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Characterization of the Extracellular Domain in Vascular Endothelial Growth Factor Receptor-1 (Flt-1 Tyrosine Kinase)
Abstract
Flt-1 tyrosine kinase, vascular endothelial growth factor (VEGF) receptor-1, binds VEGF and a new VEGF-related ligand, placenta growth factor, but KDR/Flk-1 (VEGF receptor-2) binds only VEGF. To characterize the functional regions in the Flt-1 extracellular domain such as the ligand binding region and the dimer formation of the receptor, we constructed a series of mutants of the Flt-1 extracellular domain as soluble forms in a baculovirus system. We found that a region carrying the N-terminal 1st to 3rd immunoglobulin (Ig)-like domains of Flt-1 binds both ligands with high affinity. However, for dimer formation of soluble Flt-1, a region further downstream in the Flt-1 extracellular domain was required. Mutant Flt-1 receptors expressed in COS cells confirmed the requirement of the 4th to 7th Ig region for the activation of Flt-1 tyrosine kinase. Soluble Flt-1 carrying the N-terminal 1st to 3rd Ig region suppressed VEGF-dependent endothelial proliferation in vitro to the same level as the larger forms of soluble Flt-1, suggesting that the binding of one soluble Flt-1 molecule to one subunit of the VEGF homodimer may be sufficient to block the VEGF activity.
... Proteins were separated and transferred to polyvinylidene fluoride membranes by gel electrophoresis and electroblotting, respectively. The following primary antibodies were used: anti-human FLT1 N-terminal region (1:1000) [22] and anti-β-actin (1:500; Cell Signaling Technology, Beverly, MA, USA). Bands were visualized using an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA). ...
... To construct expression vectors for sFLT1-i13 or green fluorescent protein (GFP), DNA fragments encoding these molecules were digested using pVL-6N-Flt [22] and pEGFP-N1 (Clontech, Mountain View, CA, USA), respectively, then cloned into a bovine papilloma virus-based plasmid vector pBCMGSneo [23]. To establish stable sFLT1-i13-or GFP-expressing JEG-3 cells, each vector was transfected into JEG-3 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ...
... Choriocarcinoma, a malignant trophoblastic cancer, is known to be highly pro-angiogenic [4]. It has previously been reported that the protein expression level of VEGF, a pro-angiogenic factor, is higher in choriocarcinoma cell lines than in normal trophoblastic cell lines [22]. Therefore, we first measured the mRNA expression level of VEGF-A in human primary trophoblasts, immortalized human trophoblasts (HTR-8/SVneo), and choriocarcinoma cell lines (BeWo, JAR and JEG-3) by qRT-PCR analysis. ...
Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells.
Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model.
Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells.
Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo . We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.
... The following primary antibodies were used: anti-human FLT1 N-terminal region (1:1000) [22] and anti-βactin (1:500; Cell Signaling Technology, Beverly, MA, USA). Bands were visualized using an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA). ...
... To construct expression vectors for sFLT1-i13 or green uorescent protein (GFP), DNA fragments encoding these molecules were digested using pVL-6N-Flt [22] and pEGFP-N1 (Clontech, Mountain View, CA, USA), respectively, then cloned into a bovine papilloma virus-based plasmid vector pBCMGSneo [23]. To establish stable sFLT1-i13-or GFP-expressing JEG-3 cells, each vector was transfected into JEG-3 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ...
... Choriocarcinoma, a malignant trophoblastic cancer, is known to be highly pro-angiogenic [4]. It has previously been reported that the protein expression level of VEGF, a pro-angiogenic factor, is higher in choriocarcinoma cell lines than in normal trophoblastic cell lines [22]. Therefore, we rst measured the mRNA expression level of VEGF-A in human primary trophoblasts, immortalized human trophoblasts (HTR-8/SVneo), and choriocarcinoma cell lines (BeWo, JAR and JEG-3) by qRT-PCR analysis. ...
Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells.
Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulphite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model.
Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells.
Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.
... Proteins were separated and transferred to polyvinylidene fluoride membranes by gel electrophoresis and electroblotting, respectively. The following primary antibodies were used: anti-human FLT1 N-terminal region (1:1000) [22] and anti-β-actin (1:500; Cell Signaling Technology, Beverly, MA, USA). Bands were visualized using an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA). ...
... To construct expression vectors for sFLT1-i13 or green fluorescent protein (GFP), DNA fragments encoding these molecules were digested using pVL-6N-Flt [22] and pEGFP-N1 (Clontech, Mountain View, CA, USA), respectively, then cloned into a bovine papilloma virus-based plasmid vector pBCMGSneo [23]. To establish stable sFLT1-i13-or GFP-expressing JEG-3 cells, each vector was transfected into JEG-3 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ...
... Choriocarcinoma, a malignant trophoblastic cancer, is known to be highly pro-angiogenic [4]. It has previously been reported that the protein expression level of VEGF, a pro-angiogenic factor, is higher in choriocarcinoma cell lines than in normal trophoblastic cell lines [22]. Therefore, we first measured the mRNA expression level of VEGF-A in human primary trophoblasts, immortalized human trophoblasts (HTR-8/SVneo), and choriocarcinoma cell lines (BeWo, JAR and JEG-3) by qRT-PCR analysis. ...
Background: Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells.
Methods: qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model.
Results: Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells.
Conclusions: Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo . We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.
... Proteins were separated and transferred to polyvinylidene fluoride membranes by gel electrophoresis and electroblotting, respectively. The following primary antibodies were used: anti-human FLT1 N-terminal region (1:1000) [22] and anti-β-actin (1:500; Cell Signaling Technology, Beverly, MA, USA). Bands were visualized using an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA). ...
... To construct expression vectors for sFLT1-i13 or green fluorescent protein (GFP), DNA fragments encoding these molecules were digested using pVL-6 N-Flt [22] and pEGFP-N1 (Clontech, Mountain View, CA, USA), respectively, then cloned into a bovine papilloma virus-based plasmid vector pBCMGSneo [23]. To establish stable sFLT1-i13-or GFPexpressing JEG-3 cells, each vector was transfected into JEG-3 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. ...
... Choriocarcinoma, a malignant trophoblastic cancer, is known to be highly pro-angiogenic [4]. It has previously been reported that the protein expression level of VEGF, a pro-angiogenic factor, is higher in choriocarcinoma cell lines than in normal trophoblastic cell lines [22]. Therefore, we first measured the mRNA expression level of VEGF-A in human primary trophoblasts, immortalized human trophoblasts (HTR-8/SVneo), and choriocarcinoma cell lines (BeWo, JAR and JEG-3) by qRT-PCR analysis. ...
Background:
Soluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells.
Methods:
qRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2'-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model.
Results:
Expression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells.
Conclusions:
Inhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.
... It has been approved as a third-line treatment for patients with advanced non-small-cell lung cancer (NSCLC) (28). Based on a preclinical study, anlotinib showed high selectivity for VEGF family members, especially VEGFR2 and VEGFR3, with IC50 values of 0.2 and 0.7 nmol/L, showing high selectivity and inhibitory potency to sunitinib (29). A phase 2 clinical trial involving 166 patients to evaluate anlotinib in recurrent metastatic soft tissue sarcomas found that the overall 12-week PFS and ORR rates were 68% and 13%, respectively, with easily controllable adverse events (30). ...
... The FLT1 gene encodes VEGFR1 and is a member of the VEGFR family. The point mutation c.542G>A was found in FLT1's Ig-like domain 2, which is a high-affinity ligand-binding region (29). Some studies have found that soluble FLT1 expression is associated with poor clinical outcomes and may be a biomarker of intrinsic resistance to antiangiogenic therapies that selectively inhibit the VEGFR-2 signaling pathway (39). ...
Angiosarcoma (AS) is a rare, clinically aggressive tumor with limited treatment options and a poor prognosis. Mutations involving the angiogenesis-related genesTP53, PTPRB, PLCG1, KDR as well as FLT4 amplification have been observed in AS. There is a potential therapeutic value of inhibition of the VEGF pathway against angiosarcoma. Our case first described a patient with two sites of cutaneous angiosarcomas (cASs) that responded differently to anlotinib. And genetic analysis revealed that those two sites had different FLT4 variants, suggesting that FLT4 amplification could be the cause of anlotinib non-response.
... The reason for this difference is not clear, but might be at least partly due to the use of heparin-beads at one side in our assay, which efficiently binds various sites of sFLT1. We previously showed that an artificially shortened sFLT1 peptide, which contains the first to third Ig domains of FLT1, about half of regular sFLT1, still efficiently bound with heparin-bead columns 26 . Thus, partly degraded sFLT1 peptide as well as an intact sFLT1 in the serum could be detectable in this assay. ...
... For a standard sFLT1 assay, we used 7N FLT1 protein (1-7 Ig domain- www.nature.com/scientificreports/ containing soluble FLT1), which was expressed in SF9-insect cells with a Baculo-virus vector and purified with a heparin-beads column and gel-filtration column 26 . 7N sFLT1 has a peptide 750 amino acids long, similar to 6 N i13 (688 amino acids) and e15a (734 amino acids) sFLT1. ...
In normal pregnancy, the soluble form of FMS-like tyrosine kinase-1 (sFLT1)/ vascular endothelial growth factor receptor-1 (sVEGFR-1), a VEGF-trapping protein, is expressed in trophoblasts of the placenta, suggesting that it plays an important role in the physiological barrier between fetal and maternal angiogenesis, when stimulated with VEGF-A. In pathological conditions such as preeclampsia (PE), sFLT1 protein is abnormally overexpressed in trophoblasts and secreted into the serum, which could cause hypertension and proteinuria on the maternal side and growth retardation on the fetal side. Detection of an abnormal increase in serum sFLT1 during the early to middle stages of PE is essential for proper initiation of medical care. To carry out this screening for sFLT1, we developed an easier and relatively low-cost sandwich-type ELISA method using a single mixture of human serum sample with an anti-FLT1 antibody and heparin-beads, namely heparin-beads-coupled ELISA (HB-ELISA). This method takes only about 2 h, and the sFLT1 values were similar levels with commercially available recent ELISA kits: the serum sFLT1 protein was approximately 4.3-fold increased in severe PE compared with those in normal pregnancy.
... The region spanning from the second to third Ig regions of M-CSFR/ PDGFRs is the ligand-binding site. Similarly, the same regions in VEGFRs contain the binding regions for VEGF and VEGF-family members (Keyt et al. 1996;Tanaka et al. 1997;Shinkai et al. 1998). For the ligand-dependent activation of VEGFRs, Yang et al. (2010) recently reported that a direct interaction between the extracellular domains of two receptors is required. ...
... Shorter forms of sFlt-1 that contain only the first three, four, or five Ig domains still maintain a high affinity for VEGF (Tanaka et al. 1997). In contrast, short peptides of VEGFR2, with only the first four or five Ig domains, have a lower affinity for VEGF, 10-to 50-fold lower than fulllength VEGFR2 (Shinkai et al. 1998). ...
Vascular endothelial growth factor receptors (VEGFRs) in vertebrates play essential roles in the regulation of angiogenesis and lymphangiogenesis. VEGFRs belong to the receptor-type tyrosine kinase (RTK) supergene family. They consist of a ligand-binding region with seven immunoglobulin (7 Ig) -like domains, a trans-membrane (TM) domain, and a tyrosine kinase (TK) domain with a long kinase insert (KI) (also known as a type-V RTK). Structurally, VEGFRs are distantly related to the members of the M-colony stimulating factor receptor/platelet-derived growth factor receptor (CSFR)/(PDGFR) family, which have five immunoglobulin (5 Ig)-like domains. However, signal transduction in VEGFRs significantly differs from that in M-CSFR/PDGFRs. VEGFR2, the major signal transducer for angiogenesis, preferentially uses the phospholipase Cγ-protein kinase C (PLC-γ-PKC)-MAPK pathway, whereas M-CSFR/PDGFRs use the PI3 kinase-Ras-MAPK pathway for cell proliferation. In phylogenetic development, the VEGFR-like receptor in nonvertebrates appears to be the ancestor of the 7 Ig- and 5 Ig-RTK families because most nonvertebrates have only a single 7 Ig-RTK gene. In mammals, VEGFRs are deeply involved in pathological angiogenesis, including cancer and inflammation. Thus, an efficient inhibitor targeting VEGFRs could be useful in suppressing various diseases.
... Preparation of whole cell lysates and nuclear extracts, gel electrophoresis and electroblotting were performed as previously reported (Sasagawa et al., 2018). The following primary antibodies were used: anti-HIF-1a (1:500, #3716; Cell Signaling Technology, Beverly, MA, USA), anti-HIF-2a (1:1000, NB100-122; Novus Biologicals, Littleton, CO, USA), anti-HIF-1b (1:2000, NB100-110; Novus Biologicals), antib-actin (1:1000, #4967; Cell Signaling Technology), anti-TATA binding protein (1:200, sc-421; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-human FLT1 N-terminal region (rabbit polyclonal) antibody (1:1000) (Tanaka et al., 1997;Sasagawa et al., 2018;Sasagawa et al., 2020). ...
