Biomedical subjects

I Flamme

Publications and source records attributed to I Flamme.

26 records · Page 2Linked to original sources

PubMed1992

A comparative study on the effects of tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF) and basic fibroblast growth factor (bFGF) on the chorioallantoic membrane of the chick embryo.

The chorioallantoic membrane (CAM) assay is a widely used bioassay for testing angiogenic activities. In the present study we compared the gross and micromorphological effects of three angiogenic factors applied in Elvax carriers on the CAM: Tumor necrosis factor-alpha (TNF-alpha), human angiogenic factor (h-AF), and basic fibroblast growth factor (bFGF). Our question was whether the CAM responds to these factors which have very different actions with a stereotype or with a factor specific reaction. By microangiography and light microscopy, all positive reactions appeared as a spoke-wheel vascular pattern with a bundle of small capillary blood vessels in the center. These vessels were predominantly of a distended type in h-AF and TNF experiments, while narrower capillary vessels followed bFGF application. Chorioallantoic ectoderm and endoderm were thickened by cell accumulation and the mesenchymal stroma of the CAM was edematous and infiltrated with leucocytes in all three reactions. Additionally, bFGF experiments showed areas of densely arranged fibroblasts. Observations in vivo showed chorioallantoic tissue movements as a possible mechanism for the spokewheel vascular pattern. As compared with our results from studies of cytokinetics with bromodeoxyuridine, these current findings indicate that chemotaxis is responsible for the chorioallantoic angiogenic reaction rather than cellular proliferation.

Allantois↗

PubMed1992

Induction of vasculogenesis and hematopoiesis in vitro.

Despite a large number of investigations of embryonic vascular development, in particular in avian embryos, the conditions under which the endothelial and hematopoietic cell lineages emerge remain unknown. As we demonstrate here, both endothelial and hematopoietic cells can be induced by treatment of dissociated quail epiblast with fibroblast growth factors in vitro. These cells aggregate in characteristic blood islands. In long-term culture, the induced endothelial cells gave rise to vascular structures in vitro, i.e. vasculogenesis. No induction was observed in the absence of fibroblast growth factors, and other growth factors like TGF-beta, TGF-alpha and EGF were not capable of inducing blood island formation. Thus, the dissociated quail epiblast provides a remarkably simple test system to investigate cell lineage diversification in higher vertebrates.

Animals↗

PubMed1991

Two-phase in vitro culture of explanted chick embryos.

The present study describes a method of culturing chick embryos together with their surrounding area vasculosa on two different culture media in succession. Embryos in the 2nd day of incubation (stages 13, 14, 15 according to Hamburger and Hamilton, 1951) were explanted from the yolk with the aid of a ring of filter paper and transferred dorsal side up to a silicone culture dish containing the first culture medium (89.5% L-15, 10% fetal calf serum, 0.5% Antibiotics). The paper ring was clamped onto the wall of the culture dish by a steel ring so that the embryo was fixed for the culture period. After 4 +/- 1, 8 +/- 1, 12 +/- 1 hrs, the embryos were taken from the culture dishes and transferred to others containing a yolk-albumen mixture as culture medium; 81.2% of embryos survived the first phase of culture. On the second medium 50.3% of explanted embryos were still alive at stage 20 (HH), and 7.9% of them reached the 5th day of development (St 25 HH). The average length of survival in vitro was found to be influenced by both the length of the first culture phase and the stage at which embryos were explanted. This culture method may be useful for teratological tests, since in the first phase of culture, concentrations of test substances and the time of exposure can be exactly adjusted, and in the second phase, the embryo is allowed to develop quite normally, under conditions similar to those in ovo.

Animals↗

PubMed1991

Mitogenic activity of chicken chorioallantoic fluid is temporally correlated to vascular growth in the chorioallantoic membrane and related to fibroblast growth factors.

The chorioallantoic membrane (CAM) is one of the most vascularized tissues in the chicken embryo. Capillary growth proceeds until day 10 of development and thereafter abruptly regresses. As it is generally accepted that the formation of new blood vessel is regulated by growth factors, we have investigated the presence of angiogenic and mitogenic factors in the chicken chorioallantois. In the present study, we show that chorioallantoic fluid (CAF) contains angiogenic substances that are probably synthesized in the CAM or the embryonic kidney. When applied in the chorioallantoic membrane assay, CAF from 9 day chicken embryos elicits a strong angiogenic response. This angiogenic activity of CAF is associated with pronounced mitogenic effects in vitro. Comparison of different embryonic fluids reveals that mitogenic activity is particularly evident in the CAF but not detectable in embryonic serum and amnion fluid. Expression of mitogenic activity is found to be temporally correlated with vascular growth in the CAM. High activity is detected in CAF prior to day 10 and then sharply decreases, thus preceding termination of capillary growth by one day. Heparin-sepharose affinity chromatography suggests that the biological activities of CAF probably correspond to the presence of acidic and basic fibroblast growth factor (aFGF and bFGF). In Western blot analyses of CAF, an immunoreactive bFGF-like protein of about 17 x 10(3) Mr is recognized by a monospecific anti-bFGF antiserum. This protein elutes at 2.4 M NaCl from the heparin-sepharose. The mitogenic activity of the CAF can be specifically blocked by the anti-bFGF antibody indicating bFGF to be the active mitogenic principle of the CAF. These results strongly suggest that basic and probably acidic FGF play an important role in the regulation of chorioallantoic vascular growth.

Allantois↗

PubMed1990

Reexpression of alpha-smooth muscle actin isoform in cultured adult rat cardiomyocytes.

