Selective requirement for Src kinases during VEGF-induced angiogenesis and vascular permeability

Src kinase activity was found to protect endothelial cells from apoptosis during vascular endothelial growth factor (VEGF)-, but not basic fibroblast growth factor (bFGF)-, mediated angiogenesis in chick embryos and mice. In fact, retroviral targeting of kinase-deleted Src to tumor-associated blood vessels suppressed angiogenesis and the growth of a VEGF-producing tumor. Although mice lacking individual Src family kinases (SFKs) showed normal angiogenesis, mice deficient in pp60c-src or pp62c-yes showed no VEGF-induced vascular permeability (VP), yet fyn-/- mice displayed normal VP. In contrast, inflammation-mediated VP appeared normal in Src-deficient mice. Therefore, VEGF-, but not bFGF-, mediated angiogenesis requires SFK activity in general, whereas the VP activity of VEGF specifically depends on the SFKs, Src, or Yes.     

Inhibition of endothelial FAK activity prevents tumor metastasis by enhancing barrier function

Pharmacological focal adhesion kinase (FAK) inhibition prevents tumor growth and metastasis, via actions on both tumor and stromal cells. In this paper, we show that vascular endothelial cadherin (VEC) tyrosine (Y) 658 is a target of FAK in tumor-associated endothelial cells (ECs). Conditional kinase-dead FAK knockin within ECs inhibited recombinant vascular endothelial growth factor (VEGF-A) and tumor-induced VEC-Y658 phosphorylation in vivo. Adherence of VEGF-expressing tumor cells to ECs triggered FAK-dependent VEC-Y658 phosphorylation. Both FAK inhibition and VEC-Y658F mutation within ECs prevented VEGF-initiated paracellular permeability and tumor cell transmigration across EC barriers. In mice, EC FAK inhibition prevented VEGF-dependent tumor cell extravasation and melanoma dermal to lung metastasis without affecting primary tumor growth. As pharmacological c-Src or FAK inhibition prevents VEGF-stimulated c-Src and FAK translocation to EC adherens junctions, but FAK inhibition does not alter c-Src activation, our experiments identify EC FAK as a key intermediate between c-Src and the regulation of EC barrier function controlling tumor metastasis.     

Protein tyrosine phosphatase epsilon activates Yes and Fyn in Neu-induced mammary tumor cells

The receptor-type form of protein tyrosine phosphatase epsilon (RPTP) is among the few tyrosine phosphatases that can support the transformed phenotype of tumor cells. Accordingly, cells from mammary epithelial tumors induced by activated Neu in mice genetically lacking RPTP appear morphologically less transformed and exhibit reduced proliferation. The effect of RPTP in these cells is mediated at least in part by its ability to activate Src, the prototypic member of a family of related kinases. We show here that RPTP is a physiological activator of two additional Src family kinases, Yes and Fyn. Activities of both kinases are inhibited in mammary tumor cells lacking RPTP, and phosphorylation at their C-terminal inhibitory tyrosines is increased. In agreement, opposite effects on activities and phosphorylation of Yes and Fyn are observed following increased expression of PTP. RPTP also forms stable complexes with either kinase, providing physical opportunity for their activation by RPTP. Surprisingly, expression of Yes or of Fyn does not rescue the morphological phenotype of RPTP-deficient tumor cells in contrast with the strong ability of Src to do so. We conclude that RPTP activates Src, Yes, and Fyn, but that these related kinases play distinct roles in Neu-induced mammary tumor cells.     

Rac1-regulated endothelial radiation response stimulates extravasation and metastasis that can be blocked by HMG-CoA reductase inhibitors

