VEGF-induced Endothelial Cell Migration Research Articles

Insulin-Like Growth Factor Binding Protein-Related Protein 1 Mediates VEGF-Induced Proliferation, Migration and Tube Formation of Retinal Endothelial Cells

The potential role of insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) as an endogenous angiogenesis inhibitor in the prevention of vascular endothelial growth factor (VEGF)-induced retinal angiogenesis has not been explored. Expression of IGFBP-rP1 in rhesus macaque choroid-retinal endothelial cell line (RF/6A) cells was assessed by reverse transcription polymerase chain reaction analysis and Western blotting. RF/6A cells were treated with VEGF (10 ng/ml) alone or in the presence of IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml. The proliferation, migration and capillary-like tube formation of RF/6A cells were evaluated using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium colorimetric assay, the chemotactic motility assay and the Matrigel tube formation assay, respectively. Flow cytometry was used to detect the apoptosis of RF/6A cells, and oncogenic B-Raf expression was assessed to elucidate the pathway for IGFBP-rP1-mediated induction of apoptosis in the presence of VEGF. RF/6A cells expressed both IGFBP-rP1 transcripts and IGFBP-rP1 protein. VEGF markedly stimulated proliferation, migration and capillary-like tube formation of RF/6A cells (P<0.05), whereas those VEGF-induced parameters were significantly inhibited by IGFBP-rP1 at concentrations ranging from 50 ng/ml to 200 ng/ml in a dose-dependent manner (P < 0.05). Following addition of IGFBP-rP1, expression of B-Raf was significantly decreased dose-dependently, and apoptosis occurred as evidenced by flow cytometry (P < 0.05). IGFBP-rP1 can inhibit the stimulatory effect of VEGF on retinal angiogenesis in vitro by inhibiting expression of B-Raf to induce apoptosis. It is a novel endogenous anti-angiogenic factor with potential therapeutic action in retinal neovascularization dependent disorders.

Urokinase Receptor (uPAR)-Dependent Integrin Redistribution Represents a Central Mechanism for Growth Factor Induced Endothelial Cell Migration

AbstractAbstract 2119Tumor angiogenesis is induced when the net balance of pro- and antiangiogenic molecules is tipped in favor of angiogenesis, the so called ‘angiogenic switch’. Recently, we described a mechanism how VEGF induces pro-urokinase (pro-uPA) activation, which led to uPAR-complex formation and internalization of beta-1 integrins into the endosomal compartment via LDLR-proteins such as ApoER2 or VLDLR. Thereby, uPAR plays a central role for VEGF-induced endothelial cell migration.Here, we describe that uPAR-induced integrin internalization and redistribution to the leading edge is not only limited to VEGF-induced endothelial cell migration, but plays a central role for others angiogenic growth factors such as fibroblast growth factor-2 (FGF-2), hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF). Furthermore, we found that a hitherto undescribed binding site on domain 3 of uPAR for direct LDLR-protein interaction is required and sufficient for uPAR-dependent integrin redistribution. Interference with the uPAR/integrin internalization either by the Receptor Associated Protein (RAP) or a specific LDLR-binding site mimicking peptide (P1), the migratory response of endothelial cells towards the growth factors VEGF, HGF, FGF-2, or EGF was almost blocked (20.24% ± 4.56%). Consistently, expression of a mutated uPAR lacking interaction site for LDLR-proteins in uPAR-/- endothelial cells via a retroviral construct led to reduced invasive response towards angiogenic growth factors in vitro as well as in a Matrigel plug in vivo assay.From these data we conclude that uPAR/LDLR-protein interaction represents a central molecule in growth factor-induced endothelial cell behavior, which might open a new avenue for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

Inhibition of vascular endothelial growth factor-induced angiogenesis by scopoletin through interrupting the autophosphorylation of VEGF receptor 2 and its downstream signaling pathways

Our previous studies revealed that scopoletin, the main bioactive constituent of Erycibe obtusifolia Benth stems, exerted anti-arthritic activity in vivo partly by preventing synovial angiogenesis. Herein we further investigated the anti-angiogenic potential and related mechanisms of this coumarin compound in vivo and in vitro. On chick chorioallantoic membrane (CAM) model, scopoletin (10, 30, 100nmol/egg) dose-dependently reduced the blood vessels that were quantified by counting the number of blood vessel branch points. In vitro, scopoletin at concentrations above 30μM obviously inhibited the VEGF-induced tube formation, proliferation and migration of human umbilical vein endothelial cells (HUVECs). Furthermore, scopoletin was shown to block VEGF-induced autophosphorylation of VEGFR2 but not VEGFR1, and down-regulate the following activation of ERK1/2, p38 MAPK and endothelial nitric oxide synthase (eNOS) as well as the production of nitric oxide (NO) in HUVECs. In sum, our findings further support that scopoletin is a candidate of angiogenesis inhibitors, and it functions by interrupting the autophosphorylation of VEGF receptor 2 (VEGFR2) and the downstream signaling pathways.

