Vascular endothelial cadherin is an endostatin receptor

Angiogenesis is involved in tumor growth and metastasis. Endostatin inhibits angiogenesis, but its precise mechanism is not fully understood. To clarify signal transduction involved in endostatin-induced angiogenesis inhibition (endothelial cell growth inhibition), it is important to identify an endostatin receptor, which is the aim of the present study. We hypothesized that vascular endothelial cadherin (VE-cadherin) is an endostatin receptor and found that endostatin induced apoptosis in cultured calf pulmonary artery endothelium (CPAE) cells. Immunoprecipitation and western blots revealed that endostatin specifically bound to VE-cadherin in a Ca²⁺-dependent manner. Binding of endostatin to VE-cadherin induced tyrosine phosphorylation of VE-cadherin, β-catenin recruitment, and endothelial cell death. Antisense oligonucleotides against VE-cadherin rescued endostatin-induced endothelial cell death. Inhibition of tyrosine phosphorylation of VE-cadherin inhibited endostatin-induced β-catenin recruitment and CPAE cell death. Taken together, we conclude that VE-cadherin is an endostatin receptor.     

Vascular endothelial growth factor activates the Tie family of receptor tyrosine kinases

The ability of cells to respond appropriately to changes in their environment requires integration and cross-talk between relevant signalling pathways. The vascular endothelial growth factor (VEGF) and angiopoietin families of ligands are key regulators of blood vessel formation. VEGF binds to receptor tyrosine kinases of the VEGF-receptor family to activate signalling pathways leading to endothelial migration, proliferation and survival whereas the angiopoietins interact with the Tie receptor tyrosine kinases to control vessel stability, survival and maturation. Here we show that VEGF can also activate the angiopoietin receptor Tie2. Activation of human endothelial cells with VEGF caused a four-fold stimulation of tyrosine phosphorylation of Tie2. This stimulation was not due to VEGF-induction of Tie2 ligands as soluble ligand binding domain of Tie2 failed to inhibit VEGF activation of the receptor. Immunoprecipitation analysis demonstrated no physical interaction between VEGF receptors and Tie2. However Tie2 does interact with the related receptor tyrosine kinase Tie1 and this receptor was found to be essential for VEGF activation of Tie2. VEGF stimulated proteolytic cleavage of Tie1 generating a truncated Tie1 intracellular domain. Similarly, phorbol ester also both stimulated Tie1 truncation and activated Tie2 phosphorylation. Inhibition of Tie1 cleavage with the metalloprotease inhibitor TAPI-2 suppressed VEGF- and phorbol ester-induced phosphorylation of Tie2. Truncated Tie1 formed in response to VEGF was also found to be tyrosine phosphorylated and this was independent of Tie2, though Tie2 could enhance Tie1 intracellular domain phosphorylation. Together these data demonstrate that VEGF activates Tie2 via a mechanism involving proteolytic cleavage of the associated tyrosine kinase Tie1 leading to trans-phosphorylation of Tie2. This novel mechanism of receptor tyrosine kinase activation is likely to be important in integrating signalling between two of the key receptor groups regulating angiogenesis.     

Alteration of Interendothelial Adherens Junctions Following Tumor Cell–Endothelial Cell Interactionin Vitro

The integrity of the vascular endothelium is mainly dependent upon the organization of interendothelial adherens junctions (AJ). These junctions are formed by the homotypic interaction of a transmembrane protein, vascular endothelial cadherin (VE-cadherin), which is complexed to an intracellular protein network including α-, β-, and γ-catenin. Additional proteins such as vinculin and α-actinin have been suggested to link the VE-cadherin/catenin complex to the actin-based cytoskeleton. During the process of hematogenous metastasis, circulating tumor cells must disrupt these intercellular junctions in order to extravasate. In the present study, we have investigated the influence of tumor cell–endothelial cell interaction upon interendothelial AJ. We show that human breast adenocarcinoma cells (MCF-7), but not normal human mammary epithelial cells, induce a rapid endothelial cell (EC) dissociation which correlates with the loss of VE-cadherin expression at the site of tumor cell–EC contact and with profound changes in vinculin distribution and organization. This process could not be inhibited by metalloproteinase nor serine protease inhibitors. Immunoprecipitations and Western blot analysis demonstrate that the overall expression of VE-cadherin and vinculin as well as the composition of the VE-cadherin/catenins complex are not affected by tumor cells while the tyrosine phosphorylation status of proteins within the complex is significantly altered. Our data suggest that tumor cells modulate AJ protein distribution and phosphorylation in EC and may, thereby, facilitate EC dissociation.     

