Molecular and biological properties of the vascular endothelial growth factor family of proteins.

0163-769X/92/1301-0018$03.00/0 Endocrine Reviews Copyright © 1992 by The Endocrine Society

Vol. 13, No. 1 Printed in U.S.A.

Molecular and Biological Properties of the Vascular Endothelial Growth Factor Family of Proteins NAPOLEONE FERRARA, KEITH HOUCK, LYN JAKEMAN, AND DAVID W. LEUNG Departments of Cardiovascular Research (N.F., L.J.) and Molecular Biology (K.H., D. W.L.), Genentech, Inc., South San Francisco, California 94080

I. Introduction II. Identification of VEGF Secreted by Pituitary Folliculostellate Cells III. Molecular Biology of VEGF IV. Biological and Structural Characterization of the VEGF Proteins V. The VEGF Binding Sites VI. Signal Transduction and Biochemical Responses Induced by VEGF VII. Distribution of VEGF A. Expression in cultured cells B. Tissue distribution of VEGF mRNA 1. Brain 2. Pituitary gland 3. Kidney 4. Ovary VIII. Possible Involvement of VEGF in Tumor Angiogenesis and Metastasis IX. Conclusions

Two separate processes for blood vessel development and differentiation have been identified (13). One mechanism, termed Vasculogenesis,' takes place in the embryo and consists of the initial process of in situ differentiation of mesenchymal cells into hemangioblasts. These cells are the precursors of both endothelial and blood cells, after their segregation from the mesoderm (14-16). Hemangioblasts form nests of isolated cell cords which soon develop a lumen and coalesce. Other cellular components of the vessel wall, such as smooth muscle cells, are recruited by hemangioblasts from undifferentiated mesenchyme. Primitive embryonic vessels including the dorsal aortae and the posterior cardinal veins develop by this process (13). The nature of the factors that induce vasculogenesis remains unknown (13). The other mechanism, called 'angiogenesis,' is the formation of new vessels by sprouting from a preexisting endothelium (17, 18). This process is required not only for further development and differentiation of the embryonic vascular tree but also for a wide variety of fundamental physiological processes in the postnatal life such as somatic growth, wound healing, tissue and organ regeneration, and cyclical growth of the corpus luteum and endometrium (13,17,18). In addition, uncontrolled proliferation of blood vessels is an important pathogenic component of a variety of disorders including cancer, rheumatoid arthritis, retinopathies, psoriasis, and retrolental fibroplasia (17, 18). Angiogenesis is a cascade process consisting of 1) degradation of the extracellular matrix of a local venule after the release of proteases, 2) proliferation of capillary endothelial cells, and 3) migration of capillary tubules toward the angiogenic stimulus (19-21). In view of the remarkable physiological and pathological importance of angiogenesis, much work has been dedicated in recent years to the elucidation of the factors capable of regulating this process. Certain factors of both peptide and nonpeptide nature such as epidermal growth factor (EGF)/transforming growth factor-a (TGF-a) (22), TGF-/3 (23), tumor necrosis factor-a (TNF-a) (24), angiogenin (25), prostaglandin E2 (PGE2) (26), and mon-

I. Introduction

T

HE establishment of a vascular supply is a critical requirement for cellular inflow of nutrients, outflow of waste products, and gas exchange in most tissues and organs (1). In endocrine glands, the vascularization not only serves such needs but also provides a pathway for the specific secretory products (2, 3). Furthermore, in the anterior pituitary (4-6) and in the adrenal medulla (7, 8), an unusual angioarchitecture, where a portal capillary plexus delivers venous blood originating from an adjacent gland, is intimately involved in the control of the secretory activity. In the adrenal medulla, this vascular design may even determine the ultimate secretory product (8). Not surprisingly, the cardiovascular system is the first organ system to develop and reach a functional state in an embryo (9-12). In the human, primitive blood vessels appear as early as day 15, and a circulation with a beating heart is already established by the end of the third week. Address requests for reprints to: Napoleone Ferrara, Department of Cardiovascular Research, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, California 94080.

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MOLECULAR AND BIOLOGICAL PROPERTIES OF VEGF

obutyrin (27) are able to induce a neovascular response in vivo. However, these agents have either little or no mitogenic effects on cultured endothelial cells (TGF-a, EGF, angiogenin, PGE2, monobutyrin) or, paradoxically, inhibit their growth (TGF-/3, TNF-a) (22-29). Their in vivo angiogenic effects are thought to be largely mediated by the paracrine release of direct-acting angiogenic inducers from inflammatory cells (17, 18). In contrast, acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and PD-ECGF are able to stimulate both vascular endothelial cell growth in vitro and angiogenesis in vivo (30-33). Surprisingly, PD-ECGF (33) and FGFs (34, 35) lack a signal peptide required for extracellular transport according to classic secretory pathways (36, 37). This suggests that these factors may be available to their target cells only after cell death, i.e. during embryonic and fetal development, in a variety of rapidly proliferating neoplasias, or after an injury. Alternatively, it has been proposed that FGF may be incorporated into the basement membrane and be released when specific enzymes degrade this structure (38). However, evidence from several experiments links physiological and pathological angiogenesis to the release of diffusible factors (39-41). For example, the finding that the angiogenic response induced by a transplanted melanoma was not prevented by a millipore filter interposed between the tumor and the host provided strong evidence for the diffusible nature of tumor angiogenesis factor(s) (41). In this article we review the molecular and biological properties of a recently identified family of direct-acting endothelial cell mitogens and angiogenic factors referred to as vascular endothelial growth factor (VEGF) (42-47), vascular permeability factor (VPF) (48-50), or vasculotropin (51). VEGF was identified in the media conditioned by normal bovine pituitary folliculo-stellate cells (FC) (42) and by a variety of transformed cell lines (4447). VPF was identified in the media conditioned by tumor cell lines on the basis of its ability to induce vascular leakage, rather than as a growth factor (48), and later on was found to promote endothelial cell growth (52). Molecular cloning of the complementary DNAs for these factors revealed that they are encoded by the same gene, and alternative splicing of messenger RNA is the mechanism for their generation (53, 54). The resulting four polypeptides have strikingly different secretion patterns, which suggests multiple physiological roles for this family of proteins. Two members of this family are secreted by cells, while the third and fourth are mostly cell-associated, despite the fact that all members have an identical signal sequence (53, 54).

