Basic fibroblast growth factor opens and closes the endothelial blood-brain barrier in a concentration-dependent manner

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We have recently demonstrated that autocrine secretion of basic fibroblast growth factor (bFGF) by BECs is necessary for the establishment of endothelial barrier (as demonstrated by high trans-endothelial electric resistance, TEER), whereas exogenous bFGF inhibits TEER in a concentration-dependent manner. In the present study we analysed the contribution of MAPK/ERK and STAT3 signalling pathways to the inhibitory effects of exogenous bFGF. Treatment with bFGF (8 ng/ml) for 3 days increased phosphorylation of ERK1/2 and STAT3. Treatment with FGF receptor 1 (FGFR1) inhibitor PD173074 (15 µM) suppressed both basal and bFGF-induced activation of ERK1/2 and STAT3. Suppression of STAT signalling with Janus kinase inhibitor JAKi (15 nM) alone or in the presence of bFGF did not change TEER in BEC monolayers. Exposure to JAKi affected neither proliferation, nor expression and distribution of tight junction (TJ) proteins claudin-5, occludin and zonula occludens-1 (ZO-1). In contrast, treatment with MEK 1/2 inhibitor U0126 (10 µM) partially neutralized inhibitory effect of bFGF thus increasing TEER, whereas U0126 alone did not affect resistance of endothelial barrier. Our findings demonstrate that MAPK/ERK signalling pathway does not affect autocrine bFGF signalling-dependent BECs barrier function but is largely responsible for the disruptive effects of the exogenous bFGF. We speculate that bFGF may (depending on concentration and possibly origin) dynamically regulate permeability of the endothelial blood-brain barrier. bFGF TEER blood-brain barrier ERK STAT3 tight junctions Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction The blood–brain barrier (BBB), that governs selective exchange of substances between blood and the parenchyma of the central nervous system (CNS), is formed by the continuous layer of specialised brain endothelial cells (BECs), clasped by tight and adherent junctions (TJs and AJs, respectively) [ 1 ]. Together with adjacent pericytes, smooth muscle cells, astrocytes and neurones, BECs are integrated into complex multi-cellular structure of neurogliovascular unit (NGVU) involved in the precise coordination of local blood flow, transport across the BBB, energy metabolism and formation of the brain active milieu [ 2 – 4 ]. Cellular components of the NGVU interact through multiple mechanisms, including numerous paracrine factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoetins, sonic hedgehog (SHH), retinoic acid (RA) and others [ 5 , 6 ]. Identification of precise mode of action of these paracrine factors in different physiologic and pathologic settings remains challenging. Firstly, majority of currently available in vivo models do not allow accurate control and dynamic monitoring of local fluctuations of different paracrine factors at the BBB. Secondly, defined protocols, allowing generation of BECs monocultures with high transendothelial electrical resistance (TEER) values comparable with those recorded in vivo , have been introduced only relatively recently. In our previous study, which employed a fully defined differentiation protocol [ 7 ] for producing BECs from human inducible pluripotent stem cells (iPSCs), we demonstrated a dual role for bFGF in the regulation of BECs barrier function [ 8 ]. Autocrine secretion of bFGF is required for the establishment of tight-junctions defining BECs barrier, whereas exogenous bFGF in high concentrations (> 4 ng/ml) disrupts the barrier integrity [ 8 ]. We also found that inhibitory effects of exogenous bFGF on the in vitro BBB depend neither on phosphoinositide 3-Kinase (PI-3K), nor on ROCK signalling pathways [ 8 ]. In the present study we further investigated molecular mechanisms of the inhibitory effects of exogenous bFGF. Binding of FGFs to the FGF receptor 1 (FGFR1) activates FGFR tyrosine kinase, which phosphorylates adaptor proteins and subsequent activation of rat sarcoma virus (Ras)-mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK), phosphoinositide 3-kinase (PI3K)-Akt, phospholypase Cγ (PLCγ), and signal transducer and activator of transcription (STAT) intracellular signalling pathways [ 9 ]. Since our previous study demonstrated that PI-3K cascade suppresses inhibitory effects of bFGF on TEER, we focused on the STAT and Ras-MAPK/ERK signalling. We found that inhibition of STAT signalling with Janus kinase inhibitor (JAKi) alone, or in the presence of bFGF did not change TEER of BEC monolayers. In contrast, treatment with MEK 1/2 inhibitor U0126 alone did not affect TEER, but significantly increased TEER values in bFGF-treated BECs, thus partially neutralising inhibitory effect of exogenous bFGF. Our findings demonstrate that MAPK/ERK signalling pathway does not affect autocrine bFGF signalling-dependent BECs barrier function but is largely responsible for the disruptive effects of the exogenous bFGF. The bFGF therefore acts as a dynamic regulator of the BBB which opens it at high and closes at low concentrations. Methods Maintenance of iPSCs and differentiation to the brain capillary endothelial cells (BECs) In this study two healthy donor-derived iPSC lines, MBE 2960 (male, 78 years old) and SHED-iPSC (female, 7 years old), from the passages 12 to 34, were used. Both iPSC lines were cultured on matrigel-coated (Corning) plates with Essential 8 (E8) medium exchange every 24 hours. iPSCs were differentiated to BECs according to slightly modified previously published protocol [ 7 ]. Briefly, 24 hours after splitting, the differentiation was initiated by changing the E8 to the Essential 6 (E6) medium (Thermo Fisher Scientific). E6 was fully refreshed every 24 hours for four days. On the fifth day, the E6 was changed to the human Endothelial Serum Free-Medium (hESFM, Thermo Fisher Scientific) supplemented with 20 ng/ml bFGF (Thermo Fisher Scientific), 10 µM retinoic acid (Merck Darmstadt, Germany), and 0.25× B-27 (Thermo Fisher Scientific). After 48 hours the hESFM medium was fully refreshed. The next day BECs were split for selection onto 400 µg/ml collagen IV and 100 µg/ml fibronectin-precoated (both from Merck) Transwell inserts (Corning) or 48-well plates (TPP). After four days, BECs monolayer barrier was characterised by measuring TEER and expression of claudin-5, occludin, ZO-1, VE-cadherin, CD34, and von Willebrand factor by immunocytochemistry and Western blotting (Fig. 1 ). Experimental design All treatments of BECs were performed once by refreshing the medium on the next day after the differentiation (see Fig. 2 A for experimental design). The medium administered to the different experimental groups of BECs contained one of the following: 8 ng/ml bFGF, 15 µM PD173074 (Tocris Bioscience), 15 nM JAK inhibitor I (JAKi, Santa Cruz), 10 µM U0126 (MedChemExpress), bFGF + PD173074, bFGF + JAKi, bFGF + U0126. After three days of the treatment, BECs were subjected to TEER measurement and immunocytochemistry or Western blot analysis. Additionally, in the experiment with U0126, BECs were analysed by Western blot after 2 and 24 hours of the treatment (see Fig. 4 A for experimental design). Transendothelial electrical resistance (TEER) measurements To measure TEER, BECs were grown on Transwell inserts (Corning) with 0.4 µm pore size and 0.33 cm 2 surface area polyester membrane. TEER was measured with Millicell ERS-2 Electrical Resistance System (Merck-Millipore). Each insert was measured three times in different locations. To calculate TEER (Ω*cm 2 ), the mean electrical resistance of the BECs-free inserts was subtracted from the mean readings of the inserts with BECs monolayers and then multiplied by the surface area of the insert. Immunocytochemistry Immediately after TEER measurement BECs were fixed and permeabilised by incubating them with ice-cold methanol and acetone in a 1:1 ratio