Hydroxyethylstarch (130/0.4) tightens the blood-brain barrier in vitro

Gerhartl, Anna; Hahn, Katrin; Neuhoff, Anja; Friedl, Heinz-Peter; Förster, Carola; Wunder, Christian; Schick, Martin Alexander; Burek, Małgorzata; Neuhaus, Winfried

doi:10.1016/j.brainres.2019.146560

Cited by 17 publications

(11 citation statements)

References 26 publications

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“…Western blotting was performed as recently described in Gerhartl et al (2019). 16 In brief 20 µg protein was loaded on 10% SDS gels upon determining the protein concentration using the BCA assay. Primary antibodies [P-gp (ABCB1, clone C219, ENZO, #ALX-801-002, mouse, 1:30), MRP4 (ABCC4, clone M4I-80, ENZO, #ALX-801-039, rat, 1:100), BCRP (ABCG2, clone BXP-53, Abcam, #ab24115, rat, 1:100)] and secondary antibodies [anti-mouse IgG HRP-linked (Cell signaling, #7076, goat, 1:5000) or anti-rat IgG HRP-linked (Invitrogen, #61-9520, rabbit 1:5000)] were diluted in 5% dry milk.…”

Section: Western Blotmentioning

confidence: 99%

Optimization of an oral mucosa in vitro model based on cell line TR146

et al. 2020

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During the last years, the popularity of saliva has been increasing for its applicability as a diagnostic fluid. Blood biomarker molecules have to cross the blood-saliva barrier (BSB) in order to appear in saliva. The BSB consists of all oral and salivary gland epithelial barriers. Within this context, the optimization of in vitro models for mechanistic studies about the transport of molecules across the oral mucosa is an important task. Here, we describe the optimization and comprehensive characterization of a Transwell model of the oral mucosa based on the epithelial cell line TR146. Through systematic media optimization investigating 12 different setups , a significant increase of barrier integrity upon airlift cultivation was achieved for TR146 cell layers. The distinct improvement of the paracellular barrier was shown by measurements of transepithelial electrical resistance (TEER) and carboxyfluorescein permeability assays. Histological characterization supported TEER data and showed a stratified, non-keratinized multilayer of the optimized TR146 model. High-Throughput qPCR using 96 selected markers for keratinization, cornification, epithelial-mesenchymal transition, aquaporins, mucins, tight junctions, receptors, and transporter proteins was applied to comprehensively characterize the systematic optimization of the cellular model and validate against human biopsy samples. Data revealed the expression of several genes in the oral mucosa epithelium for the first time and elucidated novel regulations dependent on culture conditions. Moreover, functional activity of ABC transporters ABCB1 and ABCC4 was shown indicating the applicability of the model for drug transport studies. In conclusion, a Transwell model of the oral mucosa epithelium was optimized being suitable for transport studies.

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Section: Western Blotmentioning

confidence: 99%

Optimization of an oral mucosa in vitro model based on cell line TR146

et al. 2020

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“…Real-time PCR was performed as previously described [26][27][28]. Briefly, RNA was isolated using the RNA isolation kit NucleoSpin ® RNA (Machery-Nagel) according to manufacturer's instruction.…”

