Synergistic induction of blood-brain barrier properties.

Blood-brain barrier (BBB) models derived from human stem cells are powerful tools to improve our understanding of cerebrovascular diseases and to facilitate drug development for the human brain. Yet providing stem cell-derived endothelial cells with the right signaling cues to acquire BBB characteristics while also retaining their vascular identity remains challenging. Here, we show that the simultaneous activation of cyclic AMP and Wnt/ß-catenin signaling and inhibition of the TGF-ß pathway in endothelial cells robustly induce BBB properties in vitro. To target this interaction, we present a small-molecule cocktail named cARLA, which synergistically enhances barrier tightness in a range of BBB models across species. Mechanistically, we reveal that the three pathways converge on Wnt/ß-catenin signaling to mediate the effect of cARLA via the tight junction protein claudin-5. We demonstrate that cARLA shifts the gene expressional profile of human stem cell-derived endothelial cells toward the in vivo brain endothelial signature, with a higher glycocalyx density and efflux pump activity, lower rates of endocytosis, and a characteristic endothelial response to proinflammatory cytokines. Finally, we illustrate how cARLA can improve the predictive value of human BBB models regarding the brain penetration of drugs and targeted nanoparticles. Due to its synergistic effect, high reproducibility, and ease of use, cARLA has the potential to advance drug development for the human brain by improving BBB models across laboratories.

17-Oxime ethers of oxidized ecdysteroid derivatives modulate oxidative stress in human brain endothelial cells and dose-dependently might protect or damage the blood-brain barrier.

20-Hydroxyecdysone and several of its oxidized derivatives exert cytoprotective effect in mammals including humans. Inspired by this bioactivity of ecdysteroids, in the current study it was our aim to prepare a set of sidechain-modified derivatives and to evaluate their potential to protect the blood-brain barrier (BBB) from oxidative stress. Six novel ecdysteroids, including an oxime and five oxime ethers, were obtained through regioselective synthesis from a sidechain-cleaved calonysterone derivative 2 and fully characterized by comprehensive NMR techniques revealing their complete 1H and 13C signal assignments. Surprisingly, several compounds sensitized hCMEC/D3 brain microvascular endothelial cells to tert-butyl hydroperoxide (tBHP)-induced oxidative damage as recorded by impedance measurements. Compound 8, containing a benzyloxime ether moiety in its sidechain, was the only one that exerted a protective effect at a higher, 10 µM concentration, while at lower (10 nM- 1 µM) concentrations it promoted tBHP-induced cellular damage. Brain endothelial cells were protected from tBHP-induced barrier integrity decrease by treatment with 10 µM of compound 8, which also mitigated the intracellular reactive oxygen species production elevated by tBHP. Based on our results, 17-oxime ethers of oxidized ecdysteroids modulate oxidative stress of the BBB in a way that may point towards unexpected toxicity. Further studies are needed to evaluate any possible risk connected to dietary ecdysteroid consumption and CNS pathologies in which BBB damage plays an important role.

Lab-on-a-chip models of the blood-brain barrier: evolution, problems, perspectives.

A great progress has been made in the development and use of lab-on-a-chip devices to model and study the blood-brain barrier (BBB) in the last decade. We present the main types of BBB-on-chip models and their use for the investigation of BBB physiology, drug and nanoparticle transport, toxicology and pathology. The selection of the appropriate cell types to be integrated into BBB-on-chip devices is discussed, as this greatly impacts the physiological relevance and translatability of findings. We identify knowledge gaps, neglected engineering and cell biological aspects and point out problems and contradictions in the literature of BBB-on-chip models, and suggest areas for further studies to progress this highly interdisciplinary field. BBB-on-chip models have an exceptional potential as predictive tools and alternatives of animal experiments in basic and preclinical research. To exploit the full potential of this technique expertise from materials science, bioengineering as well as stem cell and vascular/BBB biology is necessary. There is a need for better integration of these diverse disciplines that can only be achieved by setting clear parameters for characterizing both the chip and the BBB model parts technically and functionally.

