,

,

*School of Chinese Medicine, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

�Center of Neurology Rehabilitation, Second Affiliated Hospital of Wenzhou Medical College,

Wenzhou, China

�Department of Neurology Rehabilitation, Wenzhou Chinese Medicine Hospital, Wenzhou, China

§Department of Anatomy, The University of Hong Kong, Pokfulam, Hong Kong SAR, China

¶College of Pharmacy, The University of New Mexico, Albuquerque, New Mexico, USA

**Research Center of Heart, Brain, Hormone & Health Aging, The University of Hong Kong,

Pokfulam, Hong Kong SAR, China

Abstract

The roles of caveolin-1 (cav-1) in regulating blood–brain bar-

rier (BBB) permeability are unclear yet. We previously

reported that cav-1 was down-regulated and the production of

nitric oxide (NO) induced the loss of cav-1 in focal cerebral

ischemia and reperfusion injury. The present study aims to

address whether the loss of cav-1 impacts on BBB perme-

ability and matrix metalloproteinases (MMPs) activity during

cerebral ischemia-reperfusion injury. We found that focal

cerebral ischemia-reperfusion down-regulated the expression

of cav-1 in isolated cortex microvessels, hippocampus, and

cortex of ischemic brain. The down-regulation of cav-1 was

correlated with the increased MMP-2 and -9 activities,

decreased tight junction (TJ) protein zonula occludens (ZO)-1

expression and enhanced BBB permeability. Treatment of NG-

nitro-L-arginine methyl ester [L-NAME, a non-selective nitric

oxide synthase (NOS) inhibitor] reserved the expression of

cav-1, inhibited MMPs activity, and reduced BBB permeability.

To elucidate the roles of cav-1 in regulating MMPs and BBB

permeability, we used two approaches including cav-1

knockdown in cultured brain microvascular endothelial cells

(BMECs) in vitro and cav-1 knockout (KO) mice in vivo. Cav-1

knockdown remarkably increased MMPs activity in BMECs.

Meanwhile, with focal cerebral ischemia-reperfusion, cav-1

deficiency mice displayed higher MMPs activities and BBB

permeability than wild-type mice. Interestingly, the effects of

L-NAME on MMPs activity and BBB permeability was partly

reversed in cav-1 deficiency mice. These results, when taken

together, suggest that cav-1 plays important roles in regulating

MMPs activity and BBB permeability in focal cerebral ischemia

and reperfusion injury. The effects of L-NAME on MMPs

activity and BBB permeability are partly mediated by preser-

vation of cav-1.

Keywords: blood–brain barrier, caveolin-1, ischemia, nitric

oxide synthase.

J. Neurochem. (2012) 120, 147–156.

Read the Editorial Highlight for this article on page 4.

Received June 13, 2011; revised manuscript received August 17, 2011;accepted September 7, 2011.Address correspondence and reprint requests to Dr Jiangang Shen,

School of Chinese Medicine, The University of Hong Kong, 10 SassoonRoad, Pokfulam, Hong Kong SAR, China.E-mail: shenjg@hkucc.hku.hk

Abbreviations used: BBB, blood–brain barrier; BMECs, brainmicrovascular endothelial cells; cav-1, caveolin-1; DMEM, Dulbecco’smodified Eagle’s medium; eNOS, endothelial nitric oxide synthase; ICA,internal carotid artery; KO, knockout; L-NAME, NG-nitro-L-argininemethyl ester; MCAO, middle cerebral artery occlusion; NO, nitric oxide;NOS, nitric oxide synthase; PBS, phosphate-buffered saline; SD, Spra-gue-Daweley; TJ, tight junction; ZO-1, zonula occludens-1.

JOURNAL OF NEUROCHEMISTRY | 2012 | 120 | 147–156 doi: 10.1111/j.1471-4159.2011.07542.x

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 147–156 147

Nitric oxide plays an important role in the regulation of BBBpermeability, brain infarction and cell death during cerebralischemia-reperfusion injury. Physiological concentration ofNO (less than 10 nM) generated from endothelial NOS(eNOS) participates in neuronal communication, vasculartone regulation, synaptic transmission and plasticity, etc.(Moncada et al. 1991; Kiss and Vizi 2001; Conti et al. 2007;Lundberg et al. 2008; Forstermann 2010). During cerebralischemia and reperfusion injury, however, high concentrationof NO derived from neuronal NOS activation and de novosynthesis of inducible NOS can induce cell death (Dalkaraet al. 1998), increase infarction volume (Malinski et al.1993; Matsui et al. 1999; Murphy and Gibson 2007), andinduce BBB hyper-permeability (Gursoy-Ozdemir et al.2000, 2004). Although the roles of NO in cerebralischemia-reperfusion injury are intensively studied, themechanisms of NO in regulation of BBB permeability arenot well understood.

