Poly-L-lysine/heparin stimulates angiogenesis in chick embryo chorioallantoic membrane.

The effects of heparin on angiogenesis are controversial, with some studies claiming stimulatory and other studies claiming inhibitory effects. Since heparin in human plasma is complexed with basic peptides and proteins, we studied the angiogenic effect of complexes resulting by mixing poly-L-lysine (a basic heparin-binding polypeptide) and heparin. Angiogenesis was investigate by chorioallantoic membrane assay. In specimens treated with PBS (negative control), or poly-L-lysine, no significant vascular reaction was detectable. Heparin induced only moderate angiogenic response. However, neutral complexes purified from a mixture of poly-L-lysine and heparin (20/1, w/w) induced a very strong angiogenic response. These results demonstrate that the angiogenic effect of heparin was associated with neutralization of electric charge when the polysaccharide was complexed with a basic peptide.     

Friend erythroleukemia cells induce angiogenesis in chick embryo chorioallantoic membrane and in human umbilical vein endothelial cells

The effects of Friend erythroleukemia cells on angiogenesis were studied in chick embryo chorioallantoic membrane assay and in human umbilical vein endothelial cells. In chorioallantoic membrane assay, the conditioned medium of Friend cells stimulated in vivo angiogenesis to an extent comparable to that observed with Prostaglandin El, used as positive control. Prostaglandin El added to conditioned medium of Friend cells did not further increase angiogenesis. Conditioned medium of Friend erythroleukemia cells also stimulated proliferation of human umbilical vein endothelial cells to an extent comparable to that observed with fetal bovine serum, used as positive control. Conditioned medium and fetal bovine serum together did not affect human umbilical vein endothelial cells proliferation, as compared to that observed when tested separately. These results seem to indicate that Friend erythroleukemia cells produce and secrete factors stimulating angiogenesis. These findings extend and confirm the hypothesis that successful angiogenesis is necessary for development of leukemias.     

Corticotropin releasing factor stimulates cAMP formation in pituitary corticotropic tumor cells

Addition of corticotropin-releasing factor (CRF) to membranes from two ACTH-secreting pituitary tumors strikingly increased in a dose-dependent fashion adenylate cyclase (AC) activity. Significant stimulation was already apparent at 10(-9)M CRF. Stimulation of AC activity by CRF in membranes from non-tumoral tissue adjacent to tumoral corticotrophs was considerably lower, and was lacking in membranes from a growth hormone secreting tumor. These data correlated well with in vivo pre-surgery and post-surgery ACTH responsiveness to CRF of the tumor bearing patients. Basal AC activity was higher in pituitary adenomas than in non-tumoral adjacent tissue. It is concluded that 1) a CRF-sensitive AC exists in ACTH-secreting tumor cells and, 2) increased sensitivity to CRF, as evidenced by greater stimulation of AC activity, may be responsible for the increased ACTH output of tumoral corticotrophs.     

Prostaglandin E2 rather than lymphocyte-activating factor produced by activated human mononuclear cells stimulates increases in murine thymocyte cAMP

Culture supernatants (SUPS) of endotoxin (LPS)-activated human mononuclear cells (MNL) stimulated greater production of cAMP by thymocytes than by spleen cells of C3H/HeJ or nude ($\text{nu}\text{nu}$) mice. Similarly, the addition of prostaglandin E₂ (PGE₂) stimulated higher levels of cAMP in thymocytes and progressively lower levels in spleen cells from C3H/HeJ mice and $\text{nu}\text{nu}$ spleen cells, respectively. Partial purification on Bio-Gel P100 of the LPS-induced MNL SUPS yielded peaks of thymocyte proliferative activity characteristic of lymphocyte activation factor (LAF) but these fractions failed to stimulate cAMP levels in thymocytes. Moreover, MNL SUPS induced with LPS in the presence of indomethacin retained their LAF activity but no longer increased thymocyte cAMP levels. Radioimmunoassay of the SUPS for PGE₂ revealed significantly higher levels of PGE₂ in the media of those MNL cultures stimulated by LPS than when stimulated by phorbol myristic acetate, phytohemagglutin, or extracted cell wall fraction of Actinomyces viscosus. Thus, PGE₂ is produced by human MNL and may exert considerable immunoregulatory effects mediated by elevation of lymphocyte cAMP levels.     

A novel human macrophage-activating factor: distinction from interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GMCSF)

Culture supernatant from a human T-cell leukemia virus type I (HTLV-1)-infected cell line, DGA-1, contained a novel macrophage-activating factor (MAF). This MAF was antigenically and functionally distinct from interferon-gamma (IFN-gamma) and from granulocyte-monocyte colony-stimulating factor (GMCSF). Potential contaminants such as bacterial lipopolysaccharide (LPS), Mycoplasma spp, and HTLV-1 were not responsible for this MAF activity. The DGA-1 MAF was secreted constitutively and the cell line grew well in the absence of growth factors such as interleukin-2, mitogen, or antigen. This cell line should provide a good source of this MAF for further purification and characterization.     