Placental hypoxia and increased levels of maternal blood anti-angiogenic protein, soluble fms-like tyrosine kinase-1 (sFLT1), are associated with the pathogenesis of preeclampsia. We have demonstrated that hypoxia-inducible factor (HIF)-2α mediates the upregulation of the hypoxia-induced FLT1 gene in trophoblasts and their cell lines. Here, we investigated the involvement of HIF-1β, which acts as a dimerisation partner for HIF-α, in the upregulation of the FLT1 gene via hypoxia. We confirmed the interactions between HIF-1β and HIF-2α in the nuclei of BeWo, JAR and JEG-3 cells under hypoxia via co-immunoprecipitation. We found that hypoxia-induced upregulation of the FLT1 gene in BeWo cells and secretion of sFLT1 in human primary trophoblasts were significantly reduced by siRNAs targeting HIF-1β. Moreover, the upregulation of the FLT1 gene in BeWo cells induced by dimethyloxalylglycine (DMOG) was also inhibited by silencing either HIF-2α or HIF-1β mRNA. It was recently shown that DNA demethylation increases both basal and hypoxia-induced expression levels of the FLT1 gene in three trophoblast-derived cell lines. In the demethylated BeWo cells, siRNAs targeting HIF-2α and HIF-1β suppressed the further increase in the expression levels of the FLT1 gene due to hypoxia or treatment with DMOG. However, luciferase reporter assays and bisulfite sequencing revealed that a hypoxia response element (–966 to –962) of the FLT1 gene is not involved in hypoxia or DMOG-induced upregulation of the FLT1 gene. These findings suggest that HIF-1β is essential for the elevated production of sFLT1 in the hypoxic trophoblasts and that the HIF-2α/HIF-1β complex may be a crucial therapeutic target for preeclampsia.
... The main difference with VEGFR3 is within the extracellular domain, by replacement of the fifth Ig-like loop by a disulfide bridge that keeps the proteolytically cleaved N-terminal part of the extracellular domain connected with the remainder of the molecule [190]. VEGFR1 and VEGFR2 ligands bind to the extracellular region to the second and third Ig-like domains and show symmetrical 2∶2 complex structure ( Figure 4B) [186,191,192]. The fourth Ig-like domain appears essential for VEGFR dimerization. ...
Simple Summary
Receptor tyrosine kinases (RTKs) have long been demonstrated to play key roles in melanoma development. RTK activation requires dimerization and intracellular tyrosine trans-phosphorylation leading to downstream signaling pathways activation. As RTKs show different structures, mechanism of activation could differ. In this review, we will discuss the structure and specific mechanism of activation of each RTK, and its alteration associated with stage of the disease. Additionally, we summarize the effect of RTK inhibitors tested in preclinical and clinical melanoma studies indicating the reason, the reported results, and the rational approaches for combination strategies based on RTK inhibition in melanoma.
Abstract
MAPK (mitogen activated protein kinase) and PI3K/AKT (Phosphatidylinositol-3-Kinase and Protein Kinase B) pathways play a key role in melanoma progression and metastasis that are regulated by receptor tyrosine kinases (RTKs). Although RTKs are mutated in a small percentage of melanomas, several receptors were found up regulated/altered in various stages of melanoma initiation, progression, or metastasis. Targeting RTKs remains a significant challenge in melanoma, due to their variable expression across different melanoma stages of progression and among melanoma subtypes that consequently affect response to treatment and disease progression. In this review, we discuss in details the activation mechanism of several key RTKs: type III: c-KIT (mast/stem cell growth factor receptor); type I: EGFR (Epidermal growth factor receptor); type VIII: HGFR (hepatocyte growth factor receptor); type V: VEGFR (Vascular endothelial growth factor), structure variants, the function of their structural domains, and their alteration and its association with melanoma initiation and progression. Furthermore, several RTK inhibitors targeting the same receptor were tested alone or in combination with other therapies, yielding variable responses among different melanoma groups. Here, we classified RTK inhibitors by families and summarized all tested drugs in melanoma indicating the rationale behind the use of these drugs in each melanoma subgroups from preclinical studies to clinical trials with a specific focus on their purpose of treatment, resulted effect, and outcomes.
... VEGF-A commonly known as VEGF binds to immunoglobulin domains 2 and 3 found on extracellular ligand-binding domains of VEGFR-1 and VEGFR-2 [72,73]. VEGF-C binds and activates VEGFR-2 and VEGFR-3 [74]. ...
Purpose of review:
To provide insight on the imbalance of angiogenic and lymphangiogenic factors in pre-eclampsia, as well as highlight polymorphism in genes related to angiogenesis and lymphangiogenesis.
Recent findings:
The pregnancy-specific disorder pre-eclampsia is diagnosed by the presence of hypertension with/without proteinuria, after 20 weeks of gestation. The pathogenesis of pre-eclampsia remains ambiguous, but research over the years has identified an imbalance in maternal and foetal factors. Familial predisposition and gene variation are also linked to pre-eclampsia development. The sFlt-1/PIGF ratio has attracted great attention over the years; more recently several researchers have reported that a sFlt-1/PIGF ratio of ≤ 38 can be used to predict short-term absence of pre-eclampsia. This ratio has the potential to prevent adverse pregnancy outcomes and reduce healthcare costs significantly. Genome-wide studies have additionally identified variation in the foetal gene near Flt-1. The development of preeclampsia is not limited to the maternal interface, but foetal involvement as well as genetic interplay is associated with the disorder.
... provided by Dr. K. Ohsumi, Mitsubishi-Yuka BCL, Tokyo, Japan)43 . The bands were visualized with an ECL Western Blotting Detection System (GE Healthcare, Uppsala, Sweden) on a chemiluminescence imaging system (KETA C Plus; Wealtec Corp., Sparks, NV, USA). ...
Abstract Placental hypoxia and elevated levels of circulating soluble Fms-like tyrosine kinase-1 (sFlt-1), an anti-angiogenic factor, are closely related to the pathogenesis of preeclampsia. Although sFlt-1 secretion from the placental trophoblasts is increased under hypoxic conditions, the underlying molecular mechanism remains unclear. Previously, an authentic hypoxia response element in the Flt-1 gene promoter was shown to be a potential binding site for hypoxia-inducible factors (HIFs). Here, we investigated the roles of HIF-1α and HIF-2α in Flt-1 gene expression in trophoblast-derived choriocarcinoma cell lines and cytotrophoblasts exposed to hypoxic conditions. In the cell lines, increased expression of sFlt-1 splice variants and nuclear accumulation of HIF-1α and HIF-2α were observed after hypoxic stimulation. A specific small interfering RNA or an inhibitor molecule targeting HIF-2α decreased hypoxia-induced up-regulation of Flt-1 gene expression. Moreover, in cytotrophoblasts, increased sFlt-1 mRNA expression and elevated sFlt-1 production were induced by hypoxic stimulation. Notably, hypoxia-induced elevation of sFlt-1 secretion from the cytotrophoblasts was inhibited by silencing the HIF-2α, but not HIF-1α mRNA. These findings suggest that hypoxia-induced activation of HIF-2α is essential for the increased production of sFlt-1 proteins in trophoblasts. Targeting the HIF-2α may be a novel strategy for the treatment of preeclampsia.
... We have developed a novel multivalent drug technology platform that allows for the longterm delivery of anti-VEGF drugs into the vitreous. Using a naturally occurring biopolymer, hyaluronic acid (HyA) we synthesized drug multivalent conjugates by chemically conjugating sFlt (soluble fms-like tyrosine kinase-1 [11]), an anti-VEGF protein, to the biopolymer chain thereby significantly increasing the overall drug size. We have shown previously that these anti-VEGF multivalent conjugates, mvsFlt, maintain their ability to bind VEGF and inhibit endothelial cell proliferation, migration and tube formation in vitro. ...
Current anti-VEGF drugs for patients with diabetic retinopathy suffer from short residence time in the vitreous of the eye. In order to maintain biologically effective doses of drug for inhibiting retinal neovascularization, patients are required to receive regular monthly injections of drug, which often results in low patient compliance and progression of the disease. To improve the intravitreal residence time of anti-VEGF drugs, we have synthesized multivalent bioconjugates of an anti-VEGF protein, soluble fms-like tyrosine kinase-1 (sFlt) that is covalently grafted to chains of hyaluronic acid (HyA), conjugates that are termed mvsFlt. Using a mouse corneal angiogenesis assay, we demonstrate that covalent conjugation to HyA chains does not decrease the bioactivity of sFlt and that mvsFlt is equivalent to sFlt at inhibiting corneal angiogenesis. In a rat vitreous model, we observed that mvsFlt had significantly increased intravitreal residence time compared to the unconjugated sFlt after 2 days. The calculated intravitreal half-lives for sFlt and mvsFlt were 3.3 and 35 hours, respectively. Furthermore, we show that mvsFlt is more effective than the unconjugated form at inhibiting retinal neovascularization in an oxygen-induced retinopathy model, an effect that is most likely due to the longer half-life of mvsFlt in the vitreous. Taken together, our results indicate that conjugation of sFlt to HyA does not affect its affinity for VEGF and this conjugation significantly improves drug half-life. These in vivo results suggest that our strategy of multivalent conjugation could substantially improve upon drug half-life, and thus the efficacy of currently available drugs that are used in diseases such as diabetic retinopathy, thereby improving patient quality of life.
... Since the most important mediator of cancer angiogenesis is VEGF-A, it will be referred as VEGF from now on [10]. The angiogenic effects of VEGF ligands are mainly mediated through three separate cell membrane receptors, VEGF R-1, -2 and -3 [11]. The structure of the receptors is composed by three different domains: an extracellular domain, a transmembrane domain and an intracellular tyrosine kinase domain. ...
The cancer originated from large bowel represents a widespread cancer along the
world and a considerable percentage of new cases are diagnosed with distant disease. Recently,
the detection of “clever” medicaments such as focused therapy was the outcome of
an improved comprehension of the procedures that transform physiological tissues to tumor. The family of
focused medication includes medicaments that specifically affect specific molecular routes implicated in the
production of the tumor and its development. The new natural substances aim at inhibiting the procedure of
angiogenesis which is fundamental for cancer development and distant metastasis. This process of angiogenesis
and tumor development is inhibited by Bevacizumab a humanized recombinant antibody that prevents
vascular endothelial growth factor (VEGF) receptor binding. The usage of bevacizumab as primary therapy
for patients with metastatic colorectal cancer was approved by FDA (Food and Drug Administration) on February
26, 2004. Incorporating the focused therapies in the management of metastatic colon cancer has led to
considerable advance in efficiency results. In this review article, the effectiveness of bevacizumab given as
primary therapy in patients with large bowel tumor and distant metastases is analysed.
... It is believed that the main mediator of cancer angiogenesis is VEGF-A and will be stated as VEGF from now on in this paper [12]. VEGF ligands mediate their angiogenic results essentially through 3 different cell membrane receptors, VEGF R-1, -2 and -3 [13]. Receptors' structure is made of three different parts; an extracellular part, a transmembrane part and an intracellular tyrosine kinase part. ...
Recently, the improvement of innovative medications named focused treatments represents the consequence of a superior knowledge of the procedures implicated in the modification of physiological tissues in tumor. Focused treatment is known as the therapy which uses specific substances that affect selective mechanisms implicated in tumorigenesis and tumor development. Angiogenesis is important for tumor development and distant metastatic disease and represents a significant aim for modern biological substances. Bevacizumab belongs to humanized recombinant antibody family which obviates vascular endothelial growth factor (VEGF) receptor fastening, and suspending genesis of new vessels and tumor development. Bevacizumab represents the primary antiangiogenic treatment authorized for usage in tumor and has FDA authorization to treat the recurrent glioblastoma multiform since 2009. Bevacizumab's efficiency for treating malignant brain gliomas along with correlated patents appliances related to this agent is discussed below.
... The second Ig region of Flt-1 and its surrounding structures were previously shown to be the direct binding site for VEGF. 20,28,29 These anti-Flt-1 mAbs also recognized Flt-1 endogenously expressed on HUVECs ( Figure 2B). On the other hand, anti-KDR mAbs, KM1992 and KM1995, recognized only the KDR molecule expressed on HUVECs and NIH 3T3 cells (Figures 1B,2B). ...
Flt-1, also known as vascular endothelial growth factor receptor 1 (VEGFR-1), is a high-affinity tyrosine kinase receptor for VEGF and is expressed almost exclusively on vascular endothelial cells. As an exception, Flt-1 transcript was recently found to be expressed in human peripheral blood monocytes. However, the protein of the Flt-1 receptor on the cell surface of monocytes is yet to be identified, and whether the Flt-1 protein is expressed during the differentiation of monocyte-macrophage lineage cells has not been examined. Using monoclonal antibodies against 2 different antigenic epitopes on the Flt-1 extracellular domain, this study found that the major population of the monocyte-marker CD97⁺ cells in human peripheral blood express Flt-1 as a cell surface molecule. VEGFR-2 (KDR/Flk-1) was not expressed at detectable levels in these cells. An Flt-1 neutralizing monoclonal antibody significantly suppressed VEGF-induced migration of the monocytes, suggesting an important role for Flt-1 in the biologic function of monocytes. Furthermore, CD34⁺cells in human cord blood, originally negative for the Flt-1 expression, differentiated into Flt-1⁺ cells in association with the appearance of monocyte-macrophage markers after a 2-week culture in the presence of hematopoietic cytokines. In addition, the Flt-1⁺CD11b⁺ cell fraction from CD34⁺ cells was found to efficiently differentiate into multinuclear osteoclasts in the presence of macrophage colony-stimulating factor and osteoclast differentiation factor. These results strongly suggest that Flt-1 is a novel cell surface marker as well as a biologically functional molecule for monocyte-macrophage lineages in humans.
