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Autocrine and paracrine up-regulation of blood–brain barrier function by plasminogen activator inhibitor-1
Microvascular Research 81 (2011) 103–107
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Autocrine and paracrine up-regulation of blood–brain barrier function by plasminogen activator inhibitor-1 Shinya Dohgu a, Fuyuko Takata a,b, Junichi Matsumoto a, Masatoshi Oda a, Eriko Harada a, Takuya Watanabe a, Tsuyoshi Nishioku a, Hideki Shuto a, Atsushi Yamauchi a, Yasufumi Kataoka a,b,⁎ a b
Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan BBB Laboratory, PharmaCo-Cell Co., Ltd., 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
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Article history: Accepted 22 October 2010 Available online 29 October 2010 Keywords: Blood–brain barrier Plasminogen activator inhibitor-1 Brain endothelial cells Pericytes Permeability Transendothelial electrical resistance Co-culture
a b s t r a c t The blood–brain barrier (BBB) is the interface that separates the central nervous system (CNS) from the peripheral circulation. An increase in blood-borne substances including cytokines in plasma and brain affects BBB function, and this is associated with the development of pathogenesis of a number of diseases. Plasminogen activator inhibitor (PAI)-1 regulates the plasminogen activator/plasmin system as a serpin in the periphery and the CNS. We investigated whether PAI-1 alters BBB function using in vitro models of the BBB consisting of rat primary brain endothelial cells (RBECs) alone and co-cultured with pericytes. We found that PAI-1 increased the tightness of the brain endothelial barrier in a time- and dose-dependent manner, as shown by an increase in the transendothelial electrical resistance (TEER) and a decrease in the permeability to sodium fluorescein (Na-F). RBECs responded equally to PAI-1 in the blood-facing and brain-facing sides of the brain, leading to a decrease in Na-F permeability. In addition, RBECs constitutively released PAI-1 into the blood-facing (luminal) and brain-facing (abluminal) sides. This release was polarized in favor of the luminal side and facilitated by serum. The neutralization of PAI-1 by an antibody to PAI-1 in RBEC/pericyte co-culture more robustly reduced TEER of RBECs than in RBEC monolayers. These findings suggest that PAI-1 derived from the neurovascular unit and peripheral vascular system participates as a positive regulator of the BBB in facilitating the barrier function of the endothelial tight junctions. © 2010 Elsevier Inc. All rights reserved.
Introduction The blood–brain barrier (BBB), which is composed of brain endothelial cells, pericytes, and astrocytes, regulates plasma components from the cerebral microcirculation entering the central nervous system (CNS). The main machinery underlying barrier function is composed of tight junctions formed between brain endothelial cells and various efflux transporters. All structural components of the BBB, neurons, and non-neuronal cells (e.g., microglia) form a neurovascular unit to contribute to dynamic and continuous regulation of cerebral microvascular permeability, synaptic transmission, and neurogenesis (Hawkins and Davis, 2005; Zlokovic, 2008). Pericytes and astrocytes are required to induce and maintain barrier properties by secreting various soluble factors including neurotrophic factors (Abbott et al., 2006; Zlokovic, 2008). Moreover, brain endothelial cells, which act as an interface between blood and brain parenchyma, can directly communicate with blood-borne substances that lead to the alteration of BBB function. ⁎ Corresponding author. Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Fax: +81 92 862 2699. E-mail address: [email protected] (Y. Kataoka). 0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2010.10.004
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily and the primary inhibitor of tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). The serine protease proteolytically converts plasminogen into active plasmin in the blood. PAI-1 regulates both tPA and uPA activity, indicating that PAI-1 plays a role in tissue remodeling and fibrinolysis in the periphery. Endothelial cells and smooth muscle cells are a major source of PAI-1. Regulation of PAI-1 production is inducible rather than constitutive (Juhan-Vague et al., 2003). The proinflammatory mediators, tumor necrosis factor (TNF)α and interleukin (IL)-1, are inducers of PAI-1 and are released from adipose tissue (Juhan-Vague et al., 2003). Increased PAI-1 causes impaired fibrinolysis and enhanced thrombosis leading to microvascular thrombosis and the development of atherosclerotic lesions (Sobel, 1999). Clinical studies have suggested that PAI-1 is associated with metabolic syndrome (Ingelsson et al., 2007) and vascular diseases in diabetes (Agirbasli, 2005). While tPA is widely expressed in the CNS and plays a role in synaptic remodeling and neuronal plasticity (Adibhatla and Hatcher, 2008), exogenous tPA facilitates excitotoxic neuronal death (Lo et al., 2004) and disruption of the BBB (Yepes et al., 2003). PAI-1 prevents this tPA-induced neuronal degeneration and BBB disruption (Nagai et al., 2005; Abu Fanne et al., 2010). In contrast, elevated levels of PAI-1 in the
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brain are known to be associated with the pathogenesis of Alzheimer's disease (Jacobsen et al., 2008; Zlokovic, 2008). PAI-1 derived from astrocytes, a major source of PAI-1 in the brain (Buisson et al., 1998), protects against neuronal apoptotic death (Soeda et al., 2004). Besides astrocytes, brain endothelial cells and pericytes express PAI-1 mRNA (Kose et al., 2007). Thus, PAI-1 in the brain is highly likely to act as a mediator for cross-talk among the elements of the neurovascular unit to regulate endothelial barrier function. However, the role of PAI-1 in BBB function is incompletely understood. In the present study, we demonstrated that PA-1 released from brain endothelial cells and pericytes increases the tightness of the brain endothelial barrier.
