Hypoxia Disrupts the Barrier Function of Neural Blood Vessels through Changes in the Expression of Claudin-5 in Endothelial Cells
2007; Elsevier BV; Volume: 170; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.060693
ISSN1525-2191
AutoresTakashi Koto, Keiyo Takubo, Susumu Ishida, Hajime Shinoda, Makoto Inoue, Kazuo Tsubota, Yasunori Okada, Eiji Ikeda,
Tópico(s)Neonatal and fetal brain pathology
ResumoThe mechanisms underlying the hypoxia-induced disruption of the barrier function of neural vasculature were analyzed with reference to the expression of claudin-5, a component of tight junctions between neural endothelial cells. The movement of claudin-5 from the cytoplasm to the plasma membrane of cultured confluent brain-derived endothelial (bEND.3) cells was closely correlated with the increase in the transendothelial electrical resistance. Inhibition of the expression of claudin-5 by RNAi resulted in a reduction of transendothelial electrical resistance, indicating a critical role of claudin-5 in the barrier property. Hypoxia (1% O2) altered the location of claudin-5 in the plasma membrane and the level of claudin-5 protein in bEND.3 cells, and these changes were accompanied by a decrease in the transendothelial electrical resistance. In vivo the claudin-5 molecules were expressed under normoxia in the plasma membrane of retinal microvascular endothelial cells but were significantly reduced under hypoxic conditions. Tracer experiments revealed that the barrier function of hypoxic retinal vasculature with depressed claudin-5 expression was selectively disrupted against small molecules, which is very similar to the phenotype of claudin-5-deficient mice. These in vitro and in vivo data indicate that claudin-5 is a target molecule of hypoxia leading to the disruption of the barrier function of neural vasculature. The mechanisms underlying the hypoxia-induced disruption of the barrier function of neural vasculature were analyzed with reference to the expression of claudin-5, a component of tight junctions between neural endothelial cells. The movement of claudin-5 from the cytoplasm to the plasma membrane of cultured confluent brain-derived endothelial (bEND.3) cells was closely correlated with the increase in the transendothelial electrical resistance. Inhibition of the expression of claudin-5 by RNAi resulted in a reduction of transendothelial electrical resistance, indicating a critical role of claudin-5 in the barrier property. Hypoxia (1% O2) altered the location of claudin-5 in the plasma membrane and the level of claudin-5 protein in bEND.3 cells, and these changes were accompanied by a decrease in the transendothelial electrical resistance. In vivo the claudin-5 molecules were expressed under normoxia in the plasma membrane of retinal microvascular endothelial cells but were significantly reduced under hypoxic conditions. Tracer experiments revealed that the barrier function of hypoxic retinal vasculature with depressed claudin-5 expression was selectively disrupted against small molecules, which is very similar to the phenotype of claudin-5-deficient mice. These in vitro and in vivo data indicate that claudin-5 is a target molecule of hypoxia leading to the disruption of the barrier function of neural vasculature. Homeostasis of the microenvironment is essential for the normal functioning of the nervous system, and it is maintained in part by the blood-brain barrier and blood-retinal barrier. These barriers are formed by the endothelial cells of neural tissue-specific vasculature. 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Here, we demonstrate that the expression and location of claudin-5 in neural microvascular endothelial cells is altered by tissue hypoxia resulting in a breakdown of the neural endothelial barrier. A mouse brain endothelial cell line, bEND.3, was obtained from the American Type Culture Collection (Manassas, VA) and cultured in fibronectin-coated culture dishes (BD Biosciences, Franklin Lakes, NJ) or on fibronectin-coated cell inserts with 0.4-μm pore size (BD Biosciences). The culture medium was Dulbecco's modified Eagle's medium (4500 mg/L glucose) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 10 μg/ml streptomycin. The cells were incubated with a CO2 level of 5% either with 20% O2 (atmospheric air) for normoxia or with 1% O2 balanced with N2 for hypoxia. The hypoxic condition was generated in an oxygen-regulated incubator (Personal Multi Gas Incubator; Astec, Tokyo, Japan). All animal experiments adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Laboratory Animal Care and Use Committee at the School of Medicine, Keio University. Adult male C57BL/6J mice (7 to 10 weeks old; CLEA Japan, Tokyo, Japan) were maintained in a chamber in which the O2 concentration could be regulated by controlling the inflow rates of O2 and N2. For hypoxic condition, the O2 concentration was maintained at 7 to 9% as described,32Yu AY Shimoda LA Iyer NV Huso DL Sun X McWilliams R Beaty T Sham JS Wiener CM Sylvester JT Semenza GL Impaired physiological responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1alpha.J Clin Invest. 