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 . In this study we analysed the role of intracellular MAPK/ERK and STAT signalling pathways in mediating FGFR effects , 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. 2B, 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. 5A). 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. 2B, 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  and can be mediated by different members of MAPK family p38 , ERK1/2 [13, 14], c-Jun N-terminal Kinase (JNK) , protein kinase C (PKC) , and mammalian target of rapamycin (mTOR) . 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. 3A).
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%  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. 2B, 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. 5A, 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 . MAPK/ERK signalling cascades may promote or disrupt endothelial barriers in stimulus- and cell type-dependent manner . For example, H2O2 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 . Exposure to microwaves damages BBB through the VEGF/Flk-1-ERK-dependent Tyr phosphorylation of occludin and inhibition of its interaction with ZO-1 . 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 . However, the present study shows that MEK 1/2 inhibitor U0126 does not affect proliferation of bFGF untreated BECs (Fig. 5B) 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  affects BECs barrier integrity. FGFR kinase recruits and activates the PLCγ which produces inositol trisphosphate (InsP3) and diacylglycerol (DAG) by the hydrolysis of phosphatidylinositol (4,5) bisphosphate (PIP2). InsP3 induces calcium release from the intracellular stores while DAG activates DAG-sensitive protein kinases C (PKC) and protein kinases D (PKD) . 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 . 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 . Exosomes derived from brain microvascular endothelial cells after oxygen glucose deprivation contained increased levels of bFGF . 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 . Peripheral nerve pericytes partially modify blood-nerve barrier function through the secretion of bFGF . In the adult brain, bFGF is predominantly synthesised and secreted by the astrocytes . However, little is known about the role of bFGF in regulation of BBB in the unperturbed adult brain . In contrast, many studies demonstrated that various injuries trigger reactive astrogliosis  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 . 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).