Journal: Journal of Neuroscience

Article Title: Matrix Metalloproteinase-2-Mediated Occludin Degradation and Caveolin-1-Mediated Claudin-5 Redistribution Contribute to Blood-Brain Barrier Damage in Early Ischemic Stroke Stage

doi: 10.1523/jneurosci.6409-11.2012

Figure Lengend Snippet: Figure1. IschemiacausesrapiddisruptionoftheBBBinvitroandinvivo.A,Toppanel,Schematicrepresentationoftheinvitro BBB model (bEND3 monolayer grown on an insert) with FITC-dextran loaded in the luminal compartment. Bottom panel, The endothelial monolayer permeability was assessed by calculating the transfer rate of FITC-dextran from luminal compartment to abluminal compartment and was expressed as apparent permeability coefficient (Papp) (in centimeters per second). Exposure of bEND3 monolayer to OGD for 2 h significantly increased its permeability to FITC-dextran. *p 0.05 versus control cultures, Student’s t test; n 6. B, Cerebral ischemia rapidly induced BBB disruption in the ischemic brain of all tested rats (n 6). Representative fluorescence micrographs of brain cryosections from four different rats revealed that 2 h MCAO induced FITC- albuminextravasation(brightgreenfluorescence)inthesubcorticalregionsintheischemichemisphere.NoFITC-albuminleakage was observed in other brain regions. Error bars indicate SEM.

Article Snippet: To test whether MMP-2/9 and Cav-1 were implicated in OGD-induced endothelial barrier disruption, cells were treated with selective MMP-2/9 inhibitor 2-[[(4-phenoxyphenyl) sulfonyl]methyl]-thiirane (SB-3CT) (10 M; Calbiochem) 2 h before and during OGD treatment or pretreated with Cav-1 siRNA for 48 h before OGD treatment. siRNA transfection. bEnd3 cells at 60 –70% confluence were transfected with 80 pmol of Cav-1 siRNA (Santa Cruz Biotechnology; sc29520) or scrambled control siRNA (Santa Cruz Biotechnology; sc-37007) using siRNA Transfection Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instruction.

Techniques: Permeability, Control, Disruption, Fluorescence

Journal: Journal of Neuroscience

Article Title: Matrix Metalloproteinase-2-Mediated Occludin Degradation and Caveolin-1-Mediated Claudin-5 Redistribution Contribute to Blood-Brain Barrier Damage in Early Ischemic Stroke Stage

doi: 10.1523/jneurosci.6409-11.2012

Figure Lengend Snippet: Figure 3. OGD-induced occludin degradation is MMP-2/9 dependent. A, OGD rapidly elevated MMP-2/9 levels in conditioned media. After exposure of bEND3 cells to OGD for 2 h, a significant increase in MMP-2/9 levels was detected in the conditioned medium on gelatin zymograms when compared with the control cultures (Ctrl). Active MMP-2 (the bottom band), but no active MMP-9,wasseenonzymogramgels.MMP-2or-9levelswerequantifiedbymeasuringthesumintensityoftheirlatentandactive bands. *p 0.05 versus Ctrl, Student’s t test; n 5. Std, Standard human MMP-2/9. B, Selective MMP-2/9 inhibitor SB-3CT completelyinhibitedOGD-inducedoccludindegradation.bEND3cellsweretreatedwithSB-3CT(10M)2hbeforeandduring2h OGD. Occludin protein in total cellular extracts was detected with Western blot. -Actin served as a loading control. *p 0.05 versus vehicle (DMSO) plus Ctrl, #p 0.05 versus vehicle plus OGD, ANOVA; n 5. C, MMP-2 neutralizing antibody (M2) completelyinhibitedOGD-inducedoccludindegradation,whilenosignificanteffectswereobservedforIgGorMMP-9neutralizing antibody (M9). bEND3 cells were treated with 20 g/ml control mouse IgG, MMP-2 or MMP-9 neutralizing antibodies, or both (M2,9) during 2 h OGD. Occludin protein in total cellular extracts was detected with Western blot. -Actin served as a loading control. *p 0.05 versus control (Ctrl), #p 0.05 versus OGD alone () or OGD plus IgG, ANOVA; n 4. D, Representative confocal micrographs showed MMP-dependent degradation of occludin. Control bEND3 cells revealed a circumcellular immunostaining of occludin, which was significantly reduced after exposing cells to OGD for 2 h. SB-3CT treatment completely inhibited occludin reduction in OGD-treated cells. Experiments were repeated three times with similar results. Scale bar, 20 m. E, Inhibition of MMP-2/9 with SB-3CT had no effect on OGD-induced claudin-5 redistri- bution. Claudin-5 proteins in subcellular fractions were detected with Western blot. *p 0.05 versus vehicle plus Ctrl, ANOVA; n 5. Error bars indicate SEM.