Activation of MMPs is one of the critical pathways in theBBB opening (Gasche et al. 1999; Aoki et al. 2002;Pfefferkorn and Rosenberg 2003; Yang et al. 2007). MMPsare a group of proteases with more than 20 members. MMP-2, -3, and -9 are the main forms of MMPs in brain. Incomparison with a broad spectrum of activity of MMP-3against extracellular matrix, MMP-2 and MMP-9 shownarrow ranges of substrates but including basement mem-brane elements. MMP-2 is present in astrocytic end feetsurrounding cerebral blood vessels. As key elements inregulation of BBB permeability, TJ proteins are sensitive toMMP-9 activity (Ichiyasu et al. 2004). Activated MMPs canhydrolyze BBB extracellular matrix and TJ proteins,degrades extracellular matrix around cerebral blood vesselsand neurons, and subsequently leads to the BBB opening,brain edema, hemorrhage, and cell death. Administration ofnon-selective NOS inhibitor L-NAME significantly reducedvascular damage and MMP-9 activity in cerebral ischemia-reperfusion injury (Gursoy-Ozdemir et al. 2000, 2004).Those studies indicate that NO mediates the BBB disruptionthrough MMPs activation.

Caveolin-1, an integral membrane protein, appears toregulate the activation of NOS and MMPs as well as BBBpermeability (Gu et al. 2011). Cav-1 binds to all isoforms ofNOS that contain cav-binding motif, and inhibits NOproduction (Garcia-Cardena et al. 1997; Ju et al. 1997;Bucci et al. 2000; Sato et al. 2004). Cav-1 KO mice sufferedfrom microvascular hyper-permeability and treatment ofL-NAME reduced BBB permeability (Schubert et al. 2002).Our previous study revealed that ischemia-reperfusion down-regulated the expression of cav-1 protein and NOS inhibitorsprevented the loss of cav-1 in an experimental transientmiddle cerebral artery occlusion (MCAO) rat model (Shenet al. 2006). Treated with transient MCAO ischemia-reper-fusion, cav-1 KO mice had more serious infarction volumethan wild-type mice (Jasmin et al. 2007). Cav-1 co-localized

with MMP-2 on the surface of endothelial cells (Puyraimondet al. 2001; Chow et al. 2007). Cav-1 could modulatechemokine-induced BBB permeability by up-regulating TJand adheren junction proteins in BMECs (Song et al. 2007).Cav-1 KO mice revealed the increases of eNOS activity andNO production in endothelial cells and had endothelialhyper-permeability (Siddiqui et al. 2011). However, in ratcortical cold injury model, increased cav-1 expression andphosphorylation were associated with decreased level ofoccludin and claudin-5 and brain edema (Nag et al. 2007,2009). Therefore, it is desirable to further clarify the roles ofcav-1 in regulating MMPs activity and BBB permeabilityduring cerebral ischemia-reperfusion injury.

In this study, we hypothesized that loss of cav-1 mightcontribute to NO-induced MMPs activity and BBB disrup-tion during cerebral ischemia-reperfusion injury. We inves-tigated the expressions of cav-1 and ZO-1, activities ofMMP-2 and MMP-9, and BBB permeability in focal cerebralischemia-reperfused rats with or without L-NAME treatment.The roles of cav-1 in regulation of MMPs activity and BBBpermeability were further verified by cav-1 deletion in vivoand in vitro. This study provides direct evidence to prove that(i) Cav-1 plays a critical role in protecting BBB integritythrough inhibition of MMPs activity in brain microvesselsand other brain tissues during cerebral ischemia-reperfusionanimal injury; (ii) the effects of L-NAME on protecting BBBintegrity are partly mediated through preserving cav-1expression and inhibiting MMPs activity in brain microves-sels during cerebral ischemia-reperfusion injury.

Materials and methods

Focal cerebral ischemia-reperfusion and surgical proceduresMale adult Sprague-Daweley (SD) rats and wild-type C57BL/6Jmice were obtained from Laboratorial Animal Unit, The Universityof Hong Kong. Cav-1 KO mice (Cav-1 KO; Cav-1 tm1Mls/J) wereobtained from Jackson Laboratory, Bar harbor, ME, USA and theirheterozygote breedings were genotyped and used in the experi-ments. All of animal experimental protocols were approved andregulated by the Committee on the Use of Live Animals in Teachingand research, University of Hong Kong. Every effort was made tominimize the number of animals used and their suffering. Theanimals were maintained at a controlled temperature (20 ± 2�C) andgroup-housed (12 h light/dark cycle) with access to food and waterad libitum.