P124. 3-Polyunsaturated fatty acids may inhibit angiogenesis through suppression of ELR+ CXC chemokines expression and VEGF signaling in pancreatic cancer

Angiogenesis, the process of new blood vessel formation, is essential for tumor growth and metastasis. Although the protective role of ω3-PUFAs on cancer progression has been reported, the anti-angiogenic mechanisms remain to be further understood. The aim of this study is to investigate the anti-angiogenic mechanisms of ω3-PUFAs in pancreatic cancer. In angiogenesis protein array chip assay, only angiogenic ELR+ CXC chemokines expression, which activate endothelial cell migration and neutrophils recruitment, was significantly reduced by DHA treatment in SW1990 human pancreatic cancer cells. The reduced expression of ELR+ CXC chemokine was confirmed by RT-PCR. Chemotactic migration of HUVEC was significantly reduced in DHA-treated SW1990 conditioned medium (CM) compared to control CM. DHA also inhibited VEGF reporter activity in SW1990 cells. VEGF-induced endothelial cell proliferation and migration were markedly inhibited by DHA treatment. MMP-2 and MMP-9 promoter activities were reduced by DHA treatment, and ultimately attenuated the invasiveness of SW1990 cells as revealed by matrigel droplet assay. Moreover, NF-κB reporter activity, which is closely correlated with the expression of CXC chemokine, VEGF and MMPs, was reduced by DHA treatment. When mouse pancreatic cancer PANC02 cells were implanted into Fat-1 transgenic mice, tumor size and volume were dramatically reduced in Fat-1 mice compared to control mice. In immunohistochemical analysis of tumors of Fat-1 mice, apoptosis (TUNEL positive cells) was markedly increased and microvessel density (CD31 intensity) was significantly reduced in Fat-1 mice compared to control mice. In addition, VEGF-induced neovascularization to a subcutaneous matrigel plug was significantly inhibited in Fat-1 mice compared with control mice. Our finding suggests that ω3-PUFAs can suppress tumor angiogenesis through inhibition of ELR+ chemokines expression and VEGF signal in pancreatic cancer. Thus ω3-PUFAs supplementation may beneficial for chemoprevention and treatment of pancreatic cancer by anti-angiogenic effect. This work was supported by basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R13-2007-020-01000-0 and 2010-0016447), Korea.

PLD1-dependent PKCγ activation downstream to Src is essential for the development of pathologic retinal neovascularization

AbstractVascular endothelial growth factor (VEGF) appears to be an important mediator of pathologic retinal angiogenesis. In understanding the mechanisms of pathologic retinal neovascularization, we found that VEGF activates PLD1 in human retinal microvascular endothelial cells, and this event is dependent on Src. In addition, VEGF activates protein kinase C-γ (PKCγ) via Src-dependent PLD1 stimulation. Inhibition of Src, PLD1, or PKCγ via pharmacologic, dominant negative mutant, or siRNA approaches significantly attenuated VEGF-induced human retinal microvascular endothelial cell migration, proliferation, and tube formation. Hypoxia also induced Src-PLD1-PKCγ signaling in retina, leading to retinal neovascularization. Furthermore, siRNA-mediated down-regulation of VEGF inhibited hypoxia-induced Src-PLD1-PKCγ activation and neovascularization. Blockade of Src-PLD1-PKCγ signaling via the siRNA approach also suppressed hypoxia-induced retinal neovascularization. Thus, these observations demonstrate, for the first time, that Src-dependent PLD1-PKCγ activation plays an important role in pathologic retinal angiogenesis.

