Download - Angiopoietin-1 Induces Migration of Monocytes in a Tie-2 and Integrin-Independent Manner

Angiopoietin-1 Induces Migration of Monocytes in a Tie-2and Integrin-Independent Manner

Shakil Ahmad, Melissa J. Cudmore, Keqing Wang, Peter Hewett, Rahul Potluri,Takeshi Fujisawa, Asif Ahmed

Abstract—Angiopoietin-1 (Ang-1) is an angiogenic growth factor that activates Tie-2 and integrins to promote vessel wallremodeling. The recent finding of the potential proatherogenic effects of Ang-1 prompted us to investigate whetherAng-1 promotes monocyte chemotaxis, endothelial binding, and transendothelial migration, key events in theprogression of atherosclerosis. Here, we show that Ang-1 induces chemotaxis of monocytes in a manner that isindependent of Tie-2 and integrin binding but dependent on phosphoinositide 3-kinase and heparin. In addition, Ang-1promoted phosphoinositide 3-kinase-dependent binding of monocytes to endothelial monolayers and stimulatedtransendothelial migration. Fluorescence-activated cell sorting analysis showed that exogenous Ang-1 adheres directly tomonocytes as well as to human umbilical endothelial cells, but neither Tie-2 mRNA nor protein were expressed by primarymonocytes. Although Ang-1 binding to human umbilical endothelial cells was partially Tie-2 and integrin dependent, Ang-1binding to monocytes was independent of these factors. Finally, preincubation of monocytes with soluble heparin abrogatedAng-1 binding to monocytes and migration, and partially prevented Ang-1 binding to human umbilical endothelial cells. Insummary, Ang-1 induces chemotaxis of monocytes by a mechanism that is dependent on phosphoinositide 3-kinase andheparin but independent of Tie-2 and integrins. The ability of Ang-1 to recruit monocytes suggests it may play a role ininflammatory angiogenesis and may promote atherosclerosis. (Hypertension. 2010;56:477-483.)

Key Words: angiopoietin-1 � Tie-2 � monocytes � chemotaxis � vascular � phosphoinositide 3-kinase� endothelium

Angiopoietins (Angs) are a family of polypeptide ligandsthat bind to Tie-2, an endothelial cell-specific receptor

tyrosine kinase that is required for vascular remodeling,stabilization, and mural cell recruitment.1 Ang-1 and Ang-2are unique in that they elicit distinct responses from the sameTie-2 receptor.1 Ang-1 can also bind to and elicit functionsvia integrins and extracellular matrix proteins, such as vitro-nectin.2 Transgenic overexpression of Ang-1 in the skin orsystemic delivery of Ang-1 dramatically blocks increases invascular permeability in response to vascular endothelialgrowth factor3 and reduces microvascular leakage in inflam-matory disease.4 Ang-1 also inhibits vascular endothelialgrowth factor-mediated leukocyte adhesion to endothelialcells by preventing upregulation of cell surface adhesionmolecules.5,6 These studies led to the view that Ang-1 is avascular protective agent.

The function of Ang-1 in the context of vascular disease ismore complex than originally contemplated. First, it has beenreported that postintracoronary perfusion of the allograftswith an adenovirus encoding human Ang-1 can promote the

development of cardiac allograft arteriosclerosis in rats.7

Second, Ang-1 gene expression was strongly correlated withboth femoral and carotid-radial artery pulse-wave velocitywaveforms in the peripheral blood monocytes of hypertensivepatients, indicating a link between arterial stiffness andhypertensives.8 Arterial stiffness is an important independentpredictor of cardiovascular mortality in hypertensive patients,and pulse-wave velocity is a useful index of arterial stiffnessand an independent marker of cardiovascular adverse out-come in hypertensives.9 Finally, eosinophils,10 neutro-phils,11,12 and a subset of monocytes that contribute to tumorangiogenesis13 were reported to express Tie-2. In addition,Ang-1 was shown to induce migration of eosinophils10 andneutrophils,11 and this is highly relevant because leukocytemigration and invasion into the arterial wall is critical for thedevelopment of atherosclerotic lesions and in the develop-ment of a vulnerable plaque.14

In this study, we investigated the expression of Tie-2 onhuman peripheral blood monocytes and monocyte-derivedcell lines and their migratory responses to Ang-1 stimulation.

