From the
The vascular system is essential for providing oxygen and
nutrients, removing metabolic waste products, and furnishing efficient
access of leukocytes to tissues throughout larger animals.
Angiogenesis, the sprouting of new capillaries, is required for the
development of the vascular system and, consequently, the growth of
vertebrates. Angiogenic proteins, including several from the fibroblast
growth factor family, were identified and purified in the 1980s. They
were, however, found to be mitogenic not only for vascular endothelial
cells but also for a wide variety of other types of cells and appeared
to promote angiogenesis as part of coordinated tissue growth and
repair. In the late 1980s the first selective angiogenic growth factor
was purified on the basis of its ability to induce transient vascular
leakage (vascular permeability factor) and endothelial cell mitogenesis
(vascular endothelial growth factor (VEGF) ()or
vasculotropin). By amino acid and cDNA sequencing, these proteins were
subsequently demonstrated to be identical. The identification of VEGF (
)set the stage for a rapid expansion in the understanding
of what now appears to be one of the most important mediators of
physiologic and pathologic angiogenesis yet discovered. Previous
reviews have documented some of the initial characterization of VEGF
structure and activities (1, 2, 3) . (
)
The originally characterized form of VEGF is an approximately
34-46-kDa homodimeric glycoprotein. The amino acid sequence is
20% identical with platelet derived growth factor (PDGF) A and B
chains (4, 5, 6, 7) including 8
conserved Cys residues previously located within the minimal PDGF
receptor-binding domain defined by truncated forms of the
v-sis-derived oncogenic protein, a viral version of PDGF. In
PDGF-BB homodimers, these cysteine residues participate in 3 disulfide
bonds within each subunit and 2 symmetric intersubunit disulfide bonds.
The amino acid sequence homology implies that the VEGF secondary and
tertiary structures, arrangement of intra- and intersubunit disulfide
bonds, and relative subunit orientation are similar to those of PDGF.
Soon after the identification of VEGF a DNA sequence encoding a
close homologue was reported. Denoted placenta growth factor (PlGF) on
the basis of its original source, it shares 53% amino acid sequence
identity with VEGF, including the 8 conserved Cys residues in the
putative receptor-binding domain(8) , comparable with the 50%
identity between the mature PDGF A and B chains. In addition to PlGF
homodimers, heterodimers composed of VEGF and PlGF subunits have been
recently identified and purified (9) in analogy to PDGF-AB
heterodimers. Although the VEGFPlGF heterodimer is a potent
endothelial cell mitogen in vitro, PlGF homodimers exhibit
only weak endothelial cell mitogenic activity under similar conditions.
The cDNA sequences of VEGF and PlGF encode N-terminal hydrophobic secretory leader sequences that promote active secretion. Like the PDGF A chain, the VEGF and PlGF genes are expressed as alternatively spliced mRNAs including forms coding polycationic regions near the C-terminal ends of the translated polypeptides as shown in Fig. 1. Three major VEGFs containing mature 121-, 165-, and 189-amino acid residue sequences and a polymerase chain reaction product inferring the existence of a minor 206-amino acid residue version have been identified.
Figure 1: Structures of VEGF and PlGF subunits. Polypeptide subunits translated from alternatively spliced mRNAs are shown schematically as horizontal bars followed by amino acid residue sequence lengths of the mature processed human subunits after removal of secretory leader sequences. Horizontal lines denote the positions of N-terminal secretory leader signal peptide (SP) sequences and the minimum receptor-binding region of the homologous PDGF B sequence mapped from truncated versions of its v-sis oncogene homologue (MINIMUM V-SIS). Asn-linked oligosaccharides are shown as branched Y symbols. Amino acid residue sequence inserts encoded by alternatively spliced exons are filled by stipples and diagonal lines. The human amino acid residue sequences and net charges of the polycationic matrix targeting signal regions are listed below the corresponding insert bar segments.