Deciphering Mechanisms Controlling Placental Artery Endothelial Cell Migration Stimulated by Vascular Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) stimulated fetoplacental artery endothelial (oFPAE) cell migration and activated multiple signaling pathways including ERK2/1, p38MAPK, Jun N-terminal kinase (JNK1/2), v-Akt murine thymoma viral oncogene homolog 1 (Akt1), and c-Src in oFPAE cells. VEGF-induced cell migration was blocked by specific kinase inhibitors of JNK1/2 (SP600125), c-Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and phosphatidylinositol 3-kinase/Akt (wortmannin) but not ERK2/1 (U0126) and p38MAPK (SB203580). VEGF-induced cell migration was associated with dynamic actin reorganization and focal adhesion as evidenced by increased stress fiber formation and phosphorylation of cofilin-1 and focal adhesion kinase (FAK) and paxillin. Inhibition of JNK1/2, c-Src, and phosphatidylinositol 3-kinase/Akt suppressed VEGF-induced stress fiber formation and cofilin-1 phosphorylation. c-Src inhibition suppressed VEGF-induced phosphorylation of focal adhesion kinase, paxillin, and focal adhesion. VEGF-induced cell migration requires endogenous nitric oxide (NO) as: 1) VEGF-stimulated phosphorylation of endothelial NO synthase (eNOS) via activation of Akt, JNK1/2, and Src; 2) a NO donor diethylenetriamine-NO-stimulated cell migration; and 3) NO synthase inhibition blocked VEGF-induced cell migration. Targeted down-regulation and overexpression of caveolin-1 both inhibited VEGF-induced cell migration. Caveolin-1 down-regulation suppressed VEGF-stimulated phosphorylation of Akt, JNK, eNOS, c-Src, and FAK; however, basal activities of c-Src and FAK were elevated in parallel with increased stress fiber formation and focal adhesion. Caveolin-1 overexpression also inhibited VEGF-induced phosphorylation of Akt, JNK, c-Src, FAK, and eNOS. Thus, VEGF-induced placental endothelial cell migration requires activation of complex pathways that are paradoxically regulated by caveolin-1.

In vitro antiproliferative and antiangiogenic effects of synthetic chalcone analogues

As flavonoids, chalcones possess a wide variety of biological activities including anticancer properties. In the present study we have investigated the in vitro antiproliferative and antiangiogenic effects of four synthetic chalcones. E-2-(4′-methoxybenzylidene)-1-benzosuberone ( 3) was the most active compound with IC 50 = 10 −7 mol l −1 in Jurkat cells. In both Jurkat and HeLa chalcone 3-treated cells we found a significant increase in the proportion of cancer cells in the G 2/M phase of the cell cycle as well as an increase in cells having sub-G 0/G 1 DNA content which is considered to be a marker of apoptotic cell death. Apoptosis was also confirmed by annexin V staining and DNA fragmentation. These effects were associated with reduced expression of the anti-apoptotic gene, Bcl-2, and increased expression of the pro-apoptotic gene, Bax. Furthermore, chalcone 3 was selected to evaluate its effect on some angiogenic events. In non-toxic concentrations, chalcone 3 inhibited VEGF-induced migration of human umbilical vein endothelial cells. Moreover, it also decreased secretion of matrix metalloproteinase (mainly MMP-9) and vascular endothelial growth factor (VEGF). In conclusion, the present study has assessed the in vitro antiproliferative/antiangiogenic potential of chalcone 3. This results generate a rationale for in vivo efficacy studies with this compound in preclinical cancer models.

Abstract 2377: Aminopeptidase A plays a role in VEGF-induced migration and proliferation through direct activation by VEGFR2 kinase activity

Abstract Tyrosine kinases pose attractive targets for cancer therapy, as their abnormal signaling activity results in enhanced proliferation, migration and angiogenesis. Previously we have been able to generate a comprehensive description of the biological kinase activity present in pediatric brain tumor tissue lysate by the application of a peptide microarray containing 144 different tyrosine kinase peptide substrates. To establish which peptide substrates present on the array are most sensitive and discriminative for VEGFR activity we measured the peptide phosphorylation applying recombinant VEGFR1, 2 and 3. Furthermore, a panel of recombinant kinases with highly diverse VEGFR sequence homology was included to study substrate specificity. An aminopeptidase A (APA) derived peptide proved to be highly phosphorylated by VEGFR 1 and 2. No phosphorylation of this peptide could be observed applying any of the other kinases, arguing that this peptide is discriminative for VEGFR1 and 2 activity. In the peptide array data of pediatric brain tumors, phosphorylation of the APA peptide was observed, suggestive for VEGFR activation. Based on these results we hypothesize that APA can be activated through direct phosphorylation by VEGFR2 and exert functional effects on endothelial cell proliferation and migration. To test this hypothesis we stimulated human umbilical vein endothelial cells (HUVECs) with VEGF and observed phosphorylation of Aminopeptidase A. Literature states that inhibition of APA suppresses VEGF-induced migration and proliferation of endothelial cells and inhibits angiogenesis. Based on our data we hypothesize that these APA-mediated effects of VEGF stimulation are induced through direct activation by VEGFR2 kinase activity. Concluding, peptide microarray technology is able to provide a selective view on tyrosine kinase activation, as has been shown here for VEGFR1 and 2. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2377.