Received April 27, 2010; first decision May 14, 2010; revision accepted July 12, 2010.From the Department of Reproductive and Vascular Biology (P.H., R.P., A.A.), School of Clinical and Experimental Medicine, College of Medical

and Dental Sciences, University of Birmingham, Birmingham, United Kingdom; Birmingham Women’s Hospital (A.A.), Edgbaston, Birmingham, UnitedKingdom; Gustav Born Centre for Vascular Biology and Centre for Cardiovascular Science (S.A., M.J.C., K.W., T.F., A.A.), Queen’s Medical ResearchInstitute, University of Edinburgh, Edinburgh, United Kingdom.

S.A. and M.J.C. contributed equally to this work.Correspondence to Asif Ahmed, Gustav Born Centre for Vascular Biology, Queen’s Medical Research Institute, College of Medicine and Veterinary

Medicine, University of Edinburgh, 47 Little France Crescent, Edinburgh EH16 4TJ, UK. E-mail [email protected]© 2010 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.110.155556

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We show for the first time that Ang-1 binds to monocytes andinduces migration of these cells via a mechanism that isindependent of Tie-2 and integrins.

MethodsFull methods are described in the supplemental data, available onlineat http://hyper.ahajournals.org.

ResultsAng-1 Induces Phosphoinositide 3-Kinase(PI3K)-Dependent Monocyte MigrationAng-1 induced a concentration-dependent increase in migra-tion of monocytes (Figure 1A), whereas Ang-2 had no effect.Migration could be abrogated by prior heat-inactivation of theligand (Figure 1B). Blockade of Ang-1 through preincubationwith recombinant soluble Tie-2 receptor (rTie-2-Fc) alsoprevented migration, indicating the ligand-specific nature ofthe response (Figure 1C). Checkerboard analysis demon-strated that Ang-1 is capable of promoting both chemotaxisand chemokinesis of monocytes (Table).

Many of the functions attributed to Ang-1 are mediated bysignaling via the PI3K pathway,15,16 and monocyte chemo-taxis is reported to be PI3K dependent.17 The PI3K inhibitor,LY29004, abrogated Ang-1–induced monocyte migration(Figure 1D), whereas the MEK-1 inhibitor, PD98059, did notprevent Ang-1–mediated monocyte migration (data not

shown), suggesting that the PI3K pathway is required for thisresponse.

Monocytes, on activation, adhere firmly to the endotheliumand transmigrate through the endothelial cell monolayer. Toexamine the role of Ang-1 in monocyte transmigration,human umbilical endothelial cells (HUVECs) were pretreatedwith tumor necrosis factor (TNF)-�, and monocytes wereallowed to migrate across the endothelial monolayer towardAng-1 (400 ng/mL) for 2 hours. Ang-1 induced a significantincrease in monocyte transendothelial migration as comparedwith control treated cells (Figure 1E), suggesting that Ang-1can induce direct binding and migration of monocytes.

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Figure 1. Ang-1 induces monocyte migration via PI3K. Migration of primary monocytes was assessed using a modified Boyden cham-ber assay in response to Ang-1 or Ang-2 (A), heat-inactivated (HI) (100°C for 30 minutes) Ang-1 (200 ng/mL) and vascular endothelialgrowth factor (VEGF) (20 ng/mL) (B), Ang-1 (200 ng/mL) after 30-minute preincubation with recombinant Tie-2-Fc (1 to 5 �g/mL) (C),and Ang-1 (200 ng/mL) after cells were incubated with LY294002 (20 �mol/L) (D). E, HUVEC monolayers were treated with TNF-� (10ng/mL) and transmigration of monocytes to Ang-1 (200 ng/mL) and MCP-1 (10 ng/mL) assessed. Data are mean (�SEM) of monocytescounted per 10 fields (�200) in at least 5 experiments performed in duplicate. **P�0.01 or ***P�0.001 vs control.

Table. Checkerboard Analysis of Ang-1-StimulatedMonocyte Migration

Lower CompartmentAng-1 (ng/mL)

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0 9�1 8�1 12�3 8�1

50 13�1 8�1 19�5 13�1

100 23�2 16�1 12�3 22�3

200 68�2 33�5 20�4 34�4

A concentration range of Ang-1 was supplied to the upper and/or lowercompartments of the modified Boydens chemotaxis chamber. Data are mean(�SEM) of monocytes counted per 10 fields (�200) in at least 5 experimentsperformed in duplicate.

