Growth Factor-induced Endothelial Cell Migration Research Articles

Dihydroartemisinin inhibits vascular endothelial growth factor-induced endothelial cell migration by a p38 mitogen-activated protein kinase-independent pathway.

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin, has been demonstrated to possess a strong antiangiogenic activity. However, the molecular mechanisms underlying this effect remain unclear. Endothelial cell (EC) migration is an essential component of angiogenesis, and the p38 mitogen-activated protein kinase (MAPK) signaling pathway plays a key role in the regulation of migration induced by vascular endothelial growth factor (VEGF). The aim of the present study was to investigate the effects of DHA on EC migration and the p38 MAPK signaling pathway. Human umbilical vein ECs (HUVECs) were treated with DHA and VEGF-induced migration was analyzed. The activation of p38 MAPK was detected by western blot analysis, and the migration assays were performed with a p38-specific inhibitor, SB203850. It was revealed that 20 μM DHA significantly reduced EC migration in the transwell migration assay, wound healing assay and electrical cell-substrate impedance sensing real-time analysis. However, DHA did not affect p38 MAPK phosphorylation or expression. In the absence or presence of SB203850, DHA induced a similar proportional reduction of EC migration in the three migration assays. Therefore, the present study demonstrated that DHA inhibits VEGF-induced EC migration via a p38 MAPK-independent pathway.

CC-122 Is a Novel Pleiotropic Pathway Modifier with Potent in Vitro Immunomodulatory and Anti-Angiogenic Properties and in Vivo Anti-Tumor Activity in Hematological Cancers.

