ABSTRACT
BACKGROUND: The blood-brain barrier (BBB) is pivotal for the maintenance of brain homeostasis and it strictly regulates the cerebral transport of a wide range of endogenous compounds and drugs. While fasting is increasingly recognized as a potential therapeutic intervention in neurology and psychiatry, its impact upon the BBB has not been studied. This study was designed to assess the global impact of fasting upon the repertoire of BBB transporters. METHODS: We used a combination of in vivo and in vitro experiments to assess the response of the brain endothelium in male rats that were fed ad libitum or fasted for one to three days. Brain endothelial cells were acutely purified and transcriptionaly profiled using RNA-Seq. Isolated brain microvessels were used to assess the protein expression of selected BBB transporters through western blot. The molecular mechanisms involved in the adaptation to fasting were investigated in primary cultured rat brain endothelial cells. MCT1 activity was probed by in situ brain perfusion. RESULTS: Fasting did not change the expression of the main drug efflux ATP-binding cassette transporters or P-glycoprotein activity at the BBB but modulated a restrictive set of solute carrier transporters. These included the ketone bodies transporter MCT1, which is pivotal for the brain adaptation to fasting. Our findings in vivo suggested that PPAR δ, a major lipid sensor, was selectively activated in brain endothelial cells in response to fasting. This was confirmed in vitro where pharmacological agonists and free fatty acids selectively activated PPAR δ, resulting in the upregulation of MCT1 expression. Moreover, dosing rats with a specific PPAR δ antagonist blocked the upregulation of MCT1 expression and activity induced by fasting. CONCLUSIONS: Altogether, our study shows that fasting affects a selected set of BBB transporters which does not include the main drug efflux transporters. Moreover, we describe a previously unknown selective adaptive response of the brain vasculature to fasting which involves PPAR δ and is responsible for the up-regulation of MCT1 expression and activity. Our study opens new perspectives for the metabolic manipulation of the BBB in the healthy or diseased brain.
Subject(s)
Blood-Brain Barrier , PPAR delta , Rats , Male , Animals , Blood-Brain Barrier/metabolism , PPAR delta/metabolism , Endothelial Cells/metabolism , Membrane Transport Proteins/metabolism , Brain/metabolism , FastingABSTRACT
(1) A 24 h urinary free cortisol (UFF) is one of the first-line exams recommended for the diagnosis of Cushing's syndrome. In a hospital hormonology department, this activity can exceed several hundred dosages per week. The UFF is generally determined via an immunoassay with an automate using a chemiluminescence or electrochemiluminescence detection system. To increase the cortisol concentration in the analyzed sample, the automated analysis is preceded by urine extraction, which does not prevent there from being some interferences due to other steroids with close structures. (2) This paper describes the development of on-line solid phase extraction coupled to liquid chromatography and mass spectrometry for the analysis of urinary free cortisol. The on-line extraction was based on the TurboflowTM chromatography coupled to the analytical column by two valves, easily available for the laboratories. (3) The choice of the Accucore Polar Premium® analytical column made it possible to avoid analytical interferences with exogenous or endogenous molecules having the same SRM transition (363 â 121) as cortisol. (4) The method was fully validated in the range of clinically relevant concentrations from the lower limit of quantification (LLOQ) to 411.75 nmol·L-1.
ABSTRACT
The blood-brain barrier (BBB) protects the brain from toxins but hinders the penetration of neurotherapeutic drugs. Therefore, the blood-to-brain permeability of chemotherapeutics must be carefully evaluated. Here, we aimed to establish a workflow to generate primary cultures of human brain microvascular endothelial cells (BMVECs) to study drug brain permeability and bioavailability. Furthermore, we characterized and validated this BBB model in terms of quantitative expression of junction and drug-transport proteins, and drug permeability. We isolated brain microvessels (MVs) and cultured BMVECs from glioma patient biopsies. Then, we employed targeted LC-MS proteomics for absolute protein quantification and immunostaining to characterize protein localization and radiolabeled drugs to predict drug behavior at the Human BBB. The abundance levels of ABC transporters, junction proteins, and cell markers in the cultured BMVECs were similar to the MVs and correctly localized to the cell membrane. Permeability values (entrance and exit) and efflux ratios tested in vitro using the primary BMVECs were within the expected in vivo values. They correctly reflected the transport mechanism for 20 drugs (carbamazepine, diazepam, imipramine, ketoprofen, paracetamol, propranolol, sulfasalazine, terbutaline, warfarin, cimetidine, ciprofloxacin, digoxin, indinavir, methotrexate, ofloxacin, azidothymidine (AZT), indomethacin, verapamil, quinidine, and prazosin). We established a human primary in vitro model suitable for studying blood-to-brain drug permeability with a characterized quantitative abundance of transport and junction proteins, and drug permeability profiles, mimicking the human BBB. Our results indicate that this approach could be employed to generate patient-specific BMVEC cultures to evaluate BBB drug permeability and develop personalized therapeutic strategies.
