腺苷受体打开血脑屏障

Cellular/Molecular Adenosine Receptor Signaling Modulates Permeability of the Blood–Brain Barrier Aaron J. Carman, Jeffrey H. Mills, Antje Krenz, Do-Geun Kim, andMargaret S. Bynoe Department of Microbiology and Immunology, Cornell University, College of Veterinary Medicine, Ithaca, New York 14853 The blood–brain barrier (BBB) is comprised of specialized endothelial cells that form the capillary microvasculature of the CNS and is essential for brain function. It also poses the greatest impediment in the treatment of many CNS diseases because it commonly blocks entry of therapeutic compounds. Here we report that adenosine receptor (AR) signalingmodulates BBB permeability in vivo. A1 andA2A ARactivation facilitated the entry of intravenously administeredmacromolecules, including large dextrans andantibodies to�-amyloid, into murine brains. Additionally, treatment with an FDA-approved selective A2A agonist, Lexiscan, also increased BBB permeability in murinemodels. These changes in BBB permeability are dose-dependent and temporally discrete. Transgenicmice lacking A1 or A2A ARs showeddiminisheddextran entry into thebrain afterARagonism.Following treatmentwith abroad-spectrumARagonist, intravenously administered anti-�-amyloid antibody was observed to enter the CNS and bind �-amyloid plaques in a transgenic mouse model of Alzheimer’s disease (AD). Selective AR activation resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules. These results suggest that AR signaling can be used tomodulate BBB permeability in vivo to facilitate the entry of potentially therapeutic compounds into the CNS. AR signaling at brain endothelial cells represents a novel endogenous mechanism of modulating BBB permeability. We anticipate these results will aid in drug design, drug delivery and treatment options for neurological diseases such as AD, Parkinson’s disease, multiple sclerosis and cancers of the CNS. Introduction The blood–brain barrier (BBB) is comprised of brain endothelial cells (BECs), which form the lumen of the brain microvascula- ture (Abbott et al., 2010). The barrier function is achieved through tight junctions between endothelial cells that regulate the extravasation of molecules and cells into and out of the CNS (Abbott et al., 2010). Although the BBB serves to restrict the entry of potentially toxic substances into the CNS, it poses a tremen- dous hurdle to the delivery of therapeutic drugs into the CNS. It has been estimated that�98% of small-molecule drugs�500 Da in size do not cross the BBB (Pardridge, 2001, 2005). Current approaches aimed at altering the BBB to permit the entry of ther- apeutics are either too invasive, too painful, can result in perma- nent brain damage or result in loss of drug efficacy (Hanig et al., 1972; Broadwell et al., 1982; Rapoport, 2001; Bidros and Vogel- baum, 2009; Hynynen, 2010). There is a monumental need to modulate the BBB to facilitate the entry of therapeutic drugs into the CNS. Determining how to safely and effectively do this could greatly benefit a broad range of neurological diseases, such as Alzheimer’s disease (AD), Parkinson’s disease, multiple sclerosis, neurological manifestations of acquired immune deficiency syn- drome (AIDS), CNS cancers, and many more. Promising thera- pies are available to treat some of these disorders, but their potential cannot be fully realized due to the tremendous imped- iment posed by a functional BBB. Here, we provide novel data demonstrating that signaling through receptors for the purine nucleoside adenosine acts as a potent, endogenous modulator of BBB permeability. It is well established that adenosine has many diverse roles in mammalian physiology, including immunomodulatory roles regulating immune cell responses (Bours et al., 2006; Kobie et al., 2006; Deaglio et al., 2007) and roles in proper CNS functioning (Sebastia˜o and Ribeiro, 2009; Stone et al., 2009). The first clues to adenosine’s involvement