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The biology of vascular endothelial growth factors

Tuomas Tammela, Berndt Enholm, Kari Alitalo, Karri Paavonen
DOI: http://dx.doi.org/10.1016/j.cardiores.2004.12.002 550-563 First published online: 15 February 2005

Abstract

The discovery of the vascular endothelial growth factor (VEGF) family members VEGF, VEGF-B, placental growth factor (PlGF), VEGF-C and VEGF-D and their receptors VEGFR-1, -2 and -3 has provided tools for studying the vascular system in development as well as in diseases ranging from ischemic heart disease to cancer. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. PlGF may primarily mediate arteriogenesis, the formation of collateral arteries from preexisting arterioles, with potential future therapeutic use in for example occlusive atherosclerotic disease. VEGF-C and VEGF-D are primarily lymphangiogenic factors, but they can also induce angiogenesis in some conditions. While many studies have addressed the role of angiogenesis and the blood vasculature in human physiology, the lymphatic vascular system has until recently attracted very little attention. In this review, we will discuss recent advances in angiogenesis research and provide an overview of the molecular players involved in lymphangiogenesis.

Keywords
  • Angiogenesis
  • Lymphangiogenesis
  • Growth factors
  • Endothelial receptors
  • VEGF

1. Introduction

Many human diseases are characterized by disorders of the vasculature. In ischemic heart disease or peripheral artery disease, insufficient blood vasculature leads to tissue ischemia. In cancer, tumor angiogenesis contributes to tumor growth and metastasis. Out of the many players in the angiogenesis field, the vascular endothelial growth factors are by far the best characterized and several VEGFs have already entered clinical use in a variety of human conditions. While many studies have addressed the role of angiogenesis and the blood vasculature in human physiology and pathology, the lymphatic vascular system has until recently attracted very little attention. The vascular endothelial growth factor (VEGF) family members VEGF, VEGF-B, placental growth factor (PlGF), VEGF-C and VEGF-D bind their cognate receptors VEGFR-1, VEGFR-2 and VEGFR-3 found on the vascular endothelium. VEGF is the prime hypoxia inducible angiogenic factor. VEGF inhibition has been validated as clinical cancer therapy. Furthermore, several studies have reported beneficial effects of VEGF when used as a proangiogenic therapy in the setting of tissue ischemia. PlGF may primarily mediate arteriogenesis, the formation of collateral arteries from preexisting arterioles, and thus it has potential as a therapeutic in for example occlusive atherosclerotic disease. VEGF-C and VEGF-D are primarily lymphangiogenic factors, inducing the growth of lymphatic vessels during development as well as in the adult. Inhibition of VEGF-C and VEGF-D signal transduction may prove crucial in the inhibition of lymphatic metastasis in cancer and stimulation of this pathway may be effective in the treatment of lymphedema.

2. VEGF

VEGF binds VEGFR-1 and VEGFR-2 as well as neuropilin-1 (Nrp-1) and Nrp-2; the latter are receptors for semaphorins, molecules involved in axonal guidance during neuronal development [1,2]. VEGF induces proliferation, sprouting, migration and tube formation of endothelial cells (ECs) [3]. VEGF is also a potent survival factor for ECs during physiological and tumor angiogenesis and it has been shown to induce the expression of antiapoptotic proteins in the ECs [4,5]. VEGF was originally described as a permeability factor, as it increases permeability of the endothelium through the formation of intercellular gaps, vesico-vascular organelles, vacuoles and fenestrations [6]. VEGF also causes vasodilatation through the induction of the endothelial nitric oxide synthase (eNOS) and the subsequent increase in nitric oxide production [7,8].

Although VEGF acts mostly on ECs, it has been shown to also bind VEGF receptors on hematopoietic stem cells (HSCs), monocytes, osteoblasts and neurons [3]. Besides angiogenesis, VEGF induces HSC mobilization from the bone marrow, monocyte chemoattraction, osteoblast-mediated bone formation and neuronal protection [3,9]. Furthermore, VEGF stimulates inflammatory cell recruitment and promotes the expression of proteases implicated in pericellular matrix degradation in angiogenesis [10–12]. Many cytokines including platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor and transforming growth factors induce VEGF expression in cells [13].

At least six VEGF isoforms of variable amino acid number are produced through alternative splicing: VEGF121, VEGF145, VEGF165, VEGF183, VEGF189 and VEGF206 (Table 1) [3]. VEGF121, VEGF165 and VEGF189 are the major forms secreted by most cell types [14]. After secretion, VEGF121 may diffuse relatively freely in tissues, while approximately half of the secreted VEGF165 binds to cell surface heparan sulfate proteoglycans (HSPGs). VEGF189 remains almost completely sequestered by HSPGs in the extracellular matrix making HSPGs a reservoir of VEGF that can be mobilized via proteolysis [3].