culture plates (Costar, Corning, NY). After 2-3 days, RBECs (5 × 104 cells/well) were seeded on the upper side of a fibronectin-collagen IV (0.1 mg/ml)-coated polyester membrane (0.33 cm2, 0.4 μm pore size) of a Transwell®-Clear insert (Costar) placed in the well of a 24-well culture plate containing layers of brain pericytes (RBEC/pericyte coculture). This co-culture system allows cells to communicate with each other through soluble factors. A monolayer system was made with RBECs alone (RBEC monolayer). Cells were cultured in RBEC medium II supplemented with 500 nM hydrocortisone (Sigma) at 37 °C with a humidified atmosphere of 5% CO2/95% air until the in vitro BBB models reached confluency. Pretreatment of RBECs with PAI-1
Materials and methods Isolation of rat brain microvascular endothelial cells (RBECs) and pericytes All procedures involving experimental animals adhered to the Law (No. 105) and Notification (No. 6) of the Japanese Government, and were approved by the Laboratory Animal Care and Use Committee of Fukuoka University. Primary cultures of RBECs and pericytes were prepared from 3-week-old Wistar rats, as previously described (Dohgu et al., 2005; Takata et al., 2008; Sumi et al., 2010). In brief, the meninges were carefully removed from the forebrain and gray matter was minced into small pieces in ice-cold Dulbecco's modified Eagles medium (DMEM; Wako, Osaka, Japan), and then digested in DMEM containing collagenase type 2 (1 mg/ml; Worthington, Lakewood, NJ), DNase I (15 μg/ml; Sigma, St. Louis, MO, USA), and gentamicin (50 μg/ml; Sigma) for 1.5 h at 37 °C. The cell pellet was separated by centrifugation in 20% bovine serum albumin (BSA; Sigma)-DMEM (1000g, 20 min). The microvessels obtained in the pellet were further digested with collagenase/dispase (1 mg/ml; Roche, Mannheim, Germany) and DNase I (6.7 μg/ml) in DMEM for 1 h at 37 °C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll (GE Healthcare, Buckinghamshire, UK) gradient (1000g for 10 min), collected, and washed in DMEM before plating on culture dishes coated with collagen type IV and fibronectin (both 0.1 mg/ml; Sigma). RBEC cultures were maintained in DMEM/F12 (Sigma) supplemented with 10% bovine plasma derived serum (Animal Technologies, Tyler, TX), basic fibroblast growth factor (1.5 ng/ml; Roche), heparin (100 μg/ml; Sigma), insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml) (insulin-transferrinsodium selenite media supplement; Sigma), gentamicin (50 μg/ml), and puromycin (4 μg/ml; Sigma) (RBEC medium I) at 37 °C in a humidified atmosphere of 5% CO2/95% air, for 2 days. On the third day, the cells received a new medium that contained all the components of RBEC medium I except for puromycin (RBEC medium II). When the cultures reached confluency, the purified endothelial cells were passaged and used to construct in vitro BBB models. Brain pericytes were obtained by a prolonged culture of isolated brain microvessel fragments under selective culture conditions (Dohgu et al., 2005; Takata et al., 2009; Nakagawa et al., 2007). Briefly, the obtained brain microvessel fragments were placed in an uncoated culture flask in DMEM supplemented with 20% fetal bovine serum (FBS; Biowest, Nuaillé, France) (FBS-DMEM), 100 units/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto). After 7 days in culture, rat pericytes overgrew brain endothelial cells and typically reached 80–90% confluency. The cells were used at passage 2.
The median plasma levels of PAI-1 in healthy control subjects and patients with venous thrombosis were more than 100 ng/ml (Meltzer et al., 2010). Based on these data, we treated RBECs with various doses of PAI-1 up to 100 ng/ml. Recombinant rat PAI-1 (Calbiochem, La Jolla, CA) was supplied in solution and this original solution was diluted with a solution containing 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, pH 5.0 to obtain 100-fold concentrations for experiments. These PAI-1 solutions and an anti PAI-1 mouse monoclonal antibody (100 μg/ml; Affinity BioReagents, Golden, CO) were diluted with serum-free RBEC medium II containing 500 nM hydrocortisone in the luminal and/or abluminal side of the Transwell insert to expose RBECs. In all experiments, controls were RBECs treated with the corresponding amount of mouse IgG (Sigma) or the solvent for PAI-1. Measurement of transendothelial electrical resistance (TEER) TEER across the monolayers grown on the filter membranes was measured by an EVOM resistance meter in an EndOhm tissue resistance measurement chamber (World Precision Instruments, Sarasota, FL). Values are presented as Ω × cm2 culture insert. The TEER of cell-free inserts was subtracted from the obtained values. Measurement of transendothelial transport of sodium fluorescein (Na-F) Endothelial barrier function was evaluated by measuring permeability of RBECs to Na-F as previously described (Dohgu et al., 2004a, 2005). To initiate the transport experiments, the medium was removed and then physiological buffer (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM HEPES and 10 mM d-glucose, pH 7.4) containing 100 μg/ml Na-F (Sigma) was loaded in the luminal chamber of the insert (0.1 ml). Samples (0.4 ml) were removed from the abluminal chamber at 15, 30, 45, and 60 min and immediately replaced with fresh physiological buffer. The concentration of Na-F was determined using a fluorescence multiwell plate reader (Ex(λ) 485 nm; Em(λ) 530 nm; CytoFluor Series 4000; PerSeptive Biosystems, Framingham, MA). The permeability coefficient and clearance were calculated as previously described (Dehouck et al., 1992; Dohgu et al., 2005). Measurement of PAI-1 in culture supernatants RBEC monolayers were incubated in RBEC medium II with or without 10% bovine plasma-derived serum for 24 h. Culture supernatants were collected from the luminal and abluminal chambers, and stored at -80 °C until use. Levels of PAI-1 in culture supernatants were measured with the total rat PAI-1 antigen ELISA kit (Innovative Research Inc., Novi, MI) by following the manufacturer's instructions.
Preparation of the in vitro BBB models Statistical analysis Preparation of the in vitro BBB models co-culturing RBECs and brain pericytes has been described previously (Dohgu et al., 2005). Brain pericytes (4 × 104 cells/well) were seeded in wells of 24-well
Values are expressed as means ± SEM. The Student's t-test was applied to compare the two groups. One-way and two-way analyses
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of variance (ANOVAs) followed by Tukey–Kramer's test were applied to multiple comparisons. The differences between means were considered to be significant when p values were less than 0.05 using Prism 5.0 (GraphPad, San Diego, CA).
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chambers of the RBEC monolayer. A 24-h exposure to PAI-1 produced equal rate of decrease in Na-F permeability when PAI-1 was added to either the luminal or abluminal chambers (35.7% and 44.1% of decrease, respectively). Addition of PAI-1 to both the luminal and abluminal chambers failed to facilitate the lowered permeability of RBECs to Na-F (Fig. 1B).