Cerebral ischemia and reperfusion was induced by using MCAOmodel as our previously described (Shen et al. 2006). Briefly, adultSD rats weighting 250�280 g were anaesthetized by inhalation of5% isoflurane and maintained with 2% isoflurane in a mixture of70% N2O and 30% O2. After external carotid artery, internal carotidartery (ICA) and pterygopalatine artery of the ICA were exposed, apiece of 3/0 monofilament nylon suture (Ethicon Johnson-Johnson,Brussels, Belgium), with its tip rounded by gentle heating, wasintroduced via lumen of left external carotid artery stump and leftICA to embed into left anterior cerebral artery so that left middle

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 147–156� 2011 The Authors

148 | Y. Gu et al.

cerebral artery was occluded at its origin. After 2 h of ischemia, theintra-luminal suture was withdrawn from left anterior cerebral arteryand right ICA to permit reperfusion. After operation, rats weretransferred to intensive care incubator in which the temperature waskept at 37�C until animals woke up completely. Ketoprofen wassubcutaneously injected for post-operative care. For wild-type andcav-1 KO mice, similar surgical procedures and drug treatments torats were used, in which a 6/0 monofilament rather than 3/0 nylonsuture was used to occlude middle cerebral artery, and the suturewas removed after 15 min occlusion to induce reperfusion for 24 h.

Drug treatmentBoth rats and mice were divided into three groups: sham operationgroup, MCAO group, and MCAO with L-NAME treatment group.In the L-NAME-treated group, rats or mice were intraperitoneallyinjected with 3 mg/kg L-NAME (Sigma, St Louis, MO, USA) at15 min before suture insertion. Sham-operated and MCAO rats wereadministrated with same volume of saline.

Evens blue leakage experimentsBlood–brain barrier integrity was evaluated by measuring theextravasation of Evans blue dye (Yu et al. 2008). Briefly, 2%Evans blue was injected (2 mL/kg, i.v.) before 1 h of killing. At24 h of reperfusion, rats and mice were transcardially perfusedwith 0.9% NaCl to remove the intravascular dye until the drainagewas colorless. After that, ipsilateral hemisphere was removed andincubated in N,N¢-dimethyl formamide (1 mL/200 mg brain;Sigma) at 60�C for 24 h. Evans blue content was determined insupernatants at 632 nm using a spectrophometer (Analytica Jena,Germany). Meanwhile, gradient concentrations of Evans blue wereused for standard curve. BBB leakage was represented as lg pergram brain.

In situ zymography for measuring MMP-2/9 activity in brainsGelatinolytic activities of MMP-2/9 in frozen brain sections wereanalyzed by in situ zymography using EnzCheck collagenase kit(Invitrogen, Carlsbad, CA, USA) following the manufacturer’sinstructions. Frozen brain sections were incubated with a reactionbuffer containing 40 lg/mL of FITC-labeled DQ gelatin (Invitro-gen) at 37�C for 2 h. Gelatin-FITC was cleaved by gelatinases andyielded the peptides whose fluorescence intensity was used as therepresentatives of the net gelatinolytic activity. Fluorescenceintensity was measured with a fluorescence microscopy (Zeiss,Oberkochen, Germany) under constant exposure.

In situ immunofluorescent detections of cav-1 and ZO-1 in brainsFrozen coronal sections from normal control and ischemia-reper-fused brains were used for detections of cav-1 and ZO-1 withimmunoflorescent chemistry. The sections were treated withblocking buffer containing 0.1% Triton (Sigma) and 5% goat serum(Sigma) and 50 mM phosphate-buffered saline (PBS) for 1 h, andthen incubated with polyclonal rabbit cav-1 (1 : 500; Cell signaling,Danvers, MA, USA) or polyclonal rabbit ZO-1 (1 : 100; Invitrogen)overnight at 4�C. After rinsed with PBS, the slides were incubatedwith an appropriate Alexa 568 Goat anti-mouse secondary antibody(1 : 500; Invitrogen) or Alexa 488 Goat anti-rabbit secondaryantibody (1 : 500; Invitrogen) for 1 h at 25�C. Sections weremounted with anti-fade fluorescence mounting medium (Dako,

Glostrup, Denmark). Images were acquired using fluorescentmicroscope at a constant exposure.

Isolation of cortex microvesselsMinced cortexes isolated from normal control and ischemia-reperfused brains were digested with 1 mg/mL collagenase II(Hyclone, Lakewood, NJ, USA) in Dulbecco’s modified Eagle’smedium (DMEM; Hyclone, Erie, PA, USA) in a shaker for 1.5 h at37�C. The pellet was then separated by centrifugation in 20%bovine serum albumin-DMEM (1000 g, 20 min, 4�C). The micro-vessels were obtained in the pellet, washed with PBS, and lysed inradio-immunoprecipitation assay buffer (Sigma) with proteinaseinhibitor cocktail (Roche, Basel, Swiss) for western blot analysisand gelatin zymography.

