Regulation of endothelial cell motility by complexes of tetraspan molecules CD81/TAPA-1 and CD151/PETA-3 with alpha3 beta1 integrin localized at endothelial lateral junctions

Abstract

Cell-to-cell junction structures play a key role in cell growth rate control and cell polarization. In endothelial cells (EC), these structures are also involved in regulation of vascular permeability and leukocyte extravasation. To identify novel components in EC intercellular junctions, mAbs against these cells were produced and selected using a morphological screening by immunofluorescence microscopy. Two novel mAbs, LIA1/1 and VJ1/16, specifically recognized a 25-kD protein that was selectively localized at cell-cell junctions of EC, both in the primary formation of cell monolayers and when EC reorganized in the process of wound healing. This antigen corresponded to the recently cloned platelet-endothelial tetraspan antigen CD151/PETA-3 (platelet-endothelial tetraspan antigen-3), and was consistently detected at EC cell-cell contact sites. In addition to CD151/PETA-3, two other members of the tetraspan superfamily, CD9 and CD81/ TAPA-1 (target of antiproliferative antibody-1), localized at endothelial cell-to-cell junctions. Biochemical analysis demonstrated molecular associations among tetraspan molecules themselves and those of CD151/ PETA-3 and CD9 with alpha3 beta1 integrin. Interestingly, mAbs directed to both CD151/PETA-3 and CD81/ TAPA-1 as well as mAb specific for alpha3 integrin, were able to inhibit the migration of ECs in the process of wound healing. The engagement of CD151/PETA-3 and CD81/TAPA-1 inhibited the movement of individual ECs, as determined by quantitative time-lapse video microscopy studies. Furthermore, mAbs against the CD151/PETA-3 molecule diminished the rate of EC invasion into collagen gels. In addition, these mAbs were able to increase the adhesion of EC to extracellular matrix proteins. Together these results indicate that CD81/TAPA-1 and CD151/PETA-3 tetraspan molecules are components of the endothelial lateral junctions implicated in the regulation of cell motility, either directly or by modulation of the function of the associated integrin heterodimers.

Figure 1

Subcellular localization at cell-to-cell junctions of HUVEC confluent monolayers and cellular distribution of the antigen recognized by LIA1/1 and VJ1/16 mAbs. (a) HUVEC confluent monolayers were stained with mAbs LIA1/1 and VJ1/16 (C and D, respectively), and with mAbs specific to previously characterized markers of EC cell–cell junctions: TEA1/31 anti–VE-cadherin (A) and TP1/15 anti-CD31 (B). The antigen recognized by both mAbs LIA1/1 and VJ1/16 localizes selectively at cell-to-cell junctions. (b) Immunoprecipitation of a 25-kD protein by mAbs LIA1/1 and VJ1/16. HUVECs were metabolically labeled with [³⁵S]methionine and immunoprecipitated with LIA1/1 (lanes D and H),VJ1/16 (lanes C and G), and anti-CD31 TP1/15 (lanes B and F) mAbs coupled to Sepharose, or with Gly-Sepharose (lanes A and E). Immunoprecipitates were subjected to SDS-PAGE under either reducing (lanes A–D) or nonreducing (lanes E–H) conditions. Molecular mass markers are indicated. (c) Expression of LIA1/1 in ECs from different origins and states of activation. Flow cytometry analysis was performed on resting (A) or TNF-α–stimulated HUVECs (B), and HMEC-1 cells (C) stained with the LIA1/1 mAb. Negative control P3X63 mouse myeloma IgG1 is shown in dotted line. Bar, 20 μm.

