Kimiko Takahashi

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.

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.

Increased secretion of urokinase-type plasminogen activator by human lung microvascular endothelial cells

Human lung microvascular endothelial cells (HLMECs) secreted 1.5-15 times more urokinase-type plasminogen activator (uPA) antigen than human hepatic microvascular endothelial cells, human umbilical vein endothelial cells (HUVECs), angioma endothelial cells, and lung fibroblasts. All of these cells also secreted a 100-fold greater amount of plasminogen activator inhibitor-1 than of uPA antigen, and uPA activities were not detected in the culture medium. The expression of uPA mRNA in HLMECs was higher (100-fold) compared with HUVECs, angioma endothelial cells, and lung fibroblasts. HLMECs secreted uPA antigen on both the luminal and basal sides of the cells. On the other hand, HLMECs secreted a 10- to 15-fold lower amount of tissue-type plasminogen activator than HUVECs, mostly on the luminal side. After stimulation with interleukin (IL)-1β, HLMECs secreted a six- to ninefold amount of uPA antigen. In contrast, no stimulatory effect was observed in HUVECs even under high IL-1β concentrations. The secretion of uPA and plasminogen activator inhibitor-1 from HLMECs was also enhanced by tumor necrosis factor-α and IL-2. These results suggest that HLMECs may contribute not only to the patency of lung vessels but also to the maintenance of alveolar functions through the production and secretion of uPA, especially in the presence of inflammatory cytokines.Human lung microvascular endothelial cells (HLMECs) secreted 1.5-15 times more urokinase-type plasminogen activator (uPA) antigen than human hepatic microvascular endothelial cells, human umbilical vein endothelial cells (HUVECs), angioma endothelial cells, and lung fibroblasts. All of these cells also secreted a 100-fold greater amount of plasminogen activator inhibitor-1 than of uPA antigen, and uPA activities were not detected in the culture medium. The expression of uPA mRNA in HLMECs was higher (100-fold) compared with HUVECs, angioma endothelial cells, and lung fibroblasts. HLMECs secreted uPA antigen on both the luminal and basal sides of the cells. On the other hand, HLMECs secreted a 10- to 15-fold lower amount of tissue-type plasminogen activator than HUVECs, mostly on the luminal side. After stimulation with interleukin (IL)-1beta, HLMECs secreted a six- to ninefold amount of uPA antigen. In contrast, no stimulatory effect was observed in HUVECs even under high IL-1beta concentrations. The secretion of uPA and plasminogen activator inhibitor-1 from HLMECs was also enhanced by tumor necrosis factor-alpha and IL-2. These results suggest that HLMECs may contribute not only to the patency of lung vessels but also to the maintenance of alveolar functions through the production and secretion of uPA, especially in the presence of inflammatory cytokines.

Basic Fibroblast Growth Factor Modulates the Surface Expression of Effector Cell Molecules and Primes Respiratory Burst Activity in Human Neutrophils

Basic fibroblast growth factor (b-FGF) mediates a variety of biological responses such as angiogenesis and hematopoiesis. We examined the effect of b-FGF on human neutrophil functions in vitro. The surface expression of effector cell molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. b-FGF increased the expression of CD11b leukocyte integrin and complement receptor type 1 on neutrophils and decreased the expression of L-selectin on neutrophils in a dose- and time-dependent manner. We also examined the effect of b-FGF on the respiratory burst activity in neutrophils. Although b-FGF alone did not induce intracellular oxidative product formation by neutrophils, it enhanced H2O2 production in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These findings suggest that b-FGF may participate in the inflammatory process via modulating the surface expression of effector cell molecules and enhancing respiratory burst activity in neutrophils.

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.

