Figure 1.
The effects of AnxA2, S100A10 and the heterotetrameric AnxA22-S100A102 complex on the formation of an in vitro capillary-like network.
Co-cultures of SMCs and GFP-expressing HUVECs were untreated (A), or treated with 15 µM AnxA2 (B), 15 µM S100A10 (C), 6 µM AnxA22-S100A102 complex (12 µM AnxA2) (D) or 100 nM PTK787 (E) at 2 h after seeding. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (F) and expressed as percentage relative to the untreated EC control (100%) (A) using the Attovision and BD Image Data Explorer programmes. Results (F) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).
Figure 2.
The effects of soluble AnxA2-DI, AnxA2-DIV and lysozyme on the formation of an in vitro capillary-like network.
Co-cultures of SMCs and GFP-expressing HUVECs were treated with 5–15 µM AnxA2-DI (B–D), AnxA2-DIV (G–I), lysozyme (L–N) at 2 h after seeding. Panels A, F and K show the corresponding controls with untreated cells. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (E, J and O) and expressed as percentage relative to the corresponding untreated EC controls (100%) (A, F and K, respectively), as described for Figure 1. Results (E, J and O) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).
Figure 3.
The effect of monoclonal AnxA2 antibodies on the formation of an in vitro capillary-like network.
Co-cultures of SMCs and GFP-expressing HUVECs were untreated (A), or treated with 4 µg/200 µl monoclonal AnxA2 antibodies (BD Biosciences) (B), (C-10; Santa Cruz), (D), Tyr23 AnxA2 specific antibodies (Santa Cruz) (F) or normal mouse IgG (H) at 2 h after seeding. After 72 h incubation, images were taken at 10× magnification. The tube total length was analysed (C, E, G and I) and expressed as percentage relative to the corresponding untreated EC control (100%) (A), as described for Figure 1. Results (C, E, G and I) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).
Figure 4.
Tyr23 phosphorylated AnxA2 is present in the ECM of confluent HUVECs.
HUVECs were incubated for 30 min in the absence (control) or presence of 50 µM PP2 before harvesting. The control fraction (ECM) was obtained in the presence of 200 µM ortho-vanadate to inhibit dephosphorylation. 100 µg of EGTA-released extracellular proteins were subjected to 10% SDS-PAGE and subsequently transferred to a nitrocellulose membrane for the detection of AnxA2 by Western blot analysis using monoclonal antibodies directed against pTyr23 AnxA2 (A) or AnxA2 (BD Biosciences) (B). Selected standards are indicated by arrowheads to the left. AnxA2 and actin (as a loading control) are indicated by an arrowhead and asterisk, respectively.
Figure 5.
The effects of AnxA2, S100A10, AnxA2-DI, or AnxA2-DIV on in vitro preformed capillary-like networks.
Co-cultures with a preformed EC network were treated with 15 µM AnxA2 (B), S100A10 (C), AnxA2-DI (D), or AnxA2-DIV (E). After 72 h incubation, images were taken at 10× magnification. The degree of disruption of the mature vascular network was quantified (F) as described for Figure 1. The tube total length is expressed as percentage relative to the untreated EC control (100%) (A). Results (F) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).
Figure 6.
AnxA2-DI and AnxA2-DIV do not inhibit the migration of GFP-expressing HUVECs through a fibrin clot in a trans-well assay.
Images of GFP-expressing HUVECs migrated to the backside of a filter with a fibrin clot on its upper side, onto which the HUVECs were seeded. Control HUVECs (A); HUVECs incubated with 100 nM PTK787 (B), 15 µM lysozyme (C), AnxA2-DI (D), or AnxA2-DIV (E), which were present in both the upper and lower chambers. Migration is expressed as percentage relative to the untreated EC control (100%) (A). Results (F) are the mean ± SEM of 3 independent experiments each. Statistical significance was determined by the two-tailed Student's t-test (*P<0.05).
Figure 7.
AnxA2-DI and AnxA2-DIV do not bind to α-actin.
∼10 µg of AnxA2-DI (∼55 µM) (lane 1), AnxA2-DIV (∼55 µM) (lane 2) and 5 µg of AnxA2 (∼6 µM) (lane 3) were separated by 15% SDS-PAGE (A and B) and transferred to a nitrocellulose membrane (B). Proteins were visualised by Coomassie Brilliant Blue staining (A). Far-Western (B); after denaturation and renaturation as described in Methods, the proteins were subjected to an actin overlay assay by incubation ON with 10 µg/ml α-actin and subsequent detection of bound actin by monoclonal actin antibodies. The positions of full-length (FL) AnxA2, and the domains I and IV of AnxA2 are indicated by arrowheads to the right. Selected standards are indicated by arrowheads to the left.
Figure 8.
AnxA2 and VE-cadherin co-localise in endosome- and filopodia-like structures in sub-confluent HUVECs grown as a monolayer.
The cells were fixed in 3% paraformaldehyde and permeabilised with 0.05% Triton X-100 in PBS before further processing for dual label immunofluorescence using antibodies directed against endogenous VE-cadherin (A) and AnxA2 (B). (C) shows the merged image. Several sites of VE-cadherin and AnxA2 co-localisation are indicated by arrowheads. Bar, 40 µm.
Figure 9.
AnxA2-DI and AnxA2-DIV disrupt in vitro preformed capillary-like networks.
Co-cultures with a preformed EC network were untreated (A, D, G and J), or treated for 3 h with 15 µM AnxA2-DI (B, E, H and K) or AnxA2-DIV (C, F, I and L). The cells were fixed in 3% paraformaldehyde and permeabilised with 0.05% Triton X-100 in PBS before processing for immunofluorescence using antibodies against endogenous AnxA2 (BD Biosciences). Note the filopodia-like structures (arrowheads) particularly in Panels K and L. Inserts in Panels G, H and I are magnified and shown in Panels J, K and L, respectively. Graphical representation (D–F) of the fluorescence intensity profiles determined for cross-sections of the cell as indicated in A–C. The orientation of the sections (from I to II) corresponds to intensity profiles from left to right in D, E and F. Bars, 100 µm (A–C) or 50 µm (G–I).