Figure 1

Subcellular localization at cell-to-cell junctions of HUVEC confluent monolayers and cellular distribution of the antigen recognized by LIA1/1 and VJ1/16 mAbs. (a) HUVEC confluent monolayers were stained with mAbs LIA1/1 and VJ1/16 (C and D, respectively), and with mAbs specific to previously characterized markers of EC cell–cell junctions: TEA1/31 anti–VE-cadherin (A) and TP1/15 anti-CD31 (B). The antigen recognized by both mAbs LIA1/1 and VJ1/16 localizes selectively at cell-to-cell junctions. (b) Immunoprecipitation of a 25-kD protein by mAbs LIA1/1 and VJ1/16. HUVECs were metabolically labeled with [³⁵S]methionine and immunoprecipitated with LIA1/1 (lanes D and H),VJ1/16 (lanes C and G), and anti-CD31 TP1/15 (lanes B and F) mAbs coupled to Sepharose, or with Gly-Sepharose (lanes A and E). Immunoprecipitates were subjected to SDS-PAGE under either reducing (lanes A–D) or nonreducing (lanes E–H) conditions. Molecular mass markers are indicated. (c) Expression of LIA1/1 in ECs from different origins and states of activation. Flow cytometry analysis was performed on resting (A) or TNF-α–stimulated HUVECs (B), and HMEC-1 cells (C) stained with the LIA1/1 mAb. Negative control P3X63 mouse myeloma IgG1 is shown in dotted line. Bar, 20 μm.

Figure 1

Subcellular localization at cell-to-cell junctions of HUVEC confluent monolayers and cellular distribution of the antigen recognized by LIA1/1 and VJ1/16 mAbs. (a) HUVEC confluent monolayers were stained with mAbs LIA1/1 and VJ1/16 (C and D, respectively), and with mAbs specific to previously characterized markers of EC cell–cell junctions: TEA1/31 anti–VE-cadherin (A) and TP1/15 anti-CD31 (B). The antigen recognized by both mAbs LIA1/1 and VJ1/16 localizes selectively at cell-to-cell junctions. (b) Immunoprecipitation of a 25-kD protein by mAbs LIA1/1 and VJ1/16. HUVECs were metabolically labeled with [³⁵S]methionine and immunoprecipitated with LIA1/1 (lanes D and H),VJ1/16 (lanes C and G), and anti-CD31 TP1/15 (lanes B and F) mAbs coupled to Sepharose, or with Gly-Sepharose (lanes A and E). Immunoprecipitates were subjected to SDS-PAGE under either reducing (lanes A–D) or nonreducing (lanes E–H) conditions. Molecular mass markers are indicated. (c) Expression of LIA1/1 in ECs from different origins and states of activation. Flow cytometry analysis was performed on resting (A) or TNF-α–stimulated HUVECs (B), and HMEC-1 cells (C) stained with the LIA1/1 mAb. Negative control P3X63 mouse myeloma IgG1 is shown in dotted line. Bar, 20 μm.

Figure 2

LIA1/1 and VJ1/16 mAbs specifically recognize the CD151/PETA-3 molecule. (a) The antigen immunoprecipitated by LIA1/1 and VJ1/16 is recognized by anti–CD151/ PETA-3 mAb in Western-blot. EC lysates obtained under “stringent” detergent conditions were immunoprecipitated with LIA1/1 (lane A) and VJ1/ 16 (lane B) as well as with mAbs to other tetraspan antigens present in ECs: GR2110 anti-CD9 (lane C), and TEA3/18 anti-CD63 (lane D). Then, precipitates were subjected to SDS-PAGE, transferred and blotted with the anti–CD151/PETA-3 11B1.G4 mAb. Specific recognition is observed only with the 25 kD antigen immunoprecipitated by LIA1/1 and VJ1/16 (lanes A and B). (b) LIA1/1 and VJ1/16 specifically react with CD151/PETA-3 cDNA expressing cells. LIA1/1, VJ1/16, 11B1.G4, and 14A2.H1 anti–CD151/PETA-3 mAbs were screened on murine FDC-P1 cells infected with a retrovirus containing CD151 cDNA (FD-CD151) or empty retrovirus (FD-Ruf). All mAbs were used as 1/100 dilutions of ascites. Data correspond to the arithmetic mean of mean fluorescence intensities ± 1 SE from triplicate determinations. mAbs 1D4.5 (IgG2a) and 3D3 (IgG1) are, respectively, isotype-matched negative controls for 11B1.G4 and all other mAbs.