478 Hypertension September 2010

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Ang-1 Induces PI3K-Dependent Monocyte AdhesionDuring inflammation, monocytes are recruited to sites of endo-thelial cell injury and roll along the vascular endothelium, wherethey become activated. We assessed whether treatment ofendothelial cells with Ang-1 or Ang-2 could stimulate adhe-sion of monocytes. Both Ang-1 (200 ng/mL) and Ang-2 (200ng/mL) induced a significant increase monocyte adhesionafter 1 hour of coculture (Figure 2A and 2B). TNF-� at 10ng/mL was used as a positive control. Inhibition of Ang-1 orAng-2 with rTie-2-Fc produced a significant decrease inmonocyte adhesion (Figure 2C). Moreover, inhibition ofendothelial Tie-2 receptor using Tie-2 inhibitory peptide(NLLMAAS) also led to a decrease in Ang-1–induced mono-cyte adhesion (Figure 2D). To further elucidate the signalingpathway, inhibition of the PI3K pathway with LY294002blocked Ang-1–mediated monocyte adhesion. In contrast,heparin pretreatment had no effect on Ang-1– or Ang-2–induced adhesion, indicating a heparin-independent butPI3K-dependent monocyte adhesion event.

Ang-1 Binds Directly to the Monocyte Cell SurfaceHUVECs, primary monocytes, and the monocyte-derived celllines, U937 and THP-1, express endogenous cell surface Ang-1(data not shown). Thus, to differentiate between endogenous andexogenously administered Ang-1, a recombinant human Ang-1was used as the source of Ang-1 (Ang-1–His). Binding of

Ang-1–His was detected on THP-1, U937 cells, and HUVECs(Figure 3A through 3C), whereas binding of Ang-2 was onlydetectable on HUVECs (Figure 3C).

Ang-1–Induced Monocyte Migration Independentof Tie-2To assess the involvement of Tie-2 in the Ang-1–mediatedmonocyte migratory response, monocytes were preincubatedwith the inhibitory Tie-2 peptide or a blocking Tie-2 anti-body. Inhibition of Tie-2 did not affect Ang-1–inducedmonocyte migration (Figure 4A), whereas Tie-2 inhibitorypeptide significantly abrogated Ang-1–induced HUVEC mi-gration (Figure 4B), suggesting that the Ang-1–inducedmonocyte response is a Tie-2-independent function. More-over, the Tie-2 blocking peptide had no effect on the directbinding of Ang-1 to monocytes (Figure 4C), whereas itpartially abrogated Ang-1 binding to HUVECs, which ex-press Tie-2 (Figure 4D). RT-PCR (Figure 4E), Western blotanalysis (Figure 4F), and ELISA (Figure 4G) demonstratedthe absence of Tie-2 mRNA and protein in monocytes andmonocyte-derived cell lines. Thus, Ang-1 induces monocytemigration in a Tie-2-independent manner.

Ang-1–Induced Monocyte Migration IsIntegrin IndependentAng-1 has been shown to mediate cellular functions inendothelial and nonendothelial cells via direct binding and

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Figure 2. Ang-1 induces monocyte adhesion via PI3K. A, Representative photomicrograph of adherent monocyte treated with Ang-1(200 ng/mL), Ang-2 (200 ng/mL), or TNF-� (10 ng/mL) for 1 hour. B, Quantification of monocyte adhesion. Monocyte adhesion inresponse to Ang-1 or Ang-2 that had been preincubated with recombinant Tie-2-Fc (2 �g/mL) (C) and Tie-2 peptide, NLLMAAS (D). E,Monocytes were incubated with LY294002 (20 �mol/L) prior to Ang-1. F, Adhesion of monocyte with Ang-1 or Ang-2 prior to pretreat-ment with heparin (50 IU/mL). Data are mean (�SEM) of monocytes counted per 10 fields (�200) in at least 5 experiments performedin duplicate. **P�0.01 or ***P�0.001 vs control.