in CNS barrier permeability came from our recent findings demonstrating that extracellular adenosine, produced by the catalytic action of CD73 (a 5�-ectonucleotidase) from AMP, promotes lymphocyte entry into the CNS in experi- mental autoimmune encephalomyelitis (EAE) (Mills et al., 2008). These studies demonstrated that mice lacking CD73 (Thompson et al., 2004), which are unable to produce extracel- lular adenosine, are protected from EAE and that blockade of the A2A adenosine receptor (AR) inhibits T cell entry into the CNS (Mills et al., 2008). These observations led us to hypothesize that modulation of AR signaling at BECs might modulate BBB per- meability to facilitate the entry of molecules and cells into the CNS. Indeed, our results suggest that AR signaling represents a novel, endogenous modulator of BBB permeability. Received June 30, 2011; revised July 26, 2011; accepted July 28, 2011. Author contributions: A.J.C., J.H.M., M.S.B., and A.K. designed research; A.J.C., J.H.M., M.S.B., A.K., and D.-G.K. performed research; A.J.C., J.H.M., M.S.B., A.K., and D.-G.K. analyzed data; A.J.C., J.H.M., M.S.B., and A.K. wrote the paper. Thisworkwas supportedbyNational Institutes of HealthGrants R01NS063011 (toM.S.B.) and F32NS066682 (to J.H.M.).WeacknowledgeDr. Chris Schaffer of Cornell University for theAD transgenicmice, Dr. HelenMarquis for her critical reading of the manuscript, and Delbert Abi-Abdallah for help with Western blotting. We also acknowledge Adenios, Inc. for their kind gift of the anti-�-amyloid antibody. The authors declare no competing financial interests. Correspondence should be addressed to Margaret S. Bynoe at the above address. E-mail: msb76@cornell.edu. DOI:10.1523/JNEUROSCI.3337-11.2011 Copyright © 2011 the authors 0270-6474/11/3113272-09$15.00/0 13272 • The Journal of Neuroscience, September 14, 2011 • 31(37):13272–13280 Materials andMethods Mouse and rat models. C57BL/6 mice (Jackson Laboratories) were used as WT. A1 �/� AR mice were a gift from Dr. Jurgen Schnermann (NIH/NIDDK, Bethesda, MD) (Sun et al., 2001). A2A �/� AR were a gift from Dr. Jiang-Fan Chen (Boston University School of Medicine, Boston, MA) (Chen et al., 1999). The transgenic AD mice [B6.Cg- Tg(APPswe,PSEN1dE9)85Dbo/J] were a gift fromDr. Chris Schaffer (Cor- nell University, Ithaca, NY) (Jankowsky et al., 2004). Typically, mice were aged 7–9 weeks and weighed between 20 and 25 g. Sprague Dawley rats (Charles River Laboratories) were female, aged 8 weeks and weighed 200– 220 g. Animals were bred and housed under specific pathogen-free condi- tions at Cornell University, Ithaca, NY. All procedures were done in accordance with approved Institutional Animal Care and Use Committee protocols. Administration of drugs and tissue collection. NECA [1-(6-amino- 9H-purin-9-yl)-1-deoxy-N-ethyl-�-D-ribofuranuronamide], CCPA (2-chloro-N6-cyclopentyladenosine), CGS 21680 (4-[2-[[6-amino-9-(N- ethyl-b-D-ribofuranuronamidosyl)-9H-purin-2 yl]amino]ethyl] benzenepropanoic acid), and SCH 58261 (5-amino-7(phenylethyl)-2-(2- furyl)-pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]-pyrimidine) (Tocris Biosci- ence) were each dissolved in DMSO then diluted in PBS to the desired concentration; inmost cases finalDMSOconcentrationswere�0.5% (v/v). Lexiscan (regadenoson; TorontoResearchChemicals)was dissolved in PBS. For vehicle controls, DMSO was diluted in PBS to the same concentration. Dextrans labeled with either FITC or Texas Red (Invitrogen) were sus- pended in PBS to 10 mg/ml. Experiments involving dextran injection used 1.0 mg of dextran in PBS. When drug and dextran were injected concomi- tantly, 1.0 mg of dextran wasmixed with the drug to the desired concentra- tion ina final volumeof200�l.All injections except injectionsof SCH58261 were retro-orbital intravenous. Lexiscan was administered intravenously with 3 injections, 5 min apart, and tissues were collected at 15 min unless otherwise