Paracrine and autocrine functions of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in brain-derived endothelial cells

Brain-derived neurotrophic factor (BDNF) is expressed by endothelial cells. We investigated the characteristics of BDNF expression by brain-derived endothelial cells and tested the hypothesis that BDNF serves paracrine and autocrine functions affecting the vasculature of the central nervous system. In addition to expressing TrkB and p75NTR and BDNF under normoxic conditions, these cells increased their expression of BDNF under hypoxia. While the expression of TrkB is unaffected by hypoxia, TrkB exhibits a base-line phosphorylation under normoxic conditions and an increased phosphorylation when BDNF is added. TrkB phosphorylation is decreased when endogenous BDNF is sequestered by soluble TrkB. Exogenous BDNF elicits robust angiogenesis and survival in three-dimensional cultures of these endothelial cells, while sequestration of endogenous BDNF caused significant apoptosis. The effects of BDNF engagement of TrkB appears to be mediated via the phosphatidylinositol (PI) 3-kinase-Akt pathway. Modulation of BDNF levels directly correlate with Akt phosphorylation and inhibitors of PI 3-kinase abrogate the BDNF responses. BDNF-mediated effects on endothelial cell survival/apoptosis correlated directly with activation of caspase 3. These endothelial cells also express p75NTR and respond to its preferred ligand, pro-nerve growth factor (pro-NGF), by undergoing apoptosis. These data support a role for neurotrophins signaling in the dynamic maintenance/differentiation of central nervous system endothelia.     

IGF-1 induced vascular endothelial growth factor secretion in head and neck squamous cell carcinoma

Elevated vascular endothelial growth factor (VEGF) levels correlate with increased progression and poor prognosis of head and neck squamous cell carcinomas (HNSCC). VEGF expression is regulated by hypoxia and cytokines, including insulin-like growth factor-1 (IGF-1). In this report, we examined IGF-1 signaling and VEGF expression in SCC-9 cells. IGF-1 and the chemical hypoxia agent, cobalt chloride, each stimulated VEGF secretion and VEGF promoter activation. Cobalt chloride increased Hif-1alpha protein levels and HIF-1 dependent activation of the enolase promoter. IGF-1 increased these parameters only in the presence of cobalt chloride. IGF-1 stimulated PI-3K/Akt and Erk/MAPK pathways in SCC-9 cells, each contributing to Hif-1alpha expression and VEGF secretion. SCC-9 cells express the VEGF receptors Flk-1 and neuropilin-1, with VEGF treatment increasing VEGF promoter activity and VEGF secretion that was attenuated by the Flk-1 tyrosine kinase inhibitor, ZM 323881. These results demonstrate the presence of an IGF-1 regulated VEGF autocrine loop in HNSCC.     

Transactivation of vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is involved in sphingosine 1-phosphate-stimulated phosphorylation of Akt and endothelial nitric-oxide synthase (eNOS)

Sphingosine 1-phosphate (S1P) and vascular endothelial growth factor (VEGF) elicit numerous biological responses including cell survival, growth, migration, and differentiation in endothelial cells mediated by the endothelial differentiation gene, a family of G-protein-coupled receptors, and fetal liver kinase-1/kinase-insert domain-containing receptor (Flk-1/KDR), one of VEGF receptors, respectively. Recently, it was reported that S1P or VEGF treatment of endothelial cells leads to phosphorylation at Ser-1179 in bovine endothelial nitric oxide synthase (eNOS), and this phosphorylation is critical for eNOS activation. S1P stimulation of eNOS phosphorylation was shown to involve G(i) protein, phosphoinositide 3-kinase, and Akt. VEGF also activates eNOS through Flk-1/KDR, phosphoinositide 3-kinase, and Akt, which suggested that S1P and VEGF may share upstream signaling mediators. We now report that S1P treatment of bovine aortic endothelial cells acutely increases the tyrosine phosphorylation of Flk-1/KDR, similar to VEGF treatment. S1P-mediated phosphorylation of Flk-1/KDR, Akt, and eNOS were all inhibited by VEGF receptor tyrosine kinase inhibitors and by antisense Flk-1/KDR oligonucleotides. Our study suggests that S1P activation of eNOS involves G(i), calcium, and Src family kinase-dependent transactivation of Flk-1/KDR. These data are the first to establish a critical role of Flk-1/KDR in S1P-stimulated eNOS phosphorylation and activation.     

