RESUMO
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.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Barreira Hematoencefálica/metabolismo , Humanos , Células Endoteliais/metabolismo , Animais , Via de Sinalização Wnt , Claudina-5/metabolismo , Claudina-5/genética , AMP Cíclico/metabolismo , Camundongos , Células-Tronco/metabolismo , Células-Tronco/citologia , Junções Íntimas/metabolismo , beta Catenina/metabolismoRESUMO
As the most prescribed psychotropic drugs in current medical practice, antidepressant drugs (ADs) of the selective serotonin reuptake inhibitor (SSRI) class represent prime candidates for drug repurposing. The mechanisms underlying their mode of action, however, remain unclear. Here, we show that common SSRIs and selected representatives of other AD classes bidirectionally regulate fluid-phase uptake at therapeutic concentrations and below. We further characterize membrane trafficking induced by a canonical SSRI fluvoxamine to show that it involves enhancement of clathrin-mediated endocytosis, endosomal system, and exocytosis. RNA sequencing analysis showed few fluvoxamine-associated differences, consistent with the effect being independent of gene expression. Fluvoxamine-induced increase in membrane trafficking boosted transcytosis in cell-based blood-brain barrier models, while a single injection of fluvoxamine was sufficient to enable brain accumulation of a fluid-phase fluorescent tracer in vivo. These findings reveal modulation of membrane trafficking by ADs as a possible cellular mechanism of action and indicate their clinical repositioning potential for regulating drug delivery to the brain.
RESUMO
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.
Assuntos
Doenças Neuroinflamatórias , Pericitos , Adulto , Humanos , Encéfalo/irrigação sanguínea , Barreira Hematoencefálica , Sistema Nervoso CentralRESUMO
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.
Assuntos
Commelinaceae , Doenças Neurodegenerativas , Humanos , Ecdisteroides/farmacologia , Ecdisteroides/química , Barreira Hematoencefálica , Células Endoteliais , Commelinaceae/química , Extratos Vegetais/farmacologia , Extratos Vegetais/químicaRESUMO
BACKGROUND: Toxoplasma gondii (T. gondii) is a highly successful parasite being able to cross all biological barriers of the body, finally reaching the central nervous system (CNS). Previous studies have highlighted the critical involvement of the blood-brain barrier (BBB) during T. gondii invasion and development of subsequent neuroinflammation. Still, the potential contribution of the choroid plexus (CP), the main structure forming the blood-cerebrospinal fluid (CSF) barrier (BCSFB) have not been addressed. METHODS: To investigate T. gondii invasion at the onset of neuroinflammation, the CP and brain microvessels (BMV) were isolated and analyzed for parasite burden. Additionally, immuno-stained brain sections and three-dimensional whole mount preparations were evaluated for parasite localization and morphological alterations. Activation of choroidal and brain endothelial cells were characterized by flow cytometry. To evaluate the impact of early immune responses on CP and BMV, expression levels of inflammatory mediators, tight junctions (TJ) and matrix metalloproteinases (MMPs) were quantified. Additionally, FITC-dextran was applied to determine infection-related changes in BCSFB permeability. Finally, the response of primary CP epithelial cells to T. gondii parasites was tested in vitro. RESULTS: Here we revealed that endothelial cells in the CP are initially infected by T. gondii, and become activated prior to BBB endothelial cells indicated by MHCII upregulation. Additionally, CP elicited early local immune response with upregulation of IFN-γ, TNF, IL-6, host-defence factors as well as swift expression of CXCL9 chemokine, when compared to the BMV. Consequently, we uncovered distinct TJ disturbances of claudins, associated with upregulation of MMP-8 and MMP-13 expression in infected CP in vivo, which was confirmed by in vitro infection of primary CP epithelial cells. Notably, we detected early barrier damage and functional loss by increased BCSFB permeability to FITC-dextran in vivo, which was extended over the infection course. CONCLUSIONS: Altogether, our data reveal a close interaction between T. gondii infection at the CP and the impairment of the BCSFB function indicating that infection-related neuroinflammation is initiated in the CP.