Cell cultureRat BMECs were cultured as previously described with minormodifications (Lin and Rui 1994). Briefly, fresh rat brains wereobtained from 3- to 5-week-old SD rats, and placed in ice-coldD-Hanks solution. The cortices were isolated and rolled on dry filterpaper to remove adherent surface cells and cut into uniform 1 mm3

sections in D-Hanks. Sections were digested in 0.1% type IIcollagenase solution (Sigma) in DMEM for 1.5 h at 37�C to separatethe microvessels. The samples were centrifuged for 20 min at 1000 gin 20% bovine serum albumin (Sigma) at 4�C. After washed withDMEM, microvessel fragments were then plated on type IV collagen(Sigma) coated dishes and cultured with minimal essential mediacontaining 20% fetal bovine serum (Gibco, Grand island, NY, USA),basic fibroblast growth factor (10 ng/mL; Roche), heparin (100 lg/mL; Sigma), streptomycin (100 lg/mL; Invitrogen), and penicillin(100 U/mL; Invitrogen). Cells were incubated at 37�C in a humidatmosphere of 95% air and 5% CO2 and used for experiments whenconfluent at 7 days. Culture medium was replaced every 3 days. Cellpurity was confirmed to be more than 95% by immunostaining ofFactor VIII related antigen (1 : 100; Thermo Scientific, Rockford, IL,USA), a specific marker of endothelial cells.

Knockdown of Cav-1 in BMECsBMECs were transfected with Cav-1 Stealth� RNAi (Invitrogen).The Cav-1 Stealth� RNAi provides non-overlapping Stealth�RNAi duplex for this gene to obtain high knock-down efficiency.The duplex was transfected into the BMECs with LipofectamineRNAiMAX (Invitrogen). Each cav-1 Stealth� RNAi duplex wasadded at a concentration of 13 nM. A Stealth� RNAi NegativeControl Duplex (Invitrogen) was also used to transfect the BMECsas control group.

Gelatin zymography for measuring MMP-2/9 activity inmicrovessels and BMECsProteins from isolated microvessels and cell lysate were subject togelatin zymography. The protein concentration was determinedusing Bradford reagent (Bio-Rad Laboratories, Hercules, CA, USA).MMP-2/9 activity was analyzed by gelatin zymography as previ-ously described with minor modifications (Yang et al. 2007).Protein samples (20 lg) were loaded on 10% sodium dodecylsulfate–polyacrylamide gels, co-polymerized with 1 mg/mL gelatin(Sigma). Gels were washed in 2.5% Triton X-100 (Sigma) for 1 hand then incubated for 24 h in a developing buffer, including Tris

� 2011 The AuthorsJournal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 147–156

NOS inhibitor protects BBB through caveolin-1 | 149

50 mmol/L, pH 7.6, CaCl2 5 mmol/L, NaCl 0.2 mmol/L, and 0.02%(w/v) Brij-35 (Sigma) at 37�C followed by staining with Coomassieblue. Gels were destained until clear bands of gelatinolysisappeared.

Western blot analysisDenatured protein samples were resolved on sodium dodecylsulfate–polyacrylamide gels and transferred to polyvinylidenefluoride membranes (Millipore, Billerica, MA, USA). After block-ing, membranes were incubated overnight at 4�C with polyclonalrabbit Cav-1 (1 : 1000; Cell signaling) followed by incubation withthe goat anti-mouse horseradish peroxidase-conjugated secondaryantibodies (1 : 2000; Santa Cruz Biotechnology, Santa Cruz, CA,USA). Chemiluminescence detection was performed using ECLadvance western blotting detection reagents (BD biosciences, SanJose, CA, USA).

Statistics analysisData were presented as means ± standard deviation. Statisticanalysis was performed using SPSS 18.0 statistical programs (SPSS,Chicago, IL, USA). For multiple groups designed experiments,comparisons were made by one-way analysis of variance (ANOVA)and followed by Dunnett test for two group comparison within themultiple groups. For two groups designed experiments, comparisonswere determined using unpaired Student’s t-test. Statisticallysignificance was set as a probability level of p < 0.05.