... Later, Flt-1 was shown to bind VEGF and PlGF [242][243][244] and to be important for embryonic vascular development [243,245]. It was also revealed that the first three extracellular Ig-like domains of Flt-1 are essential for ligand-binding, while the 4-7th extracellular Iglike domains for receptor dimerization [242,[246][247][248]. In 1993, a soluble isoform of Flt-1 was identified, which is encoded by the first 13 out of 30 exons of FLT1, and is generated by skipped splicing of the Flt-1 mRNA and its premature termination due to intron 13 polyadenylation, hence it is denoted as sFlt-1-i13 ( Fig 1B) [242,243,249,250]. ...
Objective:
Most anti-angiogenic preeclampsia models in rodents utilized the overexpression of a truncated soluble fms-like tyrosine kinase-1 (sFlt-1) not expressed in any species. Other limitations of mouse preeclampsia models included stressful blood pressure measurements and the lack of postpartum monitoring. We aimed to 1) develop a mouse model of preeclampsia by administering the most abundant human placental sFlt-1 isoform (hsFlt-1-e15a) in preeclampsia; 2) determine blood pressures in non-stressed conditions; and 3) develop a survival surgery that enables the collection of fetuses and placentas and postpartum (PP) monitoring.
Methods:
Pregnancy status of CD-1 mice was evaluated with high-frequency ultrasound on gestational days (GD) 6 and 7. Telemetry catheters were implanted in the carotid artery on GD7, and their positions were verified by ultrasound on GD13. Mice were injected through tail-vein with adenoviruses expressing hsFlt-1-e15a (n = 11) or green fluorescent protein (GFP; n = 9) on GD8/GD11. Placentas and pups were delivered by cesarean section on GD18 allowing PP monitoring. Urine samples were collected with cystocentesis on GD6/GD7, GD13, GD18, and PPD8, and albumin/creatinine ratios were determined. GFP and hsFlt-1-e15a expression profiles were determined by qRT-PCR. Aortic ring assays were performed to assess the effect of hsFlt-1-e15a on endothelia.
Results:
Ultrasound predicted pregnancy on GD7 in 97% of cases. Cesarean section survival rate was 100%. Mean arterial blood pressure was higher in hsFlt-1-e15a-treated than in GFP-treated mice (∆MAP = 13.2 mmHg, p = 0.00107; GD18). Focal glomerular changes were found in hsFlt-1-e15a -treated mice, which had higher urine albumin/creatinine ratios than controls (109.3 ± 51.7 μg/mg vs. 19.3 ± 5.6 μg/mg, p = 4.4 x 10(-2); GD18). Aortic ring assays showed a 46% lesser microvessel outgrowth in hsFlt-1-e15a-treated than in GFP-treated mice (p = 1.2 x 10(-2)). Placental and fetal weights did not differ between the groups. One mouse with liver disease developed early-onset preeclampsia-like symptoms with intrauterine growth restriction (IUGR).
Conclusions:
A mouse model of late-onset preeclampsia was developed with the overexpression of hsFlt-1-e15a, verifying the in vivo pathologic effects of this primate-specific, predominant placental sFlt-1 isoform. HsFlt-1-e15a induced early-onset preeclampsia-like symptoms associated with IUGR in a mouse with a liver disease. Our findings support that hsFlt-1-e15a is central to the terminal pathway of preeclampsia, and it can induce the full spectrum of symptoms in this obstetrical syndrome.
... [17][18][19] VEGF ligands mediate their angiogenic effects mainly through 3 different cell membrane receptors, VEGF R-1, -2 and -3. [20][21][22][23][24][25] These receptors consist of an extracellular domain, a transmembrane domain and an intracellular tyrosine kinase domain. Binding of the ligand to the receptor induces the activation of intracellular signaling transduction pathways that are involved in the regulation of cellular proliferation and survival, such as the raf/MEK, mTOR and PI3K pathways (see Figure 1). ...
Colorectal cancer continues to be an important public health concern, despite improvements in screening and better systemic chemotherapy. The integration of targeted therapies in the treatment of colon cancer has resulted in significant improvements in efficacy outcomes. Angiogenesis is important for tumor growth and metastasis and is an important target for new biological agents. Bevacizumab is a humanized recombinant antibody that prevents vascular endothelial growth factor (VEGF) receptor binding, and inhibits angiogenesis and tumor growth. The addition of bevacizumab to fluoropyrimidine-based chemotherapy, with or without irinotecan or oxaliplatin, in both the first- and second-line treatment of metastatic colorectal cancer, significantly increased median progression-free survival and overall survival in select randomized phase III studies. Ongoing studies are evaluating the role of bevacizumab in the adjuvant treatment of colon cancer. Common toxicities associated with bevacizumab include hypertension, bleeding episodes, and thrombotic events. This review will focus on the integration of bevacizumab in the treatment paradigm of colon cancer and the management of its side effects.
... Although VEGF A, VEGF B and PDGF are known to bind to the receptor, de Vries et al. [290] in 1992 demonstrated that VEGFR1 has the highest affinity for VEGF165 and is required for normal blood vessel development during embryogenesis. Tanaka et al. [291] constructed soluble forms of Flk-1 which carry only the first and third Ig-like domains. The mutants of Flk-1 were generated by alternative splices and found to inhibit VEGF-dependent endothelial cell proliferation in vitro. ...
... VEGF-C und VEGF-D binden ebenfalls mit höherer Affinität an VEGFR-3 (Achen et al., 1998 (Kendall und Thomas, 1996) (Barleon et al., 1997a;Tanaka et al., 1997). ...
... They are structurally similar, with seven immunoglobulin-like domains making up the extracellular region, a transmembrane domain, and an intracellular region containing a tyrosine kinase domain divided by a long kinase-insert domain (Brekken et al., 2000;Davis-Smith et al., 1996;Hiratsuka et al., 2001;Kroll and Waltenberger, 1997). In the case of Flt-1, the first, second, and third immunoglobulin-like domains are required for ligand binding, and the fourth domain is involved in receptor dimerization (Barleon et al., 1997;Cunningham et al., 1997;Davis-Smith et al., 1996;Shibuya, 2001b;Tanaka et al., 1997). ...
... Since sFlt-1 is derived from the ligand-binding region of VEGFR-1/Flt-1, its major biochemical function is thought to be trapping of VEGF for suppression of VEGF signals (Tanaka et al., 1997;Shibuya, 2006). To our surprise, cancer patients treated with anti-VEGF signal drugs often develop side effects such as hypertension and proteinurea that are similar to the symptoms of PE (Hurwitz et al., 2004). ...
Vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) system has been shown to play central roles not only in physiological angiogenesis, but also in pathological angiogenesis in diseases such as cancer. Based on these findings, a variety of anti-angiogenic drugs, including anti-VEGF antibodies and VEGFR/multi-receptor kinase inhibitors have been developed and approved for the clinical use. While the clinical efficacy of these drugs has been clearly demonstrated in cancer patients, they have not been shown to be effective in curing cancer, suggesting that further improvement in their design is necessary. Abnormal expression of an endogenous VEGF-inhibitor sFlt-1 has been shown to be involved in a variety of diseases, such as preeclampsia and aged macular degeneration. In addition, various factors modulating angiogenic processes have been recently isolated. Given this complexity then, extensive studies on the interrelationship between VEGF signals and other angiogenesis-regulatory systems will be important for developing future strategies to suppress diseases with an angiogenic component.
... Taking the relative molecular mass of VEGF to be 45 kDa [19] and that of free sFlt-1 to be about 100 kDa [3], then the molarity of sFlt-1 still exceed that of VEGF by approx. 74 in these subjects, since one molecule of VEGF is thought to bind stochiometrically to one molecule of sFlt-1 [3,20,21]. This molar ratio is doubled if receptor dimerization occurs upon ligand binding. ...
Vascular endothelial growth factor (VEGF) mediates endothelial cell mitogenesis and enhances vascular permeability. VEGF interacts with the endothelium via two membrane-spanning receptors, fms-like tyrosine kinase (Flt)-1 and kinase domain receptor. A soluble form of Flt-1 (sFlt-1) was isolated from endothelial cell media; however, its biological significance is still unknown, with limited data on plasma sFlt-1 levels in disease states. We have developed two new ELISAs for detecting free and VEGF-complexed sFlt-1, which were tested in accordance with standard validation and assessment methodologies employed in commercial settings. The intra-and inter-assay coefficients of variation are < 5% and 10% respectively, and results are highly reproducible. Applying these ELISAs in a clinical setting, we measured levels of VEGF, free and complexed sFlt-1 in citrated plasma from 40 patients with cardiovascular disease and 40 healthy controls. Median (interquartile range) plasma levels of VEGF in patients were significantly greater than controls [403 pg/ml (158-925 pg/ml) versus 113 pg/ml (33-231 pg/ml), P less than or equal to 0.05]. Free sFlt-1 was significantly lower in patients compared with controls [8 ng/ml (2-22 ng/ml) versus 21 ng/ml (10-73 ng/ml), P less than or equal to 0.05]. There was no significant difference in the levels of complexed sFlt-1 between the two groups. Plasma levels of VEGF-complexed sFlt-1 are minimal, despite the presence of excess free sFlt-1. Thus unbound plasma VEGF detected by ELISA may represent the majority of circulating VEGF, and justifies the measurement of plasma VEGF as an indicator of circulating VEGF levels. Furthermore, these results suggest that circulating sFlt-1 may serve as a selective inhibitor of VEGF activity, and that this regulatory mechanism may be altered by pathological conditions.
... In contrast to VEGFR2, VEGFR1 has very weak kinase activity and acts as a decoy receptor, competitively reducing VEGF binding to VEGFR2 and therefore limiting the activity of VEGF pathway in the vascular endothelial cells (Park et al. 1994). In addition, alternative splicing of VEGFR1 produces a soluble isoform of this receptor (sVEGFR1) that sequesters VEGF in the extracellular medium and thereby modulates VEGF signaling in the vascular endothelium (Tanaka et al. 1997). Furthermore, the cell-surface glycoproteins neuropilin1 (Nrp1) and neuropilin2 (Nrp2), originally identified as receptors for semaphorins, also modulate the VEGF signaling output. ...
Sprouting angiogenesis is a dynamic process in which endothelial cells collectively migrate, shape new lumenized tubes, make new connections, and remodel the nascent network into a hierarchically branched and functionally perfused vascular bed. Endothelial cells in the nascent sprout adopt two distinct cellular phenotypes-known as tip and stalk cells-with specialized functions and gene expression patterns. VEGF and Notch signaling engage in an intricate cross talk to balance tip and stalk cell formation and to regulate directed tip cell migration and stalk cell proliferation. In this article, we summarize the current knowledge and implications of the tip/stalk cell concepts and the quantitative and dynamic integration of VEGF and Notch signaling in tip and stalk cell selection.
... The presence of seven immunoglobulin (Ig)-like domains characterizes the extracellular ligand-binding domain. The ligand-binding region is localized within the second and third Ig domains (Barleon et al. 1997b; Davis-Smyth et al. 1996; Shinkai et al. 1998; Tanaka et al. 1997). Although, the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule (Shinkai et al. 1998). ...
... This assumes that kinetic parameters, such as binding and unbinding rates, are similar among different experimental systems, e.g., in vitro cell culture and endothelial cells in developing vasculature. sFlt-1–VEGF binding and dimerization parameters were constrained to produce observed K d values for VEGF–sFlt-1 binding close to those that were measured assuming 1:1 binding (Tanaka et al., 1997). We assumed that the affinity of sFlt-1 dimerization with mFlt-1 was similar to that of VEGF binding mFlt-1. ...
Experimental data indicates that soluble vascular endothelial growth factor (VEGF) receptor 1 (sFlt-1) modulates the guidance cues provided to sprouting blood vessels by VEGF-A. To better delineate the role of sFlt-1 in VEGF signaling, we have developed an experimentally based computational model. This model describes dynamic spatial transport of VEGF, and its binding to receptors Flt-1 and Flk-1, in a mouse embryonic stem cell model of vessel morphogenesis. The model represents the local environment of a single blood vessel. Our simulations predict that blood vessel secretion of sFlt-1 and increased local sFlt-1 sequestration of VEGF results in decreased VEGF-Flk-1 levels on the sprout surface. In addition, the model predicts that sFlt-1 secretion increases the relative gradient of VEGF-Flk-1 along the sprout surface, which could alter endothelial cell perception of directionality cues. We also show that the proximity of neighboring sprouts may alter VEGF gradients, VEGF receptor binding, and the directionality of sprout growth. As sprout distances decrease, the probability that the sprouts will move in divergent directions increases. This model is a useful tool for determining how local sFlt-1 and VEGF gradients contribute to the spatial distribution of VEGF receptor binding, and can be used in conjunction with experimental data to explore how multi-cellular interactions and relationships between local growth factor gradients drive angiogenesis.
... VEGFR1 (also called Flt1 in the mouse) is a 180-185 kDa glycoprotein [23,24] that is activated in response to binding of VEGFA, VEGFB and PlGF ( Figure 1). The ligands bind to Igloop 2, but loops 1 and 3 are also required for high-affinity binding [25,26]. The crystal structure of the receptor-binding domain of VEGF bound to the second Ig-loop of VEGFR1 shows hydrophobic interactions stabilizing the ligand-receptor dimers [27]. ...
VEGFs (vascular endothelial growth factors) control vascular development during embryogenesis and the function of blood vessels and lymphatic vessels in the adult. There are five related mammalian ligands, which act through three receptor tyrosine kinases. Signalling is modulated through neuropilins, which act as VEGF co-receptors. Heparan sulfate and integrins are also important modulators of VEGF signalling. Therapeutic agents that interfere with VEGF signalling have been developed with the aim of decreasing angiogenesis in diseases that involve tissue growth and inflammation, such as cancer. The present review will outline the current understanding and consequent biology of VEGF receptor signalling.
