Background Excessive inflammation might activate and injure the blood-brain barrier (BBB), a common feature of many central nervous system (CNS) disorders. We previously developed an in vitro BBB injury model in which the organophosphate paraoxon (PX) affects the BBB endothelium by attenuating junctional protein expression leading to weakened barrier integrity. The objective of this study was to investigate the inflammatory cellular response at the BBB to elucidate critical pathways that might lead to effective treatment in CNS pathologies in which the BBB is compromised. We hypothesized that caspase-1, a core component of the inflammasome complex, might have important role in BBB function since accumulating evidence indicates its involvement in brain inflammation and pathophysiology. Methods An in vitro human BBB model was employed to investigate BBB functions related to inflammation, primarily adhesion and transmigration of peripheral blood mononuclear cells (PBMCs). Caspase-1 pathway was studied by measurements of its activation state and its role in PBMCs adhesion, transmigration, and BBB permeability were investigated using the specific caspase-1 inhibitor, VX-765. Expression level of adhesion and junctional molecules and the secretion of pro-inflammatory cytokines were measured in vitro and in vivo at the BBB endothelium after exposure to PX. The potential repair effect of blocking caspase-1 and downstream molecules was evaluated by immunocytochemistry, ELISA, and Nanostring technology. Results PX affected the BBB in vitro by elevating the expression of the adhesion molecules E-selectin and ICAM-1 leading to increased adhesion of PBMCs to endothelial monolayer, followed by elevated transendothelial-migration which was ICAM-1 and LFA-1 dependent. Blocking caspase-8 and 9 rescued the viability of the endothelial cells but not the elevated transmigration of PBMCs. Inhibition of caspase-1, on the other hand, robustly restored all of barrier insults tested including PBMCs adhesion and transmigration, permeability, and VE-cadherin protein levels. The in vitro inflammatory response induced by PX and the role of caspase-1 in BBB injury were corroborated in vivo in isolated blood vessels from hippocampi of mice exposed to PX and treated with VX-765. Conclusions These results shed light on the important role of caspase-1 in BBB insult in general and specifically in the inflamed endothelium, and suggest therapeutic potential for various CNS disorders, by targeting caspase-1 in the injured BBB.

Organophosphorus compounds (OPs) are highly toxic chemicals widely used as pesticides (e.g., paraoxon (PX)- the active metabolite of the insecticide parathion) and as chemical warfare nerve agents. Blood-brain barrier (BBB) leakage has been shown in rodents exposed to PX, which is an organophosphate oxon. In this study, we investigated the cellular mechanisms involved in BBB reaction after acute exposure to PX in an established in vitro BBB system made of stem-cell derived, human brain-like endothelial cells (BLECs) together with brain pericytes that closely mimic the in vivo BBB. Our results show that PX directly affects the BBB in vitro both at toxic and non-toxic concentrations by attenuating tight junctional (TJ) protein expression and that only above a certain threshold the paracellular barrier integrity is compromised. Below this threshold, BLECs exhibit a morphological coping mechanism in which they enlarge their cell area thus preventing the formation of meaningful intercellular gaps and maintaining barrier integrity. Importantly, we demonstrate that reversal of the apoptotic cell death induced by PX, by a pan-caspase-inhibitor ZVAD-FMK (ZVAD) can reduce PX-induced cell death and elevate cell area but do not prevent the induced BBB permeability, implying that TJ complex functionality is hindered. This is corroborated by formation of ROS at all toxic concentrations of PX and which are even higher with ZVAD. We suggest that while lower levels of ROS can induce compensating mechanisms, higher PX-induced oxidative stress levels interfere with barrier integrity.

Replication and assembly of hepatitis C virus (HCV) depend on the host's secretory and lipid-biosynthetic machinery. Viral replication occurs on endoplasmic reticulum (ER)-derived modified membranes, while viral assembly is thought to occur on lipid droplets (LDs). A physical association and coordination between the viral replication and assembly complexes are prerequisites for efficient viral production. Nonstructural protein 5A (NS5A), which localizes both to the ER and LDs, is an ideal candidate for this function. Here, the interaction of NS5A with host cell membranes and binding partners was characterized in living cells. The binding of NS5A to LDs is apparently irreversible, both in HCV-infected cells and when ectopically expressed. In HCV-infected cells, NS5A fluorescence was observed around the LDs and in perinuclear structures that were incorporated into a highly immobile platform superimposed over the ER membrane. Moreover, TBC1D20 and its cognate GTPase Rab1 are recruited by NS5A to LDs. The NS5A-TBC1D20 interaction was shown to be essential for the viral life cycle. In cells, expression of the Rab1 dominant negative (Rab1DN) GTPase mutant abolished steady-state LDs. In infected cells, Rab1DN induced the elimination of NS5A from viral replication sites. Our results demonstrate the significance of the localization of NS5A to LDs and support a model whereby its interaction with TBC1D20 and Rab1 affects lipid droplet metabolism to promote the viral life cycle.

The blood–brain barrier (BBB) is limiting transcellular and paracellular movement of molecules and cells, controls molecular traffic, and keeps out toxins. However, this protective function is the major hurdle for treating brain diseases such as brain tumors, Parkinson’s disease, Alzheimer’s disease, etc. It was previously demonstrated that high pulsed electrical fields (PEFs) can disrupt the BBB by inducing electroporation (EP) which increases the permeability of the transcellular route. Our goal was to study the effects of low PEFs, well below the threshold of EP on the integrity and function of the BBB. Ten low voltage pulses (5–100 V) were applied to a human in vitro BBB model. Changes in permeability to small molecules (NaF) were studied as well as changes in impedance spectrum and trans-endothelial electric resistivity. Viability and EP were evaluated by Presto-Blue and endogenous Lactate dehydrogenase release assays. The effect on tight junction and adherent junction protein was also studied. The results of low voltage experiments were compared to high voltage experiments (200–1400 V). A significant increase in permeability was found at voltages as low as 10 V despite EP only occurring from 100 V. The changes in permeability as a function of applied voltage were fitted to an inverse-exponential function, suggesting a plateau effect. Staining of VE-cadherin showed specific changes in protein expression. The results indicate that low PEFs can transiently disrupt the BBB by affecting the paracellular route, although the mechanism remains unclear.

Mesenchymal stromal cells (MSC) are used extensively in clinical trials; however, the possibility that MSCs have a potential for malignant transformation was raised. We examined the genomic stability versus the tumorforming capacity of multiple mouse MSCs. Murine MSCs have been shown to be less stable and more prone to malignant transformation than their human counterparts. A large series of independently isolated MSC populations exhibited low tumorigenic potential under syngeneic conditions, which increased in immunocompromised animals. Unexpectedly, higher ploidy correlated with reduced tumor-forming capacity. Furthermore, in both cultured MSCs and primary hepatocytes, polyploidization was associated with a dramatic decrease in the expression of the long noncoding RNA H19. Direct knockdown of H19 expression in diploid cells resulted in acquisition of polyploid cell traits. Moreover, artificial tetraploidization of diploid cancer cells led to a reduction of H19 levels, as well as to an attenuation of the tumorigenic potential. Polyploidy might therefore serve as a protective mechanism aimed at reducing malignant transformation through the involvement of the H19 regulatory long noncoding RNA. Cancer Res; 72(24); 6403-13. Ó2012 AACR.