Development of a New Conditionally Immortalized Human Liver Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs), which are specialized endothelial cells that line liver sinusoids, have been reported to participate in a variety of liver functions, such as blood macromolecule clearance and factor VIII production. In addition, LSECs play crucial roles in liver regeneration following acute liver injury, as well as the development and progression of liver diseases or drug-induced hepatotoxicity. However, the molecular mechanisms underlying their roles remain mostly unknown. Therefore, in order to contribute to the clarification of those mechanisms, herein we report on the development of a new immortalized human LSEC (HLSEC) line. To produce this cell line, two immortalized genes were introduced into the primary HLSECs, which eventually resulted in the establishment of the HLSEC/conditionally immortalized, clone-J (HLSEC/ciJ). Consistent with the two-immortalized gene expression, HLSEC/ciJ showed excellent proliferation activity. Additionally, the results of gene expression analyses showed that several LSEC (as well as pan-endothelial) marker mRNAs and proteins were clearly expressed in HLSEC/ciJ. Furthermore, we found that adherence junction proteins were localized at the cell border in the HLSEC/ciJ monolayer, and that the cells exhibited a tube-like structure formation property. Taken together, the results obtained thus far indicate that we have successfully immortalized HLSECs, resulting in creation of HLSEC/ciJ, a cell line that possesses infinite proliferation ability while retaining possession of at least some HLSEC features. We believe that the HLSEC/ciJ have the potential to provide a valuable and unlimited alternative source of HLSECs for use in liver/LSEC physiology/pathophysiology, pharmacology, and toxicology studies.

Establishment and characterization of a new conditionally immortalized human astrocyte cell line.

Astrocytes are the most abundant cell types in mammalian brains, within which they participate in various neuronal activities, partly by utilizing the numerous transporters expressed at their plasma membranes. Accordingly, detailed characterization of astrocytic functions, including transporters, are essential for understanding of mechanistic basis of normal brain functions, as well as the pathogenesis and treatment of various brain diseases. As a part of overall efforts to facilitate such studies, this study reports on the establishment of a new human astrocyte cell line, which is hereafter referred to as human astrocyte/conditionally immortalized, clone 35 (HASTR/ci35). This line, which was developed utilizing a cell immortalization method, showed excellent proliferative ability and expressed various astrocyte markers, including glial fibrillary acidic protein. When co-cultured with neuronal cells, HASTR/ci35 cells could facilitate their dendritic network formation. Furthermore, HASTR/ci35 cells not only possessed significant glutamate and adenosine transporter activities but also exhibited organic ion transporter activities. To summarize, HASTR/ci35 cells possess several key astrocytic characteristics, including various transporter functions, while simultaneously showing infinite proliferation and scalability. Based on these findings, HASTR/ci35 cells can be expected to contribute significantly to various human astrocyte study fields. In vitro astrocyte models are valuable experimental tools in various astrocyte studies. Here, we report the establishment of a new human astrocyte cell line, HASTR/ci35, which show various key astrocyte properties, including astrocytic transporter activities, glycogen storage and facilitation of neuronal cell differentiation. Thus, HASTR/ci35 is expected to significantly contribute to advances toward detailed understanding of human astrocyte functions.

Hydrocortisone enhances the barrier properties of HBMEC/ciß, a brain microvascular endothelial cell line, through mesenchymal-to-endothelial transition-like effects.

BACKGROUND: Because in vitro blood-brain barrier (BBB) models are important tools for studying brain diseases and drug development, we recently established a new line of conditionally immortalized human brain microvascular endothelial cells (HBMEC/ciß) for use in such models. Since one of the most important functional features of the BBB is its strong intercellular adhesion, in this study, we aimed at improving HBMEC/ciß barrier properties by means of culture media modifications, thus enhancing their use for future BBB studies. In addition, we simultaneously attempted to obtain insights on related mechanistic properties. METHODS: Several types of culture media were prepared in an effort to identify the medium most suitable for culturing HBMEC/ciß. The barrier properties of HBMEC/ciß were examined by determining Na(+)-fluorescein permeability and transendothelial electric resistance (TEER). Endothelial marker mRNA expression levels were determined by quantitative real-time polymerase chain reaction. Adherens junction (AJ) formation was examined by immunocytochemistry. Cell migration ability was analyzed by scratch assay. Furthermore, cellular lipid composition was examined by liquid chromatography-time-of-flight mass spectrometry. RESULTS: Our initial screening tests showed that addition of hydrocortisone (HC) to the basal medium significantly reduced the Na(+)-fluorescein permeability and increased the TEER of HBMEC/ciß monolayers. It was also found that, while AJ proteins were diffused in the cytoplasm of HBMEC/ciß cultured without HC, those expressed in cells cultured with HC were primarily localized at the cell border. Furthermore, this facilitation of AJ formation by HC was in concert with increased endothelial marker mRNA levels and increased ether-type phosphatidylethanolamine levels, while cell migration was retarded in the presence of HC. CONCLUSIONS: Our results show that HC supplementation to the basal medium significantly enhances the barrier properties of HBMEC/ciß. This was associated with a marked phenotypic alteration in HBMEC/ciß through orchestration of various signaling pathways. Taken together, it appears that overall effects of HC on HBMEC/ciß could be summarized as facilitating endothelial differentiation characteristics while concurrently retarding mesenchymal characteristics.

