Expression and role of pent(r)axin 3 (PTX3) in endothelial cell invasion and capillary morphogenesis. (a) Western blotting of 60 μg protein from cell lysates of microvascular endothelial cells (MVECs) from normal subjects (N-MVECs) and patients with systemic sclerosis (SSc-MVECs) with anti-PTX3 antibodies. Each lane represents western blotting of MVECs obtained from a single patient. Actin was used as an internal reference standard. Numbers on the right represent the molecular weight expressed in kDa. (b) Effect of PTX3 antibodies (3 μg/ml) on matrigel invasion of N-MVECs. The effect of irrelevant rat IgG is also shown. Numbers on the x-axis refer to the total number of cells migrated through the matrigel after 6 hours. The presence of anti-PTX3 antibodies is able to revert inhibition of matrigel invasion induced by SSc-MVECs conditioned medium (C.M.). Data are the mean ± standard deviation of three experiments performed in triplicate in three N-MVEC lines. An asterisk indicates that values are significantly different from the values of control (p < 0.05). (c) Effect of anti-PTX3 antibodies on capillary morphogenesis of N-MVECs. Experimental conditions were as described in the legend to Figure 1. Conditioned medium from SSc-MVECs was able to impair capillary morphogenesis observed after 6 hours from plating (panel c). The process of endothelial cell organization after 6 hours was partially restored in the presence of 3 μg/ml of anti-PTX3 rat monoclonal antibodies (panel d), which showed only a small effect when added to control N-MVECs (panel b). Irrelevant rat IgG did not show any effect (not shown). These data are representative of three different experiments performed on three N-MVEC cell lines (100× magnification). Numbers reported within each panel indicate the percent of the photographic field occupied by cells ± standard deviation. An asterisk indicates that values are significantly different from the values of control N-MVECs at 6 hours after plating on Matrigel (p < 0.05).