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.

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.

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.

Inhibition of NHE-1 Increases Smoke-Induced Proliferative Activity of Barrett's Esophageal Cell Line.

Several clinical studies indicate that smoking predisposes its consumers to esophageal inflammatory and malignant diseases, but the cellular mechanism is not clear. Ion transporters protect esophageal epithelial cells by maintaining intracellular pH at normal levels. In this study, we hypothesized that smoking affects the function of ion transporters, thus playing a role in the development of smoking-induced esophageal diseases. Esophageal cell lines were treated with cigarettesmoke extract (CSE), and the viability and proliferation of the cells, as well as the activity, mRNA and protein expression of the Na+/H+ exchanger-1 (NHE-1), were studied. NHE-1 expression was also investigated in human samples. For chronic treatment, guinea pigs were exposed to tobacco smoke, and NHE-1 activity was measured. Silencing of NHE-1 was performed by using specific siRNA. CSE treatment increased the activity and protein expression of NHE-1 in the metaplastic cells and decreased the rate of proliferation in a NHE-1-dependent manner. In contrast, CSE increased the proliferation of dysplastic cells independently of NHE-1. In the normal cells, the expression and activity of NHE-1 decreased due to in vitro and in vivo smoke exposure. Smoking enhances the function of NHE-1 in Barrett's esophagus, and this is presumably a compensatory mechanism against this toxic agent.

Biotin and Glutathione Targeting of Solid Nanoparticles to Cross Human Brain Endothelial Cells.

BACKGROUND: The blood-brain barrier restricts drug penetration to the central nervous system. Targeted nanocarriers are new potential tools to increase the brain entry of drugs. Ligands of endogenous transporters of the blood-brain barrier can be used as targeting vectors for brain delivery of nanoparticles. OBJECTIVE: We tested biotin-labeled solid nanoparticles for the first time and compared to biotinylated glutathione- labeled nanoparticles in brain endothelial cells. METHOD: Neutravidin coated fluorescent polystyrene nanoparticles were derivatized with biotin and biotinylated glutathione. As a human in vitro blood-brain barrier model hCMEC/D3 brain endothelial cells were used. Cell viability by MTT test, uptake and transfer of the nanoparticles across the endothelial monolayers were measured. The uptake of the nanoparticles was visualized by confocal microscopy. RESULTS: The tested nanoparticles caused no change in cell viability. The uptake of biotin- and glutathione-labeled nanoparticles by brain endothelial cells was time-dependent and significantly higher compared to non-labeled nanoparticles. The penetration of the glutathione-labeled nanoparticles across the endothelial monolayer was higher than the biotin-targeted ones. Biotin- and glutathione-targeted nanoparticles were visualized in hCMEC/D3 cells. We verified that hCMEC/D3 express mRNA for sodium-dependent multivitamin transporter (SMVT/SLC5A6) responsible for the blood-brain barrier transport of biotin. CONCLUSION: Biotin as a ligand increased the uptake and the transfer of nanoparticles across brain endothelial cells. Biotinylated glutathione could further increase nanoparticle permeability through endothelial monolayers supporting its use as a brain targeting vector.

Aromatase and estrogen receptor beta expression in the rat olfactory bulb: Neuroestrogen action in the first relay station of the olfactory pathway?

The expression pattern of aromatase (ARO), the enzyme converting androgens to estrogens, was analyzed in the olfactory bulb of adult male rats and was compared with the distribution of estrogen receptor beta (ERbeta), the main estrogen receptor isoform expressed in this brain region. A strong ARO immunolabeling obtained with a specificity tested antibody was observed in juxtaglomerular neurons of the glomerular layer and a weaker immunoreaction was detected in the mitral cell layer of the main olfactory bulb, while the granule cell layer of the main olfactory bulb as well as all layers in the accessory olfactory bulb showed faint immunolabeling. Fluorescence double labeling experiments revealed that ARO detected in juxtaglomerular neurons of the main olfactory bulb colocalized with tyrosine hydroxylase (TH) and glutamic acid decarboxylase 67 (GAD67), while no colocalization between ARO and the calcium binding proteins calretinin (CR) and calbindin (CB) was observed. Furthermore, the TH immunoreactive neurons expressed metabotropic glutamate receptor 1 (mGluR1) too. ERß immunoreactivity, in contrast to ARO, was detected in all layers of both the main and accessory olfactory bulb. In the glomerular layer of the main olfactory bulb it was expressed in TH and GAD67 containing juxtaglomerular neurons, and it colocalized with CR, CB and even with glial fibrillary acidic protein too. Our morphological findings suggest that ARO expression is a novel feature of dopaminergic/GABAergic juxtaglomerular neurons in the adult rat main olfactory bulb, and raise the possibility that ARO activity may change in function of olfactory input via mGluR1. In situ estrogen production in the olfactory bulb in turn may modulate interglomerular circuits through ERbeta.

