
1995
;
4
:
98
–103.
138
Vuckovic
M
, Ponting J, Terman BI et al. Expression ofthe vascular endothelial growth factor receptor, KDR, in human placenta.
J Anatomy
 .
1996
;
188
:
361
–366.
139
Katoh
O
, Tauchi H, Kawaishi K et al. Expression of the vascular endothelial growth factor (VEGF) receptor gene, KDR, in hematopoietic cells and inhibitory effect ofVEGF on apoptotic cell death caused by ionizing radiation.
Cancer Res
 .
1995
;
55
:
5687
–5692.
140
Yang
XJ
, Cepko CL. Flk-1, a receptor for vascular endothelial growth factor (VEGF), is expressed by retinalprogenitor cells.
J Neurosci
 .
1996
;
16
:
6089
–6099.
141
Waltenberger
J
, Claesson-Welsh L et al. Different signal transduction properties of KDR and Flt1, two receptors for vascular endothelial growth factor.
J Biol Chem
 .
1994
;
269
:
26988
–26995.
142
Millauer
B
, Longhi MP, Plate KH et al. Dominant-negative inhibition of Flk-1 suppresses the growth of many tumour types in vivo.
Cancer Res
 .
1996
;
56
:
1615
–1620.
143
Galland
F
, Karamysheva A, Mattei MG et al. Chromosomal localization of Flt-4, a novel receptor-type tyrosine gene.
Genomics
 .
1992
;
13
:
475
–478.
144
Hewett
PW
, Murray JC. Coexpression-1, flt-4 and KDRin freshly isolated and cultured human endothelial cells.
Biochem Biophys Res Commun
 .
1996
;
221
:
697
–702.
145
Soker
S
, Takashima S, Miao HQ et al. Neuropilin-1 is expressed by endothelial and tumour cells as an isoform-specific receptor for vascular endothelial growth factor.
Cell
 .
1998
;
92
:
735
–745.
146
Kitsukawa
T
, Shimizu M, Sanbo M et al. Neuropilin-semaphorin III/D-mediated chemorepulsive signals play a crucial role in peripheral nerve projection in mice.
Neuron
 .
1997
;
19
:
995
–1105.
147
Adamis
AP
, Shima DT, Tolentino MJ et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularisation in non-human primate.
Arch Ophthalmol
 .
1996
;
144
:
66
–71.
148
Aiello
LP
, Pierce EA, Foley ED et al. Suppression of retinal neovascularisation in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins.
Proc Natl Sci USA
 .
1995
;
92
:
10457
–10461.
149
Borgi
E
, Schatteman G, Wu T et al. Hypoxia-inducedparacrine regulation of vascular endothelial growth factor receptor expression.
J Clin Invest
 .
1996
;
97
:
469
–476.
150
Williams
B
. Factors regulating the expression of vascular permeability/vascular endothelial growth factor by human vascular tissues.
Diabetologia
 .
1997
;
40
:
S118
–S120.
151
Deroanne
CF
, Hajitou A, Calberg-Bacq CM et al. Angiogenesis by fibroblast growth factor 4 is mediated through an autocrine up-regulation of vascular endothelial growth factor expression.
Cancer Res
 .
1997
;
57
:
5590
–5597.
152
Finkenzeller
G
, Sparacio A, Technau A et al. Sp1 recognition sites in the proximal promoter of the human vascular endothelial growth factor gene are essential for platelet-derived growth factor-induced gene expression.
Oncogene
 .
1997
;
15
:
669
–676.
153
Ryuto
M
, Ono M, Izumi H et al. Induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells.
J Biol Chem
 .
1996
;
271
:
28220
–28228.
154
Stavri
GT
, Zachary IC, Baskerville PA et al. Basic fibroblast growth factor upregulates the expression of vascularendothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia.
Circulation
 .
1995
;
92
:
11
–14.
155
Williams
B
, Baker AQ, Gallacher B et al. Angio-tensin II increases vascular permeability factor gene expression by human vascular smooth muscle cells.
