Concentration‐dependent duality of bFGF in regulation of barrier properties of human brain endothelial cells

Multiple paracrine factors regulate the barrier properties of human brain capillary endothelial cells (BCECs). Understanding the precise mode of action of these factors remains a challenging task, because of the limited availability of functionally competent BCECs and the use of serum-containing medium. In the present study, we employed a defined protocol for producing BCECs from human inducible pluripotent stem cells. We found that autocrine secretion of basic fibroblast growth factor (bFGF) is necessary for the establishment a tight BCECs barrier, as revealed by measurements of transendothelial electric resistance (TEER). In contrast, addition of exogenous bFGF in concentrations higher than 4 ng/ml inhibited TEER in a concentration-dependent manner. Exogenous bFGF did not significantly affect expression and distribution of tight junction proteins claudin-5, occludin and zonula occludens (ZO)-1. Treatment with FGF receptor blocker PD173074 (15 µM) suppressed inhibitory effects of bFGF and induced nuclear translocation of protein ZO-1. Inhibition of phosphoinositide 3-Kinase (PI-3K) with LY294002 (25 µM) significantly potentiated an inhibitory effect of bFGF on TEER indicating that PI-3K signalling pathway counteracts bFGF modulation of TEER. In conclusion, we show that autocrine bFGF secretion is necessary for the proper barrier function of BCECs, whereas exogenous bFGF in higher doses suppresses barrier resistance. Our findings demonstrate a dual role for bFGF in the regulation of BCEC barrier function.</p

Basic Fibroblast Growth Factor Opens and Closes the Endothelial Blood–Brain Barrier in a Concentration-Dependent Manner

Multiple paracrine factors are 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) 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 neutralised 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.</p

Immortalised Hippocampal Astrocytes from 3xTG-AD Mice Fail to Support BBB Integrity In Vitro: Role of Extracellular Vesicles in Glial-Endothelial Communication

Impairments of the blood-brain barrier (BBB) and vascular dysfunction contribute to Alzheimer's disease (AD) from the earliest stages. However, the influence of AD-affected astrocytes on the BBB remain largely unexplored. In the present study, we created an in vitro BBB using human-immortalized endothelial cells in combination with immortalized astroglial cell lines from the hippocampus of 3xTG-AD and wild-type mice (3Tg-iAstro and WT-iAstro, respectively). We found that co-culturing endothelial monolayers with WT-iAstro upregulates expression of endothelial tight junction proteins (claudin-5, occludin, ZO-1) and increases the trans-endothelial electrical resistance (TEER). In contrast, co-culturing with 3Tg-iAstro does not affect expression of tight junction proteins and does not change the TEER of endothelial monolayers. The same in vitro model has been used to evaluate the effects of extracellular vesicles (EVs) derived from the WT-iAstro and 3Tg-iAstro. The EVs derived from WT-iAstro increased TEER and upregulated expression of tight junction proteins, whereas EVs from 3Tg-iAstro were ineffective. In conclusion, we show for the first time that immortalized hippocampal astrocytes from 3xTG-AD mice exhibit impaired capacity to support BBB integrity in vitro through paracrine mechanisms and may represent an important factor underlying vascular abnormalities during development of AD