Subject(s)
Blood-Brain Barrier , Endothelial Cells , Humans , Blood-Brain Barrier/metabolism , Endothelial Cells/metabolism , Proteomics , ATP-Binding Cassette Transporters/metabolism , PermeabilityABSTRACT
In humans, tetrahydrobiopterin (H4Bip) is the cofactor of several essential hydroxylation reactions which dysfunction cause very serious diseases at any age. Hence, the determination of pterins in biological media is of outmost importance in the diagnosis and monitoring of H4Bip deficiency. More than half a century after the discovery of the physiological role of H4Bip and the recent advent of gene therapy for dopamine and serotonin disorders linked to H4Bip deficiency, the quantification of quinonoid dihydrobiopterin (qH2Bip), the transient intermediate of H4Bip, has not been considered yet. This is mainly due to its short half-life, which goes from 0.9 to 5 min according to previous studies. Based on our recent disclosure of the specific MS/MS transition of qH2Bip, here, we developed an efficient HPLC-MS/MS method to achieve the separation of qH2Bip from H4Bip and other oxidation products in less than 3.5 min. The application of this method to the investigation of H4Bip autoxidation kinetics clearly shows that qH2Bip's half-life is much longer than previously reported, and mostly longer than that of H4Bip, irrespective of the considered experimental conditions. These findings definitely confirm that an accurate method of H4Bip analysis should include the quantification of qH2Bip.
Subject(s)
Biopterins , Tandem Mass Spectrometry , Humans , Biopterins/analysis , Biopterins/metabolism , Pterins , KineticsABSTRACT
Objective: Large response of steroid precursors, including 17-hydroxyprogesterone, to adrenocorticotropic hormone (ACTH) has been described in adrenocortical tumors, suggesting the existence of intra-tumoral enzymatic deficiencies. This study aimed to compare steroidogenesis enzymes activity in unilateral and bilateral benign tumors using serum steroid profiling in liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in the basal state and after ACTH 1-24 stimulation. Design and methods: A serum profile of seven consecutive adrenal steroids was determined in LC-MS/MS in the basal state (T0) and after ACTH 1-24 stimulation (T60) in 35 patients with bilateral adrenocortical tumors (BL), 38 patients with unilateral tumors (UL) and 37 control subjects (CT). Response amplitude of each individual steroid was evaluated by T60/T0 ratio, whereas enzymatic activity was assessed by the downstream/upstream steroid ratio. Adrenal volume was quantified by a semi-automatic segmentation method. Results: For the seven steroids assayed, the amplitude of response to ACTH was higher in BL than in UL and in CT. The difference between BL and UL persisted even after matching patients on adrenal volume. On glucocorticoids pathway, enzymatic activity of CYP11B1 was significantly decreased in BL (78.3 (43.1-199.4)) in comparison to both UL (122.7 (13.8-228.4), P = 0.0002) and CT (186.8 (42.1-1236.3), P < 0.0001). On mineralocorticoids and androgens pathways, the enzymatic activity of CYP11B2 and CYP17A1-17,20 lyase was also lower in BL than UL and CT. Conclusions: Decreased activity of distal steroidogenesis enzymes CYP11B1, CYP11B2 and CYP17A1-17,20 lyase, responsible for an explosive response to ACTH of upstream precursors in bilateral tumors, limits the synthesis of bioactive steroids, in particular cortisol, despite the increase in adrenal mass. Significance statement: Activity of distal steroidogenesis enzymes (CYP11B1, CYP11B2 and CYP17A1 on glucocorticoids, mineralocorticoids and androgens pathways, respectively) is decreased in adrenocortical benign tumors. This decrease is more pronounced in bilateral lesions and seems to depend more on the nature of the lesion than on the increase in adrenal volume. It is responsible for the explosive response to ACTH of steroid precursors located upstream of these enzymes. It probably allows bioactive steroids, particularly cortisol, to stay in the normal range for a long time despite the increase in adrenal mass.
ABSTRACT
Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood-brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm-2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.