The human VEGF gene is composed of 8 polypeptide coding
exons(10) . The shortest form of the protein is encoded by
exons 1-5 and 8. Inclusion of the cationic polypeptide sequence
encoded by exon 7 generates the apparently predominant 165-amino acid
form that, in contrast to VEGF, binds to isolated heparin
and to heparan proteoglycans distributed on cellular surfaces and
within extracellular matrices. Addition of the very cationic 24-amino
acid residue sequence (Fig. 1) encoded by exon 6 promotes even
tighter binding of VEGF
to these endogenous
polyanions(11) . The PlGF gene contains 7 coding exons (12) that can generate two alternatively spliced forms.
PlGF
differs from the shorter PlGF
by
inclusion of an exon 6-encoded 21-amino acid residue polycationic
sequence (Fig. 1) in an equivalent C-terminal location to the
VEGF inserts(12) . This cationic sequence also promotes heparin
binding in vitro(13) and presumably facilitates
heparan proteoglycan binding in vivo. Therefore, alternative
mRNA splicing appears to modulate VEGF and PlGF binding to endogenous
heparan proteoglycans, thus controlling diffusion from cellular sites
of synthesis and determining the extent of local storage.
The
heparan proteoglycan binding forms of VEGF can be released from
cellular surfaces and extracellular matrices by heparinases and by
plasmin, a protease that is proteolytically activated during tissue
remodeling by plasminogen activators(14) . Plasmin-mobilized
VEGF, a truncated active form similar in size to VEGF,
does not bind heparin, indicating that this proteolytic treatment
probably removes much of the heparin binding C-terminal sequence
encoded by the alternatively spliced exons.
VEGF Receptors and Signal Transduction
Two homologous VEGF receptors, KDR (or Flk-1 from mouse) and
Flt-1, are expressed by vascular endothelial cells in vitro and in vivo beginning during early vascular embryonic
development. As shown in Fig. 2, KDR (15) and Flt-1 (16) , each 1300 amino acid residues long, are composed of
7 extracellular Ig-like domains containing the ligand-binding region, a
single short membrane-spanning sequence, and an intracellular region
containing tyrosine kinase domains. The amino acid sequences of KDR and
Flt-1 are
45% identical to each other, which is equivalent to the
homology between the related 5 Ig-like domain PDGF
and -
receptors.
Figure 2: Structures of VEGF/PlGF receptors. The full-length VEGF-specific KDR receptor and the homologous Flt-1 receptor, which binds both VEGF and PlGF, are each composed of 7 extracellular Ig-like domains containing the ligand-binding region, a single plasma membrane-spanning sequence, and intracellular tyrosine kinase domains containing a kinase insert sequence. An alternatively expressed soluble truncated form of Flt-1, denoted sFlt-1, containing the N-terminal 6 Ig-like domains followed by a unique 31-amino acid residue C-terminal sequence functions as an inhibitor of VEGF mitogenic activity. Human amino acid (aa) residue sequence lengths are given in parentheses.
Flt-1 binds VEGF and PlGF with high affinity whereas KDR
complexes tightly with VEGF but not PlGF
homodimers(13, 17) . VEGF binding to KDR but not Flt-1
elicits an efficient (ED
0.1-1 ng/ml) DNA
synthetic and chemotactic endothelial cell response(18) . The
absence of potent PlGF mitogenic activity (9) also implies that
its binding to Flt-1 does not effectively mediate a mitogenic signal.
The mitogenic inefficiency of Flt-1 is consistent with recent mouse
receptor gene knockout results. Although eliminating either receptor is
lethal by day 8-10 of gestation, the phenotypes immediately prior
to death are different. Few, if any, vascular endothelial cells are
observed in KDR knockout mice compatible with the role of this receptor
as a critical mediator of endothelial cell mitosis(19) . In
contrast, Flt-1 knockout mice contain endothelial cells, but they exist
in poorly organized vessels. Therefore, activation of this second
receptor by VEGF and PlGF might modulate the interaction of these cells
with each other or the basement membrane on which they
reside(20) .
