Figure 2
Figure 2. EGFL7 promotes EC adhesion via αvβ3 integrin. (A) HUVECs were treated with various integrin-neutralizing antibodies and subsequently seeded on EGFL7-coated dishes. Pretreatment with integrin αvβ3-blocking antibody LM609 abolished HUVEC adhesion to EGFL7 and vitronectin (Vn). Blocking antibodies against αvβ5 or α5 did not affect adhesion to EGFL7, while blocking of integrin subunit β1 decreased HUVEC adhesion to a nonsignificant extent (mean ± standard deviation, n = 4, *P < .005). Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to EGFL7 in the presence of IgG control antibody. (B) Adhesion of HUVECs to EGFL7 or vitronectin was reduced by about 80% upon specific inhibition of integrin αvβ3 with the inhibitor cRGDfV as compared with control compound cRADfV. Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to the respective adhesion protein treated with the control compound cRADfV. (C) HEK293T cells were transfected with Flag-tagged EGFL7 and plasmids encoding for αv and β3 integrin subunits. Upon lysis, EGFL7 was immunoprecipitated using an anti-Flag antibody and the immunoprecipitates were analyzed for the presence of αv or β3 integrin by western blot. (D) HUVECs were transfected with Flag-tagged EGFL7 and, upon lysis, Flag-EGFL7 or endogenous integrin αv were immunoprecipitated using specific antibodies. Subsequently, immunoprecipitates were analyzed for the presence of Flag-EGFL7 or αv integrin by western blot. (E) HUVECs were cultured in the presence or absence of recombinant EGFL7 (rEGFL7). Immune complexes were precipitated with anti-αv antibody previously shown to coimmunoprecipitate the whole integrin complex, subjected to western blotting, and probed with anti-β3 and anti-EGFL7 antibodies. (F) HUVECs were costained with anti-EGFL7 and anti-integrin αvβ3 antibodies, which revealed partial colocalization of the endogenous proteins (white stars). Bar represents 10 µM. (G) HEK293T cells were transfected with either Flag-tagged wild-type EGFL7, EGFL7 lacking the RGD motif (EGFL7 ΔRGD) or EGFL7 having the RGD motif mutated into RAD (EGFL7 RAD). The cells were cotransfected with constructs encoding for αv and β3 integrin subunits. Immune complexes were immunoprecipitated with anti-Flag antibody and blotted for αvβ3 integrin. (H) HUVEC adhesion to dishes coated with purified wild-type EGFL7 or mutant EGFL7 ΔRGD was measured. Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to wild-type EGFL7 in the presence of immunoglobulin G control antibody. Adhesion of HUVECs to EGFL7 ΔRGD was reduced by 78.6% as compared with wild-type EGFL7. The integrin αvβ3-neutralizing antibody LM609 abrogated the binding of HUVECs to wild-type but not to mutant EGFL7 (mean ± standard deviation, n = 4, *P < .005, **P < .001). IgG, immunoglobulin G; TCL, total cell lysates.

EGFL7 promotes EC adhesion via αvβ3 integrin. (A) HUVECs were treated with various integrin-neutralizing antibodies and subsequently seeded on EGFL7-coated dishes. Pretreatment with integrin αvβ3-blocking antibody LM609 abolished HUVEC adhesion to EGFL7 and vitronectin (Vn). Blocking antibodies against αvβ5 or α5 did not affect adhesion to EGFL7, while blocking of integrin subunit β1 decreased HUVEC adhesion to a nonsignificant extent (mean ± standard deviation, n = 4, *P < .005). Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to EGFL7 in the presence of IgG control antibody. (B) Adhesion of HUVECs to EGFL7 or vitronectin was reduced by about 80% upon specific inhibition of integrin αvβ3 with the inhibitor cRGDfV as compared with control compound cRADfV. Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to the respective adhesion protein treated with the control compound cRADfV. (C) HEK293T cells were transfected with Flag-tagged EGFL7 and plasmids encoding for αv and β3 integrin subunits. Upon lysis, EGFL7 was immunoprecipitated using an anti-Flag antibody and the immunoprecipitates were analyzed for the presence of αv or β3 integrin by western blot. (D) HUVECs were transfected with Flag-tagged EGFL7 and, upon lysis, Flag-EGFL7 or endogenous integrin αv were immunoprecipitated using specific antibodies. Subsequently, immunoprecipitates were analyzed for the presence of Flag-EGFL7 or αv integrin by western blot. (E) HUVECs were cultured in the presence or absence of recombinant EGFL7 (rEGFL7). Immune complexes were precipitated with anti-αv antibody previously shown to coimmunoprecipitate the whole integrin complex, subjected to western blotting, and probed with anti-β3 and anti-EGFL7 antibodies. (F) HUVECs were costained with anti-EGFL7 and anti-integrin αvβ3 antibodies, which revealed partial colocalization of the endogenous proteins (white stars). Bar represents 10 µM. (G) HEK293T cells were transfected with either Flag-tagged wild-type EGFL7, EGFL7 lacking the RGD motif (EGFL7 ΔRGD) or EGFL7 having the RGD motif mutated into RAD (EGFL7 RAD). The cells were cotransfected with constructs encoding for αv and β3 integrin subunits. Immune complexes were immunoprecipitated with anti-Flag antibody and blotted for αvβ3 integrin. (H) HUVEC adhesion to dishes coated with purified wild-type EGFL7 or mutant EGFL7 ΔRGD was measured. Adhesion to the negative control BSA was subtracted from each value and values were normalized to adhesion of HUVECs to wild-type EGFL7 in the presence of immunoglobulin G control antibody. Adhesion of HUVECs to EGFL7 ΔRGD was reduced by 78.6% as compared with wild-type EGFL7. The integrin αvβ3-neutralizing antibody LM609 abrogated the binding of HUVECs to wild-type but not to mutant EGFL7 (mean ± standard deviation, n = 4, *P < .005, **P < .001). IgG, immunoglobulin G; TCL, total cell lysates.

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