Osteogenin (bone morphogenetic protein-3) stimulates cartilage formation by chick limb bud cells in vitro.

Osteogenin is a protein isolated from demineralized bovine bone matrix. When implanted in rats, osteogenin induces the differentiation of cartilage and formation of endochondral bone. When added to stage 24 and 25 chick limb bud mesoderm cells in culture, it stimulated synthesis of sulfated proteoglycans by over 10-fold without stimulating cell division. The increase was detected after only 2 days in culture. Morphologically, in the presence of osteogenin, all cells in the culture appeared to form cartilage, rather than the nodules of cartilage surrounded by noncartilage areas in control cultures. The distribution of type II collagen correlated with the morphological differentiation of cartilage. When nonchondrocyte and chondrocyte cell populations were separated, osteogenin stimulated sulfated proteoglycan synthesis in all populations of cells. However, the greatest stimulation (24-fold) was seen in the originally nonchondrocyte population, which apparently still had some potential to form cartilage. In this study, chick limb bud mesoderm cells in vitro responded to osteogenin, a protein derived from adult bovine bone matrix. The cells that were responsive included those that initially did not form cartilage. Osteogenin belongs to a superfamily of proteins, many of which are important in development. It is possible that osteogenin has a role in embryonic cartilage development.     

Endothelin stimulates both cAMP formation and phosphatidylinositol hydrolysis in cultured embryonic bovine tracheal cells.

Embryonic bovine tracheal (EBTr) cells were found to possess receptors for endothelin (ET) of ET-1-selective (ETA) subtype with a Kd for ET-1 of 114 pM and a Bmax of 12.9 fmol/10(5) cells. Stimulation of EBTr cells with 100 pM to 100 nM ET-1 increased the contents of both inositol phosphates and cAMP in a concentration-dependent manner, indicating that the receptors are coupled to both phosphatidylinositol hydrolysis and cAMP formation in EBTr cells.     

SK&F 96365 inhibits histamine-induced formation of endothelium-derived relaxing factor in human endothelial cells.

Formation of endothelium-derived relaxing factor (EDRF) strictly correlates with the intracellular free Ca2+ ([Ca2+]i) concentration. We now demonstrate that the histamine-induced rise in [Ca2+]i of human umbilical vein endothelial cells is mostly due to activation of a membrane current which allows Ca2+ entry. This membrane current is sensitive to the novel inhibitor of agonist-induced Ca2+ entry, SK&F 96365, which blocked the histamine-induced sustained rise in [Ca2+]i, as well as 45Ca2+ uptake and membrane currents. Inhibition of the above cellular responses to histamine was accompanied by a considerable reduction of EDRF formation and release. Thus biosynthesis and release of EDRF from human umbilical vein endothelial cells significantly depend on agonist-induced Ca2+ entry involving receptor-operated Ca(2+)-permeable channels which can be blocked by SK&F 96365.     

Ultrastructure of oral (buccopharyngeal) membrane formation and rupture in the hamster embryo

The ultrastructure of the oral (buccopharyngeal) membrane was examined during normal development in embryos removed from pregnant hamsters at intervals from 7.5–10 days postcoitum. The oral membrane is represented at 7.5 days by a region of close approximation between endoderm and surface ectoderm anterior to the neural folds. A distinct basal lamina develops subjacent to each epithelial germ layer, and the narrow extracellular space separating the epithelia contains patches of fibrillar and flocculent material. Cell processes extend from cells of one epithelium across the extracellular space to make direct contact with cells of the opposing germ layer by 7.75 days. Increased intermingling of cells subsequently occurs within the oral membrane, and some cells extend the entire width of the membrane with surfaces exposed to both foregut and stomodeum. Accumulations of presumed basal lamina and extracellular material are observed at intervals within the oral membrane, but a continuous intercellular space is no longer present. Many of these accumulations are encompassed by processes of adjacent cells containing dense intracellular bodies, indicating active phagocytosis of this material by the epithelial cells. Rupture of the oral membrane begins between 8.25 and 8.75 days, and all remnants are removed by Day 9. Possible factors involved in rupture are discussed.     

Effect of multiplication stimulating activity (MSA) on intracellular cAMP levels and adenylate cyclase activity in chick embryo fibroblasts.

Stimulation of hamster lymph node cells, splenocytes and thymocytes by the mitogen phytohemagglutinin-P (PHA) was found to be greatly enhanced by addition of 1–10 mM LiCl to the cultures. Lithium enhanced stimulation, as determined by [³H]TdR incorporation, only if added within the first 24 h of culture. The enhancing effect of lithium was specific for this monovalent cation since equivalent concentrations of KCl or NaCl did not induce a similar effect on [³H]TdR incorporation. The divalent cations Mg²⁺ (1–10 mM) and Ca²⁺ (1-1.6 mM), also had an enhancing effect on PHA stimulation. However, addition of Li⁺ to cultures enhanced with Mg²⁺ and/or Ca²⁺ led to an additional potentiation of the response to PHA. These results suggest that Li⁺ modifies a unique early event during stimulation of lymphoid cells by this mitogen.     

