Anti-HSP27 antibody and dephosphorylation inhibit hHSP27 neuroprotective effects.

<p><b><i>A,</i></b> Infarct volumes in control, hHSP27 (50 µg), hHSP27 plus HSP27 antibody cocktails, HSP27 elution peptide, recombinant HSP27, and dephosphorylated hHSP27 groups. Data are means±SEM of 3 mice in each group. **<i>P</i><0.001 vs. controls. <b><i>B– D,</i></b> Dephosphorylated and phosphorylated hHSP27 proteins were separated by SDS-PAGE (<b><i>B</i></b>) and native-PAGE (<b><i>C</i></b>), stained with Coomassie brilliant blue (<b><i>B,C</i></b>), and immunoblotted with anti-phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27 antibodies (<b><i>D</i></b>). <b><i>E</i></b>, Photomicrographs of infarct areas stained with cresyl violet in hHSP27 and dephosphorylated hHSP27 groups prepared 24 h after reperfusion. Scale bar = 1 mm. hHSP27, human heat shock protein.</p

prHSP27 reduces mortality and hemorrhagic transformation at 24 h after reperfusion.

<p>(A) At 24 h after reperfusion following MCAO, the mortality rate was 20% (6/30) in the BSA treatment group, 0% (0/15) in the prHSP27 group, 35% (11/31) in the tPA group, and 7% (2/28) in the tPA plus prHSP27 group. *<i>p</i> < 0.05: BSA vs. prHSP27, †<i>p</i> < 0.01: tPA plus prHSP27 vs. tPA. (B) Effect of administering tPA on macroscopic hemorrhages. (<i>Upper</i>) Representative coronal brain sections stained with 1% 2,3,5-triphenyl tetrazolium chloride at 24 h after reperfusion showing the five grades of hemorrhages: Grades 0–4. (<i>Lower</i>) whole, unstained brains with the same hemorrhages.</p

Characterization of hHSP27.

<p><b><i>A–B,</i></b> Isolated human heat shock protein 27 (hHSP27) or recombinant HSP27 (rHSP27) proteins (1 µg or 10 µg) were separated by SDS-PAGE (<b><i>A</i></b>) and native-PAGE (<b><i>B</i></b>) and stained with Coomassie brilliant blue. <b><i>C,</i></b> hHSP27 proteins were immunoblotted with antibodies against HSP27, phosphorylated S15 HSP27, S78 HSP27, and S82 HSP27. <b><i>D,</i></b> hHSP27 proteins were separated by native-PAGE and immunoblotted with antibodies against αβ-crystallin and HSP20. <b><i>E,</i></b> rHSP27 (10 ng), hHSP27 (10 ng), αβ-crystallin (5 ng), and HSP20 (5 ng) were separated by SDS-PAGE followed by immunoblotting with antibodies against HSP27, αβ-crystallin, and HSP20.</p

Effects of hHSP27 on oxidative stress and inflammatory response.

<p><b><i>A,</i></b> Photomicrographs of 8-OHdG-, HHE-, Iba-1-, and GFAP-immunostaining in the infarct boundary zones in the control and hHSP27 groups 24 h after reperfusion. Bars = 50 µm. <b><i>B,</i></b> Numbers of 8-OHdG-, HHE–, Iba-1-, and GFAP-positive cells in control and hHSP27-treated mice. Data are means±SEM (<b><i>B</i></b>). **<i>P</i><0.001 vs. controls. 8-OHdG, 8-hydroxydeoxyguanosine; HHE, 4-hydroxy-2-hexenal; Iba-1, ionized calcium-binding adapter molecule-1; GFAP, glial fibrillary acidic protein; hHSP27, human heat shock protein.</p

Localization of injected FITC-hHSP27 on the ischemic and non-ischemic sides of mouse brain.

<p><b><b><i>A–H,</i></b></b> FITC-hHSP27 (<i>green</i>, <b><i>A,B</i></b>); NeuN, a neuronal marker protein (<i>red</i>, <b><i>C,D</i></b>); merge (<b><i>E–H</i></b>), FITC, fluorescein isothiocyanate. Scale bar = 100 um.</p

Effects of hHSP27 on cell death.

