Single and combined effects of alphavbeta3- and alpha5beta1-integrins on capillary tube formation in a human fibrinous matrix

Abstract

The fibrinous exudate of a wound or tumor stroma facilitates angiogenesis. We studied the involvement of RGD-binding integrins during tube formation in human plasma-derived fibrin clots and human purified fibrin matrices. Capillary-like tube formation by human microvascular endothelial cells in a 3D plasma-derived fibrinous matrix was induced by FGF-2 and TNF-alpha and depended largely on cell-bound u-PA and plasmin activities. While tube formation was minimally affected by the addition of either the alphavbeta3-integrin inhibiting mAb LM609 or the alpha5-integrin inhibiting mAb IIA1, the general RGD-antagonist echistatin completely inhibited this process. Remarkably, when alphavbeta3- and alpha5beta1-integrins were inhibited simultaneously, tube formation was reduced by 78%. It was accompanied by a 44% reduction of u-PA antigen accumulation and 41% less production of fibrin degradation products. alphavbeta5-integrin-blocking antibodies further enhanced the inhibition by mAb LM609 and mAb IIA1 to 94%, but had no effect by themselves. alphav-specific cRGD only inhibited angiogenesis when alpha5beta1-integrin was simultaneously blocked. Endostatin mimicked the effect of alpha5beta1-integrin and inhibited tube formation only in the presence of LM609 or cRGD (73 and 80%, respectively). Comparable results were obtained when purified fibrin matrices were used instead of the plasma-derived fibrinous matrices. These data show that blocking of tube formation in a fibrinous exudate requires the simultaneous inhibition of alphavbeta3- and alpha5beta1-integrins. This may bear impact on attempts to influence angiogenesis in a fibrinous environment.

Fig. 1

Localization of RGD-binding integrins in endothelial cells in vivo. Immunohistochemistry was performed on paraffin sections of a recanalized mural thrombus formed in a human coronary artery as described in Materials and Methods. a Phloxin staining; b Immunostaining of endothelial cells by CD31; c Immunostaining of αvβ3-integrin by mAb LM609; d Immunostaining of αvβ5-integrin by rabbit anti-human β5-integrin antibody; e Immunostaining of α5β1-integrin by rabbit anti-human α5-integrin antibody

Fig. 2

Blocking of αvβ3- and α5β1-integrins results in a combined effect on the inhibition of capillary-like tube formation in plasma-derived fibrinous matrices. HMVECs cultured on a three-dimensional fibrinous plasma clot were not stimulated (control) or stimulated with FGF-2/TNF-α in the absence or presence of the αvβ3-blocking mAb LM609 (10 μg/ml), the αvβ5-blocking mAb P1F6 (10 μg/ml), the α5-blocking mAb IIA1 (2 μg/ml) or the combinations of these mAbs. After 6 days of culturing, the mean tube length of duplicate wells was quantified (mm/cm²) as described (a). The data represent mean % ± SEM of FGF-2/TNF-α stimulated tube formation of 3–7 independent experiments. The mean tube length of FGF-2/TNF-α in the plasma-derived fibrinous matrix was 70 ± 20 mm/cm². b After 6 days of culturing, non-phase photomicrographs were taken of hMVECs stimulated with FGF-2/TNF-α in the absence or presence of LM609, IIA1 or the combination of these mAbs in the concentrations described above. Bar represents 300 μm. * P < 0.01 as compared to 100% (=FGF-2/TNF-α stimulated condition)

Fig. 3

Combined effect of endostatin, LM609, and/or cRGD on capillary-like tube formation in fibrinous plasma clots. HMVECs cultured on a three-dimensional plasma clot were not stimulated (control) or stimulated with FGF-2/TNF-α in the presence of either endostatin (10 μg/ml), the αvβ3-blocking mAb LM609 (10 μg/ml), the α5-blocking mAb IIA1 (2 μg/ml) or cRGD (50 μg/ml), a combination of endostatin and LM609, endostatin and IIA1, cRGD and endostatin, or IIA1 and cRGD. After 6 days of culturing, the mean tube length was quantified (mm/cm²) as described. The data represent mean % ± SEM of FGF-2/TNF-α stimulated tube formation of 3–5 independent experiments. The mean tube length of FGF-2/TNF-α was 98 ± 15 mm/cm². * P < 0.05, ** P < 0.01 as compared to 100% (=FGF-2/TNF-α stimulated condition)

Fig. 4

Blocking of αvβ3- and α5β1-integrins results in a combined effect on the inhibition of capillary-like tube formation in purified fibrin matrices. HMVECs cultured on a three-dimensional fibrin matrix and were not stimulated (control) or stimulated with FGF-2/TNF-α in the absence or presence of the αvβ3-blocking mAb LM609 (10 μg/ml), the αvβ5-blocking mAb P1F6 (10 μg/ml), the α5-blocking mAb IIA1 (2 μg/ml) or the combinations of these mAbs. After 6 days of culturing, the mean tube length of duplicate wells was quantified (mm/cm²) as described (a). The data represent mean % ± SEM of FGF-2/TNF-α stimulated tube formation of 3–7 independent experiments. The mean tube length of FGF-2/TNF-α in the purified fibrin matrix was 83 ± 4 mm/cm². b After 6 days of culturing, non-phase photomicrographs were taken of hMVECs stimulated with FGF-2/TNF-α in the absence or presence of LM609, IIA1 or the combination of these mAbs in the concentrations described above. Bar represents 300 μm. *P < 0.01 as compared to 100% (=FGF-2/TNF-α stimulated condition)

Fig. 5

Analysis of the human and mouse fibrinogen. Scanning electron microscopy of human (a) and mouse (b) plasma-derived fibrin clots. Bar represents 1 μm. c Schematic representation of the fibrinogen Aα-chain in human and mouse plasma