Hypertension
 .
1995
;
25
:
913
–917.
156
Frank
S
, Hubner G, Breier G et al. Regulation of vascular endothelial growth factor expression in cultured keratinocytes. Implications for normal and impaired wound healing.
J Biol Chem
 .
1995
;
270
:
12607
–12613.
157
Goad
DL
, Rubin J, Wang H et al. Enhanced expression of vascular endothelial growth factor in human SaOS-2 osteoblast-like cells and murine osteoblasts induced by insulin-like growth factor I.
Endocrinology
 .
1996
;
137
:
2262
–2268.
158
Cohen
T
, Nahari D, Cerem LW et al. Interleukin 6 induces the expression of vascular endothelial growth factor.
J Biol Chem
 .
1996
;
271
:
736
–741.
159
Keck
PJ
, Hauser SD, Krivi G et al. Vascular permeability factor, an endothelial cell mitogen related to PDGF.
Science
 .
1989
;
246
:
1309
–1312.
160
Matsumoto
K
. Interleukin 10 inhibits vascular permeability factor release by peripheral blood mononuclear cells in patients with lipoid nephrosis.
Nephron
 .
1997
;
75
:
154
–159.
161
Matsumoto
K
, Ohi H, Kanmatsuse K. Interleukin 10 and interleukin 13 synergize to inhibit vascular permeability factor release by peripheral blood mononuclear cells from patients with lipoid nephrosis.
Nephron
 .
1997
;
77
:
212
–218.
162
Dumont
DJ
, Gradwohl G, Fong GH et al. Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.
Genes Dev
 .
1994
;
8
:
1897
–1909.
163
Shweiki
D
, Neeman M, Itin A et al. Induction of vascular endothelial growth factor expression by hypoxia and by glucose deficiency in multicell spheroids: implicationsfor tumor angiogenesis.
Proc Natl Acad Sci USA
 .
1995
;
92
:
768
–772.
164
Forsythe
JA
, Jiang BH, Iyer NV et al. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1.
Mol Cell Biol
 .
1996
;
16
:
4604
–4613.
165
Jiang
BH
, Semenza GL, Bauer C et al. Hypoxia-inducible factor 1 (HIF-1) levels vary exponentially over a physiologically relevant range of O2tension.
Am J Physiol
 .
1996
;
271
:
C1172
–C1180.
166
Gerber
HP
, Condorelli F, Park J et al. Differential transcriptional regulation of the two vascular endothelial growth factor receptor genes. Flt-1, but not Flk-1/KDR,is up-regulated by hypoxia.
J Biol Chem
 .
1997
;
272
:
23659
–23667.
167
Sandner
P
, Wolf K, Bergmaier U et al. Induction of VEGF and VEGF receptor gene expression by hypoxia: divergent regulation in vivo and in vitro.
Kidney Int
 .
1997
;
51
:
448
–453.
168
Sandner
P
, Wolf K, Bergmaier U et al. Hypoxia and cobalt stimulate vascular endothelial growth factor receptor gene expression in rats.
Pflugers Arch
 .
1997
;
433
:
803
–808.
169
Shweiki
D
, Itin A, Neufeld G et al. Patterns of expressionof vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis.
J Clin Invest
 .
1993
;
91
:
2235
–2243.
170
Battegay
EJ
. Angiogenesis: mechanistic insight, neovascular diseases, and therapeutic prospects.
J Mol Med
 .
1995
;
73
:
333
–346.
171
Gu
JW
, Adair TH. Hypoxia-induced expression of VEGF is reversible in myocardial vascular smooth muscle cells.
Am J Physiol
 .
1997
;
273
(2 Pt 2):
H628
–H633.
172
Klekamp
JG
, Jarzecka K, Perkett EA. Exposure to hyperoxia decreases the expression of vascular endothelial growth factor and its receptors in adults rat lungs.
Am J Pathol
 .
1999
;
154
:
823
–831.
173
Yue
X
, Tomanek RJ. Stimulation of coronary vasculogenesis/angiogenesis by hypoxia in cultured embryonic hearts.