... VEGFR-2 consists of 1356 and 1345 amino acids in humans and mice, respectively, and can be separated into four regions: the extracellular ligand-binding domain, transmembrane domain, tyrosine kinase domain, and downstream carboxy terminal region [3][4][5][6]. The extracellular ligand-binding domain consists of seven immunoglobulin (Ig)-like domains with the ligand-binding region being localized within the second and third Ig domains [24][25][26][27]. Shinkai et al, further demonstrated that the third Ig-like domain is critical for ligand binding, the second and fourth domains are important for ligand association, and the fifth and sixth domains are required for retention of the ligand bound to the receptor molecule [26]. ...
Investigations over the last decade have established the essential role of growth factors and their receptors during angiogenesis and carcinogenesis. The vascular endothelial growth factor receptor (VEGFR) family in mammals contains three members, VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1) and VEGFR-3 (Flt-4), which are transmembrane tyrosine kinase receptors that regulate the formation of blood and lymphatic vessels. In the early 1990s, the above VEGFR was structurally characterized by cDNA cloning. Among these three receptors, VEGFR-2 is generally recognized to have a principal role in mediating VEGF-induced responses. VEGFR-2 is considered as the earliest marker for endothelial cell development. Importantly, VEGFR-2 directly regulates tumor angiogenesis. Therefore, several inhibitors of VEGFR-2 have been developed and many of them are now in clinical trials. In addition to targeting endothelial cells, the VEGF/VEGFR-2 system works as an essential autocrine/paracrine process for cancer cell proliferation and survival. Recent studies mark the continuous and increased interest in this related, but distinct, function of VEGF/VEGFR-2 in cancer cells: the autocrine/paracrine loop. Several mechanisms regulate VEGFR-2 levels and modulate its role in tumor angiogenesis and physiologic functions, i.e.: cellular localization/trafficking, regulation of cis-elements of promoter, epigenetic regulation and signaling from Notch, cytokines/growth factors and estrogen, etc. In this review, we will focus on updated information regarding VEGFR-2 research with respect to the molecular mechanisms of VEGFR-2 regulation in human breast cancer. Investigations in the activation, function, and regulation of VEGFR-2 in breast cancer will allow the development of new pharmacological strategies aimed at directly targeting cancer cell proliferation and survival.
... Alternative Splice-Varianten ergeben eine kürzere, lösliche Form, den löslichen VEGFR-1 Rezeptor (sVEGFR-1), der inhibitorisch auf VEGF-A wirkt (Tanaka et al., 1997). VEGF-A bindet 10mal stärker an VEGFR-1 als an VEGFR-2, aber die Tyrosinkinase Aktivität des VEGFR-1 ist 10mal schwächer als die des VEGFR-2. ...
Das Neuroblastom (NB) ist der häufigste bösartige extrakranielle Tumor im Kindesalter. Es entsteht aus undifferenzierten Zellen des sympathischen Nervensystems und tritt meist im Bauchraum auf, seltener im Brustkorb bzw. Halsbereich. Bei der Therapie kommen die Chemotherapie, die Chirurgie, die Strahlentherapie und die Differenzierungstherapie mit Retinolsäure zum Einsatz. Vor allem im Stadium IV sind die Therapieergebnisse nicht befriedigend und die Überlebensrate liegt nur bei 27%. Daher ist die Entwicklung neuer Therapieansätze dringend erforderlich.
Anti-Blut-Angiogenese ist eine neue Strategie in der Tumortherapie und richtet sich direkt gegen die Neubildung von Blutgefäßen in Tumoren. Dadurch wird die Versorgung des Tumors mit Nährstoffen und Sauerstoff gestoppt, ein weiteres Wachstum des Tumors und eine hämatogene Metastasierung verhindert. Anti-Blut-Angiogenese Substanzen konnten bereits in NB Mausmodellen erfolgreich angewendet werden. Aber auch Lymphgefäße dienen der Tumorzelldisseminierung und der Bildung von Lymphknoten Metastasen. Deshalb könnte eine gegen die Lymph-Angiogenese, die Neubildung von Lymphgefäßen, gerichtete Therapie eine weitere Möglichkeit in der Krebstherapie darstellen.
Um eine mögliche Anti-Blut- und Anti-Lymph-Angiogenese Therapie im NB zu untersuchen, wurden zunächst Blut- und Lymphgefäße in NB Tumoren mittels Immunohistochemie identifiziert. Hier konnte mit Antikörpern gegen CD34 und D2_40 zum ersten mal gezeigt werden, dass Blut- und Lymphgefäße tatsächlich in NB Tumoren vorhanden sind. Desweiteren wurde festgestellt, dass in Grad III NB Tumoren statistisch häufiger Lymphgefäße fehlen als in Grad I und II NB Tumoren. Zusätzlich wurde die Expression der an der Blut- und Lymph-Angiogenese beteiligten Wachstumsfaktoren bFGF, VEGF-A sowie VEGF-C und VEGF-D und den dazugehörigen Rezeptoren VEGFR-1, -2 und -3 mittels RT-PCR in verschiedenen NB Tumorzelllinien und NB Biopsaten nachgewiesen. Untersuchungen zur Regulation dieser Faktoren ergaben, dass Retinolsäure eine verstärkte, verminderte oder unveränderte mRNA Expression bewirkt, dass VEGF-C und VEGF-D keinen autokrinen Wachstumsstimulus aufweisen und dass der Promotor von VEGF-C in Lan-5 NB Zellen hypermethyliert ist.
Um die Blut- und Lymph-Angiogenese im NB zu hemmen, wurden spezielle siRNA (small interfering RNA) Moleküle entwickelt, die sich an die mRNA der VEGF-A und VEGF-C Gene anlagern und die Expression der entsprechenden Proteine verhindern. Um die siRNA vor enzymatischen Abbau zu schützen, wurden sie mittels Liposomen in die NB Tumorzellen transportiert. Hierfür wurde ein Vergleich verschiedener kommerziell erhältlicher Liposomenpräparationen (Lipofektamin2000® und HiPerFect®) mit am Lehrstuhl hergestellten Liposomen (pH sensitive Liposomen, DOTAP/Cholesterol Liposomen, S75 Liposomen) erstellt. Es konnte gezeigt werden, dass die Transfektion der siRNA gegen VEGF-A bzw. VEGF-C zu einem Silencing der beiden Gene in der NB Zelllinie Kelly führte und dass die hierfür erforderliche siRNA Menge mit eigens hergestellten S75 Liposomen am geringsten war.
Somit zeigt liposomal verpackte siRNA gegen VEGF-A bzw. VEGF-C eine neue Behandlungsmöglichkeit in der Therapie des NB auf.
... VEGF-C und VEGF-D binden an VEGFR-2 (KDR/ Flk-1) oder an VEGFR-3 (Flt-4) und können die Lymphangiogenese induzieren (Byrne et al., 2005). Splicen entsteht eine kürzere lösliche Form des Rezeptors: sVEGFR-1 (soluble flt-1) (Tanaka et al., 1997). Die Affinität von VEGFR-1 (Flt-1) für VEGF ist zehnmal höher als die von VEGFR-2 (KDR/ Flk-1). ...
Bei Patienten mit angeborenem zyanotischen Herzfehler kommt es zur Bildung von Kollateralen in verschiedenen Formen und Varianten. Diese Kollateralbildung beruht auf einer abnormen Angiogenese (Starnes et al., 2000). Da VEGF (vascular endothelial growth factor) als ein potenter Induktor der Angiogenese bekannt ist, war es Ziel dieser Studie zu untersuchen, ob der VEGF-Spiegel im Plasma und die VEGF-Expression im Myokard bei Kindern mit angeborenem zyanotischen Herzfehler im Vergleich zu Kindern mit angeborenem azyanotischen Herzfehler erhöht sind. Weiterhin haben wir überprüft, ob die VEGF-Spiegel im Plasma mit der myokardialen Expression von VEGF korrelieren. Darüber hinaus haben wir den Verlauf der VEGF-Blutkonzentrationen im Rahmen einer Herzoperation mit EKK (extrakorporaler Kreislauf) beobachtet und diskutiert, ob VEGF einen protektiven Einfluss auf die Herz-Kreislaufverhältnisse und den postoperativenVerlauf hat. Zu diesem Zweck haben wir 27 Kinder mit einem angeborenen Herzfehler, im Median 4,5 Monate (0,3 - 19,5 Monate) alt, bei denen eine Herzoperation mit EKK durchgeführt wurde, in unsere Studie aufgenommen. Darunter waren 12 Kinder mit angeborenem azyanotischen und 15 Kinder mit angeborenem zyanotischen Herzfehler. Die VEGF-Plasmakonzentrationen haben wir prä-, intra- und postoperativ mittels ELISAs und die VEGF-Expression im Myokard aus Biopsien des rechten Vorhofs mittels Real-Time-PCR bestimmt. Die entsprechenden Ergebnisse haben wir jeweils mit den EKK-Parametern, den prä- und postoperativen klinischen Daten, der postoperativen Therapie und den postoperativen Laborwerten korreliert und analysiert. Bei der Betrachtung des Einflusses der präoperativen Hypoxämie auf die Expression von VEGF ergab sich, dass die präoperativen VEGF-Spiegel im Plasma im Mittel bei zyanotischen Patienten höher, jedoch nicht signifikant, als bei den azyanotischen Patienten waren (71,24 ± 17,95 pg/ml vs. 60,51 ± 15,16 pg/ml). Auch zwischen der präoperativen VEGF-Plasmakonzentration und der präoperativ gemessenen Sauerstoffsättigung ergab sich keine signifikante Korrelation, obwohl die SaO2-Werte der zyanotischen Patienten signifikant unter den Werten der azyanotischen Patienten lagen (85,13 ± 2,39% vs. 97,33 ± 0,60%, p < 0,001). Statistisch konnte demnach keine signifikante Korrelation zwischen dem präoperativen VEGF und dem Grad der Zyanose bzw. der Oxygenierung nachgewiesen werden. Wir stellten fest, dass es in dem von uns beobachteten Zeitraum, von präoperativ bis 24 h postoperativ, im Rahmen der Herzoperation mit EKK zu einem Abfall der VEGF-Plasmakonzentrationen (ca. um den Faktor 2) kam. Zu einem bedeutsamen Abfall kam es direkt nach Einleitung des EKKs (gesamte Patientengruppe: p < 0,005). Ein weiterer Abfall von VEGF bis zum Ende der Operation trat in der azyanotischen Gruppe auf, während in der zyanotischen Gruppe unmittelbar nach Abschalten des EKKs bzw. gegen Ende der Operation bereits wieder ein leichter Anstieg von VEGF zu detektieren war. In beiden Gruppen verfolgten wir postoperativ einen Wiederanstieg von VEGF, verbunden mit einer Zunahme des VEGF-Spiegels von 4 h bis 24 h postoperativ. Wir untersuchten die Expression von VEGF-mRNA im Myokard. Die myokardiale Expression von VEGF war in der azyanotischen Gruppe größer als in der zyanotischen Gruppe (1,77 ± 0,06 vs. 1,53 ± 0,09, p = 0,06). Der Unterschied zwischen beiden Gruppen war tendenziell signifikant. Dieses Ergebnis weist darauf hin, dass das Myokard bei Kindern mit zyanotischen Herzfehlern ein Ort mit geringer Aktivität der Transkription (Synthese von RNA anhand der DNA-Vorlage) von VEGF-mRNA im Vergleich zu anderen Organen ist. Die dadurch zu erklärenden gleichzeitig höheren Plasmakonzentrationen von VEGF bei den zyanotischen Kindern lassen eine kompensatorische Mehrproduktion von VEGF aufgrund der systemischen Hypoxämie in anderen Zellen und Organen vermuten. Unsere Analysen ergaben, dass in der gesamten Patientengruppe die Blutkonzentrationen von VEGF nicht die Expression von VEGF-mRNA im Myokard widerspiegeln. Nur bei den azyanotischen Patienten korrelierte der VEGF-Plasmaspiegel tendenziell signifikant mit der VEGF-mRNA Expression im Myokard (p = 0,05). In unserer Arbeit konnten wir keine Korrelation zwischen der VEGF-Expression und dem Alter der Patienten finden. Es lässt sich allerdings ableiten, dass sich das Alter der Patienten auf die EKK-Parameter, die Herz-Kreislaufsituation und den postoperativen Verlauf auswirkte, wobei junges Alter mit einem risikoreicheren Ablauf verbunden war. Zusammengefasst wurde in unserer Studie eine erhöhte systemische Produktion von VEGF bei den zyanotischen Kindern im Vergleich zu den azyanotischen gemessen. Hingegen war die myokardiale Expression von VEGF-mRNA bei den zyanotischen Patienten niedriger als bei den azyanotischen. Während die myokardiale mRNA-Expression mit einer gewissen myokardialen Protektion einherging, korrelierten die VEGF-Plasmaspiegel mit einem ungünstigeren Verlauf. Children with cyanotic congenital heart disease (C-CHD) often develop collateral blood vessels that could be interpreted as a form of abnormal angiogenesis (Starnes et al., 2000). Since vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis, we examined in this study whether children with cyanotic congenital heart disease have elevated plasma levels of VEGF in comparison to children with acyanotic congenital heart disease (A-CHD), and if there is a correlation between plasma VEGF levels and the expression of VEGF-mRNA in the myocardium. To test the hypothesis if VEGF has a protective influence on the cardiovascular system and on the postoperative course, we measured the plasma levels of VEGF during and after cardiac surgery. 27 patients, who underwent heart surgery with extracorporeal circulation (ECC), were divided into 2 groups: the C-CHD group (n = 15) and the A-CHD group (n = 12). Median age of the whole study population was 4,5 months (Range 0,3 - 19,5 months). Median age was 5,0 (0,3 - 19,5) months in the cyanotic group and 4,3 (2,5 - 13,5) months in the acyanotic group. Blood samples were collected pre-, intra- and postoperatively and a biopsy of myocardium (right atrium) was taken during the operation. VEGF concentrations were measured in the plasma by enzyme-linked immunosorbent assay (ELISA). The VEGF-mRNA expression in the myocardium was detected by Real-Time-Polymerase Chain Reaction (Real-Time-PCR). Results were analyzed with respect to preoperative clinical data, ECC-parameters, postoperative clinical data and postoperative laboratory findings, respectively. Mean systemic oxygen saturation (SaO2) and plasma VEGF levels were measured preoperatively to verify the influence of preoperative hypoxaemia on the expression of VEGF. Before the operation, plasma VEGF levels were higher in the C-CHD group compared to the A-CHD group, but the difference did not reach statistical significance (71,24 ± 17,95 pg/ml vs. 60,51 ± 15,16 pg/ml). No correlation was found between preoperative VEGF levels and arterial oxygen saturation, even though mean systemic oxygen saturation was significantly lower in the cyanotic group than in the acyanotic group (85,13 ± 2,39 % vs. 97,33 ± 0,60 %, p < 0,001). Postoperative plasma VEGF levels (taken 24h after surgery) were markedly reduced (factor 2) compared to the preoperative values. After starting the ECC, VEGF levels decreased significantly in both groups (C-CHD group and A-CHD group: p < 0,005). While in the A-CHD group VEGF levels continuously dropped until the end of the operation, the C-CHD group VEGF levels increased immediately after ECC was stopped. Furthermore, the plasma VEGF levels increased postoperatively in both groups. The mean value of VEGF-mRNA copies was higher in the A-CHD group than in the C-CHD group (1,77 ± 0,06 vs. 1,53 ± 0,09, p = 0,06). This suggests that the myocardium of children with C-CHD may be a location of lower transcription of VEGF-mRNA in comparison to other organs. Since the plasma levels of VEGF were higher in patients with C-CHD, a lower myocardial expression of VEGF may lead to a compensatory overexpression of VEGF in other cells and organs induced by hypoxaemia. We could not show any correlation between plasma VEGF and myocardial VEGF-mRNA for the whole group (C-CHD group and A-CHD group). However, in the A-CHD subgroup, plasma VEGF levels correlated with the expression of myocardial VEGF-mRNA (p = 0,05). In our study the expression of VEGF (in plasma and myocardium) was not correlated with patients age. However patients age had an influence on ECC-parameters, cardiovascular complications and the postoperative period. Indeed, younger children showed a greater risk for complications. The results of the present study demonstrate that the plasma VEGF levels (systemic production) were higher in the C-CHD group than in the A-CHD group. In contrast, the expression of myocardial VEGF-mRNA tended to be higher in the A-CHD group than in the C-CHD group. While the expression of myocardial VEGF-mRNA had a protective influence, the plasma levels of VEGF correlated with poor outcome.
