Sachiko Yamaguchi

A single autophosphorylation site on KDR/Flk‐1 is essential for VEGF‐A‐dependent activation of PLC‐γ and DNA synthesis in vascular endothelial cells

KDR/Flk‐1 tyrosine kinase, one of the two vascular endothelial growth factor (VEGF) receptors, induces mitogenesis and differentiation of vascular endothelial cells. To understand the mechanisms underlying the VEGF‐A‐induced growth signaling pathway, we constructed a series of human KDR mutants and examined their biological properties. An in vitro kinase assay and subsequent tryptic peptide mapping revealed that Y1175 and Y1214 are the two major VEGF‐A‐dependent autophosphorylation sites. Using an antibody highly specific to the phosphoY1175 region, we demonstrated that Y1175 is phosphorylated rapidly in vivo in primary endothelial cells. When the mutated KDRs were introduced into the endothelial cell lines by adenoviral vectors, only the Y1175F KDR, Tyr1175 to phenylalanine mutant, lost the ability to tyrosine phosphorylate phospholipase C‐γ and, significantly, reduced MAP kinase phosphorylation and DNA synthesis in response to VEGF‐A. Furthermore, primary endothelial cells microinjected with anti‐phosphoY1175 antibody clearly decreased DNA synthesis compared with control cells. These findings strongly suggest that autophosphorylation of Y1175 on KDR is crucial for endothelial cell proliferation, and that this region is a new target for anti‐angiogenic reagents.

A Novel Type of Vascular Endothelial Growth Factor, VEGF-E (NZ-7 VEGF), Preferentially Utilizes KDR/Flk-1 Receptor and Carries a Potent Mitotic Activity without Heparin-binding Domain

Vascular endothelial growth factor (VEGF) mediates endothelial cell proliferation, angiogenesis, and vascular permeability via the endothelial cell receptors, KDR/Flk-1 and Flt-1. Recently, a gene encoding a polypeptide with about 25% amino acid identity to mammalian VEGF was identified in the genome of Orf virus (OV), a parapoxvirus that affects sheep and goats and occasionally, humans, to generate lesions with angiogenesis. In this study, we examined the biological activities and receptor of OV-derived NZ-7 VEGF (VEGF-E). VEGF-E was found to be a dimer of about 20 kDa with no basic domain nor affinity for heparin column, similar to VEGF121 subtype. VEGF121 has 10–100-fold less endothelial cell mitotic activity than VEGF165 due to lack of a heparin-binding basic region. Interestingly, however, VEGF-E showed almost equal levels of mitotic activity on primary endothelial cells and vascular permeability activity as VEGF165. Furthermore, VEGF-E bound KDR/Flk-1 (VEGFR-2) and induced its autophosphorylation to almost the same extent as VEGF165, but did not bind Flt-1 (VEGFR-1) nor induce autophosphorylation of Flt-1. These results indicate that VEGF-E is a novel type of endothelial growth factor, utilizing only one of the VEGF receptors, and carrying a potent mitogenic activity without affinity to heparin.

Mapping of the Sites Involved in Ligand Association and Dissociation at the Extracellular Domain of the Kinase Insert Domain-containing Receptor for Vascular Endothelial Growth Factor

The kinase insert domain-containing receptor (KDR) for vascular endothelial growth factor (VEGF) has been shown to be involved in vasculogenesis and angiogenesis. This receptor is characterized by seven immunoglobulin (Ig)-like domains within its extracellular region. To identify the domains involved in VEGF binding, we constructed various deletion mutants of the extracellular region fused with the crystallizable fragment portion of an IgG and then examined the binding affinity with VEGF by means of the BIAcore biosensor assay. Deletion of the COOH-terminal two or three Ig-like domains out of a total of seven affected ligand dissociation rather than association. Further deletion of the fourth domain caused a drastic decrease in the association rate. Binding ability was abolished completely with removal of the third domain. The mutant KDR proteins lacking the NH2-terminal Ig-like domain exhibited a slightly higher association rate compared with those of the mutants having this domain. Deletion of the first two NH2-terminal Ig-like domains caused a drastic reduction in the association rate, but affinity to VEGF was retained. These results suggest 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. The first Ig-like domain may regulate the ligand binding.

Epithelial cell-derived IL-25, but not Th17 cell-derived IL-17 or IL-17F, is crucial for murine asthma.