Dev Dyn
 .
1999
;
216
:
28
–36.
174
Alon
T
, Hemo I, Itin A et al. Vascular growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity.
Nat Med
 .
1995
;
1
:
1024
–1028.
175
Murohara
T
, Horowitz JR, Silver M et al. Vascular endothelial growth factor/vascular permeability factor enhances vascular permeability via nitric oxide and prostacyclin.
Circulation
 .
1998
;
97
:
99
–107.
176
Ziche
M
, Morbidelli L, Choudhuri R et al. Nitric oxide synthase lies downstream from vascular endothelial growth factor-induced but not basic fibroblast growth factor-induced angiogenesis.
J Clin Invest
 .
1997
;
99
:
2625
–2634.
177
Miyazaki
H
, Matsuoka H, Cooke JP et al. Endogenous nitric oxide synthase inhibitor: a novel marker of angiogenesis.
Circulation
 .
1999
;
99
:
1141
–1146.
178
Tsurumi
Y
, Murohara T, Krasinski K et al. Reciprocal relation between VEGF and NO in the regulation of endothelial integrity.
Nat Med
 .
1997
;
3
:
879
–886.
179
Moncada
S
, Palmer RM, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology.
Pharmacol Rev
 .
1991
;
2
:
109
–142.
180
Konda
S
, Asano M, Matsuo K et al. Vascular, endothelial growth factor/vascular permeability factor is detectablein the sera of tumor-bearing mice and cancer patients.
Biochim Biophys Acta
 .
1994
;
1221
:
211
–214.
181
Belgore
FM
, Lip GYH, Wadley M et al. Plasma levelsof vascular endothelial cell growth factor (VEGF) andits receptor (sFlt-1) in haematological cancers: a comparison with breast cancer.
Am J Haematol
 .
2001
;
66
:
59
–61.
182
Kotch
LE
, Iyer NV, Laughner E et al. Defective vascularization of HIF-lalpha-null embryos is not associated with VEGF deficiency but with mesenchymal cell death.
Dev Biol
 .
1999
;
209
:
254
–267.
183
Sone
H
, Kawakami Y, Okuda Y et al. Vascular endothelial growth factor is induced by long-term high glucose concentration and up-regulation by acute glucose deprivationin cultured bovine retinal pigmented epithelial cells.
Biochem Biophys Res Commun
 .
1996
;
221
:
193
–198.
184
Satake
S
, Kuzuya M, Miura H et al. Up-regulation of vascular endothelial growth factor in response to glucose deprivation.
Biol Chem
 .
1998
;
90
:
161
–168.
185
Park
SH
, Kim KW, Lee YS et al. Hypoglycemia-induced VEGF expression is mediated by intracellular Ca2+ and protein kinase C signalling pathway in HepG2 human hapatoblastoma cells.
Int J Mol Med
 .
2001
;
7
:
91
–96.
186
Cha
DR
, Kim NH, Yoon JW et al. Role of vascular endothelial growth factor in diabetic nephropathy.
Kidney Int
 .
2000
;(Suppl 77):
S104
–S112.
187
Kim
NH
, Jung HH, Cha DR et al. Expression of vascular endothelial growth factor in response to high glucose in rat mesangial cells.
J Endocrinol
 .
2000
;
165
:
617
–624.
188
Hoshi
S
, Nomoto Ki K, Kuromitsu J. High glucose induced VEGF expression via PKC and ERK in glomerular podocytes.
Biochem Biophys Res Commun
 .
2002
;
290
:
177
–184.
189
Aiello
LP
, Avery RL, Arrigg PG et al. Vascular endothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders.
N Engl J Med
 .
1994
;
332
:
1480
–1487.
190
Breier
G
, Damert A, Plate KH et al. Angiogenesis in embryos and ischemic disease.
Thromb Haemost
 .
1997
;
78
:
678
–683.