at -20°C for 10 min. Then the cells were washed three times with PBS and blocked with 1% BSA-PBS solution at room temperature for 30 min. Afterwards, the primary antibodies against ZO-1 (1:33), claudin-5 (1:100) and occludin (1:50, all from Thermo Fisher Scientific) were diluted in blocking solution and administered to the cells for incubation at 4°C overnight. Next, cells were washed three times with PBS and incubated with the secondary antibodies conjugated with Alexa Fluor 594 (1:1000, Thermo Fisher Scientific) diluted in PBS at room temperature, in the dark, for 1 hour. Then the samples were washed three times and mounted on coverslips using an aqueous fluorescent mounting medium with DAPI (Abcam). Prepared samples were analysed with the Leica TCS SP8 confocal microscope (Leica Microsystems, Mannheim, Germany) using lasers of Diode 405 nm and DPSS 561 nm and 63x oil immersion lens. The images of confocal microscopy were used to cell count evaluation. Cells were counted according to the DAPI stain using the ImageJ program, a multi-point tool. Western blot Cells growing in a 48-well plate were washed three times with cold PBS and incubated with RIPA buffer (Thermo Fisher Scientific) containing 1× protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 15 min. Obtained lysates were collected, vortexed and placed on ice again for the next 10 min. Afterwards, the samples were centrifuged at 18 000 g at 4°C for 20 min and the remaining supernatants were collected to new tubes. The concentration of proteins was measured with the NanoPhotometer Pearl (Implen) and equalised between samples with 1x Laemmli sample buffer (Bio-Rad). Then lysates were diluted in 6x Laemmli sample buffer and denatured at 95°C for 5 min. Protein lysates were subjected to 4–10% polyacrylamide gel electrophoresis using Mini-PROTEAN Tetra cell apparatus (Bio-Rad). Separated proteins were blotted onto a polyvinylidene difluoride membrane by a semidry Trans-Blot Turbo transfer apparatus (Bio-Rad). The membrane was blocked with 5% bovine serum albumin (Applichem) diluted in PBS with 0.18% Tween-20 (hereinafter referred to as PBS-T) at room temperature on a platform rocker for 1 h. The primary antibodies against pAkt Ser473 (1:2000), pAkt Thr308 (1:1000), pERK (1:2000), pSTAT3 Ser727 (1:1000), pSTAT3 Tyr705 (1:800), β-actin (1:1000, all from Cell Signaling Technology) and α-tubulin (1:500, Thermo Fisher Scientific) were diluted in a blocking solution and the membrane was then probed with them and incubated at 4°C overnight. Then the membrane was washed three times with PBS-T on a platform rocker for 5 min and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific) diluted in PBS-T on a platform rocker at room temperature for 1 h. Washing procedure was repeated and immunoreactive bands were detected with Clarity ECL Western blotting substrate (Bio-Rad) using ChemiDoc MP system (Bio-Rad). Statistics Statistical analysis was performed from at least three biological experiments using Graph Pad Prism® software 8.0.2 (Graph Pad Software, Inc., City, State, USA). Data in the graphs is presented as the mean ± standard deviation (± SD). Differences between the groups were compared by one-way ANOVA following Tukey’s post-test. Results were considered significant at p < 0.05. Results Exogenous bFGF activates ERK 1/2, STAT3 and Akt signalling pathways in differentiated BECs We demonstrated previously that exogenous bFGF at concentrations above 4 ng/ml inhibits TEER and proliferation of BECs in a concentration-dependent manner [ 8 ]. After bFGF binding, FGF receptor 1 (FGFR1) activates several downstream signalling pathways, including Ras-MAPK, PI-3K-Akt and STAT [ 9 ]. We treated differentiated BECs with 8 ng/ml of exogenous bFGF for 3 days and monitored levels of phosphorylation of ERK 1/2, Akt (Thr308/Ser473) and STAT3 (Ser727/Tyr705) proteins. We found that treatment with bFGF significantly increased phosphorylation of ERK 1/2 and STAT3 (Ser727) and slightly increased of phosphorylation Akt at Thr308 (Fig. 2 B, C). Combined treatment with selective inhibitor of FGFR1 PD173074 (15 µM) almost completely suppressed phosphorylation of ERK 1/2 and STAT3 at Ser727 and Tyr705 but not of Akt (Fig. 2 B, C). Treatment with PD173074 alone strongly suppressed ERK 1/2 and STAT3 (Tyr705) phosphorylation (Fig. 2 B, C) highlighting the importance of autocrine/paracrine bFGF signalling for maintaining the basal activity of ERK 1/2 and STAT3 in differentiated BECs. Inhibition of FGFR1 with PD173074 did not affect Akt phosphorylation (Fig. 2 B, C). Inhibition of JAK/STAT signalling does not affect bFGF-mediated suppression of TEER in BEC monolayers We used JAKi (15 nM), a potent inhibitor of janus kinases (JAK) [ 10 ] to block STAT signalling in differentiated BECs (Suppl. Figure 1). Treatment of BECs monolayers with JAKi alone affected neither TEER nor proliferation of BECs (Fig. 3 A, B), whereas bFGF administered on its own down-regulated TEER by 89.53 ± 7.77% (n = 4, p < 0.0001, Fig. 3 A) and increased cell numbers by 90.00 ± 31.61% (n = 4, p = 0.008, Fig. 3 B). JAKi did not affect bFGF-mediated suppression of TEER and increase of BCEC proliferation (Fig. 3 A, B). Neither did it alter expression and distribution of TJ proteins claudin-5, occludin and ZO-1 (Fig. 3 C). Inhibition of ERK 1/2 signalling partially neutralised bFGF-mediated suppression of TEER in BEC monolayers Treatment with exogenous bFGF (8 ng/ml) induced long-term activation of ERK 1/2 signalling in the differentiated BECs (Fig. 4 ). ERK 1/2 phosphorylation was effectively suppressed by U0126 (10 µM), a potent inhibitor of MEK 1/2 kinases, upstream activators of ERK 1/2 (Fig. 4 B, C). We found that U0126 (10 µM) partially neutralised inhibitory effect of bFGF by increasing TEER by 40.95 ± 23.49% (n = 4, p = 0.0467), whereas U0126 alone did not affect barrier properties of BECs (Fig. 5 A). Treatment with U0126 alone or in combination with bFGF did not affect BEC proliferation and expression/distribution of TJ proteins (Fig. 5 B, C). At the same time, U0126 suppressed STAT3 (Ser727) phosphorylation in bFGF-treated and untreated BECs (Suppl. Figure 1) indicating that Ser727 represents a downstream target for ERK 1/2. Discussion As we discovered before, autocrine secretion of bFGF by BECs is necessary for the establishment of proper barrier function, whereas exogenous bFGF in concentrations exceeding 4 ng/ml inhibits TEER of BECs monolayers in a concentration-dependent manner [ 8 ]. In this study we analysed the role of intracellular MAPK/ERK and STAT signalling pathways in mediating FGFR effects [ 9 ], in particular the contribution of these cascades to the inhibitory action of the exogenous bFGF on the barrier function of BECs. We found that inhibition of bFGF autocrine/paracrine signalling with selective inhibitor of FGFR1 PD173074 completely suppressed basal ERK 1/2 phosphorylation in differentiated BECs (Fig. 2 B, C). The basal ERK 1/2 activity, however, was not required for the establishment of BECs barrier, because inhibition of ERK 1/2 phosphorylation with U0126 did not change TEER (Fig. 5 A). We therefore conclude that ERK 1/2 signalling pathway does not affect autocrine bFGF regulation of BEC barrier. We also found that PD173074 decreased both basal and bFGF-induced activity of STAT3 (Fig. 2 B, C). In this study we used antibodies against phospho-STAT3 (Ser727/Tyr705). Phosphorylation of STAT3 at Ser 727 is required for its maximal transcriptional activation [ 11 ] and can be mediated by different members of MAPK family p38 [ 12 ], ERK1/2 [ 13 , 14 ], c-Jun N-terminal Kinase (JNK) [ 15 ], protein kinase C (PKC) [ 16 ], and mammalian target of rapamycin (mTOR) [ 17 ]. We found that inhibitor of MEK 1/2 kinases U0126 suppressed basal and bFGF-induced Ser727 phosphorylation in BECs (Suppl. Figure 1B). This indicates that Ser727 of STAT3 represents a downstream (direct or indirect, which remain unknown) target for ERK 