Using the yeast two-hybrid system and an in vitro binding assay, we have identified a novel protein termed vinexin as a vinculin-binding protein. By Northern blotting, we identified two types of vinexin mRNA that were 3 and 2 kb in length. Screening for full-length cDNA clones and sequencing indicated that the two mRNA encode 82- and 37-kD polypeptides termed vinexin α and β, respectively. Both forms of vinexin share a common carboxyl-terminal sequence containing three SH3 domains. The larger vinexin α contains an additional amino-terminal sequence. The interaction between vinexin and vinculin was mediated by two SH3 domains of vinexin and the proline-rich region of vinculin. When expressed, vinexin α and β localized to focal adhesions in NIH 3T3 fibroblasts, and to cell–cell junctions in epithelial LLC-PK1 cells. Furthermore, expression of vinexin increased focal adhesion size. Vinexin α also promoted upregulation of actin stress fiber formation. In addition, cell lines stably expressing vinexin β showed enhanced cell spreading on fibronectin. These data identify vinexin as a novel focal adhesion and cell– cell adhesion protein that binds via SH3 domains to the hinge region of vinculin, which can enhance actin cytoskeletal organization and cell spreading.

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The conversion of fibrinogen into fibrin and the association of fibrin(ogen) with activated platelets play a fundamental role in hemostasis because their interaction with the injured vessel prevents blood extravasation. Platelet aggregates and fibrin also participate in the occlusion of the vascular lumen in pathological conditions. Fibrin II also promotes the formation of new blood vessels, for example, during wound healing and tumor growth. Using an in vitro assay, we have studied the mechanism by which fibrin II induces formation of capillaries. Generation of fibrin II on top of an endothelial cell monolayer rapidly rearranged the ECs into a capillary network. In contrast, neither fibrin I nor fibrin 325 induced these morphogenetic changes, indicating that exposure of the N‐terminal peptide β15–42 is involved in this process. Binding studies, using the N‐terminal fragment of fibrin (NDSK II), showed that NDSK II binds to EC with high affinity, but neither NDSK nor NDSK325 bound specifically. Binding of NDSK II to endothelial cells was blocked with an antibody to VE‐cadherin. Direct association of NDSK II and VE‐cadherin was also demonstrated in a VE‐cadherin antibody capture assay. NDSK II bound specifically with the captured VE‐cadherin but NDSK or NDSK 325 did not associate with VE‐cadherin. Moreover, fibrin II associated with EC VE‐cadherin and this interaction triggered the formation of capillary‐like structures. A better understanding of the cellular responses to fibrin, identification of the fibrin binding site within VE‐cadherin and the intracellular signaling that follows this interaction, could yield important information that may translate into better control of the angiogenic process.

When human umbilical vein endothelial cells (HUVEC) differentiate into capillary-like tubes, there is a five-fold upregulation of the mRNA for thymosin beta4 (Tbeta4) (Grant et al. J Cell Sci 1995; 108: 3685-94 [1]) and this endogenous expression plays an important role in endothelial cell attachment to and spreading on matrix components. We now show that exogenous addition of thymosin beta4 (in the ng-microg range) to HUVEC in culture can induce several biological responses. These responses include increased tube formation in vitro. Additionally, exogenous thymosin beta4 enhances vascular sprouting in the coronary artery ring angiogenesis assay. Measurements of these vascular sprouts show a doubling of the vessel area (via increased branching) with as little as 100 ng of synthetic thymosin beta4. These processes appear to involve the binding of thymosin beta4 to an unknown cell surface receptor and internalization of the protein. This cell surface-binding appears not to be mediated through the thymosin beta4-actin binding domain LKTET. An increase in thymosin beta4 cytoplasmic staining in HUVEC exposed 10 microg of the peptide appears to occur without increased mRNA translation. In summary Tbeta4 induces an increase in cell-matrix attachment, proliferation, tube formation, internalization of the peptide and rearrangement of the actin cytoskeleton. The data now defines both an autocrine and paracrine role for thymosin beta4 in vessel formation.