KDR Phosphorylation Research Articles

The heparin-binding domain of VEGF165 directly binds to integrin αvβ3 and VEGFR2/KDR D1: a potential mechanism of negative regulation of VEGF165 signaling by αvβ3.

VEGF-A is a key cytokine in tumor angiogenesis and a major therapeutic target for cancer. VEGF165 is the predominant isoform of VEGF-A, and it is the most potent angiogenesis stimulant. VEGFR2/KDR domains 2 and 3 (D2D3) bind to the N-terminal domain (NTD, residues 1-110) of VEGF165. Since removal of the heparin-binding domain (HBD, residues 111-165) markedly reduced the mitogenic activity of the growth factor, it has been proposed that the HBD plays a critical role in the mitogenicity of VEGF165. Here, we report that αvβ3 specifically bound to the isolated VEGF165 HBD but not to VEGF165 NTD. Based on docking simulation and mutagenesis, we identified several critical amino acid residues within the VEGF165 HBD required for αvβ3 binding, i.e., Arg123, Arg124, Lys125, Lys140, Arg145, and Arg149. We discovered that VEGF165 HBD binds to the KDR domain 1 (D1) and identified that Arg123 and Arg124 are critical for KDR D1 binding by mutagenesis, indicating that the KDR D1-binding and αvβ3-binding sites overlap in the HBD. Full-length VEGF165 mutant (R123A/R124A/K125A/K140A/R145A/R149A) defective in αvβ3 and KDR D1 binding failed to induce ERK1/2 phosphorylation, integrin β3 phosphorylation, and KDR phosphorylation and did not support proliferation of endothelial cells, although the mutation did not affect the KDR D2D3 interaction with VEGF165. Since β3-knockout mice are known to show enhanced VEGF165 signaling, we propose that the binding of KDR D1 to the VEGF165 HBD and KDR D2D3 binding to the VEGF165 NTD are critically involved in the potent mitogenicity of VEGF165. We propose that binding competition between KDR and αvβ3 to the VEGF165 HBD endows integrin αvβ3 with regulatory properties to act as a negative regulator of VEGF165 signaling.

Ionizing Radiation-Induced GDF15 Promotes Angiogenesis in Human Glioblastoma Models by Promoting VEGFA Expression Through p-MAPK1/SP1 Signaling.

Glioblastoma multiforme (GBM), the most aggressive cancer type that has a poor prognosis, is characterized by enhanced and aberrant angiogenesis. In addition to surgical resection and chemotherapy, radiotherapy is commonly used to treat GBM. However, radiation-induced angiogenesis in GBM remains unexplored. This study examined the role of radiation-induced growth/differentiation factor-15 (GDF15) in regulating tumor angiogenesis by promoting intercellular cross-talk between brain endothelial cells (ECs) and glioblastoma cells. Radiation promoted GDF15 secretion from human brain microvascular endothelial cells (HBMVECs). Subsequently, GDF15 activated the transcriptional promoter VEGFA in the human glioblastoma cell line U373 through p-MAPK1/SP1 signaling. Upregulation of vascular endothelial growth factor (VEGF) expression in U373 cells resulted in the activation of angiogenic activity in HBMVECs via KDR phosphorylation. Wound healing, tube formation, and invasion assay results revealed that the conditioned medium of recombinant human GDF15 (rhGDF15)-stimulated U373 cell cultures promoted the angiogenic activity of HBMVECs. In the HBMVEC-U373 cell co-culture, GDF15 knockdown mitigated radiation-induced VEGFA upregulation in U373 cells and enhanced angiogenic activity of HBMVECs. Moreover, injecting rhGDF15-stimulated U373 cells into orthotopic brain tumors in mice promoted angiogenesis in the tumors. Thus, radiation-induced GDF15 is essential for the cross-talk between ECs and GBM cells and promotes angiogenesis. These findings indicate that GDF15 is a putative therapeutic target for patients with GBM undergoing radio-chemotherapy.