Subject(s)
Endothelial Cells , Proteomics , Blood-Brain Barrier/metabolism , Brain/blood supply , Cells, Cultured , Endothelial Cells/metabolism , Humans , Stress, MechanicalABSTRACT
The blood-brain barrier (BBB) hinders the brain delivery of many anticancer drugs. In pediatric patients, diffuse intrinsic pontine glioma (DIPG) represents the main cause of brain cancer mortality lacking effective drug therapy. Using sham and DIPG-bearing rats, we analyzed 1) the brain distribution of 3-kDa-Texas red-dextran (TRD) or [14C]-sucrose as measures of BBB integrity, and 2) the role of major ATP-binding cassette (ABC) transporters at the BBB on the efflux of the irinotecan metabolite [3H]-SN-38. The unaffected [14C]-sucrose or TRD distribution in the cerebrum, cerebellum, and brainstem regions in DIPG-bearing animals suggests an intact BBB. Targeted proteomics retrieved no change in P-glycoprotein (P-gp), BCRP, MRP1, and MRP4 levels in the analyzed regions of DIPG rats. In vitro, DIPG cells express BCRP but not P-gp, MRP1, or MRP4. Dual inhibition of P-gp/Bcrp, or Mrp showed a significant increase on SN-38 BBB transport: Cerebrum (8.3-fold and 3-fold, respectively), cerebellum (4.2-fold and 2.8-fold), and brainstem (2.6-fold and 2.2-fold). Elacridar increased [3H]-SN-38 brain delivery beyond a P-gp/Bcrp inhibitor effect alone, emphasizing the role of another unidentified transporter in BBB efflux of SN-38. These results confirm a well-preserved BBB in DIPG-bearing rats, along with functional ABC-transporter expression. The development of chemotherapeutic strategies to circumvent ABC-mediated BBB efflux are needed to improve anticancer drug delivery against DIPG.
ABSTRACT
Organic cation transporters (OCTs) participate in the handling of compounds in kidneys and at the synaptic cleft. Their role at the blood-brain barrier (BBB) in brain drug delivery is still unclear. The presence of OCT1,2,3 (SLC22A1-3) in mouse, rat and human isolated brain microvessels was investigated by either qRT-PCR, quantitative proteomics and/or functional studies. BBB transport of the prototypical substrate [3H]-1-methyl-4-phenylpyridinium ([3H]-MPP+) was measured by in situ brain perfusion in six mouse strains and in Sprague Dawley rats, in primary human brain microvascular endothelial cells seeded on inserts, in the presence or absence of OCTs and a MATE1 (SLC49A1) inhibitor. The results show negligible OCT1 (SLC22A1) and OCT2 (SLC22A2) expression in either mice, rat or human brain microvessels, while OCT3 expression was identified in rat microvessels by qRT-PCR. The in vitro human cellular uptake of [3H]-MPP+ was not modified by OCTs/MATE-inhibitor. Brain transport of [3H]-MPP+ remains unchanged between 2- and 6-month old mice, and no alteration was observed in mice and rats with inhibitors. In conclusion, the evidenced lack of expression and/or functional OCTs and MATE at the BBB allows the maintenance of the brain homeostasis and function as it prevents an easy access of their neurotoxicant substrates to the brain parenchyma.
ABSTRACT
BACKGROUND: P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) are two efflux transporters expressed at the blood-brain barrier which effectively restrict the brain distribution of the majority of currently known anticancer drugs. High-grade brain tumors often possess a disrupted blood-brain tumor barrier (BBTB) leading to enhanced accumulation of magnetic resonance imaging contrast agents, and possibly anticancer drugs, as compared to normal brain. In contrast to high-grade brain tumors, considerably less information is available with respect to BBTB integrity in lower grade brain tumors. MATERIALS AND METHODS: We performed positron emission tomography imaging with the radiolabeled ABCB1 inhibitor [11C]tariquidar, a prototypical ABCB1/ABCG2 substrate, in seven patients with non-contrast -enhancing brain tumors (WHO grades I-III). In addition, ABCB1 and ABCG2 levels were determined in surgically resected tumor tissue of four patients using quantitative targeted absolute proteomics. RESULTS: Brain distribution of [11C]tariquidar was found to be very low across the whole brain and not significantly different between tumor and tumor-free brain tissue. Only one patient showed a small area of enhanced [11C]tariquidar uptake within the brain tumor. ABCG2/ABCB1 ratios in surgically resected tumor tissue (1.4 ± 0.2) were comparable to previously reported ABCG2/ABCB1 ratios in isolated human micro-vessels (1.3), which suggested that no overexpression of ABCB1 or ABCG2 occurred in the investigated tumors. CONCLUSIONS: Our data suggest that the investigated brain tumors had an intact BBTB, which is impermeable to anticancer drugs, which are dual ABCB1/ABCG2 substrates. Therefore, effective drugs for antitumor treatment should have high passive permeability and lack ABCB1/ABCG2 substrate affinity. TRIAL REGISTRATION: European Union Drug Regulating Authorities Clinical Trials Database (EUDRACT), 2011-004189-13. Registered on 23 February 2012, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2011-004189-13.