indicated. Indose–responseexperiments,drugsanddextranswere injectedconcomitantly. SCH58261 injections,1mg/kg,were intraperitoneal andmice were predosed with this concentration daily for 4 d before the day of the experiment. An additional injection was administered at the time of theexperiment.At indicated timesmicewereanesthetizedandperfusedwith cold PBS through the left ventricle of the heart. Brains were weighed and frozen for later analysis. Fluorimetric analysis.Tris-Cl, 50mM, pH 7.6, was added to brains (100 �l per 100 mg brain). Brains were homogenized with a Dounce homog- enizer and centrifuged at 16.1� g for 30 min. Supernatants were trans- ferred to new tubes and an equal volume absolute methanol was added. Samples were centrifuged at 16.1 � g for 30 min. Supernatant (200 �l) was transferred to a Corning Costar 96 well black polystyrene assay plate (clear bottom). A series of standards containing 0.001–10�g/ml dextran in 50% Tris-Cl/50% absolute methanol (v/v) was added to each plate. Absolute concentrations of dextrans were derived from these standard curves. Fluorimetric analysis was performed on a BioTek Synergy 4. Primary brain endothelial cell isolation. This method has been adapted from previously described techniques (Song and Pachter, 2003). Briefly, 12-week-old C57BL/6mice were killed and decapitated. Dissected brains were freed from the cerebellumand large surface vessels were removed by carefully rolling the brains on sterileWhatmanpaper. The tissuewas then homogenized in a Dounce homogenizer in ice-cold DMEM-F12 me- dium, supplemented with L-glutamine and Pen/Strep, and the resulting homogenate was centrifuged at 3800� g, 4°C for 5min. After discarding the supernatant, the pellet was resuspended in 18% (w/v) dextran in PBS solution, vigorously mixed, and centrifuged at 10,000 � g, 4°C for 10 min. The foamy myelin layer was carefully removed with the dextran supernatant by gentle aspiration. The pellet was resuspended in pre- warmed (37°C) digestionmedium (DMEM supplemented with 1mg/ml collagenase/dispase, 40 �g/ml DNase I, and 0.147 �g/ml of the protease inhibitor tosyllysine chloromethyl ketone) and incubated at 37°C for 75 min with occasional agitation. The suspension was centrifuged at 3800� g. The supernatant was discarded; the pellet was resuspended in prewarmed (37°C) PBS and centrifuged at 3800 � g. The pellet was suspended in full medium (DMEM-F12 medium containing 10% plasma-derived serum, L-glutamine, 1%antibiotic-antimycotic, 100mg/mlheparin, and100mg/ml endothelial cell growth supplement). The resulting capillary fragments were plated onto tissue culture dishes coatedwithmurine collagen IV (50�g/ml) at adensity corresponding toonebrainper9.5 cm2.Mediumwasexchanged after24and48h.Puromycin(8�g/ml)wasaddedto themediumfor the first 2 d. Before analysis, the primary mouse brain endothelial cells were grown until the culture reached complete confluence after 5–7 d in vitro. Cell culture and quantitative reverse transcription PCR. Bend.3 mouse BECs (ATCC) were grown in ATCC-formulated DMEM supplemented with 10% FBS. Using TRIzol (Invitrogen), RNA was isolated. cDNA was synthesized using Multiscribe reverse transcriptase (Applied Biosys- tems). Primers (available upon request) for ARs and CD73 were used to determine gene expression and standardized to TBP gene levels using Kapa Sybr Fast (Kapa Biosystems) run on a Bio-Rad CFX96 real-time quantitative PCR (qPCR) system.Melt curve analyses were performed to measure the specificity for each qPCR product. Adenosine receptor Western blotting and immunofluorescent analysis. Primary mouse brain endothelial cells and Bend.3 cell cultures were grown as described above. Cells were lysed with 1 ml of lysis buffer containing protease inhibitor and condensed with TCA solution up to 200 �l. Samples were run on a 12% SDS-PAGE and transferred to nitro- cellulose paper.Membraneswere blockedwith 1%polyvinyl pyrrolidone and incubated with anti-A1 AR (AAR-006) and -A2A AR (AAR-002) primary antibodies (Alomone Labs) overnight. The membranes were washed and then incubated with goat anti-rabbit HRP.Membranes were washed thoroughly and developed with ECL solution and exposed to x-ray film. For adenosine receptor immunostaining, anesthetized mice were perfused with PBS and brains were isolated and snap frozen in Tissue Tek-OCT medium. Sections (5 �m; brains in a sagittal orienta- tion) were affixed to Superfrost/Plus slides (Fisher Scientific), fixed in acetone, and stored at �80°C. Slides were thawed, washed in PBS, blocked with casein (Vector Laboratories) in normal goat serum (Zymed), and then incubated with anti-CD31 (MEC 13.3, BD Biosci- ences) and anti-A1 AR (A4104, Sigma) or anti-A2A AR (AAR-002, Alo- mone Labs). Slides were then incubated with goat anti-rat Ig Alexa Fluor 488 (Invitrogen) and goat anti-rabbit Ig Texas Red-X (Invitrogen). Sec- tions were mounted with Vectashield mounting medium with DAPI (Vector Laboratories). Images were obtained on a Zeiss Axio Imager M1 fluorescent microscope. Fluorescence in situ hybridization. For detection of adenosine receptor mRNA in brain endothelium, we performed fluorescence in situ hybrid- ization (FISH) using FITC-labeled CD31 and either biotin-labeled A1 or A2A DNA oligonucleotide probes (Integrated DNA Technologies, probe sequences available upon request). Anesthetizedmicewere perfusedwith PBS and brains were isolated and snap frozen in Tissue Tek-OCT me- dium. Twelve micrometer cryosections were mounted on Superfrost/ Plus slides (Fisher Scientific). After air drying on the slides for 30min, the tissue was fixed in 4% neutral buffered PFA for 20 min and rinsed for 3 min in 1� PBS. Next, the tissue was equilibrated briefly in 0.1 M trietha- nolamine and acetylated for 10 min in 0.1 M triethanolamine with 0.25% acetic anhydride. Immediately following acetylation, the sections were dehydrated through an ascending ethanol series, and stored at room temperature. The tissue was rehydrated for 2 � 15 min in PBS, and equilibrated for 15min in 5� SSC (0.75 MNaCl, 0.075 MNa-citrate). The sections were then prehybridized for 1 h at 42°C in hybridization buffer (50%deionized formamide, 4� SSC, 40�g/ml salmon spermDNA, 20% (w/v) dextran sulfate, 1�Denhardt’s solution). The probes (300 ng/ml) were denatured for 3 min at 80°C and added to the prewarmed (42°C) buffer (hybridizationmix). The hybridization reaction was performed at 42°C for 38 hwith 250�l of hybridizationmix on each slide, coveredwith Parafilm. Prehybridization and hybridization were performed in a black box saturated with a 4� SSC-50% formamide solution to avoid evapo- ration and photobleaching of FITC. After incubation, the sections were washed for 30 min in 2� SSC (room temperature), 15 min in 2� SSC (65°C), 15min in 0.2� SSC, 0.1%SDS (65°C), and equilibrated for 5min in PBS. For detection of the biotin-probes, sectionswere incubated for 30 min at room temperature with Texas Red X-conjugated streptavidin (Invitrogen, S6370, 1 �g/ml) in PBS containing 1� casein (Vector Lab- oratories). Excess streptavidin was removed by 15 min in PBS, followed by 15 min in 0.2� SSC, 0.1% SDS (65°C), and 15 min in PBS washes. Carman et al. • Adenosine Alters Blood–Brain Barrier Permeability J. Neurosci., September 14, 2011 • 31(37):13272–13280 • 13273 Sections were coverslipped with Vectashield mounting medium with DAPI (Vector Laboratories). Images were acquired using a Zeiss Axio Imager M1 fluorescent microscope. Injection of anti-�-amyloid antibodies and immunofluorescent micros- copy.WT and transgenic (AD) mice were given 0.08 mg/kg NECA (i.v.). After 3 h, 400 �g of antibody to �-amyloid (200 �l of 2 mg/ml; clone 6E10, Covance) was administered intravenously and the mice rested for 90 min. Mice were anesthetized and perfused (as described above) and brains were placed in OTC and flash-frozen for sectioning. Sagittal sec- tions (6 �m) were fixed in acetone, washed