AbstractAbstract 2963 Background:The immunomodulatory agents thalidomide (THAL), lenalidomide (LEN), and pomalidomide (POM) have significant activity in a wide range of hematologic cancers. THAL is primarily a potent anti-angiogenic agent with minimal immunomodulatory activity. LEN and POM both demonstrate significant immunomodulatory activity. Additionally, POM displays anti-myeloma activity in patients with LEN-resistant multiple myeloma (MM). Recently, modulation of cereblon (CRBN)-bound E3 ubiquitin ligase complexes has been implicated in the mechanisms of action of THAL, LEN, and POM. This has enabled rational development of a next generation of compounds. CC-122 is a non-phthalimide analog of the immunomodulatory drugs and a first in class PPM™ pleiotropic pathway modulator that binds the CRBN-DDB1-Cul4-Roc1 E3 ubiquitin ligase complex. This study investigated the anti-proliferative, immunomodulatory, and anti-angiogenic activity of CC-122 in MM and lymphoma cells. Results:CC-122 inhibited proliferation of H929 MM cells in a CRBN-dependent and dose-dependent manner (IC50 = 43 nM). CC-122 induced cell cycle arrest at G0/G1 stage, which was associated with reduced retinoblastoma protein phosphorylation, and increased CDK inhibitor p27 protein expression. CC-122 also inhibited the growth of LEN-resistant H929 cells, although the proliferation IC50 for CC-122 was relatively higher in LEN-resistant cells vs H929 control cells (Table). CC-122 has significant anti-myeloma activity, and has greater activity in LEN-resistant H929 cells vs LEN and POM. Compared with LEN, CC-122 had a greater anti-proliferative effect in diffuse-large B-cell lymphoma (DLBCL). Furthermore, CC-122 had greater anti-proliferative effects in ABC- and PBML-subtypes compared with GCB subtype lines. In ABC-subtype U2932 and OCI-Ly10-DLBCL cell lines, 10 μM CC-122 treatment significantly inhibited DNA-binding of NF-κB p65 (P < .001), p50 subunits (P < .05), and IRF4 in a CRBN-dependent manner. In vivo anticancer activity of CC-122 was demonstrated in xenograft models of human lymphoma and MM.CC-122 exhibits potent immunomodulatory activity in whole blood, T, and natural killer (NK) cells. Additionally, CC-122 enhanced the anti-CD3 mAb-stimulated T-cell production of IL-5, IL-13, GM-CSF, IFN-γ, RANTES, and TNF-α. The immunomodulatory activity of CC-122 was 10-fold more potent vs LEN.We investigated the anti-angiogenic properties of CC-122. In a human umbilical artery sprout outgrowth assay, CC-122 inhibited new vessel growth as well as endothelial cell migration and invasion. It also inhibited endothelial cell sprout formation and migration in a growth factor-induced endothelial cell migration and invasion assay. CC-122 has significantly greater anti-angiogenic activity compared with the LEN and POM in human angiogenesis assays (Table). In contrast, it has less of an anti-platelet effect as measured by megakaryocyte proliferation vs LEN and POM.CRBN binding competition assays were conducted with THAL-binding beads. As demonstrated by the higher IC50 concentration, CC-122 has less potency with regard to CRBN binding compared with LEN or POM. Conclusion:Together, these data demonstrate that the first-in-class PPM™ CC-122 has anti-proliferative, immunomodulatory, and anti-angiogenic properties that may have clinical significance in the treatment of advanced refractory lymphoproliferative disorders and is currently in Phase I studies. Furthermore the data suggest that the potency of binding to CRBN per se does not explain the broad pleiotropic activity of CC-122.Table:Activities of LEN, POM, and CC-122ActivityAssay/Cell LineLEN (μM)POM (μM)CC-122 (μM)Immune modulation (EC50)Whole blood TNF-α>100.150.14T-cell IL-20.170.0100.012NK-cell IFN-γ0.0520.00110.0015Anti-proliferative (IC50)H929 (MM)10.090.09LEN-Resistant H929 (MM)>306.0 to >300.8 to 2WSU-DLCL2 (GCB-DLBCL)>100NA2.1SUDHL4 (GCB-DLBCL)>100NA>10OCI-Ly19 (GCB DLBCL)>100NA>10OCI-Ly10 (ABC-DLBCL)0.15NA0.0085U2932 (ABC-DLBCL)2.6NA0.12TMD8 (ABC-DLBCL)75NA0.44RIVA (ABC-DLBCL)>100NA4.3Karpas-1106P (PMBL-DLBCL)>100NA0.71Anti-angiogenesis (IC50)Human Umbilical Artery1.70.330.0094Anti-platelet (IC50)Immature MK colonies0.41 to 1.30.35 to >10>10Intermediate MK colonies1.3 to >101.4 to >10>10CRBN binding (IC50)CRBN competition binding to THAL-beads2220 Disclosures:Gandhi:Celgene Corp: Employment, Equity Ownership. Mendy:Celgene Corp.: Employment, Equity Ownership. Parton:Celgene Corp: Employment, Equity Ownership. Wu:Celgene Corp: Employment, Equity Ownership. Kosek:Celgene Corp: Employment, Equity Ownership. Zhang:Celgene Corp: Employment, Equity Ownership. Capone:Celgene Corp: Employment, Equity Ownership. Lopez-Girona:Celgene Corp: Employment, Equity Ownership. Schafer:Celgene: Employment, Equity Ownership. Chopra:Celgene Corp: Employment, Equity Ownership.

Abstract 4360: CD87 is essential for mediating growth-factor induced angiogenesis via distribution of integrin beta-1

