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

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2119-2119 ◽  
Author(s):  
Rene Novotny ◽  
Matthias Unseld ◽  
Marina Poettler ◽  
Christoph Zielinski ◽  
Bernd Binder ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Binding Site ◽  
Protein Interaction ◽  
Conflicts Of Interest ◽  
Endothelial Cell Migration ◽  
Angiogenic Growth Factors

Abstract Abstract 2119 Tumor 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.

Urokinase-Type Plasminogen Activator Receptor (uPAR, CD87) Regulates Integrin Redistribution in Endothelial Cells Via Interaction with Low Density Lipoprotein Receptor- (LDLR-) Like Proteins

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5315-5315
Author(s):  
Gerald Prager ◽  
Rene Novotny ◽  
Matthias Unseld ◽  
Marina Poettler ◽  
Waclawa Kalinowska ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Protein Interaction ◽  
Conflicts Of Interest ◽  
Focal Adhesions ◽  
Matrix Proteins ◽  
Endothelial Cell Migration ◽  
Binding Motif ◽  
Wild Type

Abstract Abstract 5315 angiogenesis by degradation of extracellular matrix proteins as well as induction of intracellular signal transduction. We recently could demonstrate that in VEGF-stimulated endothelial cells pro-uPA becomes activated, which leads to uPAR-complex formation, it's internalization and redistribution of uPAR to newly formed focal adhesions ad the leading edge of migrating endothelial cells. Thereby, uPAR surface expression is tightly transcriptional regulated via the Density Enhanced Phosphatase-1 (DEP-1), but also via the LDLR-family members, which regulate subcellular uPAR distribution. Here, we describe a mechanisms by which uPAR-internalization regulates integrin redistribution. We have characterized a novel binding motif on uPAR domain 3 for LDLR-protein interaction by using affinity chromatography as well as co-immunoprecipitation experiments. To proof a functional relevance of a direct uPAR/LDLR protein interaction, we reconstituted either uPAR mutants (mutL3/uPAR), lacking the binding site for LDLR-proteins, or wild type uPAR into endothelial cells derived from uPAR−/− mice. Reconstitution of mutL3/uPAR was incapable to redistribute uPAR as well as integrins during VEGF-induced endothelial cell migration when compared to wild type uPAR reconstitutes. The functional importance of uPAR / LDLR interaction was further reflected by the use of an inhibitory peptide (P1) interfering with uPAR/LDLR-protein interaction, which functionally reverted full length uPAR reconstitution, or the chaperon Receptor Associated Protein (RAP), a high affinity ligand for LDLR-proteins, which prevents uPAR/LDLR interactions. Thus, interfering with uPAR/LDLR-protein interaction at different levels led to an impaired endothelial cell spreading behavior on integrin-adhesive matrix proteins as well as a reduced pY576 FAK phosphorylation upon endothelial cell adhesion, leading to an reduced migratory response towards VEGF. These data suggest a central role of uPAR/LDLR-protein interaction in VEGF-induced endothelial cell migration via induction of integrin redistribution. Thus, uPAR/LDLR interaction might represent a novel therapeutic target in angiogenesis-related diseases. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human endothelial cells are chemotactic to endothelial cell growth factor and heparin.

1985 ◽  
Vol 101 (6) ◽  
pp. 2330-2334 ◽  
Author(s):  
V P Terranova ◽  
R DiFlorio ◽  
R M Lyall ◽  
S Hic ◽  
R Friesel ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Cell Growth ◽  
Endothelial Cell Migration ◽  
Human Endothelial Cells ◽  
Endothelial Cell Growth Factor ◽  
Endothelial Cell Growth

The response of human endothelial cell migration to various extracellular matrix components and growth factors has been assessed. Human endothelial cells demonstrate increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with endothelial cell growth factor (ECGF) used at a concentration of 10(-9) M. Anti-ECGF antibody inhibits the chemotactic response. Heparin (10(-8) to 10(-10) M) was also chemotactic and was shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, III, IV, and V demonstrate a chemotactic response, these activities were one third to one half less than observed with ECGF. These data suggest that ECGF and heparin may play a significant role as response modifiers of human endothelial cell migration which may be relevant to tumor metastasis, wound healing, and atherogenesis.

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2304-2311
Author(s):  
Daotai Nie ◽  
Keqin Tang ◽  
Clement Diglio ◽  
Kenneth V. Honn
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Cell Line ◽  
Critical Role ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial ◽  
12 Lipoxygenase

Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.