... VEGFR-1 has a modulator role during embryogenesis, as knockout leads to overgrowth of endothelial cells [28,29] but a positive role in inflammation [30,31] and cancer growth [32], as it appears to have a more widespread expression that initially described. Soluble VEGFR-1 has a possible decoy effect [33, 34] and is increased in serum from pregnant women suffering from pre-eclampsia, manifesting itself with hypertension and proteinuria [35,36]. VEGFR-2 is the main transducing receptor for VEGF, and VEGFF-2 knockout mice show defective vasculogenesis and die during embryo day E8–8.5 [37]. ...
Anti-angiogenic therapies currently revolve around targeting vascular endothelial growth factor-A (VEGF-A) or its receptors. These therapies are effective to some degree, but have low response rates and poor side-effect profiles. Part of these problems is likely to be due to their lack of specificity between pro- and anti-angiogenic isoforms, and their nonspecific effects on proactive, pleiotropic survival and maintenance roles of VEGF-A in endothelial and other cell types. An alternative approach, and one which has recently been shown to be effective in animal models of neovascularization in the eye, is to target the mechanisms by which the cell generates pro-angiogenic splice forms of VEGF-A, its receptors and, co-incidentally, by targeting the upstream processes, other oncogenes that have antagonistic splice isoforms. The concept here is to target the splicing mechanisms that control splice site choice in the VEGF-A mRNA. Recent evidence on the pharmacological possibilities of such splice factors is described.
The Fms-like tyrosine kinase-1 (FLT1) gene is expressed in various types of cells, including vascular endothelial cells and placental trophoblasts, and regulates angiogenesis, inflammation, and pregnancy. However, the basal transcriptional machinery of FLT1 is still not well understood. In this study, we first examined FLT1 promoter activity in three different types of cells, that is, trophoblast-derived cells, vascular endothelial-related cells, and HEK293 cells, using plasmid-based luciferase reporter assays, and showed that a cAMP-response element (CRE) and an ETS-binding site (EBS) are important for FLT1 expression in all cell types. To further examine the importance of these sites at the chromosomal level using HEK293 cells, we introduced CRISPR/Cas9-mediated mutations in these sites on the genomic DNA. HEK293 cells carrying these mutations clearly showed a significant decrease in endogenous FLT1 gene expression. These results suggest that CRE and EBS transcription regulatory elements are crucial for FLT1 gene expression in human tissues.
Vascular endothelial growth factor (VEGF) is highly expressed in vascular remodeling processes and accelerates reendothelialization after mechanical denudation. Two VEGF tyrosine kinase receptors have been reported—fms-like–tyrosine kinase-1 (Flt-1) and kinase domain region (KDR). Little is known about the regulation of the expression of these receptors after vascular injury. Herein, we have analyzed the expression of Flt-1 after mechanical denudation of primary cultures of endothelial cells, which has been considered a useful in vitro model to study endothelium responses to vascular injury. After denudation, the Flt-1 protein and mRNA levels are clearly up-regulated, and transient transfection experiments showed a strong induction of theflt-1 promoter-dependent transcription. Analysis of the flt-1 promoter sequence revealed the presence of a putative binding site for the early growth response factor-1 (Egr-1) at positions −24 to −16. Electrophoretic mobility shift and supershift assays showed that Egr-1 was able to bind to this DNA sequence, and cotransfection of the flt-1 promoter reporter plasmid with an Egr-1 expression vector resulted in enhancement of its transcriptional activity. Furthermore, the mutation of the Egr-1 binding site markedly reduced the denudation-induced flt-1promoter activity. These data demonstrate that Flt-1 is up-regulated after endothelial denudation and that Egr-1 plays a relevant role in this process.
Vascular patterning is a key process during development and disease. The diffusive decoy receptor sVEGFR1 (sFlt1) is a known regulator of endothelial cell behavior, yet the mechanism by which it controls vascular structure is little understood. We propose computational models to shed light on how vascular patterning is guided by self-organized gradients of the VEGF/sVEGFR1 factors. We demonstrate that a diffusive inhibitor can generate structures with a dense branching morphology in models where the activator elicits directed growth. Inadequate presence of the inhibitor leads to compact growth, while excessive production of the inhibitor blocks expansion and stabilizes existing structures. Model predictions were compared with time-resolved experimental data obtained from endothelial sprout kinetics in fibrin gels. In the presence of inhibitory antibodies against VEGFR1 vascular sprout density increases while the speed of sprout expansion remains unchanged. Thus, the rate of secretion and stability of extracellular sVEGFR1 can modulate vascular sprout density.
Vascular endothelial growth factor and its receptor (VEGF-VEGFR) system play a critical role in the regulation of angiogenesis and lymphangiogenesis in vertebrates. Each of the VEGF has specific receptors, which it activates by binding to the extracellular domain of the receptors, and, thus, regulates the angiogenic balance in the early embryonic and adult stages. However, de-regulation of the VEGF-VEGFR implicates directly in various diseases, particularly cancer. Moreover, tumor growth needs a dedicated blood supply to provide oxygen and other essential nutrients. Tumor metastasis requires blood vessels to carry tumors to distant sites, where they can implant and begin the growth of secondary tumors. Thus, investigation of signaling systems related to the human disease, such as VEGF-VEGFR, will facilitate the development of treatments for such illnesses.
Vascular endothelial growth factor-B (VEGF-B), discovered over 15 years ago, has long been seen as one of the more ambiguous members of the VEGF family. VEGF-B is produced as two isoforms: one that binds strongly to heparan sulfate in the pericellular matrix and a soluble form that can acquire binding via proteolytic processing. Both forms of VEGF-B bind to VEGF-receptor 1 (VEGFR-1) and the neuropilin-1 (NRP-1) coreceptor, which are expressed mainly in blood vascular endothelial cells. VEGF-B-deficient mice and rats are viable without any overt phenotype, and the ability of VEGF-B to induce angiogenesis in most tissues is weak. This has been a puzzle, as the related placenta growth factor (PlGF) binds to the same receptors and induces angiogenesis and arteriogenesis in a variety of tissues. However, it seems that VEGF-B is a vascular growth factor that is more tissue specific and can have trophic and metabolic effects, and its binding to VEGFR-1 shows subtle but important differences compared with that of PlGF. VEGF-B has the potential to induce coronary vessel growth and cardiac hypertrophy, which can protect the heart from ischemic damage as well as heart failure. In addition, VEGF-B is abundantly expressed in tissues with highly active energy metabolism, where it could support significant metabolic functions. VEGF-B also has a role in neuroprotection, but unlike other members of the VEGF family, it does not have a clear role in tumor progression. Here we review what is hitherto known about the functions of this growth factor in physiology and disease.
Vascular endothelial growth factor (VEGF) activity is highly regulated via sequestering within the ECM and cell-demanded proteolysis to release the sequestered VEGF. Numerous studies have demonstrated that VEGF activity mediates cellular events leading to angiogenesis and capillary formation in vivo. This has motivated the study of biomaterials to sustain VEGF release, and in many cases, the materials are inspired by the structure and function of the native ECM. However, there remains a need for materials that can bind to VEGF with high specificity, as the in vivo environment is rich in a variety of growth factors (GFs) and GF-binding moieties. Here we describe a strategy to control VEGF release using hydrogel microspheres with tethered peptides derived from VEGF receptor 2 (VEGFR2). Using biomaterials covalently modified with varying concentrations of two distinct VEGFR2-derived peptides with varying serum stability, we analyzed both biomaterial and environmental variables that influence VEGF release and activity. The presence of tethered VEGF-binding peptides (VBPs) resulted in significantly extended VEGF release relative to control conditions, and the resulting released VEGF significantly increased the expansion of human umbilical vein endothelial cells in culture. VEGF release rates were also strongly influenced by the concentration of serum. The presence of Feline McDonough Sarcoma-like tyrosine kinase 1 (sFlt-1), a serum-borne receptor fragment derived from VEGF receptor 1, increased VEGF release rates, although sFlt-1 was not sufficient to recapitulate the release profile of VEGF in serum. Further, the influence of serum on VEGF release was not due to protease activity or non-specific VEGF interactions in the presence of serum-borne heparin. VEGF release kinetics correlated well with a generalizable mathematical model describing affinity-mediated release of VEGF from hydrogel microspheres in defined conditions. Modeling results suggest a potential mechanism whereby competition between VEGF and multiple VEGF-binding serum proteins including sFlt-1, soluble kinase insert domain receptor (sKDR), and α2-macroglobulin (α2-M) likely influenced VEGF release from microspheres. The materials and mathematical model described in this approach may be useful in a range of applications in which sustained, biologically active GF release of a specific GF is desirable.
The vascular-endothelial-growth-factor (VEGF) receptor Flt-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven immunoglobulin-like loops within the extracellular domain and the first three N-terminal immunoglobulin-like loops are involved in high-affinity binding of VEGF. The minimal extracellular domains of Flt-1 to achieve VEGF binding were screened using the yeast two-hybrid system. The result showed that the binding capacity of loop2-3 was close to that of loop1-3. The two truncated mutants consisting of loop2-3 and loop1-3 were expressed in the methylotrophic yeast Pichia pastoris at high levels (0.3 mg/litre). The corresponding proteins, named soluble (s)Flt-1(2-3) and sFlt-1(1-3), were purified. An in vitro biological activity assay showed that the binding capacity of sFlt-1(2-3) to human VEGF165 and the inhibiting effect of it on human umbilical-vein endothelial cell proliferation stimulated by human VEGF165 were close to those of sFlt-1(1-3). Animal tests showed that sFlt-1(2-3) could significantly inhibit the formation of regenerated blood vessels stimulated by hVEGF165.
Vascular endothelial growth factor (VEGF) is a principal regulator of vasculogenesis and angiogenesis. VEGF expresses its effects by binding to two VEGF receptors, Flt-1 and KDR. However, properties of Flt-1 and KDR in the signal transduction of VEGF-mediated effects in endothelial cells (ECs) were not entirely clarified. We investigated this issue by using two newly developed blocking monoclonal antibodies (mAbs) against Flt-1 and KDR. VEGF elicits DNA synthesis and cell migration of human umbilical vein endothelial cells (HUVECs). The pattern of inhibition of these effects by two mAbs indicates that DNA synthesis is preferentially mediated by KDR. In contrast, the regulation of cell migration by VEGF appears to be more complicated. Flt-1 regulates cell migration through modulating actin reorganization, which is essential for cell motility. A distinct signal is generated by KDR, which influences cell migration by regulating cell adhesion via the assembly of vinculin in focal adhesion plaque and tyrosine-phosphorylation of focal adhesion kinase (FAK) and paxillin.