Abstract CD87 (urokinase-type plasminogen activator receptor, uPAR) expression has been correlated with tumor progression most likely via its functional ole as a central receptor for extracellular proteolysis, but also via its role in initiating intracellular signal transduction. The role of CD87 in angiogenesis has recently been elucidated. Thus, we observed that in VEGF-stimulated ndothelial cells CD87 is essential for endothelial cell survival as well as for n efficient growth factor- induced endothelial cell migration. Thereby, the PI-anchored protein CD87 formed a complex with its ligand urokinase uPA) as well as the inhibitor plasminogen activator inhibitor-1 (PAI-1), which became redistributed to the leading edge via low density lipoprotein receptor- (LDLR-) like proteins. In this study, we analyzed the functional role of CD87 in integrin redistribution during growth factor-induced endothelial cell migration. CD87, thereby, directly interacted with integrin adhesion receptors (a5b1) as well as with LDLR- proteins. Either by interfering with CD87/integrin nteraction or by interfering with CD87/LDLR-protein interaction by using specific interfering peptides, we observed disruption of integrin redistribution to the leading edge in migrating cells. As a consequence diminished focal adhesion formations were observed and endothelial cell migration was diminished. In CD87-deficient HEK 293 cells as well as in endothelial cells derived from CD87 -/- mice, expression of CD87 mutants (mutL3/uPAR), which lacked the binding motif for LDLR, led to an impaired cell spreading as well as a diminished migratory response towards VEGF, while full length CD87 (wt/CD87) reconstituted this phenotype. That CD87 plays a central role in growth factor induced endothelial cell invasion and capillary-like tube formation in vivo was demonstrated by a directed in vivo angiogenesis assay (DIVAA), which was performed either in CD87 deficient or wild type animals in the presence or absence of interfering peptides. Our data give novel insights into the central role of CD87 in pro- angiogenic endothelial cell behavior as well as in its role in angiogenesis. Thus, CD87 might lead to a novel therapeutic strategies in tumor- angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4360. doi:1538-7445.AM2012-4360

The urokinase receptor (CD87) represents a central mediator of growth factor-induced endothelial cell migration

Angiogenesis, the sprouting of blood vessels form pre-existing vasculature after injury or in neoplastic diseases, is initiated by growth factor-induced endothelial cell migration. Recently, the major angiogenic growth factor VEGF165 has become the target of therapeutic interventions. However, this approach has been clinically proven to be of limited efficacy, which might be due to the fact that tumour angiogenesis is not only induced by VEGF, but also by a variety of other growth factors. Thus, the identification of a common downstream mediator of growth-factor-induced endothelial cell migration is mandatory to effectively interfere with (tumour-) angiogenesis. We found that the urokinase-type plasminogen activator (uPA)-system, which affects proteolytic as well as adhesive capacities, represents an essential regulatory mechanism in growth factor-induced endothelial cell migration and invasion. This mechanism was not limited to VEGF165, but mediated pro-angiogenic endothelial cell behaviour induced by various growth factors. Thus, VEGF165, VEGF-E, FGF-2, EGF as well as HGF induced a PI3k-dependent activation of pro-uPA when bound to uPAR, which led to an increase in cell surface fibrinolytic activity. As a consequence, uPAR became internalised and redistributed via LDLR-proteins. Interference with these events led to a reduced migratory response of endothelial cells towards VEGF in vitro as well as endothelial cell invasion in vivo. These data give first evidence that the uPA-system, which represents the only level-of-evidence-1 cancer biomarker system for prognosis and/or prediction in node negative breast cancer, might directly affect (tumour-) angiogenesis.

Regulation of Vascular Endothelial Growth Factor-induced Endothelial Cell Migration by LIM Kinase 1-mediated Phosphorylation of Annexin 1

In this study, we obtained evidence indicating that annexin 1 is a new target of the p38/MAPKAP kinase-2 pathway and that it regulates endothelial cell migration in response to vascular endothelial growth factor (VEGF). These conclusions are supported by a series of substantiating experiments. First, by two-dimensional gel electrophoresis and mass spectrometry, we identified annexin 1 as a protein whose phosphorylation is induced by VEGF and is impaired by inhibiting p38. Second, using in vitro kinase assays and in vivo phosphorylation assays, we found that VEGF-mediated activation of LIM kinase 1 downstream of the p38 pathway triggers the phosphorylation of annexin 1. Third, VEGF-induced cell migration and tube formation in Matrigel are inhibited following small interfering RNA-mediated knockdown of annexin 1. Fourth, both processes are rescued in cells expressing an annexin 1 construct insensitive to the small interfering RNA knockdown. Finally, the VEGF/annexin 1-mediated cell migration is impaired by inhibiting p38. We therefore conclude that phosphorylation of annexin 1 regulates the angiogenic effect that is associated with the activation of the p38/LIM kinase 1 axis by VEGF.

