The effect of sucrose esters on a culture model of the nasal barrier.

Sucrose esters are effective solubilizers and there is an interest to use them as pharmaceutical excipients for nasal drug delivery. We have determined for the first time the non-toxic doses of laurate and myristate sucrose esters by four independent methods, and their effects on epithelial permeability using RPMI 2650 human nasal epithelial cell line. Based on real-time cell electronic sensing, MTT dye conversion and lactate dehydrogenase release methods reference surfactant Cremophor RH40 proved to be the least toxic excipient, and could be used at 5mg/mL concentration for 1h in epithelial cells without cellular damage. The non-toxic dose of Tween 80 was 1 mg/mL, while the dose of laurate and myristate sucrose esters that could be safely used on cells for 1 h was 0.1 mg/mL. Both the reference surfactants and the sucrose esters significantly enhanced the permeability of epithelial cell layers for the paracellular marker FITC-labelled 4.4 kDa dextran at 0.1 mg/mL concentration. The effects of sucrose esters on epithelial permeability were dose-dependent. These data indicate that laurate and myristate sucrose esters can be potentially used as permeability enhancers in nasal formulations to augment drug delivery to the systemic circulation.

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.

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.

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 beta-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.

Human serum amyloid P component attenuates the bacterial lipopolysaccharide-induced increase in blood-brain barrier permeability in mice.

Endotoxin challenge leads to septic shock, multi-organ failure and death in mice. Permeability of the blood-brain barrier (BBB) is increased by endotoxemia. Serum amyloid P component (SAP) is a lipopolysaccharide (LPS)-binding protein that can modulate the host reactions during infections. It is controversial whether SAP can protect from LPS toxicity in vivo or not. We have tested the effect of human SAP on BBB permeability of Salmonella typhimurium LPS-injected mice. The animals showed signs of sickness behaviour including immobility, anorexia, and diarrhoea. Intraperitoneally administered LPS increased the BBB permeability for sodium fluorescein for about 4-fold, and for albumin for more than 2-fold in brain cortex. SAP, given intravenously, had no effect on basal BBB permeability for albumin, although it decreased sodium fluorescein extravasation to brain tissue. In LPS-treated mice, SAP administration alleviated the symptoms of septic shock, and significantly inhibited the enhanced BBB permeability for both tracers. Our data indicate that human SAP may counteract the toxic effects of LPS during septic shock.