Bidirectional apical–basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells

Siupka, Piotr; Hersom, Maria; Lykke‐Hartmann, Karin; Johnsen, Kjeld; Thomsen, Louiza Bohn; Andresen, Thomas Lars; Moos, Torben; Abbott, N. Joan; Brodin, Birger; Nielsen, Morten S.

doi:10.1177/0271678x17700665

Cited by 22 publications

(12 citation statements)

References 93 publications

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“…At this time points, the models were validated by measuring transendothelial electrical resistance. The obtained value for control (80 ± 3.5 Ω × cm 2 ), for differentiated monoculture (513 ± 22.91 Ω × cm 2 ) and for co-culture (914 ± 38.66 Ωxcm 2 ), was in the range of previously described data from other groups [36][37][38].…”

Section: In Vitro Bbb Model Constructionssupporting

confidence: 84%

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

et al. 2018

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Receptor- and adsorptive-mediated transport through brain endothelial cells (BEC) of the blood-brain barrier (BBB) involves a complex array of subcellular vesicular structures, the endo-lysosomal system. It consists of several types of vesicles, such as early, recycling, and late endosomes, retromer-positive structures, and lysosomes. Since this system is important for receptor-mediated transcytosis of drugs across brain capillaries, our aim was to characterise the endo-lysosomal system in BEC with emphasis on their interactions with astrocytes. We used primary porcine BEC in monoculture and in co-culture with primary rat astrocytes. The presence of astrocytes changed the intraendothelial vesicular network and significantly impacted vesicular number, morphology, and distribution. Additionally, gene set enrichment analysis revealed that 60 genes associated with vesicular trafficking showed altered expression in co-cultured BEC. Cytosolic proteins involved in subcellular trafficking were investigated to mark transport routes, such as RAB25 for transcytosis. Strikingly, the adaptor protein called AP1-μ1B, important for basolateral sorting in epithelial cells, was not expressed in BEC. Altogether, our data pin-point unique features of BEC trafficking network, essentially mapping the endo-lysosomal system of in vitro BBB models. Consequently, our findings constitute a valuable basis for planning the optimal route across the BBB when advancing drug delivery to the brain.

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Section: In Vitro Bbb Model Constructionssupporting

confidence: 84%

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

et al. 2018

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“…3a). Retrograde-transported receptors represent a new and exciting target for drug delivery to the brain, particularly since the retrograde-receptor mannose-6-phosphate receptor has been described in PBEC as a potential target for receptor-mediated transcytosis [35]. Our findings should be taken into consideration when choosing an appropriate in vitro model for investigation of retromer-transported ligands.…”

Section: Discussionmentioning

confidence: 85%

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

Toth

Nielsen

Tomaka

et al. 2019

Fluids Barriers CNS

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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. Electronic supplementary material The online version of this article (10.1186/s12987-019-0134-9) contains supplementary material, which is available to authorized users.

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“…A similar distribution of recombinant NPC2 produced following transfection could explain why a transfection efficiency similar to that previously observed for erythropoietin using a similar setup would result in less recombinant protein secreted to the cell media in the case of NPC2. Moreover as the endothelial cells present the M6P receptor MPR300 on the plasma membrane, for which NPC2 is a natural ligand, there might be a re‐uptake of secreted recombinant NPC2, which would decrease the extracellular levels even more (Nielsen et al, 2001; Siupka et al, 2017). The results of the NPC2 transport study strongly supports this, as only around 30% of added NPC2 protein could be retrieved from the media after 24 hr of incubation.…”

Section: Discussionmentioning

confidence: 99%

Gene therapy to the blood–brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease

Hede

Christiansen

Heegaard

et al. 2020

Journal of Neurochemistry

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Treatment of many diseases affecting the central nervous system (CNS) is complicated by the inability of several therapeutics to cross the blood–brain barrier (BBB). Genetically modifying brain capillary endothelial cells (BCECs) denotes an approach to overcome the limitations of the BBB by turning BCECs into recombinant protein factories. This will result in protein secretion toward both the brain and peripheral circulation, which is particularly relevant in genetic diseases, like lysosomal storage diseases (LSD), where cells are ubiquitously affected both in the CNS and the periphery. Here we investigated transfection of primary rat brain capillary endothelial cells (rBCECs) for synthesis and secretion of recombinant NPC2, the protein deficient in the lysosomal cholesterol storage disease Niemann Pick type C2. We demonstrate prominent NPC2 gene induction and protein secretion in 21% of BCECs in non‐mitotic monocultures with a biological effect on NPC2‐deficient fibroblasts as verified from changes in filipin III staining of cholesterol deposits. By comparison the transfection efficiency was 75% in HeLa‐cells, known to persist in a mitotic state. When co‐cultured with primary rat astrocytes in conditions with maintained BBB properties 7% BCECs were transfected, clearly suggesting that induction of BBB properties with polarized conditions of the non‐mitotic BCECs influences the transfection efficacy and secretion directionality. In conclusion, non‐viral gene therapy to rBCECs leads to protein secretion and signifies a method for NPC2 to target cells inside the CNS otherwise inaccessible because of the presence of the BBB. However, obtaining high transfection efficiencies is crucial in order to achieve sufficient therapeutic effects. Cover Image for this issue: https://doi.org/10.1111/jnc.15050.

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Bidirectional apical–basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells

Cited by 22 publications

References 93 publications

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

The Endo-Lysosomal System of Brain Endothelial Cells Is Influenced by Astrocytes In Vitro

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

Gene therapy to the blood–brain barrier with resulting protein secretion as a strategy for treatment of Niemann Picks type C2 disease

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