Results Polarized release of PAI-1 from RBECs Effect of PAI-1 on BBB permeability First, to determine the effect of PAI-1 on brain endothelial barrier function, we treated RBEC monolayers with recombinant rat PAI-1 (0.01, 1, and 100 ng/ml) for 24 h. PAI-1 was added every 6 h to both the luminal and abluminal chambers since PAI-1 in the serum-free culture medium was degraded to 8.9 ± 0.3% of the initial loaded amount within 6 h (Fig. 1A inset). PAI-1 increased TEER of RBECs time- and dosedependently (Fig. 1A) [dose: F(3, 40) = 9.121, p b 0.01; time: F(4, 40) = 15.30, p b 0.001; interaction (dose× time): F(12, 40) = 3.315, p b 0.01]. This reached a peak 12 h after the treatment. Second, to test whether RBECs showed a polarized response to PAI-1 for permeability to Na-F, PAI-1 (100 ng/ml) was added every 6 h to the luminal and/or abluminal
To determine spontaneous release of PAI-1 from RBECs in the presence or absence of serum, we measured the levels of PAI-1 in the luminal and abluminal chambers of RBEC monolayers. PAI-1 was detected in both the luminal and abluminal chambers (Fig. 2). When RBECs were cultured under serum-free conditions, 4-fold higher levels of PAI-1 were released to the luminal chamber compared with the abluminal chamber (0.81 ± 0.06 vs 0.19 ± 0.01 ng/ml). In the presence of serum (10% bovine plasma-derived serum), RBECs released 10- to 20-fold higher levels of PAI-1 to both the luminal and abluminal chambers compared with serum-free conditions. The baseline level of PAI-1 in the medium containing bovine plasmaderived serum was below detectable levels. Effect of PAI-1 inhibition on TEER of RBECs in RBEC monolayers and RBEC/pericyte co-cultures Pericytes also released PAI-1 (3.1 ±0.1 ng/ml) under serum-free conditions. We examined the effect of PAI-1 neutralizing antibody on brain endothelial barrier function in the presence or absence of pericytes. Adding PAI-1 neutralizing antibody (10 μg/ml) to both luminal and abluminal chambers significantly decreased TEER of RBECs by 41.5% and 57.6% of control (IgG) in RBEC monolayers and RBEC/pericyte cocultures, respectively (Fig. 3). A two-way ANOVA showed significant effects for the culture system (monolayer and co-culture) [F(1, 18) = 13.53, p b 0.01], treatment (IgG and PAI-1 antibody) [F(1, 18) = 33.08, p b 0.001], and interaction (culture system × treatment) [F(1, 18) = 5.746, p b 0.05]. The extent of decrease in TEER of RBECs in RBEC/pericyte co-cultures was significantly greater than that of RBEC monolayers (p b 0.05). Discussion Under pathological conditions, the cytokine profile in the blood is dramatically altered (Kunz and Ibrahim, 2009). High plasma levels of PAI-1 are considered a risk factor of vascular diseases. Brain endothelial cells face two compartments, the blood and brain. This allows cells to receive stimuli mediated by various substances from the cerebral microcirculation and CNS cells (e.g., pericytes, astrocytes,
Fig. 1. (A) Time-course of the changes in TEER of RBECs after exposure to various concentrations of PAI-1 during a 24-h period. PAI-1 was added to both the luminal and abluminal chambers of RBEC monolayers every 6 h. Data are expressed as the percentage of the time-matched control value. TEER values of control RBECs were 108.0 ± 3.4, 91.6 ± 2.9, 134.6 ± 1.8, 135.5 ± 5.5, and 191.7 ± 11.3 Ω × cm2 at 0, 1, 6, 12, and 24 h, respectively (n = 3–4). **p b 0.01, ***p b 0.001 compared with controls. The inset shows the time-course of PAI-1 levels in the medium after adding PAI-1. Data are expressed as the percentage of the initial concentration of PAI-1 (0 h; 11.5 ± 1.4 ng/ml) (n = 3). (B) Non polarized response to PAI-1 in the permeability of RBECs to Na-F. RBEC monolayers were treated with PAI-1 (100 ng/ml) added to the luminal or abluminal chambers for 24 h. PAI-1 was added to the luminal or abluminal chambers of RBEC monolayers every 6 h. Data are expressed as the percentage of controls (9.79 ± 1.12 × 10−5 cm/min) (n = 7–8). *p b 0.05, **p b 0.01 compared with controls. Values are means ± SEM.
Fig. 2. Spontaneous release of PAI-1 into the luminal or abluminal chambers of RBECs in the presence or absence of serum. After 24 h incubation, the medium was collected from both the luminal and abluminal chambers. Values are means ± SEM (n = 3). **p b 0.01, ***p b 0.001 compared with the luminal chamber.
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Fig. 3. Effect of anti-PAI-1 antibody on TEER of RBECs in RBEC monolayers and RBEC/ pericyte co-cultures. RBEC monolayers and RBEC/pericyte co-cultures were treated with mouse IgG and anti-PAI-1 antibody (10 μg/ml) added to both the luminal and abluminal chambers for 24 h. Values are means ± SEM (n = 3–8). *p b 0.05, **p b 0.01 compared with IgG; ††p b 0.01 compared with IgG-treated RBEC monolayers.
