Preparation and properties of nanoscale containers for biomedical application in drug delivery: preliminary studies with kynurenic acid.

The main purpose of this study was to facilitate the delivery of kynurenic acid (KYNA) across the blood-brain barrier (BBB) by applying micelles as nanoscale containers. Non-ionic amphiphilic molecules were used for preparation of spherical micelles for delivery of kynurenic acid in aqueous solution in physiological condition. It was established that Triton X 100 and Lutensol AP 20 non-ionic surfactants are able to produce stable nanocontainers for delivery of kynurenic acid molecules. The incorporation of KYNA molecules was investigated by dynamic light scattering and the size of micelles were calculated between 5 and 10 nm in 150 mM NaCl and pH 7.5-7.6 solutions. Encapsulated kynurenic acid showed a significantly higher blood-brain barrier permeability compared with non-encapsulated kynurenic acid. The in vivo experiments showed that the encapsulated kynurenic acid is able to display effects within the central nervous system, even after its peripheral administration.

New aspects of the molecular constituents of tissue barriers.

Epithelial and endothelial tissue barriers are based on tight intercellular contacts (Tight Junctions, TJs) between neighbouring cells. TJs are multimeric complexes, located at the most apical border of the lateral membrane. So far, a plethora of proteins locating at tight intercellular contacts have been discovered, the role of which has just partly been unraveled. Yet, there is convincing evidence that many TJ proteins exert a dual role: They act as structural components at the junctional site and they are involved in signalling pathways leading to alterations of gene expression and cell behaviour (migration, proliferation). This review will shortly summarize the classical functions of TJs and TJ-related proteins and will introduce a new category, termed the "non-classical" functions of junctional proteins. A particular focus will be directed towards the nuclear targeting of junctional proteins and the downstream effects elicited by their intranuclear activities.

Calcein assay: a high-throughput method to assess P-gp inhibition.

Transporter mediated drug-drug interactions (tDDI) mediated by ABCB1 have been shown to be clinically relevant. Hence, the assessment of the ABCB1 tDDI potential early in the drug development process has gained interest. We have evaluated the Calcein assay as a means of assessing the ABCB1 tDDI that is amenable to high throughout and compared it with the monolayer efflux assay. We found the Calcein assay, when performed in K562MDR cells using the protocol originally published more sensitive than digoxin transport inhibition in MDCKII-MDR1 cells. Application of the Calcein assay to cell lines containing different amounts of ABCB1, yielded IC(50) values that varied 10-100-fold. The differences observed for IC(50) values for the same compounds were in the following rank order: IC(50, MDCKII-MDR1) >IC(50, K562MDR)>IC(50, hCMEC/D3). Higher IC(50) values were obtained in cells with higher ABCB1 expression. The Calcein assay is a high-throughput alternative to digoxin transport inhibition as it appears to have a comparable selectivity but higher sensitivity than previously published digoxin transport inhibition in MDCKII-MDR1 cells. In addition, it can be performed in a barrier-specific manner highlighting the dependence of ABCB1 IC(50) values on different ABCB1 expression levels.

Targeting of the kynurenic acid across the blood-brain barrier by core-shell nanoparticles.

Core-shell nanoparticles (CSNPs) were developed to get over therapeutic amount of kynurenic acid (KYNA) across the blood-brain barrier (BBB). Bovine serum albumin (BSA) was used as core for encapsulation of KYNA and the BSA/KYNA composite was finally encapsulated by poly(allylamine) hydrochloride (PAH) polymer as shell. In the interest of the optimization of the synthesis the BSA and KYNA interaction was studied by two-dimensional surface plasmon resonance (SPR) technique as well. The average size of d~100 nm was proven by dynamic light scattering (DLS) and transmission electron microscopy (TEM), while the structure of the composites was characterized by fluorescence (FL) and circular dichroism (CD) spectroscopy. The in vitro release properties of KYNA were investigated by a vertical diffusion cell at 25.0 °C and 37.5 °C and the kinetic of the release were discussed. The penetration capacity of the NPs into the central nervous system (CNS) was tested by an in vitro BBB model. The results demonstrated that the encapsulated KYNA had significantly higher permeability compared to free KYNA molecules. In the neurobiological serial of in vivo experiments the effects of peripherally administered KYNA with CSNPs were studied in comparison with untreated KYNA. These results clearly proved that KYNA in the CSNPs, administrated peripherally is suitable to cross the BBB and to induce electrophysiological effects within the CNS. As the neuroprotective properties of KYNA nowadays are proven, the importance of the results is obvious.

