Mechanism Of LPS-induced Brain Microvascular Endothelia Cells' Hyperpemeability

Despite the availability of effective antibacterial therapies, bacterial meningitis in infants and children is associated with significant morbidity and mortality. Even survivors,30-50%of them, sustain neurological sequelae. The mechanisms of CNS damage during meningitis have not been conclusively identified, but several lines of evidence indicate that the inflammatory response to bacterial lipopolysaccharides (LPS) promotes increased blood brain barrier (BBB) permeability; leading to vasogenic brain edema, could play a key role in its pathogeneses.The BMECs, which are linked by tight junctions, were important structural and functional roles in maintaining the integrity of the BBB. LPS, the major component of the outer membrane of gram-negative bacteria, could ruin the BMEC barrier by promoting TJ disassembly, leading to vasogenic brain edema formation. We previously demonstrated that elevated LPS is highly correlated with the occurrence of decrease of Occluding and ZO-1as well as BBB dysfunction. However, little is known about the complex signaling events in the above process. In vivo and in vitro studies indicate that a complex network of signaling pathways is involved in regulating TJ structure and function, such as protein kinase C (PKC), tyrosine kinase, Ras or Rho GTP-binding proteins and phosphatidylinositol3-kinase (PI3Ks). Recently, it has been demonstrated that, in response to stimulus, NF-κB also can participate in regulating tight junction proteins. All the above signal molecular could participate regulating LPS induced BBB hyperpermeability, but it remains unclearly that how they regulate the TJ protein and interact with each other. Therefore, to make cleary these question will contribute to clarify the mechanism of infectious brain edema, and will be possible to design new methods to ameliorating brain edema formation.Section1The immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cellsObjective The purpose of this study is to assesse weather the immortalized mouse brain endothelial cell line Bend3displayed the comparative barrier characteristics as the primary brain microvascular endothelial cells(BEMC). Methods Bend.3cells and primary BEMC, whose restrictive characteristics were assessed by transendothelial electrical resistance (TEER) assay and HRP permeability assay. Meanwhile, Western Blot and F-actin Stain were used to detect the tight junction protein and cytoskeleton. Results The TEER over the10days Bend.3samples was82.33±6.03Ω-cm2, which was significantly increased compared to the3days samples(P<0.05). Meanwhile, the permeability rates for HRP were (2.2±0.05)%and (4.3±0.20)%in3days samples and10days samples respectively, the difference was obvious in the2groups(P<0.05). Compared to the primary BEMC, there were no difference in TEER in Bend.3cells at the same time(P<0.05). Futhermore, in Bend.3and primary BEMC, Western blot indicated high level expression of multiple tight junction proteins Occludin, Claudin-5and ZO-1. Furthermore, F-actin Stain showed that the presence of F-actin was visualized around the cell membrane and presented scrobiculate linear fluorescence. Conclusions Bend.3cell has the similar barrier characteristics with primary BEMC, whose barrier function may reach to the most advantageous state after10days culture.Section2the preparatory mechanism for LPS induced BEMC hyperpermeabilityObjective To address the impair ment of TJ are close to LPS induced-Bend.3cell barrier dysfunction and to examine the signaling networks involved in the hyperpermeability of the brain endothelial barrier caused by LPS. Methods To confirm that LPS induces endothelial barrier hyperpermeability by disrupting tight junction, Bend.3cells were exposed to LPS, and changes in endothelial permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein expression (western blot or immunofluorescence) were monitored. To make clearly that wether PKC, RhoA, PI3K and tyrosine kinase were involve in the hyperpermeability of the brain endothelial barrier caused by LPS, calphostin C (PKC inhibitor), C3transferase (Rho inhibitor), wortmannin (PI3K inhibitor), PP2(tyrosine kinase inhibitor)were pretreated with Bend.3cell, then TEER assay were used to assay the barrier function of each group cells. Result LPS caused a significant decrease in TEER in a time-dependent manner, accompanied by decrease of TJ protein ZO-1, Occludin and Claudin-5, distribution of Claudin-5, rearrangement of F-actin. Inhititors of PKC and Rho could improve LPS-induced TEER decrease, while inhibitors of PI3K and tyrosine kinase could have no effect on suppressing LPS-induced BBB hyperpermeability. Conclusions These data indicate that LPS ruined TJ protein and induced rearrangement of F-actin, which