Design, Optimization, and Application of a 3D-Printed Polymer Sample Introduction System for the ICP-MS Analysis of Nanoparticles and Cells.

Commonly used sample introduction systems for inductively coupled plasma mass spectrometry (ICP-MS) are generally not well-suited for single particle ICP-MS (spICP-MS) applications due to their high sample requirements and low efficiency. In this study, the first completely 3D-printed, polymer SIS was developed to facilitate spICP-MS analysis. The system is based on a microconcentric pneumatic nebulizer and a single-pass spray chamber with an additional sheath gas flow to further facilitate the transport of larger droplets or particles. The geometry of the system was optimized using numerical simulations. Its aerosol characteristics and operational conditions were studied via optical particle counting and a course of spICP-MS measurements, involving nanodispersions and cell suspensions. In a comparison of the performance of the new and the standard (quartz microconcentric nebulizer plus a double-pass spray chamber) systems, it was found that the new sample introduction system has four times higher particle detection efficiency, significantly better signal-to-noise ratio, provides ca. 20% lower size detection limit, and allows an extension of the upper limit of transportable particle diameters to about 25 µm.

Nogo-A is secreted in extracellular vesicles, occurs in blood and can influence vascular permeability.

Nogo-A is a transmembrane protein with multiple functions in the central nervous system (CNS), including restriction of neurite growth and synaptic plasticity. Thus far, Nogo-A has been predominantly considered a cell contact-dependent ligand signaling via cell surface receptors. Here, we show that Nogo-A can be secreted by cultured cells of neuronal and glial origin in association with extracellular vesicles (EVs). Neuron- and oligodendrocyte-derived Nogo-A containing EVs inhibited fibroblast spreading, and this effect was partially reversed by Nogo-A receptor S1PR2 blockage. EVs purified from HEK cells only inhibited fibroblast spreading upon Nogo-A over-expression. Nogo-A-containing EVs were found in vivo in the blood of healthy mice and rats, as well as in human plasma. Blood Nogo-A concentrations were elevated after acute stroke lesions in mice and rats. Nogo-A active peptides decreased barrier integrity in an in vitro blood-brain barrier model. Stroked mice showed increased dye permeability in peripheral organs when tested 2 weeks after injury. In the Miles assay, an in vivo test to assess leakage of the skin vasculature, a Nogo-A active peptide increased dye permeability. These findings suggest that blood borne, possibly EV-associated Nogo-A could exert long-range regulatory actions on vascular permeability.

Role of interleukin-6 and interleukin-10 in morphological and functional changes of the blood-brain barrier in hypertriglyceridemia.

BACKGROUND: Hypertriglyceridemia is closely linked to atherosclerosis related inflammatory processes and blood-brain barrier (BBB) dysfunction. Using apolipoprotein B-100 (APOB-100) transgenic mice, an animal model of chronic hypertriglyceridemia, we analyzed BBB function and morphology in vitro and ex vivo. Our objective was to determine which BBB characteristics are produced mainly by interleukin (IL)-6, an atherosclerosis promoting cytokine, and whether these actions can be antagonized by IL-10, an anti-inflammatory cytokine. METHODS: Brain endothelial and glial cell cultures and brain microvessels were isolated from wild type (WT) and APOB-100 transgenic mice and were treated with IL-6, IL-10 and their combination. First, IL-6 and IL-10 production was measured in WT and APOB-100 microvessels using qPCR. Then functional parameters of endothelial cell cultures were analyzed and immunocytochemistry for key BBB proteins was performed. RESULTS: IL-6 mRNA levels were higher in brain microvessels than in brain parenchyma of APOB-100 transgenic mice. Transendothelial electric resistance and P-glycoprotein activity were lower, and paracellular permeability was higher in cultured APOB-100 brain endothelial cells. These features were sensitive to both IL-6 and IL-10 treatments. A decreased P-glycoprotein immunostaining was measured in transgenic endothelial cells under control conditions and in WT cells after treating them with IL-6. This effect was antagonized by IL-10. Changes in immunostaining for tight junction proteins were observed after IL-6 exposure, which were in part antagonized by IL-10. In glial cell cultures an increase in aquaporin-4 immunolabeling in the transgenic group and an increase in microglia cell density in WT glia cultures was detected after IL-6 treatment, which was antagonized by IL-10. In isolated brain microvessels a decrease in P-glycoprotein immunolabeled area fraction was measured in APOB-100 microvessels under control conditions and in WT microvessels after every cytokine treatment. ZO-1 immunolabeling showed characteristics similar to that of P-glycoprotein. No change was seen in claudin-5 and occludin immunoreactive area fractions in microvessels. A decrease in aquaporin-4 immunoreactivity was measured in WT microvessels treated by IL-6, which was antagonized by IL-10. CONCLUSION: IL-6 produced in microvessels contributes to BBB impairment observed in the APOB-100 mice. We showed that IL-10 partly antagonizes the effects of IL-6 at the BBB.

