Csilla Fazakas

Expression and regulation of toll-like receptors in cerebral endothelial cells

Cerebral endothelial cells - the principal components of the blood-brain barrier (BBB) - fulfill several important functions in the central nervous system (CNS). They form an active interface between blood and neuronal tissue and play a key role in the maintenance of the homeostasis of the CNS. Infections caused by different pathogens are often associated with systemic symptoms and may compromise the functional integrity of the BBB as well. In the mediation of the systemic effect of pathogens Toll-like receptors (TLRs) play a significant role. TLRs are a type of pattern recognition receptor and recognize molecules that are broadly shared by pathogens but distinguishable from host molecules. TLRs are broadly distributed on cells of the immune system and function as primary sensors of invading pathogens. There is also growing experimental evidence indicating that Toll-like receptors are expressed on different non-immune cell types as well, like epithelial or endothelial cells. Here we demonstrate the expression of TLR2, TLR3, TLR4 and TLR6 on rat and human cerebral endothelial cells. Oxidative stress significantly upregulated the expression of these receptors whereas TNF-alpha upregulated the expression of TLR2 and TLR3. Furthermore we have shown, that activation of TLR2/6 leads to an increased permeability which is accompanied by a downregulation of occludin and claudin-5 expression and disappearance of these tight junction proteins from the cell membrane. Changes in occludin expression and localization could be inhibited by the ERK1/2 inhibitor U0126. Our results suggest a significant role of the cerebral endothelium in mediation of the neural effects of different inflammatory processes.

Transmigration of Melanoma Cells through the Blood-Brain Barrier: Role of Endothelial Tight Junctions and Melanoma-Released Serine Proteases

Malignant melanoma represents the third common cause of brain metastasis, having the highest propensity to metastasize to the brain of all primary neoplasms in adults. Since the central nervous system lacks a lymphatic system, the only possibility for melanoma cells to reach the brain is via the blood stream and the blood-brain barrier. Despite the great clinical importance, mechanisms of transmigration of melanoma cells through the blood-brain barrier are incompletely understood. In order to investigate this question we have used an in vitro experimental setup based on the culture of cerebral endothelial cells (CECs) and the A2058 and B16/F10 melanoma cell lines, respectively. Melanoma cells were able to adhere to confluent brain endothelial cells, a process followed by elimination of protrusions and transmigration from the luminal to the basolateral side of the endothelial monolayers. The transmigration process of certain cells was accelerated when they were able to use the routes preformed by previously transmigrated melanoma cells. After migrating through the endothelial monolayer several melanoma cells continued their movement beneath the endothelial cell layer. Melanoma cells coming in contact with brain endothelial cells disrupted the tight and adherens junctions of CECs and used (at least partially) the paracellular transmigration pathway. During this process melanoma cells produced and released large amounts of proteolytic enzymes, mainly gelatinolytic serine proteases, including seprase. The serine protease inhibitor Pefabloc® was able to decrease to 44–55% the number of melanoma cells migrating through CECs. Our results suggest that release of serine proteases by melanoma cells and disintegration of the interendothelial junctional complex are main steps in the formation of brain metastases in malignant melanoma.

Role of the Blood-Brain Barrier in the Formation of Brain Metastases

The majority of brain metastases originate from lung cancer, breast cancer and malignant melanoma. In order to reach the brain, parenchyma metastatic cells have to transmigrate through the endothelial cell layer of brain capillaries, which forms the morphological basis of the blood-brain barrier (BBB). The BBB has a dual role in brain metastasis formation: it forms a tight barrier protecting the central nervous system from entering cancer cells, but it is also actively involved in protecting metastatic cells during extravasation and proliferation in the brain. The mechanisms of interaction of cancer cells and cerebral endothelial cells are largely uncharacterized. Here, we provide a comprehensive review on our current knowledge about the role of junctional and adhesion molecules, soluble factors, proteolytic enzymes and signaling pathways mediating the attachment of tumor cells to brain endothelial cells and the transendothelial migration of metastatic cells. Since brain metastases represent a great therapeutic challenge, it is indispensable to understand the mechanisms of the interaction of tumor cells with the BBB in order to find targets of prevention of brain metastasis formation.