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

2012 ◽  
Vol 108 (08) ◽  
pp. 357-366 ◽  
Author(s):  
Marina Poettler ◽  
Matthias Unseld ◽  
Judit Mihaly-Bison ◽  
Pavel Uhrin ◽  
Florian Koban ◽  
...  
Keyword(s):  
Cell Migration ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Therapeutic Interventions ◽  
Endothelial Cell Migration ◽  
Migration And Invasion ◽  
Tumour Angiogenesis ◽  
Level Of Evidence ◽  
Upa System

SummaryAngiogenesis, 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 plas-minogen 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.

scholarly journals Tetraspanin CD9 links junctional adhesion molecule-A to αvβ3 integrin to mediate basic fibroblast growth factor–specific angiogenic signaling

2013 ◽  
Vol 24 (7) ◽  
pp. 933-944 ◽  
Author(s):  
Swetha S. D. Peddibhotla ◽  
Benjamin F. Brinkmann ◽  
Daniel Kummer ◽  
Hüseyin Tuncay ◽  
Masanori Nakayama ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Fibroblast Growth Factor ◽  
Basic Fibroblast Growth Factor ◽  
Adhesion Molecule ◽  
Endothelial Cell Migration ◽  
Αvβ3 Integrin ◽  
Fibroblast Growth

Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin family with diverse functions in epithelial cells, including cell migration, cell contact maturation, and tight junction formation. In endothelial cells, JAM-A has been implicated in basic fibroblast growth factor (bFGF)-regulated angiogenesis through incompletely understood mechanisms. In this paper, we identify tetraspanin CD9 as novel binding partner for JAM-A in endothelial cells. CD9 acts as scaffold and assembles a ternary JAM-A-CD9-αvβ3 integrin complex from which JAM-A is released upon bFGF stimulation. CD9 interacts predominantly with monomeric JAM-A, which suggests that bFGF induces signaling by triggering JAM-A dimerization. Among the two vitronectin receptors, αvβ3 and αvβ5 integrin, which have been shown to cooperate during angiogenic signaling with bFGF and vascular endothelial growth factor (VEGF), respectively, CD9 links JAM-A specifically to αvβ3 integrin. In line with this, knockdown of CD9 blocks bFGF- but not VEGF-induced ERK1/2 activation. JAM-A or CD9 knockdown impairs endothelial cell migration and tube formation. Our findings indicate that CD9 incorporates monomeric JAM-A into a complex with αvβ3 integrin, which responds to bFGF stimulation by JAM-A release to regulate mitogen-activated protein kinase (MAPK) activation, endothelial cell migration, and angiogenesis. The data also provide new mechanistic insights into the cooperativity between bFGF and αvβ3 integrin during angiogenic signaling.

Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase

Blood ◽  
2000 ◽  
Vol 95 (7) ◽  
pp. 2304-2311 ◽  
Author(s):  
Daotai Nie ◽  
Keqin Tang ◽  
Clement Diglio ◽  
Kenneth V. Honn
Keyword(s):  
Endothelial Cells ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Cell Line ◽  
Critical Role ◽  
Endothelial Cell Migration ◽  
Vascular Endothelial ◽  
12 Lipoxygenase

Abstract Angiogenesis, the formation of new capillaries from preexisting blood vessels, is a multistep, highly orchestrated process involving vessel sprouting, endothelial cell migration, proliferation, tube differentiation, and survival. Eicosanoids, arachidonic acid (AA)-derived metabolites, have potent biologic activities on vascular endothelial cells. Endothelial cells can synthesize various eicosanoids, including the 12-lipoxygenase (LOX) product 12(S)-hydroxyeicosatetraenoic acid (HETE). Here we demonstrate that endogenous 12-LOX is involved in endothelial cell angiogenic responses. First, the 12-LOX inhibitor, N-benzyl-N-hydroxy-5-phenylpentanamide (BHPP), reduced endothelial cell proliferation stimulated either by basic fibroblast growth factor (bFGF) or by vascular endothelial growth factor (VEGF). Second, 12-LOX inhibitors blocked VEGF-induced endothelial cell migration, and this blockage could be partially reversed by the addition of 12(S)-HETE. Third, pretreatment of an angiogenic endothelial cell line, RV-ECT, with BHPP significantly inhibited the formation of tubelike/cordlike structures within Matrigel. Fourth, overexpression of 12-LOX in the CD4 endothelial cell line significantly stimulated cell migration and tube differentiation. In agreement with the critical role of 12-LOX in endothelial cell angiogenic responses in vitro, the 12-LOX inhibitor BHPP significantly reduced bFGF-induced angiogenesis in vivo using a Matrigel implantation bioassay. These findings demonstrate that AA metabolism in endothelial cells, especially the 12-LOX pathway, plays a critical role in angiogenesis.

Hepatoma-derived growth factor is a pulmonary endothelial cell-expressed angiogenic factor

2004 ◽  
Vol 286 (6) ◽  
pp. L1194-L1201 ◽  
Author(s):  
Allen D. Everett ◽  
Jill V. Narron ◽  
Tamara Stoops ◽  
Hideji Nakamura ◽  
Amy Tucker
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Cell Migration ◽  
Endothelial Cell ◽  
Growth Factor ◽  
Endothelial Cell Migration ◽  
Angiogenic Factor ◽  
Microvascular Endothelial Cells ◽  
Pulmonary Microvascular Endothelial Cells ◽  
Microvascular Endothelial

