Ketamine alleviates bradykinin-induced disruption of the mouse cerebrovascular endothelial cell-constructed tight junction barrier via a calcium-mediated redistribution of occludin polymerization.

Following brain injury, a sequence of mechanisms leads to disruption of the blood-brain barrier (BBB) and subsequent cerebral edema, which is thought to begin with activation of bradykinin. Our previous studies showed that ketamine, a widely used intravenous anesthetic agent, can suppress bradykinin-induced cell dysfunction. This study further aimed to evaluate the protective effects of ketamine against bradykinin-induced disruption of the mouse cerebrovascular endothelial cell (MCEC)-constructed tight junction barrier and the possible mechanisms. Exposure of MCECs to bradykinin increased intracellular calcium (Ca2+) concentrations in a time-dependent manner. However, pretreatment of MCECs with ketamine time- and concentration-dependently lowered the bradykinin-induced calcium influx. As to the mechanisms, although exposure of MCECs to ketamine induced bradykinin R1 receptor protein and mRNA expression, this anesthetic did not change levels of the bradykinin R2 receptor, a major receptor that responds to bradykinin stimulation. Bradykinin increased amounts of soluble occludin in MCECs, but pretreatment with ketamine alleviated this disturbance in occludin polymerization. Consequently, exposure to bradykinin decreased the transendothelial electronic resistance in the MCEC-constructed tight junction barrier. However, pretreatment with ketamine attenuated the bradykinin-induced disruption of the tight junction barrier. Taken together, this study shows that ketamine at a therapeutic concentration can protect against bradykinin-induced breakage of the BBB via suppressing calcium-dependent redistribution of occludin tight junctions. Thus, ketamine has the potential for maintaining the BBB in critically ill patients with severe brain disorders.

Chen JT ，Lin YL ，Chen TL ，Tai YT ，Chen CY ，Chen RM ... -  《-》

Roles of NMDARs in maintenance of the mouse cerebrovascular endothelial cell-constructed tight junction barrier.

Glutamate can activate NMDA receptor (NMDAR) and subsequently induces excitotoxic neuron loss. However, roles of NMDARs in the blood-brain barrier (BBB) are little known. This study used a mouse cerebrovascular endothelial cell (MCEC) model to evaluate the effects of NMDAR activation on maintenance of the BBB and its possible mechanisms. Analysis of confocal microscopy revealed expressions of NMDAR subunits, GluN1 and GLUN2B, in MCECs. An immunoblot assay further showed the existence of GluN1 in plasma membranes of MCECs. In brain tissues, a confocal microscopic analysis demonstrated co-localization of GluN1 and factor VIII, a biomarker of MCECs. In addition, GluN1 mRNA was detected in MCECs and the brain. Functional assays showed that exposure of MCECs to NMDA increased calcium influx. Separately, NMDA suppressed transendothelial electrical resistance values, levels of occludin, and occludin tight junctions. As to the mechanism, NMDA stimulated sequential phosphorylations of extracellular signal-regulated kinase (ERK)1/2 and mitogen-activated ERK (MEK)1. Interestingly, amounts of matrix metalloproteinase (MMP)2 and MMP9 in MCECs were augmented by NMDA. The NMDA-induced alterations in ERK1/2 phosphorylation and occludin levels were reversed by pretreatment with PD98059, a MEK inhibitor, and MK-801, a NMDAR antagonist, respectively. Therefore, this study shows the functional presence of NMDARs in MCECs, and NMDAR activation can disrupt the MCEC-constructed tight junction barrier via activation of the MEK1/2-ERK1/2 signaling pathway and upregulation of MMP2/9 expressions.

Chen JT ，Chen TG ，Chang YC ，Chen CY ，Chen RM ... -  《-》

HIV-1 Tat protein alters tight junction protein expression and distribution in cultured brain endothelial cells.