View this table:
Table 1

A table showing the chromosomal localization, major mRNA transcript and major protein sizes of the VEGF receptors

GeneSequence homologyChromosomal localizationSplice variantsMajor mRNA transcript size (kb)Major protein size (kDa)
VEGF6p23.1121,145, 165*, 183*, 189*, 206*3.7, 4.521
VEGF-B45% homology with VEGF-A11q13167*, 1861.421, 30
VEGF-C30% homology with VEGF-A1654q342.420–21
VEGF-D61% homology with VEGF-C; 31% with VEGF-A165Xp22.312.220–21
PlGF42% homology with VEGF-A14q24131, 152*, 2191.7, 1.238, 30
  • * Denotes splice variants that bind to heparan sulfate proteoglycans (HSPGs).

VEGF is first expressed mainly in the anterior portion of mouse embryos and it directs the migration of VEGFR-1 and VEGFR-2 positive cells in embryonic tissues [15]. In general, VEGF expression is stronger at sites of active vasculogenesis and angiogenesis in embryos [16]. Homozygous VEGF knockout mice die at E8-E9 from defects in blood island formation, EC development and vascular formation [13]. The levels of VEGF protein during development appear critical as mice lacking even a single VEGF allele die at E11-E12, displaying defects in early vascular development [13]. The different biological functions of VEGF isoforms were illustrated by studies on isoform-specific VEGF knockout mice. Mice expressing only VEGF120 (homologue of human VEGF121) die soon after birth and those that survive succumb to ischemic cardiomyopathy and multiorgan failure [17]. Mice expressing only VEGF188 (human VEGF189) display impaired arteriolar development and approximately half die at birth [18]. Mice expressing only VEGF164 (human VEGF165) are viable and healthy [18]. These studies underline the importance of VEGF165 as the principal effector of VEGF action, with intermediate diffusion and matrix-binding properties.

VEGF is strongly induced in hypoxic conditions via hypoxia inducible factor (HIF) regulated elements of the VEGF gene [19]. Constitutive degradation of hypoxia inducible factor (HIF)-1α is blocked in hypoxia because of the oxygen requirement of HIF prolyl hydroxylases, followed by stabilization of HIF-1α and its heterodimerization with HIF-1β, also called the aryl hydrocarbon nuclear translocator (ARNT). These complexes then bind hypoxia-responsive elements (HREs) in the promoters of hypoxia inducible genes and initiate transcription of a set of more than a hundred genes, including genes involved in glucose transport, glycolysis, and angiogenesis [19,20]. Interestingly, Bartonella henselae, the causative agent of cat-scratch fever, can induce hypoxia via an intracellular oxygen consumption mechanism, leading to VEGF induction and an angiomatous tumor [21]. Examples of other hypoxia-regulated genes include cyclooxygenase-2 (COX-2), MMP-2, VEGF and VEGFR-1 [19]. Deletion of a HRE from the mouse VEGF gene promoter results in progressive motoneuron degeneration, presumably due to insufficient vascular perfusion of nervous tissue and impaired motoneuron survival via loss of VEGF induction [22].

The skin has been widely used as a model for studying VEGF action in vivo; for example, transgenic mice overexpressing VEGF in the skin have abundant cutaneous angiogenesis and an inflammatory skin condition resembling psoriasis [23]. Overexpression of VEGF in mouse skin also accelerates experimental tumor growth [24]. In contrast, mice with a targeted deletion of VEGF in the epidermis exhibit delayed wound healing, while chemically induced skin papillomas developed less frequently in these animals [25]. VEGF blocking monoclonal antibodies or VEGF receptor inhibition reduce the growth of experimental tumors in mice and humans [13,26,27]. In humans, VEGF is expressed in practically all solid tumors studied as well as in some hematological malignancies [3]. In fact, correlations have been found between the level of VEGF expression, disease progression and survival [28].

The effects of VEGF on the lymphatic vasculature have also been recently studied. Adenoviral overexpression of the murine VEGF164 in the skin induced formation of giant lymphatic vessels [29], while another study employing the human VEGF165 isoform reported only dilatation of cutaneous lymphatics (Fig. 3A) [30]. However, VEGF did not induce lymphangiogenesis in a number of other tissue types [31–34]. The lymphangiogenic effects of VEGF may be linked to the recruitment of inflammatory cells, such as macrophages, which express VEGFR-1 and secrete lymphangiogenic factors [35–37]. At least in midgestation mouse embryos, VEGF-C but not VEGF had the capacity to induce migration of endothelial cells committed to the lymphatic endothelial lineage [38].

Fig. 3

Blood and lymphatic vascular effects of various adenovirally expressed human VEGFs in the mouse skin two weeks after treatment. (A) VEGF165 induces a prominent angiogenic response (blood vessels stained with antibodies for PECAM-1/CD31, green), accompanied by enlargement of lymphatic vessels (stained with antibodies for the lymphatic marker LYVE-1, red). (B) VEGF-B167 does not induce either angiogenesis or lymphangiogenesis. (C) VEGF-C is a powerful inducer of lymphangiogenesis, while its angiogenic activity is weak. (D) The AdLacZ control corresponds to nonstimulated, normal skin.