The Adenomatous Polyposis Coli-associated Guanine Nucleotide Exchange Factor Asef Is Involved in Angiogenesis

Mutation of the tumor suppressor adenomatous polyposis coli (APC) is a key early event in the development of most colorectal tumors. APC promotes degradation of beta-catenin and thereby negatively regulates Wnt signaling, whereas mutated APCs present in colorectal tumor cells are defective in this activity. APC also stimulates the activity of the guanine nucleotide exchange factor Asef and regulates cell morphology and migration. Truncated mutant APCs constitutively activate Asef and induce aberrant migration of colorectal tumor cells. Furthermore, we have recently found that Asef and APC function downstream of hepatocyte growth factor and phosphatidylinositol 3-kinase. We show here that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced endothelial cell migration. We further demonstrate that Asef is required for basic fibroblast growth factor- and vascular endothelial growth factor-induced microvessel formation. Furthermore, we show that the growth as well as vascularity of subcutaneously implanted tumors are markedly impaired in Asef(-/-) mice compared with wild-type mice. Thus, Asef plays a critical role in tumor angiogenesis and may be a promising target for cancer chemotherapy.

Isoprostanes

The isoprostanes (IsoPs) are a unique series of prostaglandin-like compounds formed in vivo via a nonenzymatic mechanism involving the free radical-initiated peroxidation of arachidonic acid. This article summarizes our current knowledge of these compounds. Herein, a historical account of their discovery and the mechanism of their formation are described. A specific class of IsoPs, the F2-IsoPs, are stable, robust molecules that can be measured as indices of endogenous oxidant stress. The utility of these molecules as biomarkers and methods by which these compounds can be quantified are discussed. In addition to the F2-IsoPs, isoprostanes with other prostane ring structures as well as oxidation products with furan and dioxolane rings can be generated from arachidonic acid. And, in more recent years, isoprostane-like compounds have been shown to be formed from polyunsaturated fatty acids including eicosapentaenoic acid [C20:5, omega-3], docosahexaenoic acid [C22:6, omega-3], and adrenic acid [C22:4, omega-6]. These findings will be summarized as well.

Orally administered lenalidomide (CC-5013) is anti-angiogenic in vivo and inhibits endothelial cell migration and Akt phosphorylation in vitro

The thalidomide analogue and immunomodulatory drug (IMiD®) lenalidomide (CC-5013, REVLIMID™) is emerging as a useful treatment for a number of cancers and has recently entered phase III trials for multiple myeloma. It has been suggested that the anti-tumor effect of lenalidomide is related to its anti-angiogenic potency. In this regard, we have previously shown that lenalidomide inhibits angiogenesis in both rat and human in vitro models but does not affect endothelial cell proliferation. We now show that oral administration of lenalidomide attenuates growth factor-induced angiogenesis in vivo; the rat mesenteric window assay was utilized to show that lenalidomide significantly inhibits vascularization in a dose-dependent manner. We also found that lenalidomide significantly inhibits growth factor-induced endothelial cell migration. This correlates with the inhibitory effect of lenalidomide on growth factor-induced Akt phosphorylation, thereby providing a potential mechanism for its anti-migratory and subsequent anti-angiogenic effects. These data further support the use of lenalidomide as an orally administered drug for the effective treatment of angiogenesis-dependent conditions, including cancer, and suggest a potential mechanism of action.