Angiostatic activity and metabolism of cortisol in the chorioallantoic membrane (CAM) of the chick embryo.

There is considerable interest in the discovery of compounds which inhibit angiogenesis dependent (neovascular) diseases. The chick embryo, due to the rapid development of an extensive vascular capillary network in the chorioallantoic membrane (CAM), has been used extensively as a model for studying angiogenesis. Angiostatic steroids are a new class of compounds which inhibit the growth of new capillaries in the chick CAM and in other models of neovascularization. Despite the potential therapeutic importance of these compounds, little is known about the ability of the CAM to metabolize these steroids. We have evaluated the ability of the chick CAM to metabolize cortisol which is both an angiostatic steroid as well as a glucocorticoid. When CAM homogenate was incubated with [3H]cortisol and NADPH at 37 degrees C and pH 7.4, and the reaction products analyzed by reverse phase HPLC, [3H]cortisol was converted exclusively to 20 beta-dihydrocortisol (4-pregnen-11 beta,17 alpha,20 beta,21-tetrol-3-one). The cortisol metabolite, 20 beta-dihydrocortisol, has very little glucocorticoid activity, but shows significant angiostatic activity in the CAM comparable to cortisol. The apparent Km determined for cortisol metabolism was 12 microM and the observed Vmax was 1.4 mumol cortisol/mg protein/min. The majority of the 20 beta-reductase activity was found in the soluble (242,000 g) fraction of CAM homogenate. 20 beta-Reductase activity in chick embryo CAM has not been previously reported.     

VIP elevates platelet cyclic AMP (cAMP) levels and inhibits in vitro platelet activation induced by platelet-activating factor (PAF)

Platelet-activating factor (PAF), a potent endogenous phospholipid released by a variety of mammalian cells, induces platelet activation in vivo and in vitro. Little is known, however, about the physiological modulation of its actions. We have examined the ability of two naturally occurring compounds which stimulate cAMP production, vasoactive intestinal peptide (VIP) and prostacyclin (PGI2), to inhibit PAF-induced platelet aggregation and secretion in vitro. Washed, [3H]serotonin-labeled, rabbit platelets were incubated 60 sec in the presence of VIP, PGI2 or 3-isobutyl-1-methylxanthine (IBMX) and subsequently stimulated with PAF. In separate studies, cAMP levels were determined in similar aliquots of platelets incubated for 30 sec with VIP, PGI2 or IBMX. VIP, PGI2 and IBMX inhibited platelet aggregation and secretion in a dose-dependent manner. Fifty percent inhibition was achieved at final concentrations of 1.7 X 10(6) M VIP, 3.6 X 10(6) M PGI2 and 6.5 X 10(5) M IBMX. IBMX potentiated the inhibitory effects of VIP and PGI2 on PAF-induced platelet activation. VIP and PGI2 elevated platelet cAMP levels four-fold and 50-fold, respectively, in the presence of 10(3) M IBMX. These findings demonstrate that VIP inhibits PAF-induced platelet activation, with a potency comparable to that of PGI2.     

Interactions between somite cells: the formation and maintenance of segment boundaries in the chick embryo

We have investigated the interactions between the cells of the rostral and caudal halves of the chick somite by carrying out grafting experiments. The rostral half-sclerotome was identified by its ability to support axon outgrowth and neural crest cell migration, and the caudal half by the binding of peanut agglutinin and the absence of motor axons and neural crest cells. Using the chick-quail chimaera technique we also studied the fate of each half-somite. It was found that when half-somites are placed adjacent to one another, their interactions obey a precise rule: sclerotome cells from like halves mix with each other, while those from unlike halves do not; when cells from unlike halves are adjacent to one another, a border is formed. Grafting quail half-somites into chicks showed that the fates of the rostral and caudal sclerotome halves are similar: both give rise to bone and cartilage of the vertebral column, as well as to intervertebral connective tissue. We suggest that the rostrocaudal subdivision serves to maintain the segmental arrangement when the mesenchymal sclerotome dissociates, so that the nervous system, vasculature and possibly vertebrae are patterned correctly.     

Production of nerve growth-stimulating factor(s) from chick embryo heart cells : Use of Cytodex 3 microcarriers and serum-free media

Medium conditioned by embryonic chick heart cells is known to support extensive neurite outgrowth from autonomic and sensory neurons. In the present report we describe the use of microcarrier cell culture with serum-free media to scale up the production of the nerve growth-stimulating factors. A growth medium composed of DME /F10 supplemented with insulin, transferrin, human serum albumin and fibronectin in combination with a low molecular weight (MW) fraction of fetal calf serum (FCS) or a mixture of FGF, dexamethasone, calmodulin and thrombin supported the heart cell proliferation at a rate similar to that of medium with 10% FCS. Furthermore, the level of successively accumulated nerve growth activity measured in a bioassay with sympathetic ganglia proved to be nearly equivalent to what was obtained when cells were grown in medium containing serum. The results confirm the potential of microcarrier cell culture in serum-free media for the production and subsequent recovery of a specific cell product.