3. Placental growth factor

PlGF is predominantly expressed in the placenta, heart and lungs [39]. PlGF homodimers bind VEGFR-1 and Nrp-1, while PlGF heterodimerization with VEGF may also occur. Activation of VEGFR-1 by either PlGF or VEGF induced different gene expression profiles and phosphorylation of distinct tyrosine residues in the tyrosine kinase (TK) domain of VEGFR-1 [40], while combined administration of these factors enhanced VEGF driven angiogenesis [41]. This is probably due to the fact that PlGF/VEGF heterodimers bind VEGFR-2 and also VEGFR-1/VEGFR-2 heterodimers in vitro [42]. Of the three reported human isoforms of PlGF (PlGF-1, -2 and -3), only PlGF-2 binds HSPGs [42]. PlGF knockout mice do not have an apparent phenotype. However, these mice recover poorly from experimental myocardial infarction and exhibit impaired collateral formation in response to hind limb ischemia [43]. Overexpression of PlGF in the skin of transgenic mice results in a hypervascular phenotype with increased inflammatory and permeability responses [44,45], while local administration of PlGF with recombinant adenoviruses or as a recombinant protein induces the formation of mature, leakage-resistant vessels in a macrophage-dependent manner [46–48].

4. VEGF-B

VEGF-B (also called VEGF-related factor/VRF) is a ligand for VEGFR-1 and Nrp-1, and it can form heterodimers with VEGF (Fig. 1) [49]. In humans, VEGF-B is expressed as two different isoforms, VEGF-B167 and VEGF-B186 [50]. VEGF-B167 is nonglycosylated, binds HSPGs and is mostly sequestered in the extracellular matrix while VEGF-B186 is O-glycosylated, and freely diffusible. In vivo, VEGF-B167 is the predominant form and is abundantly expressed in brown fat, in the myocardium and skeletal muscle (Fig. 2) [49].

Fig. 2

A dendrogram showing the relationships VEGF family members estimated by the amino acid residue substitution analysis.

Fig. 1

The VEGF family ligands and their receptors. VEGF, VEGF-B and PlGF bind VEGFR-1 and VEGFR-2 on the blood vascular endothelium. VEGF-C and VEGF-D primarily bind VEGFR-3 on lymphatic endothelium.

The precise role of VEGF-B in vivo is not known. As VEGF-B is highly expressed in striated muscle, myocardium and brown fat [51,52], its function may be linked to high cellular energy metabolism. Interestingly, at least in one genetic background mice deficient in VEGF-B had smaller hearts and impaired recovery after experimental myocardial infarctions suggesting that the regeneration of coronary collaterals through arteriogenesis could at least in part be dependent on VEGF-B [53]. In another genetic background, a prolonged PQ interval was observed in the electrocardiograms of VEGF-B gene targeted mice [54]. Furthermore, one report has claimed that VEGF-B knockout mice fail to develop pulmonary hypertension in response to hypoxia [55]. Conversely, overexpression of VEGF-B in the lung protected rats from hypoxic pulmonary hypertension in a manner comparable to VEGF [56]. VEGF-B knockout mice also display reduced angiogenic responses in collagen-induced arthritis, suggesting a role for VEGF-B in inflammatory angiogenesis [57]. Correspondingly, VEGF-B may have subtle angiogenic effects, as overexpression of VEGF-B in vivo by injection of recombinant plasmids in a mouse model of hind limb ischemia induced angiogenesis [58].

5. VEGF-C

Where VEGF-A and VEGF-B isoforms are formed through alternative splicing, different forms of VEGF-C and VEGF-D are the result of proteolytic processing. VEGF-C is produced as a precursor protein, which is activated by intracellular secretory proprotein convertases furin, PC5 and PC7 [59,60]. The secreted, VEGFR-3 binding 31/29 kD subunits of VEGF-C are bound together by disulphide bonds, but the factor is further proteolyzed in the extracellular environment by plasmin and other proteases to generate a 21 kD non-disulfide-linked homodimeric protein with high affinity for both VEGFR-2 and VEGFR-3. The mature form of VEGF-C induces mitogenesis, migration and survival of ECs [61]. During development, VEGF-C is expressed along with its receptor VEGFR-3 predominantly in regions where lymphatic vessels develop [38,62]. The expression then decreases in most tissues, remaining high in the lymph nodes [63]. VEGF-C induces selective lymphangiogenesis without accompanying angiogenesis as demonstrated by early experiments in the chick chorioallantoic membrane assay and in transgenic mice overexpressing VEGF-C in the skin [64,65]. Adenoviral VEGF-C gene transduction has been shown to induce growth of functional lymphatic vessels in several different animal models (Fig. 3C) [30,66]. In vivo, VEGFR-3 activation is sufficient to induce lymphangiogenesis whereas the increase in blood vascular permeability induced by VEGF-C and VEGF-D is mediated by VEGFR-2 [30,67].

