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Prepublished online May 26, 2005; doi:10.1182/blood-2004-12-4716

Endothelial intercellular adhesion molecule (ICAM)-2 regulates angiogenesis Miao-Tzu Huang, Justin C Mason, Graeme M Birdsey, Valerie Amsellem, Nicole Gerwin, Dorian O Haskard, Anne J Ridley and Anna M Randi

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Blood First Edition Paper, prepublished online May 26, 2005; DOI 10.1182/blood-2004-12-4716

Endothelial Intercellular Adhesion Molecule (ICAM)-2 Regulates Angiogenesis Short title: Endothelial ICAM-2 regulates angiogenesis Miao-Tzu HuangΨ +; Justin C MasonΨ; Graeme M BirdseyΨ, Valerie AmsellemΨ, Nicole Gerwin♦ ++; Dorian O HaskardΨ; Anne J Ridley◊ and Anna M RandiΨ∞ Ψ

From British Heart Foundation (BHF) Cardiovascular Sciences Unit, Eric Bywaters

Centre for Vascular Inflammation, Hammersmith Hospital, Imperial College, London, UK; ◊Ludwig Institute for Cancer Research, Royal Free and University College School of Medicine, London, UK. ♦ The Center for Blood Research, Harvard Medical School, Boston, Massachusetts 02115, USA +

Present address: Department of Medical Research and Department of Paediatrics,

National Taiwan University Hospital, No 7 Chung-Shan South Road, Taipei, Taiwan 100 ++

Present address: Aventis Pharma Deutschland GmbH, Frankfurt, Germany

∞ Corresponding Author:

Anna M. Randi MD PhD Imperial College BHF Cardiovascular Sciences Unit Hammersmith Hospital Du Cane Road London W12 0NN Tel: +44 20 8383 8049 fax: +44 20 8383 1640 email: [email protected]

Word Count: Abstract, 191; Text 4986 N. of Figures: 7 Scientific heading: Hemostasis, Thrombosis, and Vascular Biology Funding: British Heart Foundation (AMR; DOH; MTH; GMB); Ludwig Institute for Cancer Research (AJR); MAIN European Consortium (VA)

1 Copyright © 2005 American Society of Hematology


Authors’ contribution to the manuscript: Miao-Tzu Huang:

performed research, generated reagents, data analysis and interpretation, contributed to design of research and writing the paper

Justin C. Mason:

performed research, contributed to data analysis and interpretation

Graeme M. Birdsey: performed research, contributed to data analysis and interpretation Valerie Amsellem:

performed research, contributed to data analysis and interpretation

Nicole Gerwin:

provided key tools

Dorian O Haskard:

contributed to design of research, data analysis and interpretation and key tools

Anne J Ridley:

contributed to design of research, data analysis and interpretation and key tools

Anna M Randi:

original idea, designed research, contributed key tools and reagents, analysed and interpreted data, wrote paper

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Abstract Endothelial junctions maintain endothelial integrity and vascular homeostasis. They modulate cell trafficking into tissues, mediate cell-cell contact and regulate endothelial survival and apoptosis. Junctional adhesion molecules such as VEcadherin and CD31/PECAM mediate contact between adjacent endothelial cells and regulate leukocyte trans-migration and angiogenesis. The leukocyte adhesion molecule ICAM-2 is expressed at the endothelial junctions. In this study we demonstrate that endothelial ICAM-2 also mediates angiogenesis. Using ICAM-2deficient mice and ICAM-2-deficient endothelial cells, we show that the lack of ICAM2 expression results in impaired angiogenesis both in vitro and in vivo. We show that ICAM-2 supports homophilic interaction, and that this may be involved in tube formation. ICAM-2 deficient cells show defective in vitro migration, as well as increased apoptosis in response to serum deprivation, anti-Fas antibody or staurosporine. ICAM-2 signalling in human umbilical vein endothelial cells (HUVEC) was found to activate the small GTPase Rac, which is required for endothelial tube formation and migration. These data indicate that ICAM-2 may regulate angiogenesis via several mechanisms including survival, cell migration and Rac activation. Our findings identify a novel pathway regulating angiogenesis through ICAM-2 and a novel mechanism for Rac activation during angiogenesis.

