Functional and targeted proteomics characterization of a human primary endothelial cell model of the blood-brain barrier (BBB) for drug permeability studies.

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.

Exposure of human cerebral microvascular endothelial cells hCMEC/D3 to laminar shear stress induces vascular protective responses.

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.

Characterization of the Blood-Brain Barrier Integrity and the Brain Transport of SN-38 in an Orthotopic Xenograft Rat Model of Diffuse Intrinsic Pontine Glioma.

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.

An Interspecies Molecular and Functional Study of Organic Cation Transporters at the Blood-Brain Barrier: From Rodents to Humans.

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.

ABC Transporters at the Blood-Brain Interfaces, Their Study Models, and Drug Delivery Implications in Gliomas.

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.

LC-MS/MS-based quantification of efflux transporter proteins at the BBB.

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.

Targeted unlabeled multiple reaction monitoring analysis of cell markers for the study of sample heterogeneity in isolated rat brain cortical microvessels.

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.

Distribution of trans-resveratrol and its metabolites after acute or sustained administration in mouse heart, brain, and liver.

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.

Resveratrol metabolism in a non-human primate, the grey mouse lemur (Microcebus murinus), using ultra-high-performance liquid chromatography-quadrupole time of flight.

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.

Detection of 28 neurotransmitters and related compounds in biological fluids by liquid chromatography/tandem mass spectrometry.

This work presents two liquid chromatography/tandem mass spectrometry (LC/MS/MS) acquisition modes: multiple reaction monitoring (MRM) and neutral loss scan (NL), for the analysis of 28 compounds in a mixture. This mixture includes 21 compounds related to the metabolism of three amino acids: tyrosine, tryptophan and glutamic acid, two pterins and five deuterated compounds used as internal standards. The identification of compounds is achieved using the retention times (RT) and the characteristic fragmentations of ionized compounds. The acquisition modes used for the detection of characteristic ions turned out to be complementary: the identification of expected compounds only is feasible by MRM while expected and unexpected compounds are detected by NL. In the first part of this work, the fragmentations characterizing each molecule of interest are described. These fragmentations are used in the second part for the detection by MRM and NL of selected compounds in mixture with and without biological fluids. Any preliminary extraction precedes the analysis of compounds in biological fluids.