Radiotherapy (RT) plays a key role in cancer treatment. Although the benefit of ionizing radiation (IR) is well established, some findings raise the possibility that irradiation of the primary tumor not only triggers a killing response but also increases the metastatic potential of surviving tumor cells. Here we addressed the question of whether irradiation of normal cells outside of the primary tumor augments metastasis by stimulating the extravasation of circulating tumor cells. We show that IR exposure of human endothelial cells (EC), tumor cells (TC) or both increases TC-EC adhesion in vitro. IR-stimulated TC-EC adhesion was blocked by the HMG-CoA reductase inhibitor lovastatin. Glycyrrhizic acid from liquorice root, which acts as a Sialyl-Lewis X mimetic drug, and the Rac1 inhibitor NSC23766 also reduced TC-EC adhesion. To examine the in vivo relevance of these findings, tumorigenic cells were injected into the tail vein of immunodeficient mice followed by total body irradiation (TBI). The data obtained show that TBI dramatically enhances tumor cell extravasation and lung metastasis. This pro-metastatic radiation effect was blocked by pre-treating mice with lovastatin, glycyrrhizic acid or NSC23766. TBI of mice prior to tumor cell transplantation also stimulated metastasis, which was again blocked by lovastatin. The data point to a pro-metastatic trans-effect of RT, which likely rests on the endothelial radiation response promoting the extravasation of circulating tumor cells. Administration of the widely used lipid-lowering drug lovastatin prior to irradiation counteracts this process, likely by suppressing Rac1-regulated E-selectin expression following irradiation. The data support the concern that radiation exposure might increase the extravasation of circulating tumor cells and recommend co-administration of lipid-lowering drugs to avoid this adverse effect of ionizing radiation.     

Human neutrophils facilitate tumor cell transendothelial migration

Tumor cell extravasation plays a key role in tumor metastasis.However, the precise mechanisms by which tumor cells migrate throughnormal vascular endothelium remain unclear. In this study, using an invitro transendothelial migration model, we show that humanpolymorphonuclear neutrophils (PMN) assist the human breast tumor cellline MDA-MB-231 to cross the endothelial barrier. We found thattumor-conditioned medium (TCM) downregulated PMN cytocidal function,delayed PMN apoptosis, and concomitantly upregulated PMNadhesion molecule expression. These PMN treated with TCM attached totumor cells and facilitated tumor cell migration through different endothelial monolayers. In contrast, MDA-MB-231 cells alone did nottransmigrate. FACScan analysis revealed that these tumor cells expressed high levels of intercellular adhesion molecule-1 (ICAM-1) butdid not express CD11a, CD11b, or CD18. Blockage of CD11b and CD18 onPMN and of ICAM-1 on MDA-MB-231 cells significantly attenuated TCM-treated, PMN-mediated tumor cell migration. These tumor cells stillpossessed the ability to proliferate after PMN-assisted transmigration.These results indicate that TCM-treated PMN may serve as a carrier toassist tumor cell transendothelial migration and suggest that tumorcells can exploit PMN and alter their function to facilitate their extravasation.

     

Induction of SPARC by VEGF in human vascular endothelial cells

SPARC/osteonectin/BM-40 is a matricellular protein that is thought to be involved in angiogenesis and endothelial barrier function. Previously, we have detected high levels of SPARC expression in endothelial cells (ECs) adjacent to carcinomas of kidney and tongue. Although SPARC-derived peptide showed an angiogenic effect, intact SPARC itself inhibited the mitogenic activity of vascular endothelial growth factor (VEGF) for ECs by the inhibiting phosphorylation of flt-1 (VEGF receptor 1) and subsequent ERK activation. Thus, the role of SPARC in tumor angiogenesis, stimulation or inhibition, is still unclear. To clarify the role of SPARC in tumor growth and progression, we determined the effect of VEGF on the expression of SPARC in human microvascular EC line, HMEC-1, and human umbilical vein ECs. VEGF increased the levels of SPARC protein and steady-state levels of SPARC mRNA in serum-starved HMEC-1 cells. Inhibitors (SB202190 and SB203580) of p38, a mitogen-activated protein (MAP) kinase, attenuated VEGF-stimulated SPARC production in ECs. Since intact SPARC inhibits phosphorylation ERK MAP kinase in VEGF signaling, it was suggested that SPARC plays a dual role in the VEGF functions, tumor angiogenesis, and extravasation of tumors mediated by the increased permeability of endothelial barrier function.     

Vascular endothelial growth factor modulates the transendothelial migration of MDA-MB-231 breast cancer cells through regulation of brain microvascular endothelial cell permeability