Figure 2

LIA1/1 and VJ1/16 mAbs specifically recognize the CD151/PETA-3 molecule. (a) The antigen immunoprecipitated by LIA1/1 and VJ1/16 is recognized by anti–CD151/ PETA-3 mAb in Western-blot. EC lysates obtained under “stringent” detergent conditions were immunoprecipitated with LIA1/1 (lane A) and VJ1/ 16 (lane B) as well as with mAbs to other tetraspan antigens present in ECs: GR2110 anti-CD9 (lane C), and TEA3/18 anti-CD63 (lane D). Then, precipitates were subjected to SDS-PAGE, transferred and blotted with the anti–CD151/PETA-3 11B1.G4 mAb. Specific recognition is observed only with the 25 kD antigen immunoprecipitated by LIA1/1 and VJ1/16 (lanes A and B). (b) LIA1/1 and VJ1/16 specifically react with CD151/PETA-3 cDNA expressing cells. LIA1/1, VJ1/16, 11B1.G4, and 14A2.H1 anti–CD151/PETA-3 mAbs were screened on murine FDC-P1 cells infected with a retrovirus containing CD151 cDNA (FD-CD151) or empty retrovirus (FD-Ruf). All mAbs were used as 1/100 dilutions of ascites. Data correspond to the arithmetic mean of mean fluorescence intensities ± 1 SE from triplicate determinations. mAbs 1D4.5 (IgG2a) and 3D3 (IgG1) are, respectively, isotype-matched negative controls for 11B1.G4 and all other mAbs.

Figure 3

Analysis of the junctional character of CD151/PETA-3 in EC monolayers. (a) Comparison by confocal microscopy of the staining in HUVEC confluent monolayers of anti–VE-cadherin and anti–CD151/PETA-3 mAbs. HUVEC confluent monolayers were double-stained with anti–VE-cadherin mAb (TEA1/31) followed by a Cy2-conjugated anti-mouse Ig secondary antibody (A–C), and with biotinylated anti–CD151/PETA-3 (VJ1/16) plus Cy3-streptavidin (D–F) and analyzed by confocal microscopy. Optical sections distanced from each other by 0.4 μm the z axis, starting at the substratum level (A and D). The fluorescence signal in both channels was almost restricted to the same optical section (B and E), indicating the vertical colocalization of both antigens. (b) CD151/PETA-3 staining in HUVEC monolayers at increasing degrees of confluence is restricted to the points of cell–cell contact and is absent from cell margins where no intercellular contact has been established. Immunofluorescence staining was performed in nonconfluent (A), and confluent (C) HUVEC monolayers. B and D show the corresponding staining with phalloidin of the same field. (c) CD151/PETA-3 redistribution during EC monolayer wound healing. Confluent monolayers were disrupted and stained with the LIA1/1 mAb, as stated in Materials and Methods. Wounded edge: (A and E) asterisk shows the position of the wound; proximal to the front (C and G). (A and C) 4 h after the lesion; (E and G) 20 h after the lesion. B, D, F, and H show the corresponding staining with phalloidin of the same field. Note that the staining of LIA1/1 disappears from the margin of the EC cell where cell–cell contact is lost within 4 h, (A) remaining at the intercellular contacts during the process of invasion of the damaged area (C and G). The normal LIA1/1 staining pattern on EC distal from the wounded area did not change during the 20 h of the experiment (not shown). Bars, 20 μm.

