1. Endothelial intercellular adhesion molecule (ICAM)–2 regulates angiogenesis
by Miao-Tzu Huang, Justin C. Mason, Graeme M. Birdsey, Valerie Amsellem, Nicole Gerwin, Dorian O. Haskard, Anne J. Ridley, and Anna M. Randi
Blood
Volume 106(5):
September 1, 2005
©2005 by American Society of Hematology

2. ICAM-2 mediates angiogenesis in vitro: tube formation on Matrigel.
ICAM-2 mediates angiogenesis in vitro: tube formation on Matrigel. (A) Primary murine cardiac endothelial cells (MCECs) isolated from control (WT) or ICAM-2–/– mice were seeded on Matrigel. In each experiment, 6 age- and sex-matched mice/group were used. The figure shows 1 representative of 3 separate experiments, each performed in triplicate. Left panels show WT; right panels, ICAM-2–/–. In WT, cells were spreading, elongating, and making contacts with neighboring cells at 3 hours, and an interconnecting tube network was formed over 4 to 5 hours. At all time points studied, the branches and tubes formed by ICAM-2–/– cells were much less developed compared with control. Scale bar equals 100 μm. Images were acquired using a Leitz Labovert inverted microscope (Leica Microsystems, Milton Keynes, United Kingdom) fitted with a 10 × phase-contrast objective lens (Leitz-Phaco 10 ×/0.25 NA). Images were captured with a digital camera model DP50-CU (Olympus) using Viewfinder Lite (v. 1) software (Olympus). Image processing was carried out using Adobe Photoshop CS (Adobe Systems). (B) Branches from each cell were counted from 3 representative 100 × field/well. Error bars indicate mean ± SEM. **P < .01; ***P < .001 analysis of variance (ANOVA).
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

3. ICAM-2 mediates angiogenesis in vivo: Matrigel plug assay.
ICAM-2 mediates angiogenesis in vivo: Matrigel plug assay. Matrigel plugs were generated by subcutaneous injection of Matrigel; the plugs were removed 7 days later and processed for H&E staining. (A) In samples from WT mice (top), formation of vascular structures within the plugs is accompanied by erythrocyte and leukocyte infiltration. In samples from ICAM-2–/– mice (bottom) a significant reduction in vascular structures and cellular infiltration within the plugs was observed. Arrowheads show blood vessels. Scale bar equals 100 μm. Images were acquired as in Figure 1A. (B) The vascular lumen area was measured using Image ProPlus software. The average vessel-containing area was calculated from 5 representative 100 × fields of 3 representative serial sections of each plug and is shown as the percentage of area occupied by blood vessel (BV)/100 × field. Compared with WT mice, a 50% reduction in vessel-containing areas were observed in samples from ICAM-2–/– mice. Data represent 1 of 2 independent experiments; 5 to 6 mice per group were used in each experiment. Error bars indicate mean ± SEM. **P < .01, unpaired t test.
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

4. ICAM-2 supports homophilic interaction.
ICAM-2 supports homophilic interaction. (A) ICAM-2 surface expression on CHO cells stably transfected with human ICAM-2 (CHO-IC2), detected by immunofluorescence using an anti–ICAM-2 mAb (clone B-T1). ICAM-2 expression is concentrated at the cell junctions, similarly to what is observed in endothelial cells. Staining of mock-transfected CHO cells with the same antibody is also shown. (B) ICAM-2 homophilic interaction. Polystyrene beads conjugated with anti–human Fc Ab, preincubated with soluble ICAM-2–Fc, were added to CHO-IC2 (top) or control cells (CHO-Esel, bottom) in 96-well plates. ICAM-1–Fc (middle) or VCAM-1–Fc (image not shown) were used as control proteins. Images were acquired as in Figure 1A. (C) The number of beads retained in the wells after washings was counted per 400 × field of each replicate. Experiments were performed in triplicate. ICAM-2–Fc significantly bound to CHO-IC2 cells, compared with ICAM-1–Fc and VCAM-1–Fc. None of the protein-Fc constructs bound to CHO-Esel. ***P < .001, compared with control protein constructs and binding to CHO-Esel, ANOVA. n = 4. (D) Two anti–ICAM-2 Abs were tested for their ability to block ICAM-2 homophilic interaction, by preincubation with the protein-Fc-beads complex before adding to CHO-IC2. The polyclonal anti–ICAM-2 Ab (pAb), but not the monoclonal anti–ICAM-2 Ab (CBR-IC2/2) or goat IgG isotype control (Ctrl), significantly inhibited binding of ICAM-2 Fc to CHO-IC2 by 50%. ***P < .001, ANOVA. n = 3. (E) Matrigel tube formation is inhibited by anti–ICAM-2 mAb. HUVECs were preincubated with pAb anti–ICAM-2 or goat IgG control for 15 minutes before plating onto Matrigel. Photos were taken hourly after seeding, and branches counted as described (see “Materials and methods”). In the presence of anti–ICAM-2 pAb, HUVEC tube formation was inhibited by approximately 30% compared with control Ab (Ctrl Ab) or untreated samples (Ctrl) at 3, 4, and 5 hours. ***P < .001, ANOVA. n = 3. (C-E) Error bars indicate mean ± SEM.
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