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor (VPF), has been shown to increase potently the permeability of endothelium and is highly expressed in breast cancer cells. In this study, we investigated the role of VEGF/VPF in breast cancer metastasis to the brain. Very little is known about the role of endothelial integrity in the extravasation of breast cancer cells to the brain. We hypothesized that VEGF/VPF, having potent vascular permeability activity, may support tumor cell penetration across blood vessels by inducing vascular leakage. To examine this role of VEGF/VPF, we used a Transwell culture system of the human brain microvascular endothelial cell (HBMEC) monolayer as an in vitro model for the blood vessels. We observed that VEGF/VPF significantly increased the penetration of the highly metastatic MDA-MB-231 breast cancer cells across the HBMEC monolayer. We found that the increased transendothelial migration (TM) of MDA-MB-231 cells resulted from the increased adhesion of tumor cells onto the HBMEC monolayer. These effects (TM and adhesion of tumor cells) were inhibited by the pre-treatment of the HBMEC monolayer with the VEGF/VPF receptor (KDR/Flk-1) inhibitor, SU-1498, and the calcium chelator 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl)ester. These treatments of the HBMEC monolayer also inhibited VEGF/VPF-induced permeability and the cytoskeletal rearrangement of the monolayer. These data suggest that VEGF/VPF can modulate the TM of tumor cells by regulating the integrity of the HBMEC monolayer. Taken together, these findings indicate that VEGF/VPF might contribute to breast cancer metastasis by enhancing the TM of tumor cells through the down-regulation of endothelial integrity.     

Deciphering vascular endothelial cell growth factor/vascular permeability factor signaling to vascular permeability. Inhibition by atrial natriuretic peptide

Vascular endothelial cell growth factor (VEGF) was originally described as a potent vascular permeability factor (VPF) that importantly contributes to vascular pathobiology. The signaling pathways that underlie VEGF/VPF-induced permeability are not well defined. Furthermore, endogenous vascular peptides that regulate this important VPF function are currently unknown. We report here that VPF significantly enhances permeability in aortic endothelial cells via a linked signaling pathway, sequentially involving Src, ERK, JNK, and phosphatidylinositol 3-kinase/AKT. This leads to the serine/threonine phosphorylation and redistribution of actin and the tight junction (TJ) proteins, zona occludens-1 and occludin, and the loss of the endothelial cell barrier architecture. Atrial natriuretic peptide (ANP) inhibited VPF signaling, TJ protein phosphorylation and localization, and VPF-induced permeability. This involved both guanylate cyclase and natriuretic peptide clearance receptors. In vivo, transgenic mice that overexpress ANP showed significantly less VPF-induced kinase activation and vascular permeability compared with non-transgenic littermates. Thus, ANP acts as an anti-permeability factor by inhibiting the signaling functions of VPF that we define here and by preserving the endothelial cell TJ functional morphology.     

Thromboxane A(2) regulation of endothelial cell migration, angiogenesis, and tumor metastasis

Prostaglandin endoperoxide H synthases and their arachidonate products have been implicated in modulating angiogenesis during tumor growth and chronic inflammation. Here we report the involvement of thromboxane A(2), a downstream metabolite of prostaglandin H synthase, in angiogenesis. A TXA(2) mimetic, U46619, stimulated endothelial cell migration. Angiogenic basic fibroblast growth factor (bFGF) or vascular endothelial growth factor (VEGF) increased TXA(2) synthesis in endothelial cells three- to fivefold. Inhibition of TXA(2) synthesis with furegrelate or CI reduced HUVEC migration stimulated by VEGF or bFGF. A TXA(2) receptor antagonist, SQ29,548, inhibited VEGF- or bFGF-stimulated endothelial cell migration. In vivo, CI inhibited bFGF-induced angiogenesis. Finally, development of lung metastasis in C57Bl/6J mice intravenously injected with Lewis lung carcinoma or B16a cells was significantly inhibited by thromboxane synthase inhibitors, CI or furegrelate sodium. Our data demonstrate the involvement of TXA(2) in angiogenesis and development of tumor metastasis.     

Genome-wide Approaches Reveal Functional Vascular Endothelial Growth Factor (VEGF)-inducible Nuclear Factor of Activated T Cells (NFAT) c1 Binding to Angiogenesis-related Genes in the Endothelium