Assuntos
Plexo Corióideo , Toxoplasmose Cerebral , Barreira Hematoencefálica/metabolismo , Plexo Corióideo/metabolismo , Células Endoteliais , Humanos , Imunidade , Toxoplasmose Cerebral/metabolismoRESUMO
Permeation is one of the most evaluated parameters using preclinical in vitro blood-brain barrier models, as it has long been considered to be one of the major factors influencing central nervous system drug delivery. Blood-brain barrier permeability can be defined as the speed at which a compound crosses the brain endothelial cell barrier and is employed to assess barrier tightness, which is a crucial feature of brain capillaries in vivo. In addition, it is used to assess brain drug penetration. We review traditionally used methods to assess blood-brain barrier permeability in vitro and summarize often neglected in vivo (e.g., plasma protein and brain tissue binding) or in vitro (e.g., culture insert materials or methodology) factors that influence this property. These factors are crucial to consider when performing BBB permeability assessments, and especially when comparing permeability data obtained from different models, since model diversification significantly complicates inter-study comparisons. Finally, measuring transendothelial electrical resistance can be used to describe blood-brain barrier tightness; however, several parameters should be considered while comparing these measurements to the blood-brain barrier permeability to paracellular markers.
Assuntos
Barreira Hematoencefálica , Células Endoteliais , Transporte Biológico , Barreira Hematoencefálica/metabolismo , Encéfalo , Células Cultivadas , Células Endoteliais/metabolismo , Humanos , PermeabilidadeRESUMO
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.
Assuntos
Aromatase , Astrócitos , Animais , Aromatase/metabolismo , Astrócitos/metabolismo , Sistema Nervoso Central/metabolismo , Estradiol/metabolismo , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Neurônios/metabolismo , RatosRESUMO
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.
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Encéfalo , Células Endoteliais , Animais , Humanos , Ratos , 2-Hidroxipropil-beta-Ciclodextrina/farmacologia , Fluoresceína-5-Isotiocianato , Células CultivadasRESUMO
BACKGROUND: Heat-shock protein B1 (HSPB1) is among the most well-known and versatile member of the evolutionarily conserved family of small heat-shock proteins. It has been implicated to serve a neuroprotective role against various neurological disorders via its modulatory activity on inflammation, yet its exact role in neuroinflammation is poorly understood. In order to shed light on the exact mechanism of inflammation modulation by HSPB1, we investigated the effect of HSPB1 on neuroinflammatory processes in an in vivo and in vitro model of acute brain injury. METHODS: In this study, we used a transgenic mouse strain overexpressing the human HSPB1 protein. In the in vivo experiments, 7-day-old transgenic and wild-type mice were treated with ethanol. Apoptotic cells were detected using TUNEL assay. The mRNA and protein levels of cytokines and glial cell markers were examined using RT-PCR and immunohistochemistry in the brain. We also established primary neuronal, astrocyte, and microglial cultures which were subjected to cytokine and ethanol treatments. TNFα and hHSPB1 levels were measured from the supernates by ELISA, and intracellular hHSPB1 expression was analyzed using fluorescent immunohistochemistry. RESULTS: Following ethanol treatment, the brains of hHSPB1-overexpressing mice showed a significantly higher mRNA level of pro-inflammatory cytokines (Tnf, Il1b), microglia (Cd68, Arg1), and astrocyte (Gfap) markers compared to wild-type brains. Microglial activation, and 1 week later, reactive astrogliosis was higher in certain brain areas of ethanol-treated transgenic mice compared to those of wild-types. Despite the remarkably high expression of pro-apoptotic Tnf, hHSPB1-overexpressing mice did not exhibit higher level of apoptosis. Our data suggest that intracellular hHSPB1, showing the highest level in primary astrocytes, was responsible for the inflammation-regulating effects. Microglia cells were the main source of TNFα in our model. Microglia isolated from hHSPB1-overexpressing mice showed a significantly higher release of TNFα compared to wild-type cells under inflammatory conditions. CONCLUSIONS: Our work provides novel in vivo evidence that hHSPB1 overexpression has a regulating effect on acute neuroinflammation by intensifying the expression of pro-inflammatory cytokines and enhancing glial cell activation, but not increasing neuronal apoptosis. These results suggest that hHSPB1 may play a complex role in the modulation of the ethanol-induced neuroinflammatory response.