Estradiol-induced synaptic remodeling of tyrosine hydroxylase immunopositive neurons in the rat arcuate nucleus.

Gonadal steroids induce synaptic plasticity in several areas of the adult nervous system. In the arcuate nucleus of adult female rats, 17beta-estradiol triggers synaptic remodeling, resulting in a decrease in the number of inhibitory synaptic inputs, an increase in the number of excitatory synapses, and an enhancement of the frequency of neuronal firing. In the present paper, we studied the specificity of hormonal effects by determining the changes in synaptic connectivity of tyrosine hydroxylase (TH) immunoreactive (IR) neurons in the arcuate nucleus. We combined pre-embedding TH and post-embedding gamma-aminobutyric acid (GABA) immunostaining, and performed unbiased stereological measurements in gonadectomized and 17beta-estradiol-treated rats. We conclude that the synaptic connectivity of the TH-IR neurons is different from the other, nonlabeled population, and the response to estradiol is not uniform. TH-IR (dopaminergic) arcuate neurons of both male and female rats have more GABAergic (inhibitory) axosomatic inputs than the nondopaminergic population. Our study shows that the effect of 17beta-estradiol is sex and cell specific in the sense that not all arcuate neurons are affected by the structural synaptic remodeling. In ovariectomized females hormone treatment decreased the numerical density of GABAergic axosomatic synapses on TH-IR, but not on nondopaminergic, neurons, whereas in orchidectomized males, 17beta-estradiol treatment increased inhibitory synapses onto nondopaminergic neurons but did not affect the number of inhibitory terminals onto TH-IR neurons. The hormone-induced plastic changes in synaptic connectivity of TH-IR neurons may serve as the morphological basis for the cyclical regulation of the anterior pituitary.

Dehydroepiandrosterone regulates astroglia reaction to denervation of olfactory glomeruli.

Effects of dehydroepiandrosterone (DHEA) on glial reactions of the peripherally denervated olfactory bulb were studied in adult male rats. Denervation was achieved by destroying the olfactory mucosa with ZnSO(4) (0.17 M) irrigation of the nasal cavities. In one series of experiments, chronic DHEA treatment was applied (daily injections for 7 days, i.p., 10 mg/kg b.w. and 25 mg/kg b.w.); in the other series of experiments, animals received a single injection of DHEA (i.p., 10 mg/kg b.w., 25 mg/kg b.w. and 50 mg/kg b.w.) 2 h following ZnSO(4) treatment. To determine whether DHEA conversion to estradiol was involved in the mechanism of DHEA action on glia, a third series of experiments was carried out in which the aromatase inhibitor fadrozole (4.16 mg/ml) was administered using subcutaneously implanted osmotic minipumps. Rats were killed on day 7 after chemical denervation, and the reaction of glial cells was monitored within the olfactory bulb, using GFAP and vimentin immunohistochemistry. Qualitative changes in GFAP expression were analyzed by Western blot. Chronic DHEA treatment with both doses (10 mg/kg b.w. and 25 mg/kg b.w.) and acute DHEA treatment with the highest dose applied (50 mg/kg b.w.), inhibited the increase in GFAP expression induced by the denervation of the olfactory bulb. Furthermore, GFAP and vimentin immunostaining in the glomerular layer of the olfactory bulb were diminished in the denervated and DHEA treated groups. However, when DHEA treatment was combined with fadrozole administration, such a decrease in GFAP expression could not be detected in the chemically denervated olfactory bulb. These findings indicate that DHEA, depending on the dose applied and the mode of administration, attenuates glial reaction to denervation and may regulate glial plasticity in the olfactory glomeruli. These effects are likely to be mediated at least in part by the conversion of DHEA to estradiol.

Transneuronal induction of the highly sialylated isoform of the neural cell adhesion molecule following nerve injury.

The polysialylated, embryonic form of the neuronal cell adhesion molecule (PSA-NCAM) is known to participate in a whole series of synaptic rearrangements even in adult animals. The possible role of this molecule in neuroplastic changes of the adult rat somatosensory cortex induced by unilateral transection of the infraorbital branch of the trigeminal nerve was studied with PSA-NCAM immunostaining at light microscopic level. Two- and three-month-old CFY albino rats were sacrificied on days 1, 4, 6, 14 and 21 following operation and PSA-NCAM immunoreaction was examined at three levels of the vibrissa-cortex neuraxis, namely, in the principal nucleus of the trigeminal nerve, in the ventral posteromedial nucleus of the thalamus and in the somatosensory cortex. The lower levels of the neuraxis remained free of PSA-NCAM labeling, similarly to control, intact animals. However, a large number of scattered small neurons became PSA-NCAM immunoreactive in layers IV-VI on both ipsi- and contralateral sides of the somatosensory cortex from day 6 onwards, suggesting a possible transynaptic regulation of NCAM sialylation state.