Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

Heparin and HS (heparan sulfate) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS binding to VEGF (vascular endothelial growth factor) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that the VEGF binding affinity likely depends on the specific structural features of these oligosaccharides, including their degree of sulfation, sugar-ring stereochemistry and conformation. Notably, the unique 3-O-sulfo group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue-specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.     

Up-regulation of vascular endothelial growth factor (VEGF) in small-for-size liver grafts enhances macrophage activities through VEGF receptor 2-dependent pathway

This study aims to investigate the potential role of vascular endothelial growth factor (VEGF) and VEGF-R2 (fetal liver kinase (Flk)-1) in mediating macrophage activities in small-for-size liver transplantation. A rat orthotopic liver transplantation model was performed using either whole, 50, or 30% liver grafts (both 50 and 30% were regarded as small-for-size) in syngeneic or allogeneic combinations, respectively. Firstly, the mRNA and protein levels of VEGF and Flk-1 in liver grafts were detected by RT-PCR and Western blot, and the number of Flk-1(+) macrophages (labeled by ED1) was determined by flow cytometry. It was found that the small-for-size isografts and allografts presented higher levels of VEGF and Flk-1 expression than the whole isograft and allograft. In addition, a higher number of Flk-1(+)ED1(+) cells were detected in the small-for-size isografts and allografts than the whole isograft and allograft. Secondly, our study revealed that macrophage cell lines did not initially express detectable Flk-1, but could be induced by VEGF, and the inducible expression of Flk-1 in macrophages was related to their migration and proliferation activities. Finally, our study demonstrated that the induction of Flk-1 expression on macrophages by VEGF was associated with the expression of NF-kappaB and heat shock protein 90. In conclusion, the present study showed that the up-regulated expression of VEGF and its interaction with Flk-1 in small-for-size liver grafts might facilitate the activities of macrophages.     

Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF)

Vascular endothelial growth factor (VEGF) was originally identified as an endothelial cell specific growth factor stimulating angiogenesis and vascular permeability. Some family members, VEGF C and D, are specifically involved in lymphangiogenesis. It now appears that VEGF also has autocrine functions acting as a survival factor for tumour cells protecting them from stresses such as hypoxia, chemotherapy and radiotherapy. The mechanisms of action of VEGF are still being investigated with emerging insights into overlapping pathways and cross-talk between other receptors such as the neuropilins which were not previously associated with angiogenesis. VEGF plays an important role in embryonic development and angiogenesis during wound healing and menstrual cycle in the healthy adult. VEGF is also important in a number of both malignant and non-malignant pathologies. As it plays a limited role in normal human physiology, VEGF is an attractive therapeutic target in diseases where VEGF plays a key role. It was originally thought that in pathological conditions such as cancer, VEGF functioned solely as an angiogenic factor, stimulating new vessel formation and increasing vascular permeability. It has since emerged it plays a multifunctional role where it can also have autocrine pro-survival effects and contribute to tumour cell chemoresistance. In this review we discuss the established role of VEGF in angiogenesis and the underlying mechanisms. We discuss its role as a survival factor and mechanisms whereby angiogenesis inhibition improves efficacy of chemotherapy regimes. Finally, we discuss the therapeutic implications of targeting angiogenesis and VEGF receptors, particularly in cancer therapy.     

Role of vascular endothelial growth factor (VEGF) in endothelial cell protection against cytotoxic agents

Autocrine expression of VEGF has been detected in endothelial cells under hypoxia or oxidative stress. However, the functional significance of this VEGF autocrine expression remains undefined. To analyze the role of autocrine VEGF in the endothelial response against injury, cultured bovine aorta endothelial cells (BAEC) were challenged with potentially cytotoxic substances with different chemical structure and pharmacologic properties, namely cytochalasin D (CyD), hydrogen peroxide (H2O2) and cyclosporine A (CsA). Our results revealed that: i. In particular conditions, exposure to potentially cytotoxic agents as CyD, H2O2 or CsA results in significant BAEC cytoprotection rather than injury. ii. The response to the 3 agents is shifted to a cell damaging pattern in the presence of a specific anti VEGF monoclonal antibody (mAb). iii. CyD and H2O2 markedly stimulate the autocrine expression of VEGF mRNA and VEGF protein. In conclusion, the present study reveals a protective mechanism of endothelial cells against injury involving autocrine VEGF production. Moreover, the occurrence of a significant increase in VEGF expression accompanying this defensive mechanism is further disclosed.     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurrs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis. (Mol Cell Biochem 264: 51–61, 2004)     

Complexity in the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF)-receptors signaling

