Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells

Krause, Petra; Markus, Peter M.; Schwartz, Peter J.; Unthan-Fechner, Kirsten; Pestel, Sabine; Fandrey, Joachim; Probst, Irmelin

doi:10.1016/s0168-8278(00)80239-1

Cited by 46 publications

(62 citation statements)

References 27 publications

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“…To determine whether recapitulation of the sinusoidal microenvironment could regulate lymphocyte adhesion to ECs, we performed flow-based adhesion assays using endothelium cocultured with hepatocytes/HepG 2 cells or cultured alone and 12 The parallel flow chamber is constructed from a Perspex upper plate (2) incorporating a machined groove or flow channel in the lower surface (3), a gasket (4), and a lower plate (5) that accommodated the tissue culture insert (B) into a receiving slot (6) and was secured with a gasket and a Perspex plate (8). (B) When the culture insert was inserted into the lower plate, it protruded through the gasket and formed the base of the flow channel defined in the upper plate.…”

Section: Resultsmentioning

confidence: 99%

“…However, paracrine interactions with other cells in the microenvironment are likely to provide important signals that can act as cues to regulate endothelial function, 6,7 as demonstrated by the dependence of liver sinusoidal EC proliferation and differentiation on the local production of vascular endothelial growth factor by hepa-tocytes. [8][9][10] This intimate relationship between intrahepatic ECs and hepatocytes led us to propose that signaling between these cell types could induce the expression of sinusoidal endothelial adhesion molecules and thereby the regulation of lymphocyte trafficking into the liver. Such a relationship may explain the unique molecular regulation of leukocyte recruitment across the sinusoidal bed and the distinct patterns of infiltration in inflammatory liver disease.…”

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confidence: 99%

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Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes

et al. 2005

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Crosstalk between hepatic sinusoidal ECs and closely juxtaposed hepatocytes via vascular endothelial growth factor is essential for the maintenance of sinusoidal endothelial growth and differentiation. We propose that paracrine interactions between endothelial cells and hepatocytes also may be responsible for the unique complement of adhesion receptors expressed on sinusoidal endothelium that regulate the recruitment of lymphocytes into the liver. To address this hypothesis, we developed an in vitro model of the hepatic sinusoid in which flowing lymphocytes could interact with hepatic endothelium conditioned by the presence of hepatocytes. Human hepatic sinusoidal endothelial cells cocultured with hepatocytes were activated so that they supported the adhesion of lymphocytes at levels equivalent to those seen on endothelium stimulated with the inflammatory cytokine tumour necrosis factor-␤.

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes

et al. 2005

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“…Because higher serum concentrations (Ͼ5%) have been reported to be harmful for rat SECs, 24 the low concentration was used throughout the in vitro studies. After 3 hours (designated as "time 0"), medium was replaced with new medium with or without OV or VEGF.…”

Section: Isolation and Culture Of Secsmentioning

confidence: 99%

Maintenance of Bad Phosphorylation Prevents Apoptosis of Rat Hepatic Sinusoidal Endothelial Cells in Vitro and in Vivo

Ohi

Nishikawa

Tokairin

et al. 2006

The American Journal of Pathology

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To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signalregulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.

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“…Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1 . Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11 .In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SECspecific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.…”

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confidence: 88%

<em>In vivo</em> Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

Gopalakrishnan

Harris

2011

JoVE

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The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (˜100 μm) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of . These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4 . Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6 . For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7 .With the exception of one report 8 , there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1 . Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11 .In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SECspecific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis. Video LinkThe video component of this article can be found at https://www.jove.com/video/3138/ Protocol 1. Excision of the liver (adopted from P.O. Seglen 12 and R. Blomhoff 13 with modifications 14,15 )1. Add 10 mL 30% isoflurane in polyethylene glycol to a petri dish in a closed 5.5 L chamber. It is optimal to wait 10-15 min after addition of isoflurane to create a stabilized atmosphere within the chamber. 2. Place a rat in the sealed chamber and allow it to become anesthetized. Full effects of the anesthesia are apparent when the animal becomes limp, unresponsive, and exhibits deep breathing. 3. Place 2 mL of isoflurane in po...

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Hepatocyte-supported serum-free culture of rat liver sinusoidal endothelial cells

Cited by 46 publications

References 27 publications

Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes

Lymphocyte traffic through sinusoidal endothelial cells is regulated by hepatocytes

Maintenance of Bad Phosphorylation Prevents Apoptosis of Rat Hepatic Sinusoidal Endothelial Cells in Vitro and in Vivo

<em>In vivo</em> Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

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