The Flt-1 receptor mRNA can be spliced to generate forms encoding either the full-length membrane-spanning receptor or a soluble form, denoted sFlt-1, that is truncated on the C-terminal side of the sixth extracellular Ig-like domain (21) as shown in Fig. 2. Pure sFlt-1 retains its specific high affinity binding for VEGF and PlGF(17) . However, the soluble receptor fully inhibits VEGF-stimulated endothelial cell mitogenesis at concentrations that are substoichiometric to VEGF so it does not appear to act simply by sequestering the growth factor.
Like other growth factor transmembrane tyrosine kinase receptors, VEGF receptors presumably undergo ligand-induced dimerization. Formation of dimers between sFlt-1 and full-length VEGF receptors could account for the ability of the truncated receptor to override the activity of membrane-spanning receptors because sFlt-1-containing heterodimers would not trigger signal transduction dependent on intracellular tyrosine kinase dimerization. For sFlt-1 to efficiently inhibit mitogenesis by such a ``dominant negative'' mechanism it would be expected to dimerize not only with Flt-1 but also with the mitogenically competent KDR receptor. Similar inhibitory heterodimerization has been shown to occur between artificially truncated tyrosine kinase-deficient KDR and full-length VEGF receptors (22) . Modulation of the relative expression of Flt-1 and sFlt-1 might provide a means by which endothelial cells could regulate their response to VEGF and PlGF.
Ligand-induced growth factor receptor dimerization triggers signal transduction by promoting either autophosphorylation or transphosphorylation of the adjacent receptor subunit and by binding and phosphorylating specific downstream signal transduction protein mediators. In several other growth factor receptors insert sequences within the tyrosine kinase domains contain tyrosine residues that upon phosphorylation generate docking sites for complexation with downstream signal transduction proteins. KDR and Flt-1 kinase domains contain 70 (15) and 66 (16) amino acid residue insert sequences, respectively. At least 4 tyrosine residues in the KDR cytoplasmic domains are subject to either auto- or transphosphorylation, two of which are within the kinase insert region(23) .
VEGF-activated endothelial cell receptors also
phosphorylate several cytoplasmic proteins including some that contain
receptor phosphotyrosine-binding SH2 domains and can participate in
downstream signal transduction. These tyrosine-phosphorylated proteins
include phosphatidylinositol 3-kinase, which phosphorylates
phosphatidylinositols at the 3-position of the inositol ring to produce
potential second messengers, and phospholipase C, an enzyme that
hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol
1,4,5-triphosphate and 1,2-diacylglycerol, which stimulate
Ca
release and activation of protein kinase C,
respectively. In addition, the Ras GTPase-activating protein GAP and
NcK, a protein containing one SH2 and three SH3 docking domains that
might couple cell surface receptors to other downstream effectors, are
phosphorylated(24) . The differences in KDR and Flt-1 signal
transduction that account for the disparity in mitogenic signal
generation are not yet known.
Distribution and Control of Expression
Angiogenesis is minimal in healthy adult males but is a prominent activity associated with the female estrus cycle. VEGF expression and neovascularization of ovarian follicles increase immediately prior to ovulation whereas the corpus luteum expresses VEGF shortly after ovulation. Repair of the endometrium at the end of each estrus cycle is dependent on vascular regeneration, and VEGF mRNA expression is elevated in estrogen-responsive epithelial cells lining the oviducts and uterus. VEGF mRNA is found in extraembryonic giant trophoblast cells at the sites of implanted fertilized eggs (25) and persists during early postimplantation. It falls to low levels through the initial stages of embryonic development and then increases during organ growth(26) .
VEGF mRNA remains detectable in several adult organs and cell types in vivo. In adult rats VEGF mRNA is present in lung alveolar cells and kidney glomerular and proximal tubules(27) . Lower levels are found in liver hepatocytes and brain. In addition, VEGF mRNA is expressed in all cells of the adrenal cortex and testosterone-producing Leydig cells of the testes(25) . PlGF mRNA is abundantly expressed within placenta and by human vascular endothelial cells(28) . It is also present in several transformed cell lines (9, 12) and in lower levels in adult heart, brain, lung, and skeletal muscle(12) , perhaps reflecting endothelial cell expression in vivo.
