Analysis of biological effects and signaling properties of Flt-1 (VEGFR-1) and KDR (VEGFR-2). A reassessment using novel receptor-specific vascular endothelial growth factor mutants

Endothelial cells express two related vascular endothelial growth factor (VEGF) receptor tyrosine kinases, KDR (kinase-insert domain containing receptor, or VEGFR-2) and Flt-1 (fms-like tyrosine kinase, or VEGFR-1). Although considerable experimental evidence links KDR activation to endothelial cell mitogenesis, there is still significant uncertainty concerning the role of individual VEGF receptors for other biological effects such as vascular permeability. VEGF mutants that bind to either KDR or Flt-1 with high selectivity were used to determine which of the two receptors serves to mediate different VEGF functions. In addition to mediating mitogenic signaling, selective KDR activation was sufficient for the activation of intracellular signaling pathways implicated in cell migration. KDR stimulation caused tyrosine phosphorylation of both phosphatidylinositol 3-kinase and phospholipase Cgamma in primary endothelial cells and stimulated cell migration. KDR-selective VEGF was also able to induce angiogenesis in the rat cornea to an extent indistinguishable from wild type VEGF. We also demonstrate that KDR, but not Flt-1, stimulation is responsible for the induction of vascular permeability by VEGF.     

Receptor chimeras indicate that the vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic activity of VEGFR-2 in endothelial cells

Vascular endothelial growth factor (VEGF) provokes angiogenesis in vivo and stimulates growth and differentiation of endothelial cells in vitro. Although VEGF receptor-1 (VEGFR-1) and VEGFR-2 are known to be high affinity receptors for VEGF, it is not clear which of the VEGFRs are responsible for the transmission of the diverse biological responses of VEGF. For this purpose we have constructed a chimeric receptor for VEGFR-1 (CTR) and VEGFR-2 (CKR) in which the extracellular domain of each receptor was replaced with the extracellular domain of human colony-stimulating factor-1 receptor (CSF-1R), and these receptors were expressed in pig aortic endothelial (PAE) cells. We show that CKR individually expressed in PAE cells is readily tyrosine-phosphorylated in vivo, autophosphorylated in vitro, and stimulates cell proliferation in a CSF-1-dependent manner. In contrast, CTR individually expressed in PAE cells showed no significant in vivo, in vitro tyrosine phosphorylation and cell growth in response to CSF-1 stimulation. The kinase activity of CKR was essential for its biological activity, since mutation of lysine 866 to arginine abolished its in vivo, in vitro tyrosine phosphorylation and mitogenic signals. Remarkably, activation of CTR repressed CKR-mediated mitogen-activate protein kinase activation and cell proliferation. Similar effects were observed for VEGFR-2 co-expressed with VEGFR-1. Collectively, these findings demonstrate that VEGFR-2 activation plays a positive role in angiogenesis by promoting endothelial cell proliferation. In contrast, activation of VEGFR-1 plays a stationary role in angiogenesis by antagonizing VEGFR-2 responses.     

Monoclonal antibodies to vascular endothelial growth factor-D block its interactions with both VEGF receptor-2 and VEGF receptor-3.

Vascular endothelial growth factor-D (VEGF-D), the most recently discovered mammalian member of the VEGF family, is an angiogenic protein that activates VEGF receptor-2 (VEGFR-2/Flk1/KDR) and VEGFR-3 (Flt4). These receptor tyrosine kinases, localized on vascular and lymphatic endothelial cells, signal for angiogenesis and lymphangiogenesis. VEGF-D consists of a central receptor-binding VEGF homology domain (VHD) and N-terminal and C-terminal propeptides that are cleaved from the VHD to generate a mature, bioactive form consisting of dimers of the VHD. Here we report characterization of mAbs raised to the VHD of human VEGF-D in order to generate VEGF-D antagonists. The mAbs bind the fully processed VHD with high affinity and also bind unprocessed VEGF-D. We demonstrate, using bioassays for the binding and cross-linking of VEGFR-2 and VEGFR-3 and biosensor analysis with immobilized receptors, that one of the mAbs, designated VD1, is able to compete potently with mature VEGF-D for binding to both VEGFR-2 and VEGFR-3 for binding to mature VEGF-D. This indicates that the binding epitopes on VEGF-D for these two receptors may be in close proximity. Furthermore, VD1 blocks the mitogenic response of human microvascular endothelial cells to VEGF-D. The anti-(VEGF-D) mAbs raised to the bioactive region of this growth factor will be powerful tools for analysis of the biological functions of VEGF-D.     