Tyrosine phosphorylation of sam68 by breast tumor kinase regulates intranuclear localization and cell cycle progression

The breast tumor kinase (BRK) is a growth promoting non-receptor tyrosine kinase overexpressed in the majority of human breast tumors. BRK is known to potentiate the epidermal growth factor (EGF) response in these cells. Although BRK is known to phosphorylate the RNA-binding protein Sam68, the specific tyrosines phosphorylated and the exact role of this phosphorylation remains unknown. Herein, we have generated Sam68 phospho-specific antibodies against C-terminal phosphorylated tyrosine residues within the Sam68 nuclear localization signal. We show that BRK phosphorylates Sam68 on all three tyrosines in the nuclear localization signal. By indirect immunofluorescence we observed that BRK and EGF treatment not only phosphorylates Sam68 but also induces its relocalization. Tyrosine 440 was identified as a principal modulator of Sam68 localization and this site was phosphorylated in response to EGF treatment in human breast tumor cell lines. Moreover, this phosphorylation event was inhibited by BRK small interfering RNA treatment, consistent with Sam68 being a physiological substrate of BRK downstream of the EGF receptor in breast cancer cells. Finally, we observed that Sam68 suppressed BRK-induced cell proliferation, suggesting that Sam68 does indeed contain anti-proliferative properties that may be neutralized in breast cancer cells by phosphorylation.     

The phosphorylation of tyrosine 332 is necessary for the caspase 3-dependent cleavage of PKCdelta and the regulation of cell apoptosis

Protein kinase C delta (PKCdelta plays a major role in the regulation of cell apoptosis and survival. PKCdelta is cleaved by caspase 3 to generate a constitutively active catalytic domain that mediates both its apoptotic and anti-apoptotic effects. The caspase cleavage site of PKCdelta in the hinge region is flanked by the two tyrosine residues, Y311 and Y332. Here, we examined the role of the phosphorylation of tyrosines 311 and 332 in the cleavage and apoptotic function of PKCdelta using the apoptotic stimuli, TRAIL and cisplatin. Tyrosine 332 was constitutively phosphorylated in the A172 and HeLa cells and was further phosphorylated by TRAIL and cisplatin. This phosphorylation was inhibited by the Src inhibitors, PP2 and SU6656, and by silencing of Src. Treatment of the A172 and HeLa cells with TRAIL induced cleavage of the WT PKCdelta and of the PKCdeltaY311F mutant, whereas a lower level of cleavage was observed in the PKCdeltaY332F mutant. Similarly, a smaller degree of cleavage of the PKCdeltaY332 mutant was observed in LNZ308 cells treated with cisplatin. Mutation of Y332F affected the apoptotic function of PKCdelta; overexpression of the PKCdeltaY332 mutant increased the apoptotic effect of TRAIL, whereas it decreased the apoptotic effect of cisplatin. Inhibition of Src decreased the cleavage of PKCdelta and modified the apoptotic responses of the cells to TRAIL and cisplatin, similar to effect of the PKCdeltaY332F mutant. These results demonstrate that the phosphorylation of tyrosine 332 by Src modulates the cleavage of PKCdelta and the sensitivity of glioma cells to TRAIL and cisplatin.     