Introduction Angiogenesis involves a cascade of events that requires the disassembly of endothelial junctions, followed by detachment, proliferation and migration of endothelial cells (EC), and finally the re-establishment of cell-cell and cell-matrix contacts 1. Adhesion molecules at the endothelial junctions, such as CD31/PECAM and junctional adhesion molecule (JAM)-A, support endothelial cell-cell contact through homophilic binding, and are involved in regulating endothelial homeostasis and angiogenesis via different mechanisms. Junctional molecules also mediate leukocyte trans-endothelial migration through homophilic or heterophilic interactions with leukocytes 2. ICAM-2 is a member of the immunoglobulin (Ig) super-family, constitutively expressed at the endothelial junctions 3. Its structure includes two extracellular Ig-

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domains, a trans-membrane region and a short intracellular tail 4. ICAM-2, originally identified as a ligand for leukocyte integrin LFA-1 4, also interacts with the integrin Mac-1 5 and the dendritic cell receptor DC-SIGN 6. Through these interactions, ICAM2 mediates leukocyte trafficking 4,6,7. Recent data indicates that ICAM-2 involvement in leukocyte transmigration may be stimulus-specific 8. However its role in inflammation is still unclear: inflammatory cytokines that up-regulate the expression of leukocyte adhesion molecules such as ICAM-1 or VCAM-1, down-regulate ICAM-2 expression and induce its redistribution away from the junctions 3. A similar patter is observed for other junctional adhesion molecules such as CD31/PECAM 9 and JAMA 10. On the basis of the similarities in function, structure and localisation between ICAM-2 and other junctional adhesion molecules involved in vascular homeostasis, we postulated that ICAM-2 may also play a role in angiogenesis. Data on the regulation of ICAM-2 expression further support this hypothesis. In endothelial cells, ICAM-2 expression can be regulated by cell-cell contact and growth factors (MTH and AMR, manuscript in preparation). ICAM-2 endothelial expression is driven by the transcription factor Erg 11, which is required for EC tube formation on Matrigel 12 and also regulates the expression of angiogenesis mediators including thrombospondin, SPARC, RhoA, and VE-Cadherin 13; 12. In this paper we demonstrate that ICAM-2 is required for in vitro and in vivo angiogenesis. We also demonstrate for the first time that ICAM-2 can engage in homophilic interaction, and is involved in endothelial survival and migration. Finally we show that ICAM-2 signalling activates the small GTPase Rac, a key regulator of signal transduction pathways to the cytoskeleton. In conclusion, we have identified a novel angiogenesis pathway, involving the adhesion molecule ICAM-2 and the activation of the small GTPase Rac. Materials and Methods Animals ICAM-2 -/-mice were generated as described 7. 129SV X C57BL/6 wild-type (WT) mice were generated at the Biological Service Unit, Hammersmith Hospital, London, UK. Experiments were performed according to the Animals Scientific Procedures Act 1986.

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Cells Murine cardiac endothelial cells (MCEC) were isolated as previously described 14. Briefly, mice heart tissue was diced and incubated with 5mL of 1mg/mL collagenase A for 30 min at 37°C followed by 5mL Trypsin/EDTA for 10 min at 37°C. The released EC were isolated by positive selection with anti-endoglin antibody (Ab) (MJ7/18, 10mg/mL) and MiniMac microbeads system (Miltenyi Biotech, UK). MCEC were cultured on 1 % gelatin in DMEM supplemented with 10% foetal calf serum (FCS), 30µg/mL endothelial cell growth supplement (ECGS, Sigma), 10U/mL heparin, 2mM L-glutamine, 100IU/mL penicillin and 0.1mg/mL streptomycin. Cells were used for experiments at passage 1 and 2. To generate the immortalized endothelial cell lines, passage 1 MCEC from WT or ICAM-2 -/- mice were transduced with a retrovirus encoding the temperature sensitive mutant SV40 large T antigen tsTA58, a gift from Prof Yuti Chernajovsky (Barts, London, UK), as described 15. After infection, the cells were left to recover for 48 hr in fresh medium. Cells were then cultured in the above medium containing G418 (400 µg/mL) at 33°C for 2-3 weeks, and resistant colonies were pooled and expanded. Cells were switched to 37°C to arrest proliferation 24 hr before experiments. The endothelial origin of the WT (WTMCEC-SV) and ICAM-2 -/- (IC2-MCEC-SV)-derived endothelioma lines was verified by FACS, using anti-endoglin and anti-CD31 Ab. The phenotype of the IC2-MCECSV endothelioma line was similar to that observed in primary MCEC, based on ICAM-2 expression, Matrigel tube formation and cell proliferation. HUVECs were isolated as described 16, and cultured in 1% gelatin-coated tissue culture ware in Medium 199 supplemented with 20% FCS, 30µg/mL ECGS, 10U/mL heparin, L-glutamine 2mM, penicillin 100IU/mL and streptomycin 0.1mg/mL. Flow Cytometry (FACS) FACS analysis was performed by standard methods 14. Cell samples were incubated with primary or isotype-matched control Ab for 30 min at 4°C and washed in phosphate buffered solution (PBS) twice followed by incubation with fluorescenceconjugated secondary Ab. After 2 more washes in PBS, samples were resuspended in 4% paraformaldehyde and analysed with Epic XL–MCL flow cytometer and System II™ software (Coulter flow, UK). The primary Ab data were normalised against isotype control and expressed as relative fluorescence intensity (RFI). In vitro Matrigel tube formation assay