II. Identification of VEGF Secreted by Pituitary FC The pituitary gland is a major regulator of physiological processes (55). Its specific hormones are mediators

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of several important bodily functions. In addition, the gland has been shown to be an abundant source of factors capable of affecting proliferation and differentiation in a variety of cell types (56, 57). A potent and versatile growth factor such as FGF was originally identified and isolated from pituitary extracts (58, 59). Even though classical hormone-secreting cells have long been considered to be the most likely origin of regulatory molecules, more recently a population of nonsecretory cells, follicular or FC, has received considerable attention as an important intrapituitary regulatory system (60-62) and also as a source of trophic factors (42, 63). FC represent a population of agranular cells in the adenohypophysis of all species examined (64-67). They have a stellate shape and send cytoplasmic processes between secretory cells. In some instances FC form clusters in which adjacent cells, connected by periapical tight junctions, project microvilli and cilia into a central follicle. Slender cytoplasmic projections of FC end in the parenchymal basal lamina, in close proximity to the perivascular space. The significance and function of FC have been long debated (67). Among several hypotheses, FC have been suggested to be neural crest-derived cells homologous to astrocytes in the central nervous system (68), paracrine regulators of hormone secretion (60,61), phagocytes (69), regulators of ion concentration and composition in the interstitial fluid (70), stem cells capable of differentiating into hormone-secreting cells (71), part of an uptake/transport system for small molecules, metabolites, or hormones (72, 73). Recent studies reported the culture of cells from bovine adenohypophyseal pars tuberalis, pars distalis, and pars intermedia which, by a variety of morphological criteria and by the lack of hormone secretion, were identified as FC (74-76). Such cells rapidly proliferate in culture and form contact-inhibited cell monolayers which develop domes, an expression of active ion transport provided by polarized epithelial cells. Domes are widely observed in cultured cells derived from ion-transporting epithelia (77, 78). Consistent with ion transport properties (77), FC monolayers display transepithelial potential difference and resistance in Ussing chambers (75, 76). Several mediators such as /3-adrenergic agonists, antidiuretic hormone, PGE2, or bradykinin are able to stimulate active ion transport across FC monolayers. These findings led to the conclusion, in agreement with other studies (70), that one of the roles played by FC is the local regulation of ion and fluid composition in the interstitial fluid surrounding the pituitary cells (75, 76). It has been suggested that FC also play an important trophic role toward secretory cells. FC appear to contribute to the survival and regeneration of secretory cells in the early stage of a hypophyseal transplant under the kidney capsule (79). FC form tube-like structures that


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FERRARA ET AL.

organize and direct the regeneration. These findings suggest that FC may produce trophic factors for the secretory cells. Furthermore, the intimate relationship existing between FC end processes and microvessels (72, 73) suggests that FC may also produce factors capable of exerting regulatory influences on the pituitary vasculature. In agreement with this hypothesis, cultured bovine FC are a rich source of the potent mitogen and angiogenic factor bFGF (63). Also, recent studies have shown that cultured FC secrete a potent inhibitor of aortic endothelial cell growth, having little inhibitory effect on capillary endothelial cells (80). This inhibitor was identified, unexpectedly, as leukemia inhibitory factor, a glycoprotein originally identified as a growth inhibitor for murine leukemia cells and subsequently found to have diverse and apparently unrelated biological effects, including the ability to promote differentiation of cholinergic neurons (81). The production of such a factor suggests that FC may play a role in organizing the peculiar vascular architecture of the pituitary, where no arteries reach the pars distalis and virtually all of the blood to this area is supplied by capillaries originating from the pituitary portal veins (4-6). In addition, medium conditioned by FC is strongly mitogenic to cultured bovine capillary endothelial cells (42). These vascular endothelial cells are stimulated to proliferate by the FGFs and, possibly, PD-ECGF, but not by a variety of other factors including insulin, platelet-derived growth factor (PDGF), TGF-a, or EGF (30). Since FGFs and PD-ECGF are intracellular proteins, the possibility that FC release into the medium a soluble ECGF was considered. This hypothesis led to the purification of a heparin-binding protein, VEGF, from serum-free medium conditioned by FC using a combination of heparin-sepharose affinity chromatography and reversed phase chromatography (42, 82). The denomination of VEGF was proposed to designate the very narrow target cell selectivity of the growth factor, apparently restricted to vascular endothelial cells.

III. Molecular Biology of VEGF Molecular cloning of a bovine VEGF cDNA was achieved from a library derived from FC mRNA (43). This provided a direct verification of the hypothesis that VEGF is a secreted protein. The NH2-terminal amino acid sequence determined by microsequencing (42, 82) is preceded by 26 amino acids corresponding to a typical signal sequence (36, 37). The mature VEGF protein is generated directly after signal sequence cleavage, without an intervening prosequence. The bovine monomer is expected to have 164 amino acids with a calculated molecular mass of 19,162 after signal sequence cleavage.

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The existence of a potential glycosylation site at Asn74 suggests that VEGF is a glycoprotein. Clusters of basic amino acid residues are present at the positions around 110, 123, and 163, and they are likely to be responsible for the binding of the molecule to heparin. The amino acid sequence of VEGF displays limited but significant (18-20%) homologies with those of the A and B chains of PDGF and the product of the sis oncogene (83-85). All eight cysteine residues found in PDGF are conserved in VEGF. However, VEGF contains eight additional cysteine residues within the COOH-terminal region. Three types of cDNA clones encoding human VEGF were isolated from a cDNA library prepared from HL 60 leukemia cells (43). The most abundant clone encodes a protein that is 95% identical to bovine VEGF. Human VEGF, which has an insertion of glycine in position 6, is expected to have 165 amino acids (VEGFi65) after signal sequence cleavage. Two clones were also identified which encode for a shorter form with a deletion of 44 amino acids between position 116 and 159 and for a longer form with an insertion of 24 amino acids highly enriched in basic residues at position 116. The mature proteins are expected to contain 121 and 189 amino acids, respectively (VEGF121, VEGFi89). In both cases Asn115 is replaced by aLys. An additional member of the VEGF family was recently identified by screening a variety of human cDNA libraries by the polymerase chain reaction technique. In a fetal liver library, a unique transcript, which did not correspond to any of the previously cloned forms of VEGF, was identified, and a full length clone was generated (53). DNA sequence analysis showed that this novel form contained an insertion of 41 amino acids compared to VEGFi65. This insertion included the highly basic 24-amino acid insertion found in VEGF18g. Therefore, the mature protein is expected to have 206 amino acids (VEGF2o6). The amino acid sequence of the four molecular species of VEGF is shown in Fig. 1A. Alternative splicing of mRNA, rather than transcription from different genes, is the most likely explanation for the existence of such multiple forms. This is supported by Southern blot analysis of human genomic DNA, which shows that the restriction pattern is identical using either a probe for VEGFi65 or one that contains the insertion in VEGF2o6- Further evidence of alternative splicing of mRNA is provided by analysis of genomic clones in the area of putative mRNA splicing. This shows an intron/ exon structure consistent with alternative splicing but with an unusual feature. There is no intron between the coding sequences for the 24-amino acid insertion in VEGF189 and the additional 17-amino acid insertion in VEGF206. The 5'-end of the 51 base pair insertion of VEGF206 begins with GT, the consensus sequence for the 5'-splice donor necessary for mRNA processing. Thus,