Background:Blocking vascular endothelial
growth factor receptor (VEGFR) is a successful
approach for inhibiting vascular endothelial
growth factor (VEGF) signaling. Small molecules
impairing the interaction of VEGF with VEGF
receptors have been synthesized and evaluated
in this research.
Methods:In this study, we amplified and
cloned the cDNA of VEGFR fragments. After
expression of the fragments in Escherichia
coli(E. coli), they were purified by immobilized
metal affinity chromatography (IMAC).
The biological activity of recombinant KDR
fragments was evaluated by human umbilical
vein endothelial cells (HUVEC) proliferation
assay.
Results:The sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PSGE)
and immune-blotting results confirmed the
production and purification of recombinant
VEGFR fragments. The purified VEGFR2-III
domain, VEGFR2-II-III domains, and VEGFR1-II
domain showed 24%, 36%, and 27%
inhibition effect in HUVEC proliferation assay,
respectively. The recombinant VEGFR2-IIIII domains strongly inhibited formation of
capillary-like structures (CLS).
Conclusions:The produced recombinant
proteins will serve as small soluble molecules
with an inhibitory effect against VEGF in antiangiogenesis researches.
Keywords:angiogenesis, VEGFR fragments,
expression
This study aimed to examine the effects of marchantin C and fucoidan on angiogenesis induced by glioma cells and monocytes, and to elucidate the role of sFlt-1 in this process.
T98G glioma cells and THP1 monocytes were pretreated with marchantin C or fucoidan, respectively. Conditioned media were used for endothelial cell tube formation assay and detection of sFlt-1 by ELISA. Depletion of sFlt-1 was achieved by a neutralizing antibody to assess its role in the process.
Marchantin C inhibited angiogenesis induced by T98G cells while fucoidan inhibited both T98G and THP1 cell-induced angiogenesis. In all three groups in which angiogenesis was inhibited, sFlt-1 level in the supernatants was elevated. Pretreatment of the conditioned media with sFlt-1 antibody restored the inhibited angiogenesis to a certain degree.
This study suggested for the first time that marchantin C and fucoidan could significantly inhibit angiogenesis induced by glioma cells or monocytes. Up-regulation of sFlt-1 played an important role in this process.
During the last decade, the development of new drugs known as targeted therapies was the result of a better understanding of the processes involved in the transformation of normal cells into cancer. The term targeted therapy refers to drugs that selectively target specific molecular pathways involved in tumourigenesis or tumour progression. Angiogenesis is important for tumour growth and metastasis and is an important target for new biological agents. Bevacizumab is a humanised recombinant antibody that prevents vascular endothelial growth factor (VEGF) receptor binding, and inhibits angiogenesis and tumour growth. On February 26, 2004, the FDA (Food and Drug Administration) approved Bevacizumab as first-line treatment for patients with metastatic colorectal cancer. The integration of targeted therapies in the treatment of colon cancer has resulted in significant improvements in efficacy outcomes. Bevacizumab was the first antiangiogenic therapy approved for use in cancer and received accelerated FDA approval for the treatment of recurrent glioblastoma multiform in 2009. The efficacy of Bevacizumab in the treatment of metastatic colorectal cancer and recurrent glioblastoma multiform is presented in this review article. The structural characteristics and selectivity profiles of this antiangiogenic drug and those disclosed in related patent applications are also summarised in this article.
During the last decade, the development of new drugs known as targeted therapies was the result of a better understanding of the processes involved in the transformation of normal cells into cancer. The term targeted therapy refers to drugs that selectively target specific molecular pathways involved in tumorigenesis or tumour progression. Angiogenesis is important for tumour growth and metastasis, and is an important target for new biological agents. Bevacizumab is a humanised recombinant antibody that prevents vascular endothelial growth factor (VEGF) receptor binding, and inhibits angiogenesis and tumour growth. On February 26, 2004, the Food and Drug Administration approved bevacizumab as first-line treatment for patients with metastatic colorectal cancer (CRC). The integration of targeted therapies in the treatment of colon cancer has resulted in significant improvements in efficacy outcomes. The efficacy of bevacizumab in the treatment of metastatic CRC is presented in this review article.
We previously isolated a novel tyrosine kinase receptor, Flt-1, now known as VEGF-receptor (VEGFR)-1. The VEGF–VEGFR system plays a pivotal role in not only physiological but also pathological angiogenesis. We examined the role of Flt-1 in carcinogenesis using Flt-1-signal-deficient (Flt-1 TK−/−) mice, and found that this receptor stimulates tumor growth and metastasis most likely via macrophages, making it an important potential target in the treatment of cancer. In addition to the full-length receptor, the Flt-1 gene produces a soluble protein, sFlt-1, an endogenous VEGF-inhibitor. sFlt-1 is expressed in trophoblasts of the placenta between fetal and maternal blood vessels, suggesting it to be a barrier against extreme VEGF-signaling. Abnormally high expression of sFlt-1 occurs in most preeclampsia patients, whose main symptoms are hypertension and proteinurea. In cancer patients, strong suppression of VEGF–VEGFR by drugs induces similar side effects including hypertension. These results indicate a close relationship between abnormal VEGF-block and hypertension/proteinurea. sFlt-1 is an attractive target for the control of preeclampsia.
(Communicated by Kumao TOYOSHIMA, M.J.A.)
Colorectal cancer continues to be an important public health concern, despite improvements in screening and better systemic chemotherapy. The integration of targeted therapies in the treatment of colon cancer has resulted in significant improvements in efficacy outcomes. Angiogenesis is important for tumor growth and metastasis and is an important target for new biological agents. Bevacizumab is a humanized recombinant antibody that prevents vascular endothelial growth factor (VEGF) receptor binding, and inhibits angiogenesis and tumor growth. The addition of bevacizumab to fluoropyrimidine-based chemotherapy, with or without irinotecan or oxaliplatin, in both the first- and second-line treatment of metastatic colorectal cancer, significantly increased median progression-free survival and overall survival in select randomized phase III studies. Ongoing studies are evaluating the role of bevacizumab in the adjuvant treatment of colon cancer. Common toxicities associated with bevacizumab include hypertension, bleeding episodes, and thrombotic events. This review will focus on the integration of bevacizumab in the treatment paradigm of colon cancer and the management of its side effects.
Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen in vitro and an angiogenic inducer in a variety of in vivo models. VEGF gene transcription is induced in particular in hypoxic cells. In developmental angiogenesis, the role of VEGF is demonstrated by the finding that the loss of a single VEGF allele results in defective vascularization and early embryonic lethality. Substantial evidence also implicates VEGF as a mediator of pathological angiogenesis. In situ hybridization studies demonstrate expression of VEGF mRNA in the majority of human tumors. Platelet-derived growth factor (PDGF) is mainly believed to be an important mitogen for connective tissue, and also has important roles during embryonal development. Its overexpression has been linked to different types of malignancies. Thus, it is important to understand the physiology of VEGF and PDGF and their receptors as well as their roles in malignancies in order to develop antiangiogenic strategies for the treatment of malignant disease.
Angiogenesis is the formation of new blood vessels from existing vasculature. Vascular Endothelial Growth Factor (VEGF), a known angiogenic protein, stimulates endothelial cell proliferation and migration via interactions with its receptors, KDR and Flt-1. A secreted form of Flt-1 (sFlt-1), derived from alternatively-spliced RNA, can inhibit actions of VEGF in vivo. It has been suggested that alterations in sFlt-1 expression could significantly change the angiogenic VEGF activity. This project focuses on characterizing intronic elements that regulate Flt-1 mRNA splicing. A "wild-type" construct (pFIN13), containing the first 13 exons, intron 13 and exons 14-30 of mouse Flt-1, was shown to produce both forms of Flt-1 mRNA after transfection into HEK293 cells. To gauge the strength of the native splicing signals in intron 13 of Flt-1, a series of point mutations were made in the polypyrimidine tract using pFIN13. After transient transfection, the levels of Flt-1 and sFlt-1 protein and mRNA were compared using quantitative PCR, RNA hybridization analysis, and protein immunoblotting. Results from quantitative PCR showed that purine substitutions were associated with 120 to 350 fold decreases in Flt-1 mRNA (normalized against neor), consistent with less efficient splicing. These large decreases in Flt-1 mRNA were accompanied by increases in sFlt-1 mRNA. Modest (20 to 100%) increases in Flt-1 mRNA, reflecting improved splicing, resulted from increasing the uridine complement in the polypyrimidine tract. These results suggest that the wild-type polypyrimidine tract is of intermediate strength and may be a regulatory locus for modulating Flt-1: sFlt-1 ratios. System requirements: PC, World Wide Web browser, and PDF reader. Available electronically via the Internet. Title from electronic submission form. Thesis (M.S.)--Virginia Polytechnic Institute and State University, 2002. Vita. Abstract. Includes bibliographical references.
To prepare a soluble human extracellular III domain of Flt1 and analyze its biological activity. The gene encoding extracellular domain III of Flt-1 was cloned into the expression vector pAZY by RT-PCR from human umbilical vein endothelial cell (HUVEC), and induced to express in Escherichia coli by low phosphoric medium, the product was purified by E-tag affinity chromatography. SDS-PAGE and Western blotting analysis showed that Flt-1 gene domain III gene was expressed in E. coli and the yield of the soluble fusion protein was about 1.10 mg/L. Enzyme-Linked ImmunoSorbent Assay (ELISA) revealed that the Flt-1 domain III was able to bind to VEGF165 dose-dependently. Monolayer denudation assay and Transwell assay showed that the fusion protein could inhibit HUVECs migration induced by conditional medium with 50 ng/mL VEGF165 and 100 ng/mL bFGF. In conclusion, Flt-1 gene domain III gene has been successfully cloned and expressed in E. coli, which will be useful in both the research on the function of Flt-1 gene domain III and preparation of anti-Flt-1 monoclonal antibody in the future.
Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is believed to be a potent mediator of peritoneal fluid accumulation and angiogenesis and of tumor growth in ascites tumor. Such roles, however, have not been generally established because of insufficient quantitative and systemic analyses. To address this, we examined the expression of VEGF in 13 mouse ascites tumors (5 sarcomas, 3 carcinomas, 2 lymphomas, 1 leukemia, 1 mastocytoma, and 1 plasmacytoma). Using a newly developed sensitive and specific radioreceptor binding assay and functional assays, we found that active VEGF was significantly accumulated (6-850 ng/ml) in the ascites fluids of all 13 tumors. VEGF concentrations are higher in the tumors of sarcoma and carcinoma origin (430.4 +/- 234.2 ng/ml) than in those of lymphoma and hematological tumor origin (19.2 +/- 10.45 ng/ml). VEGF that accumulated in the peritoneal fluids or expressed in the ascites tumor cells was easily visualized with immunoprecipitation Western blot analysis with a rough correlation to the expression levels of VEGF gene in these tumor cells, suggesting that the tumor cells, at least in part, contributed to the production of the VEGF that accumulated in the ascites fluid. Most ascites tumors expressed VEGF; the 164-amino acid isoform was predominant, the 120-amino acid isoform was less abundant, and the 188-amino acid isoform was least abundant. Several representative ascites tumors expressed similar, if not higher, levels of VEGF when they were cultured at normoxic states, suggesting that they expressed VEGF at high levels in a constitutive manner. The microvessel densities in the peritoneal walls of tumor-bearing mice, which are significantly higher than those in normal mice, basically correlated to but did not parallel the VEGF concentrations in their respective ascites fluids. Thus, a complicated relationship may exist between the VEGF production and angiogenesis associated with ascites tumor in vivo. Taken together, our observations suggest that VEGF plays a fundamental role in ascites tumor formation; however, its importance may vary according to tumor origin.
Two distinct receptors for vascular endothelial growth factor (VEGF), the tyrosine kinase receptors Flt-1 and Flk-1/KDR, have been described. In this study we show that monocytes, in contrast to endothelium, express only the VEGF receptor Flt-1, and that this receptor specifically binds also the VEGF homolog placenta growth factor (PlGF). Both VEGF and PlGF stimulate tissue factor production and chemotaxis in monocytes at equivalent doses. In contrast, endothelial cells expressing both the Flt-1 and the Flk-1/KDR receptors produce more tissue factor upon stimulation with VEGF than after stimulation with PlGF. Neutralizing antibodies to the KDR receptor reduce the VEGF- stimulated tissue factor induction in endothelial cells to levels obtained by stimulation with PlGF alone, but do not affect PlGF-induced tissue factor induction in endothelial cells nor the VEGF-dependent tissue factor production in monocytes. These findings strongly suggest Flt-1 as a functional receptor for VEGF and PlGF in monocytes and endothelial cells and identify this receptor as a mediator of monocyte recruitment and procoagulant activity.
The fms-like tyrosine kinase (Flt) is a transmembrane receptor in the tyrosine kinase family. Expression of flt complementary
DNA in COS cells conferred specific, high-affinity binding of vascular endothelial growth factor, also known as vascular permeability
factor (VEGF-VPF), a factor that induces vascular permeability when injected in the guinea pig skin and stimulates endothelial
cell proliferation. Expression of Flt in Xenopus laevis oocytes caused the oocytes to release calcium in response to VEGF-VPF.
These findings show that flt encodes a receptor for VEGF-VPF.