Hypoxia alleviates dexamethasone-induced inhibition of angiogenesis in cocultures of HUVECs and rBMSCs via HIF-1\u03b1

Background and aimInadequate vascularization is a challenge in bone tissue engineering because internal cells are prone to necrosis due to a lack of nutrient supply. Rat bone marrow-derived mesenchymal stem cells (rBMSCs) and human umbilical vein endothelial cells (HUVECs) were cocultured to construct prevascularized bone tissue in osteogenic induction medium (OIM) in vitro. The angiogenic capacity of HUVECs was limited in the coculture system. In this study, the effects of the components in the medium on HUVEC angiogenesis were analyzed.MethodsThe coculture system was established in OIM. Alizarin red staining and alkaline phosphatase staining were used to assess the osteogenic ability of MSCs. A Matrigel tube assay was used to assess the angiogenic ability of HUVECs in vitro. The proliferation of HUVECs was evaluated by cell counting and CCK-8 assays, and migration was evaluated by the streaked plate assay. The expression levels of angiogenesis-associated genes and proteins in HUVECs were measured by qRT-PCR and Western blotting, respectively.ResultsDexamethasone in the OIM suppressed the proliferation and migration of HUVECs, inhibiting the formation of capillary-like structures. Our research showed that dexamethasone stimulated HUVECs to secrete tissue inhibitor of metalloproteinase (TIMP-3), which competed with vascular endothelial growth factor (VEGF-A) to bind to vascular endothelial growth factor receptor 2 (VEGFR2, KDR). This effect was related to inhibiting the phosphorylation of ERK and AKT, which are two downstream targets of KDR. However, under hypoxia, the enhanced expression of hypoxia-inducible factor-1α (HIF-1α) decreased the expression of TIMP-3 and promoted the phosphorylation of KDR, improving HUVEC angiogenesis in the coculture system.ConclusionCoculture of hypoxia-preconditioned HUVECs and MSCs showed robust angiogenesis and osteogenesis in OIM, which has important implications for prevascularization in bone tissue engineering in the future.

The study of the treatment of non-small cell lung cancer by the combination therapy of cetuximab and ramucirumab

Objective Explore the target West to rituximab and remo combination on cell A549 Lo mAb treatment of non-small cell lung cancer and its effect mechanism. Methods Detection of epidermal growth factor receptor (EGFR)/vascular endothelial growth factor receptor 2 (VEGFR2) expression in A549 cell surface; detection effect of cetuximab and General Administration of the A549 Lo monoclonal antibody combined with cell proliferation, migration, antibody dependent cellular cytotoxicity (ADCC) and related signal pathway. Results In combination with cetuximab and A549 cells was 90.5%, antibody binding rate was 27% Lu remo. Cetuximab, monoclonal antibody, CO administration of Remo Lu had inhibitory effect on A549 cells, in a dose-dependent manner; cetuximab, Remo Lo mAb group cell migration is relatively slow, with the slowest cell migration group; drug combination can be a very good mediated cell killing effect, better than the West rituximab group and remo Lu co administration of monoclonal antibody group; simultaneously blocking EGFR and phosphorylation of KDR, protein kinase B (Akt) and p38, resulting in the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway was strongly inhibited. Conclusion After combined treatment of antibodies targeting EGFR and VEGFR2, the proliferation and cell migration of A549 cells were decreased, and the ADCC function was enhanced. Combined administration significantly improved the antitumor effect of single drug. Key words: Epidermal growth factor receptor; Vascular endothelial growth factor receptor 2; Non-small-cell lung carcinoma

Autophagy-induced KDR/VEGFR-2 activation promotes the formation of vasculogenic mimicry by glioma stem cells

ABSTRACTAntiangiogenesis with bevacizumab, an antibody against vascular endothelial growth factor (VEGF), has been used for devascularization to limit the growth of malignant glioma. However, the benefits are transient due to elusive mechanisms underlying resistance to the antiangiogenic therapy. Glioma stem cells (GSCs) are capable of forming vasculogenic mimicry (VM), an alternative microvascular circulation independent of VEGF-driven angiogenesis. Herein, we report that the formation of VM was promoted by bevacizumab-induced macroautophagy/autophagy in GSCs, which was associated with tumor resistance to antiangiogenic therapy. We established a 3-dimensional collagen scaffold to examine the formation of VM and autophagy by GSCs, and found that rapamycin increased the number of VM and enhanced KDR/VEGFR-2 phosphorylation. Treatment with chloroquine, or knockdown of the autophagy gene ATG5, inhibited the formation of VM and KDR phosphorylation in GSCs. Notably, neutralization of GSCs-produced VEGF with bevacizumab failed to recapitulate the effect of chloroquine treatment and ATG5 knockdown, suggesting that autophagy-promoted formation of VM was independent of tumor cell-derived VEGF. ROS was elevated when autophagy was induced in GSCs and activated KDR phosphorylation through the phosphoinositide 3-kinase (PI3K)-AKT pathway. A ROS inhibitor, N-acetylcysteine, abolished KDR phosphorylation and the formation of VM by GSCs. By examination of the specimens from 95 patients with glioblastoma, we found that ATG5 and p-KDR expression was strongly associated with the density of VM in tumors and poor clinical outcome. Our results thus demonstrate a crucial role of autophagy in the formation of VM by GSCs, which may serve as a therapeutic target in drug-resistant glioma.