Methylnaltrexone Potentiates the Anti-Angiogenic Effects of mTOR Inhibitors

BackgroundRecent cancer therapies include drugs that target both tumor growth and angiogenesis including mammalian target of rapamycin (mTOR) inhibitors. Since mTOR inhibitor therapy is associated with significant side effects, we examined potential agents that can reduce the therapeutic dose.MethodsMethylnaltrexone (MNTX), a peripheral mu opioid receptor (MOR) antagonist, in combination with the mTOR inhibitors temsirolimus and/or rapamycin, was evaluated for inhibition of VEGF-induced human pulmonary microvascular endothelial cell (EC) proliferation and migration as well as in vivo angiogenesis (mouse Matrigel plug assay).ResultsMNTX inhibited VEGF-induced EC proliferation and migration with an IC50 of ~100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation and migration from ~10 nM to ~1 nM and from ~50 to ~10 nM respectively. We observed similar effects with rapamycin. On a mechanistic level, we observed that MNTX increased EC plasma membrane-associated tyrosine phosphate activity. Inhibition of tyrosine phosphatase activity (3,4-dephostatin) blocked the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR Complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR Complex 1 (rapamycin and temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events.ConclusionsOur data indicate that MNTX exerts a synergistic effect with rapamycin and temsirolimus on inhibition of VEGF-induced human EC proliferation and migration and in vivo angiogenesis. Therefore, addition of MNTX could potentially lower the dose of mTOR inhibitors which could improve therapeutic index.

A serum-stable branched dimeric anti-VEGF peptide blocks tumor growth via anti-angiogenic activity

Angiogenesis is critical and indispensable for tumor progression. Since VEGF is known to play a central role in angiogenesis, the disruption of VEGF-VEGF receptor system is a promising target for anti-cancer therapy. Previously, we reported that a hexapeptide (RRKRRR, RK6) blocked the growth and metastasis of tumor by inhibiting VEGF binding to its receptors. In addition, dRK6, the D-form derivative of RK6, retained its biological activity with improved serum stability. In the present study, we developed a serum-stable branched dimeric peptide (MAP2-dRK6) with enhanced anti-VEGF and anti-tumor activity. MAP2-dRK6 is more effective than dRK6 in many respects: inhibition of VEGF binding to its receptors, VEGF- and tumor conditioned medium-induced proliferation and ERK signaling of endothelial cells, and VEGF-induced migration and tube formation of endothelial cells. Moreover, MAP2-dRK6 blocks in vivo growth of VEGF-secreting colorectal cancer cells by the suppression of angiogenesis and the subsequent induction of tumor cell apoptosis. Our observations suggest that MAP2-dRK6 can be a prospective therapeutic molecule or lead compound for the development of drugs for various VEGF-related angiogenic diseases.

Alzheimer’s β‐amyloid peptide blocks vascular endothelial growth factor mediated signaling via direct interaction with VEGFR‐2

Beta-amyloid peptides (Abeta) are the major constituents of senile plaques and cerebrovascular deposits in the brains of Alzheimer's disease patients. We have shown previously that soluble forms of Abeta are anti-angiogenic both in vitro and in vivo. However, the mechanism of the anti-angiogenic activity of Abeta peptides is unclear. In this study, we examined the effects of Abeta1-42 on vascular endothelial growth factor receptor 2 (VEGFR-2) signaling, which plays a key role in angiogenesis. Abeta inhibited VEGF-induced migration of endothelial cells, as well as VEGF-induced permeability of an in vitro model of the blood brain barrier. Consistently, exogenous VEGF dose-dependently antagonized the anti-angiogenic activity of Abeta in a capillary network assay. Abeta1-42 also blocked VEGF-induced tyrosine phosphorylation of VEGFR-2 in two types of primary endothelial cells, suggesting an antagonistic action of Abeta toward VEGFR-2 signaling in cells. Moreover, Abeta was able to directly interact with the extracellular domain of VEGFR-2 and to compete with the binding of VEGF to its receptor in a cell-free assay. Co-immunoprecipitation experiments confirmed that Abeta can bind VEGFR-2 both in vitro and in vivo. Altogether, our data suggest that Abeta acts as an antagonist of VEGFR-2 and provide a mechanism explaining the anti-angiogenic activity of Abeta peptides.

Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for anti-angiogenesis treatment. However, the side effects associated with most anti-VEGF agents limit their chronic use. Identification of naturally occurring VEGF inhibitors derived from diet is a potential alternative approach, with the advantage of known safety. To isolate natural inhibitors of VEGF, we established an in vitro tyrosine kinase assay to screen for diet-based agents that suppress VEGFR2 kinase activity. We found that a water-based extract from cinnamon (cinnamon extract, CE), one of the oldest and most popular spices, was a potent inhibitor of VEGFR2 kinase activity, directly inhibiting kinase activity of purified VEGFR2 as well as mitogen-activated protein kinase- and Stat3-mediated signaling pathway in endothelial cells. As a result, CE inhibited VEGF-induced endothelial cell proliferation, migration and tube formation in vitro, sprout formation from aortic ring ex vivo and tumor-induced blood vessel formation in vivo. Depletion of polyphenol from CE with polyvinylpyrrolidone abolished its anti-angiogenesis activity. While cinnamaldehyde, a component responsible for CE aroma, had little effect on VEGFR2 kinase activity, high-performance liquid chromatography-purified components of CE, procyanidin type A trimer (molecular weight, 864) and a tetramer (molecular weight, 1152) were found to inhibit kinase activity of purified VEGFR2 and VEGFR2 signaling, implicating procyanidin oligomers as active components in CE that inhibit angiogenesis. Our data revealed a novel activity in cinnamon and identified a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment.

Abstract 5329: ROS-dependent Cysteine Sulfenic Acid Formation of IQGAP1 and ERK1/2 in VEGF-induced Endothelial Cell Proliferation and Migration

VEGF receptor type 2 (VEGFR2) activation induces endothelial cell (EC) proliferation and migration. ROS-mediated protein modification involves oxidation of reactive cysteine residues to form a cysteine sulfenic acid (Cys-SOH) and subsequent oxidation products. However, role of Cys-SOH formation in VEGF signaling linked to angiogenesis is unknown. We previously identified IQGAP1, as a novel VEGFR2 binding protein, involved in regulating ROS-dependent EC migration and proliferation by recruiting Nox2 NADPH oxidase to activated VEGFR2 signaling complex. By using a newly-developed Cys-SOH trapping reagent we show that VEGF stimulation significantly increases protein-Cys-SOH formation in HUVECs. Immunofluorescence reveals that Cys-SOH modified proteins accumulate at the leading edge where they colocalize with NADPH oxidase, F-actin and IQGAP1 in actively migrating ECs. VEGF stimulation increases IQGAP1-Cys-SOH formation within 5min (60%) with a maximum increase at 15min (90%). VEGF-induced Cys-SOH formation of IQGAP1 is prevented by pretreatment with a GSH donor antioxidant, N-acetylcysteine. Knockdown of IQGAP1 by siRNA or trapping of Cys-SOH with dimedone, blocks wound-induced Cys-SOH formation at the leading edge and EC migration toward the injured site (80%), EC chemotaxis (90%) and matrigel angiogenesis (80%). These suggest linkage between IQGAP1-Cys-SOH formation and IQGAP1-mediated EC migration. In addition, dimedone pretreatment inhibits VEGF-stimulated EC proliferation. VEGF stimulation also induces oxidation of ERK1/2 upon 5 min (50%) and 15min (80%) stimulation. Trapping Cys-SOH by dimedone inhibits ERK phosphorylation (95%), which is associated with increasing IQGAP1/ERK complex (50%) after VEGF stimulation. Hind limb ischemia increases oxidation of tissue proteins including IQGAP1 (80%) and ERK1/2 (60%) at day 1 after injury, which is prevented in mice lacking Nox2. Thus, oxidation of IQGAP1 and ERK contributes to directional migration and proliferation of ECs and to neo-vascularization in ischemic tissue. The present study should provide insights into identifying and visualizing Cys-SOH formed proteins as important strategy for understanding mechanisms by which ROS regulate EC function such as angiogenesis.