The platelet-derived growth factor beta-receptor (PDGFr) is a 180-kDa transmembrane glycoprotein which binds BB-PDGF with high affinity. We have expressed the extracellular region of the receptor in Chinese hamster ovary cells using an expression vector that carries a dihydrofolate reductase gene as an amplifiable marker. Upon amplification of the receptor cDNA sequences by methotrexate a 110-kDa soluble form of the receptor extracellular region (XR) was secreted at 12 mg/liter. The soluble XR protein fully retained the high affinity specific binding of the intact PDGFr for BB-PDGF (apparent dissociation constant, 0.4 nM). In the presence of ligand the soluble XR protein formed complexes that migrated on sodium dodecyl sulfate gels at the size expected for dimers of the protein. When added to fibroblast cultures the soluble XR protein blocked the ability of BB-PDGF to stimulate DNA synthesis but did not alter the mitogenic effect of AA-PDGF. The XR fragment also inhibited the binding of BB-PDGF to PDGFr and the activation of PDGFr tyrosine kinase by BB-PDGF. Thus, the soluble extracellular region protein of the PDGFr binds BB-PDGF with high affinity and functions as a specific antagonist of BB-PDGF actions.
A human cDNA coding for a protein related to the vascular permeability factor (VPF) was isolated from a term placenta cDNA library; we therefore named its product placenta growth factor (PlGF). PlGF is a 149-amino-acid-long protein and is highly homologous (53% identity) to the platelet-derived growth factor-like region of human VPF. Computer analyses reveal a putative signal peptide and two probable N-glycosylation sites in the PlGF protein, one of which is also conserved in human VPF. By using N-glycosidase F, tunicamycin, and specific antibodies produced in both chicken and rabbit, we demonstrate that PlGF, derived from transfected COS-1 cells, is actually N-glycosylated and secreted into the medium. In addition, PlGF, like VPF, proves to be a dimeric protein. Finally, a conditioned medium from COS-1 cells containing PlGF is capable of stimulating specifically the growth of CPA, a line of endothelial cells, in vitro.
Embryonic stem cells (ESC) have been established previously from the inner cell mass cells of mouse blastocysts. In suspension culture, they spontaneously differentiate to blood-island-containing cystic embryoid bodies (CEB). The development of blood vessels from in situ differentiating endothelial cells of blood islands, a process which we call vasculogenesis, was induced by injecting ESC into the peritoneal cavity of syngeneic mice. In the peritoneum, fusion of blood islands and formation of an in vivo-like primary capillary plexus occurred. Transplantation of ESC and ESC-derived complex and cystic embryoid bodies (ESC-CEB) onto the quail chorioallantoic membrane (CAM) induced an angiogenic response, which was directed by nonyolk sac endoderm structures. Neither yolk sac endoderm from ESC-CEB nor normal mouse yolk sac tissue induced angiogenesis on the quail CAM. Extracts from ESC-CEB stimulated the proliferation of capillary endothelial cells in vitro. Mitogenic activity increase during in vitro culture and differentiation of ESC. Almost all growth factor activity was associated with the cells. The ESC-CEB derived endothelial cell growth factor bound to heparin-sepharose. The identification of acidic fibroblast growth factor (FGF)in heparin-sepharose-purified material was accomplished by immunoblot experiments involving antibodies against acidic and basic FGF. We conclude that vasculogenesis, the development of blood vessels from in situ differentiating endothelial cells, and angiogenesis, the sprouting of capillaries from preexisting vessels are very early events during embryogenesis which can be studied using ESC differentiating in vitro. Our results suggest that vasculogenesis and angiogenesis are differently regulated.
We developed a novel promoter system, designated SR alpha, which is composed of the simian virus 40 (SV40) early promoter
and the R segment and part of the U5 sequence (R-U5') of the long terminal repeat of human T-cell leukemia virus type 1. The
R-U5' sequence stimulated chloramphenicol acetyltransferase (CAT) gene expression only when placed immediately downstream
of the SV40 early promoter in the sense orientation. The SR alpha expression system was 1 or 2 orders of magnitude more active
than the SV40 early promoter in a wide variety of cell types, including fibroblasts and lymphoid cells, and was capable of
promoting a high level of expression of various lymphokine cDNAs. These features of the SR alpha promoter were incorporated
into the pcD-cDNA expression cloning vector originally developed by Okayama and Berg.
The recently identified placenta growth factor (PIGF) is a member of the vascular endothelial growth factor (VEGF) family of growth factors. PIGF displays a 53% identity with the platelet-derived growth factor-like region of VEGF. By alternative splicing of RNA, two PIGF isoforms are generated: PIGF131 (PIGF-1) and PIGF152 (PIGF-2). Relative to PIGF131, PIGF152 has a 21-amino acid insertion enriched in basic amino acids. Little is known at the present time about the significance and function of these proteins. To assess their potential role, we cloned the cDNAs coding for both isoforms, expressed them in mammalian cells, and purified to apparent homogeneity the recombinant proteins. Like VEGF, the PIGF isoforms are homodimeric glycoproteins. PIGF131 is a non-heparin binding protein, whereas PIGF152 strongly binds to heparin. We examined the ability of PIGF to bind to soluble VEGF receptors, Flt-1 and Flk-1/KDR, and characterized the binding of PIGF to endothelial cells. While the PIGF proteins bound with high affinity to Flt-1, they failed to bind to Flk-1/KDR. Binding of 125I-PIGF to human endothelial cells revealed two classes of sites, having high and low affinity. The high affinity site is consistent with Flt-1; the identity of the low affinity site remains to be determined. Purified PIGF isoforms had little or no direct mitogenic or permeability-enhancing activity. However, they were able to significantly potentiate the action of low concentrations of VEGF in vitro and, more strikingly, in vivo.
Vascular endothelial cell growth factor, a mitogen selective for vascular endothelial cells in vitro that promotes angiogenesis in vivo, functions through distinct membrane-spanning tyrosine kinase receptors. The cDNA encoding a soluble truncated form of one such receptor, fms-like tyrosine kinase receptor, has been cloned from a human vascular endothelial cell library. The mRNA coding region distinctive to this cDNA has been confirmed to be present in vascular endothelial cells. Soluble fms-like tyrosine kinase receptor mRNA, generated by alternative splicing of the same pre-mRNA used to produce the full-length membrane-spanning receptor, encodes the six N-terminal immunoglobulin-like extracellular ligand-binding domains but does not encode the last such domain, transmembrane-spanning region, and intracellular tyrosine kinase domains. The recombinant soluble human receptor binds vascular endothelial cell growth factor with high affinity and inhibits its mitogenic activity for vascular endothelial cells; thus this soluble receptor could act as an efficient specific antagonist of vascular endothelial cell growth factor in vivo.
Treatment of human monocytes with vascular endothelial growth factor (VEGF) isolated from tumor cell supernatants was reported to induce monocyte activation and migration. In this study we show that recombinant human VEGF165, and VEGF121 had a maximal effect on human monocyte migration at 65 to 250 pmol/L. Chemotactic activity of VEGF165 was inhibited by a specific antiserum against VEGF, by heat treatment of VEGF165, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding protein. Placenta growth factor (PlGF152), a heparin-binding growth factor related to VEGF, was also chemotactic for monocytes at concentrations between 2.5 and 25 pmol/L. In accordance with these findings, human monocytes showed specific and saturable binding for 125I-VEGF165 (half-maximal binding at 1 to 1.5 nmol/L). Using Northern blot analysis, we further could show that human monocytes express only the gene for the VEGF receptor type, flt-1, but not for the second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 to 4 hours) of human monocytes to lipopolysaccharide, a prototypic monocyte activator, led to a significant upregulation of the flt-1 mRNA level. The results presented here suggest that monocyte chemotaxis in response to VEGF and most likely to PlGF152 is mediated by flt-1 and thus show a possible function for the VEGF-receptor flt-1.
Vascular endothelial growth factor (VEGF) expression in various cell types is induced by hypoxia and other stimuli. VEGF mediates endothelial cell proliferation, angiogenesis, vascular growth, and vascular permeability via the endothelial cell receptors, kinase insert domain-containing receptor (KDR)/fetal liver kinase 1 (Flk-1) and FLT-1. Alanine-scanning mutagenesis was used to identify a positively charged surface in VEGF that mediates binding to KDR/Flk-1. Arg82, Lys84 and His86, located in a hairpin loop, were found to be critical for binding KDR/Flk-1, while negatively charged residues, Asp63, Glu64, and Glu67, were associated with FLT-1 binding. A VEGF model based on PDGFb indicated these positively and negatively charged regions are distal in the monomer but are spatially close in the dimer. Mutations within the KDR site had minimal effect on FLT-1 binding, and mutants deficient in FLT-1 binding did not affect KDR binding. Endothelial cell mitogenesis was abolished in mutants lacking KDR affinity; however, FLT-1 deficient mutants induced normal proliferation. These results suggest dual sets of determinants in the VEGF dimer that cross-link cell surface receptors, triggering endothelial cell growth and angiogenesis. Furthermore, this mutational analysis implicates KDR, but not FLT-1, in VEGF induction of endothelial cell proliferation.
Placenta Growth Factor (PIGF) is a new member of vascular endothelial growth factor (VEGF) family. Although VEGF binds Flt family Flt-1 and KDR/Flk-1 tyrosine kinases at high affinity for signal transduction, biological activities and the receptors of PIGF have not been extensively studied. Reverse transcription-PCR showed that PIGF-2, a subtype of PIGF-1 that bears a basic amino acid-rich domain, is more abundant than PIGF-1 and thus is the major subtype in human placenta. Using antibodies specific to PIGF-1 or -2 as markers, we obtained large amounts of PIGFs in the baculovirus expression system. PIGF-2 had growth-stimulatory activity on human umbilical vein endothelial cells and vascular permeability activity in the Miles assay at levels about 10-fold lower than those of VEGF. All PIGF-1 activities were lower than those of PIGF-2. Both PIGFs competed for the binding of 125I-labeled VEGF to Flt-1 receptor but not to KDR/Flk-1 expressed on NIH3T3 cells. Furthermore, 125I-labeled PIGF bound to Flt-1 at high affinity but not to KDR/Flk-1. Supporting the notion that PIGF can use only Flt-1 as a receptor, PIGF activated Flt-1 to autophosphorylate, whereas PIGF could not generate signals from KDR/Flk-1. These results indicate that Flt-1, but not KDR/Flk-1, is a receptor for PIGF, suggesting that the weak biological activities of PIGF are due to its use of only part of the available VEGF signaling. These mild characteristics of PIGF may be important for the appropriate development and maintenance of normal placental tissue.
Treatment of human monocytes with vascular endothelial growth factor (VEGF) isolated from tumor cell supernatants was reported to induce monocyte activation and migration. In this study we show that recombinant human VEGF165, and VEGF121 had a maximal effect on human monocyte migration at 65 to 250 pmol/L. Chemotactic activity of VEGF165 was inhibited by a specific antiserum against VEGF, by heat treatment of VEGF165, and by protein kinase inhibitors. In addition, we could show that VEGF-stimulated monocyte migration is mediated by a pertussis toxin-sensitive GTP-binding protein. Placenta growth factor (PlGF152), a heparin-binding growth factor related to VEGF, was also chemotactic for monocytes at concentrations between 2.5 and 25 pmol/L. In accordance with these findings, human monocytes showed specific and saturable binding for 125I-VEGF165 (half-maximal binding at 1 to 1.5 nmol/L). Using Northern blot analysis, we further could show that human monocytes express only the gene for the VEGF receptor type, flt-1, but not for the second known VEGF receptor, KDR. Resting monocytes expressed low levels of flt-1 gene only. Brief exposure (2 to 4 hours) of human monocytes to lipopolysaccharide, a prototypic monocyte activator, led to a significant upregulation of the flt-1 mRNA level. The results presented here suggest that monocyte chemotaxis in response to VEGF and most likely to PlGF152 is mediated by flt-1 and thus show a possible function for the VEGF-receptor flt-1.
Films of 1,3,4,6-tetrachloro-3a,6a-diphenylglycoluril (conveniently “plated” in reaction vessels from methylene chloride solution) react rapidly in the solid phase with aqueous mixtures of I− and proteins to yield iodinated proteins. Similarly applied, this reagent brings about iodination of cell membranes, although, apparently, at a somewhat lesser rate than in iodinations carried out with lactoperoxidase-glucose oxidase. The stability and sparing solubility of this chloroglycoluril in water can account for the minimal damage to proteins and living cells observed in these iodinations; further, these properties allow for elimination of the reduction step employed at the close of iodinations with soluble chloroamides such as chloramine-T.
Liver parenchymal cells were isolated from adult rats by digesting liver slices or perfusing liver with collagenase. The cell yields were 1.5×l0⁷ and 1.0×10⁸ cells/g liver from slices and perfused liver, respectively, and in both cases the cell viabilities and attachment efficiencies were over 90% and 60%, respectively. The cells were viable for more than one week when cultured in Williams medium E with 10% fetal bovine serum, and addition of insulin and dexamethasone enhanced the maintenance of cell viability. Various biochemical functions of freshly isolated cells and cultured cells were compared in this medium.
In freshly isolated cells, induction of tyrosine transaminase [EC 2.6.1.5] by dexamethasone was low and none of the hormones examined stimulated protein synthesis; but when the cells had been cultured for a few days, induction of tyrosine transaminase became prominent, and insulin and dexamethasone stimulated protein synthesis and glucagon inhibited their effect. About half the synthesized proteins were secreted into the medium, and among these proteins, albumin, transferrin, fibrinogen, and lipoproteins were identified immunochemically and electrophoretically. It was also shown that the polysomes in freshly isolated cells were almost completely disaggregated, but that in cells after a few days culture they were reaggregated.