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FIG. 1. A, Amino acid sequence of the four molecular species of VEGF. The arrow points to the first NH2-terminal residue in the mature protein. The bar underlines the glycosylation site. B, Schematic model for the generation of the four molecular species of VEGF by alternative splicing of mRNA. Exons are represented by boxes and introns by solid lines.

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definition of the 5'-splice donor site for removal of this 1 kilobase (kb) intron appears to be variable. Figure IB illustrates a model for the generation of all identified molecular species. Also, recent work (86) indicates that the human VEGF gene is organized in eight exons. According to these studies, VEGF165 is missing the residues encoded by exon 6, while VEGF12i is missing the residues encoded by exons 6 and 7. Analysis of the VEGF gene promoter region provided evidence for a single major transcription start which lies near a cluster of potential Sp 1 factor binding sites (86). Analysis of a variety of cDNA libraries derived from cultured cells and tissues by polymerase chain reaction technique reveals that the transcript encoding the 165amino acid species is the most abundant product of the VEGF gene, with the exception of the placenta where the transcript encoding VEGF^i is even more abundant. Very little is known about the distribution of the longer

forms. It is possible that these may be only minor molecular species expressed by all cells that express the VEGF gene, or they could be preferentially expressed in specific circumstances. Interestingly, alternative splicing of mRNA also takes place in PDGF-A. The two molecular species of PDGFA differ by 15 amino acids in the COOH terminus (87). This alternative splicing pattern is found in all species examined, including Xenopus laeuis (88, 89). The 15 amino acid extension in PDGF-A, encoded by exon 6, is rich in basic amino acids and is highly homologous to the 24-amino acid insertion found in human VEGFi89 or VEGF206 (90). These basic motifs have been termed 'nuclear targeting sites' and display homology to highly conserved basic sequences in histones (91, 92). It has been proposed that such sequences impart directional or targeting information on polypeptides. For example, recent studies on acidic FGF, which also contains a nuclear


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FERRARA ET AL.

targeting sequence in its amino-terminal domain, indicate that such a sequence may encode critical information for intracellular signaling and protein trafficking (93). In the case of PDGF it appears that nuclear targeting sequences impart on the molecule the information for remaining cell-associated. The extended form of PDGF-A would be sequestered into specific domains, while the nonextended form would be freely soluble, after secretion (94). Very recent studies (95) also provide evidence that a cell retention signal is localized to 11 amino acids in the carboxyl-terminal third of the PDGFB chain precursor (amino acids 219-229). This region contains an 8-amino acid basic sequence highly homologous to the sequence encoded by exon 6 in the extended form of PDGF-A and to the 24-amino acid insertion in VEGF. As described below, the presence of the nuclear targeting domain also confers on VEGF the property of remaining mostly cell-associated, rather than freely soluble.

IV. Biological and Structural Characterization of the VEGF Proteins FC-derived VEGF is a heat- and acid-stable, dimeric, heparin-binding glycoprotein, with a mol wt of approximately 45 K (42, 82). It is completely inactivated by reducing agents such as dithiothreitol or /?-mercaptoethanol. VEGF is a basic protein; its isoelectric point is approximately 8.5. VEGF is able to promote the growth of endothelial cells isolated from bovine adrenal cortex, cerebral cortex, fetal and adult aorta, and human umbilical vein. Half-maximal stimulation of endothelial cells growth is obtained at 100-150 pg/ml of VEGF (2-3 pM), and a maximal effect occurs at 1-4 ng/ml (22-88 pM). VEGF, however, has no mitogenic effect on cultured corneal endothelial cells, vascular smooth muscle cells, BHK-21 fibroblasts, keratinocytes, human sarcoma cells, or lens epithelial cells. This indicates that the target cell specificity of VEGF is restricted to vascular endothelial cells. In contrast, bFGF and aFGF exert mitogenic effects on virtually all of those cell types (30). VEGF is also able to induce a pronounced angiogenic response in the chick chorioallantoic membrane (43, 51). This in vivo effect demonstrates that VEGF has the ability to trigger the entire sequence of events leading to new blood vessel growth (19-21). Recombinant human VEGFi65 (rhVEGFi65) behaves similarly to native FC-derived VEGF in terms of binding to heparin, retention in reverse phase columns, as well as in vitro and in vivo bioactivity. The mol wt of rhVEGF165 is approximately 46 K. rhVEGF165 can exist both in a glycosylated and a deglycosylated species. This results in two types of monomers, having molecular mass, respectively, of 23 K and 18 K (96). Since these two