191
Gale
NW
, Yancopoulos GD. Growth factors acting viaendothelial cell-specific receptor tyrosine kinases: VEGFs, angiopoietins, and ephrins in vascular development.
Gene Dev
 .
1999
;
13
:
1055
–1066.
192
Carmeliet
P
, Ferrera V, Breier G et al. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele.
Nature
 .
1996
;
380
:
435
–439.
193
Ferrara
N
, Carver-Moore K, Chen H et al. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene.
Nature
 .
1996
;
380
:
439
–442.
194
Molica
S
, Vitelli G, Levato D et al. Increased serum levels of vascular endothelial growth factor predict risk of progression in early B-cell chronic lymphocytic leukaemia.
Br J Haematol
 .
1999
;
107
:
606
–610.
195
Zhang
H-T
, Craft P, Scott PAE et al. Enhancement of tumour growth and vascular dentistry by transfection of vascular endothelial cell growth factor into MCF-7 human breast carcinoma cells.
J Natl Cancer Inst
 .
1995
;
87
:
213
–219.
196
Relf
M
, LeJeune S, Scott PA et al. Expression of the angiogenic factors VEGF, acidic and basic FGF, tumour growth factor beta-1, PDGF, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis.
Cancer Res
 .
1997
;
57
:
963
–969.
197
Suzuki
K
, Hayashi N, Miyamoto Y et al. Expression of vascular permeability factor/vascular endothelial growth factor in human hepatocellular carcinoma.
Cancer Res
 .
1996
;
56
:
3004
–3009.
198
Viglietto
G
, Romano A, Maglione D et al. Neovascularisation in human germ cell tumors correlates with a marked increase in the expression of the vascular endothelial growth factor but not placenta-derived growth factor.
Oncogene
 .
1996
;
13
:
577
–587.
199
Viglietto
G
, Maglione D, Rambaldi M et al. Upregulation of vascular endothelial growth factor (VEGF) and down regulation of placenta growth factor (PIGF) associated with malignancy in human thyroid tumors and cell lines.
Oncogene
 .
1995
;
11
:
1569
–1579.
200
Tsurusaki
T
, Kanda S, Sakai H et al. Vascular endothelial growth factor-C expression in human prostatic carcinoma and its relationship to lymph node metastasis.
Br J Cancer
 .
1999
;
80
:
309
–313.
201
Salven
P
, Lymboussaki A, Heikkila P et al. Vascular endothelial growth factor VEGF-B and VEGF-C are expressed in human tumours.
Am J Pathol
 .
1998
;
153
:
103
–108.
202
Soker
S
, Fidder H, Neufeld G et al. Characterization of novel endothelial growth factor (VEGF) receptors ontumour cells that bind VEGF165 via exon 7-encoded domain.
J Biol Chem
 .
1996
;
271
:
5761
–5767.
203
Faller
DV
. Endothelial cell responses to hypoxic stress.
Clin Exp Pharmacol Physiol
 .
1999
;
26
:
74
–84.
204
Blann
AD
, Li J-L, Kumar S. Increased VEGF in 13 children with Wilms' tumour falls after surgery but rising levels predict mortality.
Cancer Lett
 .
2001
;
173
:
183
–186.
205
Ausprunk
DH
, Folkman J. Migration and proliferation of endothelial cells in preformed and newly formed blood vessels during angiogenesis.
Microvasc Res
 .
1997
;
14
:
53
–65.
206
Lauer
G
, Sollberg S, Cole M et al. Expression and proteolysis of vascular endothelial growth factor increased in chronic wounds.
J Invest Dermatol
 .
2000
;
1115
:
12
–18.
207
Mochida
S
, Ishikawa K, Inao M et al. Increased expression of vascular endothelial growth factor and its receptors, flt-1, and KDR/flk-1, in regenerating liver.
Biochem Biophys Res Commun
 .
1996
;
266
:
176
–179.
208
Detmar
M
, Brown LF, Berse B et al. Hypoxia regulates the expression of vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) and its receptors inhuman skin.
J Invest Derm
 .