ABSTRACT
Drug delivery into the brain is regulated by the blood-brain interfaces. The blood-brain barrier (BBB), the blood-cerebrospinal fluid barrier (BCSFB), and the blood-arachnoid barrier (BAB) regulate the exchange of substances between the blood and brain parenchyma. These selective barriers present a high impermeability to most substances, with the selective transport of nutrients and transporters preventing the entry and accumulation of possibly toxic molecules, comprising many therapeutic drugs. Transporters of the ATP-binding cassette (ABC) superfamily have an important role in drug delivery, because they extrude a broad molecular diversity of xenobiotics, including several anticancer drugs, preventing their entry into the brain. Gliomas are the most common primary tumors diagnosed in adults, which are often characterized by a poor prognosis, notably in the case of high-grade gliomas. Therapeutic treatments frequently fail due to the difficulty of delivering drugs through the brain barriers, adding to diverse mechanisms developed by the cancer, including the overexpression or expression de novo of ABC transporters in tumoral cells and/or in the endothelial cells forming the blood-brain tumor barrier (BBTB). Many models have been developed to study the phenotype, molecular characteristics, and function of the blood-brain interfaces as well as to evaluate drug permeability into the brain. These include in vitro, in vivo, and in silico models, which together can help us to better understand their implication in drug resistance and to develop new therapeutics or delivery strategies to improve the treatment of pathologies of the central nervous system (CNS). In this review, we present the principal characteristics of the blood-brain interfaces; then, we focus on the ABC transporters present on them and their implication in drug delivery; next, we present some of the most important models used for the study of drug transport; finally, we summarize the implication of ABC transporters in glioma and the BBTB in drug resistance and the strategies to improve the delivery of CNS anticancer drugs.
ABSTRACT
The effect of cannabidiol (CBD), a high-affinity agonist of the transient receptor potential vanilloid-2 (TRPV2) channel, has been poorly investigated in human brain microvessel endothelial cells (BMEC) forming the blood-brain barrier (BBB). TRPV2 expression and its role on Ca2+ cellular dynamics, trans-endothelial electrical resistance (TEER), cell viability and growth, migration, and tubulogenesis were evaluated in human primary cultures of BMEC (hPBMEC) or in the human cerebral microvessel endothelial hCMEC/D3 cell line. Abundant TRPV2 expression was measured in hCMEC/D3 and hPBMEC by qRT-PCR, Western blotting, nontargeted proteomics, and cellular immunofluorescence studies. Intracellular Ca2+ levels were increased by heat and CBD and blocked by the nonspecific TRP antagonist ruthenium red (RR) and the selective TRPV2 inhibitor tranilast (TNL) or by silencing cells with TRPV2 siRNA. CBD dose-dependently induced the hCMEC/D3 cell number (EC50 0.3 ± 0.1 µM), and this effect was fully abolished by TNL or TRPV2 siRNA. A wound healing assay showed that CBD induced cell migration, which was also inhibited by TNL or TRPV2 siRNA. Tubulogenesis of hCMEC/D3 cells in 3D matrigel cultures was significantly increased by 41 and 73% after a 7 or 24 h CBD treatment, respectively, and abolished by TNL. CBD also increased the TEER of hPBMEC monolayers cultured in transwell, and this was blocked by TNL. Our results show that CBD, at extracellular concentrations close to those observed in plasma of patients treated by CBD, induces proliferation, migration, tubulogenesis, and TEER increase in human brain endothelial cells, suggesting CBD might be a potent target for modulating the human BBB.
