Molecular response of liver sinusoidal endothelial cells on hydrogels

Bartneck, Matthias; Topuz, Fuat; Tag, Carmen G.; Sauer-Lehnen, Sibille; Warzecha, Klaudia Theresa; Weiskirchen, Ralf; Tacke, Frank

doi:10.1016/j.msec.2015.02.045

Cited by 14 publications

(15 citation statements)

References 30 publications

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“…LSECs of the liver and hepatocytes were separated as shown in Fig. S3, following the protocol previously described (Bartneck et al 2015;Meyer et al 2016). Briefly, two to three month old male mice were anesthetized with isoflurane, a catheter was inserted from the right atrium into the suprahepatic portion of the inferior vena cava, and the liver was perfused with pre-warmed wash buffer (calcium-free Hank's Balanced Salt Solution (HBSS) supplemented with 5 UI/ ml heparin, 0.1% glucose, 25 mM HEPES, 0.5 mM EGTA, 100 UI/ml penicillin and 100 μg/ml streptomycin) for 5 min at a flow rate of 5 ml/minute, then with digestion buffer (Iscove's modified eagle medium (IMDM) with Glutamax containing 80 U/ml type IV collagenase (Thermo Fisher Scientific) and 0.08 μg/ml DNase I (Sigma)) for 5 min.…”

Section: Isolation Of Lsecs From the Livermentioning

confidence: 99%

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

et al. 2020

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Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the Stab2 locus. Stab2 mRNA in the LSECs was significantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), in the promoter region of Stab2 DBA which likely interferes with normal expression in the LSECs. In contrast, in other tissues of DBA/2J mice, the IAP drives high ectopic Stab2 DBA transcription starting within the 5′ long terminal repeat of IAP in a reverse orientation and continuing through the downstream Stab2 DBA . Ectopic transcription requires the Stab2-IAP element but is dominantly suppressed by the presence of loci on 59.7-73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9-73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-finger proteins that target transposable elements-derived sequences and repress their expression. Despite the high amount of ectopic Stab2 DBA transcript in tissues other than liver, STAB2 protein was undetectable and unlikely to contribute to the plasma HA levels of DBA/2J mice. Nevertheless, the IAP insertion and its effects on the transcription of the downstream Stab2 DBA exemplify that stochastic evolutional events could significantly influence susceptibility to complex but common diseases.

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Section: Isolation Of Lsecs From the Livermentioning

confidence: 99%

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

et al. 2020

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“…However, the comparison of the LSEC populations obtained by CD146+ MACS to our technique showed that significant higher proportions of macrophages (4.85±1.11%) and unidentified cells (9.20±2.63%) were still present in the final cell population using the CD146+ MACS technique. To increase purity of LSEC isolated using this method, some authors optimized the procedure by adding a CD45magnetic sorting to remove blood cells [29,56]. Further, we observed a decreased viability in the population obtained by the CD146+ MACS technique, suggesting that the positive sorting either led to a non-specific retention of dead cells or decreased cell viability.…”

Section: Discussionmentioning

confidence: 85%

“…Of note, the presence of contaminating macrophages in the final preparation could severely impair the reliability of studies using primary cells. MACS for CD146+ [19][20][21][22][23], CD31+ [24], CD105+ [25,26], CD45- [27] cells and FACS for CD146+ [28] or CD45-CD146+ [29] cells were reported for LSEC isolation in mice.…”

Section: Introductionmentioning

confidence: 99%

An optimized method for mouse liver sinusoidal endothelial cell isolation

Meyer

Lacotte

Morel

et al. 2016

Experimental Cell Research

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The objective of the present study was to develop an accurate and reproducible method for liver sinusoidal endothelial cell (LSEC) isolation in mice. Non-parenchymal cells were isolated using a modified two-step collagenase digestion combined with Optiprep density gradient centrifugation. LSEC were further purified using two prevalent methods, short-term selective adherence and CD146+ magnetic-activated cell sorting (MACS), and compared in terms of cell yield, viability and purity to our purification technique using CD11b cell depletion combined with long-term selective adherence. LSEC purification using our technique allowed to obtain 7.07±3.80 million LSEC per liver, while CD146+ MACS and short-term selective adherence yielded 2.94±1.28 and 0.99±0.66 million LSEC, respectively. Purity of the final cell preparation reached 95.10±2.58% when using our method. In contrast, CD146+ MACS and short-term selective adherence gave purities of 86.75±3.26% and 47.95±9.82%, respectively. Similarly, contamination by non-LSEC was the lowest when purification was performed using our technique, with a proportion of contaminating macrophages of only 1.87±0.77%. Further, isolated cells analysed by scanning electron microscopy presented typical LSEC fenestrations organized in sieve plates, demonstrating that the technique allowed to isolate bona fide LSEC. In conclusion, we described a reliable and reproducible technique for the isolation of high yields of pure LSEC in mice. This protocol provides an efficient method to prepare LSEC for studying their biological functions.

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“…Differences in cell fate specification, inefficient transitions of a given cell phenotype through specific stages of development, and intrinsic heterogeneity existing within populations of progenitor cells1 can each result in complex admixtures of many distinct cell types, and identifying and characterizing individual cell types in that mixture can be challenging. Other examples include the need to identify and characterize cells isolated from primary tissues such as liver23, pancreatic islets45, brain6, cardiomyocytes7 or blood leukocytes8. Assessing cellular differences in drug toxicity within a given tissue preparation can also be confounded if, for example, a sparsely represented cell type, but not the major parenchymal cell type, is targeted and eliminated by the drug.…”

Section: A Need For Functional Assessment Of Heterogeneous Mixture Ofmentioning

confidence: 99%

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

Neal

Rountree

Radtke

et al. 2016

Sci Rep

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Functional characterization of individual cells within heterogeneous tissue preparations is challenging. Here, we report the development of a versatile imaging method that assesses single cell responses of various endpoints in real time, while identifying the individual cell types. Endpoints that can be measured include (but are not limited to) ionic flux (calcium, sodium, potassium and hydrogen), metabolic responsiveness (NAD(P)H, mitochondrial membrane potential), and signal transduction (H2O2 and cAMP). Subsequent to fluorescent imaging, identification of cell types using immunohistochemistry allows for mapping of cell type to their respective functional real time responses. To validate the utility of this method, NAD(P)H responses to glucose of islet alpha versus beta cells generated from dispersed pancreatic islets, followed by the construction of frequency distributions characterizing the variability in the magnitude of each individual cell responses were compared. As expected, no overlap between the glucose response frequency distributions for beta cells versus alpha cells was observed, thereby establishing both the high degree of fidelity and low rate of both false-negatives and false-positives in this approach. This novel method has the ability not only to resolve single cell level functional differences between cell types, but also to characterize functional heterogeneity within a given cell type.

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Molecular response of liver sinusoidal endothelial cells on hydrogels

Cited by 14 publications

References 30 publications

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice

An optimized method for mouse liver sinusoidal endothelial cell isolation

A method for high-throughput functional imaging of single cells within heterogeneous cell preparations

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