Mice lacking both VEGF-C alleles fail to develop lymphatic vessels and succumb to tissue edema at E15.5–E17.5 [38]. Loss of one VEGF-C allele results in lymphedema characterized by hypoplasia of the cutaneous lymphatic vessels indicating that the VEGF-C protein concentration is critical for the development of the lymphatic vasculature [38]. However, VEGF-C is not essential for the development of blood vessels unlike its receptors VEGFR-2 and VEGFR-3. Sprouting of endothelial cells committed to the lymphatic endothelial lineage in VEGF-C gene targeted mice could be rescued by application of recombinant VEGF-C and to a lesser degree by VEGF-D, but not with VEGF. This indicates that development of the lymphatic vessels is dependent on VEGFR-3 and VEGF-C in a nonredundant fashion [38].

VEGF-C mRNA transcription was induced in ECs in response to proinflammatory cytokines [68]. The regulation of VEGF-C mRNA transcription by these cytokines indicates that VEGF-C could regulate lymphatic vessel function during inflammation, reflecting the role of the lymphatic vasculature in the control of immune function and leukocyte trafficking. The promoter of the VEGF-C gene has been shown to contain putative NF-κB binding sites that could mediate the activation of VEGF-C mRNA transcription by proinflammatory cytokines [69]. A recent report demonstrated that prostaglandin E2 mediated activation of COX-2 increased VEGF-C mRNA transcription suggesting that prostanoids could induce VEGF-C driven lymphangiogenesis [70]. Recent reports have shown that inflammatory reactions in human kidney transplants undergoing rejection are accompanied by abundant lymphangiogenesis and that VEGFR-3 signaling contributes to reactive lymphadenitis [71]. In a rabbit cornea model of inflammatory angiogenesis and lymphangiogenesis, selective depletion of macrophages blocked lymphangiogenesis demonstrating that inflammatory cells can mediate the formation of lymphatic vessels [37]. The angiogenic and lymphangiogenic responses were blocked by a VEGF inhibitor. The recruited macrophages expressed abundant amounts of VEGF-C and VEGF-D, suggesting that the lymphangiogenic effect of VEGF is indirect and most likely mediated by inflammatory cytokines that induce the expression of lymphangiogenic factors in the inflammatory infiltrate. In this regard, it is interesting to note that COX-2 up-regulated VEGF-C and promoted lymphangiogenesis in human lung adenocarcinoma [70]. VEGF-C is also highly expressed in arthritic joint synovium in patients with rheumatoid arthritis in the absence of lymphangiogenesis per se, suggesting that the lymphangiogenic response is insufficient in this disorder or that the induced VEGF-C participates in other functions [72].

In experimental models, tumor cells overexpressing VEGF-C or VEGF-D induce peritumoral and in some cases stromal lymphangiogenesis and tumor cell invasion into lymphatic vessels [61,73,74]. Furthermore, overexpression of VEGF-C in pancreatic islet cell tumors of transgenic mice induced lymphangiogenesis and promoted lymph node metastasis [75]. Interestingly, neutralization of VEGF-C and VEGF-D in an experimental tumor model by systemic overexpression of a soluble VEGFR-3 fused to the Fc domain of immunoglobulin γ chain (VEGFR-3-Ig) inhibited lymphangiogenesis and lymph node metastasis [76,77]. Peritumoral lymphatics were recently found to originate from the preexisting lymphatic vasculature while circulating endothelial progenitor cells did not contribute to the process [78]. Although enlarged lymphatic vessels at the tumor edge have been reported in human cancers, functional intratumoral lymphatic vessels have not been reported [79]. Interestingly, several clinical studies of cancer patients have shown a positive correlation between VEGF-C expression and lymphatic invasion, lymphatic metastasis and patient survival [80]. However, although breast cancers frequently metastasize to the lymph nodes, at least one study did not detect tumor lymphangiogenesis in breast cancer [81].

6. VEGF-D

Like VEGF-C, the human VEGF-D is processed in its N-terminal and C-terminal ends; the mature form binds to and activates VEGFR-2 and VEGFR-3, is mitogenic for EC and angiogenic as well as lymphangiogenic in vivo [61]. Mouse VEGF-D binds only VEGFR-3, indicating a different role for VEGF-D in mice [82]. VEGF-D is present in most human tissues, most abundantly in the lung and skin during embryogenesis [83]. In experimental tumors VEGF-D increases lymphatic vessel growth and lymphatic metastasis [84]. VEGF-D is expressed by melanoma cells and has been proposed to have a role in tumor angiogenesis and lymphangiogenesis in this tumor [85]. VEGF-D, along with VEGF-C, has recently been shown to be involved in lymphatic metastasis in experimental intrathoracic lung cancer [86]. In humans, VEGF-D has been shown to be of prognostic value for lymphatic vessel invasion and also survival in certain human cancers [80]. The fully processed form of VEGF-D is able to induce strong angiogenesis in addition to lymphangiogenesis in the rabbit hind limb muscle [32,87].