Subject(s)
Brain Neoplasms/blood supply , Cannabidiol/pharmacology , Cell Movement/drug effects , Cell Proliferation/drug effects , Endothelial Cells/metabolism , Microvessels/pathology , TRPV Cation Channels/metabolism , Blood-Brain Barrier/metabolism , Calcium/metabolism , Cannabis/chemistry , Cell Line , Cell Survival/drug effects , Electric Impedance , Hot Temperature , Humans , Plant Extracts/pharmacology , Ruthenium Red/pharmacology , TRPV Cation Channels/antagonists & inhibitors , ortho-Aminobenzoates/pharmacologyABSTRACT
Targeted protein quantification using tandem mass spectrometry coupled to high performance chromatography (LC-MS/MS) has been used to quantify proteins involved in the absorption, distribution, metabolism and excretion (ADME) of xenobiotics to better understand these processes. At the blood-brain barrier (BBB), these proteins are particularly important for the maintenance of brain homeostasis, but also regulate the distribution of therapeutic drugs. Absolute quantification (AQUA) is achieved by using stable isotope labeled surrogate peptides specific to the target protein and analyzing the digested proteins in a triple-quadrupole mass spectrometer in multiple reaction monitoring (MRM) mode to achieve a high specificity, sensitivity, accuracy and reproducibility. The main objective in this work was to develop and validate an UHPLC-MS/MS method for quantification of the ATP-binding cassette (ABC) transporter proteins Bcrp and P-gp and Na+/K + ATPase pump at the BBB. Three isoforms of the α-subunit from this pump (Atp1a 1, 2 and 3) were quantified to evaluate the presence of non-endothelial cells in the BBB using one common and three isoform-specific peptides; while Bcrp ad P-gp were quantified using 2 and 3 peptides, respectively, to improve the confidence on their quantification. The protein digestion was optimized, and the analytical method was comprehensively validated according to the American Food and Drug Administration Bioanalytical Method Validation Guidance published in 2018. Linearity across four magnitude orders (0.125 to 510 pmol·mL-1) sub-pmol·mL-1 LOD and LOQ, accuracy and precision (deviation < 15% and CV < 15%) were proven for most of the peptides by analyzing calibration curves and four levels of quality controls in both a pure solution and a complex matrix of digested yeast proteins, to mimic the matrix effect. In addition, digestion performance and stability of the peptides was shown using standard peptides spiked in a yeast digest or mouse kidney plasma membrane proteins as a study case. The validated method was used to characterize mouse kidney plasma membrane proteins, mouse brain cortical vessels and rat brain cortical microvessels. Most of the results agree with previously reported values, although some differences are seen due to different sample treatment, heterogeneity of the sample or peptide used. Importantly, the use of three peptides allowed the quantification of P-gp in mouse kidney plasma membrane proteins which was below the limit of quantification of the previously NTTGALTTR peptide. The different levels obtained for each peptide highlight the importance and difficulty of choosing surrogate peptides for protein quantification. In addition, using isoform-specific peptides for the quantification of the Na+/K + ATPase pump, we evaluated the presence of neuronal and glial cells on rat and mouse brain cortical vessels in addition to endothelial cells. In mouse liver and kidney, only the alpha-1 isoform was detected.
Subject(s)
ATP-Binding Cassette Transporters/analysis , Blood-Brain Barrier/metabolism , Oligopeptides/chemistry , Proteomics/methods , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Animals , Carbon Isotopes , Cell Membrane/metabolism , Chromatography, High Pressure Liquid/instrumentation , Chromatography, High Pressure Liquid/methods , Isotopes , Kidney/cytology , Kidney/metabolism , Limit of Detection , Male , Mice , Mice, Inbred C57BL , Models, Animal , Nitrogen Isotopes , Protein Isoforms/analysis , Protein Isoforms/chemistry , Protein Stability , Proteomics/instrumentation , Rats , Rats, Sprague-Dawley , Reproducibility of Results , Tandem Mass Spectrometry/instrumentation , Tandem Mass Spectrometry/methodsABSTRACT
ATP-binding cassette (ABC) transporters are of major importance for the restricted access of toxins and drugs to the human body. At the body's barrier tissues like the blood-brain barrier, these transporters are highly represented. Especially, ABCB1 (P-glycoprotein) has been a priority target of pharmaceutical research, for instance, to aid chemotherapy of cancers, therapy resistant epilepsy, and lately even neurodegenerative diseases. To improve translational research, the humanization of mouse genes has become a popular tool although, like recently seen for Abcb1, not all approaches were successful. Here, we report the characterization of another unsuccessful commercially available ABCB1 humanized mouse strain. In vivo assessment of transporter activity using positron emission tomography imaging revealed a severe reduction of ABCB1 function in the brain of these mice. Analyses of brain mRNA and protein expression showed that the murine Abcb1a gene is still expressed in homozygous humanized animals while expression of the human gene is minimal. Promoter region analyses underpinned that the introduced human gene might dysregulate normal expression and provided insights into the regulation of both transcription and translation of Abcb1a. We conclude that insertion of the human coding DNA sequence (CDS) into exon 3 instead of exon 2 most probably represents a more promising strategy for Abcb1a humanization.
ABSTRACT
Liquid chromatography coupled to tandem mass spectrometry-based targeted absolute protein quantification (in fmol of the analyte protein per µg of total protein) is employed for the molecular characterization of the blood-brain barrier using isolated brain microvessels. Nevertheless, the heterogeneity of the sample regarding the levels of different cells co-isolated within the microvessels and bovine serum albumin (BSA) contamination (from buffers) are not always evaluated. We developed an unlabeled targeted liquid chromatography coupled to tandem mass spectrometry method to survey the levels of endothelial cells (ECs), astrocytes, and pericytes, as well as BSA contaminant in rat cortical microvessels. Peptide peak identities were evaluated using a spectral library and chromatographic parameters. Sprague-Dawley rat microvessels obtained on three different days were analyzed with this method complemented by an absolute quantification multiple reaction monitoring method for transporter proteins P-gp, Bcrp, and Na+ /K+ ATPase pump using stable isotope labeled peptides as internal standard. Inter-day differences in the cell markers and BSA contamination were observed. Levels of cell markers correlated positively between each other. Then, the correlation between cell marker proteins and transporter proteins was evaluated to choose the best EC marker protein for protein quantification normalization. The membrane protein Pecam-1 showed a very high correlation with the EC-specific transporter P-gp (Pearson product-moment correlation coefficient (r) > 0.89) and moderate to high with Bcrp (r ≥ 0.77), that can be found also in pericytes and astrocytes. Therefore, Pecam-1 was selected as a marker for the normalization of the quantification of the proteins of endothelial cells.