1997
;
108
:
263
–268.
209
Lee
SS
, Joo YS, Kim WU et al. Vascular endothelial growth factor levels in the serum and synovial fluid of patients with rheumatoid arthritis.
Clin Exp Rheumatol
 .
2001
;
19
:
321
–324.
210
Robak
E
, Wozniacka A, Sysa-Jedrzejowska A et al. Serum levels of angiogenic cytokines in systemic lupus erythematosus and their correlation with disease activity.
EurCytokine Netw
 .
2001
;
12
:
445
–452.
211
Mohle
R
, Green D, Moore MAS et al. Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.
Proc Natl Acad Sci USA
 .
1997
;
94
:
663
–668.
212
Fava
RA
, Olsen NJ, Spencer-Green G et al. Vascular permeability factor/endothelial growth factor (VPF/VEGF): accumulation and expression in human synovial fluids and rheumatoid synovial tissue.
J Exp Med
 .
1994
;
180
:
341
–346.
213
Nagashima
M
, Yoshino S, Ishiwata T et al. Role of vascular endothelial growth factor in angiogenesis of rheumatoid arthritis.
J Rheumatol
 .
1995
;
22
:
1624
–1630.
214
Latour
F
, Zabraniecki L, Fromer C et al. Does vascular endothelial growth factor in the rheumatoid synovium predict joint destruction? A clinical, radiological and pathological study in 12 patients monitored for 10 years.
Joint Bone Spine
 .
2001
;
68
:
493
–498.
215
Paavonen
K
, Mandelin J, Partanen T et al. Vascular endothelial growth factors C and D and their VEGFR-2 and 3 receptors in blood and lymphatic vessels in healthy and arthritic synovium.
J Rheumatol
 .
2002
;
29
:
39
–45.
216
Li
J
, Brown LF, Hibberd MG et al. VEGF, flk-1, and flt-1 expression in a rat myocardial infarction model of angiogenesis.
Am J Physiol
 .
1996
;
39
:
H1803
–H1811.
217
Hausner
EA
, Orsini JA, Foster LL et al. Vascular endothelial growth factor in porcine coronary arteries followingballoon angioplasty.
J Invest Surg
 .
1999
;
12
:
15
–23.
218
Ruef
J
, Hu ZY, Yin LY et al. Induction of vascular endothelial growth factor in balloon-injured baboon arteries. A novel role for reactive oxygen species in atherosclerosis.
Circ Res
 .
1997
;
81
:
24
–33.
219
Harada
K
, Friedman M, Lopez JJ et al. Vascular endothelial growth factor administration in chronic myocardial ischaemia.
Am J Physiol
 .
1996
;
270
:
H1791
–H1802.
220
Lee
SH
, Wolf PL, Escudero et al. Early expression of angiogenesis factors in acute myocardial ischemia and infarction.
N Engl J Med
 .
2000
;
342
:
626
–633.
221
Inoue
M
, Itoh H, Ueda M et al. Vascular endothelial growth factor (VEGF) expression in human coronary atherosclerotic lesions. Possible pathophysiological significance of VEGF in the progression of atherosclerosis.
Circulation
 .
1998
;
98
:
2108
–2116.
222
Chen
YX
, Nakashima Y, Tenaka K et al. Immunohistochemical expression of vascular endothelial growth factor/vascular permeability factor in atherosclerosis of human coronary arteries.
Arterioscler Thromb Vasc Biol
 .
1999
;
19
:
131
–139.
223
Couffinhal
C
, Kearney M, Witzenbichler B et al. Vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in normal and atheroscleortic human arteries.
Am J Pathol
 .
1997
;
150
:
1673
–1685.
224
Bobryshev
YV
, Farnsworth AE, Lord RS. Expression of vascular endothelial growth factor in aortocoronary saphenous vein bypass grafts.
Cardiovasc Surg
 .
2001
;
9
:
492
–498.
225
Fleisch
M
, Billinger M, Eberli FR et al. Physiologically assessed coronary collateral flow and intra-coronary growth factor concentrations in patients with 1- to 3-vessel coronary artery disease.