Contact inhibition of VEGF-induced proliferation requires vascular endothelial cadherin,beta-catenin,and the phosphatase DEP-1/CD148

Confluent endothelial cells respond poorly to the proliferative signals of VEGF. Comparing isogenic endothelial cells differing for vascular endothelial cadherin (VE-cadherin) expression only, we found that the presence of this protein attenuates VEGF-induced VEGF receptor (VEGFR) 2 phosphorylation in tyrosine, p44/p42 MAP kinase phosphorylation, and cell proliferation. VE-cadherin truncated in beta-catenin but not p120 binding domain is unable to associate with VEGFR-2 and to induce its inactivation. beta-Catenin-null endothelial cells are not contact inhibited by VE-cadherin and are still responsive to VEGF, indicating that this protein is required to restrain growth factor signaling. A dominant-negative mutant of high cell density-enhanced PTP 1 (DEP-1)//CD148 as well as reduction of its expression by RNA interference partially restore VEGFR-2 phosphorylation and MAP kinase activation. Overall the data indicate that VE-cadherin-beta-catenin complex participates in contact inhibition of VEGF signaling. Upon stimulation with VEGF, VEGFR-2 associates with the complex and concentrates at cell-cell contacts, where it may be inactivated by junctional phosphatases such as DEP-1. In sparse cells or in VE-cadherin-null cells, this phenomenon cannot occur and the receptor is fully activated by the growth factor.     

Detection of hepatocyte growth factor (HGF) ligand-c-MET receptor activation in formalin-fixed paraffin embedded specimens by a novel proximity assay

Aberrant activation of membrane receptors frequently occurs in human carcinomas. Detection of phosphorylated receptors is commonly used as an indicator of receptor activation in formalin-fixed paraffin embedded (FFPE) tumor specimens. FFPE is a standard method of specimen preparation used in the histological analysis of solid tumors. Due to variability in FFPE preparations and the labile nature of protein phosphorylation, measurements of phospho-proteins are unreliable and create ambiguities in clinical interpretation. Here, we describe an alternative, novel approach to measure receptor activation by detecting and quantifying ligand-receptor complexes in FFPE specimens. We used hepatocyte growth factor (HGF)-c-MET as our model ligand-receptor system. HGF is the only known ligand of the c-MET tyrosine kinase receptor and HGF binding triggers c-MET phosphorylation. Novel antibody proximity-based assays were developed and used to detect and quantify total c-MET, total HGF, and HGF-c-MET ligand-receptor interactions in FFPE cell line and tumor tissue. In glioma cells, autocrine activation of c-MET by HGF-c-MET increased basal levels of c-MET phosphorylation at tyrosine (Tyr) 1003. Furthermore, HGF-c-MET activation in glioma cell lines was verified by Surface Protein-Protein Interaction by Crosslinking ELISA (SPPICE) assay in corresponding soluble cell lysates. Finally, we profiled levels ofc-MET, HGF, and HGF-c-MET complexes in FFPE specimens of human Non-Small Cell Lung Cancer (NSCLC), Gastric Cancer, Head and Neck Squamous Cell, and Head and Neck Non-Squamous Cell carcinomas. This report describes a novel approach for the detection and quantification of ligand-receptor interactions that can be widely applied to measure receptor activation in FFPE preclinical models and archived FFPE human tissue specimens.     

Association of Csk to VE-cadherin and inhibition of cell proliferation

Vascular endothelial cadherin (VE-cadherin) mediates contact inhibition of cell growth in quiescent endothelial cell layers. Searching for proteins that could be involved in VE-cadherin signaling, we found the cytosolic C-terminal Src kinase (Csk), a negative regulator of Src family kinases. We show that Csk binds via its SH2 domain to the phosphorylated tyrosine 685 of VE-cadherin. VE-cadherin recruits Csk to cell contacts and both proteins can be co-precipitated from cell lysates of transfected cells and endothelial cells. Association of VE-cadherin and Csk in endothelial cells increased with increasing cell density. CHO cells expressing the tyrosine replacement mutant VE-cadherin-Y685F grow to higher cell densities than cells expressing wild-type VE-cadherin. Overexpression of Csk in these cells under an inducible promoter inhibits cell proliferation in the presence and absence of VE-cadherin, but not in the presence of VE-cadherin-Y685F. Reduction of Csk expression by RNA interference enhances endothelial cell proliferation. Our results suggest that the phosphorylated tyrosine residue 685 of VE-cadherin and probably the binding of Csk to this site are involved in inhibition of cell growth triggered by cell density.     

