Sinusoidal Endothelial Cells from Normal Guinea Pig Liver: Isolation, Culture and Characterization†

Shaw, R. Gideon; Johnson, Alice; Schulz, Werner; Zahlten, Rainer N.; Combes, Burton

doi:10.1002/hep.1840040403

Cited by 48 publications

(17 citation statements)

References 33 publications

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“…Hepatocytes were obtained by using a modification of a rat liver perfusion method (21). Kupffer cells were prepared as described (22), and endothelial cells were prepared by a modification of the method of Shaw et al (23). All cells were incubated in 10% fetal calf serum (GIBCO) in a medium (GIBCO) for 24 hr to regenerate protease-sensitive receptors.…”

Section: Methodsmentioning

confidence: 99%

Macrophages specifically regulate the concentration of their own growth factor in the circulation.

Bartocci¹,

Mastrogiannis²,

Migliorati³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

237

120

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The physiological mechanism of clearance of the mononuclear phagocyte growth factor, colony-stimulating factor 1 (CSF-1), from the circulation of normal mice was investigated by following the fate of a trace amount of i.v.injected '25I-labeled CSF-1. Macrophages selectively cleared CSF-1 by CSF-1 receptor-mediated endocytosis and degraded the growth factor intracellularly. This manner of clearance provides a feedback control mechanism whereby the rate of macrophage production is determined by the number of mature macrophages.

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Section: Methodsmentioning

confidence: 99%

Macrophages specifically regulate the concentration of their own growth factor in the circulation.

Bartocci¹,

Mastrogiannis²,

Migliorati³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

237

120

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“…Nonparenchymal cells (NPCs) were prepared by collagenase and protease digestion of the liver, and cultured as described previously. [57][58][59] Stellate cells were separated from the other NPCs by centrifugation on a Nycodenz gradient. 58 Kupffer cells and endothelial cells were separated on a metrizamide gradient, followed by centrifugal elutriation.…”

Section: Western Analysis Of Alr In Hepatic Extracts and Recombinant mentioning

confidence: 99%

A Fresh Look At Augmenter of Liver Regeneration in Rats

et al. 1999

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Augmenter of liver regeneration (ALR) is a hepatotrophic protein originally identified by bioassay in regenerating rat and canine livers following partial hepatectomy and in the hyperplastic livers of weanling rats, but not in resting adult livers. The ALR gene and gene product were subsequently described, but little is known about the cellular/subcellular sites of ALR synthesis in the liver, or about the release and dissemination of the peptide. To obtain this information in rats, we raised antibodies in rabbits against rat ALR for development of an enzyme-linked immunosorbent assay (ELISA). ALR concentrations were then determined in intact livers of unaltered weanling and adult rats; in regenerating residual liver after partial hepatectomy; in cultured hepatocytes and nonparenchymal cells (NPCs); and in culture medium and serum. ALR in the various liver cells was localized with immunohistochemistry. In addition, hepatic ALR and ALR mRNA were assayed with Western blotting and reversetranscriptase polymerase chain reaction (RT-PCR), respectively. The hepatocyte was the predominant liver cell in which ALR was synthesized and stored; the cultured hepatocytes secreted ALR into the medium in a timedependent fashion. Contrary to previous belief, the ALR peptide and ALR mRNA were present in comparable concentrations in the hepatocytes of both weanling and resting adult livers, as well as in cultured hepatocytes. A further unexpected finding was that hepatic ALR levels decreased for 12 hours after 70% hepatectomy in adult rats and then rose with no corresponding change in mRNA transcripts. In the meantime, circulating (serum) ALR levels increased up to 12 hours and declined thereafter. Thus, ALR appears to be constitutively expressed in hepatocytes in an inactive form, and released from the cells in an active form by unknown means in response to partial hepatectomy and under other circumstances of liver maturation (as in weanling rats) or regeneration. (HEPATOLOGY 1999;29:1435-1445.)The control of hepatic growth and regeneration has interested experimentalists for much of the 20th century. 1 Soon after the classical description in 1931 by Higgins and Anderson 2 of liver regeneration in rats following 70% hepatectomy, a search began for growth factors within the liver itself. McJunkin and Breuhaus 3 observed that the modest mitotic response to a 30% to 40% hepatectomy in rats was enhanced with an intraperitoneal injection 2 days postoperatively of homogenized homologous rat liver. Two decades later, Teir and Ravanti 4 and Blomquist 5 noted that this ''augmentation'' effect was demonstrable only when the injected homogenates were prepared from regenerating liver fragments following hepatectomy or from weanling rat livers that have a naturally heightened mitotic index. Subsequently, LaBrecque and Pesch 6 reported the same prerequisite of a hyperplastic liver source for cytosol extracts containing a putative ''hepatic stimulatory substance'' (HSS).Importantly, however, a cocondition for demonstrating a mitosis-augmenting a...