<p><b><i>A,</i></b> Photomicrographs of anti-cytochrome c, anti-cleaved caspase-9, anti-cleaved caspase-3, and TUNEL staining in the infarct boundary zones in controls and the hHSP27-treated group prepared 24 h after reperfusion. Scale bars = 50 µm. <b><i>B,</i></b> Number of cytochrome c-, cleaved caspase-9-, cleaved caspase-3-, and TUNEL-positive cells. <b><i>C,</i></b> Immunoblots of cytochrome c, Tom20 (mitochondrial marker), and actin, 24 h after reperfusion. <b><i>D,</i></b> Densitometric analysis of cytochrome c protein in cytosolic fractions of isolated hHSP27. Data are means±SEM (<b><i>B,D</i></b>). **<i>P</i><0.001 vs. controls. TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling; hHSP27, human heat shock protein.</p

Neuroprotective effects of hHSP27 against ischemic/reperfusion injury 72 h after reperfusion.

<p><b><i>A,</i></b> Photomicrographs of infarct areas stained with cresyl violet in control and hHSP27 groups 72 h after reperfusion. Infarct areas are circumscribed with dotted lines. Scale bar = 1 mm. <b><i>B,</i></b> Infarct volumes in control and hHSP27 groups. <b><i>C,</i></b> Neurological deficit scores in control and hHSP27 groups. Data are presented as mean±SEM of 3 mice (<b><i>B</i></b>) and 5 mice (<b><i>C</i></b>) in each group. *<i>P</i><0.05, **<i>P</i><0.001 vs. controls.</p

Effect of cerebral ischemia on prHSP27 in the blood-brain barrier.

<p><b>(A)</b> Electron microscopic analysis of the blood-brain barrier on the non-ischemic side of tPA- (contralateral tPA, n = 3), ischemic side of tPA- (tPA, n = 3), and ischemic side of tPA plus prHSP27- (tPA and prHSP27, n = 3) treated mice. Magnification, ×5000 (<i>left row</i>) and ×15000 (<i>right row</i>). The left row shows magnifications of the boxed areas in the right row. Scale bars, 1 μm (<i>left row</i>) and 500 nm (<i>right row</i>). a: astrocyte endfeet; e: endothelial cell; L: lumen. Arrowheads: the space between the basement membrane and astrocyte endfeet. Asterisks indicate the space between the basement membrane and astrocyte endfeet (B).</p

prHSP27 attenuates blood-brain barrier permeability.

<p>(A) Representative photomicrographs of endogenous IgG extravasation stained in BSA- (<i>n</i> = 7), prHSP27- (<i>n</i> = 6), tPA- (<i>n</i> = 7), and tPA plus prHSP27- (<i>n</i> = 7) treated mice at 24 h after reperfusion. Scale bar = 2 mm. (B) Quantitation of IgG staining in nine consecutive coronal brain slices from each mouse. Data are means ± SEM. *<i>p</i> < 0.05: tPA plus prHSP27 vs. tPA, ‡<i>p</i> < 0.005: BSA vs. prHSP27.</p

Effect of prHSP27 on the expression of matrix metalloproteinase-9 (MMP-9) on the ischemic side.

<p>(A) Zymographic (upper) and densitometric (lower) analyses of MMP-9 protein in BSA- (<i>n</i> = 7), tPA- (<i>n</i> = 7), and tPA plus prHSP27- (<i>n</i> = 7) treated mice at 24 h after reperfusion. ‡<i>p</i> < 0.005: tPA plus prHSP27 vs. tPA. (B) Photomicrographs of ionized calcium binding adapter molecule-1 (Iba-1) immunostaining (<i>upper</i>) and number of Iba-1-positive cells (<i>lower</i>) in the infarct boundary zones in BSA- (<i>n</i> = 5), tPA- (<i>n</i> = 5), and tPA plus prHSP27- (<i>n</i> = 5) treated mice at 24 h after reperfusion. prHSP27 suppressed the inflammatory response. Scale bars = 100 μm. Data are mean ± SEM. *<i>p</i> < 0.05: tPA plus prHSP27 vs. BSA, ^<b>§</b><i>p</i> < 0.001: tPA plus prHSP27 vs. tPA.</p