Phosphorylation of DEP-1/PTPRJ on threonine 1318 regulates Src activation and endothelial cell permeability induced by vascular endothelial growth factor

The protein tyrosine phosphatase DEP-1/PTPRJ positively regulates Src family kinases and critical biological functions in endothelial and hematopoietic cells. Phosphorylation of DEP-1 on Y1311/Y1320 mediates the association and activation of Src, and promotes Src-dependent angiogenic responses including endothelial cell permeability. We have identified T1318 as a phosphorylated residue proximal to Y1320. The aim of this study was to determine if T1318 phosphorylation exerts a regulatory role over the function of DEP-1. We show that phosphorylation of DEP-1 on Y1320 was reduced when T1318 was mutated. This led to the decreased association of DEP-1 T1318A with Src, and defective Src activation in both HEK 293T and VEGF-stimulated endothelial cells. Consistent with these findings, VEGF-induced tyrosine phosphorylation of VE-cadherin, its association to β-arrestin1/2, and cell permeability were impaired in cells expressing DEP-1 T1318A. Conversely, expression of the phosphomimetic mutant DEP-1 T1318E constitutively enhanced the phosphorylation of Y1320 and VE-cadherin over that induced by WT DEP-1, and resulted in increased VEGF-dependent permeability. DEP-1 T1318 is part of a CK2 consensus phosphorylation site and was identified as a CK2 substrate. Modulation of CK2 expression or activity in endothelial cells regulated T1318 phosphorylation, and correlated with the status of Y1320 phosphorylation, Src activation, and cell permeability. CK2-dependent phosphorylation of DEP-1 T1318 promotes Y1320 phosphorylation and Src activation upon VEGF stimulation. Phosphorylation of T1318 is thus part of a regulatory mechanism that channels the activity of DEP-1 towards Src to allow its optimal activation and the promotion of endothelial cell permeability.     

SHP2 association with VE-cadherin complexes in human endothelial cells is regulated by thrombin

Thrombin-mediated changes in endothelial cell adherens junctions modulate vascular permeability. We demonstrate that the nonreceptor protein-tyrosine phosphatase SHP2 co-precipitates with VE-cadherin complexes in confluent, quiescent human umbilical vein endothelial cells. Ligand-binding blots using a SHP2-glutathione S-transferase fusion peptide established that SHP2 associates selectively with beta-catenin in VE-cadherin complexes. Thrombin treatment of human umbilical vein endothelial cells promotes SHP2 tyrosine phosphorylation and dissociation from VE-cadherin complexes. The loss of SHP2 from the cadherin complexes correlates with a dramatic increase in the tyrosine phosphorylation of beta-catenin, gamma-catenin, and p120-catenin complexed with VE-cadherin. We propose that thrombin regulates the tyrosine phosphorylation of VE-cadherin-associated beta-catenin, gamma-catenin, and p120-catenin by modulating the quantity of SHP2 associated with VE-cadherin complexes. Such changes in adherens junction complex composition likely underlie thrombin-elicited alterations in endothelial monolayer permeability.     