Regulation of proteolytic activity induced by inflammatory stimuli in lung epithelial cells.

A large number of chronic lung diseases such as asthma bronchiale are associated with alveolar and/or bronchial inflammation accompanied by a damage of the alveolocapillary barrier. In this process proteolytic mechanisms may play a crucial role. The aim of the present study was to assess the role of TNF-alpha on the proteolytic activity of pulmonary epithelial cells and to find possible intracellular signaling pathways which may mediate the effect of TNF-alpha. For our studies we have used the A549 human lung epithelial cell line. Plasminogen activator and metalloproteinase activity was measured using zymography. TNF-alpha induced a time and concentration dependent activation of the urokinase type plasminogen activator (u-PA) and tissue type plasminogen activator (t-PA) activity in A549 cells. This effect could be blocked completely by dexamethasone and was reduced significantly by the Rho-kinase inhibitor Y27632. Similarly, an increased activity in the culture medium of the 72 kDa MMP-2 in response to TNF-alpha could be observed as well. This could be reduced by dexamethasone and Y27632. Our results show that TNF-alpha is at least partly responsible for an increased proteolytic activity and beside corticosteroids Rho-kinase may constitute a potential target for future therapeutical approaches.

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells.

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.

Non-conventional role of lysosomal acid phosphatase in olfactory receptor axons: co-localization with growth-associated phosphoprotein-43.

Olfactory receptor neurons undergo a continuous turnover in adult mammals. It is largely unknown how their axons invade the olfactory bulb and induce synaptic re-organization in glomeruli. Here, the cytochemical localization of lysosomal acid phosphatase has been studied in olfactory bulbs of adult rats and mice. The enzyme has been identified by specific substrate, inhibitors and absence in lysosomal acid phosphatase-knockout mice. Lysosomal acid phosphatase is located in primary and secondary lysosomes, which are unevenly distributed in the olfactory nerve layer and among olfactory glomeruli. In consecutive sections of glomeruli, the intensity of lysosomal acid phosphatase immunoreactivity co-varied with that of growth-associated phosphoprotein. Electron microscopically, differential lysosomal acid phosphatase staining in glomeruli corresponded to different proportions of labelled and unlabelled axons. Quantification revealed that lysosomal acid phosphatase labelling was strongest in non-synaptic profiles of terminal axons, while it was weak in or even missing from most synaptic profiles. Hence, growing olfactory axons apparently carry more lysosomal acid phosphatase than those which have established synaptic contacts. Following olfactory deafferentation both lysosomal acid phosphatase activity and growth-associated phosphoprotein-43 are lost from glomeruli, suggesting that both proteins are expressed in olfactory sensory axons during growth, while lysosomal acid phosphatase is apparently not a marker of anterograde terminal degeneration.

Localization and biochemical characterization of acid phosphatase isoforms in the olfactory system of adult rats.

Localization of acid phosphatases was studied with the use of beta-glycerophosphate and p-nitrophenyl phosphate as substrates in the brain with special emphasis on the olfactory system of adult rat at light and electron microscopic level. With the use of beta-glycerophosphate, a selective substrate for the lysosomal acid phosphatase, lead-containing reaction product was found in primary and secondary lysosomes of neurons, glial cells and perivascular macrophages as well as in the cytoplasm of olfactory sensory axons. Incubation with p-nitrophenyl phosphate as substrate additionally revealed a cytoplasmic isoform of acid phosphatase, which could not be inhibited by tartrate or fluoride and was predominantly located in dendrites. Acid phosphatase isoforms were biochemically characterized in samples prepared separately from the olfactory mucosa, olfactory nerve layer, olfactory bulb and its dendrodendritic synaptosomes isolated by subcellular fractionation. In the olfactory mucosa and olfactory nerve layer the lysosomal type (high molecular weight form) was the most prominent acid phosphatase form, whereas the isoform located in dendrites corresponded to the tartrate-resistant extralysosomal, cytosolic type (low molecular weight form). The functional significance of different isoforms of acid phosphatase in the olfactory sensory axons and dendritic elements is discussed.