A novel compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) screened by tissue angiogenesis model and its activity evaluation on anti-angiogenesis

A tissue model for angiogenesis that imitated new blood vessels formation in vivo had been established in the previous study. Here, it was used to screen and evaluate a series of synthesized compounds and the results indicated that compound T7 (N-{4′-[(1E)-N-hydroxyethanimidoyl]-3′,5,6-trimethoxybiphenyl-3-yl}-N′-[4-(3-morpholin-4-ylpropoxy)phenyl]urea) could effectively inhibit the blood vessels formation. Then the anti-angiogenic potential of T7 and its related molecular mechanisms against lung carcinoma in vitro and in vivo were investigated. Treatment with T7 significantly inhibited human umbilical vein endothelial cells and A549 cells proliferation and migration. T7 reduced human umbilical vein endothelial cells tube formation as well. Western blotting analysis of cell signaling molecules indicated that T7 reduced phosphorylation of KDR and its downstream signaling players AKT and ERK1/2 activation in endothelial cells and A549 cells. Moreover, T7 inhibited tumor growth in A549 xenografted model of athymic mice and reduced CD34 expression levels in tumor-bearing mice by immunohistochemistry. In sum, our findings showed that T7 was a candidate of tumor angiogenesis inhibitors, and it functioned by interrupting the autophosphorylation of KDR, AKT and ERK1/2.

Both Kdr and Flt1 play a vital role in hypoxia‐induced Src‐PLD1‐PKCγ‐cPLA2 activation and retinal neovascularization

Previously we have reported that Src‐PLD1‐PKCγ‐cPLA2 signaling plays a role in hypoxia induced retinal neovascularization. In the present study we explored the upstream mechanisms of Src‐PLD1‐PKCγ‐cPLA2 activation and retinal neovascularization. VEGFA, while having no effect on Flt1 phosphorylation, induced Kdr phosphorylation in human retinal microvascular endothelial cells (HRMVECs). Depletion of either Kdr or Flt1 attenuated VEGFA‐induced Src‐PLD1‐PKCγ‐cPLA2 activation, DNA synthesis, migration, and tube formation, albeit more robustly with Kdr downregulation. Hypoxia induced tyrosine phosphorylation of Kdr and Flt1 in mouse retina and depletion of Kdr or Flt1 blocked hypoxia‐induced Src‐PLD1‐PKCγ‐cPLA2 activation and retinal neovascularization. Hypoxia induced VEGFA and VEGFB expression in retina, and inhibition of their expression blocked hypoxia‐induced Kdr and Flt1 phosphorylation, respectively. Furthermore, depletion of VEGFA or VEGFB attenuated hypoxia‐induced Src‐PLD1‐PKCγ‐cPLA2 activation and retinal neovascularization. These findings suggest that both VEGFA and VEGFB mediate hypoxia‐induced Src‐PLD1‐PKCγ‐cPLA2 activation and retinal neovascularization via activation of Kdr and Flt1, respectively. Suported by EY014856, National Eye Institute/NIH

Both Kdr and Flt1 play a vital role in hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization

AbstractTo understand the mechanisms of Src-PLD1-PKCγ-cPLA2 activation by vascular endothelial growth factor A (VEGFA), we studied the role of Kdr and Flt1. VEGFA, while having no effect on Flt1 phosphorylation, induced Kdr phosphorylation in human retinal microvascular endothelial cells (HRMVECs). Depletion of Kdr attenuated VEGFA-induced Src-PLD1-PKCγ-cPLA2 activation. Regardless of its phosphorylation state, downregulation of Flt1 also inhibited VEGFA-induced Src-PLD1-PKCγ-cPLA2 activation, but only modestly. In line with these findings, depletion of either Kdr or Flt1 suppressed VEGFA-induced DNA synthesis, migration, and tube formation, albeit more robustly with Kdr downregulation. Hypoxia induced tyrosine phosphorylation of Kdr and Flt1 in mouse retina, and depletion of Kdr or Flt1 blocked hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization. VEGFB induced Flt1 tyrosine phosphorylation and Src-PLD1-PKCγ-cPLA2 activation in HRMVECs. Hypoxia induced VEGFA and VEGFB expression in retina, and inhibition of their expression blocked hypoxia-induced Kdr and Flt1 activation, respectively. Furthermore, depletion of VEGFA or VEGFB attenuated hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization. These findings suggest that although VEGFA, through Kdr and Flt1, appears to be the major modulator of Src-PLD1-PKCγ-cPLA2 signaling in HRMVECs, facilitating their angiogenic events in vitro, both VEGFA and VEGFB mediate hypoxia-induced Src-PLD1-PKCγ-cPLA2 activation and retinal neovascularization via activation of Kdr and Flt1, respectively.

Polyproline‐type helical‐structured low‐molecular weight heparin (LMWH)‐taurocholate conjugate as a new angiogenesis inhibitor

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low-molecular heparin (LMWH), for treating cancer are limited because of heparin's anticoagulant activity and risk of hemorrhages. LMWH-taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF(165)) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF-mediated KDR (VEGF-receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF(165) dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF(165) inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA-MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline-type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF(165). Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA-MB231 human breast cancer cell lines.

Human head and neck squamous cell carcinoma cells are both targets and effectors for the angiogenic cytokine, VEGF

Former vascular endothelial growth factor (VEGF)-head and neck squamous cell carcinoma (HNSCC) studies have focused on VEGF's contributions toward tumor-associated angiogenesis. Previously, we have shown that HNSCC cells produce high levels of VEGF. We therefore hypothesized that VEGF serves a biphasic role, that is, pro-angiogenic and pro-tumorigenic in HNSCC pathogenesis. Western blots confirmed the presence of VEGF's primary mitogenic receptors, VEGFR-2/KDR and VEGFR-1/Flt-1 in cultured HNSCC cells. Subsequent studies evaluated VEGF's effects on HNSCC intracellular signaling, mitogenesis, invasive capacities, and matrix metalloproteinases (MMPs) activities. Introduction of hrVEGF(165) initiated ROS-mediated intracellular signaling, resulting in kinase activation and phosphorylation of KDR and Erk1/2. As high endogenous VEGF production rendered HNSCC cells refractory to exogenous VEGF's mitogenic effects, siRNA was employed, inhibiting endogenous VEGF production for up to 96 h. Relative to transfection vector matched controls, siRNA treated HNSCC cells showed a significant decrease in proliferation at both 30 and 50 nM siRNA doses. Addition of exogenous hrVEGF(165) (30 and 50 ng/ml) to siRNA-silenced HNSCC cells resulted in dose-dependent increases in cell proliferation. Cell invasion assays showed VEGF is a potent HNSCC chemoattractant and demonstrated that VEGF pre-treatment enhanced invasiveness of HNSCC cells. Conditioned media from VEGF challenged HNSCC cells showed a moderate increase in gelatinase activity. Our results demonstrate, for the first time, that HNSCC cells are both targets and effectors for VEGF. These data introduce the prospect that VEGF targeted therapy has the potential to fulfill both anti-angiogenic and anti-tumorigenic functions.