Section: Real-time Pcrmentioning

confidence: 99%

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood–brain barrier model

Curtaz

Schmitt

Herbert

et al. 2020

Fluids Barriers CNS

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Background: The most threatening metastases in breast cancer are brain metastases, which correlate with a very poor overall survival, but also a limited quality of life. A key event for the metastatic progression of breast cancer into the brain is the migration of cancer cells across the blood-brain barrier (BBB). Methods: We adapted and validated the CD34 + cells-derived human in vitro BBB model (brain-like endothelial cells, BLECs) to analyse the effects of patient serum on BBB properties. We collected serum samples from healthy donors, breast cancer patients with primary cancer, and breast cancer patients with, bone, visceral or cerebral metastases. We analysed cytokine levels in these sera utilizing immunoassays and correlated them with clinical data. We used paracellular permeability measurements, immunofluorescence staining, Western blot and mRNA analysis to examine the effects of patient sera on the properties of BBB in vitro. Results: The BLECs cultured together with brain pericytes in transwells developed a tight monolayer with a correct localization of claudin-5 at the tight junctions (TJ). Several BBB marker proteins such as the TJ proteins claudin-5 and occludin, the glucose transporter GLUT-1 or the efflux pumps PG-P and BCRP were upregulated in these cultures. This was accompanied by a reduced paracellular permeability for fluorescein (400 Da). We then used this model for the treatment with the patient sera. Only the sera of breast cancer patients with cerebral metastases had significantly increased levels of the cytokines fractalkine (CX3CL1) and BCA-1 (CXCL13). The increased levels of fractalkine were associated with the estrogen/progesterone receptor status of the tumour. The treatment of BLECs with these sera selectively increased the expression of CXCL13 and TJ protein occludin. In addition, the permeability of fluorescein was increased after serum treatment. Conclusion: We demonstrate that the CD34 + cell-derived human in vitro BBB model can be used as a tool to study the molecular mechanisms underlying cerebrovascular pathologies. We showed that serum from patients with

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“…RNA isolation and real-time PCR analysis were performed as described previously (Burek et al, 2014;Gerhartl et al, 2020). Briefly, total RNA was isolated using the Nucleospin-RNAII Kit (Macherey Nagel) according to the manufacturer's instructions.…”

Section: Real-time Pcrmentioning

confidence: 99%

Kidney Ischemia/Reperfusion Injury Induces Changes in the Drug Transporter Expression at the Blood–Brain Barrier in vivo and in vitro

et al. 2020

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Ischemia/reperfusion injury is a major cause of acute kidney injury (AKI). AKI is characterized by a sudden decrease in kidney function, systemic inflammation, oxidative stress, and dysregulation of the sodium, potassium, and water channels. While AKI leads to uremic encephalopathy, epidemiological studies have shown that AKI is associated with a subsequent risk for developing stroke and dementia. To get more insights into kidney–brain crosstalk, we have created an in vitro co-culture model based on human kidney cells of the proximal tubule (HK-2) and brain microvascular endothelial cells (BMEC). The HK-2 cell line was grown to confluence on 6-well plates and exposed to oxygen/glucose deprivation (OGD) for 4 h. Control HK-2 cells were grown under normal conditions. The BMEC cell line cerebED was grown to confluence on transwells with 0.4 μm pores. The transwell filters seeded and grown to confluence with cereEND were inserted into the plates with HK-2 cells with or without OGD treatment. In addition, cerebEND were left untreated or treated with uremic toxins, indole-3-acetic acid (IAA) and indoxyl sulfate (IS). The protein and mRNA expression of selected BBB-typical influx transporters, efflux transporters, cellular receptors, and tight junction proteins was measured in BMECs. To validate this in vitro model of kidney–brain interaction, we isolated brain capillaries from mice exposed to bilateral renal ischemia (30 min)/reperfusion injury (24 h) and measured mRNA and protein expression as described above. Both in vitro and in vivo systems showed similar changes in the expression of drug transporters, cellular receptors, and tight junction proteins. Efflux pumps, in particular Abcb1b, Abcc1, and Abcg2, have shown increased expression in our model. Thus, our in vitro co-culture system can be used to study the cellular mechanism of kidney and brain crosstalk in renal ischemia/reperfusion injury.

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Hydroxyethylstarch (130/0.4) tightens the blood-brain barrier in vitro

Cited by 17 publications

References 26 publications

Optimization of an oral mucosa in vitro model based on cell line TR146

Optimization of an oral mucosa in vitro model based on cell line TR146

Serum-derived factors of breast cancer patients with brain metastases alter permeability of a human blood–brain barrier model

Kidney Ischemia/Reperfusion Injury Induces Changes in the Drug Transporter Expression at the Blood–Brain Barrier in vivo and in vitro

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