Stretch induces upregulation of key tyrosine kinase receptors in microvascular endothelial cells

We previously demonstrated that cyclic stretch of cardiac myocytes activates paracrine signaling via vascular endothelial growth factor (VEGF) leading to angiogenesis. The present study tested the hypothesis that cyclic stretch upregulates tyrosine kinase receptors in rat coronary microvascular endothelial cells (RCMEC) and human umbilical vein endothelial cells (HUVEC). VEGF receptor-2 (Flk-1) protein levels increased in HUVEC and RCMEC in a time-dependent manner, but the increase occurred much earlier in RCMEC than in HUVEC. The enhancement of Flk-1 protein level was not inhibited by addition of VEGF neutralizing antibodies, indicating that VEGF is not involved in stretch-induced Flk-1 expression. VEGF receptor-1 (Flt-1) protein and mRNA were not changed by stretch. However, Tie-2 and Tie-1 protein levels increased in RCMEC. Angiopoietin-1 and -2, the ligands for Tie-2, increased in cardiac myocytes subjected to cyclic stretch but were not affected by stretch in endothelial cells (EC). Stretch or incubation of RCMEC with VEGF increased cell proliferation moderately, whereas stretch + VEGF had an additive effect on proliferation. Mechanical stretch induces upregulation of the key tyrosine kinase receptors Flk-1, Tie-2, and Tie-1 in vascular EC, which underlies the increase in sensitivity of EC to growth factors and, therefore, facilitates angiogenesis. These in vitro findings support the concept that stretch of cardiac myocytes and EC plays a key role in coronary angiogenesis.     

Vascular endothelial growth factor-dependent down-regulation of Flk-1/KDR involves Cbl-mediated ubiquitination. Consequences on nitric oxide production from endothelial cells

Ligand-stimulated degradation of receptor tyrosine kinase (RTK) is an important regulatory step of signal transduction. The vascular endothelial growth factor (VEGF) receptor Flk-1/KDR is responsible for the VEGF-stimulated nitric oxide (NO) production from endothelial cells. Cellular mechanisms mediating the negative regulation of Flk-1 signaling in endothelial cells have not been investigated. Here we show that Flk-1 is rapidly down-regulated following VEGF stimulation of bovine aortic endothelial cells (BAECs). Consequently, VEGF pretreatment of endothelial cells prevents any further stimulation of Flk-1, resulting in decreased NO production from subsequent VEGF challenges. Ubiquitination of RTKs targets them for degradation; we demonstrate that activation of Flk-1 by VEGF leads to its polyubiquitination in BAECs. Furthermore, VEGF stimulation of BAECs or COS-7 cells transiently transfected with Flk-1 results in the phosphorylation of the ubiquitin ligase Cbl, the enhanced association of Cbl with Flk-1, and the relocalization of Cbl to vesicular structures in BAECs. Overexpression of Cbl in COS-7 cells enhances VEGF-induced ubiquitination of Flk-1, whereas a Cbl mutant lacking the ubiquitin ligase RING finger domain, 70Z/3-Cbl, does not. Moreover, expression of Cbl in contrast to 70Z/3-Cbl inhibits the Flk-1-dependent activation of eNOS and, thus, NO release. In BAEC overexpressing Cbl, the degradation of Flk-1 upon VEGF stimulation is accelerated compared with cells transfected with a control vector (green fluorescent protein). Our findings demonstrate that Flk-1 is rapidly down-regulated following sustained VEGF stimulation and identify Cbl as a negative regulator of Flk-1 signaling to eNOS. Cbl thus plays a role in the regulation of VEGF signaling by mediating the stimulated ubiquitination and, consequently, degradation of Flk-1 in endothelial cells.     