Subject(s)
Biological Transport/physiology , Biomarkers/metabolism , Blood-Brain Barrier/metabolism , Brain/metabolism , Membrane Transport Proteins/metabolism , Microvessels/metabolism , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Animals , Brain/blood supply , Endothelial Cells/metabolism , Male , Membrane Proteins/metabolism , Proteomics/methods , Rats, Sprague-Dawley , Tandem Mass Spectrometry/methodsABSTRACT
SCOPE: Trans-resveratrol is widely studied for its potentially beneficial effects on numerous disorders. It is rapidly metabolized and its metabolites can exhibit biological activity. The present study aimed to investigate whether acute or sustained trans-resveratrol administration impacted on the distribution of trans-resveratrol and its metabolites in brain, heart, and liver. METHODS AND RESULTS: We used ultra-HPLC quadrupole-TOF (UHPLC-Q-TOF) in a full-scan mode to identify and assess large numbers of resveratrol metabolites. For acute intake, mice were overfed with a single dose of trans-resveratrol (150 mg/kg) and organs were collected after 30 and 60 min. For sustained intake, trans-resveratrol was given in the chow (0.04% w/w corresponding to 40 mg/kg/day), and plasma and the organs were collected after 3 months of this resveratrol diet. We found that trans-resveratrol-3-O-glucuronide and resveratrol-3-sulfate were the main metabolites found after acute intake, and free trans-resveratrol (in the brain and heart) and dihydroresveratrol derivatives were found after sustained administration CONCLUSIONS: Our results show notable differences between acute and sustained administration of trans-resveratrol and distribution of trans-resveratrol and its metabolites in mouse heart, brain, and liver. The results suggest a strategy for development of galenic forms of resveratrol.
Subject(s)
Glucuronides/pharmacokinetics , Stilbenes/pharmacokinetics , Animals , Brain/drug effects , Brain/metabolism , Chromatography, High Pressure Liquid/methods , Heart/drug effects , Liver/drug effects , Liver/metabolism , Mass Spectrometry/methods , Mice , Resveratrol , Stilbenes/metabolism , Tissue DistributionABSTRACT
Breast cancer resistance protein (BCRP) is expressed in various tissues, such as the gut, liver, kidney and blood brain barrier (BBB), where it mediates the unidirectional transport of substrates to the apical/luminal side of polarized cells. Thereby BCRP acts as an efflux pump, mediating the elimination or restricting the entry of endogenous compounds or xenobiotics into tissues and it plays important roles in drug disposition, efficacy and safety. Bcrp knockout mice (Bcrp(-/-)) have been used widely to study the role of this transporter in limiting intestinal absorption and brain penetration of substrate compounds. Here we describe the first generation and characterization of a mouse line humanized for BCRP (hBCRP), in which the mouse coding sequence from the start to stop codon was replaced with the corresponding human genomic region, such that the human transporter is expressed under control of the murineBcrppromoter. We demonstrate robust human and loss of mouse BCRP/Bcrp mRNA and protein expression in the hBCRP mice and the absence of major compensatory changes in the expression of other genes involved in drug metabolism and disposition. Pharmacokinetic and brain distribution studies with several BCRP probe substrates confirmed the functional activity of the human transporter in these mice. Furthermore, we provide practical examples for the use of hBCRP mice to study drug-drug interactions (DDIs). The hBCRP mouse is a promising model to study the in vivo role of human BCRP in limiting absorption and BBB penetration of substrate compounds and to investigate clinically relevant DDIs involving BCRP.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Neoplasm Proteins/metabolism , Xenobiotics/pharmacokinetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/antagonists & inhibitors , ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/genetics , Animals , Biological Availability , Biotransformation/drug effects , Blood-Brain Barrier/drug effects , Blood-Brain Barrier/metabolism , Drug Interactions , Female , Gene Expression Regulation/drug effects , Gene Knock-In Techniques , Humans , Intestinal Absorption/drug effects , Male , Membrane Transport Modulators/blood , Membrane Transport Modulators/metabolism , Membrane Transport Modulators/pharmacokinetics , Membrane Transport Modulators/pharmacology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/chemistry , Neoplasm Proteins/genetics , Promoter Regions, Genetic/drug effects , RNA, Messenger/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Tissue Distribution/drug effects , Xenobiotics/blood , Xenobiotics/metabolism , Xenobiotics/pharmacologyABSTRACT