7. VEGFR-1

VEGFR-1 (fms-like tyrosine kinase, Flt-1) is composed of seven extracellular immunoglobulin (Ig) homology domains, a single transmembrane region and an intracellular tyrosine kinase (TK) domain that is interrupted by a kinase-insert domain (Fig. 1, Table 2) [88]. VEGFR-1 binds VEGF, VEGF-B and PlGF with high affinity. VEGFR-1 by itself transmits only weak mitogenic signals in ECs [3], but it can heterodimerize with VEGFR-2 forming a complex with stronger signaling properties than VEGFR-1 or VEGFR-2 homodimers [43,89] (Table 2). VEGFR-1 is expressed in ECs as well as osteoblasts, monocytes/macrophages, pericytes, placental trophoblasts, renal mesangial cells and also in some hematopoietic stem cells [3,90]. VEGFR-1 expression is up-regulated during angiogenesis and also by hypoxia, unlike that of VEGFR-2 and VEGFR-3 [91,92].

View this table:
Table 2

A table showing the chromosomal localization, major mRNA transcripts and major protein sizes of VEGF receptors

GeneChromosomal localizationMajor mRNA transcript size (kb)Major protein size (kDa)
VEGFR-113q12-q137.5, 8.0180
VEGFR-24q11-q125.8, 7.0230
VEGFR-35q33-qter4.5, 5.8195

During development, VEGFR-1 is first expressed in angioblasts and in the endothelium, although less strongly than VEGFR-2; VEGFR-1 expression subsides during later embryonic development [93–95]. VEGFR-1 gene targeted mice die at E8.5 due to disorganization of blood vessels and overgrowth of endothelial cells [3]. Although VEGFR-1 gene targeted mice do not survive, mice lacking only the intracellular kinase (TK) domain are normal except for slightly impaired angiogenesis during pathologic conditions [96,97]. Activation of the VEGFR-1 TK domain is a requisite for monocyte migration [98]. Moreover, VEGFR-1 mediated angiogenesis as well as arteriogenesis have been shown to be dependent on monocytes [47,48], and angiogenesis during experimental tumor growth is at least partially inhibited by anti-VEGFR-1 antibodies [47]. VEGFR-1 signaling is also involved in the recruitment and survival of bone marrow derived progenitor cells [99,100].

A naturally occurring soluble form of VEGFR-1 (sVEGFR-1), consisting of the extracellular part of VEGFR-1, that is able to inhibit VEGF action has been described in humans and mice [3]. sVEGFR-1 has recently been linked to the development of gestational toxemia and preeclampsia [101]. Women with preeclampsia have elevated circulating sVEGFR-1 levels and decreased circulating levels of PlGF and VEGF possibly leading to EC dysfunction in the maternal and placental vasculatures [102].

8. VEGFR-2

The overall structure of VEGFR-2 (kinase-insert domain receptor, KDR/fetal liver kinase, Flk-1) is similar to that of VEGFR-1 (Table 2) [103,104]. VEGFR-2 binds VEGF, VEGF-C and VEGF-D. Although the binding affinity of VEGF towards VEGFR-2 is lower than that for VEGFR-1, selective activation of VEGFR-1 and VEGFR-2 has shown that VEGFR-2 is the primary receptor transmitting VEGF signals in endothelial cells [105–107]. The expression of VEGFR-2 is autoregulated: VEGF, VEGF-C and VEGF-D up-regulate the expression of VEGFR-2 [84,108]. In addition to the ECs, VEGFR-2 is also expressed on neuronal cells, osteoblasts, pancreatic duct cells, retinal progenitor cells, megakaryocytes and hematopoietic stem cells [3,109]. During development VEGFR-2 is expressed by the primitive endoderm, embryonic angioblasts and in the blood islands as well as in angiogenic vessels [93]. VEGFR-2 gene targeted mice die at E8.5-E9.5 due to lack of development of the blood islands, embryonic vasculature and hematopoietic cells [110,111]. VEGFR-2 expression is down-regulated in the adult blood vascular ECs, and is again up-regulated in the endothelium of angiogenic blood vessels [92]. On the other hand, sequestration of VEGF results in down-regulation of VEGFR-2 and in apoptotic death of some capillary endothelial cells in vivo [112]. Moreover, VEGFR-2 may be associated with integrin-dependent migration of ECs, as it forms a complex with integrin αVβ3 upon binding VEGF [113,114]. An interaction between VEGFR-2 and VE-cadherin, a cell–cell adhesion molecule has also been described [115].