Ahmad et al Ang-1 Induces Monocyte Migration 479



interaction with integrins.2,18,19 Preincubation of monocyteswith RGD-based peptides, which inhibit integrin binding, didnot prevent Ang-1–induced monocyte migration (Figure 5A)but blocked Ang-1–induced HUVEC migration (Figure 5B).In addition, pretreatment of cells with EDTA, a pan-integrininhibitor, also did not affect Ang-1 binding to THP-1 cells(Figure 5C), and only partially prevented Ang-1 binding toHUVECs (Figure 5D). The results show that integrins play norole in Ang-1–induced monocyte chemotaxis.

Ang-1–Induced Monocyte Migration IsHeparin DependentIncubation of either primary human monocytes or HUVECswith soluble heparin prior to the assay abrogated Ang-1–induced migration of both cell types (Figure 6A and 6B). Inaddition, incubation of THP-1 or U937 with soluble heparinprior to Ang-1–His administration prevented Ang-1 binding(Figure 6C and 6D), suggesting a heparin dependence of thisfunction in these cell types. Incubation of HUVECs with

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Figure 3. Ang-1, but not Ang-2, binds to monocytes. THP-1 (A), U937 (B), and HUVECs (C) were incubated with His-tagged Ang-1 orAng-2. Binding was assessed with a FACScan Flow cytometer after incubation with an anti-His antibody and appropriate secondaryreagents. Data are representative of at least 5 experiments performed in duplicate. FL1-H indicates FL1-H (FITC).

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Figure 4. Ang-1 binding and monocyte migration are independent of Tie-2. A, Primary monocytes were incubated with a goat anti-Tie-2 blocking antibody (5 �g/mL) and a Tie-2 inhibitory peptide (NLLMAAS; 100 �mol/L); or B, HUVECs pretreated with Tie-2 inhibi-tory peptide prior to inclusion in the migration assay Ang-1 (200 ng/mL). Data are representative or mean (�SEM) of monocytescounted per 10 fields (�200) in at least 5 experiments performed in duplicate. Binding of exogenous Ang-1 to THP-1 cells (C) andHUVECs (D) was assessed following incubation of the cells with a Tie-2 synthetic peptide, NLLMAAS (0.5 mmol/L/106 cells) using FAC-Scan flow cytometer. E, RT-PCR for Tie-2 in primary monocytes and HUVECs using 3 different sets of Tie-2 primers. F, THP-1 andU937 lysates were resolved by SDS-PAGE and immunoblotted with anti-Tie-2. G, Presence of extracellular Tie-2 in lysates of THP-1,U937, and HUVECs measured using ELISA. HMEC indicates human microvascular endothelial cells; FL1-H, FL1-H (FITC).




heparin partially inhibited Ang-1 binding, but heparin had noeffect on the binding of Ang-2 to HUVECs (Figure 6E).Thus, the binding of Ang-1 to monocytes is via a heparin-dependent mechanism only, whereas Ang-1 binding to endo-thelial cells can be accounted for by Tie-2, integrin, andheparin-dependent mechanisms.

DiscussionThe transmigration of monocytes into the subendothelialspace in response to endothelial injury plays a significant rolein the development of early atherosclerotic lesions, as well asaugmenting progression of the disease in later stages.20 Thefinding that Ang-1 failed to protect against arterioscleroticlesion formation7 prompted us to examine the effects ofAng-1 on monocyte migration in vitro. We observed thatAng-1, but not Ang-2, induced a dose-dependent migration ofhuman primary monocytes and monocytic cell lines, whichcould be inhibited by preincubation of Ang-1 with recombi-nant soluble Tie-2. Additionally, Ang-1 also induced asignificant increase in monocyte transmigration. However,preincubation of the cells with a blocking Tie-2 antibody oran inhibitory Tie-2 peptide had no effect on the Ang-1–induced migration of monocytes or Ang-1 binding to thesecells but did partially inhibit Ang-1 binding to HUVECs.Demonstrating that the observed Ang-1 effects on monocytesare not mediated via Tie-2.