The biology of VEGF and its receptors

Vascular endothelial growth factor (VEGF) is a key regulator of physiological angiogenesis during embryogenesis, skeletal growth and reproductive functions. VEGF has also been implicated in pathological angiogenesis associated with tumors, intraocular neovascular disorders and other conditions. The biological effects of VEGF are mediated by two receptor tyrosine kinases (RTKs), VEGFR-1 and VEGFR-2, which differ considerably in signaling properties. Non-signaling co-receptors also modulate VEGF RTK signaling. Currently, several VEGF inhibitors are undergoing clinical testing in several malignancies. VEGF inhibition is also being tested as a strategy for the prevention of angiogenesis, vascular leakage and visual loss in age-related macular degeneration.     

VEGF and PlGF: two pleiotropic growth factors with distinct roles in development and homeostasis

Blood vessels are crucial for normal development and growth by providing oxygen and nutrients. As shown by genetic targeting studies in mice, zebrafish and Xenopus blood vessel formation (or angiogenesis) is a multistep process, which is highly dependent on angiogenic growth factors such as VEGF, the founding member of the VEGF family. VEGF binds to the tyrosine kinase receptors VEGFR-1 and VEGFR-2, and loss of VEGF or its receptors results in abnormal angiogenesis and lethality during development. In contrast, PlGF, another member of this family, binds only to VEGFR-1, and appears to be crucial exclusively for pathological angiogenesis in the adult. However, the expression of VEGFR-1 and VEGFR-2 on non-vascular cells suggests additional biological properties for these growth factors. Indeed, the VEGF family and its receptors determine development and homeostasis of many organs, including the respiratory, skeletal, hematopoietic, nervous, renal and reproductive system, independent of their vascular role. These new insights broaden the activity spectrum of these "angiogenic" growth factors, and may have therapeutic implications when using these growth factors for vascular and/or non-vascular purposes.     

Age-dependent expression of VEGF isoforms and receptors in the rabbit anterior cruciate ligament

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF. Those isoforms differ in biochemical and biological properties, and it has been reported that their expression patterns are tissue and age specific as well. We investigated the expression levels of VEGF isoforms (VEGF121, VEGF165, VEGF183, VEGF189) and its receptors (VEGFR-1, flt-1 and VEGFR-2, flk-1/KDR) in the anterior cruciate ligament (ACL) of 2- to 3-week-, 2-month-, and 18-month-old New Zealand White rabbits using Sybr green Real-Time RT-PCR. VEGF isoforms and both receptors were expressed in the ACL at all investigated ages. VEGF121 was found to be the most abundant isoform at the ages under investigation, followed by VEGF165, VEGF189 and VEGF183. All isoforms showed decreased expression levels with age, however the larger membrane bound isoforms, VEGF183 and VEGF189, showed the most striking age-associated decrease in expression level. VEGFR-1 expression levels increased with age, while the expression level of VEGFR-2 expression was highest at 2-3 weeks and was significantly lower at 2 and 18 months of age. Distinct age-associated differences in the expression level of VEGF isoforms as well as their receptors suggest differential physiological functions during development, maturation and ageing of the ACL.     