Expression of G-protein subtypes in cultured cerebral endothelial cells.

This paper describes Western-blotting evidence for the presence of various guanine nucleotide binding proteins, G-proteins in cultured rat cerebral endothelial cells (CECs) and two immortalized cerebral endothelial cell lines, RBE4 and GP8. By using specific antibodies raised against known sequences of appropriate G-protein types that were previously characterized, we demonstrated the presence of Gsalpha, Gi2alpha, Gi3alpha, Gq/11alpha, Goalpha and Gbeta in cell lysates of primary cultures of CECs, and plasma membranes of RBE4 and GP8 cells. The appearance of Goalpha proteins in CECs might be of special importance, since they were not detected in peripheral endothelial cells in previous studies. Isoproterenol and bradykinin displayed significant, dose-dependent stimulation of [35S]GTPgammaS binding above basal values. This assay, reflecting the GDP-GTP exchange reaction on Galpha-subunits by receptor agonists, suggested that there were functional, G-protein coupled beta-adrenergic and bradykinin receptors in these systems. No significant stimulation of [35S]GTP7gammaS binding was noted with serotonin under our experimental conditions. Since stimulation of [35S]GTPgammaS binding by isoproterenol and bradykinin was additive, it was concluded that different Galpha proteins were activated by these two ligands. In analogy to other systems, activation of Gs is most likely by isoproterenol, while Gi and/or Gq/11 proteins might be activated by bradykinin receptors. The possible significance of the receptors and G-proteins detected is being discussed in the functioning of cerebral endothelium, and thus the blood-brain barrier.

Systemic nitroglycerin increases nNOS levels in rat trigeminal nucleus caudalis.

Systemic administration of nitroglycerin, a nitric oxide donor, triggers in migraineurs a delayed attack of unknown mechanisms. Subcutaneous nitroglycerin (10 mg/kg) produced a significant increase of nitric oxide synthase (NOS)- and c-fos-immunoreactive neurons in the cervical part of trigeminal nucleus caudalis in rats after 4 h. This effect was not observed in the thoracic dorsal horn. Similar increase of NOS and c-fos was obtained in the brain stem after a somatic nociceptive stimulus, i.e. on the side of the formalin injection in the lip. Nitric oxide is thus able to increase NOS availability in second order nociceptive trigeminal neurons, which may be relevant for central sensitization and the understanding of its effect in migraine.

Modulation of the truncated GAD25 by estrogen in the olfactory bulb of adult rats.

The aim of our study was to investigate the influence of gonadal steroids on the expression of different GAD isoforms. Here we show that, in addition to the adult GAD forms, the two embryonic splice variants of GAD67 mRNA and the truncated GAD25 are present in the adult rat olfactory bulb, a brain region with high synaptic plasticity, which has preserved some features of the developing brain. By Western blot analysis, we could demonstrate that the expression of the embryonic GAD25 is cyclic in females: its quantity is higher on estrus day. Furthermore, in ovariectomized animals 17-beta-estradiol treatment induced an increase of GAD25 within 3 h, reaching a maximum at 9-12 h. Our data are compatible with the interpretation that the embryonic GAD isoforms may play a role in the neuroplastic changes induced by sexual steroids.

Vasoactive substances produced by cultured rat brain endothelial cells.

The vasoactive substances synthesized by primary cultures of rat brain endothelial cells were investigated and compared to those from two, immortalized cell lines, RBE4 and GP8. The vasoactivity of endothelium-derived substances was measured on isolated canine coronary artery. Vascular tone was significantly decreased by both primary and GP8, but not by RBE4 cells. Indomethacin pretreatment of primary and GP8 cells turned vasorelaxation into contraction while N(omega)-nitro-L-arginine pretreatment decreased the vasorelaxation induced by primary, but not by GP8 cells. Eicosanoid production was determined after incubation with [14C]arachidonic acid. The predominant vasoactive eicosanoid was prostaglandin E2 in both primary and GP8 cells. RBE4 cells synthetized mainly prostaglandin E2 and thromboxane B2 and significantly less prostaglandin E2 than did either primary or GP8 cells. The capacity of cerebral endothelium to regulate vascular tone by production of dilator and constrictor substances can be preserved under certain circumstances in immortalized cell lines.