Hyperosmotic stress induces Axl activation and cleavage in cerebral endothelial cells

Because of the relative impermeability of the blood‐brain barrier (BBB), many drugs are unable to reach the CNS in therapeutically relevant concentration. One method to deliver drugs to the CNS is the osmotic opening of the BBB using mannitol. Hyperosmotic mannitol induces a strong phosphorylation on tyrosine residues in a broad spectrum of proteins in cerebral endothelial cells, the principal components of the BBB. Previously, we have shown that among targets of tyrosine phosphorylation are β‐catenin, extracellular signal‐regulated kinase 1/2 and the non‐receptor tyrosine kinase Src. The aim of this study was to identify new signalling pathways activated by hypertonicity in cerebral endothelial cells. Using an antibody array and immunoprecipitation we identified the receptor tyrosine kinase Axl to become tyrosine phosphorylated in response to hyperosmotic mannitol. Besides activation, Axl was also cleaved in response to osmotic stress. Degradation of Axl proved to be metalloproteinase‐ and proteasome‐dependent and resulted in 50–55 kDa C‐terminal products which remained phosphorylated even after degradation. Specific knockdown of Axl increased the rate of apoptosis in hyperosmotic mannitol‐treated cells; therefore, we assume that activation of Axl may be a protective mechanism against hypertonicity‐induced apoptosis. Our results identify Axl as an important element of osmotic stress‐induced signalling.

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.

Regulation of NOD-like receptors and inflammasome activation in cerebral endothelial cells

Cerebral endothelial cells (CECs) forming the blood–brain barrier are at the interface of the immune and the central nervous systems and thus may play an important role in the functional integration of the two systems. Here, we investigated how CECs recognize and respond to pathogen‐ and damage‐associated molecular patterns to regulate the functions of the neurovascular unit. First we detected the expression of several NOD‐like receptors (NLRs) – including NOD1, NOD2, NLRC4, NLRC5, NLRP1, NLRP3, NLRP5, NLRP9, NLRP10, NLRP12, NLRA, and NLRX – in human brain endothelial cells. Inflammatory cytokines, such as interferon‐γ, tumor necrosis factor‐α, and IL‐1β had stimulatory effects on the transcription of many of these receptors. Expression of key inflammasome components (NOD2, NLRP3, and caspase 1) along with caspase‐cleaved interleukins IL‐1β and IL‐33 could be induced by priming with lipopolysaccharide and activation with muramyl dipeptide. In addition, combined treatment with lipopolysaccharide and muramyl dipeptide resulted in IL‐1β secretion in a caspase‐ and ERK1/2 kinase‐dependent manner. Our findings demonstrate that NLRs and inflammasomes can be activated in cerebral endothelial cells, which may confer a yet unexplored role to the blood–brain barrier in neuroimmune and neuroinflammatory processes.

Quinidine as an ABCB1 Probe for Testing Drug Interactions at the Blood–Brain Barrier An In Vitro In Vivo Correlation Study

This study provides evidence that quinidine can be used as a probe substrate for ABCB1 in multiple experimental systems both in vitro and in vivo relevant to the blood–brain barrier (BBB). The combination of quinidine and PSC-833 (valspodar) is an effective tool to assess investigational drugs for interactions on ABCB1. Effects of quinidine and substrate–inhibitor interactions were tested in a membrane assay and in monolayer assays. The authors compared quinidine and digoxin as ABCB1 probes in the in vitro assays and found that quinidine was more potent and at least as specific as digoxin in ATPase and monolayer efflux assays employing MDCKII-MDR1 and the rat brain microcapillary endothelial cell system. Brain exposure to quinidine was tested in dual-/triple-probe microdialysis experiments in rats by assessing levels of quinidine in blood and brain. Comparing quinidine levels in dialysate samples from valspodar-treated and control animals, it is evident that systemic/local administration of the inhibitor diminishes the pumping function of ABCB1 at the BBB, resulting in an increased brain penetration of quinidine. In sum, quinidine is a good probe to study ABCB1 function at the BBB. Moreover, quinidine/PSC-833 is an ABCB1-specific substrate/inhibitor combination applicable to many assay systems both in vitro and in vivo.