Abstract 5281: Novel Role of p66shc in ROS-dependent VEGF Signaling and Cell-Cell Adhesions in Endothelial Cells

p66shc is an adaptor protein and functions as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. We demonstrated that VEGF stimulation rapidly increases ROS via activation of NADPH oxidase, which are involved in VEGF receptor 2 (VEGFR2) autophosphorylation as well as endothelial cell (EC) proliferation and migration. However, the role of p66shc in VEGF signaling has not been investigated. Here we show that VEGF stimulation increases serine phosphorylation of p66shc at Ser36 in ECs (3.1-fold at 5mins, 5.4-fold at 15mins). Adenovirus-mediated transfer of mutant p66shc (S36A) or knockdown of p66shc with siRNA decreases both basal- and VEGF-stimulated ROS levels, which results in inhibiting VEGF-induced VEGFR2 autophosphorylation (43.6% and 64.9%, respectively) as well as phosphorylation of p38MAPK (20.2% and 58.5%) and Akt (57.7% and 76.8%). Functionally, p66shc siRNA or overexpression of p66shc (S36A) inhibits VEGF-induced EC migration (58.6% and 50.9%) and proliferation (86.2% and 100%). Of note, immunofluorescence analysis reveals that p66shc is colocalized with and binds to VE-cadherin/beta-catenin complex at cell-cell contacts in confluent ECs, which is required for stabilizing cell-cell adhesions. Depletion of p66shc with siRNA disrupts VE-cadherin-beta-catenin complex, thereby reducing VE-cadherin localization at adherens junction and increasing endothelial permeability (40.8% increase), as measured by transendothelial electric resistance. Moreover, p66shc siRNA markedly reduces basal- and VEGF-stimulated stress fiber formation, which may contribute to a mislocalization of VE-cadherin. In summary, p66shc functions as a key regulator for ROS-dependent VEGF signaling linked to angiogenesis and actin reorganization as well as stabilizing cell-cell junctions. Thus, p66shc is a potential therapeutic target for angiogenesis-dependent cardiovascular diseases. This research has received full or partial funding support from the American Heart Association, Midwest Affiliate (Illinois, Indiana, Iowa, Kansas, Michigan, Minnesota, Missouri, Nebraska, North Dakota, South Dakota &amp; Wisconsin).

Anti-angiogenic effect of caffeic acid on retinal neovascularization

Pathological angiogenesis is the most common cause of vision loss at all ages including retinopathy of prematurity (ROP), diabetic retinopathy, and age-related macular degeneration. ROP is a proliferative disease of the retinal vasculature in premature infants. Herein, we demonstrated caffeic acid (CA) has the anti-angiogenic activity to retinal endothelial cells and retinal neovascularization in a mouse model of ROP, which might be related to the suppression of ROS-induced VEGF expression. CA effectively inhibited VEGF-induced proliferation of retinal endothelial cells in concentration-dependent manner. In addition, VEGF-induced migration and tube formation of retinal endothelial cells were completely inhibited. This anti-angiogenic activity of CA on retinal endothelial cells was related to the anti-oxidant activity: the inhibitory activity of CA to H 2O 2-induced reactive oxygen species production and VEGF expression. Interestingly, CA significantly suppressed retinal neovascularization in oxygen-induced retinopathy as the animal model of ROP without retinal cytotoxicity. These data suggests that CA could be a potent anti-angiogenic agent for retinal neovascularization, and be applied in the treatment of other vaso-proliferative retinopathies.

Methylnaltrexone potentiates the antiangiogenic effects of the mTOR inhibitor, temsirolimus