1999; 103: 691-696Crossref PubMed Scopus (568) Google Scholar and the oxygen concentration was continuously monitored with an oxygen analyzer (Max O2; Maxtec, Salt Lake City, UT). After 7 days, the eyes were enucleated immediately after removing the animals from the chambers, and the retinas were isolated for immunohistochemistry or Western blotting. For immunohistochemistry, flat mounts of the retinas were prepared by removing the cornea, lens, sclera, and vitreous from eyes, which had been briefly fixed in 4% paraformaldehyde. Proteins were extracted from cultured bEND.3 cells and from isolated retinas by incubating them in phosphate-buffered saline (PBS) containing 10% Triton X-100, 0.2% sodium dodecyl sulfate, 1 mmol/L sodium vanadate, 10 mmol/L sodium fluoride, 1 mmol/L phenylmethylsulfonyl fluoride, 2 μg/ml leupeptin, and 1 μg/ml pepstatin. The proteins were separated on 12.5% sodium dodecyl sulfate-polyacrylamide by gel electrophoresis under reducing conditions and were electrophoretically transferred onto polyvinylidene fluoride membranes (ATTO, Tokyo, Japan). After blocking nonspecific reactions with Block Ace (Dainippon Pharmaceutical, Osaka, Japan), the membranes were incubated with rabbit polyclonal antibody against claudin-5 (1/250; Zymed, San Francisco, CA) or rabbit polyclonal antibody against β-actin (1/5000 dilution; Abcam, Cambridge, UK) for 1 hour at room temperature. After washing in PBS containing 0.1% Tween 20, the membranes were further reacted with horseradish peroxidase-conjugated anti-rabbit IgG (1:15,000 dilution; Amersham Biosciences Corp., Piscataway, NJ) for 30 minutes at room temperature. A chemiluminescence reagent, ECL Western blotting detection reagent (Amersham Biosciences Corp.), was used to make the labeled protein bands visible. For quantification, the density of each band was determined by the NIH Image 1.41 program (available at ftp from zippy.nimh.nih.gov/ or from http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, NIH, Bethesda, MD). The density of the claudin-5 band was standardized to that of β-actin, and the densities in normoxic and hypoxic bEND.3 cells or retinas were compared. Cultured bEND.3 cells on fibronectin-coated dishes were fixed with 100% methanol for 5 minutes at room temperature and were incubated with 5% normal swine serum in PBS for 30 minutes at room temperature to block the nonspecific binding of antibodies. For in vivo studies, flat mounts of retinas were fixed with 100% methanol for 5 minutes at room temperature and were treated with PBS containing 5% normal swine serum and 0.5% Triton X-100 for 6 hours at room temperature to block nonspecific binding of antibodies and for the permeabilization of the tissues. Subsequently, the cells and the flat mounts were reacted with rabbit polyclonal antibody against claudin-5 (1/25; Zymed) at 4°C overnight. After washing with PBS, they were incubated with fluorescein isothiocyanate-conjugated swine polyclonal antibody against rabbit immunoglobulins (DakoCytomation Denmark A/S, Glostrup, Denmark) for 6 hours at room temperature. The stained cells and retinal flat mounts were then washed with PBS and mounted in fluorescent mounting medium (DakoCytomation) for observation under a confocal microscope (FluoView FV1000; Olympus, Tokyo, Japan). bEND.3 cells were grown to confluence on fibronectin-coated cell inserts with 0.4-μm pore size, and the resistance of inserts was measured using the Millicell ERS Voltohmmeter (Millipore, Billerica, MA). The transendothelial electrical resistance (TEER) of the inserts was calculated by subtracting the resistance of blank inserts from that of the inserts with bEND.3 cells and multiplying the subtracted values by the area of the insert. The TEER was used as an index of the barrier property of the bEND.3 monolayer. The 21-oligonucleotide small interfering RNA (siRNA) with the sequence of claudin-5 (5′-AACATCGTTGTCCGCGAGTTC-3′) and control non-silencing (5′-AATTCTCCGAACGTGTCACGT-3′) oligonucleotides were chemically synthesized (QIAGEN, Germantown, MD). For annealing, 20 μmol/L siRNA in 30 mmol/L Hepes-KOH buffer, pH 7.4, containing 100 mmol/L CH3COOK, and 2 mmol/L (CH3COO)2Mg was incubated at 90°C for 1 minute and subsequently at 37°C for 1 hour. Then, using the Nucleofector kit (Amaxa, Gaithersburg, MD), 2 μg of siRNA was transfected into 1 × 106 bEND.3 cells suspended in 100 μl of Nucleofector solution V. Program T-20 was selected according to the manufacturer's instructions to achieve a high transfection efficiency. The cells were then plated onto the fibronectin-coated culture dishes or cell inserts and further incubated in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for Western blotting and the measurement of TEERs. RT-PCR was performed to determine the mRNA level of claudin-5. The level of the mRNA of β-actin was used as the standard. Total RNA was extracted from the bEND.3 cells cultured under either normoxia or hypoxia for 24 hours using Isogen (Nippon Gene, Toyama, Japan), and total RNA (2 μg) was reverse-transcribed in a 15-μl reaction volume with a First-Strand cDNA Synthesis Kit (Pharmacia Biotech, Uppsala, Sweden) as described.33Noda K Ishida S Inoue M Obata K Oguchi Y Okada Y Ikeda E Production and activation of matrix metalloproteinase-2 in proliferative diabetic retinopathy.Invest Ophthalmol Vis Sci. 2003; 44: 2163-2170Crossref PubMed Scopus (119) Google Scholar One microliter of the reaction mixture was then subjected to PCR for amplification of each molecule. PCR was performed in 50 μl containing 800 nmol/L each primer, 250 nmol/L dNTPs, and 5 U of TaqDNA polymerase (Toyobo, Tokyo, Japan) with a thermal controller (MiniCycler; MJ Research, Inc., Watertown, MA). The number of PCR cycles was 30 for claudin-5 and 25 for β-actin. The thermal cycle was 1 minute at 94°C, 2 minutes at 60°C (claudin-5) or 57°C (β-actin), and 3 minutes at 72°C, followed by final extension for 3 minutes at 72°C. The nucleotide sequences of the PCR primers were 5′-GACTGCCTTCCTGGACCAC-3′ (forward) and 5′-TGACCGGGAAGCTGAACTC-3′ (reverse) for claudin-5; and 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (forward) and 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ (reverse) for β-actin. The expected sizes of the amplified cDNA fragments of claudin-5 and β-actin were 500 bp and 837 bp, respectively. The PCR products were electrophoresed on a 1.5% agarose gel and then stained with ethidium bromide. For quantitative analysis of the level of claudin-5 mRNA, TaqMan real-time PCR assay was performed using the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) according to the manufacturer's protocol. Primers and TaqMan probes specific for claudin-5 and β-actin were purchased from Applied Biosystems (sequences not disclosed). The cycling conditions were 50°C for 2 minutes, initial denaturation at 95°C for 10 minutes, and then 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. β-Actin was used to normalize the amount of claudin-5 mRNA in each sample, and claudin-5 to β-actin mRNA ratio (claudin-5 mRNA/β-actin mRNA ratio) was compared between the bEND.3 cells under normoxia and hypoxia. The effect of hypoxic conditions on mouse retinas was determined by the Hypoxyprobe-1 Plus Kit (Chemicon International, Temecula, CA). One hour before sacrifice, pimonidazole hydrochloride (Hypoxyprobe-1; Chemicon International), which binds to the proteins in hypoxic cells, was injected into the peritoneal cavities of mice (60 mg/kg). Flat mounts of retinas were prepared and fixed with 100% methanol for 5 minutes at room temperature. After 6 hours of incubation with blocking buffer, retinal flat mounts were reacted with both the fluorescein isothiocyanate-conjugated mouse monoclonal antibody against Hypoxiprobe-1 (1/50 dilution) and the rat monoclonal antibody against CD31 (PECAM-1) (1/500 dilution; BD Biosciences, San Jose, CA) at 4°C overnight. The flat mounts were subsequently incubated with Alexa Fluor 546-conjugated goat antibodies against rat immunoglobulins (Molecular Probes, Eugene, OR) for 6 hours at room temperature. The retinas were stained with the anti-CD31 antibody to make the endothelial cells visible. The stained flat mounts were observed with a confocal microscope (FluoView FV1000; Olympus). To determine the permeability of the mouse retinal vessels, tracer experiments were performed as described.23Nitta T Hata M Gotoh S Seo Y Sasaki H Hashimoto N Furuse M Tsukita S Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice.J Cell Biol. 2003; 161: 653-660Crossref PubMed Scopus (1403) Google Scholar Under deep anesthesia with pentobarbital sodium, the mouse chest cavity was opened, and a 24-gauge cannula was inserted into the left ventricle. Each mouse was perfused with 500 μl/g body weight of PBS containing 100 μg/ml Hoechst stain H33258 (molecular mass, 534 d; Sigma) and 1 mg/ml tetramethylrhodamine-conjugated lysine-fixable dextran (molecular mass, 10 kd; Molecular Probes). The isolated retinas were flat mounted and observed with a confocal microscopy (FluoView FV1000; Olympus). All of the data are expressed as the means ± SD. The data were analyzed with Mann-Whitney tests, and differences were considered to be statistically significant at P < 0.05. bEND.3 cells that had reached confluence were cultured for an additional 11 days under normoxia, and the expression of claudin-5 was determined by Western blotting and immunocytochemistry. The barrier function of a monolayer of bEND.3 cells was also evaluated by measurements of the TEERs. After the cultured bEND.3 cells reached confluence, the cellular protein level of claudin-5 increased and reached a steady-state level at day 3 (Figure 1A). In addition, the claudin-5 molecule relocated to the plasma membranes between adjacent bEND.3 cells at day 7 (Figure 1B). The detection of claudin-5 in the plasma membrane was strongly correlated with the increase in the TEER of the bEND.3 monolayer (Figure 1C). To evaluate the role of claudin-5 in the barrier function of bEND.3 monolayer in more detail, the RNAi technique was used to inhibit the expression of claudin-5. Monolayers of bEND.3 cells that had the expression of claudin-5 depressed failed to develop the increased TEER but did so with non-silencing oligonucleotides were used (Figure 2). The expression of claudin-5 was analyzed and compared between the bEND.3 cells cultured under normoxic and hypoxic conditions. Confluent bEND.3 cells were cultured for an additional 7 days under normoxia to obtain the cells with claudin-5 exclusively located in the plasma membranes. The cells were then exposed for 24 hours to either normoxic or hypoxic conditions, and the expression of claudin-5 was evaluated by immunocytochemistry and Western blotting. Our results showed that the amount of claudin-5 molecules in the plasma membranes was decreased under hypoxic conditions, which was accompanied by a decrease in their cellular protein levels (Figure 3, A and B). Interestingly, the changes in the expression level of the mRNA of claudin-5 mRNA were not significantly different between cells cultured under normoxia and hypoxia (Figure 3C). The hypoxic bEND.3 monolayer with diminished localization of caludin-5 to plasma membranes showed weak barrier properties with significantly lower TEER than that of the normoxic monolayer (Figure 3D). In addition, bEND.3 cells recovered from these hypoxic changes in claudin-5 expression and TEER when they were further incubated under normoxia for 48 hours (data not shown). Mice were maintained in either atmospheric air or in decreased O2 concentration for 7 days, and then their retinas were processed for immunohistochemistry to investigate the in vivo expression of claudin-5 in the retinal blood vessels. Oxygenation of retinal tissues was evaluated by the intraperitoneal injection of pimonidazole hydrochloride, and it was confirmed that the retinal tissues from mice maintained under decreased O2 concentration were hypoxic as compared with those from mice in atmospheric air (Figure 4). Retinal blood vessels of mice in atmospheric air expressed claudin-5 with its distinct location at the interfaces of adjacent endothelial cells of both proximal and peripheral blood vessels. The expression of claudin-5 was reduced in the blood vessels of hypoxic retina from mice maintained in air with decreased O2 concentration (Figure 5A, a and b). A significant decrease (approximately 41%) in the claudin-5 expression of hypoxic retinas was confirmed by Western blotting (Figure 5B). Suppression of claudin-5 expression was predominantly observed in the peripheral small blood vessels, in contrast to its relatively preserved expression in the proximal blood vessels. In some of the capillaries, the signal for claudin-5 was hardly detected by immunohistochemistry (Figure 5A, c and d).Figure 5In vivo effect of tissue hypoxia on the expression of claudin-5 in retinal blood vessels. Expression of claudin-5 in retinas from mice that had been maintained under normoxia or hypoxia for 7 days was investigated by immunofluorescence (A) and Western blotting (B). The level of β-actin was used as a loading control of Western blotting. A: Claudin-5 expression in the plasma membranes of retinal blood vessels is depressed in mice under hypoxia (b, d) in contrast to the distinct expression in the mice under normoxia (a, c). The decrease in claudin-5 expression by hypoxia was distinct in the
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