RESUMO
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, progressive disorder and growing public health concern. To address this issue considerable research has been undertaken in pursuit of new NAFLD therapeutics. Development of effective, high-throughput in vitro models is an important aspect of drug discovery. Here, a micropatterned hepatocyte co-culture (MPCC) was used to model liver steatosis. The MPCC model (HEPATOPACTM) is comprised of hepatocytes and 3T3-J2 mouse stromal cells plated onto a patterned standard 96-well or 24-well plate, allowing the cultures to be handled and imaged in a standardized multi-well format. These studies employed high content imaging (HCI) analysis to assess lipid content in cultures. HCI analysis of lipid accumulation allows large numbers of samples to be imaged and analyzed in a relatively short period of time compared to manual acquisition and analysis methods. Treatment of MPCC with free fatty acids (FFA), high glucose and fructose (HGF), or a combination of both induces hepatic steatosis. MPCC treatment with ACC1/ACC2 inhibitors, as either a preventative or reversal agent, showed efficacy against FFA induced hepatic steatosis. Drug induced steatosis was also evaluated. Treatment with valproic acid showed steatosis induction in a lean background, which was significantly potentiated in a fatty liver background. Additionally, these media treatments changed expression of fatty liver related genes. Treatment of MPCC with FFA, HGF, or a combination reversibly altered expression of genes involved in fatty acid metabolism, insulin signaling, and lipid transport. Together, these data demonstrate that MPCC is an easy to use, long-term functional in vitro model of NAFLD having utility for compound screening, drug toxicity evaluation, and assessment of gene regulation.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Avaliação Pré-Clínica de Medicamentos , Técnicas de Cocultura , Ácidos Graxos não Esterificados , Frutose , HepatócitosRESUMO
Pathologies of the blood-brain barrier (BBB) have been linked to a multitude of central nervous system (CNS) disorders whose pathology is poorly understood. Cortical spreading depression (CSD) has long been postulated to be involved in the underlying mechanisms of these disease states, yet a complete understanding remains elusive. This study seeks to utilize an in vitro model of the blood-brain barrier (BBB) with brain endothelial cell (b.End3) murine endothelioma cells to investigate the role of CSD in BBB pathology by characterizing effects of the release of major pronociceptive substances into the extracellular space of the CNS. The application of trans-endothelial electrical resistance (TEER) screening, transcellular uptake, and immunoreactive methods were used in concert with global proteome and phospho-proteomic approaches to assess the effect of modeled CSD events on the modeled BBB in vitro. The findings demonstrate relocalization and functional alteration to proteins associated with the actin cytoskeleton and endothelial tight junctions. Additionally, unique pathologic mechanisms induced by individual substances released during CSD were found to have unique phosphorylation signatures in phospho-proteome analysis, identifying Zona Occludins 1 (ZO-1) as a possible pathologic "checkpoint" of the BBB. By utilizing these phosphorylation signatures, possible novel diagnostic methods may be developed for CSD and warrants further investigation.
RESUMO
BACKGROUND: Sex hormones have been implicated in pH regulation of numerous physiological systems. One consistent factor of these studies is the sodium-hydrogen exchanger 1 (NHE1). NHE1 has been associated with pH homeostasis at epithelial barriers. Hormone fluctuations have been implicated in protection and risk for breaches in blood brain barrier (BBB)/blood endothelial barrier (BEB) integrity. Few studies, however, have investigated BBB/BEB integrity in neurological disorders in the context of sex-hormone regulation of pH homeostasis. METHODS//RESULTS: Physiologically relevant concentrations of 17-ß-estradiol (E2, 294 pM), progesterone (P, 100 nM), and testosterone (T,3.12 nM) were independently applied to cultured immortalized bEnd.3 brain endothelial cells to study the BEB. Individual gonadal hormones showed preferential effects on extracellular pH (E2), 14C-sucrose uptake (T), stimulated paracellular breaches (P) with dependence on functional NHE1 expression without impacting transendothelial resistance (TEER) or total protein expression. While total NHE1 expression was not changed as determined via whole cell lysate and subcellular fractionation experiment, biotinylation of NHE1 for surface membrane expression showed E2 reduced functional expression. Quantitative proteomic analysis revealed divergent effects of 17-ß-estradiol and testosterone on changes in protein abundance in bEnd.3 endothelial cells as compared to untreated controls. CONCLUSIONS: These data suggest that circulating levels of sex hormones may independently control BEB integrity by 1) regulating pH homeostasis through NHE1 functional expression and 2) modifying the endothelial proteome.