9. VEGFR-3

VEGFR-3 (fms-like tyrosine kinase 4, Flt4) has only six Ig-homology domains as the fifth Ig-homology domain is proteolytically cleaved soon after biosynthesis and the resulting polypeptide chains remain linked via a disulfide bond (Table 2) [116,117]. In humans, alternative splicing of the VEGFR-3 gene generates two isoforms of VEGFR-3 that differ in their C-termini [118]. VEGFR-3 binds VEGF-C and VEGF-D [119,120]. VEGFR-3 is present on all endothelia during development but in the adult it becomes restricted to lymphatic ECs and certain fenestrated blood vascular ECs [121,122]. VEGFR-3 is up-regulated on blood vascular ECs in pathologic conditions such as in vascular tumors and in the periphery of solid tumors [92,123]. Interestingly, EC contact with SMCs has been shown to down-regulate VEGFR-3 in ECs [124], suggesting that VEGFR-3 signaling is important in nascent blood vessels, and it becomes redundant as the vessels mature. Indeed, VEGFR-3 gene targeted mice exhibit a dramatic blood vascular phenotype, with embryonic lethality at E9.5 from defective remodeling of the primary vascular plexus and disturbed hematopoiesis [125,126]. Transgenic mice overexpressing a soluble VEGFR-3-Ig fusion protein in the skin lack dermal lymphatic vessels and have hypoplastic deeper lymphatic vessels, although the phenotype is less pronounced as the mice age [127]. Missense mutations in VEGFR-3 have been linked to hereditary lymphedema in humans and also to a similar condition in a mouse model of lymphedema [128–130]. More recently, VEGFR-3 has also been shown to modulate adaptive immunity in an experimental model of corneal transplantation [131].

10. Neuropilins

The neuropilins, Nrp-1 and Nrp-2, have roles in immunology and neuronal development but they are also involved in angiogenesis [132,133]. Neuropilins bind class 3 semaphorins, which are secreted molecules that mediate repulsive signals during neuronal axon guidance [1,2,134]. Nrp-1 also binds VEGF, VEGF-B and PlGF while Nrp-2 binds VEGF, VEGF-C and PlGF [132]. Nrp-1 acts as a co-receptor enhancing VEGF-VEGFR-2 interactions, forming complexes with VEGFR-1 and augmenting tumor angiogenesis in vivo [135]. Overexpression of Nrp-1 in chimeric mice leads to excessive formation of capillaries and blood vessels and hemorrhages in addition to cardiac malformations [136]. In chick embryos, endothelial Nrp-1 expression is mostly restricted to arteries, whereas Nrp-2 primarily marks veins, indicating a role in mediating the arterial/venous identity of ECs [137]. Nrp-2 is expressed also on lymphatic ECs, and mutated Nrp-2 induces abnormalities in the formation of small lymphatic vessels and lymphatic capillaries in mice [138]. Nrp-1 gene targeted mice die at E13 from vascular defects such as insufficient development of yolk sac vascular networks, deficient neural vascularization and transposition of large vessels [139,140]. It is thought that Nrp-1 is required for cardiovascular development because it regulates VEGF165 levels [139]. While Nrp-2 gene targeted mice have normal blood vasculature, the compound Nrp-1/Nrp-2 knockout mouse has a phenotype resembling that of the VEGF and VEGFR-2 gene targeted mice [141].

11. Disorders of the vascular system linked to the VEGFs

11.1. Therapeutic angiogenesis/arteriogenesis in ischemic heart disease and peripheral vascular disease

Angiogenic growth factors may be useful for attempts to increase collateral vessel formation in ischemic heart disease (IHD) or critical limb ischemia/claudication or in the treatment of diabetic neuropathy of the lower extremities [142]. Reduction of blood flow in thromboembolic disease leads to collateral formation via arteriogenesis, a process during which preexisting arterioles enlarge, leading to a 10- to 20-fold increase in blood flow [143]. Arteriogenic pathways such as the PlGF-VEGFR-1 pathway recruit monocytes and act directly on ECs and SMCs by inducing the growth of these cell populations. Recently, PlGF was reported to induce formation of vascular collaterals in experimental models of myocardial infarction and lower limb ischemia in mice by amplification of VEGF signaling [40,43,47]. Thus, VEGFR-1 agonists are potentially useful in restoration of blood perfusion in settings of tissue ischemia.

Human trials utilizing proangiogenic therapy have arrived at some successful endpoints [142]. Attempts at treating inoperable IHD have been made with gene transfer of VEGF by adenovirus and naked plasmid injections [144,145]. Preliminary reports targeting ischemic skeletal muscle suggested that in patients with critical limb ischemia/claudication, injection of VEGF plasmid may induce the growth of functional collateral vessels [146]. In the recent regional angiogenesis with VEGF (RAVE) randomized trial patients with claudication were treated with adenoviral delivery of VEGF121 but the researchers failed to see clinical benefits [147]. However, VEGF driven angiogenesis in progressive ischemic disease may not be sufficient to restore the vascular perfusion because the formed vessels are not sufficiently functional, and even angiomas may develop [148]. Because of the permeability side effects of VEGF, it has been suggested that a combined approach with several growth factors such as VEGF and angiopoietins, platelet-derived growth factors, or the more arteriogenic PlGF may lead to better results [42].