Vascular endothelial growth factor stimulates organ-specific host matrix metalloproteinase-9 expression and ovarian cancer invasion

Vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP) regulate each other, contributing to tumor progression. We have previously reported that MMP9 induces the release of tumor VEGF, promoting ascites formation in human ovarian carcinoma xenografts. The aim of this study was to investigate whether tumor-derived VEGF regulated the expression of gelatinase by the stroma, influencing the invasive properties of ovarian tumors. Tumor variants derived from 1A9 human ovarian carcinoma, stably expressing VEGF(121) in the sense (1A9-VS-1) and antisense orientations (1A9-VAS-3), were used. In vivo, zymographic analysis of tumors from 1A9-VS-1 implanted in the peritoneal cavity of nude mice showed higher levels of gelatinases, particularly murine MMP9, indicating that VEGF stimulates host expression of the matrix-degrading enzyme. Murine MMP9 expression was also high in the ovaries of mice bearing 1A9-VS-1 tumors. The effect on host MMP9 activity was organ-specific. The levels of host pro-MMP9 in ovaries correlated with the plasma levels of tumor VEGF and with the selective invasion of the ovaries. Induction of host MMP9 expression in tumors and ovaries was independent of the site of tumor growth as it was seen in mice carrying both intraperitoneal and subcutaneous tumors. The anti-VEGF antibody bevacizumab (Avastin) inhibited MMP9 expression and tumor invasion in the ovaries of mice bearing 1A9-VS-1 tumors. These findings point to a complex cross-talk between VEGF and MMPs in the progression of ovarian tumor and suggest the possibility of using VEGF inhibitors to affect MMP-dependent tumor invasion.     

Abelson murine leukemia virus P120: identification and characterization of tyrosine phosphorylation sites.

Tryptic peptides containing two major in vivo P120gag-abl tyrosine phosphorylation acceptor sites were identified, phosphorylated in vitro, and purified to homogeneity. The tyrosine site in peptide a is localized at a position six residues distal to its trypsin cleavage site, whereas the tyrosine acceptor site in peptide b is at residue seven. A third peptide, c, contains an amino-terminal phosphotyrosine residue: phosphorylation of this latter peptide only occurs to a significant extent in vivo.     

Local mutagenesis of Rous sarcoma virus: The major sites of tyrosine and serine phosphorylation of p60src are dispensable for transformation

We have constructed mutants of Rous sarcoma virus expressing p60^src that are underphosphorylated on serine or tyrosine, by linker insertion or insertion/ deletion into cloned Rous sarcoma virus DNA, and recovery of mutant virus by transfection of chicken embryo fibroblasts. Cells infected with mutants whose p60^src lack the major site of either serine or tyrosine phosphorylation were morphologically transformed and formed colonies in soft agar. The tyrosine kinase activities of the mutant p60^src measured in vivo and in vitro were close to the wild type activity. Peptide mapping showed that phosphorylation on tyrosine and serine of p60^src is independent: the major phosphorylated tyrosine and the major phosphorylated serine can each be phosphorylated in the absence of phosphorylation of the other.     

Roles of tyrosine phosphorylation and cleavage of protein kinase Cdelta in its protective effect against tumor necrosis factor-related apoptosis inducing ligand-induced apoptosis

Protein kinase Cdelta (PKCdelta) regulates cell apoptosis in a cell- and stimulus-specific manner. Here, we studied the role of PKCdelta in the apoptotic effect of TRAIL in glioma cells. We found that transfection of the cells with a PKCdelta kinase-dead mutant (K376R) or with a small interfering RNA targeting the PKCdelta mRNA increased the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), whereas overexpression of PKCdelta decreased it. PKCdelta acted downstream of caspase 8 and upstream of cytochrome c release from the mitochondria. TRAIL induced cleavage of PKCdelta within 2-3 h of treatment, which was abolished by caspase 3, 8, and 9 inhibitors. The cleavage of PKCdelta was essential for its protective effect because overexpression of a caspase-resistant mutant (PKCdeltaD327A) did not protect glioma cells from TRAIL-induced apoptosis but rather increased it. TRAIL induced translocation of PKCdelta to the perinuclear region and the endoplasmic reticulum and phosphorylation of PKCdelta on tyrosine 155. Using a PKCdeltaY155F mutant, we found that the phosphorylation of PKCdelta on tyrosine 155 was essential for the cleavage of PKCdelta in response to TRAIL and for its translocation to the endoplasmic reticulum. In addition, phosphorylation of PKCdelta on tyrosine 155 was necessary for the activation of AKT in response to TRAIL. Our results indicate that PKCdelta protects glioma cells from the apoptosis induced by TRAIL and implicate the phosphorylation of PKCdelta on tyrosine 155 and its cleavage as essential factors in the anti-apoptotic effect of PKCdelta.     