Article Snippet: To test whether MMP-2/9 and Cav-1 were implicated in OGD-induced endothelial barrier disruption, cells were treated with selective MMP-2/9 inhibitor 2-[[(4-phenoxyphenyl) sulfonyl]methyl]-thiirane (SB-3CT) (10 M; Calbiochem) 2 h before and during OGD treatment or pretreated with Cav-1 siRNA for 48 h before OGD treatment. siRNA transfection. bEnd3 cells at 60 –70% confluence were transfected with 80 pmol of Cav-1 siRNA (Santa Cruz Biotechnology; sc29520) or scrambled control siRNA (Santa Cruz Biotechnology; sc-37007) using siRNA Transfection Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instruction.

Techniques: Control, Western Blot, Immunostaining, Inhibition

Journal: Journal of Neuroscience

Article Title: Matrix Metalloproteinase-2-Mediated Occludin Degradation and Caveolin-1-Mediated Claudin-5 Redistribution Contribute to Blood-Brain Barrier Damage in Early Ischemic Stroke Stage

doi: 10.1523/jneurosci.6409-11.2012

Figure Lengend Snippet: Figure4. OGDelevatesextracellularMMP-2/9levelsthroughpromotingtheirsecretionfromthepreexistingintracellularpool in bEND3 cells. A, Gelatin zymography analysis showed that 2 h OGD markedly increased MMP-2/9 levels in the CM, which was accompaniedbyasignificantreductionintheirlevelsinwholeCEs.*p0.05versuscontrol,Student’sttest;n5.Std,Human MMP-2/9standards.B,Real-timeRT-PCRanalysisshowedthat2hOGDdidnotchangeMMP-2/9mRNAexpressioninbEND3cells. n 5. C, D, Inhibition of mRNA synthesis with Act D or inhibition of protein synthesis with CHX did not significantly affect OGD-inducedMMP-2/9secretioninbEND3cells.CellsweretreatedwithActD(2g/ml)orCHX(100g/ml)orvehicle(DMSO)1hbefore andduring2hOGDtreatment.*p0.05versuscontrol(Ctrl),ANOVA;n5.E,TwohourOGDalsostimulatesMMP-2/9secretionfrom astrocyticcelllineC8-D1A(leftpanel)andneuronalcelllineSH-SY5Y(rightpanel),reflectedbyincreasedMMP-2/9inCMandconcurrent reductionoftheirlevelsinCEs.Experimentsarerepeatedfourtimeswithsimilarresults.ErrorbarsindicateSEM.

Article Snippet: To test whether MMP-2/9 and Cav-1 were implicated in OGD-induced endothelial barrier disruption, cells were treated with selective MMP-2/9 inhibitor 2-[[(4-phenoxyphenyl) sulfonyl]methyl]-thiirane (SB-3CT) (10 M; Calbiochem) 2 h before and during OGD treatment or pretreated with Cav-1 siRNA for 48 h before OGD treatment. siRNA transfection. bEnd3 cells at 60 –70% confluence were transfected with 80 pmol of Cav-1 siRNA (Santa Cruz Biotechnology; sc29520) or scrambled control siRNA (Santa Cruz Biotechnology; sc-37007) using siRNA Transfection Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instruction.