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Matrigel (300µL/well) was plated evenly in a 24-well plate, and incubated at 37°C for 30 min before adding MCEC (0.5x105 cells/well). Tube formation was studied over 8 hr and serial photographs of representative 100x fields were taken hourly. Endothelial tubes were quantified by counting branches from each EC. Where indicated, cells were pre-incubated with anti-ICAM-2 or control Ab for 15 min at RT before plating onto Matrigel. In Vivo Matrigel plug assay Matrigel (300µL) containing 40ng/mL vascular endothelial growth factor (Peprotech, UK) and 10u/mL heparin (Roche, UK) was injected subcutaneously into the abdominal area of the mouse. 7 days later, Matrigel plugs were carefully excised, fixed with 10% formalin and processed for hematoxylin-eosin (H&E) staining. The area of vascular lumen within the plugs was measured by Image ProPlus software (Berkshire, UK). Two independent experiments were performed, each with 5-6 mice per group. For each plug, 5 representative 100x fields from each of 3 serial sections were analysed. The data is shown as percentage of vessel-occupying area per 100x field area. Endothelial proliferation assay Cell proliferation was measured over 5 days using the MTT assay (Promega, UK). The assay was performed as per manufacturer’s instructions, on cells plated onto collagen-coated 96-well tissue culture plates at 2,000 cells/well. Apoptosis assays Apoptosis was induced by three different methods to investigate both the intrinsic and the extrinsic pathways17,18, using serum/growth factor deprivation or staurosporine (20nM) for the former and anti-Fas Ab for the latter. For the serum deprivation experiments, cells were maintained in 1% BSA/DMEM for the duration of the experiment. For anti-Fas-induced apoptosis, cells were pre-incubated with IFNγ100ng/mL for 16 hr to upregulate Fas expression, followed by anti-Fas Ab (10µg/mL, Clone Jo-2, BD Pharmingen, UK) for the indicated time. Apoptosis was evaluated by two methods: FACS analysis using the fluorescein isothiocyanate– conjugated annexin V(Anx-V) /propidium iodide (PI) assay 19 and acridine orange staining of pyknotic nuclei undergoing apoptosis 14. In vitro wound migration assay

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The in vitro wound assay was used to measure endothelial unidirectional migration. WT- or IC2-MCEC-SV cells were seeded at a density of 6.5x105/cm2 onto gelatincoated 10-mm dishes and allowed to form confluent monolayers. Two separate scratch wounds were generated with a rubber cell scraper (1-mm width). Images were taken at the time of the wound and at intervals up to 48 hr post wounding under phase contrast microscopy with an Olympus camera (objective x10). Migration was calculated as the distance between the migration front and the wound edge at each time point, as described

20

. For each time point, 8 measurements from 4 fields from

two independent wounds were taken.

Generation of stable ICAM-2 transfectants Full-length human ICAM-2 in pcDNA3, kindly provided by Dr. David Simmons (Oxford, UK), was used to generate ICAM-2 stable lines. Chinese hamster ovary (CHO) cells were transfected with linearised ICAM-2 plasmid DNA using Lipofectamine 2000® (Invitrogen, UK) as per manufacturer’s instructions. Surface ICAM-2 expression on each sub-clone was analysed by FACS (anti-ICAM-2 Ab, Clone B-T1, Serotec, UK) and 3 sub-clones with the highest ICAM-2 expression were selected for further experiments. Mock-transfected cells were generated and grown in parallel. CHO cells stably transfected with E selectin were generated as described 21

.