The adult vasculature results from a network of vessels that is originally derived in the embryo by vasculogenesis, a process whereby vessels are formed de novo from endothelial cell (EC) precursors, known as angioblasts. During vasculogenesis, angioblasts proliferate and come together to form an initial network of vessels, also known as the primary capillary plexus. Sprouting and branching of new vessels from the preexisting vessels in the process of angiogenesis remodel the capillary plexus. Normal angiogenesis, a well-balanced process, is important in the embryo to promote primary vascular tree as well as an adequate vasculature from developing organs. On the other hand, pathological angiogenesis which frequently occurs in tumors, rheumatoid arthritis, diabetic retinopathy and other circumstances can induce their own blood supply from the preexisting vasculature in a route that is close to normal angiogenesis. Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is perhaps the most important of pro-angiogenic cytokine because of its ability to regulate most of the steps in the angiogenic cascade. The main goal of this review article is to discuss the complex nature of the mode of action of VPF/VEGF on vascular endothelium. To this end, we conclude that more research needs to be done for completely understanding the VPF/VEGF biology with relation to angiogenesis.     

Identification of a peptide blocking vascular endothelial growth factor (VEGF)-mediated angiogenesis

Vascular endothelial growth factor (VEGF) binding to the kinase domain receptor (KDR/FLK1 or VEGFR-2) mediates vascularization and tumor-induced angiogenesis. Since there is evidence that KDR plays an important role in tumor angiogenesis, we sought to identify peptides able to block the VEGF-KDR interaction. A phage epitope library was screened by affinity for membrane-expressed KDR or for an anti-VEGF neutralizing monoclonal antibody. Both strategies led to the isolation of peptides binding KDR specifically, but those isolated by KDR binding tended to display lower reactivities. Of the synthetic peptides corresponding to selected clones tested to determine their inhibitory activity, ATWLPPR completely abolished VEGF binding to cell-displayed KDR. In vitro, this effect led to the inhibition of the VEGF-mediated proliferation of human vascular endothelial cells, in a dose-dependent and endothelial cell type-specific manner. Moreover, in vivo, ATWLPPR totally abolished VEGF-induced angiogenesis in a rabbit corneal model. Taken together, these data demonstrate that ATWLPPR is an effective antagonist of VEGF binding, and suggest that this peptide may be a potent inhibitor of tumor angiogenesis and metastasis.     

Fibulin-5 antagonizes vascular endothelial growth factor (VEGF) signaling and angiogenic sprouting by endothelial cells

Fibulin-5 (FBLN-5) is a widely expressed, integrin-binding extracellular matrix protein that mediates endothelial cell adhesion and scaffolds cells to elastic fibers. It is also a gene target of TGF-beta in fibroblasts and endothelial cells that regulates cell proliferation and motility in a context-specific manner. Whereas FBLN-5 expression is low in adult vasculature, its expression is high in developing and injured vasculature, implicating FBLN-5 in regulating angiogenesis and endothelial cell function. We show here that TGF-beta stimulates FBLN-5 expression in endothelial cells, and that this response was inhibited by coadministration of the proangiogenic factor, VEGF. FBLN-5 expression was downregulated significantly during endothelial cell tubulogenesis, implying that FBLN-5 expression antagonizes angiogenesis. Accordingly, FBLN-5 overexpression in or recombinant FBLN-5 treatment of endothelial cells abrogated their ability to undergo angiogenic sprouting, doing so by inhibiting endothelial cell proliferation and invasion through Matrigel matrices. Moreover, FBLN-5 antagonized VEGF signaling in endothelial cells, as well as enhanced their expression of the antiangiogenic factor, thrombospondin-1. Finally, the ability of FBLN-5 to antagonize angiogenic processes was determined to be independent of its integrin-binding RGD motif. Collectively, our findings establish FBLN-5 as a novel antagonist of angiogenesis and endothelial cell activities, and offer new insights into why tumorigenesis downregulates FBLN-5 expression.     

Manipulation of ovarian follicle development by injecting vascular endothelial growth factor (VEGF) gene

The genetic and molecular mechanisms that control the development of capillary blood vessels during follicular development are beginning to be elucidated. Ovarian follicles contain and produce angiogenic factors that may act alone or in concert to regulate thecal angiogenesis. These factors are ultimately controlled by endocrine, paracrine and autocrine regulation in the ovary. Our recent study indicated that vascular endothelial growth factor (VEGF) plays an important role in the thecal angiogenesis during follicular development. In this review, we focus on the vasculature and the expression of angiogenic factors during follicular development in a mammalian ovary.     