Sulfated polymannuroguluronate, a novel anti-AIDS drug candidate, inhibits HIV-1 Tat-induced angiogenesis in Kaposi's sarcoma cells

Kaposi's sarcoma (KS), a neoplasm often associated with iatrogenic and acquired immunosuppression, is characterized by prominent angiogenesis. Angiogenic factors released from KS and host cells and HIV viral products—the protein Tat are reported to be involved in angiogenesis. Mounting evidence further suggests that multiple angiogenic activities of Tat contribute to AIDS-associated Kaposi's sarcoma (AIDS-KS). Herein, we report that sulfated polymannuroguluronate (SPMG), a novel anti-AIDS drug candidate now undergoing phase II clinical trial, significantly eliminated Tat-induced angiogenesis in SLK cells both in vitro and in vivo. SPMG significantly and dose-dependently inhibits proliferation, migration, and tube formation by SLK cells. SPMG also dramatically arrested Tat-driven KDR phosphorylation and blocked the interaction between Tat and integrin β1, thus inhibiting the phosphorylation of the downstream kinases of FAK, paxillin and MAPKs. In addition, SPMG was noted to block the release of bFGF and VEGF from ECM. All these collectively favor an issue that SPMG functions as a promising therapeutic against Tat-induced angiogenesis and pathologic events relevant to AIDS-KS, which adds novel mechanistic profiling to the anti-AIDS action of SPMG.

Characterization of a Bicyclic Peptide Neuropilin-1 (NP-1) Antagonist (EG3287) Reveals Importance of Vascular Endothelial Growth Factor Exon 8 for NP-1 Binding and Role of NP-1 in KDR Signaling

Neuropilin-1 (NP-1) is a receptor for vascular endothelial growth factor-A165 (VEGF-A165) in endothelial cells. To define the role of NP-1 in the biological functions of VEGF, we developed a specific peptide antagonist of VEGF binding to NP-1 based on the NP-1 binding site located in the exon 7- and 8-encoded VEGF-A165 domain. The bicyclic peptide, EG3287, potently (K(i) 1.2 microM) and effectively (>95% inhibition at 100 microM) inhibited VEGF-A165 binding to porcine aortic endothelial cells expressing NP-1 (PAE/NP-1) and breast carcinoma cells expressing only NP-1 receptors for VEGF-A, but had no effect on binding to PAE/KDR or PAE/Flt-1. Molecular dynamics calculations, a nuclear magnetic resonance structure of EG3287, and determination of stability in media, indicated that it constitutes a stable subdomain very similar to the corresponding region of native VEGF-A165. The C terminus encoded by exon 8 and the three-dimensional structure were both critical for EG3287 inhibition of NP-1 binding, whereas modifications at the N terminus had little effect. Although EG3287 had no direct effect on VEGF-A165 binding to KDR receptors, it inhibited cross-linking of VEGF-A165 to KDR in human umbilical vein endothelial cells co-expressing NP-1, and inhibited stimulation of KDR and PLC-gamma tyrosine phosphorylation, activation of ERKs1/2 and prostanoid production. These findings characterize the first specific antagonist of VEGF-A165 binding to NP-1 and demonstrate that NP-1 is essential for optimum KDR activation and intracellular signaling. The results also identify a key role for the C-terminal exon 8 domain in VEGF-A165 binding to NP-1.

The VEGF-C/Flt-4 axis promotes invasion and metastasis of cancer cells

Flt-4, a VEGF receptor, is activated by its specific ligand, VEGF-C. The resultant signaling pathway promotes angiogenesis and/or lymphangiogenesis. This report provides evidence that the VEGF-C/Flt-4 axis enhances cancer cell mobility and invasiveness and contributes to the promotion of cancer cell metastasis. VEGF-C/Flt-4-mediated invasion and metastasis of cancer cells were found to require upregulation of the neural cell adhesion molecule contactin-1 through activation of the Src-p38 MAPK-C/EBP-dependent pathway. Examination of tumor tissues from various types of cancers revealed high levels of Flt-4 and VEGF-C expression that correlated closely with clinical metastasis and patient survival. The VEGF-C/Flt-4 axis, through upregulation of contactin-1, may regulate the invasive capacity in different types of cancer cells.