1/2. As under basal conditions U0126 does not affect TEER, we concluded that STAT3 (Ser727) signalling does not contribute to autocrine bFGF regulatory effect on BECs barrier function. Similarly, inhibition of STAT signalling with JAK inhibitor did not change TEER in BECs (Fig. 3 A). In our previous report, using the same experimental conditions, we found that treatment of BEC monolayers with PI-3K inhibitor LY294002 (25 µM) down-regulates TEER by approximately 40% [ 8 ] showing that basal PI-3K-Akt signalling contributes to the regulation of the BEC barrier. However, we did not detect down-regulation of Akt phosphorylation (Fig. 2 B, C) in response to the treatment with PD173074, therefore basal PI-3K-Akt signalling in BECs is not linked to the autocrine/paracrine bFGF signalling. Further studies are needed to elucidate the mechanisms involved in the regulation of autocrine bFGF signalling-dependent BEC barrier function. Treatment with high concentration of exogenous bFGF (8 ng/ml) induced long-term activation of ERK 1/2 signalling (Fig. 4 ) and effectively suppressed TEER in the differentiated BECs. We also found that inhibition of ERK 1/2 with U0126 partially neutralised inhibitory effects of bFGF (Fig. 5 A, B). Several studies demonstrated that MAPK/ERK signalling pathway can modulate permeability of endothelial and epithelial barriers by modulating expression and distribution of TJ proteins [ 18 ]. MAPK/ERK signalling cascades may promote or disrupt endothelial barriers in stimulus- and cell type-dependent manner [ 18 ]. For example, H 2 O 2 induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activating ERK 1/2 kinase pathway and these changes correlate with localisation of TJ proteins ZO-1 and ZO-2 [ 19 ]. Exposure to microwaves damages BBB through the VEGF/Flk-1-ERK-dependent Tyr phosphorylation of occludin and inhibition of its interaction with ZO-1 [ 20 ]. In our model similar mechanisms might be responsible for the inhibitory action of the exogenous bFGF. Therefore, the effects of bFGF/ERK signalling on the interaction between ZO and other TJ proteins in BECs are in need of systematic exploration in the future studies. Our finding that MEK 1/2 inhibitor U0126 suppressed FGF-induced STAT3 (Ser727) phosphorylation indicates that it represents a downstream target of ERK 1/2 which could be potentially responsible for the inhibitory action of the exogenous bFGF. Indeed, several studies demonstrated that IL-6 family cytokines promoted BBB breakdown through the activation of STAT3 signalling pathway [ 21 , 22 ]. Hence, the importance ERK 1/2-STAT3 signalling axis for the inhibitory action of the exogenous bFGF should be addressed in the future studies. We have previously reported that inhibition of FGFR1 and PI-3K signalling significantly decreased proliferation in bFGF untreated BECs and that these effects were paralleled with substantial reduction of TEER [ 7 ]. However, the present study shows that MEK 1/2 inhibitor U0126 does not affect proliferation of bFGF untreated BECs (Fig. 5 B) indicating that under basal conditions BEC proliferation occurs through ERK-independent mechanisms. These findings suggest possible relationship between BEC proliferation and barrier establishment. We did not test how PLCγ, another mediator of the intracellular FGFR signalling [ 9 ] affects BECs barrier integrity. FGFR kinase recruits and activates the PLCγ which produces inositol trisphosphate (InsP 3 ) and diacylglycerol (DAG) by the hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP 2 ). InsP 3 induces calcium release from the intracellular stores while DAG activates DAG-sensitive protein kinases C (PKC) and protein kinases D (PKD) [ 23 ]. Some of the PLCγ downstream targets can be potentially involved in the regulation BEC barrier. For instance, acoustic wave stimulation activated calcium-dependent activation of PKC-δ pathway that mediated dissociation of ZO-1 and occludin, promoted paracellular permeability and opening of BBB [ 16 ]. Further research is needed to establish whether similar mechanisms can be responsible for the inhibitory effects of exogenous bFGF in BCECs. Conceptually, bFGF can derive from either luminal (blood) or abluminal (parenchymal), or from both sides of the endotehlial barrier. In our experiments exogenous bFGF was added to both upper and lower compartments of the Transwell inserts and therefore the differential effects of bFGF on the luminal and abluminal sides of BEC monolayer were not distinguished. The potential differences between the effects of luminal and abluminal bFGF on the BEC barrier function should be carefully explored in the future studies. What is the source of paracrine bFGF in the NGVU? First of all, we can not exclude possibility that BECs can increase expression and secretion of the bFGF in response to the external clues. Some indirect evidence indicates that BECs can secrete substantial amounts of bFGF that act in the paracrine manner on the surrounding tissues. For instance, tumour microvascular endothelial cells secrete bFGF which promotes cancer stem cell features in differentiated glioblastoma cells [ 24 ]. Exosomes derived from brain microvascular endothelial cells after oxygen glucose deprivation contained increased levels of bFGF [ 25 ]. An in vitro BBB model could be used to explore the effects of different type of stress on the expression and secretion bFGF in BECs. Pericytes represent another potential source of bFGF in the NGVU. bFGF and FGFR1 are induced in pericytes at the periinfarct areas after brain ischemia [ 26 ]. Peripheral nerve pericytes partially modify blood-nerve barrier function through the secretion of bFGF [ 27 ]. In the adult brain, bFGF is predominantly synthesised and secreted by the astrocytes [ 28 ]. However, little is known about the role of bFGF in regulation of BBB in the unperturbed adult brain [ 1 ]. In contrast, many studies demonstrated that various injuries trigger reactive astrogliosis [ 29 ] associated with increased secretion of bFGF [ 28 , 30 ]. Nevertheless, the effects of local bFGF increase on the BBB integrity remain unclear. Studies allowing simultaneous in vivo monitoring of BBB permeability together with conditional inactivation of astrocytic bFGF release and (or) deletion of FGF receptors in BECs may reveal the role of astrocytic bFGF on BBB function. Astrocytes control permeability of the endothelial blood-brain barrier by secreting several factors such as VEGF-A, which loosens the endothelial barrier, or SHH, which stimulates barrier repair [ 31 ]. The bFGF can serve towards the same means: pulsative release of bFGF can rapidly and transiently open the barrier (acting through MAPK/ERK signalling pathway), whereas in the absence of additional input autocrine bFGF secretion restores the barrier integrity (Fig. 6 ). Declarations Acknowledgements This research has received funding from European Regional Development Fund (project No 13.1.1-LMT-K-718-05-0005 ) under grant agreement with the Research Council of Lithuania (LMTLT). Funded as European Union's measure in response to Cov-19 pandemic. Data availability statement The datasets used or analyzed during the current study are available from the corresponding author on reasonable request. References Pivoriunas A, Verkhratsky A (2021) Astrocyte-Endotheliocyte Axis in the Regulation of the Blood-Brain Barrier. Neurochem Res 46:2538–2550 Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV (2015) Establishment and Dysfunction of the Blood-Brain Barrier. Cell 163:1064–1078 Sweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV (2019) Blood-Brain Barrier: From Physiology to Disease and Back. Physiol Rev 99:21–78 Semyanov A, Verkhratsky A (2021) Astrocytic processes: from tripartite synapses to the active milieu. 