Tamotsu Goto

Spontaneous transformation and immortalization of human endothelial cells.

SummaryA new cell line from the human umbilical vein has been established and maintained for more than 5 yr (180 generations; 900 population doublings). This strain, designated ECV304, is characterized by a cobblestone monolayer growth pattern, high proliferative potential without any specific growth factor requirement, and anchorage dependency with contact inhibition. Karyotype analysis of this cell line reveals it to be of human chromosomal constitution with a high trisomic karyotype (mode 80). Ultrastructurally, endothelium-specific Weibel-Palade bodies were identified. Although one of the endothelial cell markers, Factor VIII-related antigen (VIIIR:Ag) was negative in this cell line, immunocytochemical staining for the lectin Ulex europaeus I (UEA-I), and PHM5 (anti-human endothelium as well as glomerular epithelium monoclonal antibody) was positive, and angiotensin-converting enzyme (ACE) activity was also demonstrated. In addition, ECV304 displayed negativity for alkaline and acid phosphatase and for the epithelial marker keratin. All of these findings suggest that ECV304 cells originated from umbilical vein endothelial cells by spontaneous transformation. Ultrastructurally, no viruslike particles have been detected intracellularly. Nude mouse tumorigenicity and rabbit cornea tests were both positive. This is a report on a novel case of phenotypic alteration of normal venous endothelial cells of human origin in vitro, and generation of a transformant with indefinite life spans. This line may be useful in studies of some physiologically active factors available for medical use.

Inhibition of in vitro angiogenesis by lymphotoxin and interferon-γ

The effects of lymphotoxin (LT) and interferon (IFN)-gamma on the capillary formation was examined using an in vitro angiogenesis model system. Both LT and IFN-gamma inhibited the capillary formation in a dose dependent manner. To elucidate the mode of action, effects of the lymphokines on endothelial and myofibroblastic cells were studied. We found that the lymphokines inhibited not only the growth of endothelial cells but also the production of collagen by myofibroblastic cells. These results suggest that the pleiotropic effects of the lymphokines on different types of cells might result in the inhibition of the capillary formation.

Development of capillary networks from rat microvascular fragments in vitro: The role of myofibroblastic cells

A new model useful for studying capillary growth in vitro is described. When the microvessel fragments and accompanying single cells (myofibroblastic cells) from rat epididymal fat pads were co-cultivated, the myofibroblastic cells initially began to grow and reached confluence. A few days later, endothelial cells started to sprout from the vessel fragments, forming cellular cord networks on and in the multilayered myofibroblastic cells. Ultrastructurally, the lumina, surrounded by the endothelial cells having intercellular junctions, were observed at cross-sectioned cellular cords. The growth of cellular cords from the fragments always occurred after the myofibroblastic cells had reached confluence. The medium conditioned to isolated rat myofibroblastic cells stimulated not only the proliferation of the endothelial cells from the bovine capillary and human vein but also the migration of bovine capillary endothelial cells in vitro. Moreover, the extracellular matrix produced by rat myofibroblastic cells modulated the morphology of bovine capillary endothelial cells to a cordlike shape. These observations strongly suggest that the formation of the capillary in vitro is induced by myofibroblastic cells.

Effects of tumor necrosis factor (TNF) on transplanted tumors induced by methylcholanthrene in mice. A histopathologic study.

SummaryThe effects of a highly purified tumor necrosis factor (TNF) on transplanted methylcholanthrene (Meth A)-induced murine tumors were compared with those of lipopolysaccharide (LPS). TNF caused immediate subepidermal edema and hyperemia followed 2 h later by fibrin thrombi in tumor blood vessels. Finally hemorrhagic necrosis with dispersal of tumor cells occurred. LPS produced similar hemorrhagic necrotizing changes. However, the necrotic action of LPS was delayed and complete tumor regression was not achieved with LPS. These findings suggest that tumor necrosis induced by TNF is due to circulatory disturbance associated with a microvascular injury in the tumor manifested by hyperemia and multiple fibrin thrombi.

Necrotizing activity of tumor necrosis factor: histopathological investigation using Meth A sarcoma and granulation tissue

SummaryThe action of tumor necrosis factor (TNF) was investigated histopathologically in mice using methylcholanthrene A (Meth A)-induced sarcomas and granulation tissue induced by autotransplantation of fragments of liver and spleen. Highly purified murine TNF caused hemorrhagic necrosis of both the tumors and the granulation tissue. Proliferation of tumor capillaries, demonstrated microangiographically, occurred 2 h after TNF administration and hyperemia of tumor vessels was obvious after 3–6 h. Hyperemia and capillary leakage were also observed in the granulation tissue 6 h after TNF injection and hemorrhage was noted in the epidermis after 12 h. These results strongly suggest that the in vivo necrotizing action of TNF is mainly related to capillary injury.

Cultivation of arterial endothelial cells from human umbilical cord

We have developed a simple method for the isolation of endothelial cells from human umbilical artery. The method provides a sufficient number of cells to be of experimental value. The presence of factor VIII antigen specific for endothelium has been demonstrated by immunofluorescence as well as by the peroxidase-antiperoxidase immune complex method.

Identification of non heparin-binding endothelial cell growth factor from rat myofibroblasts

Myofibroblasts (Mfs) from rat fat tissues produced a potent endothelial cell growth factor (Mf-ECGF). The growth factor activity found in the conditioned media from primary cultures of Mfs, was labile to heat (80 degrees C for 10 min) and proteinase (trypsin), and did not bind to heparin in the presence of 0.2 M NaCl. Mf-ECGF was partially purified 4760-fold with a recovery of 25% from serum-free conditioned media by sequential carboxymethyl (CM) ion-exchange column chromatography and gel filtration. This Mf-ECGF activity was recovered from the 40 kD region of a non-reducing SDS-PAGE, and from the pH region between 6.5 and 7 of isoelectric focusing, with recoveries of 20% and 65%, respectively. These results indicated that a major portion of ECGF activity in the conditioned media was clearly distinct from other well-known endothelial cell growth factors including fibroblast growth factors (FGFs).