Figure 3

Analysis of the junctional character of CD151/PETA-3 in EC monolayers. (a) Comparison by confocal microscopy of the staining in HUVEC confluent monolayers of anti–VE-cadherin and anti–CD151/PETA-3 mAbs. HUVEC confluent monolayers were double-stained with anti–VE-cadherin mAb (TEA1/31) followed by a Cy2-conjugated anti-mouse Ig secondary antibody (A–C), and with biotinylated anti–CD151/PETA-3 (VJ1/16) plus Cy3-streptavidin (D–F) and analyzed by confocal microscopy. Optical sections distanced from each other by 0.4 μm the z axis, starting at the substratum level (A and D). The fluorescence signal in both channels was almost restricted to the same optical section (B and E), indicating the vertical colocalization of both antigens. (b) CD151/PETA-3 staining in HUVEC monolayers at increasing degrees of confluence is restricted to the points of cell–cell contact and is absent from cell margins where no intercellular contact has been established. Immunofluorescence staining was performed in nonconfluent (A), and confluent (C) HUVEC monolayers. B and D show the corresponding staining with phalloidin of the same field. (c) CD151/PETA-3 redistribution during EC monolayer wound healing. Confluent monolayers were disrupted and stained with the LIA1/1 mAb, as stated in Materials and Methods. Wounded edge: (A and E) asterisk shows the position of the wound; proximal to the front (C and G). (A and C) 4 h after the lesion; (E and G) 20 h after the lesion. B, D, F, and H show the corresponding staining with phalloidin of the same field. Note that the staining of LIA1/1 disappears from the margin of the EC cell where cell–cell contact is lost within 4 h, (A) remaining at the intercellular contacts during the process of invasion of the damaged area (C and G). The normal LIA1/1 staining pattern on EC distal from the wounded area did not change during the 20 h of the experiment (not shown). Bars, 20 μm.

Figure 3

Analysis of the junctional character of CD151/PETA-3 in EC monolayers. (a) Comparison by confocal microscopy of the staining in HUVEC confluent monolayers of anti–VE-cadherin and anti–CD151/PETA-3 mAbs. HUVEC confluent monolayers were double-stained with anti–VE-cadherin mAb (TEA1/31) followed by a Cy2-conjugated anti-mouse Ig secondary antibody (A–C), and with biotinylated anti–CD151/PETA-3 (VJ1/16) plus Cy3-streptavidin (D–F) and analyzed by confocal microscopy. Optical sections distanced from each other by 0.4 μm the z axis, starting at the substratum level (A and D). The fluorescence signal in both channels was almost restricted to the same optical section (B and E), indicating the vertical colocalization of both antigens. (b) CD151/PETA-3 staining in HUVEC monolayers at increasing degrees of confluence is restricted to the points of cell–cell contact and is absent from cell margins where no intercellular contact has been established. Immunofluorescence staining was performed in nonconfluent (A), and confluent (C) HUVEC monolayers. B and D show the corresponding staining with phalloidin of the same field. (c) CD151/PETA-3 redistribution during EC monolayer wound healing. Confluent monolayers were disrupted and stained with the LIA1/1 mAb, as stated in Materials and Methods. Wounded edge: (A and E) asterisk shows the position of the wound; proximal to the front (C and G). (A and C) 4 h after the lesion; (E and G) 20 h after the lesion. B, D, F, and H show the corresponding staining with phalloidin of the same field. Note that the staining of LIA1/1 disappears from the margin of the EC cell where cell–cell contact is lost within 4 h, (A) remaining at the intercellular contacts during the process of invasion of the damaged area (C and G). The normal LIA1/1 staining pattern on EC distal from the wounded area did not change during the 20 h of the experiment (not shown). Bars, 20 μm.

Figure 4

Subcellular localization and association of different tetraspan proteins in ECs. (a) Immunofluorescence staining was performed on HUVEC confluent monolayers with GR2110 anti-CD9 (A), 5A6 anti–CD81/TAPA-1 (B), and TEA3/18 anti-CD63 (C), showing the cell–cell junctional pattern of expression of CD9, CD81/TAPA-1 and the staining of lysosomes and Weibel-Palade bodies of CD63. (b) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with 5A6 anti–CD81/ TAPA-1 (lane A), or with LIA1/1 and VJ1/16 anti–CD151/PETA-3 (lanes B and C, respectively), GR2110 anti-CD9 (lane D) and TEA3/18 anti-CD63 (lane E) coupled to Sepharose. Precipitates were subjected to SDS-PAGE and immunoblots revealed with 11B1.G4 anti–CD151/PETA-3, or 50H.19 anti-CD9 mAbs. Coprecipitation of CD81/TAPA-1, CD9 and CD151/PETA-3 was observed using both anti-CD9 and anti–CD151/PETA-3 mAbs. CD63, although expressed by ECs, did neither localize at cell-to-cell junctions nor associate to CD9 or CD151/PETA-3 in these cells. Bar, 20 μm.