Elevated expression of vascular endothelial growth factor (VEGF) 120 in parthenogenetic porcine placentas

Most mammalian parthenogenetic embryos are unable to develop to term due to placental defects, potentially caused by decreased vasculogenesis and angiogenesis of the parthenogenetic placenta. Here we have compared the expression status of vascular endothelial growth factor (VEGF) and angiopoietin family members between normally developing and parthenogenetic porcine placentas. The result showed significantly reduced expression of these genes but elevated expression of VEGF 120 in the parthenogenetic porcine placenta (p < 0.05). We postulate that the abnormal expression levels of VEGF and angiopoietin family members and, especially, the elevated expression of VEGF 120 observed in parthenogenetic porcine placentas are related to the early miscarriage of parthenogenetic embryos in pigs.     

Vascular endothelial growth factor (VEGF) receptor II-derived peptides inhibit VEGF

Vascular endothelial growth factor (VEGF) directly stimulates endothelial cell proliferation and migration via tyrosine kinase receptors of the split kinase domain family. It mediates vascular growth and angiogenesis in the embryo but also in the adult in a variety of physiological and pathological conditions. The potential binding site of VEGF with its receptor was identified using cellulose-bound overlapping peptides of the extracytosolic part of the human vascular endothelial growth factor receptor II (VEGFR II). Thus, a peptide originating from the third globular domain of the VEGFR II comprising residues 247RTELNVGIDFNWEYP261 was revealed as contiguous sequence stretch, which bound 125I-VEGF165. A systematic replacement with L-amino acids within the peptide representing the putative VEGF-binding site on VEGFR II indicates Asp255 as the hydrophilic key residue for binding. The dimerized peptide (RTELNVGIDFNWEYPAS)2K inhibits VEGF165 binding with an IC50 of 0.5 microM on extracellular VEGFR II fragments and 30 microM on human umbilical vein cells. VEGF165-stimulated autophosphorylation of VEGFR II as well as proliferation and migration of microvascular endothelial cells was inhibited by the monomeric peptide RTELNVGIDFNWEYPASK at a half-maximal concentration of 3-10, 0.1, and 0.1 microM, respectively. We conclude that transduction of the VEGF165 signal can be interrupted with a peptide derived from the third Ig-like domain of VEGFR II by blockade of VEGF165 binding to its receptor.     

Overproduction of recombinant human VEGF (vascular endothelial growth factor) in Chinese hamster ovary cells

Vascular endothelial growth factors (VEGFs) are a family of proteins that mediate angiogenesis. VEGF165 is a VEGF-A isoform and has been extensively studied owing to its potential use in therapeutic angiogenesis. This study established Chinese hamster ovary (CHO) cells overexpressing recombinant human VEGF165 (rhVEGF165) protein. The production rate of the established CHO cells was over 80 mg/l of rhVEGF165 protein from a 7-day batch culture process using a 7.5-l bioreactor with a 5-l working volume and serum-free medium. The rhVEGF165 protein was purified to homogeneity from the culture supernatant using a two-step chromatographic procedure that resulted in a 48% recovery rate. The purified rhVEGF165 protein was a glycosylated homodimeric protein with a higher molecular weight (MW) than the protein expressed from insect cells, suggesting that the glycosylation of the rhVEGF165 protein in CHO cells differed from that in insect cells. The purified rhVEGF165 protein in this study was functionally active with a half-maximal effective concentration of 3.8 ng/ ml and specific activity of 2.5 x 105 U/mg.     

Polymorphism of the vascular endothelial growth factor gene (VEGF) and aerobic performance in athletes

The subject of this study is the frequency distribution of alleles of the vascular endothelial growth factor gene (VEGF; the G-634C polymorphism) in athletes (n = 670) and in a control group (n = 1073) and the relationships of genotypes with aerobic performance in rowers (n = 90). Genetic typing was performed using the analysis of restriction fragment length polymorphism. The frequency of the VEGF C allele in the group of endurance athletes (n = 294) was significantly higher than in the control group and increased together with increasing sports qualification. In addition, a correlation of the VEGF C allele with a high aerobic performance of athletes (according to data on the maximal power and maximal oxygen consumption) and with a substantial contribution to the energy supply of aerobic metabolism (according to the values of maximal lactate content) has been found. It is inferred that the G-634C polymorphism of the VEGF gene is associated with physical performance of athletes and plays a key role in sports selection.     

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.     

Expression of vascular endothelial growth factor (VEGF) and its cognate receptors in human pheochromocytomas

Pheochromocytomas are well-vascularized tumors, suggesting that a potent angiogenic factor may be involved in the mechanism of their formation. As vascular endothelial growth factor (VEGF) is a potent mitogen for vascular endothelial cells, here we have investigated the mRNA and protein expression of VEGF and the mRNA expression of its two receptors (Flt-1 and Flk-1/KDR) in pheochromocytomas tissue. An increase in VEGF mRNA (mainly isoforms VEGF(121) and VEGF(165)) and in VEGF protein expression were observed by semi-quantitative RT-PCR and Western blot, respectively, compared to normal adrenomedullary tissue. Flk-1/KDR, and Flt-1 levels of mRNA were also increased markedly in tumors and correlated with levels of VEGF mRNA. Therefore, we speculate that upregulation of VEGF expression and its receptors might be important in the pathogenesis of pheochromocytomas.     