Role of heparan sulfate domain organization in endostatin inhibition of endothelial cell function

The anti-angiogenic activity of endostatin (ES) depends on interactions with heparan sulfate (HS). In the present study, intact HS chains of >/=15 kDa bound quantitatively to ES whereas N-sulfated HS decasaccharides, with affinity for several fibroblast growth factor (FGF) species, failed to bind. Instead, ES-binding oligosaccharides composed of mixed N-sulfated and N-acetylated disaccharide units were isolated from pig intestinal HS. A 10/12mer ES-binding epitope was identified, with two N-sulfated regions separated by at least one N-acetylated glucosamine unit (SAS-domain). Cleavage at the N-acetylation site disrupted ES binding. These findings point to interaction between discontinuous sulfated domains in HS and arginine clusters at the ES surface. The inhibitory effect of ES on vascular endothelial growth factor-induced endothelial cell migration was blocked by the ES-binding SAS-domains and by heparin oligosaccharides (12mers) similar in length to the ES-binding SAS-domains, but not by 6mers capable of FGF binding. We propose that SAS-domains modulate the biological activities of ES and other protein ligands with extended HS-binding sites. The results provide a rational explanation for the preferential interaction of ES with certain HS proteoglycan species.

Oxidized LDL inhibits vascular endothelial growth factor-induced endothelial cell migration by an inhibitory effect on the Akt/endothelial nitric oxide synthase pathway.

Oxidized LDL (oxLDL) inhibits endothelial cell (EC) migration. Stimulating ECs with vascular endothelial growth factor (VEGF) leads to the activation of Akt/protein kinase B, which in turn activates endothelial nitric oxide synthase (eNOS) by phosphorylation on serine 1177. VEGF-induced cell migration is dependent on the generation of nitric oxide (NO). Therefore, we investigated whether oxLDL affects EC migration by an inhibitory effect on the Akt/eNOS pathway. During an in vitro "scratched wound assay," oxLDL dose-dependently inhibited the VEGF-induced migration of human umbilical vein endothelial cells. Western blot analysis revealed that oxLDL dose- and time-dependently led to dephosphorylation and thus deactivation of Akt. Moreover, oxLDL inhibited the VEGF-induced generation of NO, as detected and quantified using a fluorescent NO indicator, 4,5-diaminofluorescein diacetate. Overexpression of a constitutively active Akt construct (Akt T308D/S473D) or a phosphomimetic eNOS construct (eNOS S1177D) almost completely reversed the inhibitory effect of oxLDL on VEGF-induced EC migration and NO generation. Our data indicate that oxLDL-induced dephosphorylation of Akt, followed by impaired eNOS activation, reduces the intracellular level of NO and thereby inhibits VEGF-induced EC migration.

Akt Takes Center Stage in Angiogenesis Signaling

The serine/threonine kinase Akt, also named protein kinase B, plays a central role in promoting the survival of a wide range of cell types.1 In this issue of Circulation Research , Kim et al2 report that angiopoietin-1 (Ang1) activates this survival kinase and thereby inhibits endothelial cell apoptosis. Ang1 was recently identified as the specific ligand for the Tie receptor family, which during embryonic blood vessel formation is required for vascular remodeling and vessel integrity.3 Mechanistically, Ang1 acts via stimulation of the Tie2 receptor. Upon activation, tyrosine phosphorylation of the Tie2 receptor (most probably at Tyr 11014 ) induces the association and activation of the phosphatidylinositol 3′kinase with subsequent activation of Akt.2 These findings complement previous studies, which demonstrated that vascular endothelial growth factor (VEGF) via the KDR/Flt-1 receptor also stimulates Akt and thereby promotes endothelial cell survival.5 6 Moreover, potent inhibitors of endothelial cell apoptosis such as shear stress or insulin activate Akt.7 8 Thus, activation of Akt seems to be a general antiapoptotic mechanism induced by proangiogenic stimuli. Because apoptosis of endothelial cells counteracts angiogenesis, one may …