Identification of the Ulex europaeus agglutinin-I-binding protein as a unique glycoform of the neural cell adhesion molecule in the olfactory sensory axons of adults rats.

Histochemical localization of two lectins, Ulex europaeus agglutinin-I (UEA-I) and Tetragonolobus purpureus (TPA), was studied in the olfactory bulb of adult rats. In contrast to TPA, UEA-I detected a fucosylated glycoprotein that is only present in the surface membranes of olfactory sensory cells including the whole course of their neurites up to the final arborization in glomeruli. Immunoblotting revealed that UEA-I binds specifically to a protein of 205 kDa, while TPA stains several other glycoproteins. Affinity chromatography with the use of a UEA-I column identified the 205 kDa protein as a glycoform of neural cell adhesion molecule (N-CAM), specific for the rat olfactory sensory nerves.

Signalling pathways regulating the tight junction permeability in the blood-brain barrier.

Tight junctions (TJs) of the cerebral endothelial cells play a crucial role in the regulation of BBB permeability under physiological, as well as pathological conditions. The regulation of the junctional proteins is under a complex control. In these regulatory processes signalling molecules, some of them localized to the TJ, play an important role. Among the best characterized second messengers which regulate TJ function are the cyclic nucleotides, which, as shown in our experiments, as well, decrease paracellular permeability. Another important signalling molecule involved in TJ regulation is protein kinase C, which may affect differently the formation of TJ and the function of mature TJ. Further signalling molecules known to regulate paracellular permeability are G-proteins, both conventional and small G-proteins, MAP kinases and other protein kinases. Much of our knowledge concerning second messenger regulation of TJ arises fon the study of epithelial cells of different origin, mostly from kidney, therefore the specific regulation of the junctional complex of the BBB still remains to be elucidated.

Oxidative stress in cultured cerebral endothelial cells induces chromosomal aberrations, micronuclei, and apoptosis.

There is evidence accumulating that brain microvasculature is involved critically in oxidative stress-mediated brain damage. Cultured cerebral microvascular endothelial cells were used to demonstrate the cytotoxic and genotoxic effects elicited by hypoxia/reoxygenation and DMNQ treatment in vitro. In addition, the effect of glucose deprivation during oxidative insult was assessed. The parameters determined were: 1) chromosomal aberrations; 2) induction of micronuclei; and 3) apoptosis. Our results indicate that both the exposure of the cerebral endothelial cells to 24 hr of hypoxia followed by 4 hr of reoxygenation, and treatment with the redox cycling quinone DMNQ, increased markedly the occurrence of chromosomal aberrations and micronuclei. It was found that expression of p53 was induced by oxidative stress, particularly when glucose had been omitted from the culture medium. Aglycemic culture conditions in general exacerbated the cytotoxic effects of oxidative insults, as evidenced by the increase in apoptotic cells and the decrease in the mitotic index. Interestingly, neither an elevation of cell lysis nor an increase in necrosis has been observed during our experiments. In summary, our data indicate that oxidative stress exerts considerable genotoxic and cytotoxic effects on cerebral endothelial cells, which might contribute to the progression of tissue damage in the central nervous system.

Expression of protein kinase C family members in the cerebral endothelial cells.

The protein kinase C (PKC) family is composed of at least four conventional (alpha, beta I, beta II, and gamma) and several related novel (delta, epsilon, eta, and zeta) isoforms with different distribution and sensitivity to Ca2+ and phorbol esters. The enzyme is known to be present in cerebral endothelial cells. We have investigated the occurrence of seven isoforms (alpha, beta, gamma, delta, epsilon, eta, and zeta) by using reverse transcriptase-polymerase chain reaction in rat brain, in a freshly isolated brain microvessel fraction, in primary cultures of rat brain endothelial cells, in an immortalized rat brain endothelial cell line, and in aortic endothelial cell cultures. Brain tissue contained all seven investigated isoforms. A similar expression pattern was seen in freshly purified microvessels, but the PKC-gamma isoform could not be detected. Primary cultures of endothelial cells expressed PKC-alpha, -beta, -delta, -eta, and -epsilon isoenzymes, whereas the immortalized cell line expressed PKC-alpha, -delta, -epsilon, and -eta. The rat aortic endothelium contained only PKC-alpha and -delta isoforms. The variety of expression patterns of PKC family members in endothelial cells of different type may reflect differences in the functional responsiveness to environmental stimuli. Because PKC has been shown to be involved in the regulation of the blood-brain barrier permeability, the presence of different isoforms may confer a sophisticated intracellular regulatory mechanism to the brain endothelial cells.

