Induction of Blood-Brain Barrier Differentiation in a Rat Brain-Derived Endothelial Cell Line

doi:10.1006/excr.1995.1302

Experimental Cell Research

Volume 220, Issue 1, September 1995, Pages 161-170

https://doi.org/10.1006/excr.1995.1302 Get rights and content

Abstract

An immortalized brain capillary endothelial cell line displaying blood-brain barrier characteristics may represent a useful tool for studying blood-brain barrier endothelial cell differentiation and for the in vitro prediction of drug brain penetration. In the present study, we have established a rat cerebral capillary endothelial cell line (CR3) by genomic introduction of the immortalizing SV40 large T gene under the control of the human vimentin promoter. The CR3 cell line displayed endothelial morphological and biochemical characteristics for up to 30 passages. However, the CR3 cell line did not spontaneously express the specific blood-brain barrier markers γ-glutamyl transpeptidase and mdr P-glycoprotein. However, when the cells were treated with the cell differentiating agent all-trans-retinoic acid, the blood-brain barrier markers were induced. Retinoic acid-treated CR3 cells may thus represent a useful tool for biological and pharmacological research related to the blood-brain barrier.

References (0)

Cited by (49)

P450 oxidoreductase regulates barrier maturation by mediating retinoic acid metabolism in a model of the human BBB
2022, Stem Cell Reports
The blood-brain barrier (BBB) selectively regulates the entry of molecules into the central nervous system (CNS). A crosstalk between brain microvascular endothelial cells (BMECs) and resident CNS cells promotes the acquisition of functional tight junctions (TJs). Retinoic acid (RA), a key signaling molecule during embryonic development, is used to enhance in vitro BBB models’ functional barrier properties. However, its physiological relevance and affected pathways are not fully understood. P450 oxidoreductase (POR) regulates the enzymatic activity of microsomal cytochromes. POR-deficient (PORD) patients display impaired steroid homeostasis and cognitive disabilities. Here, we used both patient-specific POR-deficient and CRISPR-Cas9-mediated POR-depleted induced pluripotent stem cell (iPSC)-derived BMECs (iBMECs) to study the role of POR in the acquisition of functional barrier properties. We demonstrate that POR regulates cellular RA homeostasis and that POR deficiency leads to the accumulation of RA within iBMECs, resulting in the impaired acquisition of TJs and, consequently, to dysfunctional development of barrier properties.
Xiao-Yao-San reduces blood-brain barrier injury induced by chronic stress in vitro and vivo via glucocorticoid receptor-mediated upregulation of Occludin
2020, Journal of Ethnopharmacology
Citation Excerpt :
The OD was measured at 450 nm and the HRP was calculated according to the standard curve. According to the Lechardeur et al., the monolayer endothelial cells were washed with PBS and scraped. After centrifuged, the cells were re-suspended in 10 mmol/L Tris-HCl (10 mmol/L Tris-HCl, pH = 7.4, 10 mmol/L NaCl, 1 mmol/L MgCl2), and break up using ultrasound.
Blood-brain barrier (BBB) is a barrier which maintains the material exchange balance of brain microenvironment and could be destroyed by chronic stress (CS). Glucocorticoids (GCs) can mimic the chronic stress induced damage to BBB. GCs induced BBB trauma models in vitro and in vivo to explore the effects of the traditional medicine Xiao-Yao-San (XYS). In this research, we found CS could injure the BBB to change the biochemical index, which could be reversed by XYS in vitro. The abilities of cell proliferation, invasion, and the expression of tight junction related genes (Occludin, Claudin, JAM-1 and ZO-1) were suppressed by CS and the trauma could be reversed by XYS partly. It was showed that GRs interacted with Occludin directly and inhibited Occluding expression. In rats BBB trauma model, the GC content was deceased and BBB permeability was repaired by XYS. The expression of Occludin, Claudin, JAM-1 and ZO-1 were increased in the treatment of XYS. In our research, it shown that XYS affect the content of the GC and GR which interacted with Occludin directly for the first time. In addition, we also found that XYS could reduce BBB injury induced by CS via GR in BBB model in vitro. Therefore, it proves that XYS is a potential BBB repair medicine and may help to elucidate mechanism of brain pathology.
Generation and Characterization of Eptesicus fuscus (Big brown bat) kidney cell lines immortalized using the Myotis polyomavirus large T-antigen
2016, Journal of Virological Methods