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species can form homodimers and heterodimers, three types of dimers are identified. Similarly to VEGFi65, VEGF m promotes endothelial cell growth and is efficiently secreted by cells, as assessed by bioactivity in the conditioned medium of transfected cells, metabolic labeling, and immunoprecipitation studies. Immunoblot (96) and immunoprecipitation (53) studies provide evidence that VEGF121 may exist in two molecular forms having mol wt of 18 K and 14 K, respectively. This heterogeneity is likely due to the existence of deglycosylated and, possibly, also decarboxylated species. Unlike VEGFi65, VEGFm binds poorly to heparin and is strongly retained by anion exchange columns. This indicates that the isoelectric point of VEGF121 is in the acidic range. Such a difference in charge can be explained on the basis of the absence in VEGF121 of the 44-amino acid region, rich in basic residues, which is present in VEGF16s. As noted above, the two longer forms, unlike the shorter forms, are mostly cell-associated. Media conditioned by transfected 293 cells transiently expressing cDNAs encoding VEGFi8g or VEGF 2o6 have little or no mitogenic activity on endothelial cells (53). Also, immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of both cell lysates and conditioned medium of metabolically labeled, transiently transfected 293 cells demonstrates the presence of strong bands of the expected size (27 and 29 K, respectively, in reducing conditions) in cell lysates, while only weak bands of lower mol wt (18 to 14 K) are precipitated in the conditioned medium (53). It appears that the information for remaining cell-associated is entirely contained within' the 24-amino acid insertion. A mutant of VEGF2o6 lacking the 24-amino acid insertion, but containing the 17- amino acid insertion unique to VEGF206, was instead efficiently secreted in the medium which, accordingly, promoted endothelial cell growth (53). Media conditioned by stable cell lines expressing a VEGF189 or VEGF206 cDNA have a very low level of mitogenic activity for endothelial cells (53, 54). The significance and function of the cell-associated species of VEGF are unknown. All four molecular species are active in a Miles-type of assay (97), i.e. they induce extravasation of Evans Blue when injected intradermally into the guinea pig skin. However, significant differences in potency may exist (53). For example, despite the low amounts of immunoprecipitable VEGF and the almost complete absence of mitogenic activity, media conditioned by cells expressing VEGF189 or VEGF206 are positive in the Miles assay. This suggests that the secreted products of VEGF189 or VEGF206 may be more potent than the other forms in inducing dye extravasation.


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V. The VEGF Binding Sites The identification and characterization of the VEGF/ VPF binding sites in cultured endothelial cells has been reported (98-100). Two classes of high affinity sites were described on the membrane of bovine endothelial cells. Their dissociation constants were 10~12 and 10~n M, respectively (98). The mol wt of the putative receptor has been estimated to be approximately 180 K (98, 99) or approximately 230 K (100). Low mol wt binding sites (~110 K) have also been described in the cell surface of endothelial cells (101). Unexpectedly, high affinity binding sites were also identified in cell types that do not display a mitogenic response to VEGF, such as lens epithelial cells or corneal endothelial cells (101). In these cells, however, only the low mol wt species of receptor was found. Recent studies (102) provide evidence that the fmslike tyrosine kinase (fit) protein (103, 104) is a receptor for VEGF. The fit protein is homologous to members of the PDGF receptor family, which includes the PDGF atype and /3-type, the CSF-1 and c-kit receptors (105). The ligand for such receptor had not been previously identified (103, 104). As predicted by cDNA sequence analysis, the fit receptor has seven immunoglobulin-like domains in its extracellular region, a single transmembrane spanning region, and a tyrosine kinase sequence that is interrupted by a 'kinase insert' domain (105). [125I]VEGF165 displays specific, high affinity (IC50 = 20 pM), binding to transfected cells expressing the fit protein (102). Furthermore, expression of fit mRNA in Xenopus laevis oocytes induces the oocyte to release calcium in response to VEGF (but not PDGF), indicating that the binding event triggers a biological response. Surprisingly, VEGF did not induce an appreciable increase in tyrosine phosphorylation of the fit receptor. However, the fit protein expressed in Xenopus oocytes is constitutively phosphorylated on tyrosine residues. Therefore, it may be difficult to detect ligand-dependent phosphorylation of specific tyrosines in the presence of such a high background. Whether the fit receptor mediates the proliferative/permeability enhancing effects of the VEGF/

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VPF polypeptides and whether it represents the only receptor for this family of factors remains to be established. To elucidate the distribution of cells responsive to VEGF in the context of intact tissues, binding to sections from normal adult rats with biologically active, iodinated rhVEGFi65 was performed (106). Quantitative autoradiography was employed to analyze the binding kinetics and simultaneously localize binding sites at both macroscopic and microscopic levels. Specific, high affinity, VEGF binding sites were identified in the vast majority of tissues and organs. The highest density of binding sites was in brain and spinal cord, adrenal cortex, lung, glandular stomach, spleen, and pancreas. Interestingly, such distribution of binding correlates remarkably well with the known organ distribution of the fit mRNA (104). Scatchard analysis of saturation isotherms in sections from several organs revealed a single class of binding sites with high affinity (Kd = 16 to 35 pM) and low capacity (1.9-6.8 fmol/mg protein). Colocalization of binding with factor VHI-like immunoreactivity demonstrated that the binding was associated with vascular endothelial cells. Microvascular endothelium of both fenestrated and nonfenestrated capillaries and also large vessel endothelium exibited specific VEGF binding. However, no displaceable binding was evident on nonendothelial cell types. An especially high density of binding sites was identified in the endothelium lining the atrioventricular valves in the heart or the aortic valve. Among endocrine glands, particularly high VEGF binding was measured in the adrenal cortex. This structure is extremely well vascularized, and the ratio of parenchymal cells to capillary endothelial cells approaches 1:1 (107). Figure 2 illustrates the specific binding of [125I]rhVEGF to adult rat pituitary. The intense binding in the pars distalis and pars nervosa correlates well with the rich microvascular bed present in these areas (4-6). In contrast, very little or no binding is detectable in the pars intermedia, the least vascularized region of the adenohypophysis (108). It is believed that interstitial fluid circulating through a network of canaliculi, rather

FIG. 2. Distribution of VEGF binding sites in rat pituitary sections. Brightfield (A) and darkfield (B) micrographs of a 16 jim pituitary section after incubation in the presence of 80 pM [125I]rhVEGF165 and exposure in photographic emulsion. Binding reflects the microvascular distribution throughout pars nervosa (pn) and pars distalis (pd), while little binding is associated with the poorly vascularized pars intermedia (pi). Hematoxylin stain; scale bar = 100 jtm.