Transcription of VEGF mRNA is
induced by a variety of factors. Serum-derived and paracrine growth
factors and cytokines, including PDGF-BB(29) , keratinocyte
growth factor (fibroblast growth factor-7), epidermal growth factor,
tumor necrosis factor (30) , transforming growth
factor-
1(29, 30, 31) , and
interleukin-1
(32) , can each induce expression of VEGF
from 3- to 20-fold in a variety of cultured cells. With the exception
of PDGF-BB, none of these factors are directly mitogenic for
microvascular endothelial cells in culture; thus their observed
angiogenic activities could reflect induction of VEGF expression.
In
addition to protein growth factors, some small mediators have been
shown to modulate VEGF expression. Phorbol esters increase VEGF protein
levels more than 5-fold in human keratinocytes. Prostaglandin E increases VEGF mRNA and protein levels in a preosteoblastic cell
line in a manner promoted by the differentiation inducer retinoic acid
and inhibited by dexamethasone, which suppresses bone formation in
vivo(33) . Thus one of the mechanisms of prostaglandin
promotion of bone growth, a process dependent on angiogenesis, could be
its induction of VEGF in osteoblasts.
Hypoxia is known to induce
angiogenesis, thereby providing a compensatory mechanism by which
tissues can increase oxygenation. Therefore, diminished O is one of the most intriguing transcriptional inducers of VEGF (34) and its receptors (35) in normal and transformed
cells. Hypoxic induction of VEGF appears to be a general response since
many types of cultured cells have been observed to increase VEGF mRNA
levels by approximately 10-50-fold as a consequence of lowering
the percent O
from ambient 21% to the range of 0-3%.
Similar induction of VEGF at reduced pO levels is seen in vivo including within hypoxic regions of
tumors(34) . Occlusion of coronary arteries induces myocardial
ischemia and VEGF mRNA expression in porcine hearts(36) . In
response to exposure of rats to chronic hypobaric hypoxia for 1 month
VEGF mRNA and protein are elevated in lung alveolar cells, and both KDR
and Flt-1 mRNA levels increase in lung vascular endothelial cells along
with DNA synthesis indicative of mitosis(35) .
The mechanism
by which hypoxia increases expression of VEGF is only partially
understood. Induction of VEGF is stimulated by CoCl and
inhibited by CO(37) . An analogous CoCl
stimulation
of hypoxia-inducible erythropoietin expression is proposed to act by
replacement of iron with cobalt in the porphyrin ring of a putative
heme-containing protein oxygen sensor, decreasing its affinity for
O
and favoring the deoxy conformation. In contrast, CO
could bind tightly at the O
site and lock the sensor in the
oxy conformation, thus inhibiting hypoxic responses. Either the same or
similar regulatory proteins could modulate transcription of VEGF and
erythropoietin. In fact, two potential regulatory DNA enhancer
sequences that are 90% homologous with the human erythropoietin
hypoxia-response element are located 5` to the transcriptional start
site of the VEGF gene(37) . In addition, a functional 5`
enhancer of apparently unique sequence has been mapped to a 100-base
pair segment
800 base pairs upstream of the VEGF transcriptional
start site(38) .
Endothelial cells throughout the vascular system can respond mitogenically to VEGF. No other normally differentiated major cell types have been confirmed to divide in response to VEGF, consistent with the restricted endothelial cell expression of the mitogenically functional KDR receptor. Even if other less commonly studied cells are eventually shown to respond to VEGF, it remains the most selective vascular endothelial cell mitogen known.