Assuntos
Lesões Encefálicas/induzido quimicamente , Lesões Encefálicas/metabolismo , Etanol/toxicidade , Proteínas de Choque Térmico/biossíntese , Mediadores da Inflamação/metabolismo , Chaperonas Moleculares/biossíntese , Animais , Lesões Encefálicas/genética , Células Cultivadas , Etanol/administração & dosagem , Expressão Gênica , Proteínas de Choque Térmico/genética , Humanos , Injeções Subcutâneas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Chaperonas Moleculares/genéticaRESUMO
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.
Assuntos
Esôfago de Barrett/genética , Proliferação de Células/genética , Esôfago/metabolismo , Interferência de RNA , Fumaça , Trocador 1 de Sódio-Hidrogênio/genética , Animais , Esôfago de Barrett/metabolismo , Esôfago de Barrett/patologia , Linhagem Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Esôfago/patologia , Expressão Gênica , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Masculino , Pessoa de Meia-Idade , Fumar , Trocador 1 de Sódio-Hidrogênio/metabolismo , Nicotiana/químicaRESUMO
Quercetin-3-glucuronide (Q3GA), the main phase II metabolite of quercetin (Q) in human plasma, is considered to be a more stable form of Q for transport with the bloodstream to tissues, where it can be potentially deconjugated by ß-glucuronidase (ß-Gluc) to Q aglycone, which easily enters the brain. This study evaluates the effect of lipopolysaccharide (LPS)-induced acute inflammation on ß-Gluc gene expression in the choroid plexus (ChP) and its activity in blood plasma, ChP and cerebrospinal fluid (CSF), and the concentration of Q and its phase II metabolites in blood plasma and CSF. Studies were performed on saline- and LPS-treated adult ewes (n = 40) receiving Q3GA intravenously (n = 16) and on primary rat ChP epithelial cells and human ChP epithelial papilloma cells. We observed that acute inflammation stimulated ß-Gluc activity in the ChP and blood plasma, but not in ChP epithelial cells and CSF, and did not affect Q and its phase II metabolite concentrations in plasma and CSF, except Q3GA, for which the plasma concentration was higher 30 min after administration (p < 0.05) in LPS- compared to saline-treated ewes. The lack of Q3GA deconjugation in the ChP observed under physiological and acute inflammatory conditions, however, does not exclude its possible role in the course of neurodegenerative diseases.
Assuntos
Plexo Corióideo/metabolismo , Glucuronidase/metabolismo , Quercetina/metabolismo , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Plexo Corióideo/efeitos dos fármacos , Células Epiteliais/metabolismo , Feminino , Glucuronidase/sangue , Glucuronidase/líquido cefalorraquidiano , Humanos , Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Masculino , Cultura Primária de Células , Quercetina/análogos & derivados , Quercetina/sangue , Quercetina/líquido cefalorraquidiano , Ratos , Ratos Wistar , OvinosRESUMO
The brain insulin metabolism alteration has been addressed as a pathophysiological factor underlying Alzheimer's disease (AD). Insulin can be beneficial in AD, but its macro-polypeptide nature negatively influences the chances of reaching the brain. The intranasal (IN) administration of therapeutics in AD suggests improved brain-targeting. Solid lipid nanoparticles (SLNs) and poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) are promising carriers to deliver the IN-administered insulin to the brain due to the enhancement of the drug permeability, which can even be improved by chitosan-coating. In the present study, uncoated and chitosan-coated insulin-loaded SLNs and PLGA NPs were formulated and characterized. The obtained NPs showed desirable physicochemical properties supporting IN applicability. The in vitro investigations revealed increased mucoadhesion, nasal diffusion, and drug release rate of both insulin-loaded nanocarriers over native insulin with the superiority of chitosan-coated SLNs. Cell-line studies on human nasal epithelial and brain endothelial cells proved the safety IN applicability of nanoparticles. Insulin-loaded nanoparticles showed improved insulin permeability through the nasal mucosa, which was promoted by chitosan-coating. However, native insulin exceeded the blood-brain barrier (BBB) permeation compared with nanoparticulate formulations. Encapsulating insulin into chitosan-coated NPs can be beneficial for ensuring structural stability, enhancing nasal absorption, followed by sustained drug release.