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than blood, provides nutrients to pars intermedia cells as well as a transport channel to hormones and secretagogues (108). In contrast to pituitary and adrenal, where the endothelium is normally quiescent, follicular development in the ovary is accompanied by an active cycle of angiogenesis during luteal formation and vascular degeneration during its regression (109). The VEGF binding pattern observed in adult rat ovary (Fig. 3) reflects these dynamic vascular changes (106). In the medulla, specific binding was found in association with numerous blood vessels. In secondary and mature follicles, binding was virtually absent in the avascular granulosa cells, while intense binding was associated with vessels in the theca. Intense binding was localized in the mature corpora lutea. In degenerating corpora lutea, binding of VEGF was either limited to the periphery or was undetectable.

VI. Signal Transduction and Biochemical Responses Induced by VEGF Little is known at the present time about the intracellular responses induced by VEGF after its interaction with its receptor(s) on the cell surface of target cells. It was shown that VEGF/VPF induces a rapid Ca2+ entry into cultured endothelial cells from bovine aorta, human umbilical vein, or bovine adrenal cortex (110, 111). The addition of purified guinea pig-derived VPF induces a Ca2+ inflow within 10-15 sec in endothelial cells but not in cultured smooth muscle cells (110). The ED50 to induce such effects was 0.4 pM; a maximal effect was observed

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at 22 pM. These concentrations are very similar to those required to promote endothelial cell growth. Human VEGF165 also promotes calcium entry in cultured bovine capillary endothelial cells (111). Like other mediators that increase Ca2+ entry in cultured endothelial cells, VEGF/VPF induces expression and release of von Willebrand factor (110). The addition of rhVEGFi65 induced within 60 sec inositol (I) (1,4)P3 with a concomitant increase in I(1,3,4,5)P4, I(1,3,4)P3, I(1,4)P2, and I(1)P (111). These findings indicate that VEGF can stimulate phospholipase C activation in cultured endothelial cells and also that stimulation of second messenger formation by inositol lipid hydrolysis may contribute, at least in part, to the mitogenic and angiogenic signals produced by this growth factor (112). According to a recent report, a VEGF-like molecule purified from A-431 human epidermoid carcinoma cells induces tyrosine phosphorylation of an approximately 190 K polypeptide in human umbilical vein endothelial cell lysates (113). Even though phosphorylation of certain cellular proteins other than the receptor, such as phospholipase C, may occur in response to a growth factor (114), these studies raise the possibility that VEGF is able to induce phosphorylation of its own receptor(s) and that such an event may be involved in mediating the mitogenic response to the growth factor (105, 115). VEGF165 has been shown to induce the metalloproteinase interstitial collagenase, both at mRNA and protein levels, in human umbilical endothelial cells, but not in dermal fibroblasts (116). Also, VEGF has been found to induce the serine proteases urokinase type (u)-plasmin-

FiG.3. [126I]rhVEGF binding sites within the rat ovary, shown in brightfield (A and C) and darkfield (B and D). In sections through the entire ovary (A and B), binding corresponds to the pattern of vascularization during the dynamic processes of luteal development and regression. Intense binding is associated with the corpora lutea (cl) and theca externa (th). Note the absence of binding sites within the granulosa cells of developing follicles (gr). A regressing corpus luteum (asterisk) is shown in detail (C and D). Binding is associated with the vasculature remaining in the periphery, but little binding is present in the central region of the degenerating corpus luteum. Hematoxylin stain; scale bar in A and B = 200 //m; scale bar in C and D = 50 fim.


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ogen activator (PA) and tissue type (t)-PA, both at mRNA and protein level, and also PA inhibitor (PAI)-l in microvascular endothelial cells (117). VEGF was a more potent inducer of t-PA mRNA than bFGF, while bFGF was a more potent inducer of u-PA and PAI-1 mRNAs. The induction of these enzymes appears to be particularly relevant to the process of angiogenesis (118120). As previously indicated, extracellular proteolysis and the degradation of the basement membrane are integral parts of the angiogenesis cascade (19). Endothelial cells are freed from the constraints of the extracellular matrix and may therefore proliferate and migrate in response to the angiogenic signal. The expression of protease inhibitors such as PAI-1 serves to regulate and balance the process (120). VII. Distribution of VEGF A. Expression in cultured cells Several human and rodent tumor cell lines secrete soluble endothelial cell mitogens and Evans Blue extravasation-inducing factors which are now known to belong to the VEGF/VPF family (44,47-49). A variety of human tumor cell lines including sarcoma and carcinoma cells show a 3.7 kb RNA transcript that hybridizes with the VEGF probe in a Northern blot. A minor transcript having the size of 4.2 kb is also observed. Mouse sarcoma 180 cells express the VEGF mRNA and secrete a VEGFlike mitogen (121). Interestingly, media conditioned by these cells were used in the first successful long-term culturing of capillary endothelial cells (21). Little is known regarding the expression of VEGF in normal cells. An untransformed cell type other than pituitary FC has been recently shown to express VEGF (86, 96). Fetal human aortic smooth muscle (ASM) cells express and release VEGF (86). Adult bovine ASM cells also express the VEGF RNA transcript and secrete a VEGF-like endothelial cell mitogen (96). The localization of VEGF in ASM cells might have significant implications for maintaining the integrity of the endothelial cells in the intima. Large vessel endothelial cells have the ability to grow and repair tears after an intimal injury, provided that the extent of the damage is limited (122,123). B. Tissue distribution of VEGF mRNA To gain insight into the physiological role of VEGF, the cellular localization of the VEGF mRNA was investigated in various tissues and organs by in situ hybridization. The distribution of the VEGF mRNA was examined in brain, pituitary, kidney, and ovary (124-126). 1. Brain. The ingrowth of blood vessel into the neural primordia is a prerequisite for the development and