11.2. The role of VEGF family growth factors in atherosclerotic plaque formation

Atherosclerotic plaque progression is associated with inflammatory angiogenesis through increased secretion of angiogenic growth factors such as VEGF and basic fibroblast growth factor (bFGF) [149]. Plaque angiogenesis results in atherosclerotic plaque growth, increased plaque instability and with the increase in the number of the adventitial vasa vasora, an increased risk of intraplaque hemorrhage [150]. These factors contribute to the growing instability of the atherosclerotic plaque.

It is well known that plaque progression is associated with inflammatory cell recruitment and deposition of oxidized low-density lipoprotein (OxLDL). OxLDL increases VEGF production and VEGF secretion by inflammatory cells in vitro [151]. The number of VEGF positive cells has been shown to correlate with the degree of atherosclerotic plaque vascularization [152]. Experimental atherosclerosis can be induced with VEGF and experimental atherosclerotic changes can be reduced with antiangiogenic therapy [153,154]. In a model of experimental cardiac allografts, VEGF was found to mediate the progression of arteriosclerosis [155]. However, recent results have suggested that the angiopoietins, another family of angiogenic molecules that bind and activate Tie2 tyrosine kinase receptors on ECs, could actually prevent some of the inflammatory changes in the vessel wall [156].

11.3. The role of endothelial progenitor cells in vascular pathology

The blood circulation has been suggested to contain endothelial progenitor cells (EPCs) that incorporate into newly formed vessels at sites of active angiogenesis [157]. The EPCs are recruited to sites of angiogenesis where they differentiate in situ into mature endothelial cells much like during the embryonic vasculogenesis. EPCs express a variety of endothelial cell surface markers including platelet endothelial cell adhesion molecule-1, von Willebrand factor (vWF) and VEGF receptors. EPCs are mobilized from the bone marrow by cytokines such as macrophage chemoattractant protein-1 (MCP-1), transforming growth factor-beta (TGF-β), VEGF and PlGF [157]. Skeletal muscle has also been suggested to contain cells with stem cell properties that can contribute to angiogenesis, raising the possibility that tissues can contain resident angioblasts [158]. EPCs have been reported to incorporate into forming blood vessels during physiological angiogenesis such as during the female reproductive cycle, during pathologic angiogenesis in experimental solid tumors or in the limb muscles or the myocardium during ischemia [157]. However, the capacity of bone marrow derived EPCs to incorporate into the vessel wall and transdifferentiate into mature ECs in vivo has also been questioned [159–161].

11.4. Tumor angiogenesis

Blood vessels in tumors display several distinct features that have implications for tumor biology and treatment. Blood vessels in tumors have a chaotic structure and are leaky [162]. As a consequence, blood flow in the tumor is sluggish and the interstitial fluid pressure is high [162]. The sluggish blood flow results in hypoxic regions and perpetuates VEGF production while high interstitial fluid pressure within the tumor hampers delivery of therapeutic agents [162]. Since tumor growth is angiogenesis dependent, therapeutic targeting of the tumor vasculature is an attractive alternative or adjunct to conventional therapy. ECs in tumors express a number of molecules that are unique to blood vessels undergoing angiogenesis, such as VEGFR-2 and integrin αVβ3 that can be targeted for therapeutic purposes. Furthermore, work by Ruoslahti et al. has demonstrated that angiogenic blood vessel endothelium expresses certain peptides/integrins on their cell surface and these markers may serve as “zip codes” for targeting antiangiogenic molecules to tumor vessel endothelium [163].

An important step in antiangiogenic cancer therapy was recently taken when the anti-VEGF blocking antibody bevacizumab (Genentech, USA) showed remarkable results in the treatment of metastatic colorectal cancer, and it has recently been approved by the FDA for treatment of metastatic colorectal cancer [27,164]. This may only mark the beginning of antiangiogenic therapy for tumors, as at present bevacizumab is being evaluated in Phase III trials of metastatic breast cancer, nonsmall cell lung cancer, pancreatic cancer and renal cell carcinoma (see www.clinicaltrials.gov).

11.5. Some other human diseases involving angiogenesis

Excessive ocular neovascularization contributes to visual loss in retinopathy of prematurity and diabetic retinopathy as well as in age-related macular degeneration. Retinal hypoxia and excessive secretion of angiogenic factors like VEGF leads to inappropriate retinal neovascularization and hemorrhages [165]. Experimental retinal neovascularization can be inhibited by antiangiogenic therapy [166–168]. Phase III clinical trials are underway to treat wet age-related macular degeneration with ranibizumab, an antibody fragment to VEGF (Genentech USA). An aptamer inhibitor specific for the VEGF165 isoform (pegaptanib, EyeTech, USA) has also entered phase II–III clinical trials with similar indications [169].