Assuntos
Encéfalo/citologia , Quitosana/química , Insulina/farmacologia , Nariz/citologia , Encéfalo/metabolismo , Linhagem Celular , Liberação Controlada de Fármacos , Células Endoteliais/química , Células Endoteliais/citologia , Insulina/química , Lipossomos/química , Nanopartículas/química , Nariz/química , Tamanho da Partícula , Ácido Poliglicólico/químicaRESUMO
The blood-brain barrier (BBB) maintains the optimal microenvironment for brain function. Tight junctions (TJs) allow endothelial cells to adhere to each other, leading to the formation of a barrier that prevents the penetration of most molecules via transcellular routes. Evidence has indicated that seizure-induced vascular endothelial growth factor (VEGF) type 2 receptor (VEGFR-2) pathway activation weakens TJs, inducing vasodilatation and increasing vascular permeability and subsequent brain injury. The present study focused on investigating the expression levels of VEGF-related (VEGF-A and VEGFR-2) and TJ-related proteins (claudin-5, occludin and ZO-1) in the neocortical microvasculature of patients with drug-resistant temporal lobe epilepsy (TLE). The results obtained from hippocampal sclerosis TLE (HS-TLE) patients were compared with those obtained from patients with TLE secondary to lesions (lesion-TLE) and autopsy samples. The Western blotting and immunofluorescence results showed that VEGF-A and VEGFR-2 protein expression levels were increased in HS-TLE and lesion-TLE patients compared to autopsy group. On the other hand, claudin-5 expression was higher in HS-TLE patients and lesion-TLE patients than autopsies. The expression level of occludin and ZO-1 was decreased in HS-TLE patients. Our study described modifications to the integrity of the BBB that may contribute to the pathogenesis of TLE, in which the VEGF system may play an important role. We demonstrated that the same modifications were present in both HS-TLE and lesion-TLE patients, which suggests that seizures modify these systems and that they are not associated with the establishment of epilepsy.
Assuntos
Barreira Hematoencefálica/metabolismo , Epilepsia Resistente a Medicamentos/metabolismo , Epilepsia do Lobo Temporal/metabolismo , Microvasos/metabolismo , Neocórtex/irrigação sanguínea , Proteínas de Junções Íntimas/metabolismo , Junções Íntimas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Adolescente , Adulto , Barreira Hematoencefálica/patologia , Claudina-5/metabolismo , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia Resistente a Medicamentos/patologia , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/patologia , Feminino , Humanos , Masculino , Microvasos/patologia , Pessoa de Meia-Idade , Ocludina/metabolismo , Transdução de Sinais , Junções Íntimas/patologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Adulto Jovem , Proteína da Zônula de Oclusão-1/metabolismoRESUMO
Metastasis of lung cancer to the brain is associated with poor outcomes and limited therapeutic options. The blood-brain barrier (BBB) plays a major role in brain metastasis. However, little is known about the role of pericytes in brain metastasis formation. This study aimed to reveal the interaction between pericytes and cancer cells. We established in vitro BBB models with rat primary cultured BBB-related cells (endothelial cells, astrocytes, and pericytes) and investigated the relationship between BBB-related cells and metastatic cancer cell lines. We observed a significant decrease in transendothelial electrical resistance with metastatic cancer cells in monolayer and coculture models with astrocytes. In contrast, the coculture model with pericytes showed inhibition of the decrease in transendothelial electrical resistance with metastatic cancer cells. In addition, the expression of tight junction protein was preserved only in the coculture model with pericytes. The conditioned medium of pericytes with metastatic cancer cells suppressed the proliferation of the cancer cells significantly. This study revealed that brain pericytes are the major regulators of the resistance of the BBB to lung cancer metastasis to the brain. Pericytes exert an anti-metastatic effect and thus have potential for the preventive treatment of brain metastasis.