In situ hybridization with digoxigenin labeled oligonucleotide probes: detection of CAMK-II gene expression in primary cultures of cerebral endothelial cells.

A better understanding of the regulation of gene expression under physiological and experimental conditions is one of the most important goals of today's neurobiology. In order to accomplish this task a number of in-situ hybridization methods have been elaborated. In the past few years the use of nonradioactive procedures have gained more and more space among these efforts. One of the most promising methods in this field is the application of digoxigenin-labeled probes, which have been used successfully in several laboratories. Here we present a rapid method providing good spatial resolution and low background labeling for the detection of messenger RNAs in various cell culture systems using digoxigenin-labeled probes. By using oligonucleotides complementary to the alpha and beta subunit of the calcium/calmodulin dependent protein kinase II (CAMK-II) we were able to demonstrate the presence of this enzyme in cultured cerebral endothelial cells, an enzyme that plays an important role in mediating the effects of extracellular signals.

Calcium/calmodulin-stimulated protein kinase II is present in primary cultures of cerebral endothelial cells.

Calcium/calmodulin-stimulated protein kinase II (CaM-PK II), a major kinase in brain, has been established to play an important role in neurotransmitter release and organization of postsynaptic receptors, and it is known to be involved in long-term potentiation and memory. Less is known about the function of this enzyme in nonneural cells. Here we report on the production, presence, and phosphorylation of the alpha-subunit of CaM-PK II in primary cultures of cerebral endothelial cells. These results raise the possibility that alpha-CaM-PK II can act as one of the key enzymes of calcium-mediated intracellular signaling in the cerebral endothelial cells and suggest that alpha-CaM-PK II may participate in such basic cellular processes as permeability in physiological and pathological conditions.

Functional expression of the serotonin transporter in immortalized rat brain microvessel endothelial cells.

There is evidence from recent studies that the brain endothelium (of capillaries and/or larger vessels) may serve as a specific target for serotonin [5-hydroxytryptamine (5-HT)]. This neurotransmitter is expected to be involved in the regulation of the blood-brain barrier (BBB) permeability and/or of the cerebral blood flow via receptor-mediated mechanisms. Effective control of these processes depends on a speedy uptake and metabolism of released 5-HT molecules. To realize this, a similar mechanism of 5-HT uptake as in brain may exist at the BBB. In this study, we have demonstrated using RT-PCR that 5-HT transporter mRNA is present in the brain endothelium and that a saturable transport system for 5-HT is functionally expressed in immortalized rat brain endothelial cells (RBE4 cells). These cells take up [3H]5-HT by an active saturable process with a Km value of 397 +/- 64 nmol/L and a transport capacity of 51.7 +/- 3.5 pmol x g(-1) x min(-1). The 5-HT uptake depends on Na+, as indicated by the replacement of NaCl by LiCl. The 5-HT uptake was sensitive to specific 5-HT transport inhibitors such as paroxetine, clomipramine, fluoxetine, and citalopram but not to inhibitors of the vesicular amine transporter such as reserpine or tetrabenazine. Our results demonstrate that cerebral endothelial cells are able to participate actively in the removal and metabolism of the released 5-HT, which supports the concept of direct serotoninergic regulation of the BBB function.

Use of genetically modified glial cells overexpressing laminin alpha1-chain peptides in neurite outgrowth studies.

1. C6 glioma cells were transfected with two constructs carrying C-terminal laminin alpha1-chain sequences of 117 and 114 bp length, respectively. These sequences are specifically known to code for peptides which have neurite-promoting activity. 2. The stable expression and secretion of the two peptides was detected by Northern and Western blot analysis. 3. Primary neuronal cultures derived from embryonic mouse forebrain were cocultured with these transfected cells and exhibited a substantial increase in neurite outgrowth and in survival time. Conditioned media from the transfected cells generated similar effects. 4. Organotypic cultures from embryonic mouse brain were used as a second system as being closer to the in vivo situation. Again, coculture of brain slices with transfected cells or treatment with laminin peptide-containing media increased neuronal outgrowth.