Psoriasis is a chronic inflammatory skin disorder characterized by dermal angiogenesis and overexpression of angiogenic factors such as VEGF [170]. In fact, transgenic mice with sustained overexpression of murine VEGF in the skin have many of the classical features of psoriasis, and treatment with VEGF Trap, a VEGF antagonist (Regeneron, USA), was shown to reverse this phenotype [23]. High VEGF levels are also involved in the pathologic angiogenesis observed in endometriosis and possibly also other gynecological disorders such as uterine bleeding disorders [171].

11.6. Lymphedemas

In lymphedema, lymphatic drainage is impaired and protein-rich fluid accumulates in the subcutaneous tissue leading to fibrosis, impaired immune responses and fatty degeneration of the connective tissue. Lymphedemas are classified as primary, congenital lymphedemas and secondary or acquired lymphedemas. Secondary lymphedema is usually caused by filariasis or by iatrogenic trauma such as radiation therapy, surgery or infection [172]. Primary lymphedemas develop at birth or during adolescence. Congenital hereditary lymphedema, or Milroy disease, is characterized by hypoplastic superficial lymphatic vessels, while the lymphatics in Meige disease, or late-onset congenital lymphedema, appear enlarged and are dysfunctional [173]. The genetic defects underlying several primary lymphedemas have recently been characterized. In some patients with Milroy disease, missense mutations found in the TK domain of VEGFR-3 inhibit the function of this receptor with resulting lymphedema [129]. In lymphedema distichiasis, mutations in the FOXC2 gene have been identified that lead to abnormal mural cell coating of lymphatic vessels and lack of lymphatic valves [174,175]. Other human diseases involving lymphedema with putative, as yet uncharacterized molecular mechanisms include Turner syndrome and cholestasis-lymphedema.

11.7. Tumor lymphangiogenesis

The intravasation of tumor cells into lymphatic vessels is one of the first steps in lymphatic metastasis. Lymphangiogenesis around solid tumors could promote lymphatic metastasis by providing a larger target for the intravasation of tumor cells [80]. Although enlarged lymphatic vessels at the tumor edge have occasionally been reported in human cancers, functional intratumoral lymphatic vessels have not been reported [79,176]. One explanation could be that the high interstitial fluid pressure in tumors compresses lymphatic vessels. According to another hypothesis, compressed lymphatic vessels seen in the tumor stroma did not arise by lymphangiogenesis but are preexisting lymphatic vessels that were engulfed by the expanding tumor [177]. Although lymphatic metastasis is not necessarily dependent on lymphangiogenesis [176], tumor lymphangiogenesis has been reported to be a prognostic factor in cutaneous melanoma and squamous cell cancers of the head and neck [178–180]. Several reports have documented a correlation between expression levels of the lymphangiogenic factors VEGF-C and VEGF-D and lymphatic metastasis in cancer patients [80]. Thus the blocking of VEGF-C and VGEF-D signals with for example a soluble VEGFR-3-Ig fusion protein could be an attractive approach for inhibition of lymph node metastasis in human cancer patients, as already indicated by the results from preclinical studies [76,77].

12. Conclusion

The vascular endothelial growth factors and their receptors play a paramount role in the development of the vascular system, via vasculogenic and angiogenic mechanisms, as well as in the formation of the lymphatic vascular system. Later in life, these molecules are required for processes involving tissue repair, such as wound healing and the cyclic reconstitution of the female endometrium. Aberrant angiogenesis is a key mechanism in the pathophysiology of, e.g., atherosclerosis as well as tumor growth, while both angiogenesis and lymphangiogenesis may contribute to tumor metastasis. Controlling these processes with targeted molecular therapies is therefore at the heart of research directed at disease treatment. While proangiogenic therapies could be employed in the treatment of various ischemic diseases, generation of new lymphatic vessels could help patients with lymphedema. Meanwhile, a renaissance seems to be unraveling around the VEGF growth factors, as novel functions for them continue to be discovered outside the scope of vascular biology.

Acknowledgements

The authors wish to thank Dr. Marika Kärkkäinen for helping with the graphics. The authors have been supported by the Human Frontier Science Program, EU (Lymphangiogenomics LSHG-CT-2004-503573 and AngioNet QLG1-CT-2001-01172), the Novo Nordisk Foundation, the Finnish Academy of Sciences, the Farmos Research Foundation, the Ida Montin Foundation, the Paulo Foundation, the Biomedicum Helsinki Foundation and the Finnish Medical Foundation.

Footnotes

  • Time for primary review 18 days

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View Abstract