lead to BEMC hyperpermeability, Moreover, PKC and Rho but not PI3K and tyrosine kinase mediates LPS disruptive effect on BEMC barrier.Section3PKC and RhoA signals cross-talk in LPS induced alterations in brain endothelial permeabilityObjective to explore how PKC a, β, ζ, to mediated the LPS disruptive signal to BEMC permeability and to detected how they interact with RhoA in the process. Methods1. pull-down assay and enzyme assay were used to detected the LPS activated effect on RhoA and PKC (α,β和ζ).2. PcDNA3.1hygro-N19RhoA (the dominant negative mutant of RhoA), and PcDNA3.1hygro vector plasmids (vector-1) were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The remarkable inhibitory effect of N19RhoA was confirmed by pull-down assay. Meanwhile, he Bend.3cells transfected with PLKO.1-puro-PKCa-shRNA, PLKO.1-puro-PKCp-shRNA, PLKO.1-puro-PKCζ-shRNA and empty PLKO.1-puro vector respectively. Stably transfected cells were used for experiments after selection by Puromycin (300ug/ml). The inhibitory levels of PKC-a, β, ζ were confirmed by Western blot. To detected whether PKC-a, β, ζ, and RhoA were involved in regulating LPS induced TJ dysfunction in BEMC, Cells were divided to5groups relate to their different transfected plasmids, and each group cells were exposed to LPS, and changes in F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored.3. The interactions between the PKC and Rho pathways were therefore examined. Experiments were performed in which RhoA activation was inhibited (by N19RhoA plasmid or C3transferase) and the activity of PKC-a, PKC-β and PKC-ζ was observed by enzyme assay in Bend.3cells. Then opposite experiments were performed where ShRNA was used to deplete PKC (PKC-a, PKC-β and PKC-ζ,respectively), and RhoA activity was observed. To further detect the relationship between PKC-a and RhoA in regulating BMEC TJ dysfunction induced by LPS, Bend.3/N19RhoA cells and Bend.3/vector-1cells were pretreated with inhibitor of PKC-a, and then their TEER was detected. to confirm whether PKC-ζ acts as downstream molecular target of RhoA signals in the process, Bend.3/Sh-PKCζ and Bend.3/vector-2cells were pretreated with inhibitor of Rho, then TEER detection was performed as mentioned before. Result1. LPS active RhoA, PKC-a, PKC-β and PKC-ζ at5min.2. Established stably transfected cells for N19RhoA and PKCa, β,ζ-ShRNA. Inhibition of RhoA, PKC-a, PKC-p and PKC-ζ could improve LPS-induced TJ destruction.3. PKC-a and PKC-ζ, but not PKC-β, interact with RhoA in Bend.3cells stimulated by LPS; moreover, PKC-a and PKC-ζ act as the upstream and downstream targets for Rho, respectively. Conclusions These data indicate that LPS induced activation of PKC (a, β, ζ and RhoA mediated TJ destruction and BEMC barrier dysfunction, PKC-a and PKC-ζ, respectively, act as the upstream and downstream regulators for RhoA in the process.Section4RhoA/NF-KB/MLCK pathway involved in LPS-induced BEMC hyperpermeabilityObjective to explore whether RhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC hyperpermeability. Methods DNMu-IκBαplasmids were transfected into Bend.3cells by Lipofectamine2000. Stably transfected cells were obtained using the Hygromycin B (400ug/ml) selection method after transfection. The inhibitory effect was confirmed by Reporter gene analysis. To ensure that RhoA and NF-κB participated in the regulatory mechanisms of LPS mediated BBB dysfunction, pull down assay and Reporter gene analysis were used to detected LPS active effect on RhoA and NF-κB. Men while, Cells were divided to4groups:Bend.3, Bend.3/vector-1, Bend.3/N19RhoA and Bend.3/DNMu-IκBα, and each group cells were exposed to LPS, and changes in permeability (TEER assay), F-actin dynamics (Rhodamine-phalloidin staining) and tight junction protein (western blot or immunofluorescence) were monitored. To clarify the relationship between RhoA and NF-κB in the process, the activities of NF-κB (via luciferase reporter assays) and RhoA (via pull-down assays) were detected in the above4group cells. Lastly, to investigate whether NF-Kb/MLCK signal regulate LPS induced MLC phosphorylation, we measured changes in myosin light chain (MLC) phosphorylation and MLCK transcription by western blot and RT-PCR respectively. Result LPS caused RhoA active earlier than NF-κB. And LPS induced F-actin rearrangement, tight junction disruption and barrier dysfunction were suppressed by inhibitors of RhoA or NF-κB, in which inhibiting RhoA was more efficient. Inactivating RhoA prohibited LPS-induced NF-KB activation, but the inverse was not true. LPS increase MLCK transcription and MLC phosphorylation at30min and3h respectively, and inhibition of NF-KB could suppressed these incensement. Conclusions PhoA/NF-κB/MLCK pathway were involved in LPS-induced BEMC TJ disruption and hyperpermeability by phosphorylated MLC.