microglia, and neurons), leading to alterations in endothelial barrier function. In the present study, we found that BBB cell-produced and exogenous PAI-1 enhanced barrier properties of the BBB. To examine the effect of PAI-1 derived from the blood and brain on BBB function, we first exposed RBECs to PAI-1 added to both luminal (blood side) and abluminal (brain side) chambers of Transwell® inserts. PAI-1 in the medium was rapidly degraded during a 6 hperiod and remained less than 10% of the initial amount 6 h after addition (Fig. 1A inset). A similar finding was also reported by McIlroy et al. (2006). Therefore, we added PAI-1 to the luminal and/or abluminal chambers of RBEC monolayers every 6 h in the experiments. PAI-1 increased TEER of RBECs dose- and time-dependently (Fig. 1A). We also confirmed that PAI-1 (100 ng/ml) added to both the luminal and abluminal chambers decreased RBEC permeability to NaF, which is a paracellular permeability marker (Fig. 1B). Passage of molecules across brain endothelial cells of the BBB occurs through intercellular tight junctions (paracellular pathway) or through the cells (transcellular pathway). The permeability of the BBB to molecules is determined by the overall transendothelial transport through these two pathways. The paracellular pathway allows ions and small hydrophilic solutes to diffuse between adjacent cells according to their concentration gradient. The transcellular pathway involves various transport mechanisms such as passive diffusion of lipophilic compounds, transporter-mediated transport, and receptormediated transcytosis. Ions and hydrophilic solutes mainly permeate across the BBB through paracellular pathways. The TEER and Na-F permeability of RBECs evaluated in our experiments indicate the permeability of RBECs to ions and small hydrophilic solutes, respectively. These are associated with the integrity of the paracellular barrier sealed with tight junctions in RBECs. In addition, it has been reported that Na-F is a substrate for organic anion transporter (OAT)-3 and multidrug resistance-associated protein (MRP)-2 (Hawkins et al., 2007). Therefore, PAI-1 may regulate not only the paracellular barrier function but also OAT-3- and MRP-2-mediated transcellular permeability. To determine the functional polarity of RBECs in response to PAI-1, we added PAI-1 to either luminal or abluminal chambers. The PAI-1induced decrease in the permeability of RBECs to Na-F was independent of the PAI-1-injected site. This result indicated that RBECs are capable of responding to stimulation of PAI-1 derived from either the blood or brain. Although PAI-1 in the medium was almost degraded within 6 h, TEER of RBECs continuously increased over a period of 6 h. The possibility that PAI-1 induces the release of other substances maintaining barrier function from RBECs cannot be excluded. Various intracellular signaling pathways are involved in
the expression and phosphorylation of tight junction proteins, and these effects are associated with alterations to the barrier function (Cardoso et al., 2010). Although the PAI-1 receptor on brain endothelial cells and its subsequent signaling pathway have remained obscure, the possibility that PAI-1 could directly regulate the expression and/or phosphorylation of tight junction proteins to enhance BBB functions should be considered. These findings suggest that PAI-1 is a positive regulator of BBB function. We found that RBECs spontaneously released PAI-1 into both the luminal and abluminal sides (Fig. 2). This release of PAI-1 was polarized, and its release into the luminal (blood) compartment was superior to that in the abluminal side. This is consistent with the release of IL-1α, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and TNF-α from mouse brain endothelial cells (Verma et al., 2006). Stimulation of RBECs with serum significantly increased the release of PAI-1 into both the luminal and abluminal chambers. Oxidized very-low-density lipoprotein increases PAI-1 expression in endothelial cells (Zhao et al., 2008). These findings suggest that PAI-1 released from brain endothelial cells is inducible by a number of unknown substances in the serum. We examined whether spontaneous release of PAI-1 from RBECs maintained barrier function (Fig. 3). Anti-PAI-1 neutralizing antibody significantly lowered TEER in RBEC monolayers, suggesting that PAI-1 up-regulates BBB function in an autocrine and paracrine manner. When RBECs were co-cultured with pericytes, the anti-PAI-1 antibody-lowered TEER of RBECs was aggravated. We found that pericytes also released PAI-1. Previous studies have shown that PAI-1 expression is increased in brain endothelial-pericyte co-culture (Kim et al., 2006). These findings indicate that PAI-1 mediates pericyte-induced up-regulation of BBB function. Our previous study showed that pericytes up-regulate BBB function by secreting transforming growth factor-β1 (TGF-β1) (Dohgu et al., 2005). TGF-β1 facilitates the barrier function of brain endothelial cells, and this effect may be related to the regulation of the expression of tight junction proteins (occludin, claudins, and ZO-1) induced by the TGF-β/ALK5 signaling pathway (Dohgu et al., 2004b; Ronaldson et al., 2009). Furthermore, TGF-β1 induces mRNA expression of PAI-1 in brain endothelial cells and pericytes (Kose et al., 2007). Therefore, it is conceivable that PAI-1 cooperates with TGF-β1 to maintain BBB function. It has been shown that tPA aggravates ischemic brain damage (Nagai et al., 1999; Wang et al., 1998) and directly triggers the disruption of brain endothelial barrier function (Hiu et al., 2008). PAI1 exerts a protective effect against ischemic brain injury by inhibiting tPA activity (Yang et al., 2009). Although the direct mechanism by which PAI-1 up-regulates BBB function has not been elucidated, constitutive expression of both tPA and PAI-1 in the cellular components of the BBB (brain endothelial cells, pericytes, and astrocytes) is considered to contribute, at least in part, to pericyteinduced up-regulation of BBB function. Pericytes decrease the mRNA and protein expression of tPA and increase those of PAI-1 in brain endothelial cells (Kim et al., 2006). PAI-1 expression in the monolayers of brain endothelial cells or astrocytes was facilitated by co-culturing with these cells (Hultman et al., 2010). These findings suggest that brain endothelial cells, pericytes, and astrocytes cooperatively regulate BBB permeability through their expression of tPA and PAI-1. Thus, the balance between tPA and PAI-1 in the elements of the BBB is considered to be crucial for the integrity of BBB function. In addition, PAI-1 released from astrocytes is thought to act as a neurotrophic factor, and this phenomenon is independent of the ability for inhibition of tPA and uPA (Kimura et al., 2000; Soeda et al., 2001, 2004, 2006). Taken together, the present findings are important in the understanding of cross-talk mediated by PAI-1 in the neurovascular unit. In conclusion, we demonstrated that PAI-1 participates as a positive regulator of the BBB in facilitating the barrier function of the endothelial tight junctions.
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Acknowledgments This work was supported in part by Grants-in-Aid for Young Scientists ([Start-up] 20800066), and Grants-in-Aid for Young Scientists ([B] 21790102, [B] 21790255, [B] 21790257 and [B] 21790526) from JSPS, Japan, the Ministry of Health, Labour and Welfare of Japan (H19nanchi-ippan-006), and Kakihara Science and Technology Foundation.