Interleukin-6 triggers human cerebral endothelial cells proliferation and migration: The role for KDR and MMP-9

Interleukin-6 (IL-6) is involved in angiogenesis. However, the underlying mechanisms are unknown. Using human cerebral endothelial cell (HCEC), we report for the first time that IL-6 triggers HCEC proliferation and migration in a dose-dependent manner, specifically associated with enhancement of VEGF expression, up-regulated and phosphorylated VEGF receptor-2 (KDR), and stimulated MMP-9 secretion. We investigated the signal pathway of IL-6/IL-6R responsible for KDR’s regulation. Pharmacological inhibitor of PI3K failed to inhibit IL-6-mediated VEGF overexpression, while blocking ERK1/2 with PD98059 could abolish IL-6-induced KDR overexpression. Further, neutralizing endogenous VEGF attenuated KDR expression and phosphorylation, suggesting that IL-6-induced KDR activation is independent of VEGF stimulation. MMP-9 inhibitor GM6001 significantly decreases HCEC proliferation and migration ( p < 0.05), indicating the crucial function of MMP-9 in promoting angiogenic changes in HCECs. We conclude that IL-6 triggers VEGF-induced angiogenic activity through increasing VEGF release, up-regulates KDR expression and phosphorylation through activating ERK1/2 signaling, and stimulates MMP-9 overexpression.

AZD2171: A Highly Potent, Orally Bioavailable, Vascular Endothelial Growth Factor Receptor-2 Tyrosine Kinase Inhibitor for the Treatment of Cancer

Inhibition of vascular endothelial growth factor-A (VEGF) signaling is a promising therapeutic approach that aims to stabilize the progression of solid malignancies by abrogating tumor-induced angiogenesis. This may be accomplished by inhibiting the kinase activity of VEGF receptor-2 (KDR), which has a key role in mediating VEGF-induced responses. The novel indole-ether quinazoline AZD2171 is a highly potent (IC50 < 1 nmol/L) ATP-competitive inhibitor of recombinant KDR tyrosine kinase in vitro. Concordant with this activity, in human umbilical vein endothelial cells, AZD2171 inhibited VEGF-stimulated proliferation and KDR phosphorylation with IC50 values of 0.4 and 0.5 nmol/L, respectively. In a fibroblast/endothelial cell coculture model of vessel sprouting, AZD2171 also reduced vessel area, length, and branching at subnanomolar concentrations. Once-daily oral administration of AZD2171 ablated experimental (VEGF-induced) angiogenesis in vivo and inhibited endochondral ossification in bone or corpora luteal development in ovary; physiologic processes that are highly dependent upon neovascularization. The growth of established human tumor xenografts (colon, lung, prostate, breast, and ovary) in athymic mice was inhibited dose-dependently by AZD2171, with chronic administration of 1.5 mg per kg per day producing statistically significant inhibition in all models. A histologic analysis of Calu-6 lung tumors treated with AZD2171 revealed a reduction in microvessel density within 52 hours that became progressively greater with the duration of treatment. These changes are indicative of vascular regression within tumors. Collectively, the data obtained with AZD2171 are consistent with potent inhibition of VEGF signaling, angiogenesis, neovascular survival, and tumor growth. AZD2171 is being developed clinically as a once-daily oral therapy for the treatment of cancer.

Expression and purification of the catalytic domain of human vascular endothelial growth factor receptor 2 for inhibitor screening

Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, can act in tumor-induced angiogenesis by binding to specific receptors on the surface of endothelial cells. One such receptor, VEGFR-2/KDR, plays a key role in VEGF-induced angiogenesis. Here, we expressed the catalytic domain of VEGFR-2 as a soluble active kinase using Bac-to-Bac expression system, and investigated correlations between VEGFR-2 activity and enzyme concentration, ATP concentration, substrate concentration and divalent cation type. We used these data to establish a convenient, effective and non-radioactive ELISA screening technique for the identification and evaluation of potential inhibitors for VEGFR-2 kinase. We screened 200 RTK target-based compounds and identified one (TKI-31) that potently inhibited VEGFR-2 kinase activity (IC50=0.596 microM). Treatment of NIH3T3/KDR cells with TKI-31 blocked VEGF-induced phosphorylation of KDR in a dose-dependent manner. Moreover, TKI-31 dose-dependently suppressed HUVEC tube formation. Thus, we herein report a novel, efficient method for identifying VEGFR-2 kinase inhibitors and introduce one, TKI-31, that may prove to be a useful new angiogenesis inhibitor.

Endostatin Inhibits VEGF-Induced Lung Edema by Reducing Vascular Permeability.

