Transforming growth factor-β1 (TGFβ1), a major promoter of myofibroblast differentiation, induces α-smooth muscle (sn) actin, modulates the expression of adhesive receptors, and enhances the synthesis of extracellular matrix (ECM) molecules including ED-A fibronectin (FN), an isoform de novo expressed during wound healing and fibrotic changes. We report here that ED-A FN deposition precedes α-SM actin expression by fibroblasts during granulation tissue evolution in vivo and after TGFβ1 stimulation in vitro. Moreover, there is a correlation between in vitro expression of α-SM actin and ED-A FN in different fibroblastic populations. Seeding fibroblasts on ED-A FN does not elicit per se α-SM actin expression; however, incubation of fibroblasts with the anti-ED-A monoclonal antibody IST-9 specifically blocks the TGFβ1-triggered enhancement of α-SM actin and collagen type I, but not that of plasminogen activator inhibitor-1 mRNA. Interestingly, the same inhibiting action is exerted by the soluble recombinant domain ED-A, but neither of these inhibitory agents alter FN matrix assembly. Our findings indicate that ED-A–containing polymerized FN is necessary for the induction of the myofibroblastic phenotype by TGFβ1 and identify a hitherto unknown mechanism of cytokine-determined gene stimulation based on the generation of an ECM-derived permissive outside in signaling, under the control of the cytokine itself.

In vertebrates, networks of capillary vessels supply tissues with nutrients. Capillary patterns are closely mimicked by endothelial cells cultured on basement membrane proteins that allow single randomly dispersed cells to self‐organize into vascular networks. Here we provide a model including chemoattraction as the fundamental mechanism for cell‐to‐cell communication in order to identify key parameters in the complexity of the formation of vascular patterns. By flanking biological experiments, theoretical insights and numerical simulations, we provide strong evidence that endothelial cell number and the range of activity of a chemoattractant factor regulate vascular network formation and size. We propose a mechanism linking the scale of formed endothelial structures to the range of cell‐to‐cell interaction mediated by the release of chemoattractants.

Neuropilin 1 (Nrp1) is a coreceptor for vascular endothelial growth factor A165 (VEGF-A165, VEGF-A164 in mice) and semaphorin 3A (SEMA3A). Nevertheless, Nrp1 null embryos display vascular defects that differ from those of mice lacking either VEGF-A164 or Sema3A proteins. Furthermore, it has been recently reported that Nrp1 is required for endothelial cell (EC) response to both VEGF-A165 and VEGF-A121 isoforms, the latter being incapable of binding Nrp1 on the EC surface. Taken together, these data suggest that the vascular phenotype caused by the loss of Nrp1 could be due to a VEGF-A164/SEMA3A-independent function of Nrp1 in ECs, such as adhesion to the extracellular matrix. By using RNA interference and rescue with wild-type and mutant constructs, we show here that Nrp1 through its cytoplasmic SEA motif and independently of VEGF-A165 and SEMA3A specifically promotes α5β1-integrin-mediated EC adhesion to fibronectin that is crucial for vascular development. We provide evidence that Nrp1, while not directly mediating cell spreading on fibronectin, interacts with α5β1 at adhesion sites. Binding of the homomultimeric endocytic adaptor GAIP interacting protein C terminus, member 1 (GIPC1), to the SEA motif of Nrp1 selectively stimulates the internalization of active α5β1 in Rab5-positive early endosomes. Accordingly, GIPC1, which also interacts with α5β1, and the associated motor myosin VI (Myo6) support active α5β1 endocytosis and EC adhesion to fibronectin. In conclusion, we propose that Nrp1, in addition to and independently of its role as coreceptor for VEGF-A165 and SEMA3A, stimulates through its cytoplasmic domain the spreading of ECs on fibronectin by increasing the Rab5/GIPC1/Myo6-dependent internalization of active α5β1. Nrp1 modulation of α5β1 integrin function can play a causal role in the generation of angiogenesis defects observed in Nrp1 null mice. The vascular system is a hierarchical network of blood vessels lined by endothelial cells that, by means of the transmembrane integrin proteins, bind to the surrounding proteinaceous extracellular matrix (ECM). Integrins are required for proper cardiovascular development and exist in bent (inactive) and extended (active) shapes that are correspondingly unable and able to attach to the ECM. Extracellular guidance cues, such as vascular endothelial growth factor and semaphorins, bind the transmembrane protein neuropilin-1 (Nrp1) and then activate biochemical signals that, respectively, activate or inactivate endothelial integrins. Here, we show that Nrp1, via its short cytoplasmic domain and independently of vascular endothelial growth factor and semaphorins, specifically promotes endothelial cell attachment to the ECM protein fibronectin, which is known to be crucial for vascular development. Notably, Nrp1 favors cell adhesion by associating with fibronectin-binding integrins and promoting the fast vesicular traffic of their extended form back and forth from the endothelial cell-to-ECM contacts. Binding of the Nrp1 cytoplasmic domain with the adaptor protein GIPC1, which in turn associates with proteins required for integrin internalization and vesicle motility, is required as well. It is likely that such an integrin treadmill could act as a major regulator of cell adhesion in general.

Experiments of in vitro formation of blood vessels show that cells randomly spread on a gel matrix autonomously organize to form a connected vascular network. We propose a simple model which reproduces many features of the biological system. We show that both the model and the real system exhibit a fractal behavior at small scales, due to the process of migration and dynamical aggregation, followed at large scale by a random percolation behavior due to the coalescence of aggregates. The results are in good agreement with the analysis performed on the experimental data.