Yoshio Sawasaki

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.

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.

Close similarity between cultured human omental mesothelial cells and endothelial cells in cytochemical markers and plasminogen activator production.

SummaryThe mesothelial cells obtained from human omental adipose tissue showed a typical cobblestone monolayer and reacted strongly with keratin, but did not have Von Willebrand factor. Ultrastructurally these cells revealed the existence of desmosome-like cell junctions as well as intracellular canaliculi, tubular structures surrounded by microvilli, and tonofilament-like filaments.The mesothelial cells grew much faster in the medium containing epidermal growth factor, actively took up acetylated-low density lipoprotein into their cytoplasm, and released angiotensin-converting enzyme. They also released urokinase-type plasminogen activator, but only half as much as do human umbilical vein endothelial cells; release of tissue-type plasminogen activator was not observed. Inasmuch as the mesothelial cells also released plasminogen activator inhibitor-1, as do human umbilical vein endothelial cells, we could not detect u-PA activity in culture medium. u-PA may play a role in the protection against adhesion among visceral organs. These observations indicate that cultured human mesothelial cells have characteristics closely related to those found in human endothelial cells.

The restoration of the functions of serially passaged calf hepatocytes by spheroid formation

SummaryA serial cultivation system of hepatocytes was established for the first time using calf liver as a cell source and, repeating passage of more than 30 cumulative population doublings (PDs), was obtained in the presence of long-acting ascorbic acid derivative (L-ascorbic acid 2-phosphate) and epidermal growth factor. The complete purification of hepatocytes was achieved by repeating ethylenediaminetetraacetic acid (EDTA) treatment, by which hepatocytes were easily detached from the culture dish, leaving most of the nonparenchymal cells on the dish. As the population cumulatively doubled, the cell density and albumin-synthesizing ability decreased gradually, and doubling time has exceeded 120 h at about 30 cumulative PDs. In serially passaged cells, the hepatocyte-specific histochemical and biochemical markers—including glucose-6-phosphatase, ornithine carbamoyltransferase, glutamate hydrogenase, and ammonia-metabolizing activities—have been lost after 20 cumulative PDs. However, when these passaged cells were allowed to form spheroids, the morphologic and biochemical characteristics of hepatocytes have rapidly been restored to levels comparable to those in younger generations. Because no extrinsic factor was needed for this restoration, three-dimensional cell-cell interaction would be indispensable for the differentiation of the hepatocytes. The routine serial cultivation of hepatocytes and their redifferentiation by spheroid formation will be useful for studying metabolism, gene regulation, and transplantation of hepatocytes.

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.

EFFECTS OF LOW-FREQUENCY ELECTRIC FIELDS ON THE INTRACELLULAR Ca2+ RESPONSE INDUCED IN HUMAN VASCULAR ENDOTHELIAL CELLS BY VASOACTIVE SUBSTANCES

Human vascular endothelial cells (HUVEC) were exposed to air electric fields of 30 kV/m, 50 Hz for 24 hr. After exposure, the change in intracellular Ca2+ concentration ([Ca2+]i) induced by the vasoactive substances ATP and histamine were determined using confocal laser scanning microscopy. A statistically significant increase in [Ca2+]i was observed following stimulation with 100 μM ATP, compared with the results in sham-exposed cells (22%, p<0.05, n=20). No changes in [Ca2+]i due to the field were observed after stimulation with histamine, showing that the effect of the field on the change in [Ca2+]i was agonist-specific. The Ca2+ concentration in the medium was low, suggesting that the ATP-induced increase in [Ca2+]i in cells exposed to electric fields was due to the release of Ca2+ from intracellular stores. The results suggested that the applied low-frequency electric field affected the function of HUVEC via a change in [Ca2+]i.

Culture of Porcine Fetal Pancreatic Neurons

Objectives: Pancreatic neurons have not been cultured commonly. Cultured neurons can be continuously observed, and their external environment is easy to be controlled. We report here a simple method for separating and cultivating neuronal cells from pancreas. Methods: Pancreata of fetal swine were digested with collagenase. Clusters were collected with a sieve and digested with trypsin. Digested clusters were collected and cultured in Dulbecco modified Eagle medium containing serum and basic fibroblast growth factor. Cultured cells were investigated morphologically. Results: Cultured cells formed spiderweblike colonies. These cells were distinguished into Schwann cells and 2 types of neurons. The neurons were positive on immunocytochemical staining with antigrowth-associated protein-43 and cytochemical staining for cholinesterase. One type of neuron was located in the central cluster and had very long processes extending radially. The other type of neuron was sparsely scattered, had long processes, and was connected to other neurons. The neurotransmitter of these neurons was concluded to be acetylcholine. Conclusions: Using our method, neuronal cells were readily cultured from pancreatic tissue. These cells will be useful in elucidating the physiology and pharmacology of pancreatic neurons.