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First published online 18 May 2004
doi: 10.1242/jcs.01138

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Involvement of the VEGF receptor 3 in tubular morphogenesis demonstrated with a human anti-human VEGFR-3 monoclonal antibody that antagonizes receptor activation by VEGF-C

¹ ImClone Systems Incorporated, New York, NY 10014, USA
² Department of Morphology, University Medical Center, Geneva, Switzerland

View larger version (22K): [in a new window]   Fig. 1. In vitro characterization of soluble human VEGFR-3 and mAb hF4-hF4-3C5. (A) Conditioned media from cells expressing human sR3-AP (), human sR2-AP () or human sR1-AP () were normalized for AP activity and added to 96-well plates coated with VEGF-CNC. Bound receptors were detected with a rabbit antibody to AP and peroxidase-labeled secondary antibodies. sR3-AP binds to VEGF-CNC with an EC50 about ten times lower than sR2-AP whereas sR1-AP does not show binding activity. (B) Conditioned media were normalized for AP activity and added to 96-well plates coated with either VEGF-CNC or VEGF-A. The amount of bound receptor was measured using AP activity. R3-AP (clear bars) binds strongly to VEGF-CNC but not to VEGF-A. Control media from cells expressing untagged AP shows no binding (black bars). (C) Purified sR3-AP (µ), sR2-AP () or sR1-AP () were added to 96-well plates coated with hF4-3C5. The amount of bound receptor was measured using AP activity. sR3-AP binds to the mAb hF4-3C5 whereas sR1-AP or sR2-AP show no detectable binding. (D) Inhibition of sR3-AP binding to VEGF-CNC by the mAb hF4-3C5. Saturating amounts of sR3-AP were mixed with various amounts of mAbs or Fab fragments and added to 96-well plates coated with VEGF-CNC. Blocking of the receptor binding is evident with full-length hF4-3C5 (, IC50=1.3 nM), and Fab fragments of hF4-3C5 (, IC50=2 nM) but not with the irrelevant human mAb IMC-2F8 ().

View larger version (26K): [in a new window]   Fig. 2. Inhibition of VEGFR-3 function by the mAb hF4-hF4-3C5. (A) Expression of chimeric VEGFR-3-cFms receptor by NIH-3T3 cells (clone 3.1.2, lines 2 and 4) or control NIH 3T3 cells (lanes 1 and 3) was determined by western blotting with rabbit antibodies to cFms (lanes 1 and 2) or to the extracellular domain of VEGFR-3 (lanes 3 and 4). Lysates in lanes 2 and 4 contain a single polypeptide that is recognized by the antibody to the C-terminus of cFms and an antibody to the extracellular domain of VEGFR-3. (B) Clone 3.1.2 cells were analyzed by FACS with either mAb hF4-3C5 or the control mAb 2F8. The plasma membrane expression of VEGFR-3-cFms is evident. (C) Stimulation of mitogenic response of the clone 3.1.2 cells with VEGF-CNC results in dose-dependent [3H] thymidine uptake with an EC50 value of 3.3 ng/ml. (D) Clone 3.1.2 cells were stimulated with 80 ng/ml VEGF-CNC in the presence of variable amounts of mAb hF4-3C5 or the control antibody 2F8. The maximum inhibition of the response is seen between 10 and 15 nM hF4-3C5 with an IC50 of 5 nM (750 ng/ml). mAb 2F8 had no effect on [3H] thymidine uptake.

View larger version (20K): [in a new window]   Fig. 3. Expression of vascular and lymphatic endothelial markers by BAE cells and LECs. (A) BAE cells and LECs were analyzed by FACS for the expression of VEGFR-3, VEGFR-2 and Prox-1. The gating was set using cells stained with isotype-matched human monoclonal antibodies (for VEGFR-2 and VEGFR-3) and a rabbit antibody to GFP (for Prox-1). LECs were strongly positive for both VEGFR-3 and VEGFR-2. About half of the BAE cells expressed significant surface VEGFR-3 as shown by staining with hF4-3C5, whereas the entire BAE cell population was positive for VEGFR-2. Staining of BAE cells for Prox-1 resulted in a minor shift relative to control antibody (RFI=5) whereas LECs were strongly positive for this marker (RFI=32). (B) cDNA obtained from BAE cells and LECs was analyzed by PCR for expression of lymphatic markers. Primers used in this analysis were located in regions of sequence identity in bovine and human sequences. LECs strongly express the lymphatic markers podoplanin (lane 4) and Prox-1 (lane 5). No expression of podoplanin (lane 1) or Prox-1 (lane 2) is detected in the cDNA from BAE cells. The control sequence G3PDH was amplified from both BAE cell and LEC cDNA. The lower panel shows the result of identical PCR reactions using cDNA prepared in the absence of reverse transcriptase. The predicted lengths of the amplified fragments are: podoplanin, 357 bp; Prox-1, 163 bp; and G3PDH, 261 bp. Together, the results of A and B demonstrate that the VEGFR-3-positive population of BAE cells is not lymphatic in origin.

View larger version (11K): [in a new window]   Fig. 4. Partial inhibition of VEGF-CNC-induced in vitro angiogenesis by blocking antibody to VEGFR-3. Confluent BAE cells monolayers on three-dimensional collagen gels were treated with VEGF-CNC (100 ng/ml). Monoclonal anti-human VEGFR-3 antibody hF4-3C5 was added at the concentrations indicated. Invasion was measured after 4 days and is expressed as percent of sprouting induced by VEGF-CNC alone. Results are shown as the means±s.d. of three wells.