Disruption of the blood-brain barrier (BBB) is widely believed to be the main route of human immunodeficiency virus (HIV) entry into the central nervous system (CNS). Although mechanisms of this process are not fully understood, alterations of tight junction protein expression can contribute, at least in part, to this phenomenon. Tight junctions are critical structural and functional elements of cerebral microvascular endothelial cells and the BBB. The aim of the present study was to examine the effects of HIV-1 Tat protein on expression of tight junction proteins. Primary cultures of brain microvascular endothelial cells (BMEC) were employed in these experiments. A 24-hr exposure of BMEC to Tat(1-72) resulted in a decrease of claudin-1, claudin-5, and zonula occludens (ZO)-2 expression, whereas total levels of occludin and ZO-1 remained unchanged. In addition, a short (3-hr) exposure of BMEC to Tat(1-72) induced cellular redistribution of claudin-5 immunoreactivity. Tat(1-72)-induced alterations of claudin-5 expression also were confirmed in vivo where Tat(1-72) was injected into the right hippocampus of mice. These findings indicate that HIV-1 Tat protein can markedly affect expression and distribution of specific tight junction proteins in brain endothelium. Alterations of only distinct tight junction proteins suggest a finely tuned effect of Tat(1-72) on the BBB. Because tight junction proteins are critical for the barrier function of the BBB, such alterations can lead to disturbances of the BBB integrity and contribute to HIV trafficking into the brain.

András IE ，Pu H ，Deli MA ，Nath A ，Hennig B ，Toborek M ... -  《-》

Plant-derived triterpene celastrol ameliorates oxygen glucose deprivation-induced disruption of endothelial barrier assembly via inducing tight junction proteins.

The integrity and functions of blood-brain barrier (BBB) are regulated by the expression and organization of tight junction proteins. The present study was designed to explore whether plant-derived triterpenoid celastrol could regulate tight junction integrity in murine brain endothelial bEnd3 cells. We disrupted the tight junctions between endothelial bEnd3 cells by oxygen glucose deprivation (OGD). We investigated the effects of celastrol on the permeability of endothelial monolayers by measuring transepithelial electrical resistance (TEER). To clarify the tight junction composition, we analyzed the expression of tight junction proteins by RT-PCR and Western blotting techniques. We found that celastrol recovered OGD-induced TEER loss in a concentration-dependent manner. Celastrol induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells. As a result, celastrol effectively maintained tight junction integrity and inhibited macrophage migration through endothelial monolayers against OGD challenge. Further mechanistic studies revealed that celastrol induced the expression of occludin and ZO-1) via activating MAPKs and PI3K/Akt/mTOR pathway. We also observed that celastrol regulated claudin-5 expression through different mechanisms. The present study demonstrated that celastrol effectively protected tight junction integrity against OGD-induced damage. Thus, celastrol could be a drug candidate for the treatment of BBB dysfunction in various diseases.

Luo D ，Zhao J ，Rong J 《-》

Gold Nanoparticles Increase Endothelial Paracellular Permeability by Altering Components of Endothelial Tight Junctions, and Increase Blood-Brain Barrier Permeability in Mice.

Gold nanoparticles (Au-NPs) are being increasingly used as constituents in cosmetics, biosensors, bioimaging, photothermal therapy, and targeted drug delivery. This elevated exposure to Au-NPs poses systemic risks in humans, particularly risks associated with the biodistribution of Au-NPs and their potent interaction with biological barriers. We treated human umbilical vein endothelial cells with Au-NPs and comprehensively examined the expression levels of tight junction (TJ) proteins such as occludin, claudin-5, junctional adhesion molecules, and zonula occludens-1 (ZO-1), as well as endothelial paracellular permeability and the intracellular signaling required for TJ organization. Moreover, we validated the effects of Au-NPs on the integrity of TJs in mouse brain microvascular endothelial cells in vitro and obtained direct evidence of their influence on blood-brain barrier (BBB) permeability in vivo. Treatment with Au-NPs caused a pronounced reduction of PKCζ-dependent threonine phosphorylation of occludin and ZO-1, which resulted in the instability of endothelial TJs and led to proteasome-mediated degradation of TJ components. This impairment in the assembly of TJs between endothelial cells increased the permeability of the transendothelial paracellular passage and the BBB. Au-NPs increased endothelial paracellular permeability in vitro and elevated BBB permeability in vivo. Future studies must investigate the direct and indirect toxicity caused by Au-NP-induced endothelial TJ opening and thereby address the double-edged-sword effect of Au-NPs.

Li CH ，Shyu MK ，Jhan C ，Cheng YW ，Tsai CH ，Liu CW ，Lee CC ，Chen RM ，Kang JJ ... -  《-》