[15] Quantitative high throughput endothelial cell migration and invasion assay system

During endochondral bone formation, avascular cartilage differentiates to hypertrophic cartilage that then undergoes erosion and vascularization leading to bone deposition. Resting cartilage produces inhibitors of angiogenesis, shifting to production of angiogenic stimulators in hypertrophic cartilage. A major protein synthesized by hypertrophic cartilage both in vivo and in vitro is transferrin. Here we show that transferrin is a major angiogenic molecule released by hypertrophic cartilage. Endothelial cell migration and invasion is stimulated by transferrins from a number of different sources, including hypertrophic cartilage. Checkerboard analysis demonstrates that transferrin is a chemotactic and chemokinetic molecule. Chondrocyte-conditioned media show similar properties. Polyclonal anti-transferrin antibodies completely block endothelial cell migration and invasion induced by purified transferrin and inhibit the activity produced by hypertrophic chondrocytes by 50–70% as compared with controls. Function-blocking mAbs directed against the transferrin receptor similarly reduce the endothelial migratory response. Chondrocytes differentiating in the presence of serum produce transferrin, whereas those that differentiate in the absence of serum do not. Conditioned media from differentiated chondrocytes not producing transferrin have only 30% of the endothelial cell migratory activity of parallel cultures that synthesize transferrin. The angiogenic activity of transferrins was confirmed by in vivo assays on chicken egg chorioallantoic membrane, showing promotion of neovascularization by transferrins purified from different sources including conditioned culture medium. Based on the above results, we suggest that transferrin is a major angiogenic molecule produced by hypertrophic chondrocytes during endochondral bone formation.

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Saxatilin, a Snake Venom Disintegrin, Regulates Platelet Activation Associated with Human Vascular Endothelial Cell Migration and Invasion

Journal of Vascular Research ◽

10.1159/000098519 ◽

2007 ◽

Vol 44 (2) ◽

pp. 129-137 ◽

Cited By ~ 10

Author(s):

Yoon-Jung Jang ◽

Ok-Hee Jeon ◽

Doo-Sik Kim

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Platelet Activation ◽

Snake Venom ◽

Vascular Endothelial Cell ◽

Endothelial Cell Migration ◽

Migration And Invasion ◽

Vascular Endothelial ◽

Human Vascular Endothelial Cell ◽

Cell Migration And Invasion

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MgCl2 and ZnCl2 promote human umbilical vein endothelial cell migration and invasion and stimulate epithelial‑mesenchymal transition via the Wnt/β‑catenin pathway

Experimental and Therapeutic Medicine ◽

10.3892/etm.2017.5144 ◽

2017 ◽

Cited By ~ 1

Author(s):

Shuang Pan ◽

Liwen An ◽

Xin Meng ◽

Liming Li ◽

Fu Ren ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Epithelial Mesenchymal Transition ◽

Umbilical Vein ◽

Endothelial Cell Migration ◽

Migration And Invasion ◽

Mesenchymal Transition ◽

Human Umbilical Vein ◽

Cell Migration And Invasion

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Thymidine phosphorylase in cancer cells stimulates human endothelial cell migration and invasion by the secretion of angiogenic factors

British Journal of Cancer ◽

10.1038/bjc.2011.74 ◽

2011 ◽

Vol 104 (7) ◽

pp. 1185-1192 ◽

Cited By ~ 43

Author(s):

I V Bijnsdorp ◽

F Capriotti ◽

F A E Kruyt ◽

N Losekoot ◽

M Fukushima ◽

...