Functional analysis of purine nucleoside phosphorylase as a key enzyme in ribavirin metabolism.

Ribavirin is a purine nucleoside analogue that possesses potent anti-hepatitis C virus activity, and it has long been considered likely that ribavirin undergoes a first-pass metabolism at the small intestine. Although purine nucleoside phosphorylase (PNP) is assumed to be involved in this metabolism, this has not been conclusively demonstrated. Furthermore, no pharmacogenomic studies related to PNP-mediated ribavirin phosphorolysis have previously been conducted. In this study, we sought to identify the role of PNP in ribavirin phosphorolysis in the human small intestine, and to clarify the effect of the single nucleotide polymorphism (rs1049564) on PNP's ribavirin phosphorolysis activity. The results of our investigations show that PNP is abundantly expressed in the human small intestine, and that intestinal ribavirin phosphorolysis is severely inhibited by ganciclovir, a PNP-inhibitor. Therefore, PNP is likely to play a primary role in the ribavirin phosphorolysis in the human small intestine. On the other hand, the results of our attempt to clarify the function of rs1049564 show that it does not affect PNP's ribavirin phosphorolysis activity. We believe that the present study will facilitate further pharmacogenomic and biochemical characterization of PNP as a key metabolic enzyme of ribavirin.

Unique expression features of cancer-type organic anion transporting polypeptide 1B3 mRNA expression in human colon and lung cancers.

BACKGROUND: We have previously identified the cancer-type organic anion transporting polypeptide 1B3 (Ct-OATP1B3) mRNA in several human colon and lung cancer tissues. Ct-OATP1B3 is a variant of the liver-type OATP1B3 (Lt-OATP1B3) mRNA, which is a hepatocyte plasma membrane transporter with broad substrate specificity. However, in cancer tissues, both the detailed characteristics of Ct-OATP1B3 mRNA expression and its biological functions remain unclear. With this point in mind, we sought to characterize Ct-OATP1B3 mRNA expression in colon and lung cancer tissues. In addition, we attempted to obtain functional implication of Ct-OATP1B3 in cancer cells. METHODS: Matched pairs of cancer and normal tissues were collected from 39 colon cancer and 28 lung cancer patients. The OATP1B3 mRNA expression levels in each of these tissues were separately determined by quantitative real-time polymerase chain reaction. Mann-Whitney U test and Fisher's exact test were used in statistical analysis. The Ct-OATP1B3 functional expression in colon cancer cells was then examined by Western blotting and transport analyses. RESULTS: Ct-OATP1B3 mRNA, but not Lt-OATP1B3 mRNA, was abundantly expressed in colon cancer tissues at a higher detection frequency (87.2%) than that of the adjacent normal tissues (2.6%). Furthermore, it was found that Ct-OATP1B3 mRNA expression was often detected in early colon cancer stages (88.9%, n = 18), and that its expression was associated with well-differentiated colon cancer statuses. On the other hand, Ct-OATP1B3 mRNA also showed a predominant and cancer-associated expression profile in lung tissues, although at frequencies and expression levels that were lower than those obtained from colon cancer. As for attempts to clarify the Ct-OATP1B3 functions, neither protein expression nor transport activity could be observed in any of the cell lines examined. CONCLUSIONS: Based on the unique characteristics of the Ct-OATP1B3 mRNA expression profile identified in this study, Ct-OATP1B3 mRNA can be expected to become a biomarker candidate for use in colon (and lung) cancer diagnosis. Simultaneously, our results advance the possibility that Ct-OATP1B3 might play yet unidentified roles, in addition to transporter function, in cancer cell biology.

Establishment of a new conditionally immortalized cell line from human brain microvascular endothelial cells: a promising tool for human blood-brain barrier studies.

The blood-brain barrier (BBB) is formed by brain microvascular endothelial cells (BMEC) working together with astrocytes and pericytes, in which tight junctions and various transporters strictly regulate the penetration of diverse compounds into the brain. Clarification of the molecular machinery that provides such regulation using in vitro BBB models has provided important insights into the roles of the BBB in central nervous system (CNS) disorders and CNS drug development. In this study, we succeeded in establishing a new cell line, hereinafter referred to as human BMEC/conditionally immortalized, clone ß (HBMEC/ciß), as part of our ongoing efforts to develop an in vitro human BBB model. Our results showed that HBMEC/ciß proliferated well. Furthermore, we found that HBMEC/ciß exhibited the barrier property of restricting small molecule intercellular penetration and possessed effective efflux transporter functions, both of which are essential to a functioning BBB. Because higher temperatures are known to terminate immortalization signals, we specifically examined the effects of higher temperatures on the HBMEC/ciß differentiation status. The results showed that higher temperatures stimulated HBMEC/ciß differentiation, marked by morphological alteration and increases in several mRNA levels. To summarize, our data indicates that the newly established HBMEC/ciß offers a promising tool for use in the development of a practical in vitro human BBB model that could make significant contributions toward understanding the molecular biology of CNS disorders, as well as to CNS drug development. It is also believed that the development of a specific culture method for HBMEC/ciß will add significant value to the HBMEC/ciß-based BBB model.