Dataset Information

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells.

ABSTRACT:

Background

Brain endothelial cell-based in vitro models are among the most versatile tools in blood-brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial cell lines of in vitro blood-brain barrier models has not been investigated in detail, our aim was to characterize this system in different models.

Methods

For the investigation, we have chosen two widely-used models for in vitro drug transport studies: the bEnd.3 mouse and the hCMEC/D3 human brain endothelial cell line. We compared the structures and attributes of their endo-lysosomal system to that of primary porcine brain endothelial cells.

Results

We detected significant differences in the vesicular network regarding number, morphology, subcellular distribution and lysosomal activity. The retromer-positive vesicles of the primary cells were distinct in many ways from those of the cell lines. However, the cell lines showed higher lysosomal degradation activity than the primary cells. Additionally, the hCMEC/D3 possessed a strikingly unique ratio of recycling endosomes to late endosomes.

Conclusions

Taken together our data identify differences in the trafficking network of brain endothelial cells, essentially mapping the endo-lysosomal system of in vitro blood-brain barrier models. This knowledge is valuable for planning the optimal route across the blood-brain barrier and advancing drug delivery to the brain.

SUBMITTER: Toth AE

PROVIDER: S-EPMC6542060 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Publications

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells.

Toth Andrea E AE Nielsen Simone S E SSE Tomaka Weronika W Abbott N Joan NJ Nielsen Morten S MS

Fluids and barriers of the CNS 20190530 1

<h4>Background</h4>Brain endothelial cell-based in vitro models are among the most versatile tools in blood-brain barrier research for testing drug penetration to the central nervous system. Transcytosis of large pharmaceuticals across the brain capillary endothelium involves the complex endo-lysosomal system. This system consists of several types of vesicle, such as early, late and recycling endosomes, retromer-positive structures, and lysosomes. Since the endo-lysosomal system in endothelial c ...[more]

PMID: 31142333

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Project description:Ischemic stroke (IS) is a major neurological disease with high fatality and residual disability burdens. Long noncoding RNAs (lncRNAs) have been found to play an important role in IS. However, the roles and significance of most lncRNAs in IS are still unknown. This study was performed to identify differentially expressed (DE) lncRNAs using a lncRNA microarray in whole blood samples of patients suffering from acute cerebral ischemia. Bioinformatics analyses, including GO, KEGG pathway enrichment analysis, and proximity to putative stroke risk location analysis were performed. The novel lncRNA, ENST00000530525, significantly decreased after IS. Furthermore, we evaluated lncRNA ENST00000530525 expression in cultured hCMEC/D3 cells under oxygen-glucose deprivation/reoxygenation (OGD/R) conditions using fluorescent in situ hybridization (FISH) and quantitative real-time polymerase chain reaction (RT-qPCR) analysis. To investigate the function of lncRNA ENST00000530525, its over-expression (OE) and negative control (NC) plasmids were transfected into hCMEC/D3 cells, and cell viability was detected by a cell counting kit-8 (CCK-8) assay after OGD/R. LncRNA ENST00000530525 and ANO1 expression were investigated using RT-qPCR and immunofluorescence. For blood-brain barrier (BBB) permeability, FITC-dextran transendothelial permeability assay and tight junction (TJ) protein immunofluorescence assays were performed. There were 3352 DE lncRNAs in the blood samples of acute IS patients. The validation results were consistent with the gene chip data. The GO and KEGG results showed that these lncRNAs were mainly related to oxygen and glucose metabolism, leukocyte transendothelial migration, mitophagy and cellular senescence. Among these, lncRNA ENST00000530525 was the most highly downregulated lncRNA and it was mapped within the IS-associated gene anoctamin-1 (ANO1). We further found that lncRNA ENST00000530525 was downregulated in hCMEC/D3 cells under 4 h OGD and 20 h reoxygenation (OGD4/R20) conditions. Upregulating lncRNA ENST00000530525 by plasmid transfection decreased cell viability while increasing ANO1 expression and it contributed to BBB injury in hCMEC/D3 cells after OGD4/R20. The lncRNA ENST00000530525 might play deleterious roles in post-stroke pathogenesis. These results show that some DE lncRNAs in humans participate through characteristic roles in post-stroke pathogenesis; thus, the roles and significance of some novel lncRNAs in IS warrant further study.

Dataset Information

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells.

Background

Methods

Results

Conclusions

Publications

The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells.

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