Keyword(s):

Cell Migration ◽

Endothelial Cell ◽

Cancer Cells ◽

Thymidine Phosphorylase ◽

Angiogenic Factors ◽

Endothelial Cell Migration ◽

Human Endothelial Cell ◽

Migration And Invasion ◽

Cell Migration And Invasion

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CRIF1 deficiency suppresses endothelial cell migration via upregulation of RhoGDI2

PLoS ONE ◽

10.1371/journal.pone.0256646 ◽

2021 ◽

Vol 16 (8) ◽

pp. e0256646

Author(s):

Harsha Nagar ◽

Seonhee Kim ◽

Ikjun Lee ◽

Su-Jeong Choi ◽

Shuyu Piao ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Signaling Pathway ◽

Umbilical Vein ◽

Vascular Endothelial Cell ◽

Cellular Migration ◽

Endothelial Cell Migration ◽

Migration And Invasion ◽

Mitochondrial Functions

Rho GDP-dissociation inhibitor (RhoGDI), a downregulator of Rho family GTPases, prevents nucleotide exchange and membrane association. It is responsible for the activation of Rho GTPases, which regulate a variety of cellular processes, such as migration. Although RhoGDI2 has been identified as a tumor suppressor gene involved in cellular migration and invasion, little is known about its role in vascular endothelial cell (EC) migration. CR6-interacting factor 1 (CRIF1) is a CR6/GADD45-interacting protein with important mitochondrial functions and regulation of cell growth. We examined the expression of RhoGDI2 in CRIF1-deficient human umbilical vein endothelial cells (HUVECs) and its role in cell migration. Expression of RhoGDI2 was found to be considerably higher in CRIF1-deficient HUVECs along with suppression of cell migration. Moreover, the phosphorylation levels of Akt and CREB were decreased in CRIF1-silenced cells. The Akt-CREB signaling pathway was implicated in the changes in endothelial cell migration caused by CRIF1 downregulation. In addition to RhoGDI2, we identified another factor that promotes migration and invasion of ECs. Adrenomedullin2 (ADM2) is an autocrine/paracrine factor that regulates vascular tone and other vascular functions. Endogenous ADM2 levels were elevated in CRIF1-silenced HUVECs with no effect on cell migration. However, siRNA-mediated depletion of RhoGDI2 or exogenous ADM2 administration significantly restored cell migration via the Akt-CREB signaling pathway. In conclusion, RhoGDI2 and ADM2 play important roles in the migration of CRIF1-deficient endothelial cells.

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X-Linked Inhibitor of Apoptosis Protein (XIAP) Supports Urokinase (uPA)-Induced Endothelial Cell Survival

Blood ◽

10.1182/blood.v112.11.5461.5461 ◽

2008 ◽

Vol 112 (11) ◽

pp. 5461-5461

Author(s):

Gerald W Prager ◽

Judit Mihaly ◽

Patrick Brunner ◽

Christoph Zielinski ◽

Bernd Binder

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Cell Survival ◽

Endothelial Cell Migration ◽

Migration And Invasion ◽

Inhibitor Of Apoptosis ◽

Inhibitor Of Apoptosis Protein ◽

Endothelial Cell Survival ◽

Apoptosis Protein

Abstract High uPA expressing tumors are associated with poor prognosis. While a direct effect on tumor cell behavior is described, uPA has especially been shown to mediate (tumor-) angiogenesis. Originally, the urokinase system has been implicated to assist the angiogenic process by it’s proteolytic activities. It is now becoming increasingly evident that uPA additionally elicits a whole array pro-angiogenic responses like differentiation, proliferation and cell migration, independent of its proteolytic activity by inducing intracellular signal transduction. Here we show that uPA induces upregulation of inhibitor of apoptosis proteins (IAPs), which protects endothelial cells against apoptosis. Thereby, uPA-induced endothelial cell survival is mediated by transcriptional upregulation the X-linked inhibitor of apoptosis protein (XIAP), because downregulation of XIAP by small interfering RNA techniques significantly reduces uPA mediated cell survival efficiencies of uPA in endothelial cells. The antiapoptotic activity of uPA was dependent on the presence of uPAR and LRP, but independent of the PI3kinase pathway, while VEGF-dependent antiapoptosis is mainly PI3kinase dependent. uPA-induced cell survival is dependent on the type of extracellular matrix on which cells are attached used indicating the involvement of integrin adhesion receptors. TherebyConsistently, uPA induces phosphorylation of the CDC42 downstream effector p21-activated kinase 1 (PAK1), which leads to IkappaB kinase alpha (IKKa) phosphorylation, a prerequisite for NFkappaB activation. As a consequence, p52/p50 but not p65 is are translocated into the nucleus. Blocking NFkappaB by using the specific NFkappaB inhibitor BAY 11–7082 or by adenoviral-mediated overexpression of its inhibitor, IkB, inhibits uPA-induced XIAP expression as well as uPA-induced cell survival. From these data we conclude that uPA, which is a main player in endothelial cell migration and invasion, provides an additional, PI3-kinase independent but NFkappaB dependent cell survival mechanism.