Tie-2 expression has been demonstrated on eosinophils10

and neutrophils,11 and Ang-1 has been shown to induceTie-2-dependent migration of these cells.11 Tie-2 expressionwas also observed on a subset of monocytes, designatedTie-2-expressing monocytes, that contribute to tumor angio-genesis13 and on the murine macrophage RAW 264.7 cell line

by immunofluorescent staining.21 We undertook RT-PCR,Western blot analysis, and fluorescence-activated cell sorter(FACS) for Tie-2, but did not detect it in primary monocytesor monocytic cell lines. Functional assays assessing themigratory capacity of monocytes in response to Tie-2 inhibi-tion showed that neither the anti-Tie-2 antibody nor the Tie-2inhibitory peptide could block Ang-1–induced monocytemigration. These findings led us to conclude that Tie-2 is notinvolved in the observed Ang-1–dependent migration ofmonocytes. However, pretreatment of Ang-1 with recombi-nant soluble Tie-2 protein, particularly at higher concentra-tions, markedly inhibited Ang-1–induced monocyte migra-tion in a concentration-dependant manner, confirming theeffect to be Ang-1 specific.

Several studies have shown that the functional effects ofAng-1 can be achieved without ligation of the Tie-2 receptor.Signaling through integrins is one such alternative mecha-nism. Ang-1 has been shown to induce adhesion and spread-ing of endothelial cells via �5�1 and �5�5 integrins, andAng-1 adhesion in Tie-2-negative fibroblasts was mediatedby these integrins.2 Ang-1 can also induce integrin-dependentsurvival of Tie-2-negative cardiac and skeletal myocytes,18

and recently, �5�1 integrin was shown to directly and stablyinteract with the Tie-2 receptor itself to promote Ang-1–dependent angiogenesis.19 In our current study, integrininhibitory peptides were used to determine whether integrinswere involved in the monocyte response to Ang-1. Thesepeptides did not affect migration, suggesting that integrins arenot mediating this response. FACS analysis showed thatAng-1 binding was unaffected by pan-integrin inhibitorEDTA, suggesting that Ang-1 fails to interact with monocyteintegrins.

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Figure 5. Monocyte migration and Ang-1binding are independent of integrins. Migra-tion of monocytes (A) and HUVECs (B) toAng-1 (200 ng/mL) in the presence of EDTA(10 mmol/L), RGD (10 nmol/L), or RAD (10nmol/L). Binding of Ang-1 to THP-1 (C) cellsand HUVECs (D) in the presence of EDTA(10 mmol/L). Data are representative or mean(�SEM) of monocytes counted per 10 fields(�200) in at least 5 experiments performed induplicate. FL1-H indicates FL1-H (FITC).




More recently, low-shear flow conditions were identifiedto increase the expression of Ang-2 in human endothelialcells,22 and Ang-2 potentiates TNF-�-induced monocyteadhesion.23 In FACS-based binding, we show that Ang-1, butnot Ang-2, binds to monocytes, whereas both Ang-1 andAng-2 bind to HUVECs. Ang-1 binding was unaffected bythe preincubation of the cells with Tie-2 peptide or thepan-integrin inhibitor EDTA. Soluble heparin abrogatedthe binding of Ang-1 to monocytes but only partly inhib-ited the binding to HUVECs. Preincubation of HUVECswith Tie-2 inhibitory peptide, EDTA, and heparin abrogatedthe binding of Ang-1, indicating that Ang-1 binding toHUVECs is mediated by 3 separate entities. Future work willattempt to identify the heparin-dependent receptor by pro-teomic analysis.

Activation of the PI3K pathway has been shown to beessential for transendothelial monocyte migration and as aconsequence is implicated in chronic inflammatory diseases,such as atherosclerosis.24 Ang-1 promotes endothelial cellsurvival via activation of the PI3K signal transduction path-way. Pharmacological inhibition of the PI3K pathway inhib-ited monocyte migration, demonstrating for the first time afunction that is Ang-1 and PI3K dependent but not mediatedvia Tie-2. However, the precise cell surface structures ligatedby Ang-1 to activate the PI3K signal transduction pathway, topromote monocyte chemotaxis, remain unknown.

PerspectiveAng-1 is an angiogenic growth factor that activates Tie-2 andintegrins to promote vessel wall remodeling. This studydemonstrates, for the first time, that Ang-1 binds to mono-cytes and induces migration of these cells via a mechanismthat is independent of Tie-2 and integrins, but requiresactivation of PI3K pathway. In addition, it shows that themonocyte binding and migration is abrogated by solubleheparin and that Ang-1 binding to endothelial cells is alsopartially heparin dependent. Collectively, these findings in-dicate that Ang-1 can function as a proinflammatory media-tor. High levels of Ang-1 may contribute to vascular/inflam-matory disorders, such as atherosclerosis, and we advisecaution when considering using Ang-1 as a therapeutic agentto tackle compromised vascular status in individuals withcardiovascular disease.