Hepatoma-derived growth factor (HDGF) was previously identified as a developmentally regulated cardiovascular and renal gene that is mitogenic for vascular smooth muscle and aortic endothelial cells. As reciprocal interactions of smooth muscle and endothelial cells are necessary for vascular formation, we examined whether HDGF plays a role in angiogenesis. According to immunohistochemistry, HDGF was highly expressed in endothelial cells of nonmuscularized, forming blood vessels of the fetal lung. HDGF was also expressed in endothelial cells of small (20 μm) mature arteries and veins. By Western immunoblotting, HDGF was highly expressed by human pulmonary microvascular endothelial cells in vitro. Adenoviral overexpression of HDGF was mitogenic for human pulmonary microvascular endothelial cells in serum-free medium, stimulating a 1.75-fold increase in bromodeoxyuridine (BrdU) uptake and a twofold increase in cell migration. With the chick chorioallantoic membrane (CAM), a biologic assay for angiogenesis, exogenous recombinant HDGF significantly stimulated blood vessel formation and a dose-dependent reorganization of cells within the CAM into a more compact, linear alignment reminiscent of tube formation. According to double immunostaining for endothelial cells with a transforming growth factor-βII receptor antibody and BrdU as a marker of cell proliferation, exogenous HDGF selectively stimulated endothelial cell BrdU uptake. HDGF also activated specific ERK1/2 signaling and did not overlap with VEGF SAPK/JNK, Akt-mediated pathways. We conclude that HDGF is a highly expressed vascular endothelial cell protein in vivo and is a potent endothelial mitogen and regulator of endothelial cell migration by mechanisms distinct from VEGF.

scholarly journals A Quantitative High-Throughput Endothelial Cell Migration Assay

2004 ◽  
Vol 9 (8) ◽  
pp. 712-718 ◽  
Author(s):  
Vladimir Mastyugin ◽  
Elizabeth McWhinnie ◽  
Mark Labow ◽  
Frank Buxton
Keyword(s):  
Cell Migration ◽  
Growth Factors ◽  
Endothelial Cell ◽  
Growth Factor ◽  
High Throughput ◽  
Chemical Compounds ◽  
Endothelial Cell Migration ◽  
Inhibitory Effects ◽  
Migration Assay ◽  
Sensitive Method

By combining the use of BD Biosciences Fluoro Blok ™ membrane-based Boyden chambers with the Cellomics HCS Array Scan, a more sensitive method formeasuring cellmigration has been developed. This assay is based on counting nuclei ofmigrated cells on the bottomof the filter rather than conventional approaches, which usemeasurement of totalwell fluorescence. This cellmigration assay provides approximately 10-fold increased signal/background compared to conventional approaches and can be used to assess the effects of growth factors on endothelial cell migration and to screen chemical compounds for inhibitory effects on growth factor–mediated endothelial cell migration.

Reactive oxygen species mediate Rac-induced loss of cell-cell adhesion in primary human endothelial cells

2002 ◽  
Vol 115 (9) ◽  
pp. 1837-1846 ◽  
Author(s):  
Sandra van Wetering ◽  
Jaap D. van Buul ◽  
Safira Quik ◽  
Frederik P. J. Mul ◽  
Eloise C. Anthony ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Endothelial Cells ◽  
Cell Migration ◽  
Cell Adhesion ◽  
Endothelial Cell ◽  
Reactive Oxygen ◽  
Small Gtpases ◽  
Endothelial Cell Migration ◽  
Oxygen Species ◽  
Cell Cell

The integrity of the endothelium is dependent on cell-cell adhesion, which is mediated by vascular-endothelial (VE)-cadherin. Proper VE-cadherin-mediated homotypic adhesion is, in turn, dependent on the connection between VE-cadherin and the cortical actin cytoskeleton. Rho-like small GTPases are key molecular switches that control cytoskeletal dynamics and cadherin function in epithelial as well as endothelial cells. We show here that a cell-penetrating, constitutively active form of Rac (Tat-RacV12) induces a rapid loss of VE-cadherin-mediated cell-cell adhesion in endothelial cells from primary human umbilical veins (pHUVEC). This effect is accompanied by the formation of actin stress fibers and is dependent on Rho activity. However,transduction of pHUVEC with Tat-RhoV14, which induces pronounced stress fiber and focal adhesion formation, did not result in a redistribution of VE-cadherin or an overall loss of cell-cell adhesion. In line with this observation, endothelial permeability was more efficiently increased by Tat-RacV12 than by Tat-RhoV14. The loss of cell-cell adhesion, which is induced by Tat-RacV12, occurred in parallel to and was dependent upon the intracellular production of reactive oxygen species (ROS). Moreover, Tat-RacV12 induced an increase in tyrosine phosphorylation of a component the VE-cadherin-catenin complex, which was identified as α-catenin. The functional relevance of this signaling pathway was further underscored by the observation that endothelial cell migration, which requires a transient reduction of cell-cell adhesion, was blocked when signaling through ROS was inhibited. In conclusion, Rac-mediated production of ROS represents a previously unrecognized means of regulating VE-cadherin function and may play an important role in the (patho)physiology associated with inflammation and endothelial damage as well as with endothelial cell migration and angiogenesis.

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