Phosphorylation of the MET receptor on juxtamembrane tyrosine residue 1001 inhibits its caspase-dependent cleavage

The MET tyrosine kinase is the hepatocyte growth factor/scatter factor (HGF/SF) receptor, which elicits multiple biological responses in epithelial cells, including cell survival. We previously demonstrated that in stress conditions, the MET receptor is cleaved by caspases within its juxtamembrane region, generating a pro-apoptotic intracellular fragment of 40 kDa. The caspase cleavage site at aspartic acid D1000 is adjacent to tyrosine Y1001, which when phosphorylated upon MET activation, is involved in CBL recruitment, allowing receptor ubiquitination and down regulation. Scanning mutagenesis of the MET juxtamembrane region led us to demonstrate that V999 and D1000 are essential for the caspase cleavage, while D1000 and Y1001 are essential for CBL recruitment. By examining whether overlapping of these sites leads to a functional interference, an inverse relationship was found between generation of p40 MET and phosphorylation of MET, with a direct involvement of phosphorylated Y1001 in protecting MET against its caspase cleavage. A molecular modeling analysis of caspase 3 interaction with the juxtamembrane region of MET confirmed that phosphorylation of this tyrosine is not compatible with its recognition by active caspase 3. These data demonstrate a direct protection mechanism of an activated phosphorylated MET receptor, against its caspase-dependent cleavage.     

Glioma cells under hypoxic conditions block the brain microvascular endothelial cell death induced by serum starvation

Angiogenesis is one of essential components for the growth of neoplasms, including malignant gliomas. However, tumor vascularization is often poorly organized and marginally functional due to tumor structural abnormalities, inducing regional or temporal hypoxic conditions and nutritional shortages in tumor tissues. We investigated how during angiogenesis migrating endothelial cells survive in these hypoxic and reduced nutritional conditions. Human brain microvascular endothelial cells (HBMECs) underwent apoptosis and necrosis after serum withdrawal. This endothelial cell death was blocked by recombinant VEGF protein or the culture medium of U251 glioma cells exposed to hypoxia (H-CM). Hypoxic treatment increased vascular endothelial growth factor (VEGF) and tumor necrosis factor alpha (TNF-alpha) expression in U251 glioma cells. H-CM activated nuclear factor-kappaB (NFkappaB) protein and increased the gene expression of antiapoptotic factors including Bcl-2, Bcl-X(L), survivin and X-chromosome-linked inhibitor of apoptosis protein (XIAP) in endothelial cells. The survival activity of H-CM for endothelial cells was abolished by two kinds of VEGF inhibitors {Cyclopeptidic VEGF inhibitor and a VEGF receptor tyrosine kinase inhibitor (4-[(4'-chloro-2'-fluoro) phenylamino]-6, 7-dimethoxyquinazoline)} or NFkappaB inhibitors (ALLN and BAY 11-7082). These VEGF inhibitors did not block the activation of NFkappaB induced by H-CM in endothelial cells. On the contrary, TNF-alpha antagonist WP9QY enhanced the survival activity of H-CM for endothelial cells and blocked NFkappaB activation induced by H-CM under serum-starved conditions. Taken together, our data suggest that both the secretion of VEGF from glioma cells and activation of NFkappaB in endothelial cells induced by TNF-alpha are necessary for endothelial cell survival as they increase the expression of antiapoptotic genes in endothelial cells under conditions of serum starvation. These pathways may be one of the mechanisms by which angiogenesis is maintained in glioma tissues.     