These results showed that freshly isolated cells have impaired functions, but that after culture for a few days the cells recover various liver functions and thus become more suitable for use in biochemical studies on liver functions.
A new human gene encoding a putative receptor-type tyrosine kinase (RTK) was isolated by screening a placenta cDNA library with a mouse Flt3 probe. The deduced amino acid sequence of the intracellular region of the molecule showed that it was strongly related to the FLT1 and KDR/FLK1 gene products and to a lesser degree to members of the class III RTKs: FMS/CSF1R, PDGFRA/B, KIT, and FLT3. The gene was named FLT4. Cosmid clones of the mouse Flt4 gene were isolated. The human gene was localized to bands q34-q35 of chromosome 5, i.e., slightly telomeric to the CSF1R/PDGRFB tandem of genes, and the mouse homolog to chromosome 11, region A5-B1.
The fms-like tyrosine kinase 4 (FLT4) complementary DNA was cloned from a human HEL erythroleukemia cell library by polymerase chain reaction-amplification. We previously reported a partial sequence of FLT4 and showed that the FLT4 gene maps to chromosomal region 5q33-qter (O. Aprelikova, K. Pajusola, J. Partanen, E. Armstrong, R. Alitalo, S. Bailey, J. McMahon, J. Wasmuth, K. Huebner, and K. Alitalo, Cancer Res., 52: 746-748, 1992). Here we present the full-length sequence of the predicted FLT4 protein. The extracellular domain of FLT4 consists of 7 immunoglobulin-like loops, including 12 potential glycosylation sites. On the basis of structural similarities FLT4 and the previously known FLT1 and kinase insert domain-containing receptor tyrosine kinase/fetal liver kinase 1 (KDR/FLK1) receptors constitute a subfamily of class III tyrosine kinases. FLT4 was expressed as 5.8- and 4.5-kilobase mRNAs which were found to differ in their 3' sequences and to be differentially expressed in the HEL and DAMI leukemia cells. Interestingly, a Wilms' tumor cell line, a retinoblastoma cell line, and a nondifferentiated teratocarcinoma cell line expressed FLT4, whereas differentiated teratocarcinoma cells were negative. Most fetal tissues also expressed the FLT4 mRNA, with spleen, brain intermediate zone, and lung showing the highest levels. In in situ hybridization the FLT4 autoradiographic grains decorated bronchial epithelial cells of fetal lung. No evidence was obtained for the expression of FLT4 in the endothelial cells of blood vessels.
Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, encodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of M(r) = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, flt, in a manner reminiscent of the similarities between the alpha and beta forms of the PDGF receptors.
We have cloned a receptor tyrosine kinase cDNA, designated fetal liver kinase 1 (Flk-1), from mouse cell populations enriched for hematopoietic stem and progenitor cells. Sequence analysis of this clone reveals strong homology to the c-Kit subfamily of receptor kinases, and in particular to the Flt gene product. Chromosomal mapping shows that the Flk-1, Kit, and Pdgfra genes are closely linked. Flk-1 mRNA is expressed in primitive and more mature hematopoietic cells as well as in a wide variety of nonhematopoietic tissues.
Vascular endothelial growth factor (VEGF) was identified as a heparin-binding polypeptide mitogen with a target cell specificity restricted to vascular endothelial cells. Molecular cloning reveals the existence of four species of VEGF having 121, 165, 189, and 206 amino acids. These have strikingly different secretion patterns, which suggests multiple physiological roles for this family of polypeptides. The two shorter forms are efficiently secreted, while the longer ones are mostly cell-associated. Alternative splicing of mRNA, rather that transcription from different genes, is the mechanism for their generation. In situ hybridization reveals that the VEGF mRNA is widely distributed in most tissues and organs and expressed at particularly high levels in areas of active vascular proliferation, like the ovarian corpus luteum. Ligand autoradiography on rat tissue sections demonstrates that VEGF binding sites are associated with vascular endothelial cells of both fenestrated and non-fenestrated capillaries and with the endothelium of large vessels, while no displaceable binding is evident on non-endothelial cell types. These findings support the hypothesis that VEGF plays a highly specific role in the maintenance and in the induction of growth of vascular endothelial cells.
A new human gene encoding a receptor-type tyrosine kinase was isolated by a weak cross-hybridization with v-ros oncogene. A cDNA of about 7.7 kb carried a 4.2 kb open reading frame, and the predicted amino acid sequence of 1338 residues contained extracellular, transmembrane and tyrosine kinase domains. Although its extracellular domain is approximately 220 amino acids longer than those of the products of the fms family, i.e. c-fms, c-kit and platelet-derived growth factor receptor genes, the overall structure including cysteine motifs in its extracellular domain and a long peptide insertion in its tyrosine kinase domain indicates that this new gene is closely related to the fms family. Consequently, the gene was designated as flt (fms-like tyrosine kinase) gene. The expression of the flt gene was strongly suppressed in most of the tumor cell lines examined so far, whereas this mRNA was expressed in a variety of normal tissues of adult rat.
DNA synthesis of adult rat parenchymal hepatocytes alone in primary culture can be stimulated only by the addition of humoral growth factors to the culture medium. However, when parenchymal hepatocytes were cocultured with nonparenchymal liver cells from adult rats, their DNA synthesis was markedly stimulated in the absence of added growth factors or calf serum. DNA synthesis of parenchymal hepatocytes was not stimulated by conditioned medium from nonparenchymal liver cells and was greatest when the parenchymal cells were plated on 24-h cultures of nonparenchymal liver cells. A dead feeder layer of nonparenchymal cells was almost as effective as a feeder layer of viable nonparenchymal cells. These results suggest that the stimulation of DNA synthesis in parenchymal hepatocytes was not due to some soluble factors secreted by nonparenchymal liver cells but to an insoluble material(s) produced by the nonparenchymal liver cells. This insoluble material(s) was collagenase- and acid-sensitive, suggesting that it was a protein containing collagen. The effect of nonparenchymal liver cells was specific: coculture with hepatoma cells, liver epithelial cells, or Swiss 3T3 cells did not stimulate DNA synthesis in parenchymal hepatocytes. Added insulin and epidermal growth factor showed additive effects with nonparenchymal cells in the cocultures. These results suggest that DNA synthesis in parenchymal hepatocytes is stimulated not only by various humoral growth factors but also by cell-cell interaction between parenchymal and nonparenchymal hepatocytes, possibly endothelial cells. This cell-cell interaction may be important in repair of liver damage and liver regeneration.
Receptor dimerization is ubiquitous to the action of all receptor tyrosine kinases, and in the case of dimeric ligands, such as the stem cell factor (SCF), it was attributed to ligand bivalency. However, by using a dimerization-inhibitory monoclonal antibody to the SCF receptor, we confined a putative dimerization site to the nonstandard fourth immunoglobulin-like domain of the receptor. Deletion of this domain not only abolished ligand-induced dimerization and completely inhibited signal transduction, but also provided insights into the mechanism of the coupling of ligand binding to dimer formation. These results identify an intrinsic receptor dimerization site and suggest that similar sites may exist in other receptors.
Examination of flk-1 receptor tyrosine kinase mRNA expression by in situ hybridization analysis revealed specific association with endothelial cells at all stages of mouse development, including the blood islands in the yolk sac of day 8.5-10.5 embryos, in which the early progenitors of this lineage originate. flk-1 transcripts were abundant in proliferating endothelial cells of vascular sprouts and branching vessels of embryonic and early postnatal brain, but were drastically reduced in adult brain, where proliferation has ceased. Identification of the angiogenic mitogen, vascular endothelial growth factor (VEGF), as the high affinity ligand of Flk-1 and correlation of the temporal and spatial expression pattern of Flk-1 and VEGF suggest a major role of this ligand-receptor signaling system in vasculogenesis and angiogenesis.
Flt-1 (fms-like tyrosine kinase-1), a receptor-type tyrosine kinase of sharing similar features with two other flt-family encoded proteins KDR/Flk-1 and Flt-4, has been recently identified as a receptor for Vascular Endothelial Growth Factor (VEGF) known to induce the proliferation of vascular endothelial cells. In this study, we demonstrate that Flt-1 encodes for a 180 kDa glycoprotein, binds VEGF with high affinity, undergoes autophosphorylation but does not generate any mitogenic response in transfected NIH3T3 fibroblasts. Interestingly, the immediate early gene c-myc was not induced, whereas the c-fos was induced very weakly in Flt-1 expressing NIH3T3 cells. A comparative analysis of the Flt-1 signal cascade in the environment of endothelial cells with that of Flt-1 expressing NIH3T3 cells showed that VEGF induced phosphorylation of PLC gamma and GAP complex on tyrosine in both type of cells. However, a strong activation of MAP kinases was observed only in endothelial cells. Further, different from many other receptor tyrosine kinases, tyrosine phosphorylation of Shc protein, an important adaptor for signal transduction from many receptor kinases, was very weak in both Flt-1-NIH3T3 cells and endothelial cells. These results suggest that Flt-1 kinase utilizes a unique signal transduction system in endothelial cells, and the activation of the Flt-1 kinase is insufficient to trigger a mitogenic response in NIH3T3 fibroblasts.
Hepatocyte Growth Factor (HGF)/Scatter Factor secreted from sinusoidal endothelial cells and Kupffer cells in liver activates the c-Met tyrosine kinase receptor expressed on hepatocytes. Here we report yet another possible communication system through a different ligand and tyrosine kinase receptor in an opposite direction. We isolated and determined the primary structure of the entire coding region of rat flt-1 (fms-like tyrosine kinase), a receptor for Vascular Endothelial Growth Factor (VEGF). Using rat flt-1 cDNA as a probe we found that the flt-1 mRNA was expressed at very high levels in sinusoidal endothelial cells in normal rat liver, but was hardly detectable in hepatocytes. The transcripts of another VEGF receptor KDR/Flk-1 structurally related to Flt-1 was also expressed specifically in sinusoidal endothelial cells. On the other hand, VEGF mRNA was expressed weakly in hepatocytes, but not in the nonparenchymal cell fraction. Furthermore, in an in vitro culture system, VEGF demonstrated a remarkably specific growth-stimulatory activity as well as maintenance activity on the sinusoidal endothelial cells. These results suggest that hepatocytes regulate the proliferation and survival of the sinusoidal endothelial cells in liver in a paracrine manner. Therefore two reciprocal communication systems, VEGF-Flt receptor family and HGF-Met receptor, may exist in hepatic tissue.
Vascular endothelial cell growth factor binds with high affinity to FLT and KDR, two homologous tyrosine kinase receptors expressed on vascular endothelial cells. Placental growth factor, a vascular endothelial cell growth factor homologue, also binds with high affinity to the extracellular domains of FLT but not to the extracellular region of KDR. Vascular endothelial cell growth factor binds competitively with placental growth factor to the extracellular ligand binding domains of FLT, indicating that both ligands probably complex to overlapping or identical regions of this receptor.
This chapter focuses on the role of the vascular endothelial growth factor (VEGF)-Flt receptor system in normal and tumor angiogeneses. The vascular network in the body is crucial for the development and maintenance of a variety of normal tissues in higher organisms. Vascular formation is important not only for the establishment of stable circular systems during embryogenesis in vertebrates but also for the rapid and transient angiogenesis under physiological conditions of the adult, such as the formation of the corpus luteum. Angiogenesis is widely known to be associated with various pathological conditions: inflammatory lesions, wound healing, endocrine diseases, particularly diabetes mellitus, and growing tumors in vivo. To understand the mechanisms underlying angiogenesis during pathological conditions, an extensive analysis of the physiological processes of vasculogenesis and angiogenesis at molecular levels is essential. The VEGF-Flt receptor family system is utilized under normal angiogenic conditions; it appears to be one of the major signal transduction systems in reciprocal communication between endothelial cells and the surrounding parenchymal cells and is involved in a wide variety of tumor angiogeneses in vivo. VEGF was originally isolated from ascites-generating tumors. The contribution of VEGF to ascites or pleural effusion is different in every patient with tumor.
Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.
Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor
- D Maglione
- V Guerriero
- G Viglietto
- P Delli-Bovi
- M G Persico
A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit
- W Matthews
- C T Jordan
- M Gavin
- N A Jenkins
- N G Copeland
- I R Lemischka
A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF
- L Seetharam
- N Gotoh
- Y Maru
- G Neufeld
- S Yamaguchi
- M Shibuya
FLT4 receptor tyrosine kinase contains seven immunoglobulin-like loops and is expressed in multiple human tissues and cell lines
- K Pajusola
- O Aprelikova
- J Korhonen
- A Kaipainen
- L Pertovaara
- R Alitalo
- K Alitalo
Growth factor signaling by receptor tyrosine kinases
- Schlessinger
Nucleotide sequence and expression of a novel human receptor‐type tyrosine kinase gene (fit) closely related to the fins family
- Shibuya M.
A receptor tyrosine kinase cDNA isolated from a population of enriched primitive hematopoietic cells and exhibiting close genetic linkage to c-kit
- Matthews
A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF
- Seetharam L.
FLT4 receptor tyrosine kinase contains seven immunoglobulin‐like loops and is expressed in multiple human tissues and cell lines
- Pajusola K.
High affinity VEGF binding and developmental expression suggest Flk-1 as a major regulator of vasculogenesis and angiogenesis
- Millauer
Isolation of a human placenta cDNA coding for a protein related to the vascular permeability factor
- Maglione
The vascular endothelial growth factor receptor Flt-1 mediates biological activities
- Clauss