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Nat Neurosci 24:312–325 Xie C, Shen X, Xu X, Liu H, Li F, Lu S, Gao Z, Zhang J, Wu Q, Yang D, Bao X, Zhang F, Wu S, Lv Z, Zhu M, Xu D, Wang P, Cao L, Wang W, Yuan Z, Wang Y, Li Z, Teng H, Huang Z (2020) Astrocytic YAP Promotes the Formation of Glia Scars and Neural Regeneration after Spinal Cord Injury. J Neurosci 40:2644–2662 Sofroniew MV (2020) Astrocyte Reactivity: Subtypes, States, and Functions in CNS Innate Immunity. Trends Immunol 41:758–770 Additional Declarations No competing interests reported. Supplementary Files SupplementaryFig.tif Supplementary figure 1. Selective MEK 1/2 and JAK inhibitors decrease basal and bFGF-induced phosphorylation of STAT3.A: Representative Western blots of pSTAT3 (Ser727/Tyr705) and b-actin in BECs after treatment with bFGF (8ng/ml) alone or in combination with JAKi (15 nM)/PD173074 (15 μM).B: Representative Western blots of pSTAT3 (Ser727) and a-tubulin in BECs after treatment with bFGF (8ng/ml) alone or in combination with U0126 (10 µM)/PD173074 (15 μM). Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 05 Jul, 2022 Reviews received at journal 04 Jul, 2022 Reviewers agreed at journal 29 Jun, 2022 Reviewers agreed at journal 29 Jun, 2022 Reviewers invited by journal 29 Jun, 2022 Editor assigned by journal 26 Jun, 2022 Submission checks completed at journal 24 Jun, 2022 First submitted to journal 22 Jun, 2022 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-1784689","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":116242100,"identity":"ab78734a-5a64-4058-9ec5-0fb2660853ba","order_by":0,"name":"Karolina Kriaučiūnaitė","email":"","orcid":"","institution":"State Research Institute Centre for Innovative Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Karolina","middleName":"","lastName":"Kriaučiūnaitė","suffix":""},{"id":116242101,"identity":"5098d472-4e5c-4538-acb3-f2350d24111f","order_by":1,"name":"Agnė Pociūtė","email":"","orcid":"","institution":"State Research Institute Centre for Innovative Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Agnė","middleName":"","lastName":"Pociūtė","suffix":""},{"id":116242102,"identity":"48c88125-9652-4bba-a618-7e86d89fc58d","order_by":2,"name":"Aida Kaušylė","email":"","orcid":"","institution":"State Research Institute Centre for Innovative Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Aida","middleName":"","lastName":"Kaušylė","suffix":""},{"id":116242103,"identity":"5555d785-6222-429b-b796-7c318331421e","order_by":3,"name":"Alexei Verkhratsky","email":"","orcid":"","institution":"State Research Institute Centre for Innovative Medicine","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Alexei","middleName":"","lastName":"Verkhratsky","suffix":""},{"id":116242104,"identity":"b7beeb95-0add-42d5-8078-d6efb3514940","order_by":4,"name":"Augustas Pivoriūnas","email":"data:image/png;base64,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","orcid":"","institution":"State Research Institute Centre for Innovative Medicine","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Augustas","middleName":"","lastName":"Pivoriūnas","suffix":""}],"badges":[],"createdAt":"2022-06-22 14:14:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-1784689/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-1784689/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":23460830,"identity":"3c54a15f-9dfd-4a15-9f3c-ed72817b2c26","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2341819,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterisation of BECs derived from iPSCs\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Schematic timeline of differentiation and characterisation of BECs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e The scheme of TEER measurements of BECs monolayers growing on a Transwell inserts on the fourth day following the differentiation, n = 4.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eC:\u003c/strong\u003e Representative confocal microscopy images of BECs labelled with antibodies against VE-cadherin, CD34, von Willebrand factor and tight junction proteins, including claudin-5, occludin and ZO-1.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/86f1834ad4142032c11ae337.png"},{"id":23460832,"identity":"74270b85-fbeb-45a5-a1e9-997355cbf884","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1634552,"visible":true,"origin":"","legend":"\u003cp\u003eAn exogenous bFGF induces ERK 1/2, STAT3 and Akt signalling in BECs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Experimental protocol.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Representative Western blots of pSTAT, pAkt and pERK in the cultures of BECs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Quantified protein phosphorylation determined by ratio of pSTAT3, pAkt, and pERK to β-actin or a-tubulin band intensity.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/b8ba49e4a53f55721627b85e.png"},{"id":23460831,"identity":"a18aa84f-4942-4523-80b7-dd91bc9d7da6","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6594808,"visible":true,"origin":"","legend":"\u003cp\u003eA selective JAK inhibitor does not change bFGF-induced reduction of TEER or increased cell number in BEC cultures.\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e TEER readouts after treatment with bFGF (8ng/ml) alone or in combination with JAKi (15 nM) relative to control, n = 4.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Cell count in BEC cultures after treatment with bFGF (8ng/ml) alone or in combination with JAKi (15 nM) relative to control, n = 3.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eC:\u003c/strong\u003e Representative confocal microscopy images of BEC cultures stained with antibodies against claudin-5, occludin and ZO-1.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/b03ec52e57f36e0d20e44fe1.png"},{"id":23460834,"identity":"55d953b9-026d-4c67-bcea-636abd968ce7","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":973144,"visible":true,"origin":"","legend":"\u003cp\u003eSelective MEK1/2 inhibitor, U0126, partially reduces bFGF-induced reduction of TEER but does not change the cell numbers in BEC cultures.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e TEER readouts after treatment with bFGF (8ng/ml) alone or in combination with U0126 (10 mM) relative to control, n = 4.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Cell count in a BEC cultures after treatment with bFGF (8 ng/ml) alone or in combination with U0126 (10 µM) relative to control, n = 3.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eC\u003c/strong\u003e Representative confocal microscopy images of BECs stained with antibodies against claudin-5, occludin and ZO-1.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/08a36f0464282a3e82e6e524.png"},{"id":23460833,"identity":"8790d43a-982b-41f5-a6c0-500daa75d7f5","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":6524130,"visible":true,"origin":"","legend":"\u003cp\u003eHigh concentration of bFGF induces long-term MAPK/ERK signalling in BECs.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Experimental protocol.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB:\u003c/strong\u003e Representative Western blots of pERK after 2-hours, 1- and 3-days treatment with bFGF (8ng/ml) alone or in combination with U0126 (10 µM), n = 2-4.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eC:\u003c/strong\u003e Quantified protein phosphorylation determined by ratio of pERK1/2 to α-tubulin band intensity.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/548c0ac558dc8c88e5bce587.png"},{"id":23460836,"identity":"de20a434-a78f-4aa5-a1ae-d89dfbc533f7","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1342968,"visible":true,"origin":"","legend":"\u003cp\u003ePotential role of autocrine and paracrine bFGF in regulation of permeability of the endothelial blood-brain barrier.