Assuntos
Neoplasias Encefálicas/secundário , Neoplasias Pulmonares/patologia , Pericitos/patologia , Células A549 , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Impedância Elétrica , Humanos , Pericitos/efeitos dos fármacos , Ratos , Proteínas de Junções Íntimas/metabolismoRESUMO
Sleep loss in the rat increases blood-brain barrier permeability to circulating molecules by disrupting interendothelial tight junctions. Despite the description of the ultrastructure of cerebral microvessels and the evidence of an apparent pericyte detachment from capillary wall in sleep restricted rats the effect of sleep loss on pericytes is unknown. Here we characterized the interactions between pericytes and brain endothelial cells after sleep loss using male Wistar rats. Animals were sleep-restricted 20 h daily with 4 h sleep recovery for 10 days. At the end of the sleep restriction, brain microvessels (MVs) were isolated from cerebral cortex and hippocampus and processed for Western blot and immunocytochemistry to evaluate markers of pericyte-endothelial cell interaction (connexin 43, PDGFR-ß), tight junction proteins, and proinflammatory mediator proteins (MMP9, A2A adenosine receptor, CD73, NFκB). Sleep restriction reduced PDGFR-ß and connexin 43 expression in MVs; in addition, scanning electron microscopy micrographs showed that pericytes were detached from capillary walls, but did not undergo apoptosis (as depicted by a reduced active caspase-3 expression). Sleep restriction also decreased tight junction protein expression in MVs and increased BBB permeability to low- and high-molecular weight tracers in in vivo permeability assays. Those alterations seemed to depend on a low-grade inflammatory status as reflected by the increased expression of phosphorylated NFκB and A2A adenosine receptor in brain endothelial cells from the sleep-restricted rats. Our data show that pericyte-brain endothelial cell interaction is altered by sleep restriction; this evidence is essential to understand the role of sleep in regulating blood-brain barrier function.
Assuntos
Barreira Hematoencefálica , Pericitos , Animais , Encéfalo , Comunicação Celular , Células Endoteliais , Masculino , Ratos , Ratos Wistar , Sono , Junções ÍntimasRESUMO
Clinical and experimental results with inhaled sodium bicarbonate as an adjuvant therapy in cystic fibrosis (CF) are promising due to its mucolytic and bacteriostatic properties, but its direct effect has not been studied on respiratory epithelial cells. Our aim was to establish and characterize co-culture models of human CF bronchial epithelial (CFBE) cell lines expressing a wild-type (WT) or mutant (deltaF508) CF transmembrane conductance regulator (CFTR) channel with human vascular endothelial cells and investigate the effects of bicarbonate. Vascular endothelial cells induced better barrier properties in CFBE cells as reflected by the higher resistance and lower permeability values. Activation of CFTR by cAMP decreased the electrical resistance in WT but not in mutant CFBE cell layers confirming the presence and absence of functional channels, respectively. Sodium bicarbonate (100 mM) was well-tolerated by CFBE cells: it slightly reduced the impedance of WT but not that of the mutant CFBE cells. Sodium bicarbonate significantly decreased the more-alkaline intracellular pH of the mutant CFBE cells, while the barrier properties of the models were only minimally changed. These observations indicate that sodium bicarbonate is beneficial to deltaF508-CFTR expressing CFBE cells. Thus, sodium bicarbonate may have a direct therapeutic effect on the bronchial epithelium.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Mutação , Mucosa Respiratória/efeitos dos fármacos , Mucosa Respiratória/metabolismo , Bicarbonato de Sódio/farmacologia , Biomarcadores , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fibrose Cística/tratamento farmacológico , Fibrose Cística/metabolismo , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Mucosa Respiratória/patologia , Transdução de Sinais , Bicarbonato de Sódio/uso terapêutico , Junções Íntimas/metabolismoRESUMO