The Use of Sensors in Blood-Brain Barrier-on-a-Chip Devices: Current Practice and Future Directions.

The application of lab-on-a-chip technologies in in vitro cell culturing swiftly resulted in improved models of human organs compared to static culture insert-based ones. These chip devices provide controlled cell culture environments to mimic physiological functions and properties. Models of the blood-brain barrier (BBB) especially profited from this advanced technological approach. The BBB represents the tightest endothelial barrier within the vasculature with high electric resistance and low passive permeability, providing a controlled interface between the circulation and the brain. The multi-cell type dynamic BBB-on-chip models are in demand in several fields as alternatives to expensive animal studies or static culture inserts methods. Their combination with integrated biosensors provides real-time and noninvasive monitoring of the integrity of the BBB and of the presence and concentration of agents contributing to the physiological and metabolic functions and pathologies. In this review, we describe built-in sensors to characterize BBB models via quasi-direct current and electrical impedance measurements, as well as the different types of biosensors for the detection of metabolites, drugs, or toxic agents. We also give an outlook on the future of the field, with potential combinations of existing methods and possible improvements of current techniques.

Targeting Human Endothelial Cells with Glutathione and Alanine Increases the Crossing of a Polypeptide Nanocarrier through a Blood-Brain Barrier Model and Entry to Human Brain Organoids.

Nanoparticles (NPs) are the focus of research efforts that aim to develop successful drug delivery systems for the brain. Polypeptide nanocarriers are versatile platforms and combine high functionality with good biocompatibility and biodegradability. The key to the efficient brain delivery of NPs is the specific targeting of cerebral endothelial cells that form the blood-brain barrier (BBB). We have previously discovered that the combination of two different ligands of BBB nutrient transporters, alanine and glutathione, increases the permeability of vesicular NPs across the BBB. Our aim here was to investigate whether the combination of these molecules can also promote the efficient transfer of 3-armed poly(l-glutamic acid) NPs across a human endothelial cell and brain pericyte BBB co-culture model. Alanine and glutathione dual-targeted polypeptide NPs showed good cytocompatibility and elevated cellular uptake in a time-dependent and active manner. Targeted NPs had a higher permeability across the BBB model and could subsequently enter midbrain-like organoids derived from healthy and Parkinson's disease patient-specific stem cells. These results indicate that poly(l-glutamic acid) NPs can be used as nanocarriers for nervous system application and that the right combination of molecules that target cerebral endothelial cells, in this case alanine and glutathione, can facilitate drug delivery to the brain.

Highly Oxidized Ecdysteroids from a Commercial Cyanotis arachnoidea Root Extract as Potent Blood-Brain Barrier Protective Agents.

Ecdysteroid-containing herbal extracts, commonly prepared from the roots of Cyanotis arachnoidea, are marketed worldwide as a "green" anabolic food supplement. Herein are reported the isolation and complete 1H and 13C NMR signal assignments of three new minor ecdysteroids (compounds 2-4) from this extract. Compound 4 was identified as a possible artifact that gradually forms through the autoxidation of calonysterone. The compounds tested demonstrated a significant protective effect on the blood-brain barrier endothelial cells against oxidative stress or inflammation at a concentration of 1 µM. Based on these results, minor ecdysteroids present in food supplements may offer health benefits in various neurodegenerative disease states.

