Specialised cell types in the chorioallantoic membrane express carbonic anhydrase during chick embryogenesis.
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The expression of carbonic anhydrase in the chorioallantoic membrane (CAM) of the chick embryo was investigated by means of the histochemical localisation of the enzyme catalytic sites and the immunohistochemical identification of its isoenzymatic forms. The results show that carbonic anhydrase is developmentally expressed in a subset of cells both in the ectodermal and the endodermal epithelium. The distribution patterns from both methodological approaches indicated that carbonic anhydrase is a marker of the villus cavity cells and the mitochondria-rich cells in the ectodermal and the endodermal epithelium, respectively. Such a cell-specific pattern of the enzyme expression provides a further contribution to characterising the heterogeneous cell population of the chick CAM and supports specific functional involvement for the distinct cell types in CAM-mediated processes, such as calcium transport, maintenance of acid-base balance and water and electrolyte reabsorption, during chick embryogenesis. (+info)
Development of endometriosis-like lesions after transplantation of human endometrial fragments onto the chick embryo chorioallantoic membrane.
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The chick embryo chorioallantoic membrane (CAM) bioassay was used to investigate the early pathogenesis of endometriosis. Endometrial fragments were explanted onto the CAM. The grafts including the surrounding CAM were excised at 24, 48 or 72 h after explantation, fixed and embedded in paraffin. Immunohistochemical analysis was used to distinguish endometrial cells. To identify cells of human origin, in-situ hybridization was performed using a probe specific for human chromosome 1. After 24 h, direct contact between endometrial stromal as well as epithelial cells and the mesenchymal layer of the CAM was observed. Invasion of both stromal cells and intact endometrial glands into the mesenchymal layer was observed after 48 h. At 72 h, endometriosis-like lesions were observed in the mesenchymal layer. Positive staining with antibodies to vimentin and pan-cytokeratin was observed in the invading cells as well as in the lesions. In the lesions these positively stained cells showed in-situ hybridization signals for human chromosome 1, confirming their human origin. In conclusion, after 3 days of incubation, endometriosis-like lesions consisting of human endometrial glands and stromal cells were found in the mesenchymal layer of the CAM. These lesions apparently resulted from the invasion of intact human epithelial structures and stromal cells. (+info)
Inhibition of angiogenesis and tumor growth by SCH221153, a dual alpha(v)beta3 and alpha(v)beta5 integrin receptor antagonist.
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New blood vessel formation is essential for tumor growth and metastatic spread. Integrins alpha(v)beta3 and alpha(v)beta5 are arginine-glycine-aspartic acid-dependent adhesion receptors that play a critical role in angiogenesis. Hence, selective dual alpha(v)beta3 and alpha(v)beta5 antagonists may represent a novel class of angiogenesis and tumor-growth inhibitors. Here, an arginine-glycine-aspartic acid-based peptidomimetic library was screened to identify alpha(v)beta3 antagonists. Selected compounds were then modified to generate potent and selective dual inhibitors of alpha(v)beta3 and alpha(v)beta5 receptors. One of these compounds, SCH 221153, inhibited the binding of echistatin to alpha(v)beta3 (IC50 = 3.2 nM) and alpha(v)beta5 (IC50 = 1.7 nM) with similar potency. Its IC50 values for related alpha(IIb)beta3 and alpha5beta1 receptors were 1294 nM and 421 nM, respectively, indicating that SCH 221153 is highly selective for alpha(v)beta3 and alpha(v)beta5 receptors. In cell-based assays, SCH 221153 inhibited the binding of echistatin to alpha(v)beta3- and alpha(v)beta5-expressing 293 cells and blocked the adhesion of endothelial cells to immobilized vitronectin and fibroblast growth factor 2 (FGF2). SCH 221153, but not the inactive analogue SCH 216687, was effective in inhibiting FGF2 and vascular endothelial growth factor-induced endothelial cell proliferation in vitro with an IC50 equal to 3-10 microM. Angiogenesis induced by FGF2 in the chick chorioallantoic membrane assay was also inhibited by SCH 221153. Finally, SCH 221153 exerted a significant inhibition on tumor growth induced by intradermal or s.c. injection of human melanoma LOX cells in severe combined immunodeficient mice. (+info)
Growth factor-induced angiogenesis in vivo requires specific cleavage of fibrillar type I collagen.
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The contribution of specific type I collagen remodeling in angiogenesis was studied in vivo using a quantitative chick embryo assay that measures new blood vessel growth into well-defined fibrillar collagen implants. In response to a combination of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), a strong angiogenic response was observed, coincident with invasion into the collagen implants of activated fibroblasts, monocytes, heterophils, and endothelial cells. The angiogenic effect was highly dependent on matrix metalloproteinase (MMP) activity, because new vessel growth was inhibited by both a synthetic MMP inhibitor, BB3103, and a natural MMP inhibitor, TIMP-1. Multiple MMPs were detected in the angiogenic tissue including MMP-2, MMP-13, MMP-16, and a recently cloned MMP-9-like gelatinase. Using this assay system, wild-type collagen was compared to a unique collagenase-resistant collagen (r/r), with regard to the ability of the respective collagen implants to support cell invasion and angiogenesis. It was found that collagenase-resistant collagen constitutes a defective substratum for angiogenesis. In implants made with r/r collagen there was a substantial reduction in the number of endothelial cells and newly formed vessels. The presence of the r/r collagen, however, did not reduce the entry into the implants of other cell types, that is, activated fibroblasts and leukocytes. These results indicate that fibrillar collagen cleavage at collagenase-specific sites is a rate-limiting event in growth factor-stimulated angiogenesis in vivo. (+info)
Angiogenic activity of pyruvic acid in in vivo and in vitro angiogenesis models.