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Microvascular Research j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y m v r e
Regular Article
Autocrine and paracrine up-regulation of blood–brain barrier function by plasminogen activator inhibitor-1 Shinya Dohgu a, Fuyuko Takata a,b, Junichi Matsumoto a, Masatoshi Oda a, Eriko Harada a, Takuya Watanabe a, Tsuyoshi Nishioku a, Hideki Shuto a, Atsushi Yamauchi a, Yasufumi Kataoka a,b,⁎ a b
Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan BBB Laboratory, PharmaCo-Cell Co., Ltd., 1-12-4 Sakamoto, Nagasaki 852-8523, Japan
a r t i c l e
i n f o
Article history: Accepted 22 October 2010 Available online 29 October 2010 Keywords: Blood–brain barrier Plasminogen activator inhibitor-1 Brain endothelial cells Pericytes Permeability Transendothelial electrical resistance Co-culture
a b s t r a c t The blood–brain barrier (BBB) is the interface that separates the central nervous system (CNS) from the peripheral circulation. An increase in blood-borne substances including cytokines in plasma and brain affects BBB function, and this is associated with the development of pathogenesis of a number of diseases. Plasminogen activator inhibitor (PAI)-1 regulates the plasminogen activator/plasmin system as a serpin in the periphery and the CNS. We investigated whether PAI-1 alters BBB function using in vitro models of the BBB consisting of rat primary brain endothelial cells (RBECs) alone and co-cultured with pericytes. We found that PAI-1 increased the tightness of the brain endothelial barrier in a time- and dose-dependent manner, as shown by an increase in the transendothelial electrical resistance (TEER) and a decrease in the permeability to sodium fluorescein (Na-F). RBECs responded equally to PAI-1 in the blood-facing and brain-facing sides of the brain, leading to a decrease in Na-F permeability. In addition, RBECs constitutively released PAI-1 into the blood-facing (luminal) and brain-facing (abluminal) sides. This release was polarized in favor of the luminal side and facilitated by serum. The neutralization of PAI-1 by an antibody to PAI-1 in RBEC/pericyte co-culture more robustly reduced TEER of RBECs than in RBEC monolayers. These findings suggest that PAI-1 derived from the neurovascular unit and peripheral vascular system participates as a positive regulator of the BBB in facilitating the barrier function of the endothelial tight junctions. © 2010 Elsevier Inc. All rights reserved.
Introduction The blood–brain barrier (BBB), which is composed of brain endothelial cells, pericytes, and astrocytes, regulates plasma components from the cerebral microcirculation entering the central nervous system (CNS). The main machinery underlying barrier function is composed of tight junctions formed between brain endothelial cells and various efflux transporters. All structural components of the BBB, neurons, and non-neuronal cells (e.g., microglia) form a neurovascular unit to contribute to dynamic and continuous regulation of cerebral microvascular permeability, synaptic transmission, and neurogenesis (Hawkins and Davis, 2005; Zlokovic, 2008). Pericytes and astrocytes are required to induce and maintain barrier properties by secreting various soluble factors including neurotrophic factors (Abbott et al., 2006; Zlokovic, 2008). Moreover, brain endothelial cells, which act as an interface between blood and brain parenchyma, can directly communicate with blood-borne substances that lead to the alteration of BBB function. ⁎ Corresponding author. Department of Pharmaceutical Care and Health Sciences, Faculty of Pharmaceutical Sciences, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan. Fax: +81 92 862 2699. E-mail address: [email protected] (Y. Kataoka). 0026-2862/$ – see front matter © 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.mvr.2010.10.004
Plasminogen activator inhibitor-1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily and the primary inhibitor of tissue- and urokinase-type plasminogen activator (tPA and uPA, respectively). The serine protease proteolytically converts plasminogen into active plasmin in the blood. PAI-1 regulates both tPA and uPA activity, indicating that PAI-1 plays a role in tissue remodeling and fibrinolysis in the periphery. Endothelial cells and smooth muscle cells are a major source of PAI-1. Regulation of PAI-1 production is inducible rather than constitutive (Juhan-Vague et al., 2003). The proinflammatory mediators, tumor necrosis factor (TNF)α and interleukin (IL)-1, are inducers of PAI-1 and are released from adipose tissue (Juhan-Vague et al., 2003). Increased PAI-1 causes impaired fibrinolysis and enhanced thrombosis leading to microvascular thrombosis and the development of atherosclerotic lesions (Sobel, 1999). Clinical studies have suggested that PAI-1 is associated with metabolic syndrome (Ingelsson et al., 2007) and vascular diseases in diabetes (Agirbasli, 2005). While tPA is widely expressed in the CNS and plays a role in synaptic remodeling and neuronal plasticity (Adibhatla and Hatcher, 2008), exogenous tPA facilitates excitotoxic neuronal death (Lo et al., 2004) and disruption of the BBB (Yepes et al., 2003). PAI-1 prevents this tPA-induced neuronal degeneration and BBB disruption (Nagai et al., 2005; Abu Fanne et al., 2010). In contrast, elevated levels of PAI-1 in the
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brain are known to be associated with the pathogenesis of Alzheimer's disease (Jacobsen et al., 2008; Zlokovic, 2008). PAI-1 derived from astrocytes, a major source of PAI-1 in the brain (Buisson et al., 1998), protects against neuronal apoptotic death (Soeda et al., 2004). Besides astrocytes, brain endothelial cells and pericytes express PAI-1 mRNA (Kose et al., 2007). Thus, PAI-1 in the brain is highly likely to act as a mediator for cross-talk among the elements of the neurovascular unit to regulate endothelial barrier function. However, the role of PAI-1 in BBB function is incompletely understood. In the present study, we demonstrated that PA-1 released from brain endothelial cells and pericytes increases the tightness of the brain endothelial barrier.
