The Mouse Aorta Model: Influence of Genetic Background and Aging on bFGF- and VEGF-Induced Angiogenic Sprouting

Angiogenesis can be studied ex vivo by culturing rat or mouse aortic rings in collagen gels. Unlike rat aorta explants, unstimulated mouse aortic rings were unable to spontaneously produce an angiogenic response under serum-free conditions. They, however, responded to bFGF and VEGF, generating networks of branching neovessels. Aortic rings from GFP-Tie2-transgenic mice generated GFP-labeled neovessels that could be easily identified by their distinctly green fluorescence. Aortic rings from 1- to 2-month-old mice produced microvessels faster, more uniformly and in greater number than aortic rings from 6- to 10-month-old mice, particularly in VEGF-treated cultures. Aortic rings from 129/SVJ mice were capable of a much stronger and sustained angiogenic response to bFGF than those of C57BL/6 or BALB/c mice, which were in turn more angiogenic than aortic rings from FVB mice. The same strains of mice responded differently to VEGF, as C57BL/6 mouse aortic rings produced more microvessels than those of BALB/c, FVB, and 129/SVJ mice, which were capable of only a limited response. The significant impact that aging and genetic background have on mouse aortic angiogenesis should be taken into account when the aortic-ring assay is used to evaluate function of genes that have been deleted or overexpressed in genetically modified mice.     

Antiangiogenesis is produced by nontoxic doses of vinblastine

The effects of vinblastine (VBL) on endothelial cell functions involved in angiogenesis, namely proliferation, chemotaxis, spreading on fibronectin (FN), secretion of matrix-metalloproteinase-2 (MMP-2) and MMP-9, and morphogenesis on Matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo by using the chick embryo chorioallantoic membrane (CAM) model. In vitro, at noncytotoxic doses (0.1, 0.25, 0. 5, 0.75, and 1 pmol/L), VBL impacted all these functions, except secretion of MMPs, in a dose-dependent fashion. By contrast, proliferation of other primary cells such as fibroblasts and lymphoid tumor cells was not impacted. In vivo, VBL at 0.5, 0.75, and 1 pmol/L again displayed a dose-dependent antiangiogenic activity. Lack of cytotoxicity in vitro and in vivo was shown both morphologically, and also because the antiangiogenic effects were rapidly abolished when VBL was removed. Apoptosis was not induced. At the ultrastructural level, impairment of cell functions in vitro was associated with thin disturbance of the cytoskeleton, in the form of slight depolymerization and accumulation of microfilaments, which was equally reversible. Results suggest that VBL has an antiangiogenic component at very low, noncytotoxic doses, and that antiangiogenesis by VBL could be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases, Kaposi's sarcoma, and cancer.     

Human lymphoblastoid cells produce extracellular matrix-degrading enzymes and induce endothelial cell proliferation, migration, morphogenesis, and angiogenesis

Human lymphoproliferative diseases can be hypothesized to invade locally and to metastatize via mechanisms similar to those developed by a variety of solid tumors, i.e., the secretion of extracellular matrix-degrading enzymes and stimulation of angiogenesis. To assess this hypothesis, Namalwa, Raji, and Daudi cell lines (Burkitt’s lymphoma), LIK and SB cell lines (B-cell lymphoblastic leukemia), CEM and Jurkat cell lines (T-cell lymphoblastic leukemia), and U266 cell line (multiple myeloma) were evaluated for their capacity to produce matrix metalloproteinase-2 and -9, and urokinase-type plasminogen activator. These cell lines were also assessed for their ability: (1) to produce the angiogenic basic fibroblast growth factor and vascular endothelial growth factor; (2) to induce an angiogenic phenotype in cultured endothelial cells, represented by cell proliferation, chemotaxis, and morphogensis; (3) to stimulate angiogenesis in different in vivo experimental models. All cell lines expressed the mRNA for one or both metalloproteinases. Namalwa, Raji, LIK, SB, and U266 cells secreted the active form of both metalloproteinases, while Daudi, CEM, and Jurkat cells produced metalloproteinase-2 but not -9. In contrast, urokinase-type plasminogen activator was secreted only by SB cells. While Raji, LIK, SB, CEM, and Jurkat cells secreted both basic fibroblast growth factor and vascular endothelial growth factor, Daudi and U266 cells produced only the former, and Namalwa cells only the latter. Accordingly, the conditioned medium of all cell lines stimulated cell proliferation and/or chemotaxis in cultured endothelial cells, with the exception of that of Namalwa cells which was ineffective. The conditioned medium of CEM and Jurkat cells induced morphogenesis in cultured endothelial cells grown on a reconstituted basement membrane (Matrigel). Lastly, Namalwa, Raji, LIK, SB, U266, CEM, and Jurkat cells induced angiogenesis and mononuclear cell recruitment in the murine Matrigel sponge model and in a chick embryo chorioallantoic membrane assay. The extent of angiogenesis in both models was strictly correlated with the density of the mononuclear cell infiltrate. The results indicate that human lymphoproliferative disease cells possess both local and remote invasive ability via the secretion of matrix-degrading enzymes and the induction of angiogenesis which is fostered by host inflammatory cells and by an intervening ensemble of angiogenic factors.     