Differential regulation of vascular endothelial growth factor receptors (VEGFR) revealed by RNA interference: interactions of VEGFR-1 and VEGFR-2 in endothelial cell signaling

Vascular endothelial growth factor (VEGF) plays a central role in vascular homeostasis. VEGF receptors (VEGFRs) include several subtypes that may have a differential role in endothelial signal transduction, but interactions among these receptors are incompletely understood. In these studies, we designed small interfering RNA (siRNA) duplexes that targeted specific VEGFR subtypes in bovine aortic endothelial cells (BAEC). siRNA-mediated downregulation of VEGFR-2 by its cognate siRNA resulted in a significant attenuation of VEGF-mediated signaling. Compared to control siRNA-treated cells, VEGFR-2 siRNA markedly inhibited VEGF-mediated activation of PI3K/Akt/GSK3-beta as well as MAP kinase and PKC pathways. VEGFR-2 siRNA also blocked VEGF-stimulated phosphorylation and dephosphorylation of endothelial nitric oxide synthase (eNOS) at Ser(1179) and Ser(116), respectively. VEGFR-2-specific siRNA had no effect on the abundance of VEGFR-1 protein. By contrast, VEGFR-1-specific siRNA markedly not only downregulated the abundance of VEGFR-1 but also significantly reduced VEGFR-2 protein and mRNA abundance. VEGFR-1 siRNA had no effect on the stability of VEGFR-2 protein or mRNA. However, VEGFR-1 siRNA significantly inhibited VEGFR-2 promoter activity, as determined in luciferase assays using VEGFR-2 promoter fusion constructs in transfected BAEC. Deletion of either the 5' E box or the 3' E box and the GATA element in the VEGFR-2 promoter completely abolished the inhibition of VEGFR-2 promoter activity elicited by VEGFR-1 siRNA. Taken together, our data suggest that VEGFR-1 receptor is a critical determinant of VEGFR-2 abundance, while VEGFR-2 is the key receptor directly responsible for endothelial cell signaling stimulated by VEGF.     

Signaling through the vascular endothelial growth factor receptor VEGFR-2 protects hippocampal neurons from mitochondrial dysfunction and oxidative stress

Vascular endothelial growth factor VEGF (VEGF-A or VEGF₁₆₅) is a potent angiogenic factor that also signals neuroprotection through activation of its cognate receptor VEGFR-2. In this capacity, VEGF signaling can rescue neurons from the damage induced by stressful stimuli many of which elicit oxidative stress. However, the regulatory role that VEGFR-2 plays in providing neuroprotection remains elusive. Therefore, we investigated the effects of VEGFR-2 inhibition on primary cultures of mature hippocampal neurons undergoing nutritional stress. We found that neurons cultured under nutritional stress had increased expression of VEGF and its receptors, VEGFR-1, VEGFR-2, and NP-1, as well as enhanced levels of VEGFR-2 phosphorylation. These neurons also showed increased activation of the prosurvival pathways for MEK/ERK1/2 and PI3K/Akt, enhanced phosphorylation (inactivation) of the proapoptotic BAD, and higher levels of the antiapoptotic protein Bcl-xL, all of which were augmented by treatments with exogenous VEGF and blocked by VEGFR-2 inhibition. The blockade of VEGFR-2 function also elicited a cytotoxicity that was accompanied by caspase-3 activation, induction of hemeoxygenase-1 (HO-1), oxidative stress, and a collapse in the mitochondrial membrane potential (ΔΨ_m). Knockdown of VEGFR-2 by siRNA generated a similar pattern of redox change and mitochondrial impairment. Pretreatments with VEGF, VEGF-B, or the antioxidant N-acetylcysteine (NAC) rescued SU1498 or siRNA-treated neurons from the mitochondrial dysfunction and oxidative stress induced by VEGFR-2 inhibition in a timely fashion. These findings suggested that VEGF or VEGF-B can provide neuroprotection by signaling through an alternate VEGF receptor. Together, our findings suggest that VEGF signaling through VEGFR-2 plays a critical regulatory role in protecting stressed hippocampal neurons from the damaging effects of an oxidative insult. These findings also implicate VEGFR-1 or NP-1 as compensatory receptors that mediate neuroprotection when VEGFR-2 function is blocked.     