Soluplus® promotes efficient transport of meloxicam to the central nervous system via nasal administration.

In our present series of experiments, we investigated the nasal applicability of the previously developed Soluplus® - meloxicam polymeric micelle formulation. Utilizing the nasal drug investigations, moderately high mucoadhesion was experienced in nasal conditions which alongside the appropriate physicochemical properties in liquid state, contributed to rapid drug absorption through human RPMI 2650 cell line. Ex vivo studies also confirmed that higher nasal mucosal permeation could be expected with the polymeric micelle nanoformulation compared to a regular MEL suspension. Also, the nanoformulation met the requirements to provide rapid drug permeation in less 1 h of our measurement. The non-toxic, non-cell barrier damaging formulation also proved to provide a successful passive transport across excides human nasal mucosa. Based on our in vivo investigations, it can be concluded that the polymeric micelle formulation provides higher meloxicam transport to the central nervous system followed by a slow and long-lasting elimination process compared to prior results where physical particle size reduction methods were applied. With these results, a promising solution and nanocarrier is proposed for the successful transport of non-steroidal anti-inflammatory drugs with acidic character to the brain.

The Active Role of Pericytes During Neuroinflammation in the Adult Brain.

Microvessels in the central nervous system (CNS) have one of the highest populations of pericytes, indicating their crucial role in maintaining homeostasis. Pericytes are heterogeneous cells located around brain microvessels; they present three different morphologies along the CNS vascular tree: ensheathing, mesh, and thin-strand pericytes. At the arteriole-capillary transition ensheathing pericytes are found, while mesh and thin-strand pericytes are located at capillary beds. Brain pericytes are essential for the establishment and maintenance of the blood-brain barrier, which restricts the passage of soluble and potentially toxic molecules from the circulatory system to the brain parenchyma. Pericytes play a key role in regulating local inflammation at the CNS. Pericytes can respond differentially, depending on the degree of inflammation, by secreting a set of neurotrophic factors to promote cell survival and regeneration, or by potentiating inflammation through the release of inflammatory mediators (e.g., cytokines and chemokines), and the overexpression of cell adhesion molecules. Under inflammatory conditions, pericytes may regulate immune cell trafficking to the CNS and play a role in perpetuating local inflammation. In this review, we describe pericyte responses during acute and chronic neuroinflammation.

Effects of Hydroxypropyl-Beta-Cyclodextrin on Cultured Brain Endothelial Cells.

The application of 2-hydroxypropyl-beta-cyclodextrin (HPBCD) in the treatment of the rare cholesterol and lipid storage disorder Niemann-Pick disease type C opened new perspectives in the development of an efficient therapy. Even if the systemic administration of HPBCD was found to be effective, its low permeability across the blood-brain barrier (BBB) limited the positive neurological effects. Nevertheless, the cellular interactions of HPBCD with brain capillary endothelial cells have not been investigated in detail. In this study, the cytotoxicity, permeability, and cellular internalization of HPBCD on primary rat and immortalized human (hCMEC/D3) brain capillary endothelial cells were investigated. HPBCD shows no cytotoxicity on endothelial cells up to 100 µM, measured by impedance kinetics. Using a fluorescent derivative of HPBCD (FITC-HPBCD) the permeability measurements reveal that on an in vitro triple co-culture BBB model, FITC-HPBCD has low permeability, 0.50 × 10-6 cm/s, while on hCMEC/D3 cell layers, the permeability is higher, 1.86 × 10-5 cm/s. FITC-HPBCD enters brain capillary endothelial cells, is detected in cytoplasmic vesicles and rarely localized in lysosomes. The cellular internalization of HPBCD at the BBB can help to develop new strategies for improved HPBCD effects after systemic administration.

Effects of sub-chronic, in vivo administration of sigma-1 receptor ligands on platelet and aortic arachidonate cascade in streptozotocin-induced diabetic rats.