Circulation
 .
1999
;
100
:
1945
–1950.
226
Burton
PBJ
, Owen VJ, Hafizi S et al. Vascular endothelial growth factor release following coronary artery bypass surgery: extracorporeal circulation versus ‘beating heart’ surgery.
Eur Heart J
 .
2000
;
21
:
1708
–1713.
227
Hojo
Y
, Ikeda U, Zhu Y et al. Expression of vascular endothelial growth factor in patients with acute myocardial infarction.
J Am Coll Cardiol
 .
2000
;
35
:
968
–973.
228
Roller
RE
, Renner W, Tischler R et al. Vascular endothelial growth factor in plasma of patients undergoing peripheral angioplasty.
Thromb Haemost
 .
2001
;
85
:
1119
–1120.
229
O'Brien
KD
, Chait A. The biology of the artery wall in atherogenesis.
Med Clin North Am
 .
1994
;
78
:
41
–67.
230
Barger
AC
, Beeuwkes R, Lainey LL et al. Hypothesis: vasa vasorum and noevascularisation of human coronaryarteries. A possible role in the Pathophysiology of atherosclerosis.
N Engl J Med
 .
1984
;
310
:
175
–177.
231
Reyer
JA
, Myers PC, Appleberg M. Carotid intraplaque hemorrhage: the significance of neovascularity.
J Vasc Surg
 .
1987
;
6
:
341
–349.
232
Henry
TD
. Therapeutic angiogenesis.
Br Med J
 .
1999
;
318
:
1536
–1539.
233
Folkman
J
. Angiogenic therapy of the heart.
Circulation
 .
1998
;
97
:
628
–629.
234
Folkman
J
. Angiogenic therapy of the ischaemic limb.
Circulation
 .
1998
;
97
:
1108
–1110.
235
Helisch
A
, Ware JA. Therapeutic angiogenesis in ischemic heart disease.
Thromb Haemost
 .
1999
;
82
:
772
–780.
236
Kornowski
R
, Fuchs S, Leon MB et al. Delivery strategies to achieve therapeutic myocardial angiogenesis.
Circulation
 .
2000
;
101
:
454
–458.
237
Henry
TD
, Rocha-Singh K, Isner JM et al. Intra-coronary administration of recombinant human vascular endothelial growth factor to patients with coronary artery disease.
Am Heart J
 .
2001
;
142
:
872
–880.
238
Rosengart
TK
, Lee LY, Patel SR et al. Phase 1 asessment of direct intramyocardial administration of an adenovirisexpressing VEGF 121 cDNA to individuals with clinically significant severe coronary artery disease.
Circulation
 .
1999
;
100
:
468
–474.
239
Isner
JM
, Pieczek A, Schainfeld R et al. Clinical evidence of angiogenesis after arterial gene transfer of phVEGF 165 in paitent with ischaemic limb.
Lancet
 .
1996
;
348
:
370
–374.
240
Celletti
FL
, Waugh JM, Amabile PG et al. Vascular endothelial growth factor enhances atherosclerotic plaque progression.
Nat Med
 .
2001
;
7
:
425
–429.
241
Brenchley
PEC
. Antagonising angiogenesis in rheumatoid arthritis.
Ann Rheum Dis
 .
2001
;
60
(Suppl 3):
iii71
–iii74.
242
Braybrooke
JP
, O'Byrne K, Propper DJ et al. A phase II study of Razoxane, an anti-angiogenic topoisomerase II inhibitor, in renal cell cancer with assessment of potential surrogate markers of angiogenesis.
Clin Cancer Res
 .
2000
;
6
:
4697
–4704.
243
Lederman
RJ
, Mendelsohn FO, Anderson RD et al. for the TRAFFIC Investigators. Therapeutic angiogenesis with recombinant fibroblastic growth factor-2 for intermittent claudication (the TRAFFIC study): a randomized trial.
Lancet
 .
2002
;
359
:
2053
–2058.

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