PRODUCTION OF HEPATOCYTE GROWTH FACTOR FROM HUMAN LUNG MICROVASCULAR ENDOTHELIAL CELLS INDUCED BY INTERLEUKIN-1β

To investigate the possible role of hepatocyte growth factor (HGF)in the reconstruction process following inflammatory damage in lung tissue, we compared HGF production of human lung microvascular endothelial cells (HL MECs) and human umbilical vein endothelial cells (HUVECs) after stimulation by interleukin(IL)-1 β. In an HL MEC-conditioned medium, largeamounts of total (single and 2-chain) HGF were detected, and were 26- to 28-fold higher than those in HUVECs or human lung fibroblasts. The production of total HGF increased in a dose-dependent manner (4.7 to 9.2 times) with IL-1 β. In contrast, the amount of HGF in an HUVEC-conditioned medium was unaffected by IL-1 β treatment. The amount of cell-associated HGF also showed a dose-related increase (140% to 160%) in HL MECs, but not in HUVECs with IL-1 β. In addition, HGF and c-met (HGF receptor) mRNAs in HL MECs and HUVECs were examined by the RT-PCR method. HGF and c-met mRNAs were clearly detected in HL MECs before and after treatment with IL-1 β, but not in HUVECs. These results suggest that increases in HGF production from HL MECs may play a role in the reconstruction process following inflammatory damage in lung tissue.To investigate the possible role of hepatocyte growth factor (HGF) in the reconstruction process following inflammatory damage in lung tissue, we compared HGF production of human lung microvascular endothelial cells (HLWECs) and human umbilical vein endothelial cells (HUVECs) after stimulation by interleukin(IL)-1beta. In an HLMEC-conditioned medium, large amounts of total (single and 2-chain) HGF were detected, and were 26- to 28-fold higher than those in HUVECs or human lung fibroblasts. The production of total HGF increased in a dose-dependent manner (4.7 to 9.2 times) with IL-1beta. In contrast, the amount of HGF in an HUVEC-conditioned medium was unaffected by IL-1beta treatment. The amount of cell-associated HGF also showed a dose-related increase (140% to 160%) in HLMECs, but not in HUVECs with IL1beta. In addition, HGF and c-met (HGF receptor) mRNAs in HLMECs and HUVECs were examined by the RT-PCR method. HGF and c-met mRNAs were clearly detected in HLMECs before and after treatment with IL-1beta, but not in HUVECs. These results suggest that increases in HGF production from HLMECs may play a role in the reconstruction process following inflammatory damage in lung tissue.

Transcytosis of lipid microspheres by human endothelial cells.

Human endothelial cells were cultivated on microporous membranes mimicking the luminal and basal spaces of blood vessels. When fluorescence-labeled lipid microspheres (LM) were added to the upper chambers of the model cultures, confluent monolayers of endothelial cells transported considerable levels of fluorescence to lower chambers. The transport was time dependent and was diminished by the addition of cytochalasin B. The uptake of LM into the endothelial cytoplasm was confirmed by electron microscopy and laser scanning confocal imaging. The amounts of fluorescence in the lower chamber were reduced when the endothelial cell layer was fixed with formaldehyde. These observations suggest that endothelial cells can transport LM by transcytosis. Endothelial cells seem to carry the LM without processing, since only minimal amounts of free fluorescence were detected even after longer cultivation periods. The fluorescence in the lower chambers of cell cultures treated with interleukin 1 beta was 3.7-fold higher than that of untreated cells; interleukin 2 and tumor necrosis factor alpha treatments had no discernible effect on LM transport. The interleukin 1 beta induced increase of transcytosis in endothelial cells would explain why LM preferentially accumulate in inflammatory tissues.

Subthreshold Hyperoxia Potentiates Tnf-α-Induced Icam-1 Expression on Cultured Pulmonary Microvascular Endothelial Cells

The effects of combined exposure to subthreshold hyperoxia and the inflammatory cytokine tumor necrosis factor alpha (TNF-alpha) on the expression of intercellular adhesion molecule-1 (ICAM-1) were examined in bovine lung microvascular endothelial cells (BLuEC). The expression of total ICAM-1 was not affected by 50% hyperoxia conditions alone, indicating that this level is subthreshold for BLuEC. In the presence of 5 ng/mL TNF-alpha, which has minimal influence on BLuEC alone, the amount of total ICAM-1 expression under 50% hyperoxia was higher than that in normoxic conditions (approximately 30%) throughout the culture period. The amount of soluble ICAM-1 that has been released into the culture medium increased after joint exposure to hyperoxia and TNF-alpha. These results suggest that exposure to subthreshold hyperoxia, which does not by itself cause damage to the endothelial cells or induce ICAM-1 expression, potentiates the effects of low-level TNF-alpha exposure.

INFECTION OF HUMAN LUNG FIBROBLASTS WITH EPSTEIN-BARR VIRUS CAUSES INCREASED IL-1ß AND bFGF PRODUCTION

An association between Epstein-Barr virus (EBV) infection and fibroblast proliferation in the interstitial spaces of the lung has been suggested in idiopathic interstitial pneumonia. In this study we show that EBV can infect human lung fibroblasts in vitro. A primary-cultured human lung fibroblast cell line, designated CCD-32Lu, expressed EBV nuclear antigen 1 after coculture with lethally irradiated EBV producing cells. The infection further induced CCD-32Lu cells to produce the fibrogenic cytokines basic fibroblast growth factor (bFGF) and interleukin-1beta. These findings indicate that lung fibroblasts may be a target for EBV infection and suggest that EBV may play a role in increased production of these cytokines and induce fibroblast proliferation in idiopathic interstitial pneumonia.