in PBS, blocked with casein and incubated with goat anti-mouse Ig Cy5 (Abcam), and then washed with PBS. Sections were mounted with Vectashield Hardset mounting medium with DAPI (Vector Laboratories). Images were obtained on a Zeiss Axio Imager M1 fluorescent microscope. Transendothelial cell electrical resistance assays.Bend.3 cells were grown in ATCC-formulated DMEM supplemented with 10% FBS on 24-well Transwell inserts, 8 �m pore size (BD Falcon, BD Biosciences) until a monolayer was established. Transendothelial cell electrical resistance (TEER) was assessed using a Voltohmmeter (EVOMX, World Precision Instruments). Background resistance from unseeded Transwells was subtracted from recorded values to determine absolute TEER values. Change in absolute TEER from time 0 (t0) for each individual Transwell was expressed as percentage change and then averaged for each treatment group. F-actin staining of endothelial cells. Bend.3 cells were grown (as de- scribed above) on circular coverslips in 24-well plates. Cells were treated for 3 or 30 min with 1 �M CCPA, 1 �M Lexiscan, DMSO or media alone. Coverslips were washed with PBS, fixed in 4% paraformaldehyde, washed again in PBS and then permeabilized with 0.5% Triton X-100 in PBS. After washing in PBS/1% BSA, coverslips were blocked with 1% BSA then stained with phalloidin-Alexa Fluor 568. Coverslips were washed and mounted on slides with ProlongGold containing DAPI (In- vitrogen). Images were obtained on an Olympus BX51 fluorescent microscope. Albumin uptake assay. Bend.3 cells grown on collagen-coated cover- slips were incubated with albumin-Alexa Fluor 594 (50mg/ml) (Invitro- gen) and either medium alone, DMSO vehicle, NECA (1 �M), or Lexiscan (1 �M) for 30 min. Albumin uptake was visualized (albumin� red) using the Zeiss Axio ImagerM1 fluorescent microscope. Total albu- min fluorescence was recorded using Zeiss AxioVision software, and measured using ImageJ (NIH) software. Tight junction molecule staining. Bend.3 cells grown on collagen- coated coverslips were incubated with DMSO vehicle, NECA (1 �M), or Lexiscan (1 �M) for 1 h. Cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton-X in PBS. Cells were blockedwith PBS/BSA/goat serum and then stainedwith antibodies (Invitrogen) against either ZO-1 (1A12), Claudin-5 (34-1600), orOcclu- din (3F10). Following a wash step, cells were incubated with either goat anti-rabbit Ig Texas Red-X or goat anti-mouse Ig Cy5 (Invitrogen). Cov- erslips werewashed andmounted on slides with ProlongGold containing DAPI. Images were obtained on a Zeiss Axio Imager M1 fluorescent microscope. Statistical analyses. Statistical differences, assessed using the Student’s t test, are indicated where p� 0.05. Results The broad-spectrum AR agonist NECA increases BBB permeability to macromolecules We established that intravenous administration of NECA, which activates all ARs (A1, A2A, A2B, A3), resulted in a dose-dependent increase in extravasation of intravenously administered fluores- cently labeled dextrans into the CNS of mice (Fig. 1). Impor- tantly, varying the dose of NECA resulted in dose-dependent increases in CNS entry of both 10 kDa dextrans (Fig. 1A) and 70 kDadextrans (Fig. 1B) comparedwith treatment of vehicle alone. Maximum entry of dextrans into the CNS occurred with 0.08 mg/kg NECA. Higher concentrations of NECA had no additional effect or show diminished efficacy, possibly due to receptor desensi- tization (Ferguson et al., 2000). These results demonstrate that AR activation increases BBB permeability. We next determined the duration and kinetics of increased BBB permeability after NECA administration. In time course ex- periments using the maximum effective dose of NECA deter- mined by our dose–response experiments (0.08 mg/kg), we observed that increased barrier permeability following NECA treatment is temporally discrete (Fig. 1C), with maximum entry of labeled dextran into the CNS between 4 and