Low molecular weight fucoidan increases VEGF165-induced endothelial cell migration by enhancing VEGF165 binding to VEGFR-2 and NRP1

Therapeutic induction of angiogenesis is a potential treatment for chronic ischemia. Heparan sulfate proteoglycans are known to play an important role by their interactions with proangiogenic growth factors such as vascular endothelial growth factor (VEGF). Low molecular weight fucoidan (LMWF), a sulfated polysaccharide from brown seaweeds that mimic some biological activities of heparin, has been shown recently to promote revascularization in rat critical hindlimb ischemia. In this report, we first used cultured human endothelial cells (ECs) to investigate the possible ability of LMWF to enhance the actions of VEGF(165). Data showed that LMWF greatly enhances EC tube formation in growth factor reduced matrigel. LMWF is a strong enhancer of VEGF(165)-induced EC chemotaxis, but not proliferation. In addition, LMWF has no effect on VEGF(121)-induced EC migration, a VEGF isoform that does not bind to heparan sulfate proteoglycans. Then, with binding studies using (125)I-VEGF(165), we observed that LMWF enhances the binding of VEGF(165) to recombinant VEGFR-2 and Neuropilin-1 (NRP1), but not to VEGFR-1. Surface plasmon resonance analysis showed that LMWF binds with high affinity to VEGF(165) (1.2 nm) and its receptors (5-20 nm), but not to VEGF(121). Pre-injection of LMWF on immobilized receptors shows that VEGF(165) has the highest affinity for VEGFR-2 and NRP1, as compared with VEGFR-1. Overall, the effects of LMWF were much more pronounced than those of LMW heparin. These findings suggested an efficient mechanism of action of LMWF by promoting VEGF(165) binding to VEGFR-2 and NRP1 on ECs that could help in stimulating therapeutic revascularization.     

Vascular endothelial growth factor receptor-1 and neuropilin-2 form complexes

The products of the neuropilin-1 (Np-1) and neuropilin-2 (Np-2) genes are receptors for factors belonging to the class 3 semaphorin family and participate in the guidance of growing axons to their targets. In the presence of heparin-like molecules, both receptors also function as receptors for the heparin-binding 165-amino acid isoform of vascular endothelial growth factor (VEGF(165)). Both receptors are unable to bind to the 121-amino acid isoform of vascular endothelial growth factor (VEGF(121)), which lacks a heparin-binding domain. Interestingly, complexes corresponding in size to (125)I-VEGF(121).neuropilin complexes are formed when (125)I-VEGF(121) is bound and cross-linked to porcine aortic endothelial cells co-expressing VEGFR-1 and either Np-1 or Np-2. These complexes do not seem to represent complexes of (125)I-VEGF(121) with a truncated form of VEGFR-1, presumably formed as a result of the presence of Np-1 or Np-2 in the cells, because such truncated forms could not be detected with anti-VEGFR-1 antibodies. Antibodies directed against VEGFR-1 co-immunoprecipitated the (125)I-VEGF(121).Np-2 sized cross-linked complex along with (125)I-VEGF(121).VEGFR-1 complexes from cells expressing both VEGFR-1 and Np-2 but not from control cells, indicating that VEGFR-1 and Np-2 associate with each other. To perform the reciprocal experiment we have expressed in porcine aortic endothelial cells a Np-2 receptor containing an in-frame myc epitope at the C terminus. Surprisingly, the myc-tagged Np-2 receptor lost most of its VEGF(165) binding capacity but not its semaphorin-3F binding ability. Nevertheless, when Np-2myc was co-expressed in cells with VEGFR-1, it partially regained its VEGF(165) binding ability. Antibodies directed against the myc epitope co-immunoprecipitated (125)I-VEGF(165).Np-2myc and (125)I- VEGF(165).VEGFR-1 complexes from cells co-expressing VEGFR-1 and Np-2myc, indicating again that VEGFR-1 associates with Np-2. Our experiments therefore indicate that Np-2, and possibly also Np-1, associate with VEGFR-1 and that such complexes may be part of a cell membrane-associated signaling complex.     