VEGF receptor-2 (Flk-1) overexpression in mice counteracts focal epileptic seizures

Vascular endothelial growth factor (VEGF) was first described as an angiogenic agent, but has recently also been shown to exert various neurotrophic and neuroprotective effects in the nervous system. These effects of VEGF are mainly mediated by its receptor, VEGFR-2, which is also referred to as the fetal liver kinase receptor 1 (Flk-1). VEGF is up-regulated in neurons and glial cells after epileptic seizures and counteracts seizure-induced neurodegeneration. In vitro, VEGF administration suppresses ictal and interictal epileptiform activity caused by AP4 and 0 Mg(2+) via Flk-1 receptor. We therefore explored whether increased VEGF signaling through Flk-1 overexpression may regulate epileptogenesis and ictogenesis in vivo. To this extent, we used transgenic mice overexpressing Flk-1 postnatally in neurons. Intriguingly, Flk-1 overexpressing mice were characterized by an elevated threshold for seizure induction and a decreased duration of focal afterdischarges, indicating anti-ictal action. On the other hand, the kindling progression in these mice was similar to wild-type controls. No significant effects on blood vessels or glia cells, as assessed by Glut1 and GFAP immunohistochemistry, were detected. These results suggest that increased VEGF signaling via overexpression of Flk-1 receptors may directly affect seizure activity even without altering angiogenesis. Thus, Flk-1 could be considered as a novel target for developing future gene therapy strategies against ictal epileptic activity.     

Rapid transactivation of the vascular endothelial growth factor receptor KDR/Flk-1 by the bradykinin B2 receptor contributes to endothelial nitric-oxide synthase activation in cardiac capillary endothelial cells.

Bradykinin (BK) and vascular endothelial growth factor (VEGF)-165 stimulate vasodilatation, microvascular permeability, and angiogenesis via the activation of the B2-type and KDR/Flk-1 receptors. To delineate the signal transduction pathways distal to the receptor activation in microvascular permeability, we compared their effects on two downstream targets, i.e. endothelial nitric-oxide (NO) synthase (eNOS) and F-actin, in primary cultures of cardiac capillary endothelial cells. The two mediators induced a similar cytoskeletal reorganization and both the translocation and activation of eNOS, leading to NO release within the first minutes of cell exposure. At the same time, BK produced the tyrosine phosphorylation and internalization of KDR/Flk-1 as did VEGF itself. This transactivation was blocked by the selective inhibitor of VEGF receptor tyrosine kinase activity but not by inhibitors of epidermal growth factor receptor or protein kinase C activity. The selective inhibitor of VEGF receptor tyrosine kinase activity totally prevented the effects of VEGF but only partially inhibited NO release induced by BK without affecting the concomitant cytoskeletal reorganization. Thus, BK transactivated KDR/Flk-1 through an intrinsic kinase activity of KDR/Flk-1, resulting in a further eNOS activation in endothelial cells. This represents a novel mechanism whereby a G protein-coupled receptor activates a receptor tyrosine kinase to generate biological response.     

Flt-1, but not Flk-1 mediates hyperpermeability through activation of the PI3-K/Akt pathway

Vascular endothelial growth factor (VEGF), a potent mediator of endothelial proliferation and migration, has an important role also in brain edema formation during hypoxia and ischemia. VEGF binds to the tyrosine kinase receptors Flt-1 and Flk-1. Yet, their relative importance for hypoxia-induced hyperpermeability is not well understood. We used an in vitro blood-brain barrier (BBB) model consisting of porcine brain microvascular endothelial cells (BMEC) to determine the role of Flt-1 in VEGF-induced endothelial cell (EC) barrier dysfunction. Soluble Flt-1 abolished hypoxia/VEGF-induced hyperpermeability. Furthermore, selective antisense oligonucleotides to Flt-1, but not to Flk-1, inhibited hypoxia-induced permeability changes. Consistent with these data, addition of the receptor-specific homolog placenta-derived growth factor, which binds Flt-1 but not Flk-1, increased endothelial permeability to the same extent as VEGF, whereas adding VEGF-E, a viral VEGF molecule from the orf virus family activating Flk-1 and neuropilin-1, but not Flt-1, did not show any effect. Using the carcinoma submandibular gland cell line (CSG), only expressing Flt-1, it was demonstrated that activation of Flt-1 is sufficient to induce hyperpermeability by hypoxia and VEGF. Hyperpermeability, induced by hypoxia/VEGF, depends on activation of phosphatidylinositol 3-kinase/Akt (PI3-K/Akt), nitric oxide synthase (NOS) and protein kinase G (PKG). The activation of the PI3-K/Akt pathway by hypoxia was confirmed using an in vivo mice hypoxia model. These results demonstrate that hypoxia/VEGF-induced hyperpermeability can be mediated by activation of Flt-1 independently on the presence of Flk-1 and indicate a central role for activation of the PI3-K/Akt pathway, followed by induction of NOS and PKG activity.     