\u0026nbsp;\u003c/p\u003e\u003cp\u003eThe hypothesis (which is still in need of further corroboration) posits that at the low concentrations bFGF derived from autocrine secretion up-regulates expression of tight junction proteins thus increasing the resistance of the barrier. At high concentration, bFGF derived from paracrine secretion from astrocytes or pericytes decreases expression of the tight junctional proteins (acting though MEK1/2-ERK1/2 cascade) consequently 'opening' the endothelial barrier. Thus, the very same signalling molecule can be employed for rapid opening and closing the blood-brain barrier.\u0026nbsp;\u0026nbsp;\u003c/p\u003e\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"Fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/c40d302e5a797acacbba616d.png"},{"id":23460837,"identity":"b4ec2621-b542-4ff7-a330-eb4db0e8e4ef","added_by":"auto","created_at":"2022-07-05 16:16:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":365011,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/8e078354-884b-46fb-a0ed-7a611c79c0dc.pdf"},{"id":23460835,"identity":"305b894c-73db-4004-a03e-4b5e97a12e6d","added_by":"auto","created_at":"2022-07-05 16:16:52","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4212660,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSupplementary figure 1\u003c/strong\u003e. Selective MEK 1/2 and JAK inhibitors decrease basal and bFGF-induced phosphorylation of STAT3.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eA:\u003c/strong\u003e Representative Western blots of pSTAT3 (Ser727/Tyr705) and b-actin in BECs after treatment with bFGF (8ng/ml) alone or in combination with JAKi (15 nM)/PD173074 (15 μM).\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eB: \u003c/strong\u003eRepresentative Western blots of pSTAT3 (Ser727) and a-tubulin in BECs after treatment with bFGF (8ng/ml) alone or in combination with U0126 (10 µM)/PD173074 (15 μM).\u003c/p\u003e","description":"","filename":"SupplementaryFig.tif","url":"https://assets-eu.researchsquare.com/files/rs-1784689/v1/32e9022d11c363cc2822fba1.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Basic fibroblast growth factor opens and closes the endothelial blood-brain barrier in a concentration-dependent manner","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe blood\u0026ndash;brain barrier (BBB), that governs selective exchange of substances between blood and the parenchyma of the central nervous system (CNS), is formed by the continuous layer of specialised brain endothelial cells (BECs), clasped by tight and adherent junctions (TJs and AJs, respectively) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Together with adjacent pericytes, smooth muscle cells, astrocytes and neurones, BECs are integrated into complex multi-cellular structure of neurogliovascular unit (NGVU) involved in the precise coordination of local blood flow, transport across the BBB, energy metabolism and formation of the brain active milieu [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Cellular components of the NGVU interact through multiple mechanisms, including numerous paracrine factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), angiopoetins, sonic hedgehog (SHH), retinoic acid (RA) and others [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Identification of precise mode of action of these paracrine factors in different physiologic and pathologic settings remains challenging. Firstly, majority of currently available \u003cem\u003ein vivo\u003c/em\u003e models do not allow accurate control and dynamic monitoring of local fluctuations of different paracrine factors at the BBB. Secondly, defined protocols, allowing generation of BECs monocultures with high transendothelial electrical resistance (TEER) values comparable with those recorded \u003cem\u003ein vivo\u003c/em\u003e, have been introduced only relatively recently. In our previous study, which employed a fully defined differentiation protocol [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] for producing BECs from human inducible pluripotent stem cells (iPSCs), we demonstrated a dual role for bFGF in the regulation of BECs barrier function [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Autocrine secretion of bFGF is required for the establishment of tight-junctions defining BECs barrier, whereas exogenous bFGF in high concentrations (\u0026gt;\u0026thinsp;4 ng/ml) disrupts the barrier integrity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. We also found that inhibitory effects of exogenous bFGF on the \u003cem\u003ein vitro\u003c/em\u003e BBB depend neither on phosphoinositide 3-Kinase (PI-3K), nor on ROCK signalling pathways [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the present study we further investigated molecular mechanisms of the inhibitory effects of exogenous bFGF. Binding of FGFs to the FGF receptor 1 (FGFR1) activates FGFR tyrosine kinase, which phosphorylates adaptor proteins and subsequent activation of rat sarcoma virus (Ras)-mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinases (ERK), phosphoinositide 3-kinase (PI3K)-Akt, phospholypase Cγ (PLCγ), and signal transducer and activator of transcription (STAT) intracellular signalling pathways [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Since our previous study demonstrated that PI-3K cascade suppresses inhibitory effects of bFGF on TEER, we focused on the STAT and Ras-MAPK/ERK signalling. We found that inhibition of STAT signalling with Janus kinase inhibitor (JAKi) alone, or in the presence of bFGF did not change TEER of BEC monolayers. In contrast, treatment with MEK 1/2 inhibitor U0126 alone did not affect TEER, but significantly increased TEER values in bFGF-treated BECs, thus partially neutralising inhibitory effect of exogenous bFGF. Our findings demonstrate that MAPK/ERK signalling pathway does not affect autocrine bFGF signalling-dependent BECs barrier function but is largely responsible for the disruptive effects of the exogenous bFGF. The bFGF therefore acts as a dynamic regulator of the BBB which opens it at high and closes at low concentrations.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaintenance of iPSCs and differentiation to the brain capillary endothelial cells (BECs)\u003c/h2\u003e \u003cp\u003eIn this study two healthy donor-derived iPSC lines, MBE 2960 (male, 78 years old) and SHED-iPSC (female, 7 years old), from the passages 12 to 34, were used. Both iPSC lines were cultured on matrigel-coated (Corning) plates with Essential 8 (E8) medium exchange every 24 hours. iPSCs were differentiated to BECs according to slightly modified previously published protocol [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Briefly, 24 hours after splitting, the differentiation was initiated by changing the E8 to the Essential 6 (E6) medium (Thermo Fisher Scientific). E6 was fully refreshed every 24 hours for four days. On the fifth day, the E6 was changed to the human Endothelial Serum Free-Medium (hESFM, Thermo Fisher Scientific) supplemented with 20 ng/ml bFGF (Thermo Fisher Scientific), 10 \u0026micro;M retinoic acid (Merck Darmstadt, Germany), and 0.25\u0026times; B-27 (Thermo Fisher Scientific). After 48 hours the hESFM medium was fully refreshed. The next day BECs were split for selection onto 400 \u0026micro;g/ml collagen IV and 100 \u0026micro;g/ml fibronectin-precoated (both from Merck) Transwell inserts (Corning) or 48-well plates (TPP). After four days, BECs monolayer barrier was characterised by measuring TEER and expression of claudin-5, occludin, ZO-1, VE-cadherin, CD34, and von Willebrand factor by immunocytochemistry and Western blotting (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eAll treatments of BECs were performed once by refreshing the medium on the next day after the differentiation (see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA for experimental design). The medium administered to the different experimental groups of BECs contained one of the following: 8 ng/ml bFGF, 15 \u0026micro;M PD173074 (Tocris Bioscience), 15 nM JAK inhibitor I (JAKi, Santa Cruz), 10 \u0026micro;M U0126 (MedChemExpress), bFGF\u0026thinsp;+\u0026thinsp;PD173074, bFGF\u0026thinsp;+\u0026thinsp;JAKi, bFGF\u0026thinsp;+\u0026thinsp;U0126. After three days of the treatment, BECs were subjected to TEER measurement and immunocytochemistry or Western blot analysis. Additionally, in the experiment with U0126, BECs were analysed by Western blot after 2 and 24 hours of the treatment (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eA for experimental design).