Immunofluorescence Cells were washed in PBS and fixed in freshly prepared 3% (w/v) paraformaldehyde for 20 min at room temperature, followed by 50mM NH4Cl for 10 min to neutralise paraformaldehyde. The cells were then processed for indirect immunofluorescence using the anti-human ICAM-2 monoclonal Ab (clone B-T1, 10µg/mL) followed by a goat anti-mouse Alexa Fluor 555 Ab (Molecular Probes) at 10µg/mL. All incubations were performed at room temperature for 15 min in PBS containing 3% BSA. After each incubation the cells were washed in PBS. Slides were mounted onto coverslips using VectorShield (Vector Laboratories). Cells were visualized with a Zeiss LSM510-META confocal laser-scanning microscope and images were processed using Adobe Photoshop CS (Adobe Systems Inc.)

Homophilic ICAM-2 binding assay

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CHO-IC2 cells were grown to confluence in 96-well tissue culture plates. A recombinant chimeric molecule with the ICAM-2 extracellular region fused to the Fc portion of human (h) IgG1 (ICAM-2-Fc, R&D Systems, Minneapolis, US), was preincubated with polystyrene bead conjugated with anti-hFc Ab (HBP-30-5, 1% w/v, Spherotech, USA), at 37°C for 1.5 hr. 50µL of the mixture were added to CHO-IC2 cells, to reach a final concentration of 10µg/mL of protein-Fc construct and 0.15% w/v of beads. After 1.5 hr incubation at 37°C, unbound beads were carefully removed with 6 washes in warm medium. CHO cells stably transfected with E-selectin (CHOEsel), ICAM-1-Fc and VCAM-1-Fc (R&D Systems, US) were used as control. Experiments were carried out in triplicates. Images from three representative 400X fields from each replicate were taken and bound beads counted. In indicated experiments, anti-ICAM-2 Ab (polyclonal goat anti-ICAM-2 Ab (pAb) (R&D Systems, USA), monoclonal anti-ICAM-2 Ab (mAb), CBR-IC2/2 (Research Diagnostics, USA) or goat IgG isotype control Ab (R&D system) was added to the protein-beads mixture in the final 30 min of the pre-incubation step. Rac pull-down assay The ability of Rac-GTP to bind to the p21-binding domain (PBD) of p21-activated kinase (PAK) (PAK1-PBD) was used to study Rac activation 22. To evaluate Rac activation during MCEC tube formation on Matrigel, cells extracts were prepared at 40 min and 4 hr after plating on Matrigel (see below). For the ICAM-2 cross-linking experiments, HUVEC were grown in full medium until 80-90% confluent, and subsequently maintained in 1% BSA/M199 for 16 hours. Cells were then incubated with polyclonal anti-ICAM-2 Ab (15µg/mL, pAb, R&D systems) for 40min at 37°C, followed by incubation with secondary cross-linking Ab (30µg/mL) for the indicated time. Control cells were treated with primary or secondary Ab alone. For total cell extracts, cells were washed twice with HBSS and lysed with ice-cold buffer containing phosphatase and protease inhibitors. After sonication for 30 sec and centrifugation at 14,000xg for 5 min at 4˚C, the supernatants were incubated with GST-PBD agarose beads (Upstate Biotechnology, UK) for 1 hr at 4˚C. The bound fraction was separated onto a SDS-polyacrylamide gel and the presence of Rac in the bound fraction was detected by Western blotting using primary anti-Rac Ab (clone 23A8, Upstate Biotechnology, UK). The amount of activated Rac was normalized against total Rac in the corresponding lysate. Integrated density values of the bands were measured using Alpha Innotech Chemilmager 5500 program (Alpha Innotech, CA).