VEGF is a key regulator of endothelial cell migration, proliferation, and inflammation, which leads to activation of several signaling cascades, including the calcineurin-nuclear factor of activated T cells (NFAT) pathway. NFAT is not only important for immune responses but also for cardiovascular development and the pathogenesis of Down syndrome. By using Down syndrome model mice and clinical patient samples, we showed recently that the VEGF-calcineurin-NFAT signaling axis regulates tumor angiogenesis and tumor metastasis. However, the connection between genome-wide views of NFAT-mediated gene regulation and downstream gene function in the endothelium has not been studied extensively. Here we performed comprehensive mapping of genome-wide NFATc1 binding in VEGF-stimulated primary cultured endothelial cells and elucidated the functional consequences of VEGF-NFATc1-mediated phenotypic changes. A comparison of the NFATc1 ChIP sequence profile and epigenetic histone marks revealed that predominant NFATc1-occupied peaks overlapped with promoter-associated histone marks. Moreover, we identified two novel NFATc1 regulated genes, CXCR7 and RND1. CXCR7 knockdown abrogated SDF-1- and VEGF-mediated cell migration and tube formation. siRNA treatment of RND1 impaired vascular barrier function, caused RhoA hyperactivation, and further stimulated VEGF-mediated vascular outgrowth from aortic rings. Taken together, these findings suggest that dynamic NFATc1 binding to target genes is critical for VEGF-mediated endothelial cell activation. CXCR7 and RND1 are NFATc1 target genes with multiple functions, including regulation of cell migration, tube formation, and barrier formation in endothelial cells.     

EMT phenotype is induced by increased Src kinase activity via Src-mediated caspase-8 phosphorylation

Caspase-8 governs multiple cell responses to the microenvironmental cues. However, its integration of "death-life" signalings remains elusive. In our study, the role of caspase-8-Src is well-established as a promoter for migration or metastasis in Casp8(+)Src(+) A549/H226 cells in vivo and in vitro. In particular for nude mice models, mice implanted with Casp8(+)Src(+) A459/H226 cells remarkably increased spontaneous tumor metastatic burden with a significant survival disadvantage. Additionally, we detect that Src-mediated caspase-8 phosphorylation stimulates Src phosphorylation at Tyr-416 via the linkage of Src SH2 domain with phosph-Tyr-380 site of caspase-8. In turn, activated Src can efficiently induce epithelial-mesenchymal transition (EMT) phenotypic features to promote tumor cells metastasis. Surprisingly, RXDLL motif deletion in the DEDa of caspase-8 attenuates tumor cell migration or metastasis via impairing the recruitment of caspase-8 into the cellular periphery where activated Src is subject to caspase-8 phosphorylation. Together, a simple model is that the peripherization of caspase-8 is well-poised to facilitate Src-mediated caspase-8 phosphrylation at Tyr-380, then binding of phospho-Tyr380 of caspase-8 to Src SH2 domain may maintain Src in an active conformation to induce EMT phenotype, a key step toward cancer metastasis.     

Functional activation of Src family kinase yes protein is essential for the enhanced malignant properties of human melanoma cells expressing ganglioside GD3

The possible roles of Src family kinases in the enhanced malignant properties of melanomas related to GD3 expression were analyzed. Among Src family kinases only Yes, not Fyn or Src, was functionally involved in the increased cell proliferation and invasion of GD3-expressing transfectant cells (GD3+). Yes was located upstream of p130Cas and paxillin and at an equivalent level to focal adhesion kinase. Yes underwent autophosphorylation even before serum treatment and showed stronger kinase activity in GD3+ cells than in GD3- cells following serum treatment. Coimmunoprecipitation experiments revealed that Yes bound to focal adhesion kinase or p130Cas more strongly in GD3+ cells than in GD3- cells. As a possible mechanism for the enhancing effects of GD3 on cellular phenotypes, it was shown that majority of Yes was localized in glycolipid-enriched microdomain/rafts in GD3+ cells even before serum treatment, whereas it was scarcely detected in glycolipid-enriched microdomain/rafts in GD3- cells. An in vitro kinase assay of Yes revealed that coexistence of GD3 with Yes in membranous environments enhances the kinase activity of GD3- cell-derived Yes toward enolase, p125, and Yes itself. Knockdown of GD3 synthase resulted in the alleviation of tumor phenotypes and reduced activation levels of Yes. Taken together, these results suggest a role of GD3 in the regulation of Src family kinases.     

Src deficiency or blockade of Src activity in mice provides cerebral protection following stroke

Vascular endothelial growth factor (VEGF), an angiogenic factor produced in response to ischemic injury, promotes vascular permeability (VP). Evidence is provided that Src kinase regulates VEGF-mediated VP in the brain following stroke and that suppression of Src activity decreases VP thereby minimizing brain injury. Mice lacking pp60c-src are resistant to VEGF-induced VP and show decreased infarct volumes after stroke whereas mice deficient in pp59c-fyn, another Src family member, have normal VEGF-mediated VP and infarct size. Systemic application of a Src-inhibitor given up to six hours following stroke suppressed VP protecting wild-type mice from ischemia-induced brain damage without influencing VEGF expression. This was associated with reduced edema, improved cerebral perfusion and decreased infarct volume 24 hours after injury as measured by magnetic resonance imaging and histological analysis. Thus, Src represents a key intermediate and novel therapeutic target in the pathophysiology of cerebral ischemia where it appears to regulate neuronal damage by influencing VEGF-mediated VP.     