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eTransendothelial electrical resistance (TEER) measurements\u003c/h2\u003e \u003cp\u003eTo measure TEER, BECs were grown on Transwell inserts (Corning) with 0.4 \u0026micro;m pore size and 0.33 cm\u003csup\u003e2\u003c/sup\u003e surface area polyester membrane. TEER was measured with Millicell ERS-2 Electrical Resistance System (Merck-Millipore). Each insert was measured three times in different locations. To calculate TEER (Ω*cm\u003csup\u003e2\u003c/sup\u003e), the mean electrical resistance of the BECs-free inserts was subtracted from the mean readings of the inserts with BECs monolayers and then multiplied by the surface area of the insert.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eImmunocytochemistry\u003c/h2\u003e \u003cp\u003eImmediately after TEER measurement BECs were fixed and permeabilised by incubating them with ice-cold methanol and acetone in a 1:1 ratio at -20\u0026deg;C for 10 min. Then the cells were washed three times with PBS and blocked with 1% BSA-PBS solution at room temperature for 30 min. Afterwards, the primary antibodies against ZO-1 (1:33), claudin-5 (1:100) and occludin (1:50, all from Thermo Fisher Scientific) were diluted in blocking solution and administered to the cells for incubation at 4\u0026deg;C overnight. Next, cells were washed three times with PBS and incubated with the secondary antibodies conjugated with Alexa Fluor 594 (1:1000, Thermo Fisher Scientific) diluted in PBS at room temperature, in the dark, for 1 hour. Then the samples were washed three times and mounted on coverslips using an aqueous fluorescent mounting medium with DAPI (Abcam). Prepared samples were analysed with the Leica TCS SP8 confocal microscope (Leica Microsystems, Mannheim, Germany) using lasers of Diode 405 nm and DPSS 561 nm and 63x oil immersion lens.\u003c/p\u003e \u003cp\u003eThe images of confocal microscopy were used to cell count evaluation. Cells were counted according to the DAPI stain using the ImageJ program, a multi-point tool.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003eWestern blot\u003c/h2\u003e \u003cp\u003eCells growing in a 48-well plate were washed three times with cold PBS and incubated with RIPA buffer (Thermo Fisher Scientific) containing 1\u0026times; protease inhibitor cocktail (Thermo Fisher Scientific) on ice for 15 min. Obtained lysates were collected, vortexed and placed on ice again for the next 10 min. Afterwards, the samples were centrifuged at 18 000 \u003cem\u003eg\u003c/em\u003e at 4\u0026deg;C for 20 min and the remaining supernatants were collected to new tubes. The concentration of proteins was measured with the NanoPhotometer Pearl (Implen) and equalised between samples with 1x Laemmli sample buffer (Bio-Rad). Then lysates were diluted in 6x Laemmli sample buffer and denatured at 95\u0026deg;C for 5 min. Protein lysates were subjected to 4\u0026ndash;10% polyacrylamide gel electrophoresis using Mini-PROTEAN Tetra cell apparatus (Bio-Rad). Separated proteins were blotted onto a polyvinylidene difluoride membrane by a semidry Trans-Blot Turbo transfer apparatus (Bio-Rad). The membrane was blocked with 5% bovine serum albumin (Applichem) diluted in PBS with 0.18% Tween-20 (hereinafter referred to as PBS-T) at room temperature on a platform rocker for 1 h. The primary antibodies against pAkt Ser473 (1:2000), pAkt Thr308 (1:1000), pERK (1:2000), pSTAT3 Ser727 (1:1000), pSTAT3 Tyr705 (1:800), β-actin (1:1000, all from Cell Signaling Technology) and α-tubulin (1:500, Thermo Fisher Scientific) were diluted in a blocking solution and the membrane was then probed with them and incubated at 4\u0026deg;C overnight. Then the membrane was washed three times with PBS-T on a platform rocker for 5 min and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2000, Thermo Fisher Scientific) diluted in PBS-T on a platform rocker at room temperature for 1 h. Washing procedure was repeated and immunoreactive bands were detected with Clarity ECL Western blotting substrate (Bio-Rad) using ChemiDoc MP system (Bio-Rad).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003eStatistics\u003c/h2\u003e \u003cp\u003eStatistical analysis was performed from at least three biological experiments using Graph Pad Prism\u0026reg; software 8.0.2 (Graph Pad Software, Inc., City, State, USA). Data in the graphs is presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (\u0026plusmn;\u0026thinsp;SD). Differences between the groups were compared by one-way ANOVA following Tukey\u0026rsquo;s post-test. Results were considered significant at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eExogenous bFGF activates ERK 1/2, STAT3 and Akt signalling pathways in differentiated BECs\u003c/h2\u003e \u003cp\u003eWe demonstrated previously that exogenous bFGF at concentrations above 4 ng/ml inhibits TEER and proliferation of BECs in a concentration-dependent manner [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. After bFGF binding, FGF receptor 1 (FGFR1) activates several downstream signalling pathways, including Ras-MAPK, PI-3K-Akt and STAT [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. We treated differentiated BECs with 8 ng/ml of exogenous bFGF for 3 days and monitored levels of phosphorylation of ERK 1/2, Akt (Thr308/Ser473) and STAT3 (Ser727/Tyr705) proteins. We found that treatment with bFGF significantly increased phosphorylation of ERK 1/2 and STAT3 (Ser727) and slightly increased of phosphorylation Akt at Thr308 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Combined treatment with selective inhibitor of FGFR1 PD173074 (15 \u0026micro;M) almost completely suppressed phosphorylation of ERK 1/2 and STAT3 at Ser727 and Tyr705 but not of Akt (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). Treatment with PD173074 alone strongly suppressed ERK 1/2 and STAT3 (Tyr705) phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C) highlighting the importance of autocrine/paracrine bFGF signalling for maintaining the basal activity of ERK 1/2 and STAT3 in differentiated BECs. Inhibition of FGFR1 with PD173074 did not affect Akt phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of JAK/STAT signalling does not affect bFGF-mediated suppression of TEER in BEC monolayers\u003c/h2\u003e \u003cp\u003eWe used JAKi (15 nM), a potent inhibitor of janus kinases (JAK) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] to block STAT signalling in differentiated BECs (Suppl. Figure\u0026nbsp;1). Treatment of BECs monolayers with JAKi alone affected neither TEER nor proliferation of BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B), whereas bFGF administered on its own down-regulated TEER by 89.53\u0026thinsp;\u0026plusmn;\u0026thinsp;7.77% (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and increased cell numbers by 90.00\u0026thinsp;\u0026plusmn;\u0026thinsp;31.61% (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.008, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). JAKi did not affect bFGF-mediated suppression of TEER and increase of BCEC proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Neither did it alter expression and distribution of TJ proteins claudin-5, occludin and ZO-1 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of ERK 1/2 signalling partially neutralised bFGF-mediated suppression of TEER in BEC monolayers\u003c/h2\u003e \u003cp\u003eTreatment with exogenous bFGF (8 ng/ml) induced long-term activation of ERK 1/2 signalling in the differentiated BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e). ERK 