Assuntos
Barreira Hematoencefálica/metabolismo , Estradiol/fisiologia , Progesterona/fisiologia , Trocador 1 de Sódio-Hidrogênio/metabolismo , Testosterona/fisiologia , Animais , Transporte Biológico , Células Endoteliais/metabolismo , Estradiol/sangue , Concentração de Íons de Hidrogênio , Progesterona/sangue , Proteoma/metabolismo , Ratos , Testosterona/sangueRESUMO
Cerebral malaria (CM), results from Plasmodium falciparum infection, and has a high mortality rate. CM survivors can retain life-long post CM sequelae, including seizures and neurocognitive deficits profoundly affecting their quality of life. As the Plasmodium parasite does not enter the brain, but resides inside erythrocytes and are confined to the lumen of the brain's vasculature, the neuropathogenesis leading to these neurologic sequelae is unclear and under-investigated. Interestingly, postmortem CM pathology differs in brain regions, such as the appearance of haemorragic punctae in white versus gray matter. Various host and parasite factors contribute to the risk of CM, including exposure at a young age, parasite- and host-related genetics, parasite sequestration and the extent of host inflammatory responses. Thus far, several proposed adjunctive treatments have not been successful in the treatment of CM but are highly needed. The region-specific CM neuro-pathogenesis leading to neurologic sequelae is intriguing, but not sufficiently addressed in research. More attention to this may lead to the development of effective adjunctive treatments to address CM neurologic sequelae.
Assuntos
Malária Cerebral , Malária Falciparum , Plasmodium falciparum/fisiologia , Eritrócitos/parasitologia , Humanos , Malária Cerebral/complicações , Malária Cerebral/fisiopatologia , Malária Falciparum/complicações , Malária Falciparum/fisiopatologia , Qualidade de VidaRESUMO
Cortical spreading depression (CSD) in the CNS is suggested as a common mechanism contributing to headache. Despite strong evidence for CNS involvement in headache disorders, drug development for headache disorders remains focused on peripheral targets. Difficulty in delivering drugs across the blood-brain barrier (BBB) may partially account for this disparity. It is known, however, that BBB permeability is increased during several CNS pathologies. In this study, we investigated BBB changes in response to KCl-induced CSD events and subsequent allodynia in rats. Cortical KCl injection in awake, freely moving rats produced facial allodynia with peak intensity between 1.5 and 3 h and CSD induction within 0.5-2 h postinjection. Brain perfusion of 14C-sucrose as a marker of BBB paracellular permeability revealed increased leak in the cortex, but not brainstem, beginning 0.5 h post-KCl injection and resolving within 6 h; no changes in tight junction (TJ) proteins occludin or claudin-5 expression were observed. Acute pretreatment with topiramate to inhibit CSD did not prevent the increased BBB paracellular permeability. CNS delivery of the abortive anti-migraine agent sumatriptan was increased in the cortex 1.5 h post-KCl injection. Surprisingly, sumatriptan uptake was also increased in the brainstem following CSD induction, suggesting regulation of active transport mechanisms at the BBB. Together, these results demonstrate the ability of CSD events to produce transient, time-dependent changes in BBB permeability when allodynia is present and to mediate access of clinically relevant therapeutics (i.e., sumatriptan) to the CNS.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Tronco Encefálico/efeitos dos fármacos , Fármacos do Sistema Nervoso Central/farmacologia , Córtex Cerebral/efeitos dos fármacos , Depressão Alastrante da Atividade Elétrica Cortical/fisiologia , Cefaleia/tratamento farmacológico , Hiperalgesia/fisiopatologia , Sumatriptana/farmacologia , Animais , Fármacos do Sistema Nervoso Central/farmacocinética , Córtex Cerebral/fisiopatologia , Modelos Animais de Doenças , Feminino , Cefaleia/fisiopatologia , Hiperalgesia/induzido quimicamente , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Sumatriptana/farmacocinética , Topiramato/farmacologiaRESUMO
AIMS: Test the putative contribution of 17-ß-estradiol in the development of spreading depression (SD) events and head pain in awake, non-restrained rats. MAIN METHODS: Female, Sprague-Dawley rats were intact or underwent ovariectomy followed one week later by surgery to place electrodes onto the dura to detect epidural electroencephalographic activity (dEEG). dEEG activity was recorded two days later for 12 hours after systemic administration of 17-ß-estradiol (180 µg/kg, i.p.). A separate set of rats were observed for changes in exploratory, ambulatory, fine, and rearing behaviors; periorbital allodynia was also assessed. KEY FINDINGS: A bolus of 17-ß-estradiol significantly elevated serum estrogen levels, increased SD episodes over a 12-hour recording period and decreased rearing behaviors in ovariectomized rats. Pre-administration of ICI 182,780, an estrogen receptor antagonist, blocked 17-ß-estradiol-evoked SD events and pain behaviors; similar results were observed when the antimigraine therapeutic sumatriptan was used. SIGNIFICANCE: These data indicate that an estrogen receptor-mediated mechanism contributes to SD events in ovariectomized rats and pain behaviors in both ovariectomized -and intact- rats. This suggests that estrogen plays a different role in each phenomenon of migraine where intense fluctuations in concentration may influence SD susceptibility. This is the first study to relate estrogen peaks to SD development and pain behaviors in awake, freely moving female rats, establishing a framework for future preclinical migraine studies.