BACKGROUND: Diabetes mellitus is a chronic metabolic disorder which induces endothelial dysfunction and platelet activation. Eicosanoids produced from arachidonic acid regulate cellular and vascular functions. Sigma-1 receptors (S1R) are expressed in platelets and endothelial cells and S1R expression is protective in diabetes. OBJECTIVES: Our aim was to examine the influence of sub-chronic, in vivo administered S1R ligands PRE-084, (S)-L1 (a new compound) and NE-100 on the ex vivo arachidonic acid metabolism of platelets and aorta in streptozotocin-induced diabetic rats. METHODS: The serum level of the S1R ligands was detected by LC-MS/MS before the ex vivo analysis. Sigma-1 receptor and cyclooxygenase gene expression in platelets were determined by RT-qPCR. The eicosanoid synthesis was examined with a radiolabelled arachidonic acid substrate and ELISA. RESULTS: One month after the onset of STZ-induced diabetes, in vehicle-treated, diabetic rat platelet TxB2 and aortic 6-k-PGF1α production dropped. Sub-chronic in vivo treatment of STZ-induced diabetes in rats for one week with PRE-084 enhanced vasoconstrictor and platelet aggregator and reduced vasodilator and anti-aggregator cyclooxygenase product formation. (S)-L1 reduced the synthesis of vasodilator and anti-aggregator cyclooxygenase metabolites and promoted the recovery of physiological platelet function in diabetic rats. The S1R antagonist NE-100 produced no significant changes in platelet arachidonic acid metabolism. (S)-L1 decreased the synthesis of vasoconstrictor and platelet aggregator cyclooxygenase metabolites, whereas NE-100 increased the quantity of aortic vasodilator and anti-aggregator cyclooxygenase products and promoted the recovery of diabetic endothelial dysfunction in the aorta. The novel S1R ligand, (S)-L1 had similar effects on eicosanoid synthesis in platelets as the agonist PRE-084 and in aortas as the antagonist NE-100. CONCLUSIONS: S1R ligands regulate cellular functions and local blood circulation by influencing arachidonic acid metabolism. In diabetes mellitus, the cell-specific effects of S1R ligands have a compensatory role and aid in restoring physiological balance between the platelet and vessel.

Cellular Therapy Using Epitope-Imprinted Composite Nanoparticles to Remove α-Synuclein from an In Vitro Model.

Several degenerative disorders of the central nervous system, including Parkinson's disease (PD), are related to the pathological aggregation of proteins. Antibodies against toxic disease proteins, such as α-synuclein (SNCA), are therefore being developed as possible therapeutics. In this work, one peptide (YVGSKTKEGVVHGVA) from SNCA was used as the epitope to construct magnetic molecularly imprinted composite nanoparticles (MMIPs). These composite nanoparticles were characterized by dynamic light scattering (DLS), high-performance liquid chromatography (HPLC), isothermal titration calorimetry (ITC), Brunauer-Emmett-Teller (BET) analysis, and superconducting quantum interference device (SQUID) analysis. Finally, the viability of brain endothelial cells that were treated with MMIPs was measured, and the extraction of SNCA from CRISPR/dCas9a-activated HEK293T cells from the in vitro model system was demonstrated for the therapeutic application of MMIPs.

Distinct Uptake Routes Participate in Silver Nanoparticle Engulfment by Earthworm and Human Immune Cells.