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“…Medium was changed at 24 hours. During culture, the purity of sinusoidal endothelial cells was at least 93% as determined by transmission electron microscopy 22 and absence of 0.81 µm latex bead phagocytosis (a property of Kupffer cells). 22 All experiments on sinusoidal endothelial cells were performed on day 2.…”

Section: Methodsmentioning

confidence: 99%

“…During culture, the purity of sinusoidal endothelial cells was at least 93% as determined by transmission electron microscopy 22 and absence of 0.81 µm latex bead phagocytosis (a property of Kupffer cells). 22 All experiments on sinusoidal endothelial cells were performed on day 2. In other work, we have shown the viability of endothelial cells cultured this way to continue well into the end of day 3, as shown by trypan blue exclusion (86 Ϯ 4% of sinusoidal endothelial cells excluded trypan blue).…”

Section: Methodsmentioning

confidence: 99%

Role of oxidative stress in hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells

2000

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To characterize the role of oxidative stress in cultured rat sinusoidal endothelial cells, we studied the production of superoxide after reoxygenation, the relationship of reduced glutathione (GSH) levels to cell injury, and the protective efficacy of antioxidants. Hypoxia (pO 2 1-2 mm Hg) was achieved by culturing cells under 95% N 2 5% CO 2 for 4 hours. Reoxygenation was then reestablished, and viability was determined at 24 hours by trypan blue exclusion; putative protective agents were added at the time of reoxygenation (4 hours). As previously reported, reoxygenation after 4 hours hypoxia accentuated sinusoidal cell death fourfold compared with hypoxic or normoxic controls (P F .0001). Superoxide was not produced on reoxygenation, and superoxide dismutase provided no protection against reoxygenation injury. Cellular levels of GSH fell to 37 ؎ 4% of normoxic controls (P F .0001) following reoxygenation. These changes were essentially abrogated by Trolox (Aldrich Chemical Co., Milwaukee, WI) and dimethyl sulfoxide, both of which also completely protected against reoxygenation injury. When cellular GSH levels were lowered by addition of diethylmaleate (which conjugates GSH), this reduced the viability of endothelial cells cultured under normoxic conditions and greatly augmented reoxygenation injury. Conversely, addition of exogenous GSH partially protected endothelial cells against hypoxia-reoxygenation injury. Desferrioxamine also protected against reoxygenation injury, but catalase was only partly protective. It is concluded that sinusoidal endothelial cells undergo significant intracellular oxidative stress following reoxygenation, and their viability is critically dependent on GSH levels. Reactive oxygen species are likely mediators of oxidative stress in hepatic sinusoidal endothelial cells. (HEPATOLOGY 2000;31:160-165.)In the intact liver, oxidative stress appears to be part of the mechanism for ischemia-reperfusion injury and is related to the generation of reactive oxygen species (ROS) 1-4 and possibly nitric oxide. 5 However, the role of individual cell types in the production of these potentially cytotoxic mediators remains uncertain. Conversely, sinusoidal endothelial cells appear to be a major target of ischemia-reperfusion (or hypoxia-reoxygenation) injury. [6][7][8] The resultant endothelial injury disrupts the microcirculation, reducing blood flow and enhancing further tissue necrosis. Neutrophils adhere to damaged endothelial cells and their resultant activation is likely to be one source of ROS. In the present studies, we have explored the possibility that sinusoidal endothelial cells could themselves generate oxidative stress.Earlier in vitro studies on the effects of hypoxia-reoxygenation on endothelial cells have usually been performed on cultures of endothelial cells derived from large blood vessels, such as the pulmonary artery and umbilical vein. 9,10 From this work, it is apparent that nonhepatic endothelial cells are susceptible to hypoxia-reoxygenation injury through the generation of...

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Sinusoidal Endothelial Cells from Normal Guinea Pig Liver: Isolation, Culture and Characterization†

Cited by 48 publications

References 33 publications

Macrophages specifically regulate the concentration of their own growth factor in the circulation.

Macrophages specifically regulate the concentration of their own growth factor in the circulation.

A Fresh Look At Augmenter of Liver Regeneration in Rats

Role of oxidative stress in hypoxia-reoxygenation injury to cultured rat hepatic sinusoidal endothelial cells

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