25

differentiation of the nervous system. The neural plate appears as early as the cardiovascular primordia, but the latter reach a functional state while the neural elements remain relatively undifferentiated (11, 12). The proliferation of blood vessels into the early anlagen of mesencephalon and telencephalon correlates remarkably well with the onset of neuroectodermal proliferation (127). Previous studies have demonstrated that extracts from fetal mesencephalic and telencephalic structures have angiogenic properties and provided evidence for the presence of FGF in such extracts (128). The VEGF mRNA is expressed in the rat brain (124). In the adult rat, a diffuse hybridization signal was detected throughout the cerebral cortex. Examination of a series of coronal sections of adult rat brain at the level of the hypothalamus again revealed a diffuse low level hybridization that was appreciably higher than sections incubated with a control sense probe. A particularly high signal was identified in the supraoptic and paraventricular (PV) nuclei. In both supraoptic and paraventricular nuclei, the hybridization signal was clearly associated with cells consistent with the morphology of the magnocellular neurons, which produce antidiuretic hormone and oxytocin (129). This suggests that the expression of VEGF mRNA, at least in some brain areas, is cellspecific. Significant hybridization was also identified in the median eminence, in the choroid plexus, and in the subfornical organ. In sections of fetal bovine cerebral cortex, a low level hybridization signal was detected throughout. However, certain subpopulations of cells in layers II and III gave a particularly strong signal. 2. Pituitary gland. In the adult rat pituitary-specific hybridization to the VEGF probe was observed in approximately 20% of pars distalis cells (124). Since FC account for only 5-10% of the total cell population of the pars distalis (64-67), it is likely that certain populations of secretory cells also have the ability to express the VEGF mRNA. Very low hybridization signal was observed in the poorly vascularized pars intermedia. The low VEGF mRNA expression correlates well with the almost complete absence of specific VEGF binding sites in this area of the adenohypophysis (106). Intense hybridization signal was detected instead in the pars nervosa. A similar pattern was observed in the fetal bovine pituitary. It is tempting to speculate that VEGF may play a role in the development and differentiation of the pituitary portal vessels during fetal and early postnatal life and in the maintenance of their differentiated state in the adult animal. Capillaries of the primary portal plexus are observed as early as the 18th fetal day in the rat (130). The proliferation and maturation of these microvessels are essential for the establishment of a vascular link between


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median eminence and pituitary, which allows hypothalamic neurohormones to affect secretory cells (4-6). The link is complete by the fourth postnatal day in the rat (130) and the 11th week of gestation in the human (131). Whether VEGF plays a role in the dramatic proliferation of blood vessels that characterizes estrogen-induced PRL-secreting tumors in the rat (132) remains to be established. Interestingly, FC in these tumors undergo hypertrophy and remarkable ultrastructural changes (133, 134). 3. Kidney. The maintenance of homeostasis is critically dependent on the integrity of kidney function (135). Despite the fact that the water movement across glomerular capillaries is extremely high, normally only the smallest plasma proteins are able to cross the glomerular barrier. Therefore, an understanding of the factors that maintain the integrity of glomerular capillaries has profound implications both for kidney physiology and the pathogenesis of glomerulopathies (136, 137). A diffuse hybridization signal to the VEGF probe was observed throughout cortex and medulla in adult kidney sections (124). A strikingly higher signal was associated with the glomeruli. Silver grains were seen in the external surface of the glomerulus, and most of the hybridization signal appeared to be associated with the podocytes, the visceral epithelial lining of Bowman's capsule. These cells project foot-like processes that closely embrace the entire network of glomerular capillaries (138). Therefore, podocytes are uniquely suited to exert trophic/regulatory effects on glomerular capillaries. High VEGF expression in this site, however, does not appear to be consistent with the hypothesis that a major action of the growth factor is the induction of protein extravasation (48, 49). In fact, one of the major roles of the glomerulus is to prevent protein loss in the urine (135, 136). Whether VEGF/VPF may be overexpressed in the glomerulus and lead to proteinuria remains to be established. 4. Ovary. Angiogenesis is a significant component of the process of development and differentiation of the corpus luteum (CL) (109). In the course of follicular growth, the theca interna becomes richly vascularized. After ovulation, the thecal vessels invade the ruptured follicle and form a rich network of microvessels that nourish the developing CL. These changes in the vascular pattern suggest the local release of angiogenic factors (39, 40, 139). Accordingly, the adult rat ovary was examined for VEGF mRNA expression (125). Intense hybridization to the VEGF probe was found in the developed CL, where most of the hybridization signal was associated with luteal cells. In contrast, minimal hybridization was detected in the avascular granulosa cells. These dynamic changes in VEGF mRNA are consistent with a temporal

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relation between VEGF expression and growth of capillary vessels into the CL (109). Recent studies provide evidence for the expression of the VEGF mRNA, as assessed by Northern blot analysis and in situ hybridization, also in the ovary of a primate, Macaca fascicularis (126). Levels of VEGF mRNA were very high during the formation of the CL. A significant decline in the mRNA levels was seen during the late luteal phase, and essentially no message was detected in the postmenses CL. These mRNA changes correlate remarkably well with the pattern of vasculature changes in the primate ovary (140, 141). These studies also show that the deprivation of LH induced by the treatment with a GnRH antagonist resulted in a decline in VEGF mRNA, indicating that VEGF expression in the CL is subjected to the regulatory control of gonadotropins (142). These findings suggest that VEGF plays a role in the development and differentiation of the CL through its angiogenic properties. A defective production of VEGF by luteinizing granulosa cells could potentially be a pathogenic factor in luteal phase defects. Such conditions are characterized by insufficient progesterone production and are frequently associated with infertility and miscarriage (143, 144).

VIII. Possible Involvement of VEGF in Tumor Angiogenesis and Metastasis Substantial experimental evidence links tumor growth and metastasis with blood vessel growth (17,18, 41, 145, 146). The neovascularization provides nourishment to the growing tumor and also allows tumor cells to be in contact with the vascular bed of the host. Tumor angiogenesis is induced by factors released either by the tumor cells themselves or by macrophages attracted into the tumor proper (17, 18). Also, the acquisition of an angiogenic phenotype appears to be a crucial aspect in the transition from hyperplasia to neoplasia (147). Further evidence for the dependence of tumor spreading on angiogenesis is provided by the demonstration of a correlation between number and density of microvessels in histological sections of invasive human breast carcinoma and actual presence of distant metastases (148). Therefore, the extent of the neovascularization in a tumor may be a predictor of metastatic disease (148). As previously indicated, a variety of transformed cell lines express the VEGF transcript and secrete a VEGFlike protein, suggesting that this soluble endothelial cell mitogen and angiogenic factor may be one of the mediators of the process of tumor angiogenesis. Previous studies also suggested that this growth factor may be involved in another important aspect of tumor biology, the abnormal permeability properties of tumor vessels (48, 49). It has been proposed that excessive leakiness of tumor