INTRODUCTION: Acute lung injury is a severe condition developing in patients with acute sepsis characterized by lung edema with extravasation of plasma proteins, infiltration by inflammatory cells and pulmonary hemorrhage. Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is known to not only promote angiogenesis but also increase vascular permeability and has been linked to pathological conditions like retinopathy and acute lung injury. Patients with sepsis and acute lung injury have increased VEGF levels in their plasma. As shown recently, mice treated with VEGF develop histological changes comparable to acute lung injury in septic patients. Endostatin (ES), a endogenous inhibitor of angiogenesis, has been shown to inhibit tumor growth in various models. The mechanisms by which ES inhibits endothelial cell proliferation and function are not clear, yet. It was proposed that one mechanism is the inhibition of VEGF-induced activation of VEGF receptor 2 (VEGFR-2). This study was performed in order to examine whether ES may antagonize VEGF-induced effects leading to increased permeability and lung injury.METHODS: We established an in vitro permeability model using transwell chambers with human dermal microvascular endothelial cells (HDMEC) seeded in the upper chamber on a porous membrane. VEGF-induced permeability was determined by measuring methylene blue to the lower chamber. In order to test the effect of ES on VEGFR-2 activation porcine aortic endothelial cells expressing human VEGFR-2 were incubated with 50 ng/ml VEGF with or without 5 ug/ml ES for 30 min ice. Cell lysate was precipitated with conacavalin A, separated by SDS-PAGE, blotted and analyzed for KDR phosphorylation using phosphotyrosine specific and KDR antibodies. We generated cells with strong expresssion of either VEGF or mouse ES. The cells were encapsulated in alginate beads and injected s.c. to SCID mice divided in the following groups: A) control, B) VEGF, C) ES, D) VEGF + ES with 5 animals per group. After 5 days lungs were harvested and analyzed by H&E staining of tissue sections.RESULTS: In an in vitro permeability assay VEGF enhanced permeation of dye through a monolayer of endothelial cells. ES significantly inhibited VEGF induced permeability (fig.1). ES alone had no effect compared to controls. On the molecular level VEGF causes phosphorylation of its receptor VEGFR-2 as seen in VEGFR-2 expressing cells (fig.2). This effect was abolished by coincubation with ES showing a direct antagonism of VEGF signalling by ES. In a SCID mouse model animals were treated with VEGF, ES or the combination of both (fig.3). Animals in the VEGF group developed general edema and lung injury resembling acute lung injury with extravasation, accumulation of inflammatory cells and hemorrhage. The animals treated with a combination of VEGF and ES had less generalized edema. The lung sections showed alterations less pronounced than in the VEGF group. ES alone had no effect.CONCLUSIONS: Our results demonstrate that ES inhibits VEGF-induced permeability by blocking VEGFR-2 activation. ES treatment partially restored VEGF-induced lung injury in vivo. The incomplete inhibition may be due to excess VEGF protein levels. Taken together, VEGF blocking with ES may serve as a useful new treatment option for conditions with increased vascular permeability like acute lung injury.

Reactive oxygen species as mediators of angiogenesis signaling. Role of NAD(P)H oxidase

Angiogenesis, a process of new blood vessel growth, contributes to various pathophysiologies such as cancer, diabetic retinopathy and atherosclerosis. Accumulating evidence suggests that cardiovascular diseases are associated with increased oxidative stress in blood vessels. Reactive oxygen species (ROS) such as superoxide and H2O2 cause blood vessels to thicken, produce inflammation in the vessel wall, and thus are regarded as "risk factors" for vascular disease, whereas ROS also act as signaling molecules in many aspects of growth factor-mediated physiological responses. Recent reports suggest that ROS play an important role in angiogenesis; however, its underlying molecular mechanisms remain unknown. Vascular endothelial growth factor (VEGF) induces angiogenesis by stimulating endothelial cell (EC) proliferation and migration primarily through the receptor tyrosine kinase VEGF receptor2 (Flk1/KDR). VEGF binding initiates tyrosine phosphorylation of KDR, which results in activation of downstream signaling enzymes including ERK1/2, Akt and eNOS, which contribute to angiogenic-related responses in EC. Importantly, the major source of ROS in EC is a NAD(P)H oxidase and EC express all the components of phagocytic NAD(P)H oxidase including gp91phox, p22phox, p47phox, p67phox and the small G protein Rac1. We have recently demonstrated that ROS derived from NAD(P)H oxidase are critically important for VEGF signaling in vitro and angiogenesis in vivo. Furthermore, a peptide hormone, angiotensin II, a major stimulus for vascular NAD(P)H oxidase, also plays an important role in angiogenesis. Because EC migration and proliferation are primary features of the process of myocardial angiogenesis, we would like to focus on the recent progress that has been made in the emerging area of NAD(P)H oxidase-derived ROS-dependent signaling in ECs, and discuss the possible roles in angiogenesis. Understanding these mechanisms may provide insight into the components of NAD(P)H oxidase as potential therapeutic targets for treatment of angiogenesis-dependent diseases such as cancer and atherosclerosis and for promoting myocardial angiogenesis in ischemic heart diseases.