Figure 4

Subcellular localization and association of different tetraspan proteins in ECs. (a) Immunofluorescence staining was performed on HUVEC confluent monolayers with GR2110 anti-CD9 (A), 5A6 anti–CD81/TAPA-1 (B), and TEA3/18 anti-CD63 (C), showing the cell–cell junctional pattern of expression of CD9, CD81/TAPA-1 and the staining of lysosomes and Weibel-Palade bodies of CD63. (b) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with 5A6 anti–CD81/ TAPA-1 (lane A), or with LIA1/1 and VJ1/16 anti–CD151/PETA-3 (lanes B and C, respectively), GR2110 anti-CD9 (lane D) and TEA3/18 anti-CD63 (lane E) coupled to Sepharose. Precipitates were subjected to SDS-PAGE and immunoblots revealed with 11B1.G4 anti–CD151/PETA-3, or 50H.19 anti-CD9 mAbs. Coprecipitation of CD81/TAPA-1, CD9 and CD151/PETA-3 was observed using both anti-CD9 and anti–CD151/PETA-3 mAbs. CD63, although expressed by ECs, did neither localize at cell-to-cell junctions nor associate to CD9 or CD151/PETA-3 in these cells. Bar, 20 μm.

Figure 5

Association of TM4 proteins to α3β1 integrin, which is also localized to lateral cell junctions from ECs. (a) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with TS2/16 anti-β1 integrin chain mAb (lane A) and with rabbit polyclonal antibodies against αv, α5, α3, and α2 integrin chains (lanes B–E, respectively). Precipitates were subjected to SDS-PAGE and immunoblots revealed with biotinylated TS2/ 16 anti-β1 integrin, 11B1.G4 anti–CD151/PETA-3, or 50H.19 anti-CD9 mAbs. (b) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with 5A6 anti-CD81 (lane A), LIA1/1 anti–CD151/ PETA-3 (lane B), GR2110 anti-CD9 (lane C) and TEA3/18 anti-CD63 (lane D) mAbs coupled to Sepharose and revealed with anti-α3 integrin polyclonal Ab. EC lysates were also precipitated with VJ1/16 and LIA1/1 anti–CD151/PETA-3 (lanes E and F, respectively), GR2110 anti-CD9 (lane G) and TEA3/18 anti-CD63 (lane H) and blotted with biotinylated TS2/16 anti-β1 integrin mAb. Coprecipitation of α3β1 integrin with CD9 and CD151/PETA-3 is evident in both tetraspan and integrin immunoprecipitates, whereas CD81/TAPA-1 and CD63 do not directly associate to this integrin. (c) Immunofluorescence staining was performed on HUVEC confluent monolayers with rabbit polyclonal antibodies against integrin chains β1 (A), α2 (B), α5 (C), or with the anti-α3 P1B5 (D) and anti-αv ABA-6D1 mAbs (E). F shows the same optical section of a sample doubled stained with P1B5 anti-α3 (green) and LIA1/1 anti–CD151/PETA-3 (red) mAbs. This section showed the maximal staining intensity of both antigens. Bars, 20 μm.

Figure 5

Association of TM4 proteins to α3β1 integrin, which is also localized to lateral cell junctions from ECs. (a) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with TS2/16 anti-β1 integrin chain mAb (lane A) and with rabbit polyclonal antibodies against αv, α5, α3, and α2 integrin chains (lanes B–E, respectively). Precipitates were subjected to SDS-PAGE and immunoblots revealed with biotinylated TS2/ 16 anti-β1 integrin, 11B1.G4 anti–CD151/PETA-3, or 50H.19 anti-CD9 mAbs. (b) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with 5A6 anti-CD81 (lane A), LIA1/1 anti–CD151/ PETA-3 (lane B), GR2110 anti-CD9 (lane C) and TEA3/18 anti-CD63 (lane D) mAbs coupled to Sepharose and revealed with anti-α3 integrin polyclonal Ab. EC lysates were also precipitated with VJ1/16 and LIA1/1 anti–CD151/PETA-3 (lanes E and F, respectively), GR2110 anti-CD9 (lane G) and TEA3/18 anti-CD63 (lane H) and blotted with biotinylated TS2/16 anti-β1 integrin mAb. Coprecipitation of α3β1 integrin with CD9 and CD151/PETA-3 is evident in both tetraspan and integrin immunoprecipitates, whereas CD81/TAPA-1 and CD63 do not directly associate to this integrin. (c) Immunofluorescence staining was performed on HUVEC confluent monolayers with rabbit polyclonal antibodies against integrin chains β1 (A), α2 (B), α5 (C), or with the anti-α3 P1B5 (D) and anti-αv ABA-6D1 mAbs (E). F shows the same optical section of a sample doubled stained with P1B5 anti-α3 (green) and LIA1/1 anti–CD151/PETA-3 (red) mAbs. This section showed the maximal staining intensity of both antigens. Bars, 20 μm.