Expression of alpha-smooth muscle (sm) actin in regenerating adult cardiomyocytes in culture was investigated. No alpha-sm-actin could be detected in adult ventricular tissue or in newly dissociated rod-shaped cells, whereas a fraction of the polymorphic flattened out adult cardiac cells in culture did express the protein. Immunofluorescence studies revealed a characteristic staining pattern, suggesting the preferential presence of alpha-sm-actin in stress fiber-like structures, while newly formed myofibrils contained only little alpha-sm-actin isoprotein. Cell-cell contacts were resumed, but formation of new gap junctions, as revealed by microinjecting Lucifer yellow, was not dependent on alpha-sm-actin expression. The behavior corresponds to fetal cardiomyocytes either in tissue or as single cells in culture where expression of alpha-sm-actin can be observed. Such immunofluorescence staining patterns with corresponding immunoblot data can be expected when a return to a less differentiated, more fetal state of the adult cardiomyocyte in culture is assumed. The possible role of the alpha-sm-actin and alpha-sarcomeric actin isoforms during reformation of myofibrillar sarcomeres is discussed.

Actins↗

PubMed1989

Is extraembryonic angiogenesis in the chick embryo controlled by the endoderm? A morphology study.

The area vasculosa of the chick embryo is subdivided into two concentric zones: the inner transparent area pellucida vasculosa (AVP) and the less transparent surrounding area opaca vasculosa (AOV). The different optical properties of these zones are caused by the different morphology of the endoderm, which consists of flat cells in the APV and of high-prismatic cells containing large yolk vacuoles in the AOV. The present study describes how this endodermal subdivision of the area vasculosa is related to the development of the extraembryonic vascular pattern. By injection of ink into the vascular system of chick embryos at stages 12 to 20 (Hamburger and Hamilton 1951 "HH"), it has been demonstrated that the vascular net of the area vasculosa from stage 14 (HH) onwards develops into different patterns in APV and AOV. The small loops of uniform capillary vessels of stage 13 (HH) are widened due to the rapid expansion of the extraembryonic mesoderm. In the AOV from stage 14 (HH) onwards numerous small blood vessels sprout into the enlarged intervascular spaces. This process is maximal at stage 17 (HH). In contrast, the blood vessels of the APV remain largely unbranched. These findings suggest that the development of the extraembryonic vascular pattern is controlled by the endodermal pattern. To test this hypothesis, both zones (APV and AOV) were examined by light microscopy, transmission and scanning electron microscopy, in vivo observations and by treatment with bromodeoxyuridine (BrdU). TEM examinations show that the ultrastructural organization of the APV mesoderm is different from that of the AOV: The splanchnopleuric cells of the APV form a continuous cover around the endothelial cells connected by numerous desmosomes, whereas the splanchnopleuric cells of the AOV are frequently separated by gaps. The largest gaps are seen in the small blood vessels at stage 17 (HH). These results should be considered in relation to the dynamic changes in the vascular pattern of the AOV. The endodermal cells of APV and AOV are two different populations. In vivo observation of the endodermal transition from APV to AOV detected no transformations of APV cells into AOV cells or vice versa. The borderline between the zones is stable. The AOV endoderm, having been overgrown by the expanding mesoderm, stops proliferating almost completely, whereas the proliferation of the APV endoderm is unaffected by contact with the mesoderm. The rate of its proliferation is approximately as high as that of the AOV prior to contact with the expanding mesoderm (results after treatment with BrdU).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1987

Prolonged and simplified in vitro culture of explanted chick embryos.

The method described in this paper allows explanted chick embryos to develop normally in vitro from the 2nd to the 5th day of incubation. The embryos are explanted with the aid of a ring of filter-paper and are fixed between two steel-rings dorsal side up for cultivation. The lower ring contains a mixture of yolk and albumen as culture medium. 40% of the explanted embryos reach the 5th day of development (stage 25, Hamburger and Hamilton). The maximum time of culture is 70 h after explantation.

Amnion↗

PubMed1987

Edge cell migration in the extraembryonic mesoderm of the chick embryo. An experimental and morphological study.

The expansion of the extraembryonic mesoderm was investigated in chick embryos of 2 and 3 days incubation with special regard to the mesodermal edge cells. These cells are lying immediately distal to the sinus terminalis and have the shape of migrating cells. By SEM examination they appear to be linked together to form a uniform edge which extends numerous spike-like filopodia. The shape of these filopodia corresponds to their microtubule pattern, as shown by immunofluorescence staining. Filopodia contain strong bundles of microtubules. By in vivo observation at high magnification, the migration of edge cells was demonstrated, and the results of SEM and immunofluorescence studies could be confirmed. By local application of cytochalasin D, distal to the region of the sinus terminalis, the migration of edge cells was inhibited selectively. Subsequent to the inhibition of migration, the expansion of the mesoderm stopped although the interstitial growth of the mesoderm in drug-treated regions remained unaffected. Thus the edge cells have a promotor function in the expansive growth of the extraembryonic mesoderm. The proliferating mesoderm, located proximally to the edge cells, has no expansive tendency of its own. The selectivity of the cytochalasin effect was checked by examination of the phalloidin stained actin pattern. Furthermore, by in vivo observations at low magnification and by transplantation of endoderm from quail to chick it could be confirmed that the extraembryonic mesoderm spreads out invasively between ectoderm and endoderm separating the two sheets. The promotion of this invasion can be regarded as an additional function of the edge cells. An expansion of the mesoderm can also be observed after endoderm removal. In regions freed from endoderm the mesoderm expands faster than in adjacent regions still covered by endoderm. There is no promoting influence of endoderm on mesodermal expansion. On the contrary, expansion itself is facilitated, when the conditions for invasion are abolished by removing the endoderm.

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