e14636 Background: Angiogenesis is important in cancer growth and metastasis. Recent therapeutic interventions cancer, include drugs such as temsirolimus that target both tumor growth and angiogenesis including the mammalian target of rapamycin (mTOR) inhibitor. Previously, we have demonstrated that the peripheral opioid antagonist methylnaltrexone (MNTX) potentiates the effects of bevacizumab and 5FU on endothelial cell (EC) migration and proliferation. Since mTOR inhibitor therapy is also associated with significant side effects, we examined potential adjunctive agents that could reduce the therapeutic dose and improve the therapeutic window for this approach. Methods: We determined the effect(s) of MNTX, naloxone and/or temsirolimus on VEGF-induced human pulmonary microvascular EC proliferation and migration assays. We used siRNA and specific inhibitors to analyze Src, Akt and mTOR regulation. Results: MNTX inhibited EC VEGF-induced proliferation and migration with an IC50 of ∼100 nM. Adding 10 nM MNTX to EC shifted the IC50 of temsirolimus inhibition of VEGF-induced proliferation from ∼10 nM to ∼1 nM. Further, adding 10 nM MNTX shifted the IC50 of temsirolimus on inhibition of EC migration from ∼50 nM to ∼10 nM. These synergistic effects were not observed with naltrexone, a tertiary mu opioid receptor antagonist. On a mechanistic level, we observed that treatment of human EC with MNTX, but not naltrexone, increased protein tyrosine phosphatase (PTP) activity which was independent of mu opioid receptor expression. Treatment of human EC with the PTP inhibitor, 3,4-Dephostatin, inhibited both the synergy between MNTX and temsirolimus and increased VEGF-induced tyrosine phosphorylation of Src with enhanced PI3 kinase and mTOR complex 2-dependent phosphorylation of Akt and subsequent activation of mTOR complex 1 (temsirolimus target), while silencing Src, Akt or mTOR complex 2 components blocked VEGF-induced angiogenic events. Conclusions: Synergy of MNTX with mTOR inhibitors may have therapeutic implications which await clinical proof-of-concept. [Table: see text]

CCL23 up-regulates expression of KDR/Flk-1 and potentiates VEGF-induced proliferation and migration of human endothelial cells

CCL23 is a CC chemokine and exerts its biological activities on endothelial cells as well as on immune cells through CCR1. We investigated the potential effect of CCL23 on expression of KDR/Flk-1 receptor in endothelial cells. PCR, confocal microscope and Western blot analysis revealed that CCL23 up-regulated KDR/Flk-1 mRNA and protein levels in endothelial cells. A reporter assay indicated that CCL23-induced KDR/Flk-1 expression primarily occurred at the transcriptional level. In addition, CCL23 stimulated phosphorylation of SAPK/JNK, and an inhibitor of SAPK/JNK blocks the CCL23-induced KDR/Flk-1 expression. Furthermore, VEGF-induced ERK phosphorylation was stimulated by CCL23. Finally, CCL23 promoted VEGF-induced endothelial proliferation and migration, which were correlated with the maximal stimulation of KDR/Flk-1 expression by CCL23. Taken together, these findings suggest that CCL23 results in up-regulation of KDR/flk-1 receptor gene transcription and protein expression and that KDR/Flk-1 up-regulation induced by CCL23 may contribute to potentiation of VEGF action in angiogenesis.

Endothelial monocyte activating polypeptide II interferes with VEGF-induced proangiogenic signaling

Endothelial monocyte activating polypeptide II (EMAP II) is a proinflammatory cytokine with antiangiogenic properties. EMAP II functions as a potent inhibitor of primary and metastatic tumor growth, has strong inhibitory effects on endothelial cells (ECs), and can reduce intratumoral expression of the angiogenesis inducer vascular endothelial growth factor (VEGF). VEGF influences EC functions such as proliferation, migration, survival and tube formation. Therapeutic strategies that target VEGF have been demonstrated to reduce the tumor growth. We investigated the effects of EMAP II on VEGF-induced angiogenesis signaling. Primary human fetal lung ECs (HFLECs) and human umbilical vein ECs (HUVECs) were grown in E-Stim medium. Protein binding was analyzed using enzyme-linked immunosorbent assay (ELISA). Protein expression was determined by western blot analysis. EC proliferation and migration was determined using WST-1 reagent and transwell membrane, respectively. EMAP II efficiently and dose dependently binds to VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) as observed by ELISA. Bmax values for VEGFR1 and VEGFR2 were 0.45 and 0.17, respectively. In addition, EMAP II inhibited binding of VEGF to VEGFR1 and VEGFR2. EMAP II significantly reduced VEGF-induced expression of phosphorylated VEGFR1 (in HFLEC and HUVEC) by >50%, and of phosphorylated VEGFR2 (in HUVEC) by 66%. EMAP II also inhibited downstream VEGF signaling. Although VEGF-induced phosphorylation of Akt, Erk1/2, p38 and Raf 2.8-, 1.5-, 2.2- and 3.6-fold, respectively, EMAP II preincubation blocked this induction in phosphorylation to control levels. VEGF-induced EC proliferation 2.5-fold, and EMAP II pretreatment abrogated this effect. Similarly, VEGF-induced EC migration (2.5-fold) was significantly inhibited by EMAP II. These finding suggest that inhibition of VEGF signaling is one possible antiangiogenic mechanism of EMAP II, which may explain its in vivo antitumor activity and delineate therapeutic strategies to enhance anti-VEGF therapy to inhibit tumor growth.