Alzheimer's disease is one of the most common chronic neurodegenerative disorders. Despite several in vivo and clinical studies, the cause of the disease is poorly understood. Currently, amyloid ß (Aß) peptide and its tendency to assemble into soluble oligomers are known as a main pathogenic event leading to the interruption of synapses and brain degeneration. Targeting neurotoxic Aß oligomers can help recognize the disease at an early stage or it can be a potential therapeutic approach. Unnatural ß-peptidic foldamers are successfully used against many different protein targets due to their favorable structural and pharmacokinetic properties compared to small molecule or protein-like drug candidates. We have previously reported a tetravalent foldamer-dendrimer conjugate which can selectively bind Aß oligomers. Taking advantage of multivalency and foldamers, we synthesized different multivalent foldamer-based conjugates to optimize the geometry of the ligand. Isothermal titration calorimetry (ITC) was used to measure binding affinity to Aß, thereafter 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) based tissue viability assay and impedance-based viability assay on SH-SY5Y cells were applied to monitor Aß toxicity and protective effects of the compounds. Important factors for high binding affinity were determined and a good correlation was found between influencing the valence and the capability of the conjugates for Aß binding.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides/química , Dendrímeros/química , Fragmentos de Peptídeos/química , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/antagonistas & inibidores , Precursor de Proteína beta-Amiloide/química , Animais , Calorimetria , Dendrímeros/uso terapêutico , Humanos , Ligantes , Neurônios/química , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/uso terapêutico , Ligação Proteica , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína/efeitos dos fármacosRESUMO
The indolo[2,1-b]quinazoline alkaloid tryptanthrin was previously identified as a potent anti-inflammatory compound with a unique pharmacological profile. It is a potent inhibitor of cyclooxygenase-2, 5-lipooxygenase-catalyzed leukotriene synthesis, and nitric oxide production catalyzed by the inducible nitric oxide synthase. To characterize the pharmacokinetic properties of tryptanthrin, we performed a pilot in vivo study in male Sprague-Dawley rats (2 mg/kg bw i.âv.). Moreover, the ability of tryptanthrin to cross the blood-brain barrier was evaluated in three in vitro human and animal blood-brain barrier models. Bioanalytical UPLC-MS/MS methods used were validated according to current international guidelines. A half-life of 40.63 ± 6.66 min and a clearance of 1.00 ± 0.36 L/h/kg were found in the in vivo pharmacokinetic study. In vitro data obtained with the two primary animal blood-brain barrier models showed a good correlation with an immortalized human monoculture blood-brain barrier model (hBMEC cell line), and were indicative of a high blood-brain barrier permeation potential of tryptanthrin. These findings were corroborated by the in silico prediction of blood-brain barrier penetration. P-glycoprotein interaction of tryptanthrin was assessed by calculation of the efflux ratio in bidirectional permeability assays. An efflux ratio below 2 indicated that tryptanthrin is not subjected to active efflux.
Assuntos
Barreira Hematoencefálica/metabolismo , Quinazolinas/farmacocinética , Animais , Linhagem Celular , Cromatografia Líquida de Alta Pressão/métodos , Humanos , Isatis/química , Masculino , Estrutura Molecular , Extratos Vegetais/farmacocinética , Quinazolinas/síntese química , Quinazolinas/química , Ratos , Ratos Sprague-DawleyRESUMO
The nasal route receives a great deal of attention as a non-invasive method for the systemic administration of drugs. For nasal delivery, specific formulations containing excipients are used. Because of the sensitive respiratory mucosa, not only the active ingredients, but also additives need to be tested in appropriate models for toxicity. The aim of the study was to measure the cytotoxicity of six pharmaceutical excipients, which could help to reach larger residence time, better permeability, and increased solubility dissolution rate. The following excipients were investigated on RPMI 2650 human nasal septum tumor epithelial cells: ß-d-mannitol, sodium hyaluronate, α and ß-cyclodextrin, polyvinyl alcohol and methylcellulose. 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye conversion assay and real-time impedance analysis were used to investigate cytotoxicity. No excipient showed toxicity at 0.3% (w/v) concentration or below while 1% concentration a significantly reduced metabolic activity was measured by MTT assay for methylcellulose and cyclodextrins. Using impedance measurements, only ß-cyclodextrin (1%) was toxic to cells. Mannitol at 1% concentration had a barrier opening effect on epithelial cells, but caused no cellular damage. Based on the results, all additives at 0.3%, sodium hyaluronate and polyvinyl alcohol at 1% concentrations can be safely used for nasal formulations.