Differential expression of VEGF isoforms and receptors in knee joint menisci under systemic hypoxia

Vascular endothelial growth factor (VEGF) gene gives rise to several distinct isoforms of VEGF, which differ in their expression patterns as well as their biochemical and biological properties. We examined the expression levels of VEGF isoforms and their receptors in the medial and lateral meniscus of rabbits under normal physiologic conditions as well their expression levels after 8 and 24 h of systemic normobaric hypoxia (13%). VEGF121 is the most abundant VEGF isoform in the medial and lateral meniscus, followed by VEGF165, VEGF189, and VEGF183. While the soluble VEGF121 and VEGF165 are only upregulated at 8 h of hypoxia, the membrane-bound VEGF183 and VEGF189 are further increased at 24 h. VEGFR-2 is expressed at a much higher level than VEGFR-1 under normal conditions, and both receptors are upregulated under hypoxia. Differential expression levels under normoxia as well as a differential response to hypoxia may indicate different functions of VEGF isoforms in the meniscus.     

Activation of VEGFR-2 signaling in response to moderate dose of ultraviolet B promotes survival of normal human keratinocytes

Mounting evidence indicates that signaling via VEGF receptors (VEGFRs) extends beyond blood vessel formation. Recently, VEGFRs are also found to be constitutively expressed in keratinocytes and epidermal appendages. Here, we show that the expression of VEGFRs (including VEGFR-1, VEGFR-2, and NRP-1) was significantly enhanced by moderate dose of ultraviolet B (UVB) in normal human keratinocytes and epidermis. The elevated expression of VEGFRs by UVB was independent of autocrine stimulation by their natural ligand, VEGF, but mainly mediated through hypoxia and oxidative stress. Moderate dose UVB also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, this effect was again VEGF independent. Both α and δ isoforms of protein kinase C (PKC) were required for UVB-induced phosphorylation of VEGFR-1, but only the δ isoform was required for VEGFR-2 phosphorylation. The phosphorylation of VEGFRs or isoforms of PKC was completely inhibited by PP2, a specific inhibitor for Src family kinases (SFKs), indicating that SFKs are upstream of PKC and VEGFRs. Moderate dose UVB-induced VEGF exerted an anti-apoptotic effect for keratinocytes, whereas high dose UVB-induced VEGF played as an inflammatory factor. Of note, neutralization of VEGFR-2 but not VEGFR-1 exacerbated UVB-induced cell death and reduced survival of keratinocytes. Furthermore, VEGFR-2 neutralization inhibited the activation of ERK1/2 and Akt by UVB, suggesting that VEGFR-2 signaling was involved in the pro-survival mechanism via ERK1/2 and PI3-K/Akt pathway. Taken together, we demonstrate for the first time that VEGFR-2 signaling is activated and promotes survival of keratinocytes under moderate dose of UVB irradiation.     

Parallel solid-phase synthesis of a small library of linear and hydrocarbon-bridged analogues of VEGF(81-91): potential biological tools for studying the VEGF/VEGFR-1 interaction

The design, synthesis and binding affinity for VEGFR-1 receptors of a small library of linear and cyclic analogues of the VEGF(81-91) fragment are described. Cyclic 11- and 10-mer peptide derivatives were prepared using parallel solid-phase protocols. The formation of hydrocarbon alkene-bridged cyclic peptides was achieved through optimized ring-closing metathesis reactions from linear derivatives with conveniently located allylGly residues. Alkane-bridged analogues were successfully obtained by ulterior on-resin hydrogenation. Binding assays showed that some of these compounds were able to compete with labeled VEGF for interaction with the VEGFR-1 receptor. Several peptide derivatives, 2, 7 and 8, showed modest but significant binding affinity, indicating that the designed peptide could mimic the VEGF(81-91) fragment and therefore disrupt the VEGF/VEGFR-1 interaction. This fact opens the way for using these peptides as the starting point for biological/pharmacological tools to deeply investigate this protein-protein system.     