View larger version (130K): [in a new window]   Fig. 5. Inhibition of VEGF-CNC-induced BAE cell in vitro angiogenesis by blocking VEGFR-2 and VEGFR-3 simultaneously. Confluent BAE cell monolayers on three-dimensional collagen gels (Control) were treated for 4 days with VEGF-CNC (100 ng/ml) alone or in combination with a neutralizing anti-human VEGFR-3 monoclonal antibody (hF4-3C5, 5 µg/ml), a neutralizing anti-human VEGFR-2 monoclonal antibody (IMC-1C11, 20 µg/ml) or a control antibody (anti-KHL, 20 µg/ml). Cell cord formation within the collagen gel was viewed by phase-contrast microscopy. Co-addition of the neutralizing anti-VEGFR-3 or anti-VEGFR-2 antibodies alone partially inhibited invasion induced by VEGF-CNC, whereas co-addition of both antibodies completely blocked invasion induced by VEGF-CNC. Cells treated with IMC-1C11 or hF4-3C5 alone showed no signs of cytotoxicity. No invasion occurred in untreated cultures. Bar, 100 µm.

View larger version (14K): [in a new window]   Fig. 6. Quantitation of anti-VEGFR-3 and anti-VEGFR-2 antibody-mediated inhibition of in vitro angiogenesis in BAE cells. Confluent BAE cell monolayers on three-dimensional collagen gels were treated with VEGF-CNC (100 ng/ml) (A) or VEGF165 (100 ng/ml) (B). Anti-human VEGFR-3 antibody (hF4-3C5), anti-human VEGFR-2 monoclonal antibody (IMC-1C11) and control antibodies (anti-KLH for hF4-3C5, IMC-C225 for IMC-1C11) were added. Invasion was measured after 4 days and is expressed as percent of sprouting induced by cytokine alone. (A) hF4-3C5 and IMC-1C11 inhibited VEGF-CNC-induced BAE cell invasion by 68% at 5 µg/ml and 66% at 20 µg/ml, respectively. A complete inhibition of VEGF-CNC-induced BAE cell invasion was obtained by the co-addition of both antibodies, whereas the control antibody (KLH) had no effect on invasion at 25 µg/ml. (B) When added to VEGF-A-treated BAE cells, hF4-3C5 antibody had no effect at 5 µg/ml, whereas IMC-1C11 at 20 µg/ml totally blocked invasion. The isotope control antibody for IMC-C225 had no effect on invasion. Results are shown as the means±s.e.m. from at least three experiments per condition.

View larger version (30K): [in a new window]   Fig. 7. Inhibition of HUVEC in vitro angiogenesis by anti-VEGFR-3 and anti-VEGFR-2 antibodies. (A) HUVECs were analyzed by FACS for expression of surface VEGFR-2 and VEGFR-3 using monoclonal antibodies IMC-1121 and hF4-3C5. Both receptors were detected, with approximately equal staining intensity. (B) HUVECs were grown for 24 hours on a solid gel of basement matrix proteins (Chemicon In Vitro Angiogenesis Kit) in the presence or absence of monoclonal antibodies. The images were captured using a 4x objective of a Nikon TE2000 inverted microscope. Blocking of tube formation is evident with either mAb IMC-1C11 or hF4-3C5. Simultaneous addition of both antibodies increases the inhibitory effect. Bar, 100 µM. (C) Total sprouting response was measured in replicate wells using digital imaging (see Materials and Methods). Branching was counted manually as the number of tube branch points per field. The results are shown as percent inhibition relative to the average of untreated controls. The extent of tube-forming inhibition is the same whether total sprouting or branching is measured.

View larger version (16K): [in a new window]   Fig. 8. Effect of VEGFR-3 inhibition on the transmigration of BAE cells in response to VEGF-CNC. BAE cells were induced to transmigrate through 8 µm polycarbonate filters by the addition of 5 ng/ml ligand to the bottom well; transmigrating cells were stained with Hoechst stain, imaged under epifluorescence using a 20x lens and counted using automatic image analysis (see Materials and Methods). (A) Chemotaxis towards VEGF-CNC is inhibited in a dose-dependent manner by mAb hF4-3C5 () whereas mAb IMC-1C11 () has no effect. Combination of the two antibodies showed no additive effect (). Results are shown as the means±s.e.m. (Inserts) Examples of transmigrated cells stained by Hoechst and used in automated cell counting; (A1) no antibody; (A2) 100 nM each of hF4-3C5 and IMC-1C11. (B) Saturating amounts of VEGFR-2-AP were mixed with various amounts of mAbs and added to 96-well plates coated with VEGF-CNC. Bound receptor was measured using AP activity. Blocking of the receptor binding is evident with the mAb IMC-1C11 () but not with the irrelevant mAb IMC-C225 ().

View larger version (23K): [in a new window]   Fig. 9. The VEGFR-3 (+) subpopulation of BAE cells transmigrates more efficiently in response to VEGF-CNC. BAE cells were induced to transmigrate as described in Fig. 8. Non-migrating cells were removed from the upper aspect of the membrane by scraping and the transmigrated cells were released from the lower surface with protease-free cell dissociation buffer. Expression of VEGFR-3 was assayed by FACS as described in Fig. 3, and the RFI of each peak is shown in yellow. Each panel shows the staining in the absence (green) and presence (blue) of hF4-3C5. The cells that did not migrate in response to VEGF-CNC (non-migrated) exhibit the bimodal pattern of VEGFR-3 expression with about 50% of positive cells. The BAE cells that transmigrated in response to VEGF-CNC (Migrated) are approximately 90% positive for VEGFR-3. This shift is reversed seen when the BAE cells transmigrate in response to VEGF165 with the majority of cells being negative for VEGFR-3.