Expression and role of plexin B1 (PLXNB1) 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-PLXNB1 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 anti-PLXNB1 antibodies (3 μg/ml) on matrigel invasion of N-MVECs. The effect of irrelevant rabbit IgG is also shown. Numbers on the x-axis refer to the total number of cells migrated through the matrigel after 6 hours. Data are the mean ± standard deviation of three experiments performed in triplicate in three N-MVEC lines. The asterisk indicates that values are significantly different from the values of control (p < 0.05). (c) Effect of anti-PLXNB1 antibodies on capillary morphogenesis of N-MVECs. N-MVECs were plated on Matrigel (60 × 103/ml), in complete MCDB medium, supplemented with 30% fetal calf serum, and 20 μg/ml endothelial cells growth supplement. N-MVEC spontaneously form anastomosing cords of cells resembling a capillary plexus, which are well organized by 6 hours. The process of endothelial cell organization after 6 hours is impaired in the presence of 3 μg/ml of anti-PLXNB1 rabbit polyclonal antibodies. Irrelevant rabbit IgG gave results similar to control untreated N-MVECs (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. The asterisk indicates that values are significantly different from the values of control N-MVECs at 6 hours after plating on Matrigel (p < 0.05).