The consequences of engineered silver nanoparticle (AgNP) exposure and cellular interaction with the immune system are poorly understood. The immunocytes of the Eisenia andrei earthworm are frequently applied in ecotoxicological studies and possess functional similarity to vertebrate macrophages. Hence, we characterized and compared the endocytosis mechanisms for the uptake of 75 nm AgNPs by earthworm coelomocytes, human THP-1 monocytes, and differentiated THP-1 (macrophage-like) cells. Our results indicate that microtubule-dependent, scavenger-receptor, and PI3K signaling-mediated macropinocytosis are utilized during AgNP engulfment by human THP-1 and differentiated THP-1 cells. However, earthworm coelomocytes employ actin-dependent phagocytosis during AgNPs uptake. In both human and earthworm immunocytes, AgNPs were located in the cytoplasm, within the endo-/lysosomes. We detected that the internalization of AgNPs is TLR/MyD88-dependent, also involving the bactericidal/permeability-increasing protein (BPI) in the case of human immunocytes. The exposure led to decreased mitochondrial respiration in human immunocytes; however, in coelomocytes, it enhanced respiratory parameters. Our findings provide more data about NP trafficking as nano-carriers in the nanomedicine field, as well as contribute to an understanding of the ecotoxicological consequences of nanoparticle exposure.

Immunofluorescent Evidence for Nuclear Localization of Aromatase in Astrocytes in the Rat Central Nervous System.

Estrogens regulate a variety of neuroendocrine, reproductive and also non-reproductive brain functions. Estradiol biosynthesis in the central nervous system (CNS) is catalyzed by the enzyme aromatase, which is expressed in several brain regions by neurons, astrocytes and microglia. In this study, we performed a complex fluorescent immunocytochemical analysis which revealed that aromatase is colocalized with the nuclear stain in glial fibrillary acidic protein (GFAP) positive astrocytes in cell cultures. Confocal immunofluorescent Z-stack scanning analysis confirmed the colocalization of aromatase with the nuclear DAPI signal. Nuclear aromatase was also detectable in the S100ß positive astrocyte subpopulation. When the nuclear aromatase signal was present, estrogen receptor alpha was also abundant in the nucleus. Immunostaining of frozen brain tissue sections showed that the nuclear colocalization of the enzyme in GFAP-positive astrocytes is also detectable in the adult rat brain. CD11b/c labelled microglial cells express aromatase, but the immunopositive signal was distributed only in the cytoplasm both in the ramified and amoeboid microglial forms. Immunostaining of rat ovarian tissue sections and human granulosa cells revealed that aromatase was present only in the cytoplasm. This novel observation suggests a new unique mechanism in astrocytes that may regulate certain CNS functions via estradiol production.

Effects of fasudil on blood-brain barrier integrity.

BACKGROUND: Cerebral infarction accounts for 85% of all stroke cases. Even in an era of rapid and effective recanalization using an intravascular approach, the majority of patients have poor functional outcomes. Thus, there is an urgent need for the development of therapeutic agents to treat acute ischemic stroke. We evaluated the effect of fasudil, a Rho kinase inhibitor, on blood brain barrier (BBB) functions under normoxia or oxygen-glucose deprivation (OGD) conditions using a primary cell-based in vitro BBB model. METHODS: BBB models from rat primary cultures (brain capillary endothelial cells, astrocytes, and pericytes) were subjected to either normoxia or 6 h OGD/24 h reoxygenation. To assess the effects of fasudil on BBB functions, we evaluated real time impedance, transendothelial electrical resistance (TEER), sodium fluorescein permeability, and tight junction protein expression using western blotting. Lastly, to understand the observed protective mechanism on BBB functions by fasudil we examined the role of cyclooxygenase-2 and thromboxane A2 receptor agonist U-46619 in BBB-forming cells. RESULTS: We found that treatment with 0.3-30 µM of fasudil increased cellular impedance. Fasudil enhanced barrier properties in a concentration-dependent manner, as measured by an increased (TEER) and decreased permeability. Fasudil also increased the expression of tight junction protein claudin-5. Reductions in TEER and increased permeability were observed after OGD/reoxygenation exposure in mono- and co-culture models. The improvement in BBB integrity by fasudil was confirmed in both of the models, but was significantly higher in the co-culture than in the monoculture model. Treatment with U-46619 did not show significant changes in TEER in the monoculture model, whereas it showed a significant reduction in TEER in the co-culture model. Fasudil significantly improved the U-46619-induced TEER reduction in the co-culture models. Pericytes and astrocytes have opposite effects on endothelial cells and may contribute to endothelial injury in hyperacute ischemic stroke. Overall, fasudil protects the integrity of BBB both by a direct protective effect on endothelial cells and by a pathway mediated via pericytes and astrocytes. CONCLUSIONS: Our findings suggest that fasudil is a BBB-protective agent against acute ischemic stroke.