culture plates (Costar, Corning, NY). After 2-3 days, RBECs (5 × 104 cells/well) were seeded on the upper side of a fibronectin-collagen IV (0.1 mg/ml)-coated polyester membrane (0.33 cm2, 0.4 μm pore size) of a Transwell®-Clear insert (Costar) placed in the well of a 24-well culture plate containing layers of brain pericytes (RBEC/pericyte coculture). This co-culture system allows cells to communicate with each other through soluble factors. A monolayer system was made with RBECs alone (RBEC monolayer). Cells were cultured in RBEC medium II supplemented with 500 nM hydrocortisone (Sigma) at 37 °C with a humidified atmosphere of 5% CO2/95% air until the in vitro BBB models reached confluency. Pretreatment of RBECs with PAI-1
Materials and methods Isolation of rat brain microvascular endothelial cells (RBECs) and pericytes All procedures involving experimental animals adhered to the Law (No. 105) and Notification (No. 6) of the Japanese Government, and were approved by the Laboratory Animal Care and Use Committee of Fukuoka University. Primary cultures of RBECs and pericytes were prepared from 3-week-old Wistar rats, as previously described (Dohgu et al., 2005; Takata et al., 2008; Sumi et al., 2010). In brief, the meninges were carefully removed from the forebrain and gray matter was minced into small pieces in ice-cold Dulbecco's modified Eagles medium (DMEM; Wako, Osaka, Japan), and then digested in DMEM containing collagenase type 2 (1 mg/ml; Worthington, Lakewood, NJ), DNase I (15 μg/ml; Sigma, St. Louis, MO, USA), and gentamicin (50 μg/ml; Sigma) for 1.5 h at 37 °C. The cell pellet was separated by centrifugation in 20% bovine serum albumin (BSA; Sigma)-DMEM (1000g, 20 min). The microvessels obtained in the pellet were further digested with collagenase/dispase (1 mg/ml; Roche, Mannheim, Germany) and DNase I (6.7 μg/ml) in DMEM for 1 h at 37 °C. Microvessel endothelial cell clusters were separated on a 33% continuous Percoll (GE Healthcare, Buckinghamshire, UK) gradient (1000g for 10 min), collected, and washed in DMEM before plating on culture dishes coated with collagen type IV and fibronectin (both 0.1 mg/ml; Sigma). RBEC cultures were maintained in DMEM/F12 (Sigma) supplemented with 10% bovine plasma derived serum (Animal Technologies, Tyler, TX), basic fibroblast growth factor (1.5 ng/ml; Roche), heparin (100 μg/ml; Sigma), insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml) (insulin-transferrinsodium selenite media supplement; Sigma), gentamicin (50 μg/ml), and puromycin (4 μg/ml; Sigma) (RBEC medium I) at 37 °C in a humidified atmosphere of 5% CO2/95% air, for 2 days. On the third day, the cells received a new medium that contained all the components of RBEC medium I except for puromycin (RBEC medium II). When the cultures reached confluency, the purified endothelial cells were passaged and used to construct in vitro BBB models. Brain pericytes were obtained by a prolonged culture of isolated brain microvessel fragments under selective culture conditions (Dohgu et al., 2005; Takata et al., 2009; Nakagawa et al., 2007). Briefly, the obtained brain microvessel fragments were placed in an uncoated culture flask in DMEM supplemented with 20% fetal bovine serum (FBS; Biowest, Nuaillé, France) (FBS-DMEM), 100 units/ml penicillin, and 100 μg/ml streptomycin (Nacalai Tesque, Kyoto). After 7 days in culture, rat pericytes overgrew brain endothelial cells and typically reached 80–90% confluency. The cells were used at passage 2.
The median plasma levels of PAI-1 in healthy control subjects and patients with venous thrombosis were more than 100 ng/ml (Meltzer et al., 2010). Based on these data, we treated RBECs with various doses of PAI-1 up to 100 ng/ml. Recombinant rat PAI-1 (Calbiochem, La Jolla, CA) was supplied in solution and this original solution was diluted with a solution containing 100 mM sodium acetate, 100 mM NaCl, 1 mM EDTA, pH 5.0 to obtain 100-fold concentrations for experiments. These PAI-1 solutions and an anti PAI-1 mouse monoclonal antibody (100 μg/ml; Affinity BioReagents, Golden, CO) were diluted with serum-free RBEC medium II containing 500 nM hydrocortisone in the luminal and/or abluminal side of the Transwell insert to expose RBECs. In all experiments, controls were RBECs treated with the corresponding amount of mouse IgG (Sigma) or the solvent for PAI-1. Measurement of transendothelial electrical resistance (TEER) TEER across the monolayers grown on the filter membranes was measured by an EVOM resistance meter in an EndOhm tissue resistance measurement chamber (World Precision Instruments, Sarasota, FL). Values are presented as Ω × cm2 culture insert. The TEER of cell-free inserts was subtracted from the obtained values. Measurement of transendothelial transport of sodium fluorescein (Na-F) Endothelial barrier function was evaluated by measuring permeability of RBECs to Na-F as previously described (Dohgu et al., 2004a, 2005). To initiate the transport experiments, the medium was removed and then physiological buffer (141 mM NaCl, 4 mM KCl, 2.8 mM CaCl2, 1.0 mM MgSO4, 1.0 mM NaH2PO4, 10 mM HEPES and 10 mM d-glucose, pH 7.4) containing 100 μg/ml Na-F (Sigma) was loaded in the luminal chamber of the insert (0.1 ml). Samples (0.4 ml) were removed from the abluminal chamber at 15, 30, 45, and 60 min and immediately replaced with fresh physiological buffer. The concentration of Na-F was determined using a fluorescence multiwell plate reader (Ex(λ) 485 nm; Em(λ) 530 nm; CytoFluor Series 4000; PerSeptive Biosystems, Framingham, MA). The permeability coefficient and clearance were calculated as previously described (Dehouck et al., 1992; Dohgu et al., 2005). Measurement of PAI-1 in culture supernatants RBEC monolayers were incubated in RBEC medium II with or without 10% bovine plasma-derived serum for 24 h. Culture supernatants were collected from the luminal and abluminal chambers, and stored at -80 °C until use. Levels of PAI-1 in culture supernatants were measured with the total rat PAI-1 antigen ELISA kit (Innovative Research Inc., Novi, MI) by following the manufacturer's instructions.
Preparation of the in vitro BBB models Statistical analysis Preparation of the in vitro BBB models co-culturing RBECs and brain pericytes has been described previously (Dohgu et al., 2005). Brain pericytes (4 × 104 cells/well) were seeded in wells of 24-well
Values are expressed as means ± SEM. The Student's t-test was applied to compare the two groups. One-way and two-way analyses
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of variance (ANOVAs) followed by Tukey–Kramer's test were applied to multiple comparisons. The differences between means were considered to be significant when p values were less than 0.05 using Prism 5.0 (GraphPad, San Diego, CA).