Changes in Vascular Permeability and Expression of Different Angiogenic Factors Following Anti-Angiogenic Treatment in Rat Glioma

Background

Anti-angiogenic treatments of malignant tumors targeting vascular endothelial growth factor receptors (VEGFR) tyrosine kinase are being used in different early stages of clinical trials. Very recently, VEGFR tyrosine kinase inhibitor (Vetanalib, PTK787) was used in glioma patient in conjunction with chemotherapy and radiotherapy. However, changes in the tumor size, tumor vascular permeability, vascular density, expression of VEGFR2 and other angiogenic factors in response to PTK787 are not well documented. This study was to determine the changes in tumor size, vascular permeability, fractional plasma volume and expression of VEGFR2 in PTK787 treated U-251 glioma rat model by in vivo magnetic resonance imaging (MRI) and single photon emission computed tomography (SPECT). The findings were validated with histochemical and western blot studies.

Methodologies and Principal Findings

Seven days after implantation of U251 glioma cells, animals were treated with either PTK787 or vehicle-only for two weeks, and then tumor size, tumor vascular permeability transfer constant (K^trans), fractional plasma volume (fPV) and expression of VEGFR2 and other relevant angiogenic factors were assessed by in vivo MRI and SPECT (Tc-99-HYNIC-VEGF), and by immunohistochemistry and western blot analysis. Dynamic contrast-enhanced MRI (DCE-MRI) using a high molecular weight contrast agent albumin-(GdDTPA) showed significantly increased K^trans at the rim of the treated tumors compared to that of the central part of the treated as well as the untreated (vehicle treated) tumors. Size of the tumors was also increased in the treated group. Expression of VEGFR2 detected by Tc-99m-HYNIC-VEGF SPECT also showed significantly increased activity in the treated tumors. In PTK787-treated tumors, histological staining revealed increase in microvessel density in the close proximity to the tumor border. Western blot analysis indicated increased expression of VEGF, SDF-1, HIF-1α, VEGFR2, VEGFR3 and EGFR at the peripheral part of the treated tumors compared to that of central part of the treated tumors. Similar expression patters were not observed in vehicle treated tumors.

Conclusion

These findings indicate that PTK787 treatment induced over expression of VEGF as well as the Flk-1/VEGFR2 receptor tyrosine kinase, especially at the rim of the tumor, as proven by DCE-MRI, SPECT imaging, immunohistochemistry and western blot.     

A novel role of vascular endothelial cadherin in modulating c-Src activation and downstream signaling of vascular endothelial growth factor

Vascular endothelial growth factor (VEGF) is a potent mediator of angiogenesis and vascular permeability, in which c-Src tyrosine kinase plays an essential role. However, the mechanisms by which VEGF stimulates c-Src activation have remained unclear. Here, we demonstrate that vascular endothelial cadherin (VE-cadherin) plays a critical role in regulating c-Src activation in response to VEGF. In vascular endothelial cells, VE-cadherin was basally associated with c-Src and Csk (C-terminal Src kinase), a negative regulator of Src activation. VEGF stimulated Csk release from VE-cadherin by recruiting the protein tyrosine phosphatase SHP2 to VE-cadherin signaling complex, leading to an increase in c-Src activation. Silencing VE-cadherin with small interference RNA significantly reduced VEGF-stimulated c-Src activation. Disrupting the association of VE-cadherin and Csk through the reconstitution of Csk binding-defective mutant of VE-cadherin also diminished Src activation. Moreover, inhibiting SHP2 by small interference RNA and adenovirus-mediated expression of a catalytically inactive mutant of SHP2 attenuated c-Src activation by blocking the disassociation of Csk from VE-cadherin. Furthermore, VE-cadherin and SHP2 differentially regulates VEGF downstream signaling. The inhibition of c-Src, VE-cadherin, and SHP2 diminished VEGF-mediated activation of Akt and endothelial nitric-oxide synthase. In contrast, inhibiting VE-cadherin and SHP2 enhanced ERK1/2 activation in response to VEGF. These findings reveal a novel role for VE-cadherin in modulating c-Src activation in VEGF signaling, thus providing new insights into the importance of VE-cadherin in VEGF signaling and vascular function.     