1/2 phosphorylation was effectively suppressed by U0126 (10 \u0026micro;M), a potent inhibitor of MEK 1/2 kinases, upstream activators of ERK 1/2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). We found that U0126 (10 \u0026micro;M) partially neutralised inhibitory effect of bFGF by increasing TEER by 40.95\u0026thinsp;\u0026plusmn;\u0026thinsp;23.49% (n\u0026thinsp;=\u0026thinsp;4, p\u0026thinsp;=\u0026thinsp;0.0467), whereas U0126 alone did not affect barrier properties of BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Treatment with U0126 alone or in combination with bFGF did not affect BEC proliferation and expression/distribution of TJ proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C). At the same time, U0126 suppressed STAT3 (Ser727) phosphorylation in bFGF-treated and untreated BECs (Suppl. Figure\u0026nbsp;1) indicating that Ser727 represents a downstream target for ERK 1/2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs we discovered before, autocrine secretion of bFGF by BECs is necessary for the establishment of proper barrier function, whereas exogenous bFGF in concentrations exceeding 4 ng/ml inhibits TEER of BECs monolayers in a concentration-dependent manner [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In this study we analysed the role of intracellular MAPK/ERK and STAT signalling pathways in mediating FGFR effects [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], in particular the contribution of these cascades to the inhibitory action of the exogenous bFGF on the barrier function of BECs. We found that inhibition of bFGF autocrine/paracrine signalling with selective inhibitor of FGFR1 PD173074 completely suppressed basal ERK 1/2 phosphorylation in differentiated BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). The basal ERK 1/2 activity, however, was not required for the establishment of BECs barrier, because inhibition of ERK 1/2 phosphorylation with U0126 did not change TEER (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). We therefore conclude that ERK 1/2 signalling pathway does not affect autocrine bFGF regulation of BEC barrier.\u003c/p\u003e \u003cp\u003eWe also found that PD173074 decreased both basal and bFGF-induced activity of STAT3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C). In this study we used antibodies against phospho-STAT3 (Ser727/Tyr705). Phosphorylation of STAT3 at Ser 727 is required for its maximal transcriptional activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and can be mediated by different members of MAPK family p38 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], ERK1/2 [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], c-Jun N-terminal Kinase (JNK) [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], protein kinase C (PKC) [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], and mammalian target of rapamycin (mTOR) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We found that inhibitor of MEK 1/2 kinases U0126 suppressed basal and bFGF-induced Ser727 phosphorylation in BECs (Suppl. Figure\u0026nbsp;1B). This indicates that Ser727 of STAT3 represents a downstream (direct or indirect, which remain unknown) target for ERK 1/2. As under basal conditions U0126 does not affect TEER, we concluded that STAT3 (Ser727) signalling does not contribute to autocrine bFGF regulatory effect on BECs barrier function. Similarly, inhibition of STAT signalling with JAK inhibitor did not change TEER in BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eIn our previous report, using the same experimental conditions, we found that treatment of BEC monolayers with PI-3K inhibitor LY294002 (25 \u0026micro;M) down-regulates TEER by approximately 40% [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] showing that basal PI-3K-Akt signalling contributes to the regulation of the BEC barrier. However, we did not detect down-regulation of Akt phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, C) in response to the treatment with PD173074, therefore basal PI-3K-Akt signalling in BECs is not linked to the autocrine/paracrine bFGF signalling. Further studies are needed to elucidate the mechanisms involved in the regulation of autocrine bFGF signalling-dependent BEC barrier function.\u003c/p\u003e \u003cp\u003eTreatment with high concentration of exogenous bFGF (8 ng/ml) induced long-term activation of ERK 1/2 signalling (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and effectively suppressed TEER in the differentiated BECs. We also found that inhibition of ERK 1/2 with U0126 partially neutralised inhibitory effects of bFGF (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, B). Several studies demonstrated that MAPK/ERK signalling pathway can modulate permeability of endothelial and epithelial barriers by modulating expression and distribution of TJ proteins [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. MAPK/ERK signalling cascades may promote or disrupt endothelial barriers in stimulus- and cell type-dependent manner [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. For example, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e induces paracellular permeability of porcine brain-derived microvascular endothelial cells by activating ERK 1/2 kinase pathway and these changes correlate with localisation of TJ proteins ZO-1 and ZO-2 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Exposure to microwaves damages BBB through the VEGF/Flk-1-ERK-dependent Tyr phosphorylation of occludin and inhibition of its interaction with ZO-1 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In our model similar mechanisms might be responsible for the inhibitory action of the exogenous bFGF. Therefore, the effects of bFGF/ERK signalling on the interaction between ZO and other TJ proteins in BECs are in need of systematic exploration in the future studies. Our finding that MEK 1/2 inhibitor U0126 suppressed FGF-induced STAT3 (Ser727) phosphorylation indicates that it represents a downstream target of ERK 1/2 which could be potentially responsible for the inhibitory action of the exogenous bFGF. Indeed, several studies demonstrated that IL-6 family cytokines promoted BBB breakdown through the activation of STAT3 signalling pathway [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Hence, the importance ERK 1/2-STAT3 signalling axis for the inhibitory action of the exogenous bFGF should be addressed in the future studies.\u003c/p\u003e \u003cp\u003eWe have previously reported that inhibition of FGFR1 and PI-3K signalling significantly decreased proliferation in bFGF untreated BECs and that these effects were paralleled with substantial reduction of TEER [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, the present study shows that MEK 1/2 inhibitor U0126 does not affect proliferation of bFGF untreated BECs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) indicating that under basal conditions BEC proliferation occurs through ERK-independent mechanisms. These findings suggest possible relationship between BEC proliferation and barrier establishment.\u003c/p\u003e \u003cp\u003eWe did not test how PLCγ, another mediator of the intracellular FGFR signalling [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] affects BECs barrier integrity. FGFR kinase recruits and activates the PLCγ which produces inositol trisphosphate (InsP\u003csub\u003e3\u003c/sub\u003e) and diacylglycerol (DAG) by the hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP\u003csub\u003e2\u003c/sub\u003e). InsP\u003csub\u003e3\u003c/sub\u003e induces calcium release from the intracellular stores while DAG activates DAG-sensitive protein kinases C (PKC) and protein kinases D (PKD) [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Some of the PLCγ downstream targets can be potentially involved in the regulation BEC barrier. For instance, acoustic wave stimulation activated calcium-dependent activation of PKC-δ pathway that mediated dissociation of ZO-1 and occludin, promoted paracellular permeability and opening of BBB [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Further research is needed to establish whether similar mechanisms can be responsible for the inhibitory effects of exogenous bFGF in BCECs.