Transfection of brain capillary endothelial cells in primary culture with defined blood–brain barrier properties

BackgroundPrimary brain capillary endothelial cells (BCECs) are a promising tool to study the blood–brain barrier (BBB) in vitro, as they maintain many important characteristics of the BBB in vivo, especially when co-cultured with pericytes and/or astrocytes. A novel strategy for drug delivery to the brain is to transform BCECs into protein factories by genetic modifications leading to secretion of otherwise BBB impermeable proteins into the central nervous system. However, a huge challenge underlying this strategy is to enable transfection of non-mitotic BCECs, taking a non-viral approach. We therefore aimed to study transfection in primary, non-mitotic BCECs cultured with defined BBB properties without disrupting the cells’ integrity.MethodsPrimary cultures of BCECs, pericytes and astrocytes were generated from rat brains and used in three different in vitro BBB experimental arrangements, which were characterised based on a their expression of tight junction proteins and other BBB specific proteins, high trans-endothelial electrical resistance (TEER), and low passive permeability to radiolabeled mannitol. Recombinant gene expression and protein synthesis were examined in primary BCECs. The BCECs were transfected using a commercially available transfection agent Turbofect™ to express the red fluorescent protein HcRed1-C1. The BCECs were transfected at different time points to monitor transfection in relation to mitotic or non-mitotic cells, as indicated by fluorescence-activated cell sorting analysis after 5-and 6-carboxylfluorescein diacetate succinidyl ester incorporation.ResultsThe cell cultures exhibited important BBB characteristics judged from their expression of BBB specific proteins, high TEER values, and low passive permeability. Among the three in vitro BBB models, co-culturing with BCECs and astrocytes was well suited for the transfection studies. Transfection was independent of cell division and with equal efficacy between the mitotic and non-mitotic BCECs. Importantly, transfection of BCECs exhibiting BBB characteristics did not alter the integrity of the BCECs cell layer.ConclusionsThe data clearly indicate that non-viral gene therapy of BCECs is possible in primary culture conditions with an intact BBB.

Role of Rho/ROCK signaling in the interaction of melanoma cells with the blood-brain barrier

We have investigated the role of the Rho/ROCK signaling pathway in the interaction of metastatic melanoma cells with the brain endothelium. ROCK inhibition induced a shift of melanoma cells to the mesenchymal phenotype, increased the number of melanoma cells attached to the brain endothelium, and strengthened the adhesion force between melanoma and endothelial cells. Inhibition of ROCK raised the number of melanoma cells migrating through the brain endothelial monolayer and promoted the formation of parenchymal brain metastases in vivo. We have shown that inhibition of the Rho/ROCK pathway in melanoma, but not in brain endothelial cells, is responsible for this phenomenon. Our results indicate that the mesenchymal type of tumor cell movement is primordial in the transmigration of melanoma cells through the blood–brain barrier.

PACAP enhances barrier properties of cerebral microvessels.

Cerebral microvascular endothelial cells—coming in contact with pericytes and astrocytes—constitute the structural basis of the blood-brain barrier (BBB). The continuous belt of interendothelial tight junctions (TJs) and the presence of specific transport systems, enzymes, and receptors in the brain endothelium regulate the molecular and cellular traffic into the central nervous system. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having several cellular protective effects. However, little is known about the effects of PACAP on the cerebral endothelium and BBB functions. Here, we show that PACAP has no significant pro-survival role in cerebral microvascular endothelial cells; however, it improves the barrier properties of the brain endothelium. PACAP induces an increase in the transendothelial electrical resistance, which is the most important marker of the tightness of the TJs. Moreover, PACAP has a protective role against glucose deprivation- and oxidative stress-induced junctional damage in microvascular brain endothelial cells.