OBJECTIVES: Chronic increases in blood flow in resistance arteries induce outward remodeling associated with increased wall thickness and endothelium-mediated dilatation. This remodeling is essential for collateral arteries growth following occlusion of a large artery. As estrogens have a major role in this remodeling, we hypothesized that resveratrol, described as possessing phytoestrogen properties, could improve remodeling in ovariectomized rats. METHODS: Blood flow was increased in vivo in mesenteric arteries after ligation of adjacent arteries in 3-month old ovariectomized rats treated with resveratrol (5 or 37.5 mg/kg per day: RESV5 or RESV37.5) or vehicle. After 2 weeks arterial structure and function were measured in vitro in high flow (HF) and normal flow (NF) arteries isolated from each rat. RESULTS: Arterial diameter was greater in HF than in NF arteries in ovariectomized rats treated with RESV5 or RESV37.5, not in vehicle-treated rats. In mice lacking estrogen receptor alpha diameter was equivalent in HF and NF arteries whereas in mice treated with RESV5 diameter was greater in HF than in NF vessels. A compensatory increase in wall thickness and a greater phenylephrine-mediated contraction were observed in HF arteries. This was more pronounced in HF arteries from RESV37.5-treated rats. ERK1/2 phosphorylation, involved in hypertrophy and contraction, were higher in RESV37.5-treated rats than in RESV5- and vehicle-treated rats. Endothelium-dependent relaxation was greater in HF than in NF arteries in RESV5-treated rats only. In HF arteries from RESV37.5-treated rats relaxation was increased by superoxide reduction and markers of oxidative stress (p67phox, GP91phox) were higher than in the 2 other groups. CONCLUSION: Resveratrol improved flow-mediated outward remodeling in ovariectomized rats thus providing a potential therapeutic tool in menopause-associated ischemic disorders. This effect seems independent of the estrogen receptor alpha. Nevertheless, caution should be taken with high doses inducing excessive contractility and hypertrophy in association with oxidative stress in HF arteries.
Subject(s)
Atrial Remodeling/drug effects , Mesenteric Arteries/drug effects , Stilbenes/pharmacology , Vasodilator Agents/pharmacology , Animals , Endothelium, Vascular/metabolism , Female , Membrane Glycoproteins/metabolism , Mesenteric Arteries/physiology , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Muscle Contraction/drug effects , NADH, NADPH Oxidoreductases/metabolism , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Nitric Oxide Synthase Type III/metabolism , Ovariectomy , Oxidative Stress/drug effects , Phenylephrine/pharmacology , Phosphorylation/drug effects , Rats , Rats, Wistar , Resveratrol , Stilbenes/analysis , Superoxides/metabolism , Vasodilator Agents/analysisABSTRACT
Chronic morphine regimen increases P-glycoprotein (P-gp) and breast cancer-resistance protein (Bcrp) expressions at the rat bloodbrain barrier (BBB) but what drives this effect is poorly understood. The objective of this study is to assess subchronic continuous morphine infusion and naloxone-precipitated morphine withdrawal effects on P-gp/Bcrp contents and activities at the rat BBB. Rats were treated either with (i) a continuous i.v. morphine for 120 h, (ii) escalating morphine dosing (10-40 mg/kg, i.p., 5 days), (iii) a chronic morphine regimen (10 mg/kg s.c., 5 days) followed by a withdrawal period (2 days) and treatment for 3 additional days. Animal behavior was assessed after naloxone-precipitated withdrawal (1 mg/kg, s.c.). P-gp/Bcrp expressions and activities were determined in brain microvessels by qRT-PCR, Western blot, UHPLCMS/MS, and in situ brain perfusion of P-gp or Bcrp substrates. Results show continuous i.v. morphine did not change P-gp/Bcrp protein levels in rat brain microvessels, whereas naloxone-precipitated withdrawal after escalating or chronic morphine dose regimen increased Mdr1a and Bcrp mRNA levels by 1.4-fold and 2.4-fold, respectively. Conversely, P-gp/Bcrp protein expressions remained unchanged after naloxone administration, and brain uptake of [3H]-verapamil (P-gp) and [3H]-mitoxantrone (Bcrp) was not altered. The study concludes subchronic morphine infusion and naloxone-precipitated morphine withdrawal have poor effect on P-gp/Bcrp levels at the rat BBB.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/metabolism , ATP-Binding Cassette Transporters/metabolism , Analgesics, Opioid/pharmacology , Blood-Brain Barrier/metabolism , Morphine/pharmacology , Naloxone/pharmacology , Narcotic Antagonists/pharmacology , Substance Withdrawal Syndrome/metabolism , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , ATP Binding Cassette Transporter, Subfamily G, Member 2 , ATP-Binding Cassette Transporters/genetics , Analgesics, Opioid/administration & dosage , Analgesics, Opioid/blood , Animals , Blood-Brain Barrier/drug effects , Cerebral Cortex/drug effects , Cerebral Cortex/metabolism , Infusions, Intravenous , Male , Morphine/administration & dosage , Morphine/blood , Proteomics , Rats , Rats, Sprague-DawleyABSTRACT