Results

L-NAME treatment prevented ischemia-reperfusioninduced down-regulation of cav-1 in brain microvesselsand brain tissuesOur previous study revealed that ischemia-reperfusion down-regulated the expression of cav-1 in ischemic brain tissues(Shen et al. 2006). To elucidate whether the down-regulationof cav-1 is associated with BBB hyper-permeability inischemic brains, we investigated the expression of cav-1 inbrain microvessels isolated from ipsilateral hemispheres offocal cerebral ischemia-reperfused rats. The rats were subjectto 2 h of ischemia plus 6, 24, and 72 h of reperfusion. Theresults were showed in Fig. 1. After rats were exposed to 2 hof ischemia following 6, 24, and 72 h of reperfusion, therewere remarkably down-regulation in the expression of cav-1in brain microvessels (Fig. 1a–c). To elucidate the roles ofNO in down-regulating cav-1, we investigated the effects ofL-NAME on the expression of cav-1 in the microvesselsisolated from ischemia-reperfused rat brains. Since 2 h ofischemia following 24 h of reperfusion resulted in aremarkably down-regulation of cav-1 in brain microvessels,we particularly selected this time point for further studies.L-NAME (3 mg/kg) was intraperitoneally administered intothe rats at 15 min prior to MCAO ischemic treatment. L-NAME treatment rescued the expression of cav-1 in themicrovessels (Fig. 1b and d). To explore whether ischemia-reperfusion affects brain tissues of ipsilateral hemispheres,

we also investigated the expression of cav-1 in cortex,hippocampus, and whole brain coronal sections. Similarly,the expression of cav-1 was down-regulated in those areasand L-NAME treatment prevented the loss of cav-1 in thecortex (Fig. 1e and g), hippocampus (Fig. 1f and h), andwhole brain coronal sections of the ischemia-reperfusedbrains (Fig. 2c). The results indicate that L-NAME canreserve the expression of cav-1 in brain microvessels andtissues during focal cerebral ischemia-reperfusion injury.

L-NAME treatment decreased MMPs activity, protected TJprotein, and reduced BBB permeability in rat cerebralischemia-reperfusion injuryPrevious studies reported that L-NAME treatment decreasedinfarction volume, neurological deficit, and BBB disruptionin a cerebral ischemic mouse brain in vivo (Ding-Zhou et al.2002), and in an oxygen glucose deprivation model of thehuman BBB in vitro (Cowan and Easton 2010). MMPsactivation contributes to TJ impairment and BBB disruption(Gasche et al. 2006; Sandoval and Witt 2008). L-NAMEreduced Evans blue extravasation and MMP-9 expression inMCAO mice brains (Gursoy-Ozdemir et al. 2000, 2004).However, direct evidence is still lack regarding to the effectsof NOS inhibitors on the activation of MMP-2, -9, andimpairment of TJ proteins in brain microvessels and the linksof these changes to BBB permeability. Thus, we investigatedthe activities of MMP-2 and -9 and the expression of ZO-1, aTJ protein, in cortex microvessels and coronal brain sectionsisolated from the focal cerebral ischemia-reperfused rats withor without L-NAME treatment (3 mg/kg, i.p.). Evans blueleakage assay was used to evaluate the BBB permeabilityand gelatinase activity for detecting MMPs activity. Theresults were shown in Figs 2 and 3. Ischemia-reperfusionremarkably increased BBB permeability whereas L-NAMEtreatment significantly reduced BBB permeability in thebrain microvessels (Fig. 3a). In the meantime, ischemia-reperfusion increased activities of MMP-2, -9, whereasL-NAME treatment remarkably inhibited the ischemia-reperfusion-induced activities of MMP-2,-9 in the brainmicrovessels and cortex (Figs 2a, d and 3b–d). Moreover,immunofluorescent studies revealed that ischemia-reperfu-sion decreased the expression of ZO-1, whereas L-NAMErestored the expression of ZO-1 in the ischemia-reperfusedbrains (Fig. 2b and e). The results indicate that L-NAME caninhibit MMPs activity, protect TJ protein, and reduce BBBpermeability in cerebral ischemia-reperfusion injury.

Cav-1 deficiency increased MMPs activity and BBBpermeability in focal cerebral ischemia-reperfusion micewith or without L-NAME treatmentTo address whether loss of cav-1 contributes to MMPsactivity in brain microvessels, we artificially manipulatedcav-1 level with Cav-1 RNAi techniques in primary culturedrat BMECs. BMECs were transfected with Cav-1 Stealth�

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 147–156� 2011 The Authors

150 | Y. Gu et al.

RNAi to knock down the expression of cav-1 or treated withStealth� RNAi Negative Control Duplex as negativecontrol. Medium and cell lysate were collected at 24 h aftertransfection and the declined level of cav-1 in cell lysate wasconfirmed with western blot analysis. MMPs activity inmedium was examined with gelatinolytic activity in gelatingel. As showed in Fig. 4, cav-1 RNAi induced up-regulatedactivities of MMP-2 and MMP-9 in the medium. Theseresults indicate that cav-1 could negatively regulate MMPsactivity in BMECs.