Grape Seed Extract Inhibits Angiogenesis via Suppression of the Vascular Endothelial Growth Factor Receptor Signaling Pathway

Blockade of angiogenesis is an important approach for cancer treatment and prevention. Vascular endothelial growth factor (VEGF) is one of the most critical factors that induce angiogenesis and has thus become an attractive target for antiangiogenesis treatment. However, most current anti-VEGF agents often cause some side effects when given chronically. Identification of naturally occurring VEGF inhibitors derived from diet would be one alternative approach with an advantage of known safety. Grape seed extract (GSE), a widely used dietary supplement, is known to have antitumor activity. In this study, we have explored the activity of GSE on VEGF receptor and angiogenesis. We found that GSE could directly inhibit the kinase activity of purified VEGF receptor 2, a novel activity of GSE that has not been characterized. GSE could also inhibit the VEGF receptor/mitogen-activated protein kinase-mediated signaling pathway in endothelial cells. As a result, GSE could inhibit VEGF-induced endothelial cell proliferation and migration as well as sprout formation from aorta ring. In vivo assay further showed that GSE could inhibit tumor growth and tumor angiogenesis of MDA-MB-231 breast cancer cells in mice. Consistent with the in vitro data, GSE treatment of tumor-bearing mice led to concomitant reduction of blood vessel density and phosphorylation of mitogen-activated protein kinase. Depletion of polyphenol with polyvinylpyrrolidone abolished the antiangiogenic activity of GSE, suggesting a water-soluble fraction of polyphenol in GSE is responsible for the antiangiogenic activity. Taken together, this study indicates that GSE is a well-tolerated and inexpensive natural VEGF inhibitor and could potentially be useful in cancer prevention or treatment.

Abstract 3664: Mitochondrial p27 Is Required For Endothelial Energy Metabolism And Cell Migration

The migratory capacity of endothelial cells (EC) is a key prerequisite for the growth of new blood vessels during neovascularisation of ischemic and/or tumor tissues. Recently, mitochondria have been identified as signalling organelles in EC, which may contribute to migratory capacity. Therefore, the aim of this study was to investigate whether a molecular link exists between mitochondrial energy metabolism and migratory capacity of EC. To determine whether mitochondria are required for migratory capacity, we incubated EC with 50 ng/ml ethidiume bromide (EtBr) in the presence of uridine to deplete mitochondrial DNA (mtDNA) and, thereby, destroy active mitochondria. EC, which were depleted of mtDNA, did not migrate in a scratch wound assay (− EtBr: 22 +/− 3 migrated cells; + EtBr: 0.15 +/− 0.1 migrated cells). Since protein translocation is a major mechanism for modulating the functional capacity of the mitochondria, we hypothesized that induction of migration by the promigratory stimulus vascular endothelial growth factor (VEGF) leads to translocation of proteins to mitochondria. Recent studies demonstrated that a non-nuclear pool of p27 exerts biological activities including increase of migratory capacity independent of its cell cycle inhibitory activity. Stimulation of EC with VEGF induced a 2.5 +/− 0.4-fold increase in mitochondrially localized p27 protein. Reducing p27 expression by two different siRNAs completely abrogated VEGF-induced migration of EC (98 +/− 2 % inhibition). Next, we cloned p27 into a mitochondrial targeted vector (p27mito) to exclusively overexpress p27 in the mitochondria. Overexpression of p27mito stimulated mitochondrial ATP biosynthesis (3.24 +/− 1.06-fold empty vector), increased mitochondrial membrane potential and enhanced migratory capacity of EC to a similar extent as compared to VEGF. Cells overexpressing p27mito did not demonstrate further increased migratory capacity in response to VEGF. Finally, only p27mito but not nuclear-targeted p27 overexpression rescued the impaired migratory capacity after siRNA-mediated genetic ablation of endogenous p27 expression. These data demonstrate a novel role of mitochondrial p27 as a molecular link between cellular energy metabolism and migratory capacity of EC.