IL-17A, IL-17F, and IL-25 are ligands for IL-17RA. In the current study, we demonstrated that IL-25–deficient mice—but not IL-17A–, IL-17F–, IL-17A/F–, IL-23p19–, or retinoic acid-related orphan receptor (ROR)-γt–deficient mice—showed significant suppression of 1) the number of eosinophils and the levels of proinflammatory mediators in bronchoalveolar lavage fluids, 2) airway hyperresponsiveness to methacholine, and 3) OVA-specific IgG1 and IgE levels in the serum during OVA-induced Th2-type/eosinophilic airway inflammation. The IL-25 deficiency did not affect lung dendritic cell migration or Ag-specific memory–Th2 cell expansion during Ag sensitization. Adoptive transfer of T cells, mast cells, or bone marrow cells from IL-25–deficient mice revealed that induction of Th2-type/eosinophilic airway inflammation was dependent on activation of lung epithelial cells and eosinophils by IL-25 produced by airway structural cells such as epithelial cells but not by such hematopoietic stem-cell-origin immune cells as T cells and mast cells. Therefore, airway structural cell-derived IL-25—rather than Th17 cell-derived IL-17A and IL-17F—is responsible for induction of local inflammation by promoting activation of lung epithelial cells and eosinophils in the elicitation phase of Th2-type/eosinophilic airway inflammation. It is not required for Ag-specific Th2 cell differentiation in the sensitization phase.

Characterization of the extracellular domain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosine kinase).

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.

Flt-1, a receptor for vascular endothelial growth factor, has transforming and morphogenic potentials.

A paradox of Flt-1, a tyrosine kinase receptor for vascular endothelial growth factor (VEGF), is that the ligand cannot activate the receptor to stimulate growth of cells that exogenously overexpress the receptor. In order to find Flt-1 kinase-dependent biological systems, we obtained for the first time activated forms of the Flt-1 kinase in a ligand-independent manner. Replacement of the ABL sequences in the human leukemia oncoprotein BCR–ABL with the cytoplasmic domain of Flt-1 (BCR–FLT) followed by a retroviral random mutagenesis scheme gave constitutively active artificial chimera BCR–FLTm with mutations within the Flt-1 sequence. Like BCR–ABL it could, but not the original BCR–FLT, transform Rat1 fibroblasts, abrogate cytokine dependence in Ba/F3 cells, and induce neurite-like structures in neuronal PC12 cells. Interestingly, Rat1 cells transformed by BCR–FLTm formed tube-like structures in basement membrane matrix. BCR–FLTm retroviruses may be a very useful tool to investigate an as yet uncovered functions of the Flt-1 kinase.

Chimeric VEGF-ENZ7/PlGF Promotes Angiogenesis Via VEGFR-2 Without Significant Enhancement of Vascular Permeability and Inflammation

Objective—Vascular endothelial growth factor (VEGF) plays critical roles in the regulation of angiogenesis and lymphangiogenesis. However, tissue edema, hemorrhage, and inflammation occur when VEGF-A is used for angiogenic therapy. To design a novel angiogenic factor without severe side effects, we examined the biological function of chimeric VEGF-ENZ7/placental growth factor (PlGF), which is composed of Orf-VirusNZ7-derived VEGF-ENZ7 and human PlGF1, in a transgenic (Tg) mouse model. Methods and Results—A strong angiogenic response was observed in both VEGF-ENZ7/PlGF and VEGF-A165 Tg mice. Notably, the vascular leakage of VEGF-ENZ7/PlGF-induced blood vessels was 4-fold lower than that of VEGF-A165–induced blood vessels. Furthermore, the monocyte/macrophage recruitment in the skin of VEGF-ENZ7/PlGF Tg mice was ≈8-fold decreased compared with that of VEGF-A165 Tg mice. In addition, the lymphatic vessels in VEGF-ENZ7/PlGF Tg mice were structurally normal, whereas they were markedly dilated in VEGF-A165 Tg mice, possibly because of the high vascular leakage. Receptor binding assay demonstrated that VEGF-ENZ7/PlGF was the ligand only activating VEGF receptor (VEGFR)-2. Conclusion—These results indicated that neither the hyperpermeability in response to simultaneous stimulation of VEGFR-1 and VEGFR-2 nor VEGFR-1–mediated severe inflammation was associated with VEGF-ENZ7/PlGF-induced angiogenesis. The unique receptor binding property may shed light on VEGF-ENZ7/PlGF as a novel candidate for therapeutic angiogenesis.

Virally activated Ras cooperates with integrin to induce tubulogenesis in sinusoidal endothelial cell lines.