Appearance of vascular endothelial growth factor (VEGF) in femoral head in the growing rat

In this study, we examined the appearance of vascular endothelial growth factor (VEGF) in the femoral head of the growing rat using an immunocytochemical technique. Our results showed VEGF-immunopositive cells existed in the inner region and peripheral region of the femoral head at each developmental stage. In the 19-day-old fetus, immunopositive mesenchymal cells were demonstrated in the peripheral region of the femoral head. At 1 to 10 days after birth, VEGF immunoreactivities were observed in the osteoblasts, osteoclasts, periosteum, perichondrium and cartilage matrix of the femur. At 15 days after birth, VEGF immunoreactive chondrocytes appeared in the apex area of the femoral head. In this stage, the femoral head is still constituted by chondrocytes and no apparent vascular formation has been observed. Thereafter, the immunopositive chondrocytes in the femoral head increased in number. The penetration of capillaries was recognized within the ligament of the femoral head at 60 days after birth. The results indicate that some chondrocytes in the femoral head produce VEGF before the beginning of ossification, and that VEGF may play an important role in the penetration of blood vessels into the femoral head from the ligament of the femoral head.     

A novel snake venom vascular endothelial growth factor (VEGF) predominantly induces vascular permeability through preferential signaling via VEGF receptor-1

Vascular endothelial growth factor (VEGF)/vascular permeability factor induces both angiogenesis and vascular permeability mainly through VEGF receptor (VEGFR)-2 activation. VEGF binds VEGFR-1 as well, but the importance of VEGFR-1 signaling in vascular permeability has been largely neglected. Here, we report the purification and characterization of a novel VEGF-like protein from Trimeresurus flavoviridis Habu snake venom. The Habu snake has a venom-specific VEGF-like molecule, T. flavoviridis snake venom VEGF (TfsvVEGF), in addition to VEGF-A. TfsvVEGF has almost 10-fold less mitotic activity than VEGF(165), a predominant isoform of human VEGF-A, but a similar effect on vascular permeability. TfsvVEGF bound VEGFR-1 and induced its autophosphorylation to almost the same extent as VEGF(165), but bound VEGFR-2 weakly and induced its autophosphorylation almost 10-fold less effectively than VEGF(165). This unique binding affinity for VEGFR-1 and VEGFR-2 leads to the vascular permeability-dominant activity of TfsvVEGF. These results suggest that Habu snakes have acquired a highly purposive molecule for a toxin, which enhances the toxicity in envenomation without inducing effective angiogenesis and the following regeneration of damaged tissues, taking advantage of the difference in signaling properties involving VEGFR-1 and VEGFR-2 between vascular permeability and angiogenesis. TfsvVEGF is thus a potent inducing factor selective for vascular permeability through preferential signaling via VEGFR-1. These data strongly indicate the importance of VEGFR-1 signaling in vascular permeability.     

Alternative splicing of vascular endothelial growth factor (VEGF)-R1 (FLT-1) pre-mRNA is important for the regulation of VEGF activity

Angiogenesis is essential for normal mammalian development and is controlled by the local balance of pro- and antiangiogenic factors. Here we describe a novel mouse cDNA sequence encoding sFLT-1 that is a potent antagonist to vascular endothelial growth factor (VEGF) and show for the first time its in vivo production. In situ hybridization and Northern blot analysis with probes specific for sFLT-1 or FLT-1 showed that the relative abundance of their mRNAs changed markedly in spongiotrophoblast cells in the placenta as gestation progressed. On day 11 of pregnancy, sFLT-1 mRNA was undetectable but FLT-1 readily apparent, and by day 17 sFLT-1 mRNA was abundant but FLT-1 barely detectable. sFLT-1 was identified in conditioned medium of cultured placenta from day 17 pregnant mice and likely to be present in the circulation, as there is a substantial increase of VEGF-binding activity in the serum from day 13 of pregnancy, which coincides with the abundant sFLT-1 expression in placenta. Expression of sFLT-1 was also observed in adult lung, kidney, liver, and uterus. These data suggest a novel mechanism of regulation of angiogenesis by alternative splicing of FLT-1 pre-mRNA. Treatment of pregnant mice with exogenous VEGF from day 9 to 17 of pregnancy, which alters the ratio of VEGF to sFLT-1, resulted in an increase in the number of resorption sites and fibrin deposition in the placenta of ongoing pregnancies. These findings have important implications for understanding placental function and may be relevant in a range of disease states.