BACKGROUND: Pregnancy-associated placental protein-A (PAPP-A) is a metalloprotease which circulates as an hetero-tetramer in maternal blood. Its maternal serum concentration in fetal trisomy 21 is decreased during the first trimester, so that PAPP-A is a useful screening biomarker. However, the regulation of PAPP-A placental secretion is unclear. We therefore investigated the secretion of PAPP-A in pregnancies complicated by fetal aneuploidies, both in vivo and in vitro. METHODS: Maternal serum collected between 10 WG and 33 WG during 7014 normal pregnancies and 96 pregnancies complicated by fetal trisomy 21, 18, and 13 were assayed for PAPP-A using the Immulite 2000xpi system®. The pregnancies were monitored using ultrasound scanning, fetal karyotyping and placental analysis. Villous cytotrophoblasts were isolated from normal and trisomic placenta and cultured to investigate PAPP-A secretion in vitro (n=6). RESULTS: An increased nuchal translucency during the first trimester is a common feature of many chromosomal defect but each aneuploidy has its own syndromic pattern of abnormalities detectable at the prenatal ultrasound scanning and confirmed at the fetal examination thereafter. PAPP-A levels rise throughout normal pregnancy whereas in trisomy 21, PAPP-A levels were significantly decreased, but only during the first trimester. PAPP-A levels were decreased in trisomy 13 and sharply in trisomy 18, whatever the gestational age. In vitro, PAPP-A secretion was decreased in aneuploidy, and associated with decreased hCG secretion in Trisomy 21 and 18. These biochemical profiles did not appear to be linked to any specific histological lesions affecting the placenta. CONCLUSIONS: These profiles may reflect different quantitative and qualitative placental dysfunctions in the context of these aneuploidies.
Subject(s)
Fetal Diseases/genetics , Pregnancy Complications , Pregnancy-Associated Plasma Protein-A/metabolism , Trisomy/genetics , Cells, Cultured , Chorionic Gonadotropin/blood , Chorionic Gonadotropin/metabolism , Chromosome Disorders/genetics , Chromosomes, Human, Pair 13/genetics , Chromosomes, Human, Pair 18/genetics , Cohort Studies , Down Syndrome/genetics , Female , Fetal Diseases/diagnosis , Fetal Diseases/diagnostic imaging , Gestational Age , Humans , Nuchal Translucency Measurement/methods , Placenta/cytology , Placenta/metabolism , Pregnancy , Trisomy/diagnosis , Trisomy 13 Syndrome , Trophoblasts/cytology , Trophoblasts/metabolismABSTRACT
The grey mouse lemur (Microcebus murinus) is a non-human primate used to study the ageing process. Resveratrol is a polyphenol that may increase lifespan by delaying age-associated pathologies. However, no information about resveratrol absorption and metabolism is available for this primate. Resveratrol and its metabolites were qualitatively and quantitatively analyzed in male mouse-lemur plasma (after 200 mg.kg-1 of oral resveratrol) by ultra-high performance liquid chromatography (UHPLC), coupled to a quadrupole-time-of-flight (Q-TOF) mass spectrometer used in full-scan mode. Data analyses showed, in MSE mode, an ion common to resveratrol and all its metabolites: m/z 227.072, and an ion common to dihydro-resveratrol metabolites: m/z 229.08. A semi-targeted study enabled us to identify six hydrophilic resveratrol metabolites (one diglucurono-conjugated, two monoglucurono-conjugated, one monosulfo-conjugated and two both sulfo- and glucurono-conjugated derivatives) and three hydrophilic metabolites of dihydro-resveratrol (one monoglucurono-conjugated, one monosulfo-conjugated, and one both sulfo- and glucurono-conjugated derivatives). The presence of such metabolites has been already detected in the mouse, rat, pig, and humans. Free resveratrol was measurable for several hours in mouse-lemur plasma, and its two main metabolites were trans-resveratrol-3-O-glucuronide and trans-resveratrol-3-sulfate. Free dihydro-resveratrol was not measurable whatever the time of plasma collection, while its hydrophilic metabolites were present at 24 h after intake. These data will help us interpret the effect of resveratrol in mouse lemurs and provide further information on the inter-species characteristics of resveratrol metabolism.