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chambers of the RBEC monolayer. A 24-h exposure to PAI-1 produced equal rate of decrease in Na-F permeability when PAI-1 was added to either the luminal or abluminal chambers (35.7% and 44.1% of decrease, respectively). Addition of PAI-1 to both the luminal and abluminal chambers failed to facilitate the lowered permeability of RBECs to Na-F (Fig. 1B).
Results Polarized release of PAI-1 from RBECs Effect of PAI-1 on BBB permeability First, to determine the effect of PAI-1 on brain endothelial barrier function, we treated RBEC monolayers with recombinant rat PAI-1 (0.01, 1, and 100 ng/ml) for 24 h. PAI-1 was added every 6 h to both the luminal and abluminal chambers since PAI-1 in the serum-free culture medium was degraded to 8.9 ± 0.3% of the initial loaded amount within 6 h (Fig. 1A inset). PAI-1 increased TEER of RBECs time- and dosedependently (Fig. 1A) [dose: F(3, 40) = 9.121, p b 0.01; time: F(4, 40) = 15.30, p b 0.001; interaction (dose× time): F(12, 40) = 3.315, p b 0.01]. This reached a peak 12 h after the treatment. Second, to test whether RBECs showed a polarized response to PAI-1 for permeability to Na-F, PAI-1 (100 ng/ml) was added every 6 h to the luminal and/or abluminal
To determine spontaneous release of PAI-1 from RBECs in the presence or absence of serum, we measured the levels of PAI-1 in the luminal and abluminal chambers of RBEC monolayers. PAI-1 was detected in both the luminal and abluminal chambers (Fig. 2). When RBECs were cultured under serum-free conditions, 4-fold higher levels of PAI-1 were released to the luminal chamber compared with the abluminal chamber (0.81 ± 0.06 vs 0.19 ± 0.01 ng/ml). In the presence of serum (10% bovine plasma-derived serum), RBECs released 10- to 20-fold higher levels of PAI-1 to both the luminal and abluminal chambers compared with serum-free conditions. The baseline level of PAI-1 in the medium containing bovine plasmaderived serum was below detectable levels. Effect of PAI-1 inhibition on TEER of RBECs in RBEC monolayers and RBEC/pericyte co-cultures Pericytes also released PAI-1 (3.1 ±0.1 ng/ml) under serum-free conditions. We examined the effect of PAI-1 neutralizing antibody on brain endothelial barrier function in the presence or absence of pericytes. Adding PAI-1 neutralizing antibody (10 μg/ml) to both luminal and abluminal chambers significantly decreased TEER of RBECs by 41.5% and 57.6% of control (IgG) in RBEC monolayers and RBEC/pericyte cocultures, respectively (Fig. 3). A two-way ANOVA showed significant effects for the culture system (monolayer and co-culture) [F(1, 18) = 13.53, p b 0.01], treatment (IgG and PAI-1 antibody) [F(1, 18) = 33.08, p b 0.001], and interaction (culture system × treatment) [F(1, 18) = 5.746, p b 0.05]. The extent of decrease in TEER of RBECs in RBEC/pericyte co-cultures was significantly greater than that of RBEC monolayers (p b 0.05). Discussion Under pathological conditions, the cytokine profile in the blood is dramatically altered (Kunz and Ibrahim, 2009). High plasma levels of PAI-1 are considered a risk factor of vascular diseases. Brain endothelial cells face two compartments, the blood and brain. This allows cells to receive stimuli mediated by various substances from the cerebral microcirculation and CNS cells (e.g., pericytes, astrocytes,
Fig. 1. (A) Time-course of the changes in TEER of RBECs after exposure to various concentrations of PAI-1 during a 24-h period. PAI-1 was added to both the luminal and abluminal chambers of RBEC monolayers every 6 h. Data are expressed as the percentage of the time-matched control value. TEER values of control RBECs were 108.0 ± 3.4, 91.6 ± 2.9, 134.6 ± 1.8, 135.5 ± 5.5, and 191.7 ± 11.3 Ω × cm2 at 0, 1, 6, 12, and 24 h, respectively (n = 3–4). **p b 0.01, ***p b 0.001 compared with controls. The inset shows the time-course of PAI-1 levels in the medium after adding PAI-1. Data are expressed as the percentage of the initial concentration of PAI-1 (0 h; 11.5 ± 1.4 ng/ml) (n = 3). (B) Non polarized response to PAI-1 in the permeability of RBECs to Na-F. RBEC monolayers were treated with PAI-1 (100 ng/ml) added to the luminal or abluminal chambers for 24 h. PAI-1 was added to the luminal or abluminal chambers of RBEC monolayers every 6 h. Data are expressed as the percentage of controls (9.79 ± 1.12 × 10−5 cm/min) (n = 7–8). *p b 0.05, **p b 0.01 compared with controls. Values are means ± SEM.
Fig. 2. Spontaneous release of PAI-1 into the luminal or abluminal chambers of RBECs in the presence or absence of serum. After 24 h incubation, the medium was collected from both the luminal and abluminal chambers. Values are means ± SEM (n = 3). **p b 0.01, ***p b 0.001 compared with the luminal chamber.
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Fig. 3. Effect of anti-PAI-1 antibody on TEER of RBECs in RBEC monolayers and RBEC/ pericyte co-cultures. RBEC monolayers and RBEC/pericyte co-cultures were treated with mouse IgG and anti-PAI-1 antibody (10 μg/ml) added to both the luminal and abluminal chambers for 24 h. Values are means ± SEM (n = 3–8). *p b 0.05, **p b 0.01 compared with IgG; ††p b 0.01 compared with IgG-treated RBEC monolayers.