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The excessive growth of a tumor requires high rates of glucose uptake and glycolysis and continuous recruitment of new blood vessels. Here, we provide several lines of evidence showing that pyruvic acid, the end product of glycolysis, exhibits strong angiogenic activity. Pyruvic acid promoted angiogenesis in chorioallantoic membrane assay and in vivo mouse Matrigel plug assay. Pyruvic acid also positively affects angiogenic cascade, DNA synthesis, migration, and tube formation in bovine aortic endothelial cells. Furthermore, mRNA expression of fibroblast growth factor receptor-2 and vascular endothelial growth factor was enhanced by pyruvic acid. These results strongly suggest that pyruvic acid plays an important role in angiogenesis for tumor growth and metastasis. (+info)
Study of the murine allantois by allantoic explants.
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The murine allantois will become the umbilical artery and vein of the chorioallantoic placenta. In previous studies, growth and differentiation of the allantois had been elucidated in whole embryos. In this study, the extent to which explanted allantoises grow and differentiate outside of the conceptus was investigated. The explant model was then used to elucidate cell and growth factor requirements in allantoic development. Early headfold-stage murine allantoises were explanted directly onto tissue culture plastic or suspended in test tubes. Explanted allantoises vascularized with distal-to-proximal polarity, they exhibited many of the same signaling factors used by the vitelline and cardiovascular systems, and they contained at least three cell types whose identity, gene expression profiles, topographical associations, and behavior resembled those of intact allantoises. DiI labeling further revealed that isolated allantoises grew and vascularized in the absence of significant cell mingling, thereby supporting a model of mesodermal differentiation in the allantois that is position- and possibly age-dependent. Manipulation of allantoic explants by varying growth media demonstrated that the allantoic endothelial cell lineage, like that of other embryonic vasculatures, is responsive to VEGF(164). Although VEGF(164) was required for both survival and proliferation of allantoic angioblasts, it was not sufficient to induce appropriate epithelialization of these cells. Rather, other VEGF isoforms and/or the outer sheath of mesothelium, whose maintenance did not appear to be dependent upon endothelium, may also play important roles. On the basis of these findings, we propose murine allantoic explants as a new tool for shedding light not only on allantoic development, but for elucidating universal mechanisms of blood vessel formation, including vascular supporting cells, either in the intact organism or in existing in vitro systems. (+info)
Stimulation of tumor growth by human soluble intercellular adhesion molecule-1.
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Because serum levels of soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in cancer and sICAM-1 is angiogenic, we tested the ability of sICAM-1 to promote tumor growth. Our preliminary experiments showed that exogenous sICAM-1 significantly stimulated the growth of human tumors in vivo. Human fibrosarcoma transfectants, which express ICAM-1, produce ICAM-1 on the cell surface and release sICAM-1 into the medium without any apparent effect on cell growth in vitro. We found that conditioned medium from sense ICAM-1 transfectants compared with mock or antisense ICAM-1 transfectants stimulates endothelial cell migration in vitro and neovascularization in the chick chorioallantoic membrane assay. Tumor cells transfected with sense constructs form faster growing tumors than mock- and antisense-transfected cells in both chick embryos and nude mice models. Serum levels of human sICAM-1 from nude mice bearing sense ICAM-1 transfectants correlate positively with tumor weight. Sense ICAM-1 transfectants are more proliferative and induce more blood vessel formation than mock and antisense transfectants in nude mice. Because expression of ICAM-1 does not affect tumor cell growth in vitro, the angiogenic activity of sICAM-1 produced by sense ICAM-1 transfectants may be involved in the stimulation of tumor growth. Therefore, sICAM-1 may perform dual functions that are essential for tumor growth: angiogenesis and escape from immune surveillance. (+info)
Eotaxin (CCL11) induces in vivo angiogenic responses by human CCR3+ endothelial cells.
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Chemokines are attractants and regulators of cell activation. Several CXC family chemokine members induce angiogenesis and promote tumor growth. In contrast, the only CC chemokine, reported to play a direct role in angiogenesis is monocyte-chemotactic protein-1. Here we report that another CC chemokine, eotaxin (also known as CCL11), also induced chemotaxis of human microvascular endothelial cells. CCL11-induced chemotactic responses were comparable with those induced by monocyte-chemotactic protein-1 (CCL2), but lower than those induced by stroma-derived factor-1alpha (CXCL12) and IL-8 (CXCL8). The chemotactic activity was consistent with the expression of CCR3, the receptor for CCL11, on human microvascular endothelial cells and was inhibited by mAbs to either human CCL11 or human CCR3. CCL11 also induced the formation of blood vessels in vivo as assessed by the chick chorioallantoic membrane and Matrigel plug assays. The angiogenic response induced by CCL11 was about one-half of that induced by basic fibroblast factor, and it was accompanied by an inflammatory infiltrate, which consisted predominantly of eosinophils. Because the rat aortic sprouting assay, which is not infiltrated by eosinophils, yielded a positive response to CCL11, this angiogenic response appears to be direct and is not mediated by eosinophil products. This suggests that CCL11 may contribute to angiogenesis in conditions characterized by increased CCL11 production and eosinophil infiltration such as Hodgkin's lymphoma, nasal polyposis, endometriosis, and allergic diathesis. (+info)