Novel cyclic C5-curcuminoids penetrating the blood-brain barrier: Design, synthesis and antiproliferative activity against astrocytoma and neuroblastoma cells.

Novel series of cyclic C5-curcuminoids 17a-j and 19-22 were prepared as cytotoxic agents and evaluated against human neuroblastoma (SH-SY5Y) or human grade IV astrocytoma (CCF-STTG1) cell lines in low (∼0.1 nM - 10 nM) concentrations. Among the tested 21 derivatives, 16 displayed potent antiproliferative activity with IC50 values in the low nanomolar to picomolar range (IC50 = 7.483-0.139 nM). Highly active compounds like N-monocarboxylic derivative 19b with IC50 = 0.139 nM value against neuroblastoma and N-alkyl substituted 11 with IC50 = 0.257 nM against astrocytoma proved some degree of selectivity toward non-cancerous astrocytes and kidney cells. This potent anticancer activity did not show a strong correlation with experimental logPTLC values, but the most potent antiproliferative molecules 11-13 and 19-22 are belonging to discrete subgroups of the cyclic C5-curcuminoids. Compounds 12, 17c and 19b were subjected to blood-brain barrier (BBB) penetration studies, too. The BBB was revealed to be permeable for all of them but, as the apparent permeability coefficient (Papp) values mirrored, in different ratios. Lower toxicity of 12, 17c and 19b was observed toward primary rat brain endothelial cells of the BBB model, which means they remained undamaged under 10 µM concentrations. Penetration depends, at least in part, on albumin binding of 12, 17c and 19b and the presence of monocarboxylic acid transporters in the case of 19b. Permeation through the BBB and albumin binding, we described here, is the first example of cyclic C5-curcuminoids as to our knowledge.

Effects of sub-chronic, in vivo administration of sigma non-opioid intracellular receptor 1 ligands on platelet and aortic arachidonate cascade in rats.

Platelets regulate cell-cell interactions and local circulation through eicosanoids from arachidonic acid. Sigma non-opioid intracellular receptor 1 (sigma-1 receptor) expressed in platelets and endothelial cells can regulate intracellular signalization. Our aim was to examine the influence of sub-chronic, in vivo-administered sigma-1 receptor ligands 2-morpholin-4-ylethyl 1-phenylcyclohexane-1-carboxylate (PRE-084); N-benzyl-2-[(1S)-6,7-dimethoxy-1,2,3,4-tetrahydroisoquinolin-1-yl]ethan-1-amine; dihydrochloride, a new compound ((S)-L1); and N-[2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethyl]-N-propylpropan-1-amine (NE-100) on the ex vivo arachidonic acid metabolism of the platelets and aorta of male rats. The serum level of sigma-1 receptor ligands was determined by liquid chromatography-mass spectrometry. Sigma-1 receptor and cyclooxygenase gene expression in the platelets were determined by a reverse transcription-coupled quantitative polymerase chain reaction. The eicosanoid synthesis was examined using a radiolabeled arachidonic acid substrate and enzyme-linked immunosorbent assay. We confirmed the absorption of sigma-1 receptor ligands and confirmed that the ligands were not present during the ex vivo studies, so their acute effect could be excluded. We detected no changes in either sigma-1 receptor or cyclooxygenase mRNA levels in the platelets. Nevertheless, (S)-L1 and NE-100 increased the quantity of cyclooxygenases there. Both platelet and aortic eicosanoid synthesis was modified by the ligands, although in different ways. The effect of the new sigma-1 receptor ligand, (S)-L1, was similar to that of PRE-084 in most of the parameters studied but was found to be more potent. Our results suggest that sigma-1 receptor ligands may act at multiple points in arachidonic acid metabolism and play an important role in the control of the microcirculation by modulating the eicosanoid synthesis of the platelets and vessels.