RESUMO
Targeting proteins within the N-type voltage-gated calcium channel (CaV2.2) complex has proven to be an effective strategy for developing novel pain therapeutics. We describe a novel peptide aptamer derived from the collapsin response mediator protein 2 (CRMP2), a CaV2.2-regulatory protein. Addition of a 14-carbon myristate group to the peptide (myr-tat-CBD3) tethered it to the membrane of primary sensory neurons near surface CaV2.2. Pull-down studies demonstrated that myr-tat-CBD3 peptide interfered with the CRMP2-CaV2.2 interaction. Quantitative confocal immunofluorescence revealed a pronounced reduction of CaV2.2 trafficking after myr-tat-CBD3 treatment and increased efficiency in disrupting CRMP2-CaV2.2 colocalization compared with peptide tat-CBD3. Consequently, myr-tat-CBD3 inhibited depolarization-induced calcium influx in sensory neurons. Voltage clamp electrophysiology experiments revealed a reduction of Ca, but not Na, currents in sensory neurons after myr-tat-CBD3 exposure. Current clamp electrophysiology experiments demonstrated a reduction in excitability of small-diameter dorsal root ganglion neurons after exposure to myr-tat-CBD3. Myr-tat-CBD3 was effective in significantly attenuating carrageenan-induced thermal hypersensitivity and reversing thermal hypersensitivity induced by a surgical incision of the plantar surface of the rat hind paw, a model of postoperative pain. These effects are compared with those of tat-CBD3-the nonmyristoylated tat-conjugated CRMP2 peptide as well as scrambled versions of CBD3 and CBD3-lacking control peptides. Our results demonstrate that the myristoyl tag enhances intracellular delivery and local concentration of the CRMP2 peptide aptamer near membrane-delimited calcium channels resulting in pronounced interference with the calcium channel complex, superior suppression of calcium influx, and better antinociceptive potential.
Assuntos
Aptâmeros de Peptídeos/metabolismo , Canais de Cálcio Tipo N/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Dor Pós-Operatória/tratamento farmacológico , Dor Pós-Operatória/metabolismo , Transporte Proteico/fisiologia , Sequência de Aminoácidos , Animais , Aptâmeros de Peptídeos/genética , Aptâmeros de Peptídeos/uso terapêutico , Células Cultivadas , Feminino , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Inflamação/tratamento farmacológico , Inflamação/genética , Inflamação/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/uso terapêutico , Dor Pós-Operatória/genética , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
Impaired programmed cell death is an important contributing mechanism in the development of chronic inflammatory and autoimmune diseases. Overexpression of Bcl-2 family proteins in such diseases has led to the concept of targeted suppression of these proteins as a primary therapeutic strategy. However, limited success with this approach has prompted pharmacologists to look at the other side of the coin, with the aim of reactivating jeopardized pro-apoptotic proteins that may neutralize Bcl-2 or other anti-apoptotic molecules. In this effort, BH3-only proteins have gained recent attention as endogenous molecules for the sensitization of resistant cells to undergo apoptosis. Among the BH3-only family, Noxa stands out as exceptional for its specificity to bind Mcl-1 and Bcl-2 and blunt their biological properties. Noxa is now being tested as a promising therapeutic target in cancer biology. Nonetheless, its role and clinical application still lack validation in autoimmune diseases, including rheumatic conditions. This is partly attributed to the significant gap in our understanding of its regulatory role and how either overexpression of Noxa or delivery of BH3 mimetics could be therapeutically exploited. In this review we highlight some recent studies in RA, OA, SLE and SS suggesting that Noxa may be used as a potential therapeutic target to circumvent invasive and tissue destructive processes in these rheumatic diseases.