Figure 5

Association of TM4 proteins to α3β1 integrin, which is also localized to lateral cell junctions from ECs. (a) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with TS2/16 anti-β1 integrin chain mAb (lane A) and with rabbit polyclonal antibodies against αv, α5, α3, and α2 integrin chains (lanes B–E, respectively). Precipitates were subjected to SDS-PAGE and immunoblots revealed with biotinylated TS2/ 16 anti-β1 integrin, 11B1.G4 anti–CD151/PETA-3, or 50H.19 anti-CD9 mAbs. (b) EC lysates obtained under “mild” detergent conditions were immunoprecipitated with 5A6 anti-CD81 (lane A), LIA1/1 anti–CD151/ PETA-3 (lane B), GR2110 anti-CD9 (lane C) and TEA3/18 anti-CD63 (lane D) mAbs coupled to Sepharose and revealed with anti-α3 integrin polyclonal Ab. EC lysates were also precipitated with VJ1/16 and LIA1/1 anti–CD151/PETA-3 (lanes E and F, respectively), GR2110 anti-CD9 (lane G) and TEA3/18 anti-CD63 (lane H) and blotted with biotinylated TS2/16 anti-β1 integrin mAb. Coprecipitation of α3β1 integrin with CD9 and CD151/PETA-3 is evident in both tetraspan and integrin immunoprecipitates, whereas CD81/TAPA-1 and CD63 do not directly associate to this integrin. (c) Immunofluorescence staining was performed on HUVEC confluent monolayers with rabbit polyclonal antibodies against integrin chains β1 (A), α2 (B), α5 (C), or with the anti-α3 P1B5 (D) and anti-αv ABA-6D1 mAbs (E). F shows the same optical section of a sample doubled stained with P1B5 anti-α3 (green) and LIA1/1 anti–CD151/PETA-3 (red) mAbs. This section showed the maximal staining intensity of both antigens. Bars, 20 μm.

Figure 6

Effects of anti-TM4 mAbs on EC migration. (a) Confluent EC monolayers treated with 20 μg/ml of different purified mAbs, or a 1/50 dilution of ascitis fluid of P1B5 anti-α3 integrin mAb, were scrapped and migration of the front of the wound was followed for 28 h. A shows the migration of ECs treated with anti– VE-cadherin TEA1/31, anti-α3 integrin P1B5, and anti-β1 integrin TS2/16 mAb. In panel B, cells were treated with 5A6 anti–CD81/TAPA-1. In panel C cells were preincubated with mAbs anti– CD151/PETA-3 LIA1/1 and VJ1/16. Results correspond to the arithmetic mean ± 1 SE of two experiments performed by duplicate. (b) Antibodies to TM4 molecules reduce EC motility. Videorecords of endothelial cells were generated as described under Materials and Methods in the presence of the following monoclonal antibodies: (A) TEA1/31 anti–VE-cadherin; (B) TS2/16 proactivatory anti-β1 integrin; (C) 5A6 anti–CD81/TAPA-1; and (D) LIA1/1 anti–CD151/PETA-3. The tracks of random migration of six individual cells were followed in each condition. The end point of each cell track are indicated by a filled circle.