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Results 1. Endothelial ICAM-2 is required for in vitro angiogenesis To determine whether ICAM-2 is involved in angiogenesis, we studied the ability of primary ICAM-2-deficient EC to form tubes in an in vitro tube formation assay on Matrigel. MCEC were isolated from mouse hearts by positive endoglin selection. The endothelial origin of MCEC was confirmed by FACS analysis of endoglin and CD31/PECAM-1 expression; the lack of ICAM-2 expression in the ICAM-2-/- cells was also confirmed (not shown). MCEC from ICAM-2 -/- or WT mice were seeded on Matrigel and images of representative fields were taken hourly. In WT MCEC samples, network formation took place between 3 and 6 hr after seeding. Tube formation was quantified after 3, 4 and 5 hr by counting branches from three representative fields per replicate. As shown in Figure 1A, ICAM-2 -/- cells showed a significantly impaired ability to form tubes on Matrigel compared to WT MCEC. A significant reduction in tube formation in the ICAM-2 -/- cells compared to WT was observed at all time points studied (Figure 1B). The difference was most marked at 3 and 4 hr, when a tubular network of interconnecting branches was well established in WT samples, whilst few contacts between cells with shorter protrusions and few branches were observed in the ICAM-2 -/- samples. These data indicate that ICAM-2 is required for endothelial tube formation on Matrigel. 2. Endothelial ICAM-2 is required for in vivo angiogenesis To validate the defect observed in vitro, we studied angiogenesis in vivo in using the Matrigel plug assay 23. Matrigel was injected subcutaneously into the abdominal area of WT and ICAM-2 -/- mice; after 7 days, the vascularised plugs were removed and processed for H&E staining to identify the area covered by vessels. As shown in Figure 2, new vessel formation within the plugs, accompanied by erythrocyte and leukocyte infiltration, was observed in plug samples from WT mice (left panel). Samples from ICAM-2 KO mice (right panel) showed significantly less vascular and cellular infiltration within the plugs. Measurement of the neo-vascularized area showed a 50% reduction in the vessel-containing area in the ICAM-2 -/- mice compared to WT mice (23.32 ± 5.99% vs 11.8 ± 0.42%; Figure 2). Therefore, both in vitro and in vivo assays demonstrated defective angiogenesis in the ICAM-2 -/- mice. 3. ICAM-2 supports homophilic interaction

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Having demonstrated that endothelial ICAM-2 is involved in angiogenesis, we investigated the underlying mechanism. The assembly and disassembly of endothelial junctions is critical in the regulation of angiogenesis. Other junctional molecules, such as CD31/PECAM and JAM-A, engage in homophilic interaction between adjacent EC 24,25. Because of its similarities to these molecules, we speculated that ICAM-2 could also engage in homophilic binding. To test this hypothesis, stable CHO cell lines expressing ICAM-2 (CHO-IC2) were generated for ligand binding studies. Surface ICAM-2 expression in these clones was verified by FACS (not shown) and immunofluorescent staining, which showed that ICAM-2 expression was mainly concentrated at the cell junctions (Figure 3A). The recombinant soluble chimeric protein ICAM-2-Fc, coupled to anti-Fc-conjugated polystyrene beads, was used as ligand. CHO cells stably expressing E-selectin (CHO-Esel) and bead-coupled recombinant chimeric molecules ICAM-1-Fc and VCAM-1-Fc were used as controls. Figure 3B shows that ICAM-2-Fc bound to CHOIC2 (top) but not CHO-Esel cells (bottom), indicating that ICAM-2 supports homophilic interaction. The interaction was specific to ICAM-2, since neither ICAM-1Fc beads (Figure 3B, middle) nor VCAM-1-Fc beads (not shown) bound to CHO-IC2 cells. Quantification of bound protein-coupled beads to CHO-IC2 or CHO-Esel is shown in Figure 3C. Only ICAM-2-Fc coupled beads, but nor ICAM-1-Fc or VCAM-1Fc coupled beads, bound to CHO-IC2 cells. No binding to CHO-Esel was detected with any of the recombinant chimeric proteins. ICAM-2-Fc also bound to ICAM-2 on resting HUVEC (data not shown). Therefore ICAM-2, like other endothelial junctional molecules, can engage in homophilic interaction. To determine whether ICAM-2 homophilic interaction was required for endothelial tube formation, we first searched for an antibody that could inhibit this interaction. Two anti-ICAM-2 antibodies were screened in the bead binding assay. ICAM-2-Fc binding to CHO-IC2 cells was specifically inhibited by a polyclonal (pAb) anti-ICAM-2 antibody but not by the monoclonal antibody (mAb) CBR/IC2-2 or control Ab (Figure 3D). Interestingly, this mAb is known to inhibit ICAM-2/LFA-1 interaction 26, suggesting that the epitope(s) involved in ICAM-2 homophilic interaction may be different from those involved in binding to integrins. We then addressed the question whether ICAM-2 homophilic interaction is required for angiogenesis, by testing the effect of the anti-ICAM-2 pAb which inhibits ICAM-2 homophilic interaction (Figure 3D) on in vitro endothelial tube formation on Matrigel. In the presence of the ICAM-2 pAb, tube formation on Matrigel was significantly inhibited by 30% compared to untreated control samples, or to control Ab (Figure 3E). These results suggest that