Finally, we compared MMPs activity and BBB perme-ability between wild-type mice and cav-1 KO mice afterfocal cerebral ischemia-reperfusion injury. In situ gelatinasezymography and Evans blue leakage studies revealed thatcav-1 KO mice had remarkably higher MMPs activity andBBB permeability, respectively, than wild-type mice after15 min of focal cerebral ischemia plus 24 h of reperfusion(Fig. 5). L-NAME treatment almost completely inhibited

MMPs activity in wild-type mice but slightly reducedMMPs activity in cav-1 KO mice. Moreover, underL-NAME treatment, cav-1 KO mice revealed higher BBBpermeability than wild-type mice after cerebral ischemia-reperfusion. Taken together, the results suggest that cav-1plays an important role in inhibition of MMPs activity andprotection of BBB permeability. L-NAME could prevent theloss of cav-1, inhibit MMPs activity, and reduce BBBpermeability in focal cerebral ischemia and reperfusioninjury. The effects of L-NAME on regulating MMPsactivity and BBB permeability are partly mediated throughpreventing the loss of cav-1 in focal cerebral ischemia andreperfusion injury.

Discussion

Roles of cav-1 in modulating BBB integrity are stillconfusing in current literatures. Early study revealed that

Fig. 1 Western blot analysis on expression

of cav-1 at isolated cortex microvessels,

ipsilateral cortex, and hippocampus in focal

cerebral ischemia-reperfusion rats. Rats

were subjected to 2 h of MCAO ischemia

plus 6, 24, and 72 h of reperfusion with or

without intraperitoneally injection of 3 mg/

kg of L-NAME. (a)–(d) Expression of cav-1

in isolated brain microvessels. (a) and (b)

Representative immunoblot results,

whereas (c) and (d) are statistic results

(mean ± SD, n = 3). Sham, sham control;

2hI6hR, 2 h of ischemia plus 6 h reperfu-

sion; 2hI24hR, 2 h of ischemia plus 24 h

reperfusion; 2hI72hR, 2 h of ischemia plus

72 h reperfusion. (e)–(h) The expression of

cav-1 in whole ipsilateral cortex (e and g),

and whole hippocampus (f and h). Among

them, (e) and (f) are immunoblot results,

whereas (g) and (h) are statistic results

(mean ± SD, n = 3). Data were repre-

sented as mean ± SD (*p < 0.05,

**p < 0.01). The results showed that

ischemia-reperfusion induced the down-

regulation of cav-1 and L-NAME reserved

the expression of cav-1 in isolated cortex

microvessels, cortex, and hippocampus of

ipsilateral side of ischemia-reperfused rat

brains.

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NOS inhibitor protects BBB through caveolin-1 | 151

cav-1 KO mice suffered from microvascular hyper-perme-ability (Schubert et al. 2002). The expression of cav-1 wasremarkable down-regulated in MCAO ischemia-reperfusionrat brains in our previous report (Shen et al. 2006). Cav-1peptide prevented chemokine-induced BBB permeability byup-regulating TJ and adheren junction proteins in BMECs(Song et al. 2007). Cav-1 KO mice had larger infarctionvolumes and higher apoptotic cell death rates than wild-typemice in focal cerebral ischemia model (Jasmin et al. 2007).However, immunohistofluorescent study revealed an in-creased cav-1 expression in MCAO ischemic rat brains(Jasmin et al. 2007). Increased cav-1 expression and phos-phorylation were correlated with the decreased expression ofoccludin and claudin-5 in rat cortical cold injury model (Naget al. 2007, 2009). Green tea polyphenols reduced theexpression of cav-1 in microvessel fragments and amelio-rated BBB permeability in cerebral ischemic rats (Zhanget al. 2010). The discrepancy in previous studies might dueto different ischemia protocols used, such as permanentocclusion versus occlusion plus reperfusion, or different timeof ischemia-reperfusion. The differences in sample sources

and protein homogeneity could also be a reason since cav-1is heterogeneously distributed in neurons, astroglial cells,and endothelial cells of brain tissues (Jasmin et al. 2007). Toaddress the casual relationship between the expression ofcav-1 and BBB permeability, we designed following exper-iments: (i) we dynamically determined the change of cav-1 inisolated cortex microvessels from the rat brains subjected to2 h of MCAO ischemia with 6, 24, and 72 h of reperfusion.The expression of cav-1 in the brain microvessels was down-regulated at all of the observed time points. The expressionof cav-1 at the ischemic brain microvessels showed similarpattern to that at ischemic brain tissues in our previous report(Shen et al. 2006). Since the expression of cav-1 wasconsistently down-regulated at all of observed time pointsand early BBB opening generally occurs within 24 h aftercerebral ischemia, we selected 2 h of MCAO ischemia plus24 h of reperfusion to further evaluate the BBB permeability.The down-regulation of cav-1 was not only found in thebrain microvessels but also in cortex, hippocampus andwhole brain coronal sections of ipsilateral hemispheres.Evan’s blue leakage experiments revealed that 2 h of

Fig. 2 Representative MMPs activity, ZO-1

expression and cav-1 in focal rat brain

coronal frozen sections. Rats were sub-

jected to 2 h of MCAO ischemia plus 24 h

of reperfusion with or without L-NAME

treatment. Sham represents sham opera-

tion group (n = 3); 2hI24hR represents

ischemia 2 h plus 24 h reperfusion.