Phenotypic and gene expression modification with normal brain aging in GFAP-positive astrocytes and neural stem cells

Astrocytes secrete growth factors that are both neuroprotective and supportive for the local environment. Identified by glial fibrillary acidic protein (GFAP) expression, astrocytes exhibit heterogeneity in morphology and in the expression of phenotypic markers and growth factors throughout different adult brain regions. In adult neurogenic niches, astrocytes secrete vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF-2) within the neurogenic niche and are also a source of special GFAP-positive multipotent neural stem cells (NSCs). Normal aging is accompanied by a decline in CNS function and reduced neurogenesis. We asked whether a decreased availability of astrocyte-derived factors may contribute to the age-related decline in neurogenesis. Determining alterations of astrocytic activity in the aging brain is crucial for understanding CNS homeostasis in aging and for assessing appropriate therapeutic targets for an aging population. We found region-specific alterations in the gene expression of GFAP, VEGF, and FGF-2 and their receptors in the aged brain corresponding to changes in astrocytic reactivity, supporting astrocytic heterogeneity and demonstrating a differential aging effect. We found that GFAP-positive NSCs uniquely coexpress both VEGF and its key mitotic receptor Flk-1 in both young and aged hippocampus, indicating a possible autocrine/paracrine signaling mechanism. VEGF expression is lost once NSCs commit to a neuronal fate, but Flk-1-mediated sensitivity to VEGF signaling is maintained. We propose that age-related astrocytic changes result in reduced VEGF and FGF-2 signaling, which in turn limits NSC and progenitor cell maintenance and contributes to decreased neurogenesis.     

Expression and distribution of vascular endothelial growth factor receptor Flk-1 in the rat pituitary.

Vascular endothelial growth factor (VEGF) acts primarily as an endothelial cell mitogen via the specific receptors Flk-1 and Flt-1. To help further define the possible role of VEGF in the control of pituitary cell function, we examined Flk-1 expression in normal rat pituitaries and in GH3 cells. Flk-1 expression was studied by immunohistochemistry, in situ hybridization, and double-labeling immunofluorescence combined with confocal laser microscopy. In normal rat pituitaries, Flk-1-immunoreactive cells appeared widely distributed only in the anterior lobe and were not detected in the intermediate or posterior lobe. Apart from the adenohypophysial cells, Flk-1 immunopositivity was also evident in endothelial cells of many capillaries distributed throughout the gland. Immunohistochemistry also showed that majority of GH3 cells expressed Flk-1 protein. In situ hybridization showed conclusive staining with the antisense probe and confirmed the immunohistochemical results. The double immunofluorescence method revealed Flk-1 expression in all types of hormone-producing adenohypophysial cells but not in folliculostellate cells. The percentage of immunopositive cells varied among the various cell types. The present study demonstrates that pituitary cells are not only sources of VEGF but also targets of this multifunctional substance, supporting the concept that VEGF functions as an autocrine/paracrine factor in the pituitary.     