Four cell lines, named nonparenchymal 11 (NP11), NP26, NP31, and NP32, were established from sinusoidal endothelial cells (SECs) of rat liver. They still retained expression of receptors for vascular endothelial growth factor (VEGF), Flt‐1, and kinase domain‐containing receptor (KDR). NP31 and NP32 turned out to be incapable of tubulogenesis in basement membrane matrix (Matrigel), which belongs to endothelial properties, as shown by SECs in primary culture. Expression of temperature‐sensitive, virally activated Ras (ts‐v‐Ras) restored tubulogenic behaviors back to NP31 only at permissive temperature. Matrigel induced long‐lasting tyrosine phosphorylation of Shc, with recruitment of Grb‐2 and microtubule‐associated protein kinase (MAPK) activation in both parental NP31 and NP31 transformed by ts‐v‐Ras, which was blocked by anti‐β1 integrin antibody. Tubulogenesis was inhibited by adenovirus‐mediated expression of dominant‐negative Ras in human umbilical vein endothelial cells (HUVECs). PD 098059, a selective inhibitor of MAPK kinase (MEK), nearly perfectly blocked tubulogenesis by ts‐v‐Ras‐expressing NP31 cells at permissive temperature. Furthermore, the botulinum C3 toxin, an inhibitor for Rho, caused fragmentation of branching cords in networks formed by NP31 that expressed ts‐v‐Ras at permissive temperature. These data suggest that the integrin‐mediated Ras signals may be necessary but are not sufficient for tubulogenesis and that an artificial expression of v‐Ras might substitute for the second signal required in this system. J. Cell. Physiol. 176:223–234, 1998.

The importance of bacterial and viral infections associated with adult asthma exacerbations in clinical practice.

Background Viral infection is one of the risk factors for asthma exacerbation. However, which pathogens are related to asthma exacerbation in adults remains unclear. Objective The relation between various infections and adult asthma exacerbations was investigated in clinical practice. Methods The study subjects included 50 adult inpatients due to asthma exacerbations and 20 stable outpatients for comparison. The pathogens from a nasopharyngeal swab were measured by multiplex PCR analysis. Results Asthma exacerbations occurred after a common cold in 48 inpatients. The numbers of patients with viral, bacterial, or both infections were 16, 9, and 9, respectively. The dominant viruses were rhinoviruses, respiratory syncytial virus, influenza virus, and metapneumovirus. The major bacteria were S. pneumoniae and H. influenzae. Compared to pathogen-free patients, the patients with pathogens were older and non-atopic and had later onset of disease, lower FeNO levels, lower IgE titers, and a higher incidence of comorbid sinusitis, COPD, or pneumonia. Compared to stable outpatients, asthma exacerbation inpatients had a higher incidence of smoking and comorbid sinusitis, COPD, or pneumonia. Viruses were detected in 50% of stable outpatients, but a higher incidence of rhinovirus, respiratory syncytial virus, and metapneumovirus infections was observed in asthma exacerbation inpatients. H. influenzae was observed in stable asthmatic patients. Other bacteria, especially S. pneumoniae, were important in asthma exacerbation inpatients. Conclusion Viral or bacterial infections were observed in 70% of inpatients with an asthma exacerbation in clinical practice. Infection with S. pneumoniae was related to adult asthma exacerbation.

Involvement of VEGF and its receptors in ascites tumor formation.

Abstract Vascular endothelial growth factor (VEGF) has potent endothelial cell mitotic and vascular permeability activity. Several reports have suggested that VEGF may be one of the major factors regulating ascites formation, although no quantitative and systematic analyses have been carried out. To determine the role of VEGF in ascites formation, we examined the expression of VEGF in 13 mouse ascites tumors (5 sarcomas, 3 carcinomas, and 5 hematopoietic malignancies). We found that significant amounts (6–850 ng/mL) of biologically active VEGF accumulated in the ascites fluid of all 13 tumors, particularly in tumors of sarcoma and carcinoma origin (430 ± 234 ng/mL). The microvessel densities in the peritoneal walls of tumor-bearing mice, which are significantly higher than those in healthy mice, basically correlated with but did not parallel VEGF concentrations, suggesting the existence of an additional modulator(s) of the angiogenic process. Administration of anti-mouse VEGF-neutralizing antibody to mice bearing the carcinoma-derived ascites tumor MM2 suppressed ascites accumulation, tumor growth, and tendency to bleed. These results directly demonstrate the crucial role of VEGF in carcinoma-derived ascites tumor formation in vivo.