Techniques: Zymography, Inhibition

Journal: Journal of Neuroscience

Article Title: Matrix Metalloproteinase-2-Mediated Occludin Degradation and Caveolin-1-Mediated Claudin-5 Redistribution Contribute to Blood-Brain Barrier Damage in Early Ischemic Stroke Stage

doi: 10.1523/jneurosci.6409-11.2012

Figure Lengend Snippet: Figure 5. Cav-1mediatesOGD-inducedclaudin-5dissociationfromthecytoskeletoninendothelialcells.A,B,Westernblotanalysis showedthatexposingbEND3cellstoOGDfor2hhadnoeffectsonthetotalproteinlevelsofCav-1,buttriggereditsredistributionamong subcellularfractions,asreflectedbyincreasedCav-1levelintheCFanddecreasedlevelintheACF.*p0.05versuscontrol,Student’sttest; n5.C,Cav-1siRNAeffectivelyknockeddownCav-1proteinexpressioninbEND3cells.Westernblotanalysisshowedthatincubationcells withCav-1siRNA(Cav-1siR)for48hsignificantly(90%)reducedCav-1proteinlevels.*p0.05versuscontrolsiRNA(CtrlsiR),Student’s ttest;n5.D,KnockdownofCav-1withsiRNApreventedOGD-inducedclaudin-5redistributioninbEND3cells.FollowingOGDtreatment, claudin-5 levels in CF, MF were increased, while its level in ACF was markedly reduced, which was inhibited by Cav-1 siRNA. *p 0.05 versuscontrol(Ctrl); #p0.05versusCtrlsiRplusOGD,ANOVA;n5.E,Representativeconfocalmicroscopeimagesrevealedacircum- cellular immunostaining of claudin-5 in control bEND3 cells. Two hour OGD treatment did not change the immunostaining intensity of claudin-5, but significantly disturbed its normal distribution pattern. Knockdown of Cav-1 with siRNA inhibited claudin-5 redistribution inducedbyOGD.Experimentswererepeatedthreetimeswithsimilarresults.Scalebar,20m.F,Representativeimmunoblotofcoim- munoprecipitation of Cav-1 and claudin-5 from whole-cell lysates of control cultures or OGD-treated cells with anti-Cav-1 antibody or normalIgG(toppanel).Bottompanel,OGDenhancedinteractionofCav-1withclaudin-5.*p0.05versuscontrol,Student’sttest;n 4.ErrorbarsindicateSEM.

Article Snippet: To test whether MMP-2/9 and Cav-1 were implicated in OGD-induced endothelial barrier disruption, cells were treated with selective MMP-2/9 inhibitor 2-[[(4-phenoxyphenyl) sulfonyl]methyl]-thiirane (SB-3CT) (10 M; Calbiochem) 2 h before and during OGD treatment or pretreated with Cav-1 siRNA for 48 h before OGD treatment. siRNA transfection. bEnd3 cells at 60 –70% confluence were transfected with 80 pmol of Cav-1 siRNA (Santa Cruz Biotechnology; sc29520) or scrambled control siRNA (Santa Cruz Biotechnology; sc-37007) using siRNA Transfection Reagent (Santa Cruz Biotechnology) according to the manufacturer’s instruction.

Techniques: Immunostaining, Control, Knockdown

Journal: Metals

Article Title: Titanium Implant Osseointegration Problems with Alternate Solutions Using Epoxy/Carbon-Fiber-Reinforced Composite

doi: 10.3390/met4040549

Figure Lengend Snippet: Biomaterial Properties

Article Snippet: Unidirectional Photocure 3-pt Bend QF [ ] , , , 1118.8 (±207.6) , , 76.6 (±13.3) .

Techniques: Polymer