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ICAM-2 homophilic interaction may be involved in endothelial tube formation and angiogenesis. 4. ICAM-2 protects endothelial cells from apoptosis Apoptosis is an important regulator of angiogenesis1 and junctional adhesion molecules can provide survival signals to EC. ICAM-2 has been shown to protect T cells and ICAM-2-over-expressing fibroblasts from apoptosis27. Therefore we speculated that ICAM-2 may support angiogenesis in part by providing survival signals to EC. To test this hypothesis, the ability of WT and ICAM-2 -/- MCEC to withstand apoptotic stimulation was investigated. Apoptosis was induced in primary MCEC isolated from ICAM-2 -/- or WT mice by serum and growth factor deprivation (Figure 4A), anti-Fas Ab (Figure 4B) or staurosporine (Figure 4C). Cell death was measured by Annexin V (Anx-V)/ propidium iodide (PI) staining (Figure 4A, insert) or by acridine orange staining (Figure 4C, insert). With all stimuli used, ICAM-2 -/- cells were significantly more prone to apoptosis than WT cells. Serum and growth factor deprivation induced a significant increase in apoptosis in the ICAM-2 -/- cells after 48 hr, compared to WT (Figure 4A). A similar patter was observed when apoptosis was induced by anti-Fas antibody (Figure 4B), with a significantly increased apoptosis in the ICAM2 -/- cells after 48 hr stimulation, compared to WT cells. Staurosporineinduced apoptosis was significantly higher in the ICAM-2 -/- cells compared to WT cells at both 3 hr and 6 hr after stimulation (Figure 4c). These data indicate that the lack of ICAM-2 expression predisposes endothelial cells to apoptosis and that ICAM2 provides protection against both intrinsic and extrinsic pathways of apoptosis. The susceptibility of the ICAM-2 -/- cells to apoptosis may, at least in part, be responsible for the defect in angiogenesis observed in the ICAM-2 -/- mice. 5. ICAM-2 is not involved in endothelial proliferation Another possible mechanism by which ICAM-2 may be involved in angiogenesis is through regulation of endothelial cell proliferation. To test this hypothesis, proliferation of primary MCEC from ICAM-2 -/- or WT mice was measured in vitro using the MTT assay over 5 days. As shown in Figure 5, no difference in proliferation over 5 days was observed between ICAM-2 -/- and WT cells, suggesting that the defect in angiogenesis exhibited by the ICAM-2 -/- mice is independent of endothelial cell proliferation. 6. ICAM-2 is involved in endothelial cell migration

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Endothelial migration is a key step in angiogenesis, and involves major reorganisation of the actin cytoskeleton. ICAM-2 is linked to the actin cytoskeleton via the ezrin-radixin-moesin (ERM) family, which act as linkers between plasma membrane proteins and the cytoskeleton 28, and have been shown to regulate cell migration 29. We speculated that the absence of ICAM-2 could affect endothelial migration. In order to test this hypothesis, unidirectional migration of endothelial cells was assessed using an in vitro wound healing assay. Confluent monolayers of MCEC-SV from WT or ICAM-2 -/- mice were wounded using a thick rubber scraper. The average width of the wound was similar in WT-MCEC-SV compared to IC2MCEC-SV monolayers (mean +/- SD: 897.7 µm +/- 188.7 vs 971.4 µm +/-115 respectively, p=0.645). Pictures of the wound were taken over 48 hr. ICAM2-/MCEC-SV migrated more slowly than WT cells; the difference in migration rate was significant at 14, 18, 22.5 and 23.5 hr (Figure 6). At 40 hr however both WT and ICAM2-/- cells filled the wound area (not shown). These results indicate that ICAM-2 is involved in EC migration, and this is likely to contribute to the angiogenesis defect observed in the ICAM-2 -/- mice. 7. ICAM-2 regulates Rac activation during endothelial tube formation The defect in cell migration observed in the ICAM-2-deficient cells suggests that ICAM-2 may be required to trigger signal transduction pathways involved in migration. Rho-like GTPases are key regulators of the cytoskeletal changes required for cell migration. We therefore speculated that ICAM-2 may regulate small GTPases and mediate angiogenesis through this mechanism. Of the best characterised small GTPases, Rac was shown to regulate capillary tube formation on Matrigel 30. Therefore we tested whether ICAM-2 is required for Rac activation during tube formation on Matrigel. Rac activity in MCEC from WT or ICAM2 -/- mice seeded on Matrigel was measured by GST-PBD pull-down assay 22. As shown in Figure 7A, Rac was activated in WT cells during spreading (40 min), and the activation was sustained at 4 hr, coinciding with maximal tubular network formation. In the ICAM-2 /- cells, Rac activation at the spreading stage (40 min) was similar to WT. However ICAM-2 -/- cells failed to sustain Rac activation at 4 hr and formed significantly reduced tubular networks. These results show that ICAM-2 is required to sustain Rac activation during tube formation. To investigate further the relationship between ICAM-2 signalling and Rac activation, Rac activation in HUVEC following cross-linking of ICAM-2 with the anti-ICAM-2 pAb, which blocks ICAM-2 homophilic interaction and HUVEC tube formation on Matrigel,