Immunolocalization and expression of vascular endothelial growth factor receptors (VEGFRs) and neuropilins (NRPs) on keratinocytes in human epidermis

Vascular endothelial growth factor (VEGF) plays an important role in normal and pathological angiogenesis. VEGF receptors (VEGFRs, including VEGFR-1, VEGFR-2, and VEGFR-3) and neuropilins (NRPs, including NRP-1 and NRP-2) are high-affinity receptors for VEGF and are typically considered to be specific for endothelial cells. Here we showed expression of VEGFRs and NRPs on cultured epidermal keratinocytes at both mRNA and protein levels. We further localized these receptors by immunofluorescence (IF) staining in the epidermis of surgical skin specimens. We found positive staining for VEGFRs and NRPs in all layers of the epidermis except for the stratum corneum. VEGFR-1 and VEGFR-2 are primarily expressed on the cytoplasmic membrane of basal cells and the adjacent spinosum keratinocytes. All layers of the epidermis except for the horny cell layer demonstrated a uniform pattern of VEGFR-3, NRP-1, and NRP-2. Sections staining for NRP-1 and NRP-2 also showed diffuse intense fluorescence and were localized to the cell membrane and cytoplasm of keratinocytes. In another panel of experiments, keratinocytes were treated with different concentrations of VEGF, with or without VEGFR-2 neutralizing antibody in culture. VEGF enhanced the proliferation and migration of keratinocytes, and these effects were partially inhibited by pretreatment with VEGFR-2 neutralizing antibody. Adhesion of keratinocytes to type IV collagen-coated culture plates was decreased by VEGF treatment, but this reduction could be completely reversed by pretreatment with VEGFR-2 neutralizing antibody. Taken together, our results suggest that the expression of VEGFRs and NRPs on keratinocytes may constitute important regulators for its activity and may possibly be responsible for the autocrine signaling in the epidermis.     

Bevacizumab (Avastin), a humanized anti-VEGF monoclonal antibody for cancer therapy

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in vivo. The tyrosine kinases Flt-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2) are high affinity VEGF receptors. VEGF plays an essential role in developmental angiogenesis and is important also for reproductive and bone angiogenesis. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice. Clinical trials with VEGF inhibitors in a variety of malignancies are ongoing. Recently, a humanized anti-VEGF monoclonal antibody (bevacizumab; Avastin) has been approved by the FDA as a first-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration.     

Neuropilin-1-VEGFR-2 complexing requires the PDZ-binding domain of neuropilin-1

Vascular endothelial growth factor (VEGF) acts as a hierarchically high switch of the angiogenic cascade by interacting with its high affinity VEGF receptors and with neuropilin co-receptors. VEGF(165) binds to both Neuropilin-1 (NP-1) and VEGFR-2, and it is believed that ligand binding forms an extracellular bridge between both molecules. This leads to complex formation, thereby enhancing VEGFR-2 phosphorylation and subsequent signaling. We found that inhibition of VEGF receptor (VEGFR) phosphorylation reduced complex formation between NP-1 and VEGFR-2, suggesting a functional role of the cytoplasmic domain of VEGFR-2 for complex formation. Correspondingly, deleting the PDZ-binding domain of NP-1 decreased complex formation, indicating that extracellular VEGF(165) binding is not sufficient for VEGFR-2-NP-1 interaction. Synectin is an NP-1 PDZ-binding domain-interacting molecule. Experiments in Synectin-deficient endothelial cells revealed reduced VEGFR-2-NP-1 complex formation, suggesting a role for Synectin in VEGFR-2-NP-1 signaling. Taken together, the experiments have identified a novel mechanism of NP-1 interaction with VEGFR-2, which involves the cytoplasmic domain of NP-1.     

Vascular endothelial growth factor is involved in neoangiogenesis in Hirudo medicinalis (Annelida,Hirudinea)

Vascular endothelial growth factor (VEGF) is fundamental in vertebrates for correct development of blood vessels. However, there are only few data about the presence of VEGF in invertebrates. In this study the role of VEGF in neovessel formation is investigated in Hirudo medicinalis. The leech is able to respond to administration of human VEGF by formation of new vessels. The response of H. medicinalis to this growth factor is explained by the presence of two specific VEGF-like receptors (Flt-1/VEGFR-1 and Flk-1/VEGFR-2) as demonstrated by immunohistochemistry and biochemical analysis. The VEGF-like produced by this annelid following surgical stimulation determines not only blood vessel formation, proliferation of vascular endothelial cells but also an increase of cytoplasmic calcium levels. The administration of specific VEGF receptor antibodies can inhibit angiogenesis in leeches previously stimulated with VEGF.