Upregulation of Flk-1 by bFGF via the ERK pathway is essential for VEGF-mediated promotion of neural stem cell proliferation

Neural stem cells （NSCs） constitute the cellular basis for embryonic brain development and neurogenesis. The process is regulated by NSC niche including neighbor cells such as vascular and glial cells. Since both vascular and glial cells secrete vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, we assessed the effect of VEGF and bFGF on NSC proliferation using nearly homogeneous NSCs that were differentiated from mouse embryonic stem cells. VEGF alone did not have any significant effect. When bFGF was added, however, VEGF stimulated NSC proliferation in a dose-dependent manner, and this stimulation was inhibited by ZM323881, a VEGF receptor （Flk-1）- specific inhibitor. Interestingly, ZM323881 also inhibited cell proliferation in the absence of exogenous VEGF, suggesting that VEGF autocrine plays a role in the proliferation of NSCs. The stimulatory effect of VEGF on NSC proliferation depends on bFGF, which is likely due to the fact that expression of Flk-1 was upregulated by bFGF via phosphorylation of ERK1/2. Collectively, this study may provide insight into the mechanisms by which microenvironmental niche signals regulate NSCs.     

Enhancement of ischemia-induced tyrosine phosphorylation of Kv1.2 by vascular endothelial growth factor via activation of phosphatidylinositol 3-kinase

Our studies observed that, consistent with the literature, ischemic/hypoxic insults increased the expression of voltage-gated potassium channel (Kv) 1.2 potassium channel as well as elevating the endogenous level of vascular endothelial growth factor (VEGF) in neurons of adult rat brain following middle cerebral artery occlusion and in SH-SY5Y cells after hypoxia and glucose deprivation. Concomitantly, we also observed that ischemic injury increased the tyrosine phosphorylation of Kv 1.2 in in vivo and in vitro; the introduction of exogenous VEGF could attenuate cell death in in vitro models. Furthermore, we found that the protective effect of VEGF is mediated through its up-regulative actions on the tyrosine phosphorylation of Kv 1.2, which in turn has a direct influence on cell viability after ischemic insult. In substantiation of this result, we used anti-sense methodology to suppress the expression of endogenous VEGF, which significantly inhibited the tyrosine phosphorylation of Kv 1.2 and increased cell death elicited by ischemic/hypoxic injury. Finally, the enhancement of the tyrosine phosphorylation of the channel by VEGF in neuronal cells was significantly attenuated in the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), or genestin, an inhibitor of tyrosine kinase, thus suggesting that the phosphorylation of Kv 1.2 induced by VEGF is mechanistically linked to the PI3-K pathway.     

Differential roles of vascular endothelial growth factor receptors 1 and 2 in dendritic cell differentiation

Impaired Ag-presenting function in dendritic cells (DCs) due to abnormal differentiation is an important mechanism of tumor escape from immune control. A major role for vascular endothelial growth factor (VEGF) and its receptors, VEGFR1/Flt-1 and VEGFR2/KDR/Flk-1, has been documented in hemopoietic development. To study the roles of each of these receptors in DC differentiation, we used an in vitro system of myeloid DC differentiation from murine embryonic stem cells. Exposure of wild-type, VEGFR1(-/-), or VEGFR2(-/-) embryonic stem cells to exogenous VEGF or the VEGFR1-specific ligand, placental growth factor, revealed distinct roles of VEGF receptors. VEGFR1 is the primary mediator of the VEGF inhibition of DC maturation, whereas VEGFR2 tyrosine kinase signaling is essential for early hemopoietic differentiation, but only marginally affects final DC maturation. SU5416, a VEGF receptor tyrosine kinase inhibitor, only partially rescued the mature DC phenotype in the presence of VEGF, suggesting the involvement of both tyrosine kinase-dependent and independent inhibitory mechanisms. VEGFR1 signaling was sufficient for blocking NF-kappaB activation in bone marrow hemopoietic progenitor cells. VEGF and placental growth factor affect the early stages of myeloid/DC differentiation. The data suggest that therapeutic strategies attempting to reverse the immunosuppressive effects of VEGF in cancer patients might be more effective if they specifically targeted VEGFR1.     