VEGF also elicits non-mitogenic responses by vascular endothelial cells including chemotaxis (39) and the expression of plasminogen activators (40) and collagenases (41) that facilitate penetration of growing capillaries into tissues. A single intradermal injection of VEGF, but not PlGF, can induce vascular leakage in 5 min that is largely eliminated within 20-30 min. Endogenous paracrine expression of VEGF adjacent to fenestrated endothelium could contribute to a persistent increase in vascular permeability such as is observed in kidney and brain choroid plexus(27) . However, vascular leakage has not been detected in response either to intravascular injections of VEGF, expression from transfected cells(42) , or expression in vascularized neural tissue such as the cerebellar granule cell layer containing an intact vascular blood brain barrier that is not associated with persistent permeability(27) . Therefore, additional unknown factors might modulate VEGF-induced permeability.
Angiogenesis is an integral feature of normal tissue repair. In rodents, VEGF mRNA is maximally expressed by surface epidermal keratinocytes soon after dermal injury. However, in healing-impaired diabetic rodents it is abnormally low when highly vascular new tissue would develop in normal animals(30) . Exogenous VEGF can induce new blood vessel formation and increase perfusion in ischemic rabbit limbs (43) and in response to decreased blood flow in porcine coronary arteries(44) . VEGF also promotes the repair of damaged rat carotid artery endothelial monolayers concomitantly inhibiting pathological thickening of the underlying smooth muscle layers, thereby maintaining lumen diameter and blood flow(45) .
Elevated expression of VEGF can also contribute to progression of several diseases. The sustained growth of solid tumors appears to be dependent on angiogenesis. Human tumor biopsies exhibit enhanced expression of VEGF mRNAs by malignant cells and VEGF receptor mRNAs in adjacent endothelial cells. VEGF expression appears to be greatest in regions of tumors adjacent to avascular areas of necrosis (34) consistent with the possibility that tumor angiogenesis might be driven, at least in part, by hypoxic induction of VEGF regardless of the particular genetic mutations leading to transformation. Monoclonal anti-VEGF antibodies substantially inhibit the vascularization and growth of human tumors in nude mice but do not influence growth of the same tumor cells in culture(46) . Therefore, the tumor growth advantage conferred by VEGF expression appears to be a consequence of paracrine stimulation of angiogenesis. Viral expression of a VEGF-binding construct of the mouse KDR receptor, truncated to eliminate the cytoplasmic tyrosine kinase domains, virtually abolishes the growth of a transplantable tumor in mice(22) , presumably by the previously described dominant negative mechanism of heterodimer formation with membrane-spanning VEGF receptors.
Pathological neoangiogenesis is a defining feature of a family of human ocular diseases in which vascular growth in the retina leads to visual degeneration culminating in blindness. VEGF accounts for most of the angiogenic activity produced in or near the retina in diabetic retinopathy(47) . Elevated VEGF expression also has been observed in several inflammatory conditions typically characterized by increased angiogenesis. Rheumatoid arthritic synovial tissues contain high levels of VEGF mRNA and protein associated with macrophages along the synovial lining(39, 48) . Increased VEGF, KDR, and Flt-1 mRNAs are also detected in psoriatic skin (49) and in contact dermatitis, a delayed dermal hypersensitivity reaction(50) .
A VEGF gene has been found integrated into the Orf virus, a member of the poxvirus family that causes contagious pustular dermatitis with extensive capillary proliferation in sheep, goats, and occasionally humans(51) . The deduced VEGF amino acid sequences of two isolated viral strains retain only 24 and 44% identity with their putative mammalian ancestor and are merely 42% identical to each other, probably reflecting divergence of rapidly evolving viral genes. An analogous situation was previously recognized in the homologous PDGF system in which the primate retroviral v-sis oncogene presumably arose by the integration and divergence of a host PDGF B gene.
The unique vascular endothelial cell selectivity and hypoxic induction of VEGF have contributed to the growing recognition of its physiologic importance as an angiogenic agent. Molecular characterization of the VEGF system has already revealed selective molecular agents and targets that could provide specific therapeutic tools for either enhancing or inhibiting angiogenesis. Further elucidation of the factors and conditions that modulate the expression of VEGF, PlGF, and their receptors will increase our understanding of not only the biological chemistry of this specific molecular system but also of vascular development, growth, and repair.