\u003c/p\u003e \u003cp\u003eConceptually, bFGF can derive from either luminal (blood) or abluminal (parenchymal), or from both sides of the endotehlial barrier. In our experiments exogenous bFGF was added to both upper and lower compartments of the Transwell inserts and therefore the differential effects of bFGF on the luminal and abluminal sides of BEC monolayer were not distinguished. The potential differences between the effects of luminal and abluminal bFGF on the BEC barrier function should be carefully explored in the future studies.\u003c/p\u003e \u003cp\u003eWhat is the source of paracrine bFGF in the NGVU? First of all, we can not exclude possibility that BECs can increase expression and secretion of the bFGF in response to the external clues. Some indirect evidence indicates that BECs can secrete substantial amounts of bFGF that act in the paracrine manner on the surrounding tissues. For instance, tumour microvascular endothelial cells secrete bFGF which promotes cancer stem cell features in differentiated glioblastoma cells [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Exosomes derived from brain microvascular endothelial cells after oxygen glucose deprivation contained increased levels of bFGF [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. An \u003cem\u003ein vitro\u003c/em\u003e BBB model could be used to explore the effects of different type of stress on the expression and secretion bFGF in BECs. Pericytes represent another potential source of bFGF in the NGVU. bFGF and FGFR1 are induced in pericytes at the periinfarct areas after brain ischemia [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Peripheral nerve pericytes partially modify blood-nerve barrier function through the secretion of bFGF [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In the adult brain, bFGF is predominantly synthesised and secreted by the astrocytes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, little is known about the role of bFGF in regulation of BBB in the unperturbed adult brain [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In contrast, many studies demonstrated that various injuries trigger reactive astrogliosis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] associated with increased secretion of bFGF [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Nevertheless, the effects of local bFGF increase on the BBB integrity remain unclear. Studies allowing simultaneous \u003cem\u003ein vivo\u003c/em\u003e monitoring of BBB permeability together with conditional inactivation of astrocytic bFGF release and (or) deletion of FGF receptors in BECs may reveal the role of astrocytic bFGF on BBB function. Astrocytes control permeability of the endothelial blood-brain barrier by secreting several factors such as VEGF-A, which loosens the endothelial barrier, or SHH, which stimulates barrier repair [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The bFGF can serve towards the same means: pulsative release of bFGF can rapidly and transiently open the barrier (acting through MAPK/ERK signalling pathway), whereas in the absence of additional input autocrine bFGF secretion restores the barrier integrity (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has received funding from European Regional Development Fund (project No 13.1.1-LMT-K-718-05-0005 ) under grant agreement with the Research Council of Lithuania (LMTLT). Funded as European Union\u0026apos;s measure in response to Cov-19 pandemic.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe datasets used or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePivoriunas A, Verkhratsky A (2021) Astrocyte-Endotheliocyte Axis in the Regulation of the Blood-Brain Barrier. Neurochem Res 46:2538\u0026ndash;2550\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhao Z, Nelson AR, Betsholtz C, Zlokovic BV (2015) Establishment and Dysfunction of the Blood-Brain Barrier. Cell 163:1064\u0026ndash;1078\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSweeney MD, Zhao Z, Montagne A, Nelson AR, Zlokovic BV (2019) Blood-Brain Barrier: From Physiology to Disease and Back. 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Trends Immunol 41:758\u0026ndash;770\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"bFGF, TEER, blood-brain barrier, ERK, STAT3, tight junctions","lastPublishedDoi":"10.21203/rs.3.rs-1784689/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-1784689/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMultiple paracrine factors have been implicated in the regulation of barrier properties of human brain endothelial cells (BECs) in different physiologic and pathologic settings. We have recently demonstrated that autocrine secretion of basic fibroblast growth factor (bFGF) by BECs is necessary for the establishment of endothelial barrier (as demonstrated by high trans-endothelial electric resistance, TEER), whereas exogenous bFGF inhibits TEER in a concentration-dependent manner. In the present study we analysed the contribution of MAPK/ERK and STAT3 signalling pathways to the inhibitory effects of exogenous bFGF. Treatment with bFGF (8 ng/ml) for 3 days increased phosphorylation of ERK1/2 and STAT3. Treatment with FGF receptor 1 (FGFR1) inhibitor PD173074 (15 µM) suppressed both basal and bFGF-induced activation of ERK1/2 and STAT3. Suppression of STAT signalling with Janus kinase inhibitor JAKi (15 nM)\u0026nbsp;alone or in the presence of bFGF did not change TEER in BEC monolayers. Exposure to JAKi affected neither proliferation, nor expression and distribution of tight junction (TJ) proteins claudin-5, occludin and zonula occludens-1 (ZO-1). In contrast, treatment with MEK 1/2\u0026nbsp;inhibitor U0126 (10 µM) partially neutralized inhibitory effect of bFGF thus increasing TEER, whereas U0126 alone did not affect resistance of endothelial barrier. Our findings demonstrate that MAPK/ERK signalling pathway does not affect autocrine bFGF signalling-dependent BECs barrier function but is largely responsible for the disruptive effects of the exogenous bFGF. We speculate that bFGF may (depending on concentration and possibly origin) dynamically regulate permeability of the endothelial blood-brain barrier.\u0026nbsp;\u003c/p\u003e","manuscriptTitle":"Basic fibroblast growth factor opens and closes the endothelial blood-brain barrier in a concentration-dependent manner","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2022-07-05 16:16:50","doi":"10.21203/rs.3.rs-1784689/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2022-07-05T17:03:00+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2022-07-04T12:18:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"0773e025-4b91-476a-9da9-7c4f51947a6a","date":"2022-06-29T11:10:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55869a09-8e39-4879-a9c3-a72c3a17114b","date":"2022-06-29T09:01:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2022-06-29T07:52:26+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2022-06-26T18:29:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2022-06-25T01:08:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2022-06-22T14:04:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c45bc07b-6fe0-4bc9-abbc-5abebf717af6","owner":[],"postedDate":"July 5th, 2022","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2022-07-05T17:14:15+00:00","versionOfRecord":[],"versionCreatedAt":"2022-07-05 16:16:50","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-1784689","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-1784689","identity":"rs-1784689","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}