Sources of FundingThis work was supported by grants from the Medical ResearchCouncil (G0601295 and G0700288) and British Heart Foundation(RG/09/001/25940, PG/06/114 and BHF CoRe).

DisclosuresNone.

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Vascular-specific growth factors and blood vessel formation. Nature.2000;407:242–248.

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Figure 6. Monocyte migration and Ang-1 binding is heparin dependent. Primary monocytes (A) and HUVECs (B) were incubated withheparin (50 IU/mL) prior to migration to Ang-1 (200 ng/mL). Binding of exogenous Ang-1 to THP-1 (C), U937 (D), and HUVECs (E) wasmeasured in the presence of heparin (50 IU/mL) using FACScan Flow cytometer. Data are representative or mean (�SEM) of mono-cytes counted per 10 fields (�200) in at least 5 experiments performed in duplicate. FL1-H indicates FL1-H (FITC).




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Fujisawa and Asif AhmedShakil Ahmad, Melissa J. Cudmore, Keqing Wang, Peter Hewett, Rahul Potluri, Takeshi

MannerAngiopoietin-1 Induces Migration of Monocytes in a Tie-2 and Integrin-Independent

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2010 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.110.155556

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Online Data Supplement

Angiopoietin-1 induces migration of monocytes in a Tie-2 and integrin

independent manner

Shakil Ahmad1*, Melissa J. Cudmore1*, Keqing Wang1, Peter Hewett1, Rahul Potluri1,

Takeshi Fujisawa1,3, and Asif Ahmed1,2,3

* Equal contribution

1Department of Reproductive and Vascular Biology, School of Clinical and

Experimental Medicine, College of Medical and Dental Sciences, University of

Birmingham, B15 2TT, UK, 2Birmingham Women’s Hospital, Edgbaston,

Birmingham, B15 2TG, UK and 3Gustav Born Centre for Vascular Biology and

Centre for Cardiovascular Science, Queen's Medical Research Institute, University of

Edinburgh, Edinburgh, UK.

Methods Reagents Recombinant human C-terminal 6-His tag Ang-1, Ang-2, Tie2-Fc and Anti-Tie-2 antibodies were purchased from R&D Systems (Abingdon, UK). VEGF was obtained from RELIATech (Brauschweig, Germany). Recombinant human TNF-α was purchased from Pepro Tech (London, UK). The PI3K inhibitor LY294002 and integrin peptides RGD, RAD were obtained from Merck Chemicals (Nottingham, UK). All other cell culture reagents and chemicals were obtained from Sigma Aldrich (Poole, UK). Preparation of human monocytes Primary human monocytes were isolated from 40 ml of heparinized venous blood by adhesion to plastic dishes as published.1 In brief, density centrifugation was performed using the Lymphoprep separation solution with a density of 1.077 g/ml (Nycomed, UK) to isolate mononuclear cells. Monocytes were isolated by adherence to tissue culture flasks. Non-adherent cells were removed by vigorous washing and RPMI containing 0.2 % BSA was added. The purity of the adherent cells was >90 % monocytes as detected by non-specific esterase staining and immunofluorescence positivity with the anti-CD14 mAb (Becton-Dickinson, UK) and less than one platelet per 10 monocytes was detected. The viability of the isolated monocytes was assessed by trypan blue exclusion and was >90 %. Cell culture Human umbilical vein endothelial cells (HUVEC) were used as previously described.2 Both monocyte-derived cell lines (U937 and THP-1) were grown in RPMI containing 10 % FCS. Cells were maintained in suspension at 1 x 106 cells/ml. Chemotaxis Assay Cell migration was evaluated using a modified Boyden chamber assay as described.2 Gelatin-coated 5 μm PVP free membranes were used to separate chambers, with an incubation time of 2 hours after stimulation with the agonists. After staining of the membranes, with haematoxylin and eosin, cell migration was quantified by counting 8-10 fields of view at x10 magnification. Monocyte adhesion assay Monocyte adhesion assays were performed on confluent HUVEC grown on 24 well plates. HUVEC monolayers were stimulated in triplicate as indicated with Ang-1 (400 ng/ml), Ang-2 (400 ng/ml) or TNF-α (10 ng/ml) in M199 medium supplemented with 5% FCS at 37°C, 5% CO2. Where indicated, cells were pre-treated with rTie2-Fc (1 mg/ml), Tie2 peptide (100 μM) or LY294002 (20 uM) for 30 minutes prior to stimulation. THP-1 cells (2x105) were added to the HUVEC monolayers for further 1 hour after 6 hours of stimulation with agonists. Cells were then washed twice with PBS, fixed in 1% glutaraldehyde and stained with Mayer’s hematoxylin to visualize bound monocytes. Digital images of nine random at x200 power fields per well were counted to determine the mean number of adherent monocytes.