Tyrosine phosphorylation of VE-cadherin and claudin-5 is associated with TGF-β1-induced permeability of centrally derived vascular endothelium

Breakdown of the inner blood-retinal barrier and the blood-brain barrier is associated with changes in tight and adherens junction-associated proteins that link vascular endothelial cells. This study aimed to test the hypothesis that transforming growth factor (TGF)-β1 increases the paracellular permeability of vascular endothelial monolayers through tyrosine phosphorylation of VE-cadherin and claudin-5. Bovine retinal and human brain capillary endothelial cells were grown as monolayers on coated polycarbonate membranes. Paracellular permeability was studied by measuring the equilibration of (14)C-inulin or fluorescence-labelled dextran. Changes in VE-cadherin and claudin-5 expression were studied by immunocytochemistry (ICC) and quantified by cell-based enzyme linked immunosorbent assays (ELISA). Tyrosine phosphorylation of VE-cadherin and claudin-5 was studied by ICC, immunoprecipitation and Western blotting. We found that exposure of endothelial cells to TGF-β1 caused a dose-dependent increase in paracellular permeability as reflected by increases in the equilibration of (14)C-inulin. This effect was enhanced by the tyrosine phosphatase inhibitor orthovanadate and attenuated by the tyrosine kinase inhibitor lavendustin A. ICC and cell-based ELISA revealed that TGF-β1 induced both dose- and time-dependent decreases in VE-cadherin and claudin-5 expression. Assessment of cell viability indicated that changes in these junction-associated proteins were not due to endothelial death or injury. ICC revealed that tyrosine phosphorylation of endothelial monolayers was greatly enhanced by TGF-β1 treatment, and immunoprecipitation of cell lysates showed increased tyrosine phosphorylation of VE-cadherin and claudin-5. Our results suggest that tyrosine phosphorylation of VE-cadherin and claudin-5 is involved in the increased paracellular permeability of central nervous system-derived vascular endothelium induced by TGF-β1.     

Amlodipine,a Ca2+ channel blocker,suppresses phosphorylation of epidermal growth factor receptor in human epidermoid carcinoma A431 cells

AimsAmlodipine, a dihydropyridine Ca²⁺ channel blocker, inhibits the proliferation of human epidermoid carcinoma A431 cells in vitro and in vivo. This study examined the underlying mechanism of this antiproliferative effect in relation to epidermal growth factor receptor (EGFR) signaling.Main methodsThe tyrosine phosphorylated active state of EGFR in A431 cells incubated with the test agents was evaluated by western blot with anti-phosphotyrosine antibody. EGFR phosphorylation levels in A431 xenograft tumors were assessed by immunostaining of matrigel plug sections and western blotting for phosphoEGFR in A431 xenograft tumor homogenates.Key findingsIn vitro treatment of exponentially growing A431 cells with amlodipine decreased the tyrosine phosphorylation states of EGFR. Amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR and a membrane scaffolding protein, caveolin-1, in serum-starved A431 cells. Amlodipine attenuated the EGF-stimulated phosphorylation of EGFR coimmunoprecipitated with caveolin-1 without affecting the EGFR/caveolin-1 interaction. Crosslinking experiments showed that amlodipine also suppressed the EGF-stimulated phosphorylation of EGFR predimers. Addition of cholesterol abolished these inhibitory effects of amlodipine plus its inhibition of cell growth. Furthermore, treatment of mice with amlodipine (10 mg/kg/day × 7 days, i.p.) decreased the levels of phosphorylated EGFR in A431 xenograft tumors.SignificanceThe results indicated that amlodipine inhibits tyrosine phosphorylation of EGFR in vitro and in vivo, possibly via modulating cholesterol-rich, caveolin-1-containing membrane microdomains.