Platelet factor 4 disrupts the intracellular signalling cascade induced by vascular endothelial growth factor by both KDR dependent and independent mechanisms.

The mechanism by which the CXC chemokine platelet factor 4 (PF-4) inhibits endothelial cell proliferation is unclear. The heparin-binding domains of PF-4 have been reported to prevent vascular endothelial growth factor 165 (VEGF(165)) and fibroblast growth factor 2 (FGF2) from interacting with their receptors. However, other studies have suggested that PF-4 acts via heparin-binding independent interactions. Here, we compared the effects of PF-4 on the signalling events involved in the proliferation induced by VEGF(165), which binds heparin, and by VEGF(121), which does not. Activation of the VEGF receptor, KDR, and phospholipase Cgamma (PLCgamma) was unaffected in conditions in which PF-4 inhibited VEGF(121)-induced DNA synthesis. In contrast, VEGF(165)-induced phosphorylation of KDR and PLCgamma was partially inhibited by PF-4. These observations are consistent with PF-4 affecting the binding of VEGF(165), but not that of VEGF(121), to KDR. PF-4 also strongly inhibited the VEGF(165)- and VEGF(121)-induced mitogen-activated protein (MAP) kinase signalling pathways comprising Raf1, MEK1/2 and ERK1/2: for VEGF(165) it interacts directly or upstream from Raf1; for VEGF(121), it acts downstream from PLCgamma. Finally, the mechanism by which PF-4 may inhibit the endothelial cell proliferation induced by both VEGF(121) and VEGF(165), involving disruption of the MAP kinase signalling pathway downstream from KDR did not seem to involve CXCR3B activation.

Basic fibrobrast growth factor induces the secretion of vascular endothelial growth factor by human aortic smooth muscle cells but not by endothelial cells

Endothelial cell dysfunction and smooth muscle cell (SMC) proliferation are major events in atherogenesis. Both cells are a source of growth factors that mediate cellular proliferation and chemotaxis. Inappropriate production of, and/or response to, these growth factors (such as vascular endothelial growth factor, VEGF, and basic fibroblast growth factor (bFGF)) may contribute to atherogenesis and therefore to disease progression. Production of VEGF and its soluble receptor (sFlt-1) by human SMCs and human umbilical endothelial cells (HUVECs) after stimulation with bFGF were examined by ELISA of cell culture media and by Western blotting. Smooth muscle cells produced significantly more VEGF than HUVECs (P<0.05) after 24 h of culture with bFGF levels > or =0.001 microg mL(-1). bFGF induced dose-dependent production of VEGF by SMCs, where maximum production was present in 1 microg mL(-1) of bFGF. Conversely, the SMCs produced less sFlt-1 than HUVECs (P<0.05). However, bFGF induced dose-dependent phosphorylation of Flt1 and another VEGF receptor, KDR, in HUVECs but not SMCs. There was no VEGF or sFLT-1 after 6 h of culture in any dose of bFGF in either type of cell. Differences in the production of VEGF and sFlt-1 by SMCs and HUVECs are consistent with the role of these cells in angiogenesis. Induction of VEGF production and expression by bFGF in these cells indicates that this growth factor may participate in angiogenesis indirectly by the induction of VEGF. The production of sFlt-1 by both cell types is in agreement with the notion that sFlt-1 may be involved in the regulation of VEGF activity. Additionally, the ability of bFGF to induce dose-dependent phosphorylation of KDR in HUVECs highlights the important role of bFGF in VEGF-mediated angiogenic processes.