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was investigated. As shown in Figure 7B, ICAM-2 cross-linking resulted in Rac activation, with a peak at 20 min post cross-linking and returned to baseline after 30 min. These results indicate that ICAM-2 homophilic interaction directly activates Rac and identify a novel pathway for Rac activation during endothelial junction formation and angiogenesis.

Discussion Endothelial junctions govern vascular homeostasis by regulating leukocyte transmigration, permeability, endothelial cell survival and proliferation. Adhesion molecules expressed at the endothelial junctions mediate contact with leukocytes and with neighbouring endothelial cells. Ligand binding, both homophilic and heterophilic, results in the activation of signal transduction pathways. Examples are CD31/PECAM, which is involved in regulating both leukocyte trafficking into tissue and endothelial survival 31, and JAM-A, which has been shown to regulate monocyte migration 32 as well as endothelial cell motility 33. Therefore it appears that leukocyte adhesion molecules play multiple roles in endothelial cells. ICAM-2 is concentrated at endothelial cell-cell contacts 3 and is known as a leukocyte adhesion molecule. In this paper we demonstrate that ICAM-2 can also regulate angiogenesis, and identify several new functions for ICAM-2 which are related to its role in angiogenesis. Using ICAM-2 -/- mice and primary endothelial cells derived from these mice, we show that lack of ICAM-2 expression on EC results in defective angiogenesis, both in vitro and in vivo. A role for ICAM-2 in angiogenesis in vivo is further supported by data from Melero et al, which showed that an anti-ICAM-2 Ab eradicated established murine colon carcinoma through mechanisms involving cytotoxic T cell activation and, to a lesser extent, inhibition of angiogenesis 34. In our study, defective angiogenesis in the ICAM-2 -/-mouse in vivo was reflected in the inability of ICAM-2 deficient endothelial cells to form tubes on Matrigel in vitro, indicating a primary endothelial defect. Several studies have shown that ICAM-2 expression on endothelial cells is concentrated at the junctions, although the exact location of ICAM-2 within junctions has not yet been determined. Adhesion between adjacent endothelial cells is mediated by homophilic interactions between junctional molecules forming a zipperlike structure 35. We therefore investigated whether ICAM-2 could support homophilic

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interaction. Using CHO transfectants stably over-expressing ICAM-2, we showed that ICAM-2 is capable of forming homophilic interaction. This is the first evidence of the presence of an endothelial ligand for ICAM-2, suggesting that ICAM-2 may contribute to the formation of junctional structures. Tube formation on Matrigel was inhibited by an anti-ICAM-2 antibody that also blocked homophilic interaction, suggesting that the homophilic interaction between ICAM-2 on opposing EC may be involved in angiogenesis. Further experiments with purified F(ab)’ fragments and recombinant ICAM-2-Fc will be required to define the role of ICAM-2 homophilic interaction in angiogenesis. Our results do not rule out the possibility that ICAM-2 may interact with other endothelial ligands, or that ICAM-2 may engage with ICAM-2 on leukocytes or platelets. Several mechanisms could be responsible for the angiogenesis defect in the ICAM-2 -/- mice. Cross-linking of ICAM-2 has been shown to protect a variety of cells, including T / B lymphocytic cell lines, primary human B cells and ICAM-2 overexpressing fibroblasts, from apoptosis 27, however no data is available on the role of ICAM-2 in endothelial apoptosis. Inhibition of EC apoptosis is an essential step in angiogenesis and the induction of apoptosis counteracts angiogenesis 36,37. In this paper we show that ICAM-2 deficient endothelial cells are more susceptible to apoptosis. The lack of ICAM-2-dependent survival signals on the ICAM-2 deficient EC could therefore contribute to the defect in angiogenesis shown here. The ICAM-2dependent anti-apoptotic effects described in other cell types were shown to be linked to the PI-3K/Akt pathway 27, which plays a critical role in regulating EC survival, cell migration and tube formation 38. Studies are underway to determine whether this pathway is involved in ICAM-2-mediated survival signals in EC. Angiogenesis involves endothelial cell spreading, migration and regulation of cell-cell and cell-matrix adhesion 39, all of which require significant reorganisation of the actin cytoskeleton. The cytoplasmic tail of ICAM-2 is linked to the actin cytoskeleton by the ezrin-radixin-moesin (ERM) family, which act as linkers between plasma membrane proteins and the cytoskeleton 28. The absence of ICAM-2 could alter ERM distribution and/or function and, as a consequence, the actin cytoskeleton itself, hence affecting cell shape change, migration and junction formation. We found no difference in ezrin distribution between ICAM-2-deficient EC and control EC (MTH and AMR, unpublished observation). However the distribution of other members of the ERM family, or indeed their activation state, was not investigated; therefore it is possible that a defect in ERM function in these cells may exist. ERM control cell adhesion and