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vessels may lead to the extravasation of fibrinogen and other plasma proteins (149). The formation of a fibrin gel would serve as a substrate for both tumor and endothelial cell growth. Based on the finding that some malignant cells, but not their untransformed counterpart, have the ability to secrete VPF (48, 49), it was proposed that such a factor is a specific product of tumor cells and is also a major mediator of the abnormal permeability of tumor vessels. This hypothesis, however, is not consistent with the widespread distribution of the VEGF/VPF mRNA in normal tissues (124). Furthermore, a normal, untransformed, cell type like FC secretes even higher amounts of VEGF than a variety of tumor cell lines, providing further evidence that the level of expression/ secretion of this growth factor is not necessarily related to malignant behavior. This, however, does not exclude the possibility that the pathological overexpression of the growth factor may lead to abnormal vascular permeability and ascites as well as to pathological angiogenesis. It was also reported (150) that VEGF/VPF induces the expression and synthesis of tissue factor, a procoagulant protein (151), in cultured endothelial cells and in monocytes and synergizes with TNF in such induction. It was proposed that tissue factor induction may have significant relevance for the abnormal coagulative properties of tumor vessels in response to mediators such as TNF (152). According to these studies, the release of VEGF/VPF by tumor cells would facilitate the intravascular fibrin deposition, thrombosis, and decline in blood flow after systemic infusion of low concentrations of TNF, a response that occurs in tumor vessels but not in normal vessels. However, the induction of t-PA (117), a protein involved in intravascular thrombolysis, by the growth factor is expected to counteract, at least in part, such procoagulant effects. These studies also suggest that VEGF/VPF promotes chemotaxis of monocytes across collagen membranes and collagen gel (150). The halfmaximal concentration required to elicit these effects (~300 pM) was, however, substantially higher than that required to induce endothelial cell growth, suggesting that these effects may be pharmacological or pathological, rather than physiological, and also that they may be mediated by a different type of receptor. It is interesting to note that the induction of tissue factor might actually limit the growth of a tumor, since it is expected to result in thrombosis and occlusion of the tumor vascular bed. Recently, the hypothesis was tested that the overexpression of VEGF may confer a growth advantage in vivo on a nontumorigenic cell type such as Chinese hamster ovary (CHO) cells (Ferrara, N., et al. submitted). Nude mice were injected subcutaneously with untransfected cells, cells transfected with expression vectors encoding VEGF m or VEGFi65, or cells transfected with vector alone. Neither parental CHO cells nor cells

27

transfected with the vector alone formed any tumors. In contrast, cells expressing either form of VEGF developed small tumors. No evidence of metastasis was found. Histological examination revealed that these tumors were highly cellular and well vascularized. In situ hybridization and immunocytochemistry demonstrated that at the time of death the tumor cells were expressing very high levels of VEGF mRNA and were also synthesizing the protein. An unexpected finding was the lack of histological evidence of edema, a cardinal manifestation of increased vascular permeability (153), in tumor sections. Furthermore, ultrastuctural analysis failed to show any abnormalities in the morphology of capillaries within the tumors. Also, these microvessels did not display ultrastructural features consistent with high permeability, such as fenestrations or a large number of micropinocytotic vesicles (153,154). These findings are consistent with the hypothesis that the overexpression of VEGF may facilitate the growth of a tumor. It is likely that such action is mostly indirect, via an increase in the vascular supply, since VEGF has no mitogenic affect on CHO cells. In contrast, pleiotropic factors such as TGF-a (155) or FGFs (156, 157) are thought to facilitate tumorigenesis not only through their angiogenic properties but also by inducing autocrine stimulation of tumor cell growth (158-161). It is, however, premature to draw any definitive conclusions on the role of VEGF in the process of tumor angiogenesis. Further studies are undoubtedly required to assess the tumorigenic properties of cell types other than CHO cells, expressing the secreted as well as the intracellular forms of VEGF, and also to establish whether a temporal and spatial correlation exists between expression of VEGF and proliferation of blood vessels in human tumors. IX. Conclusions The widespread distribution of VEGF mRNA in normal tissues and the ubiquitous presence of its receptors in vascular endothelial cells suggest that VEGF is primarily a regulator of normal function, rather than a mediator of pathological processes. The existence of a temporal relation between expression of VEGF mRNA and proliferation of capillary vessels in the rat and primate CL is clearly consistent with the hypothesis that one of the physiological roles of VEGF is to promote neovascularization. Unexpectedly, high expression of VEGF mRNA is also detected around microvessels in areas where endothelial cells are normally quiescent (154), such as the adult kidney glomerulus, the pituitary, or the brain (124). Likewise, VEGF binding sites are consistently found in association with the vasculature of these areas (106). These findings raise


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the possibility that the tonic presence of the growth factor may be required to maintain the differentiated state of those vessels, which otherwise might undergo involution. It is therefore tempting to speculate that the suppressed expression of VEGF and/or its receptors may be responsible, at least partially, for a variety of regressive phenomena involving blood vessels, both in developing and adult animals (11, 12, 154). The finding that VEGF binding sites in adult rat tissues are localized to endothelial cells provides a direct verification of the unique target cell specificity of this growth factor, which had been inferred from previous in vitro studies (106). Even though one cannot rule out the possibility that cell types not included in the sections examined (e.g. circulating blood cells) may bind VEGF or that VEGF may also bind to nonendothelial cells in pathological situations, the vast majority of VEGF binding clearly appears to be associated with endothelial cells (106). Interestingly, particularly high density of binding sites for VEGF is associated with the endothelium lining the atrioventricular valves in the heart (106). These highly dynamic components of the cardiovascular system are subjected to a considerable degree of hemodynamic stress. The maintenance of the integrity of the endothelium lining these structures is essential for the prevention of thrombosis or bacterial endocarditis (162, 163). The presence of high VEGF binding in these regions might relate to a need for continual repair and maintenance of the endothelium, mediated by the growth factor. Unlike other endothelial cell mitogens such as FGF or PD-ECGF, members of the VEGF/VPF family also have the ability to induce extravasation of Evans Blue when applied in the guinea pig skin. All four molecular species have such activity, although significant quantitative differences might exist (53). The significance of this effect, however, is unclear. An attractive possibility is that one of the physiological roles of these factors is the regulation of vascular permeability. The verification of this hypothesis would probably require a correlation between expression of mRNA and/or receptors and actual permeability properties of different types of microvessels. However, binding sites for VEGFi65 are expressed both in fenestrated (e.g. endocrine glands, kidney) and tight capillaries (e.g. brain) (106). Likewise, VEGF mRNA is expressed around both types of microvessels (124). Another puzzling finding is the lack of appreciable edema in histological sections of tumors derived from transfected cells expressing high levels of VEGF (see previous paragraph). A possibility is that the induction of vascular leakage may be dependent, at least in part, on the route or the site of administration/action of VEGF/VPF. In this context it is of interest to observe that interleukin 8 induces marked polymorphonuclear leukocytes adhesion