L-NAME (3 mg/kg) was intraperitoneally

administrated at 15 min prior to ischemia

(each group n = 3). (a) In situ MMPs

activity; (b) ZO-1 expression; (c) cav-1

expression; (d)–(f) quantitative analysis

for the results of (a), (b), and (c). *p < 0.05,

**p < 0.01. The results revealed that ische-

mia-reperfusion induced the MMPs activity

but down-regulated the expression of ZO-1

and cav-1, and L-NAME decreased MMPs

activity, reserved the expression of ZO cav-1

in the ischemia-reperfused brain sections.

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152 | Y. Gu et al.

ischemia following 24 h of reperfusion resulted in theincrease of BBB permeability. (ii) We compared thedifferences of BBB permeability in wild-type and cav-1KO mice. As 2 h of MCAO ischemia had high rates ofmortality in mice, we treated the mice with 15 min of MCAOischemia plus 24 h of reperfusion. The results showed thatcav-1 KO mice had remarkably higher BBB permeabilitythan wild-type mice, indicating that cav-1 plays a role inmaintaining BBB integrity and loss of cav-1 in the cerebral

microvessels contributes to the BBB hyper-permeability inthe ischemia-reperfused brains.

Activated MMPs is responsible for degradation of theextracellular matrix around cerebral blood vessels andneurons and increase of the BBB permeability throughhydrolyzing BBB extracellular matrix and TJ proteins. AsMMPs have cav-binding motif, MMPs might be the bindingtargets of cav-1. We conducted sequence alignment andfound that caveolin scaffold domains exist in gelatin-bindingdomain and Hemopexin-like domain of both MMP-2 andMMP-9 (data not shown; for MMPs structures, referMorgunova et al. 1999). The inhibition of cav-1 on MMP-2 activity was confirmed in cav-1 KO mice heart in vivo andcav-1 scaffold domain ex vivo (Chow et al. 2007). Cav-1 wasfound to co-localize with MMP-2 in the endothelial cellsurface (Puyraimond et al. 2001). Cav-1 inhibited expressionof MMP-2 and -9 in pancreatic carcinoma cells and regulatedtumor invasion (Han and Zhu 2010). In our studies, the

Fig. 3 Evans blue leakage experiments and gelatinase zymography

for determining BBB permeability and MMPs activity respectively in rat

focal cerebral ischemia-reperfusion injury. (a) Evans Blue leakage

assay for BBB permeability. 2hI24hR, 2 h of ischemia plus 24 h rep-

erfusion; Data were represented as mean ± SD and n = 7 in each

group. The contents of Evans blue leakage were expressed as lg per

gram brain. (**p < 0.01). The results revealed that the L-NAME treat-

ment markedly reduced BBB hyper-permeability in ischemia-reperfu-

sion brains. (b) Results of gelatinase zymography for activities of

MMP-9 and MMP-2. Activities of MMP-2 and MMP-9 were detected in

gelatin gel. Protein samples were obtained from isolated ipsilateral

cortex microvessels (each group, n = 6). (c and d) Statistical results

for MMP-9 and MMP-2 activities respectively. The results showed that

ischemia-reperfusion-induced MMP-9 and MMP-2 activity was signif-

icantly inhibited by L-NAME treatment (*p < 0.05).

Fig. 4 Effects of Cav-1 knockdown on activities of MMP-2 and MMP-9

in cultured BMECs. BMECs were transfected with Cav-1 Stealth�RNAi to knockdown the expression of cav-1 (siCav-1, n = 9). Stealth�RNAi Negative Control Duplex was also used to transfect to BMECs

as control group (Ctrl siRNA, n = 9). Medium and cell lysate of each

group were collected at 24 h after transfection. Western blot analysis

revealed the declined level of cav-1 in cell lysate of siCav-1 group

compared with negative control group (a). MMPs activity in medium,

which was examined with gelatin gel, was increased in siCav-1 group

compared with control group (b). MMP-9 and MMP-2 activity was

quantified as in (c) and (d), respectively (*p < 0.05).