Determination of vascularization of B-cell non-Hodgkin's lymphomas with four markers

We evaluated the microvessel area (an index of angiogenesis) by using the factor VIII-related antigen (factor VIII-RA), and the expression of three components of the subendothelial basement membrane, namely: tenascin, laminin and type IV collagen. The four markers were assessed by immunohistochemical methods in 57 B-cell non-Hodgkin's lymphomas (B-NHL) and 28 benign lymphadenopathies. We found that microvessel area and the basement membrane markers had a different distribution pattern in relation to the histological type and degree of malignancy. In reactive and atypical lymphoid hyperplasias and in follicular low- and intermediate-grade B-NHL, microvessels were distributed within the interfollicular zones. By contrast, diffuse intermediate-grade and high-grade B-NHL were highly vascularized and microvessels were closely related to the neoplastic cells. Microvessel area was significantly associated with tenascin expression in all histological grades. Conversely, it was not correlated to laminin and type IV collagen expression, especially in diffuse intermediate-grade and high-grade B-NHL, where the expression of these markers was poor and fragmented. Our study suggests that angiogenesis and tenascin expression are associated phenomena in B-NHL, and that both increase with the malignancy grade. The prognostic value of angiogenesis and tenascin in B-NHL warrants further assessment in follow-up studies.     

Angiogenesis extent and macrophage density increase simultaneously with pathological progression in B-cell non-Hodgkin's lymphomas

Node biopsies of 30 benign lymphadenopathies and 71 B-cell non-Hodgkin's lymphomas (B-NHLs) were investigated for microvessel and macrophage counts using immunohistochemistry and morphometric analysis. Both counts were significantly higher in B-NHL. Moreover, when these were grouped into low-grade and high-grade lymphomas, according to the Kiel classification and Working Formulation (WF), statistically significant higher counts were found in the high-grade tumours. Immunohistochemistry and electron microscopy revealed a close spatial association between microvessels and macrophages. Overall, the results suggest that, in analogy to what has already been shown in solid tumours, angiogenesis occurring in B-NHLs increases with tumour progression, and that macrophages promote the induction of angiogenesis via the release of their angiogenic factors.     

VE-cadherin expression and clustering maintain low levels of survivin in endothelial cells

Survivin is strongly expressed in embryonic organs and in tumor cells but is low or absent in differentiated normal tissues. Resting endothelium expresses low levels of survivin but can up-regulate its synthesis on activation to proliferate. The mechanisms responsible for survivin down-regulation in resting conditions are still unknown. We report here that confluence and vascular endothelial-cadherin (VE-cadherin) expression induce contact inhibition of cell growth and survivin down-regulation in the endothelium. Using beta-catenin null and positive isogenic endothelial cell lines we found that the effect requires beta-catenin expression and its association to VE-cadherin cytoplasmic tail. Furthermore, in allantois organ cultures, survivin expression is up-regulated in areas of growing vessels where VE-cadherin is partially dismantled from junctions or in VE-cadherin -/- specimens. Overall, these data indicate that VE-cadherin and beta-catenin may negatively regulate survivin synthesis in endothelial cells. Consistently, in epidermal and pancreatic cell lines or ovarian tumors, epithelial-cadherin (E-cadherin) and survivin expression is inversely related, suggesting a non-cell-specific role of cadherins in reducing survivin synthesis.     

TNP-470 and recombinant human interferon-alpha2a inhibit angiogenesis synergistically

The hypothesis that the combination of two known antiangiogenic agents TNP-470 and interferon (IFN)-alpha exerts synergistic effects has been investigated in vitro and in vivo. In vitro, TNP-470 and recombinant human IFN-alpha2a (rhIFN-alpha2a) resulted in a dose-dependent inhibition of proliferation of human umbilical vein endothelial cells (HUVECs) and EA.hy926 endothelial cells. Compared with the two agents used singly at their lowest or ineffective doses, combined treatment with the same doses inhibited more intensely in the absence of cytotoxicity and displayed similar behaviour on cell chemotaxis and capillary morphogenesis on Matrigel. However, the secretion of matrix metalloproteinase 2 (MMP-2) and MMP-9 was not influenced by the two agents, either alone or in combination, even when they were applied at their lowest efficacious doses or at higher cytotoxic doses. Experiments in vivo with the chick embryo chorioallantoic membrane (CAM)-sponge assay revealed the same dose-dependent inhibition and synergy. As the basic fibroblast growth factor (bFGF)-induced angiogenesis in the CAM-sponge model was strongly inhibited by the combined treatment, TNP-470 and rhIFN-alpha2a would appear to exert antiangiogenesis synergistically, perhaps by interfering with the bFGF-mediated pathway.     

Two symptomatic cases of cluster headache associated with internal carotid artery dissection

Neurological Sciences - Cluster headache is a clinical entity characterised by strictly unilateral head pain attacks accompanied by ipsilateral autonomic phenomena. We report two patients who had...