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and emigration across endothelial cells when applied intradermally (164, 165) and therefore has been characterized as a proinflammatory molecule. However, interleukin 8 paradoxically prevents polymorphonuclear leukocyte emigration and exerts potent antiinflammatory effects when injected intravenously (166). Interestingly, recent studies provide evidence for the existence of a novel member of the VEGF/VPF family (167). A cDNA encoding a protein having an approximately 53% identity with the PDGF-like region of VEGF/VPF has been isolated from a placental cDNA library. The encoded protein, named placenta growth factor, has been reported to be an N-glycosylated dimer and to promote endothelial cell growth. Whether placenta growth factor shares other properties with the VEGF/VPF polypeptides, such as the specificity for endothelial cells or the ability to induce dye extravasation, remains to be established. Finally, the study of VEGF is expected to provide important insight into the pathogenesis of disorders other than cancer (which are characterized by excessive angiogenesis), such as atherosclerosis (168), diabetes mellitus (169), or rheumatoid arthritis (170). A recent report (171) already describes increased expression of VEGF mRNA in human coronary artery atherosclerotic plaques in comparison with unaffected regions of the vessel.

Acknowledgment We wish to thank Mike Cronin for helpful comments and for critically reading this manuscript.

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involvement in endocarditis and the blood pressure resting on the valve. Am J Med Sci 224:318 Baggiolini M, Walz A, Kunkel SL 1989 Neutrophil-activating peptide-1/interleukin 8, a novel cytokine that activates neutrophils. J Clin Invest 84:1045 Thelen M, Peveri P, Kernan P, Von Tscharner V, Walz A, Baggiolini M 1988 Mechanism of neutrophil activation by NAF, a novel monocyte-derived agonist. FASEB J 2:2702 Hechtman DH, Cybulsky MI, Fuchs HJ, Baker JB, Gimbrone MA 1991 Intravascular IL-8. Inhibitor of polymorphonuclear leukocyte accumulation at sites of acute inflammation. J Immunol 147:883 Maglione D, Guerriero V, Viglietto G, Delli-Bovi P, Persico MG 1991 Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor. Proc Natl Acad Sci USA 88:9267 Barger AC, Beekwkes A, Lainey L, Silverman KJ 1984 Hypothesis: vasa vasorum and neovascularization of human coronary arteries. N Engl J Med 310:175 L'Esperance FA, James SA 1983 The eye and diabetes mellitus. In: Ellemberg M, Rifkin H (eds) Diabetes Mellitus. Theory and Practice, ed 3. New Hyde Park Medical Examination Publishing, New York, p 727 Fassbender HG 1986 Normal and abnormal synovial tissue with emphasis on rheumatoid arthritis. In: Cohen AS, Bennett JC (eds) Rheumatology and Immunology, ed 2. Grune & Stratton, Orlando, FL, p 356 Gemperlein I, Grund Wellershof S, Graf H 1991 Vascular endothelial growth factor is expressed in sclerotic human coronary artery. J Cell Biochem 15F:CF410 (Abstract)

The 49th Laurentian Hormone Conference, Palmas de Mar, Puerto Rico, November 15-18, 1992 Topics will include Prostate, Neuroendocrine Growth Factors, Intercellular Signalling, Glucose Regulation. Contact: Laurentian Hormone Conference Box 273-JES 1230 York Avenue New York, NY 10021


The vascular endothelial growth factor family of polypeptides.

The vascular endothelial growth factor family: identification of a fourth molecular species and characterization of alternative splicing of RNA.

Expression of vascular endothelial growth factor-C and vascular endothelial growth factor receptor-3 in esophageal squamous cell carcinoma.

Levels of vascular endothelial growth factor and matrix metalloproteinase-9 proteins in patients with glioma.

Cerebrospinal fluid vascular endothelial growth factor.

vascular endothelial cell growth factor with human umbilical vein endothelial cells: binding, internalization, degradation, and biological effects.

Levels of angiogenesis-related vascular endothelial growth factor family in neovascular glaucoma eyes.

Vascular endothelial growth factor receptor-1 expression in breast cancer and its correlation to vascular endothelial growth factor a.

Effects of vascular endothelial growth factor on osteoblasts and osteoclasts.

Intrinsic bioactivity of insulin-like growth factor-binding proteins from vascular endothelial cells.

Vascular endothelial cadherin and vascular endothelial growth factor in periodontitis and smoking.

Ultrasound Molecular Imaging of Angiogenesis Using Vascular Endothelial Growth Factor-Conjugated Microbubbles.

Ultrasound molecular imaging of vascular endothelial growth factor receptor 2 expression for endometrial receptivity evaluation.

Three cases of organized hematoma of the maxillary sinus: clinical features and immunohistological studies for vascular endothelial growth factor and vascular endothelial growth factor receptor 2 expressions.

Expression of vascular endothelial growth factor and basic fibroblast growth factor in extramammary Paget disease.

Porcine placental immunoexpression of vascular endothelial growth factor, placenta growth factor, Flt-1 and Flk-1.

basic fibroblast growth factor complex peptide.

Dual delivery of vascular endothelial growth factor and hepatocyte growth factor coacervate displays strong angiogenic effects.

Pancreatic islet hepatocyte growth factor and vascular endothelial growth factor A signaling in growth restricted fetuses.

Apoptosis of vascular endothelial cells by fibroblast growth factor deprivation.

Regulation of the migration of endothelial cells by a gradient density of vascular endothelial growth factor.

Specificity of vascular endothelial growth factor treatment for radiation necrosis.

Vascular endothelial growth factor antagonist therapy for retinopathy of prematurity.

Antifibrotic role of vascular endothelial growth factor in pulmonary fibrosis.