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migration through the Rho family of small GTPases 40, which play a critical role in regulating cytoskeletal dynamics, cell migration and junction formation 41. We have previously shown that cross-linking of ICAM-2 on HUVEC does not activate Rho A 16. The small GTPase Rac has been shown to be required for tube formation on Matrigel 30

. In this study, we demonstrate that ICAM-2 cross-linking activates Rac in HUVEC,

and that endothelial ICAM-2 is required for Rac activation during tube formation. Although during spreading Rac activation was similar in ICAM-2- deficient cells and WT cells, ICAM-2-deficient cells failed to sustain Rac activation during formation of the tube network. These results indicate that ICAM-2 is required to maintain Rac activation during tube formation. The defect in Rac activation could also explain the delayed endothelial cell migration of ICAM-2 -/- cells observed in the in vitro wound healing assay, compared to WT. It is worth noting that ICAM-2 expression in the WTMCEC-SV line was low, hence other ICAM-2-independent mechanisms contributing to this phenotype cannot be ruled out. Our findings suggest that ICAM-2-dependent activation of Rac results in defective endothelial cell migration. ICAM-2 therefore may regulate angiogenesis at least in part via its ability to activate Rac at the migrating/branching stage. Rac plays multiple roles in endothelial cells, therefore the finding that ICAM-2 regulates Rac suggests that other mechanisms may also contribute to the overall defect in angiogenesis observed in the ICAM-2 deficient mice. Rac regulates endothelial junction assembly and function as well as cadherin adhesiveness 42. VECadherin itself can regulate Rac activation via the guanosine exchange factor Tiam 43

, indicating a bi-directional cross-talk between junctional signalling molecules and

small GTPases of the Rho family. Our results suggest that during the establishment of endothelial junctions Rac activation requires at least two distinct junctional signalling events, one VE-Cadherin dependent and another ICAM-2 dependent. The VE-cadherin dependent signals seem to be constitutively required, as in VE-Cadherin null cells basal levels of active Rac were found to be lower than in WT cells 44. In the ICAM-2 null cells, on the other hand, Rac activation was normal at baseline but failed to increase during tube formation, suggesting that ICAM-2 provides a secondary signal required for junction stability via Rac activation. Preliminary results suggest that this may be the case: staining for JAM-A was reduced in monolayers from ICAM2 -/- migrating cells compared to WT cells (GMB, VA and AMR, unpublished). Detailed studies of the morphology of ICAM-2 -/- cells during migration and junction formation and on the relationship between junctional pathways are in progress.

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In summary, this study demonstrates that the leukocyte adhesion molecule ICAM-2 is involved in angiogenesis. We have identified a new pathway in which ICAM-2mediated signalling activates the small GTPase Rac and regulates angiogenesis. We have described several new functions for ICAM-2, namely regulation of survival and cell migration and the ability to support homophilic interaction. This study also reveals a novel pathway for Rac activation in endothelial cells. Our results provide new insight in the regulation of endothelial homeostasis and angiogenesis by junctional adhesion molecules.

Acknowledgements We thank Prof. Britta Engelhardt (Theodor Kocher Institute, University of Bern, Switzerland) and Dr. Sussan Nourshargh (Imperial College London, UK) for access to the ICAM-2 -/- mice; Prof Yuti Chernajovsky ( Barts and The London, UK) and Dr. Charlotte Lawson (Imperial College London) for the retrovirus-expressing cell line SVU19.5; Dr Alan Entwistle and Dr Priam Villalonga (Ludwig Institute, University College London, UK) and Dr. Elaine Lidington (Imperial College London) for helpful scientific and technical advice.

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