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NOS inhibitor protects BBB through caveolin-1 | 153

in vitro experiments revealed that cav-1 RNAi remarkablyup-regulated the activities of MMP-2 and MMP-9 in theBMECs, and the in vivo experiments showed that cav-1 KOmice had remarkably higher MMPs activity and BBBpermeability than wild-type mice after 15 min of focalcerebral ischemia plus 24 h of reperfusion. This is the directevidence that cav-1 can negatively regulate MMPs activitiesand BBB permeability in BMECs during cerebral ischemia-reperfusion injury.

Cav-1 physically interacts and inhibits a rage of proteinsvia the caveolin scaffold domain (Rothberg et al. 1992). Thebinding proteins contain the cav-binding site: ‘uXuXXX-Xu’ or ‘uXXuXXXXu’, where u is Phenylalanine, Tyro-sine or Tryptophan and X is any amino acid residue. All threeisoforms of NOS have these character domains. Cav-1 is wellknown to negatively regulate NO production through bindingNOS and inhibiting NOS activity. L-NAME significantlyameliorated vascular damage, inhibited MMP-9 expression,and the BBB disruption in cerebral ischemia-reperfusioninjury (Gursoy-Ozdemir et al. 2000, 2004). Our previousstudy revealed that non-selective NOS inhibitor L-NAME

and selective inducible NOS and neuronal NOS inhibitorsrescued the expression of cav-1 in MCAO ischemia-reper-fused rat brains (Shen et al. 2006). Thus, we logicallyaddressed whether the protective effects of NOS inhibitorson cav-1 contribute to modulation of BBB permeability. Inthis study, L-NAME treatment reserved the expression ofcav-1 in brain microvessels and other brain tissues after focalischemia-reperfusion injury. Simultaneously, L-NAME treat-ment inhibited the activities of MMP-2 and -9, up-regulatedthe expression of ZO-1, and ameliorated BBB disruption inthe ischemia-reperfused rat brain microvessels and otherbrain tissues. The effects of L-NAME on MMPs activity andBBB permeability were partly reversed in cav-1 KO mice,indicating that the inhibitions of L-NAME on MMPs activityand BBB permeability are at least in part mediated by theprotection of cav-1. It is valuable to mention that the BBBopening is complex. In addition to cav-1, other cellularsignaling cascades may be also involved in the NO-inducedMMPs activity and TJ degradation during cerebral ischemia-reperfusion injury. Finally, given low concentration NOderived from eNOS has benefit effects on regulations of

Fig. 5 Evans blue leakage experiments and in situ gelatinase

zymography for determining BBB permeability and MMPs activity

respectively in wild-type and cav-1 KO mice with focal cerebral

ischemia-reperfusion injury. Both wild-type and cav-1 KO mice were

subjected to 15 min ischemia plus 24 h reperfusion. MCAO mice were

divided into four groups: wild-type mice injected with saline (n = 4);

Cav-1 KO mice injected with saline (n = 5); Cav-1 KO mice injected

with L-NAME (n = 4); Cav-1 KO mice injected with L-NAME (n = 4).

L-NAME (3 mg/kg) or saline was intraperitoneally administrated at

15 min prior to ischemia. Data were represented as mean ± SD,

*p < 0.05, **p < 0.01. (a) MMPs activity; (b) in situ MMPs activity; (c)

Evans blue leakage for BBB permeability. Compared with wild-type

mice, cav-1 KO mice exhibited higher MMPs activity after ischemia-

reperfusion treatment. L-NAME treatment completely abolished MMPs

activity in wild-type mice but partially inhibited MMPs activity in cav-1

KO mice. Meanwhile, cav-1 KO mice displayed greater extent of BBB

leakage than wild-type mice. The extent of Evans blue leakage in wild-

type mice was remarkably attenuated by L-NAME treatment, but

L-NAME had less effects of on Evans blue leakage in cav-1 KO mice

than in wild-type mice.

Journal of Neurochemistry � 2011 International Society for Neurochemistry, J. Neurochem. (2012) 120, 147–156� 2011 The Authors

154 | Y. Gu et al.

endothelial functions and blood supply, our results may notbe applied for the experimental conditions in which NO hasphysiological functions.

In conclusion, we summarized a novel pathway of BBBdisruption in cerebral ischemia-reperfusion injury as showedin Fig. 6. Our experimental results suggest that cav-1 playsan important role in maintaining BBB integrity throughinhibition of MMPs activity and protection of TJ protein. TheMMPs activities and BBB leakage are partially mediatedthrough the down-regulation of cav-1 during cerebralischemia-reperfusion injury. This study provides a valuablestep for understanding the molecular mechanisms of BBBdisruption during cerebral ischemia-reperfusion injury.

Sources of Funding

This study was mainly supported by General Research Fund fromUniversity Grants Committee (774408M, 777611M), Hong KongSpecial Administration Region and Seed fund for Basic Researchfrom The University of Hong Kong.

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