Monocyte transendothelial migration assay Monocyte transendothelial migration under static conditions was assessed using 24-well format Transwell filters (8 mm). HUVEC were seeded in the upper chambers of the filters to form confluent monolayers and stimulated with TNF-α 10 U/ml for 4h. Cells were then washed to remove residual cytokines. Primary human monocytes (1x105 per well) were resuspended in M199 containing 0.2% BSA and placed in the upper compartment of the Transwell filters. Ang-1 at 400 ng/ml was added to lower chamber and monocytes were allowed to migrate for 2 hours. After incubation, non-migrated cells were removed and transmigrated cells in lower compartment were collected and counted using flow cytometry. FACS binding assay Cells were incubated with Ang-1 or Ang-2 containing a 6 x Histidine Tag (Ang-1-His; Ang-2-His) at 1 μg/ml x 106 cells/ml for 1 hour with rotation at 4°C, then washed twice and incubated with anti-His-Tag or mouse IgG1 for 1 hour at 4°C. Cells were then washed twice and incubated with secondary anti-mouse FITC (1:50) for 1 hour at 4°C. After two subsequent washing steps binding was quantified using flow cytometry. For inhibitor studies, EDTA (5 mM), heparin (50 IU/m1 x 106 cells) or Tie-2 inhibitory peptide (0.5 mM x 106 cells) were added for 30 minutes prior to addition of Ang-1. Flow cytometry was performed using a FACScan flow cytometer with CELLQuest software (Becton Dickinson, UK) and the 488 nm argon laser. Typically, 15,000 cells were collected from each sample. All data were acquired in a list mode and three parameters checked; forward scatter (FSC), side scatter (SSC) and one fluorescence channel (FL-1). RT-PCR Total RNA was isolated from cells using the RNAqueousTM-4PCR kit (Ambion, Warrington, UK) and ~ 1 μg RNA reverse transcribed as described previously.3 PCR was performed using three sets of oligonucleotides to Tie-2. Set 1, as previously described 4, Set 2 (sense: 5’-CC ATG GCC ATG GAC TTG ATC TTG GAT C-3’; antisense: 5’-GC GGC CGC TTC ACA TCT CCG GAC T-3’) and Set 3 (sense: 5’-CC ATG GCT TGT GAA CTG CAC ACG-3’; antisense: 5’-CC GGC CGC AAA GTT ATG TCC AGT G-3’) and glyceraldehyde-3 phosphate dehydrogenase as a loading control. Samples were denatured (96°C, 5 min) and 1 U of BIOTAQTM polymerase (Bioline, London, UK) added at 58°C and PCR amplification carried out as follows: 94°C, 1 min, 58°C, 1 min and 72°C for 2 min, for 35 cycles. Full length Tie-2 cDNA control template was PCR-amplified from HUVEC cDNA cloned into pPCR Blunt (Invitrogen, Paisley, UK) and sequenced. Western Blotting Cell lysates were prepared in ice-cold RIPA buffer (Upstate, Hampshire, UK) and a total of 20 μg protein of each sample run on 10% SDS-PAGE gels, and Western-blotted onto nitrocellulose as described.5 Primary antibody used was a monoclonal antibody against rTie-2 (Regeneron, USA). ELISA

Tie-2 in cell lysates was quantified using a commercially available ELISA kit (R&D Systems, Abingdon, UK), according to the manufacturer's instructions. Statistical Analysis All data are expressed as the mean (±SEM). Statistical comparisons were performed using 1-way ANOVA followed by the Student–Newman–Keuls test as appropriate. Statistical significance was set at a value of p<0.05.

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