5. ICAM-2 protects endothelial cells from apoptosis.
ICAM-2 protects endothelial cells from apoptosis. (A) Serum starvation-induced apoptosis. Cells were maintained in DMEM with 1% BSA for 24 or 48 hours. Apoptosis was quantified by FACS analysis using the annexin V (AnxV) and propidium iodide (PI) method (A, inset) and shown as fold increase of the AnxV (+)/PI (–) cells compared with control. (B) Anti-Fas–induced apoptosis. MCECs were treated with 100 ng/mL IFN-γ for 16 hours followed by 10 μg/mL anti-Fas Ab for 24 or 48 hours. Data are shown as fold increase of the AnxV (+)/PI (–) cells compared with control. (C) Staurosporine-induced apoptosis. MCECs were incubated with 20 nM staurosporine for 3 or 6 hours. Apoptosis was measured by acridine orange staining (C, inset) and pyknotic nuclei count. Image was acquired as in Figure 1A. Data are shown as average percentage of apoptotic cells/200 × field. Scale bar equals 20 μm. With all 3 stimuli, MCECs from ICAM-2–/– mice were significantly more susceptible to apoptosis compared with WT MCECs. *Comparison with WT cells at the same time point. †Comparison with ICAM-2–/– cells at 24 hours. *P < .05; ***P < .001; †P < 0.05, ‡P < .01, ANOVA. n = 3. (A-C) Error bars indicate mean ± SEM.
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

6. ICAM-2 does not affect endothelial cell proliferation.
ICAM-2 does not affect endothelial cell proliferation. Cell proliferation was measured over 5 days on WT and ICAM-2–/– MCECs plated onto collagen-coated 96-well tissue culture using the MTT assay. No difference in the proliferation rate of MCECs was observed in the ICAM-2–deficient cells. Data are presented as absorbance at optical density (OD) 490 nm (SEM ± SD of 3 replicate experiments).
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

7. ICAM-2 is involved in endothelial-cell migration.
ICAM-2 is involved in endothelial-cell migration. WT-MCEC-SV and IC2-MCEC-SV cells were used in a wound-healing assay. Confluent monolayers were scratch-wounded and incubated for up to 48 hours. (A) Photographs of wounded monolayers taken at 1 and 23.5 hours. The vertical lines indicate the wound edge and the migration front (dotted line). The horizontal arrows indicate the migration distance. Scale bar equals 50 μm. Images were acquired as in Figure 1A. (B) Migration distance, measured at 14, 18, 22.5, and 23.5 hours. IC2-MCEC-SVs show decreased migration at all time points, compared with WT. Values represent mean ± SD of 8 measurements. *P < .001, unpaired t test.
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology

8. ICAM-2 regulates activation of the small GTPase Rac during endothelial tube formation.
ICAM-2 regulates activation of the small GTPase Rac during endothelial tube formation. (A) Rac activation during Matrigel tube formation. MCECs plated onto Matrigel were analyzed for Rac activation at spreading (40 minutes) and tube formation stage (4 hours) by GST-PBD pull-down assays. During spreading (40 minutes), ICAM-2–/– and WT cells had comparable levels of Rac activation; however, during tube formation (4 hours), ICAM-2–/– cells were unable to sustain Rac activation. *P < .05, unpaired t test. n = 4. (B) ICAM-2 signaling induces Rac activation. HUVECs grown to 80% confluence were starved for 16 hours and incubated with goat anti–ICAM-2 Ab at 15 μg/mL (pAb) for 30 minutes, followed by cross-linking with anti–goat IgG Ab. Rac activation was measured at 10, 20, 30, and 90 minutes following cross-linking. Negative control samples were incubated with either the cross-linking Ab or primary Ab alone. ICAM-2 cross-linking induced Rac activation, which peaked at 20 minutes and returned to baseline after 30 minutes. *P < .05, unpaired t test. n = 5. (A-B) Error bars indicate mean ± SEM.
Miao-Tzu Huang et al. Blood 2005;106:
©2005 by American Society of Hematology