It is speculated that bats are important reservoir hosts for numerous viruses, with 27 viral families reportedly detected in bats. Majority of these viruses have not been isolated and there is little information regarding their biology in bats. Establishing a well-characterized bat cell line supporting the replication of bat-borne viruses would facilitate the analysis of virus-host interactions in an in vitro model. Currently, few bat cell lines have been developed and only Tb1-Lu, derived from Tadarida brasiliensis is commercially available. Here we describe a method to establish and immortalize big brown bat (Eptesicus fuscus) kidney (Efk3) cells using the Myotis polyomavirus T-antigen. Subclones of this cell line expressed both epithelial and fibroblast markers to varying extents. Cell clones expressed interferon beta in response to poly(I:C) stimulation and supported the replication of four different viruses, namely, vesicular stomatitis virus (VSV), porcine epidemic diarrhea coronavirus (PED-CoV), Middle-East respiratory syndrome coronavirus (MERS-CoV) and herpes simplex virus (HSV). To our knowledge, this is the first bat cell line from a northern latitude insectivorous bat developed using a novel technology. The cell line has the potential to be used for isolation of bat viruses and for studying virus-bat interactions in culture.
Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review
2016, Brain Research
Citation Excerpt :
Thirty-six studies (24.5%) reported both TEER measurements and characterization of protein expression (Davis et al., 2010; Koto et al., 2007; Brown et al., 2007; Mendes et al., 2015; Sano et al., 2010; Ketabi-Kiyanvash et al., 2007; Paolinelli et al., 2013; Hun Lee et al., 2015; Furihata et al., 2015; Shin et al., 2015; Al-Shehri et al., 2015; Li et al., 2015; Chen et al., 2015; Ilina et al., 2015; Liu et al., 2014; Booth and Kim, 2014; Labus et al., 2014; Sajja et al., 2014; Stephan et al., 2013; Griep et al., 2013; Shimizu et al., 2013; Li et al., 2013; Eigenmann et al., 2013; Shimizu et al., 2012; Alabanza and Bynoe, 2012; Neuhaus et al., 2011; Peng et al., 2011; Xiaolu et al., 2011; Zhang et al., 2011; Manda et al., 2011; Schreibelt et al., 2008; Silwedel and Förster, 2006; Pan et al., 2006; Weksler et al., 2005; Hori et al., 2004; Omidi et al., 2003; Hosoya et al., 2000 Jan; Hurst and Fritz, 1996). Another 107 studies (69%) reported characterization only (Rust et al., 2012; Kumar et al., 2014; Bamji-Mirza et al., 2014; Vukic et al., 2009; Patak et al., 2011; Reuter et al., 2014; Lindner et al., 2012; Robertson et al., 2011; Robertson et al., 2009; Yan et al., 2008; Qosa et al., 2014; Hosoya et al., 2000; Kamiichi et al., 2012; Cestelli et al., 2001; Theumer et al., 2015; Fernandes et al., 2015; Lee et al., 2015; Jacob et al., 2015; Kito et al., 2015; Zhao et al., 2015; Reinitz et al., 2015; Yasutaka et al., 2015; Das and Felty, 2014; Fonseca et al., 2014; Naik et al., 2014; Augustine et al., 2014; Fernandes et al., 2014; Mokgokong et al., 2014; Vilas-Boas et al., 2013; Karlstedt et al., 2013; Siamwala et al., 2013; Dickens et al., 2013; Fonseca et al., 2013; Martins et al., 2013; Dickens et al., 2012; Lee et al., 2012; Bintig et al., 2012; De Bock et al., 2012; Lee et al., 2012; Smith et al., 2012; Patel et al., 2012; Schrade et al., 2012; Watson et al., 2012; Shen et al., 2011; Steiner et al., 2011; Kania et al., 2011; Poller et al., 2010; Pifferi et al., 2010; Carl et al., 2010; Ambroziak et al., 2010; Anfuso et al., 2009; Betzen et al., 2009; Tai et al., 2009; Dauchy et al., 2009; Li et al., 2009; Lim et al., 2008; Jung et al., 2008; Méthy et al., 2008; Nazer et al., 2008; Akanuma et al., 2008; Eum et al., 2008; Bélanger et al., 2007; Scott et al., 2007; Babakhanian et al., 2007; Holloway et al., 2007; Klotz et al., 2007; Okura et al., 2007; Pan et al., 2007; Biswas et al., 2006; Hackel et al., 2006; Hori et al., 2005; Drees et al., 2005; Kusch-Poddar et al., 2005; Moser et al., 2004; Jeong et al., 2004; Theron et al., 2003; Shen et al., 2003 Nov; Song and Pachter, 2003; Friedrich et al., 2003; Yokel et al., 2002 Mar 15; Lupo et al., 2002; Deguchi et al., 2002; Ohtsuki et al., 2002; Wakayama et al., 2002; Romero et al., 2002; Takanaga et al., 2001; Régina et al., 2001; Yang et al., 2001; Kido et al., 2001; Kido et al., 2001; Kido et al., 2000; Bergmann et al., 2000; Brust et al., 2000; Pham et al., 2000; Sporbert et al., 1999; Dobbie et al., 1999; Kannan et al., 1999; Sobue et al., 1999; Chat et al., 1998; Fábián et al., 1998; Regina et al., 1998; Muruganandam et al., 1997; Rist et al., 1997; Teifel and Friedl, 1996; Begley et al., 1996; Lechardeur et al., 1995; Krizbai et al., 1995), and the other 10 studies (6.5%) focused on TEER measurements (Grab et al., 2009; Cucullo et al., 2008; Lauer et al., 2004; Davidson et al., 2009; Zhang et al., 2009; Kraus et al., 2008; Neuhaus et al., 2006; Tan et al., 2001; Kannan et al., 2000; Ramsohoye and Fritz, 1998). There are 36 immortalized endothelial cell lines that have been applied to in vitro BBB models (Appendix 2).