Angiotensin II facilitates breast cancer cell migration and metastasis

Breast cancer metastasis is a leading cause of death by malignancy in women worldwide. Efforts are being made to further characterize the rate-limiting steps of cancer metastasis, i.e. extravasation of circulating tumor cells and colonization of secondary organs. In this study, we investigated whether angiotensin II, a major vasoactive peptide both produced locally and released in the bloodstream, may trigger activating signals that contribute to cancer cell extravasation and metastasis. We used an experimental in vivo model of cancer metastasis in which bioluminescent breast tumor cells (D3H2LN) were injected intra-cardiacally into nude mice in order to recapitulate the late and essential steps of metastatic dissemination. Real-time intravital imaging studies revealed that angiotensin II accelerates the formation of metastatic foci at secondary sites. Pre-treatment of cancer cells with the peptide increases the number of mice with metastases, as well as the number and size of metastases per mouse. In vitro, angiotensin II contributes to each sequential step of cancer metastasis by promoting cancer cell adhesion to endothelial cells, trans-endothelial migration and tumor cell migration across extracellular matrix. At the molecular level, a total of 102 genes differentially expressed following angiotensin II pre-treatment were identified by comparative DNA microarray. Angiotensin II regulates two groups of connected genes related to its precursor angiotensinogen. Among those, up-regulated MMP2/MMP9 and ICAM1 stand at the crossroad of a network of genes involved in cell adhesion, migration and invasion. Our data suggest that targeting angiotensin II production or action may represent a valuable therapeutic option to prevent metastatic progression of invasive breast tumors.     

VEGF controls endothelial-cell permeability by promoting the beta-arrestin-dependent endocytosis of VE-cadherin

How vascular endothelial growth factor (VEGF) induces vascular permeability, its first described function, remains poorly understood. Here, we provide evidence of a novel signalling pathway by which VEGF stimulation promotes the rapid endocytosis of a key endothelial cell adhesion molecule, VE-cadherin, thereby disrupting the endothelial barrier function. This process is initiated by the activation of the small GTPase Rac by VEGFR-2 through the Src-dependent phosphorylation of Vav2, a guanine nucleotide-exchange factor. Rac activation, in turn, promotes the p21-activated kinase (PAK)-mediated phosphorylation of a highly conserved motif within the intracellular tail of VE-cadherin. Surprisingly, this results in the recruitment of beta-arrestin2 to serine-phosphorylated VE-cadherin, thereby promoting its internalization into clathrin-coated vesicles and the consequent disassembly of intercellular junctions. Ultimately, this novel biochemical route by which VEGF promotes endothelial permeability through the beta-arrestin2-dependent endocytosis of VE-cadherin may help identify new therapeutic targets for the treatment of many human diseases that are characterized by vascular leakage.     

Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.     

Vascular endothelial growth factor receptor-3 activity is modulated by its association with caveolin-1 on endothelial membrane

Vascular endothelial growth factor receptor-3 (VEGFR-3) is constitutively expressed in lymphatic vessels and transiently in endothelial cells of blood vessels during angiogenesis. Here we report that VEGFR-3 localizes in the caveolae membrane of endothelial cells and co-immunoprecipitates with caveolin-1. Caveolin-1 silencing or its depletion from the cell membrane by cholesterol increases VEGFR-3 autophosphorylation, suggesting that caveolin acts as a negative regulator of VEGFR-3 activity. Receptor activation induces caveolin-1 phosphorylation on tyrosine residues including tyrosine 14. Cell treatment with Src or Abl inhibitors PP2 or STI571, prior to receptor stimulation, affects caveolin-1 phosphorylation without affecting receptor autophosphorylation, suggesting that both Src and Abl are involved in VEGFR-3-dependent caveolin-1 phosphorylation. Caveolin-1 phosphorylation in Src/Fyn/Yes knockout cells demonstrated that Abl phosphorylates caveolin-1 independently from Src family members. These results suggest a functional interaction between VEGFR-3 and caveolin-1 to modulate endothelial cell activation during angiogenesis.