Abstract-The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between ␤ 3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and ␤ 3 integrin appears to be synergistic, because VEGFR-2 activation induces ␤ 3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesionand growth factor-induced ␤ 3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent ␤ 3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and ␤ 3 integrin. Moreover, c-Src mediates growth factor-induced ␤ 3 integrin activation, ligand binding, ␤ 3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro-and antiangiogenic strategies. Key Words: angiogenesis Ⅲ endothelial cell Ⅲ ␤ 3 integrin signaling Ⅲ vascular endothelial growth factor receptor Ⅲ extracellular matrix proteins A ngiogenesis, the process of new blood vessel formation from preexisting vasculature, plays critical roles in tissue regeneration, postischemic tissue repair on myocardial infarction and stroke, and in the pathogenesis of cancer, rheumatoid arthritis, and diabetic microvascular disease. 1 Angiogenesis is triggered by angiogenic growth factors and their receptors in coordination with extracellular matrix (ECM) receptors known as integrins. 2 On integrin engagement, ECM triggers activation of numerous intracellular signaling pathways essential for endothelial cell (EC) survival, proliferation, migration, morphogenesis, and organization of ECs into blood vessels. 3 There are several manifestations of a tightly collaborative relationship between integrins and receptors for growth factors. 4,5 On ECs, engagement of ␣ v ␤ 3 integrin promotes phosphorylation and activation of vascular endothelial growth factor (VEGF) receptor (VEGFR)-2, thereby augmenting the mitogenic activity of VEGFs. 6 Among several integrins on ECs, ␣ v ␤ 3 is the most abundant and influential receptor regulating angiogenesis. 7 The upregulation of ␣ v ␤ 3 during angiogenesis suggests that this integrin might play a crucial role during this process. Indeed, antagonists of ␣ v ␤ 3 , including blocking monoclonal antibody (LM609) and RGD cyclic peptides, were shown ...

Apolipoprotein J (apoJ)/clusterin was first identified as an 80 kDa secretory glycoprotein present in most body fluids. It has been implicated in a variety of physiological processes including cellular differentiation and apoptosis. We demonstrate here that in addition to the well characterized secreted form of the protein, there exists an intracellular, nuclear form of apoJ. This intracellular form of the protein is induced to accumulate in the nucleus of two epithelial cell lines (HepG2 and CCL64) in response to treatment with transforming growth factor beta (TGF beta). We demonstrate in vitro that apoJ protein can be translated from two in-frame ATG sites. Initiation from the first ATG encodes for the secretory form of apoJ and initiation from the second ATG, located 33 amino acids downstream of the first and lacking the hydrophobic signal sequence, encodes for a truncated apoJ protein. This shorter form of apoJ is not recognized by microsomes and therefore not glycosylated, and we postulate that it is retained intracellularly and targeted to the nucleus due to the presence of an SV40-like nuclear localization sequence (NLS). This mechanism of nuclear targeting of apoJ occurs in cells since the protein isolated from nuclei of TGF beta-treated cells and the in vitro-translated truncated form are identical by V8 protease analysis. These results suggest that the diverse physiological responses attributed to apoJ may be elicited through a common molecular mechanism involving a previously uncharacterized intracellular form of the protein.

In platelets, ␣ IIb ␤ 3 exists in a form that cannot bind adhesive proteins in the plasma; although it can interact with immobilized fibrinogen it cannot interact with immobilized von Willebrand factor in the vessel wall. Soluble agonists such as thrombin convert ␣ IIb ␤ 3 to a form that recognizes soluble and immobilized ligands. Attempts to reconstitute ␣ IIb ␤ 3 activation in a non-hematopoietic, nucleated cell system have been unsuccessful. In the present study, we have developed a transfected Chinese hamster ovary cell model in which ␣ IIb ␤ 3 activation is induced by signaling across glycoprotein (GP) Ib-IX by its ligand, von Willebrand factor. GPIb-IX activates not only the transfected ␣ IIb ␤ 3 but also endogenous ␣ v ␤ 3 . Activation of the pathways leading to integrin activation occurred even in cells transfected with GPIb-IX lacking the domain on GPIb␣ that binds 14-3-3 or that which binds actin-binding protein. These studies demonstrate that signals induced by interaction of GPIb-IX with von Willebrand factor lead to ␣ IIb ␤ 3 activation and suggest that the signaling pathways by which GPIb-IX induces ␣ IIb ␤ 3 activation are different to those used by thrombin. Elucidation of these differences may provide insights into therapeutic ways in which to inhibit integrin activation in selective clinical settings.

Recent studies have shown that Src-family kinases (SFKs) play an important role in mediating integrin signalling, and the β3 subunit of αIIbβ3 integrin has been shown to interact with multiple SFK members. Here, we analyzed the interactions and functional consequences of Fyn and Src binding to αIIbβ3. Fyn associated with the β3 subunit in resting and thrombin-aggregated platelets, whereas interaction between Src and αIIbβ3 was seen predominantly in resting but not in thrombin-aggregated platelets. We have also observed that Fyn but not Src localized to focal adhesions in CHO cells adherent to fibrinogen through αIIbβ3. On the basis of these differences, we wanted to determine the sequence requirements for the interaction of Fyn and Src within the β3-cytoplasmic domain. Whereas Src association required the C-terminal region of β3, Fyn continued to interact with mutants that could no longer associate with Src and that contained as few as 13 membrane-proximal amino acids of the β3-cytoplasmic tail. Using deletion mutants of β3-cytoplasmic tails expressed as GST-fusion proteins, we narrowed down the Fyn-binding site even further to the amino acid residues 721-725 (IHDRK) of the β3-cytoplasmic domain. On the basis of these observations, we explored whether Fyn–/– mice exhibited any abnormalities in hemostasis and platelet function. We found that Fyn–/– mice significantly differed in their second bleeding times compared with wild-type mice, and platelets from Fyn–/– mice exhibited delayed spreading on fibrinogen-coated surfaces. Using mutant forms of Fyn, it appears that its kinase activity is required for its localization to focal adhesions and to mediate αIIbβ3-dependent cell spreading. Our results suggest that Fyn and Src have distinct requirements for interaction with αIIbβ3; and, consequently, the two SFK can mediate different functional responses.