Endothelial cells play the most important role in construction of the blood–brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood–brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood–brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood–brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood–brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood–brain barrier.
Retinoic acid ameliorates blood-brain barrier disruption following ischemic stroke in rats
2015, Pharmacological Research
The intact blood–brain barrier (BBB) is essential in maintaining a stabilized milieu for synaptic and neuronal functions. Disruptions of the BBB have been observed following ischemia and reperfusion, both in patients and in animal models. Retinoic acid (RA), which plays crucial roles during vertebrate organogenesis, has been reported to participate in BBB development. However, it remains unclear whether RA could prevent BBB disruption in ischemic stroke. In this study, we determined that the injection of RA for 4 consecutive days resulted in increases in zonula occludens-1 (ZO-1) and vascular endothelial cadherin (VE-cadherin) expression, which are crucial components of the BBB structure. We demonstrated that RA pretreatment could alleviate the ischemic stroke-induced enlargement of vascular permeability, which is related to the up-regulated expression of ZO-1 and VE-cadherin proteins in rat models of middle cerebral artery occlusion (MCAO). Our findings further corroborated that the RA protective effect on BBB is dependent on RA receptor α in vitro oxygen–glucose deprivation (OGD) treatment. Significantly, RA administration immediately after MCAO reduced tissue plasminogen activator (tPA)-induced intracerebral hemorrhage (ICH) and ameliorated neurological deficits 24 h after ischemic stroke. Taken together, our results suggest that RA may become a new therapeutic approach to prevent BBB dysfunction and tPA-induced ICH in ischemic stroke.
P-Glycoprotein expression in human retinal pigment epithelium cell lines
2006, Experimental Eye Research
Citation Excerpt :
However, P-gp expression in h1RPE cells was variable and the rhodamine uptake assay failed to demonstrate significant P-gp activity. Cell culture conditions have been shown to significantly influence P-gp expression in a variety of cell types (Tatsuta et al., 1994; Hirsch-Ernst et al., 1995; Lechardeur et al., 1995; Regina et al., 1998; Gaillard et al., 2000). Although Kennedy and Mangini (2002) were able to show constitutive P-gp expression in human RPE primary cultures, an earlier study found that, although mRNA for MDR1 was present, P-gp protein was only detected after pre-treating the cells with the anti-mitotic agent daunomycin (Esser et al., 1998).
P-Glycoprotein (P-gp), an active efflux transporter encoded by the MDR1 gene, has recently been identified in the human and pig retinal pigment epithelium (RPE) in situ. Efflux pumps such as P-gp are major barriers to drug delivery in several tissues. We wished to establish whether human RPE cell lines express P-gp under the culture conditions recommended for each cell line so as to determine their suitability as in vitro models for predicting drug transport across the outer blood–retinal barrier. Three human RPE cell lines, ARPE19, D407 and h1RPE were investigated. Reverse transcriptase–polymerase chain reaction (RT-PCR) was carried out to determine the expression of MDR1 mRNA. Immunocytochemistry using the P-gp-specific antibody C219 was undertaken to investigate the presence of P-gp protein in each cell type. Uptake of rhodamine 123, a P-gp substrate, in the presence or absence of pre-treatment with a P-gp inhibitor, verapamil, was measured in each cell line to determine functional expression of P-gp. For all experiments, MDCK cells stably transfected with the human MDR1 gene (MDCK-MDR1) were used as a positive control. ARPE19 cells were consistently negative for P-gp as assessed by RT-PCR and immunocytochemistry. By contrast, RT-PCR of D407 and h1RPE samples yielded weak bands corresponding to MDR1; P-gp protein expression, as demonstrated by C219 immunoreactivity, was also present. Rhodamine uptake after treatment with verapamil was significantly greater in D407 and MDCK-MDR1, indicating functional expression of P-gp in these two cell lines. No evidence of functional P-gp was found in ARPE19 and h1RPE. In conclusion, D407 and h1RPE cells express P-gp, though functional activity was demonstrable only in D407 cells. ARPE19 cells do not express P-gp. Of these human RPE cells lines D407 could be considered as a suitable model for in vitro drug transport studies, particularly those involving P-gp substrates, without modification of their usual culture conditions.

View all citing articles on Scopus

View full text

Regular ArticleInduction of Blood-Brain Barrier Differentiation in a Rat Brain-Derived Endothelial Cell Line

Abstract

Regular Article
Induction of Blood-Brain Barrier Differentiation in a Rat Brain-Derived Endothelial Cell Line