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

Thrombosis and Haemostasis ◽

10.1160/th11-12-0868 ◽

2012 ◽

Vol 108 (08) ◽

pp. 357-366 ◽

Cited By ~ 21

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.

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Density enhanced phosphatase-1 down-regulates urokinase receptor surface expression in confluent endothelial cells

Blood ◽

10.1182/blood-2010-09-307694 ◽

2011 ◽

Vol 117 (15) ◽

pp. 4154-4161 ◽

Cited By ~ 21

Author(s):

Patrick M. Brunner ◽

Patricia C. Heier ◽

Judit Mihaly-Bison ◽

Ute Priglinger ◽

Bernd R. Binder ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Migration ◽

Endothelial Cell ◽

Endothelial Cell Migration ◽

Cell Surface Receptor ◽

Migration And Invasion ◽

Cell Behavior ◽

Vegf Receptor ◽

Receptor System ◽

Upar Expression

Abstract VEGF165, the major angiogenic growth factor, is known to activate various steps in proangiogenic endothelial cell behavior, such as endothelial cell migration and invasion, or endothelial cell survival. Thereby, the urokinase-type plasminogen activator (uPA) system has been shown to play an essential role not only by its proteolytic capacities, but also by induction of intracellular signal transduction. Therefore, expression of its cell surface receptor uPAR is thought to be an essential regulatory mechanism in angiogenesis. We found that uPAR expression on the surface of confluent endothelial cells was down-regulated compared with subconfluent proliferating endothelial cells. Regulation of uPAR expression was most probably affected by extracellular signal-regulated kinase 1/2 (ERK1/2) activation, a downstream signaling event of the VEGF/VEGF-receptor system. Consistently, the receptor-like protein tyrosine phosphatase DEP-1 (density enhanced phosphatase-1/CD148), which is abundantly expressed in confluent endothelial cells, inhibited the VEGF-dependent activation of ERK1/2, leading to down-regulation of uPAR expression. Overexpression of active ERK1 rescued the DEP-1 effect on uPAR. That DEP-1 plays a biologic role in angiogenic endothelial cell behavior was demonstrated in endothelial cell migration, proliferation, and capillary-like tube formation assays in vitro.

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A high-throughput 3D bioprinted cancer cell migration and invasion model with versatile and broad biological applicability

10.1101/2021.12.28.474387 ◽

2021 ◽

Author(s):

MoonSun Jung ◽

Joanna Skhinas ◽

Eric Y Du ◽

Maria Kristine Tolentino ◽

Robert Utama ◽

...

Keyword(s):

Cell Migration ◽

High Throughput ◽

Drug Screening ◽

Cancer Biology ◽

Cell Movement ◽

Migration And Invasion ◽

Cell Migration And Invasion ◽

Drop On Demand

Understanding the underlying mechanisms of migration and metastasis is a key focus of cancer research. There is an urgent need to develop in vitro 3D tumor models that can mimic physiological cell-cell and cell-extracellular matrix interactions, with high reproducibility and that are suitable for high throughput (HTP) drug screening. Here, we developed a HTP 3D bioprinted migration model using a bespoke drop-on-demand bioprinting platform. This HTP platform coupled with tunable hydrogel systems enables (i) the rapid encapsulation of cancer cells within in vivo tumor mimicking matrices, (ii) in situ and real-time measurement of cell movement, (iii) detailed molecular analysis for the study of mechanisms underlying cell migration and invasion, and (iv) the identification of novel therapeutic options. This work demonstrates that this HTP 3D bioprinted cell migration platform has broad applications across quantitative cell and cancer biology as well as drug screening.

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