Figure 6

Effects of anti-TM4 mAbs on EC migration. (a) Confluent EC monolayers treated with 20 μg/ml of different purified mAbs, or a 1/50 dilution of ascitis fluid of P1B5 anti-α3 integrin mAb, were scrapped and migration of the front of the wound was followed for 28 h. A shows the migration of ECs treated with anti– VE-cadherin TEA1/31, anti-α3 integrin P1B5, and anti-β1 integrin TS2/16 mAb. In panel B, cells were treated with 5A6 anti–CD81/TAPA-1. In panel C cells were preincubated with mAbs anti– CD151/PETA-3 LIA1/1 and VJ1/16. Results correspond to the arithmetic mean ± 1 SE of two experiments performed by duplicate. (b) Antibodies to TM4 molecules reduce EC motility. Videorecords of endothelial cells were generated as described under Materials and Methods in the presence of the following monoclonal antibodies: (A) TEA1/31 anti–VE-cadherin; (B) TS2/16 proactivatory anti-β1 integrin; (C) 5A6 anti–CD81/TAPA-1; and (D) LIA1/1 anti–CD151/PETA-3. The tracks of random migration of six individual cells were followed in each condition. The end point of each cell track are indicated by a filled circle.

Figure 7

Effect of anti-TM4 mAbs on invasion of collagen gels by PMA-treated HUVECs. HUVECs were seeded on the surface of collagen gels, in the presence or not of 20 ng/ml PMA alone (control) or in combination with 20μg/ml of anti–HLA-A,B W6/ 32, anti–VE-Cadherin TEA1/31, anti-β1 integrin TS2/16, anti– CD81/TAPA-1 5A6, or anti–CD151/PETA-3 LIA1/1 and VJ1/16 mAbs for 24 h. (a) Phase-contrast micrographs of migrating cells invading the collagen gels forming tube-like structures. (A) Unstimulated cells; (B) cells treated for 24 h with PMA alone; (C and D) cells treated for 24 h with PMA plus TEA1/31 or LIA1/1, respectively. (b ) Quantitation of migrating cells under the different conditions respect to control cells in the presence of PMA (100%). Experiments were performed by duplicate and represented as the arithmetic mean of the number of migrating cells ± 1 SD of four fields per well from two different experiments.

Figure 7

Effect of anti-TM4 mAbs on invasion of collagen gels by PMA-treated HUVECs. HUVECs were seeded on the surface of collagen gels, in the presence or not of 20 ng/ml PMA alone (control) or in combination with 20μg/ml of anti–HLA-A,B W6/ 32, anti–VE-Cadherin TEA1/31, anti-β1 integrin TS2/16, anti– CD81/TAPA-1 5A6, or anti–CD151/PETA-3 LIA1/1 and VJ1/16 mAbs for 24 h. (a) Phase-contrast micrographs of migrating cells invading the collagen gels forming tube-like structures. (A) Unstimulated cells; (B) cells treated for 24 h with PMA alone; (C and D) cells treated for 24 h with PMA plus TEA1/31 or LIA1/1, respectively. (b ) Quantitation of migrating cells under the different conditions respect to control cells in the presence of PMA (100%). Experiments were performed by duplicate and represented as the arithmetic mean of the number of migrating cells ± 1 SD of four fields per well from two different experiments.

Figure 8

Effect of anti-TM4 mAbs in EC adhesion to different extracellular matrix proteins. Cells were incubated on the coated plates for 15 or 30 min either in the presence or not of 10 μg/ml of the following purified mAbs: anti–HLA-A,B W6/32, anti–CD151/PETA-3 LIA1/1, and VJ1/16, anti–CD81/ TAPA-1 5A6, anti-β1 activatory TS2/16, and anti-β1 blocking VJ1/14. Anti-α3 mAb P1B5 was used as ascitis fluid diluted 1:100. Experiments were performed by triplicate. Data correspond to the arithmetic mean of the percent of cell adhesion ± 1 SD. Levels of basal adhesion were 39.26% ± 1.82 for fibronectin, 33.53% ± 2.85 for collagen, and 25.68% ± 1.55 for laminin. Asterisks indicate significant difference (P < 0.01) compared to basal cell adhesion (Wilcoxon sum rank test).