Tumor angiogenesis is regulated by a dynamic cross-talk between tumor cells and the host microenvironment. Because membrane vesicles shed by tumor cells are known to mediate several tumor-host interactions, we determined whether vesicles might also stimulate angiogenesis. Vesicles shed by human ovarian carcinoma cell lines CABA I and A2780 stimulated the motility and invasiveness of endothelial cells in vitro. Enzyme-linked immunosorbent assay and Western blot analysis revealed relevant amounts of vascular endothelial growth factor (VEGF) and the two matrix metalloproteinases MMP-2 and MMP-9, but not fibroblast growth factor-2, contained in shed vesicles. An A2780 cell-derived clone transfected to overexpress VEGF shed the same amount of vesicles as did a control clone, but contained significantly more VEGF within the vesicles. Despite a greater amount of VEGF in vesicles of the overexpressing clone, vesicles of both clones stimulated endothelial cell motility to comparable levels, suggesting that VEGF was stored within the vesicle and was unavailable. Only following vesicle burst induced by acidic pH (a characteristic of the tumor microenvironment) was VEGF released, leading to significantly higher stimulation of cell motility. Thus, tumor-shed membrane vesicles carry VEGF and release it in a bioactive form in conditions typical of the tumor microenvironment.

Hypoplastic left heart syndrome (HLHS) is one of the most severe congenital heart malformations, characterized by underdevelopment of the structures in the left heart-aorta complex. The majority of cases are sporadic. Although multiple genetic loci have been tentatively implicated in HLHS, no gene or pathway seems to be specifically associated with the disease. To elucidate the genetic basis of HLHS, we analyzed 53 well-characterized patients with isolated HLHS using an integrated genomic approach that combined DNA sequencing of five candidate genes (NKX2-5, NOTCH1, HAND1, FOXC2 and FOXL1) and genome-wide screening by high-resolution array comparative genomic hybridization. In 30 patients, we identified two novel de novo mutations in NOTCH1, 23 rare patients inherited gene variants in NOTCH1, FOXC2 and FOXL1, and 33 rare patients mostly inherited copy-number variants. Some of the identified variations coexisted in the same patient. The biological significance of such rare variations is unknown, but our findings strengthen the role of NOTCH pathway in cardiac valve development, indicating that HLHS is, at least in part, a 'valve' disease. This is the first report of de novo mutations associated with isolated HLHS. Moreover, the coexistence of multiple rare variants suggests in some cases a cumulative effect, as shown for other complex disease.

The antiangiogenic factor thrombospondin 1 (TSP-1) binds with high affinity to several heparin-binding angiogenic factors, including fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor (VEGF), and hepatocyte growth factor/ scatter factor (HGF/SF). The aim of this study was to investigate whether TSP-1 affects FGF-2 association with the extracellular matrix (ECM) and its bioavailability. TSP-1 prevented the binding of free FGF-2 to endothelial cell ECM. It also IntroductionAngiogenesis, sprouting of new blood vessels from pre-existing ones, is a crucial event in physiologic and pathologic processes, including tumor growth and metastasis. 1 Angiogenesis is a complex process, regulated by pro-and antiangiogenic factors, that involves dynamic interaction between a variety of cells, growth factors, and the extracellular matrix (ECM). 2 As in other morphogenic processes, the ECM acts not just as a structural support, but also as a direct modulator of cellular functions. Matrix components and bioactive fragments released by limited proteolysis can directly regulate endothelial cell functions. 3 In addition, the matrix contributes to angiogenesis by acting as a reservoir for angiogenic factors. Growth factors are stored in the matrix through binding to heparansulfate proteoglycans (HSPGs). Binding to HSPGs is reversible, 4 and biologically active factors can be released from the matrix by different agents. 5,6 Fibroblast growth factor 2 (FGF-2, basic FGF) is the most extensively studied example of matrix-stored angiogenic factor. 5,7 As do other members of the FGF family, FGF-2 has a high affinity for the glycosaminoglycan heparin and for HSPGs. Cell-surface HSPGs are required for the formation of an active FGF/FGF receptor signaling complex, 8 for internalization, and hence for internalization-dependent activities such as endothelial cell proliferation. 7,9 Conversely, matrix-associated HSPGs allow the storage of FGF-2 within the ECM. 10 Matrix-associated FGF-2 can then act locally, or be released as a soluble, biologically active factor. 6 Mobilization of active FGF-2 from the matrix is an important mechanism of induction of angiogenesis. Biologically active FGF-2 is released by heparin, 11 matrix-degrading proteases, 12 heparanase, 13 and the FGF-binding protein (FGF-BP). 14 Conversely, other endogenous or pharmacologic agents that prevent FGF-2 interaction with HSPGs exert an antiangiogenic activity, as in the case of platelet factor 4 (PF-4), 15 a soluble syndecan ectodomain, 16 suramin, 17 chemically modified heparins, and heparin-mimicking polyanionic compounds. [18][19][20] Other important angiogenic factors, including vascular endothelial growth factor (VEGF) and hepatocyte growth factor/scatter factor (HGF/SF), bind to HSPGs that again contribute to growth factor binding to cell receptors and to the ECM. [21][22][23] Thrombospondin 1 (TSP-1) is the most studied member of a family of at least 5 related proteins. 24,25 TSP-1 is a matricellular molecule, a modular glycoprotein composed of mult...

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