microglia, and neurons), leading to alterations in endothelial barrier function. In the present study, we found that BBB cell-produced and exogenous PAI-1 enhanced barrier properties of the BBB. To examine the effect of PAI-1 derived from the blood and brain on BBB function, we first exposed RBECs to PAI-1 added to both luminal (blood side) and abluminal (brain side) chambers of Transwell® inserts. PAI-1 in the medium was rapidly degraded during a 6 hperiod and remained less than 10% of the initial amount 6 h after addition (Fig. 1A inset). A similar finding was also reported by McIlroy et al. (2006). Therefore, we added PAI-1 to the luminal and/or abluminal chambers of RBEC monolayers every 6 h in the experiments. PAI-1 increased TEER of RBECs dose- and time-dependently (Fig. 1A). We also confirmed that PAI-1 (100 ng/ml) added to both the luminal and abluminal chambers decreased RBEC permeability to NaF, which is a paracellular permeability marker (Fig. 1B). Passage of molecules across brain endothelial cells of the BBB occurs through intercellular tight junctions (paracellular pathway) or through the cells (transcellular pathway). The permeability of the BBB to molecules is determined by the overall transendothelial transport through these two pathways. The paracellular pathway allows ions and small hydrophilic solutes to diffuse between adjacent cells according to their concentration gradient. The transcellular pathway involves various transport mechanisms such as passive diffusion of lipophilic compounds, transporter-mediated transport, and receptormediated transcytosis. Ions and hydrophilic solutes mainly permeate across the BBB through paracellular pathways. The TEER and Na-F permeability of RBECs evaluated in our experiments indicate the permeability of RBECs to ions and small hydrophilic solutes, respectively. These are associated with the integrity of the paracellular barrier sealed with tight junctions in RBECs. In addition, it has been reported that Na-F is a substrate for organic anion transporter (OAT)-3 and multidrug resistance-associated protein (MRP)-2 (Hawkins et al., 2007). Therefore, PAI-1 may regulate not only the paracellular barrier function but also OAT-3- and MRP-2-mediated transcellular permeability. To determine the functional polarity of RBECs in response to PAI-1, we added PAI-1 to either luminal or abluminal chambers. The PAI-1induced decrease in the permeability of RBECs to Na-F was independent of the PAI-1-injected site. This result indicated that RBECs are capable of responding to stimulation of PAI-1 derived from either the blood or brain. Although PAI-1 in the medium was almost degraded within 6 h, TEER of RBECs continuously increased over a period of 6 h. The possibility that PAI-1 induces the release of other substances maintaining barrier function from RBECs cannot be excluded. Various intracellular signaling pathways are involved in
the expression and phosphorylation of tight junction proteins, and these effects are associated with alterations to the barrier function (Cardoso et al., 2010). Although the PAI-1 receptor on brain endothelial cells and its subsequent signaling pathway have remained obscure, the possibility that PAI-1 could directly regulate the expression and/or phosphorylation of tight junction proteins to enhance BBB functions should be considered. These findings suggest that PAI-1 is a positive regulator of BBB function. We found that RBECs spontaneously released PAI-1 into both the luminal and abluminal sides (Fig. 2). This release of PAI-1 was polarized, and its release into the luminal (blood) compartment was superior to that in the abluminal side. This is consistent with the release of IL-1α, IL-6, IL-10, granulocyte-macrophage colony-stimulating factor, and TNF-α from mouse brain endothelial cells (Verma et al., 2006). Stimulation of RBECs with serum significantly increased the release of PAI-1 into both the luminal and abluminal chambers. Oxidized very-low-density lipoprotein increases PAI-1 expression in endothelial cells (Zhao et al., 2008). These findings suggest that PAI-1 released from brain endothelial cells is inducible by a number of unknown substances in the serum. We examined whether spontaneous release of PAI-1 from RBECs maintained barrier function (Fig. 3). Anti-PAI-1 neutralizing antibody significantly lowered TEER in RBEC monolayers, suggesting that PAI-1 up-regulates BBB function in an autocrine and paracrine manner. When RBECs were co-cultured with pericytes, the anti-PAI-1 antibody-lowered TEER of RBECs was aggravated. We found that pericytes also released PAI-1. Previous studies have shown that PAI-1 expression is increased in brain endothelial-pericyte co-culture (Kim et al., 2006). These findings indicate that PAI-1 mediates pericyte-induced up-regulation of BBB function. Our previous study showed that pericytes up-regulate BBB function by secreting transforming growth factor-β1 (TGF-β1) (Dohgu et al., 2005). TGF-β1 facilitates the barrier function of brain endothelial cells, and this effect may be related to the regulation of the expression of tight junction proteins (occludin, claudins, and ZO-1) induced by the TGF-β/ALK5 signaling pathway (Dohgu et al., 2004b; Ronaldson et al., 2009). Furthermore, TGF-β1 induces mRNA expression of PAI-1 in brain endothelial cells and pericytes (Kose et al., 2007). Therefore, it is conceivable that PAI-1 cooperates with TGF-β1 to maintain BBB function. It has been shown that tPA aggravates ischemic brain damage (Nagai et al., 1999; Wang et al., 1998) and directly triggers the disruption of brain endothelial barrier function (Hiu et al., 2008). PAI1 exerts a protective effect against ischemic brain injury by inhibiting tPA activity (Yang et al., 2009). Although the direct mechanism by which PAI-1 up-regulates BBB function has not been elucidated, constitutive expression of both tPA and PAI-1 in the cellular components of the BBB (brain endothelial cells, pericytes, and astrocytes) is considered to contribute, at least in part, to pericyteinduced up-regulation of BBB function. Pericytes decrease the mRNA and protein expression of tPA and increase those of PAI-1 in brain endothelial cells (Kim et al., 2006). PAI-1 expression in the monolayers of brain endothelial cells or astrocytes was facilitated by co-culturing with these cells (Hultman et al., 2010). These findings suggest that brain endothelial cells, pericytes, and astrocytes cooperatively regulate BBB permeability through their expression of tPA and PAI-1. Thus, the balance between tPA and PAI-1 in the elements of the BBB is considered to be crucial for the integrity of BBB function. In addition, PAI-1 released from astrocytes is thought to act as a neurotrophic factor, and this phenomenon is independent of the ability for inhibition of tPA and uPA (Kimura et al., 2000; Soeda et al., 2001, 2004, 2006). Taken together, the present findings are important in the understanding of cross-talk mediated by PAI-1 in the neurovascular unit. In conclusion, we demonstrated that PAI-1 participates as a positive regulator of the BBB in facilitating the barrier function of the endothelial tight junctions.
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Acknowledgments This work was supported in part by Grants-in-Aid for Young Scientists ([Start-up] 20800066), and Grants-in-Aid for Young Scientists ([B] 21790102, [B] 21790255, [B] 21790257 and [B] 21790526) from JSPS, Japan, the Ministry of Health, Labour and Welfare of Japan (H19nanchi-ippan-006), and Kakihara Science and Technology Foundation.
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