Human herpesvirus 8 (HHV-8)-encoded cytokines induce expression of and autocrine signaling by vascular endothelial growth factor (VEGF) in HHV-8-infected primary-effusion lymphoma cell lines and mediate VEGF-independent antiapoptotic effects

The potential roles of human herpesvirus 8 (HHV-8) cytokines in HHV-8 pathogenesis were investigated by determining the expression of the HHV-8 chemokines viral macrophage inflammatory protein 1A (vMIP-1A) and vMIP-1B in primary effusion lymphoma (PEL)-derived cell lines and examining the signaling activities of these chemokines and HHV-8-encoded vIL-6 in these cells. Secreted vMIP-1A and vMIP-1B were detected in biologically significant concentrations following tetradecanoyl phorbol acetate treatment, which induces productive replication. vIL-6 and vMIP-1A, added exogenously to cultures of four different PEL cell lines, induced the expression of vascular endothelial growth factor type B (VEGF-B) and VEGF-A, respectively. These cells were found to express VEGF receptor 1 (Flt-1) protein, and signaling by recombinant VEGF-A(165) was demonstrated for two of the PEL cell lines, indicating the potential for autocrine, as well as paracrine, effects of viral cytokine-induced VEGF. In addition, vMIP-1A and vMIP-1B, but not VEGF-A(165), were found to inhibit chemically induced apoptosis in PEL cells. Our data suggest that vIL-6 and vMIP-1A may influence PEL through VEGF autocrine and paracrine signaling that promotes PEL cell growth and extravascular effusion and that vMIP-1A and vMIP-1B can act independently of VEGF as antiapoptotic factors.     

Lipocortin V may function as a signaling protein for vascular endothelial growth factor receptor-2/Flk-1.

Binding of vascular endothelial growth factor (VEGF) to its receptor, VEGFR-2 (Flk-1/KDR), induces dimerization and activation of the tyrosine kinase domain of the receptor, resulting in autophosphorylation of cytoplasmic tyrosine residues used as docking sites for signaling proteins that relay the signals for cell proliferation, migration, and permeability enhancement. We explored the VEGF/receptor signaling pathway by performing a two-hybrid screen of a rat lung cDNA library in yeast using the intracellular domain of rat VEGFR-2 as bait. Two clones encoding lipocortin V were isolated. Subsequent studies with the yeast two-hybrid assay showed that the complete intracellular domain of VEGFR-2 was required for the interaction. Co-immunoprecipitation of translated proteins confirmed the interaction between the VEGF receptor and lipocortin V. VEGF induced a rapid tyrosine phosphorylation of lipocortin V in human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with antisense oligodeoxyribonucleotide (ODN) for lipocortin V significantly inhibited VEGF-induced cell proliferation, which was accompanied by a decrease in protein synthesis and tyrosine phosphorylation of lipocortin V. Our results indicate that lipocortin V may function as a signaling protein for VEGFR-2 by directly interacting with the intracellular domain of the receptor and appears to be involved in regulation of vascular endothelial cell proliferation mediated by VEGFR-2.     

Endostatin blocks vascular endothelial growth factor-mediated signaling via direct interaction with KDR/Flk-1

Endostatin, a fragment of collagen XVIII, is a potent anti-angiogenic protein, but the molecular mechanism of its action is not yet clear. We examined the effects of endostatin on the biological and biochemical activities of vascular endothelial growth factor (VEGF). Endostatin blocked VEGF-induced tyrosine phosphorylation of KDR/Flk-1 and activation of ERK, p38 MAPK, and p125(FAK) in human umbilical vein endothelial cells. Endostatin also inhibited the binding of VEGF(165) to both endothelial cells and purified extracellular domain of KDR/Flk-1. Moreover, the binding of VEGF(121) to KDR/Flk-1 and VEGF(121)-stimulated ERK activation were blocked by endostatin. The direct interaction between endostatin and KDR/Flk-1 was confirmed by affinity chromatography. However, endostatin did not bind to VEGF. Our findings suggest that a direct interaction of endostatin with KDR/Flk-1 may be involved in the inhibitory function of endostatin toward VEGF actions and responsible for its potent anti-angiogenic and anti-tumor activities in vivo.