KN93 treatment attenuates CaMKII activity in the neonatal rat brain exposed to hypoxia-ischemia.

<p>P7 neonatal rats were pre-treated with KN93 (0.3 nmol and 0.6 nmol), the inactive analog KN92 (0.3 nmol), or vehicle then exposed to HI. Two hours after HI, CaMKII-like activity was determined in the right hemisphere of the brain and compared to the contralateral side. There is a significant increase in the right hemisphere that is dose-dependently attenuated by KN93, but not KN-92. Values are presented as mean ± S.E from 6 animals per group. *p<0.05 vs. sham, †p<0.05 vs. HI+vehicle, ‡P<0.05 vs. HI+KN92, § P<0.05 vs. HI+0.6 nmol KN93.</p

KN93 attenuates p38MAP kinase activation and p47^phox membrane translation in the neonatal rat brain exposed to hypoxia-ischemia.

<p>P7 neonatal rats were pre-treated with KN93 (0.3 nmol), the inactive analog KN92 (0.3 nmol), or vehicle then exposed to HI. Two hours after HI the brain was removed and the right hemisphere subjected to Western blotting to determine the effect on p38MAPK activation (estimated by ratio of phospho-p38MAPK to total p38MAPK) and p47^phox membrane translocation. KN93 pretreatment attenuates both the increase in phospho-p38MAPK (A) and the membrane translocation of p47^phox (B). Values are presented as mean ± S.E from 6 animals per group. *p<0.05 vs. sham, †p<0.05 vs. HI + vehicle, ‡P<0.05 vs. HI+KN92.</p

KN93 attenuates infract volume in the neonatal rat brain exposed to hypoxia-ischemia.

<p>P7 neonatal rats were pre-treated with KN93 (0.3- or 0.6 nmol), the inactive analog KN92 (0.3- or 0.6 nmol), or vehicle then exposed to HI. Twenty-four hours after HI the brains were removed, sectioned and subjected to TTC staining to determine changes in the infarct volume in the right hemisphere. Representative TTC stained sections are shown (A). KN93 (0.3 nmol), but not KN93 (0.6 nmol) or KN92 (either dose), reduces both the absolute infract volume (B) and relative infarct volume (C) associated with HI. Values are presented as mean ± S.E from 6 animals per group. *P<0.05 vs. left hemisphere, †p<0.05 vs. HI+vehicle, ‡P<0.05 vs. HI+0.3 nmol KN92, § P<0.05 vs. HI+0.3 nmol KN93, ψ P<0.05 vs. HI+0.6 nmol KN93.</p

Oxygen glucose deprivation increases CaMKII activity which is correlated with cell death in rat hippocampal slice cultures.

<p>Rat hippocampal slice cultures were exposed to the CaMKII inhibitor, KN93 (10- or 20 µM) or its inactive analog, KN92 (10 µM) 2 h prior to OGD. Slices were harvested 2 h after OGD to determine effects on CaMKII activity. OGD increases CaMKII-like activity (A). The increase in CaMKII-like activity is dose-dependently attenuated by KN93, but not by KN92 (A). In addition the effect of OGD on cell death (B) and LDH release (C) were evaluated at 8 h after OGD. The effect on cell injury was quantified by measuring mean changes fluorescence due to PI uptake in the whole slice (B). KN93 attenuated both PI uptake and LDH release (B & C). The LDH absorbance at 490 nm was normalized by protein content. Data are presented as mean ± S.E from 4–8 independent experiments using 24 pooled slices per experiment. * <i>P</i><0.05 vs. control, † <i>P</i><0.05 vs. OGD alone, ‡P<0.05 vs. OGD+KN-92, § P<0.05 vs. OGD+10 µM KN93; #P<0.05 vs. no OGD+KN93 10 µM.</p

Autocamitide 2-related inhibitor peptide reduces cell death in rat hippocampal slice cultures expose to OGD.

<p>Rat hippocampal slice cultures were exposed to the CaMKII inhibitor, AIP (5 µM), 2 h prior to OGD. AIP significantly attenuated the OGD-mediated increase in CaMKII-like activity (A). AIP also significantly attenuated the increase in cell death (B) and LDH release (C) associated with OGD. Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * <i>P</i><0.05 vs. control, † <i>P</i><0.05 vs. OGD alone.</p

CaMKII inhibition attenuates apoptosis in rat hippocampal slice cultures exposed to oxygen glucose deprivation.

<p>Rat hippocampal slice cultures were exposed to OGD in the presence of the CaMKII inhibitor, KN93 or its inactive analog, KN92 (10 µM, 2 h prior to OGD). Slices were harvested 8 h after OGD and subjected to Western blot analysis to determine effects on cleaved caspase-3 (A). A representative blot is shown (A). OGD increases cleaved caspase-3 levels and this is attenuated by KN93, but not by KN92 (A). Slices were also subjected to TUNEL analysis. Representative images are shown demonstrating TUNEL staining of apoptotic cells (green) co-localized with PI staining of all the nuclei (red) (B). The magnification used was 10×. Quantification of the percentage of apoptotic nuclei to total nuclei was also carried out indicating that KN-93 pretreatment decreased the level of apoptotic nuclei in response to OGD (C). Data are presented as mean ± S.E from 4 independent experiments using 24 pooled slices per experiment. * <i>P</i><0.05 vs. no OGD, † <i>P</i><0.05 vs. OGD alone, ‡P<0.05 vs. OGD+KN92.</p

KN93 attenuates neural cell death in the neonatal rat brain exposed to hypoxia-ischemia.

<p>P7 neonatal rats were pre-treated with KN93 (0.3 nmol), the inactive analog KN92 (0.3 nmol), or vehicle then exposed to HI. Twenty-four hours after HI the brains were removed, the right hemisphere was subject to Western blot analysis to determine effects on cleaved caspase-3. A representative blot is shown (A). HI increases cleaved caspase-3 levels and this is attenuated by KN93, but not by KN92 (A). The right hemisphere was also sectioned and subjected to TUNEL staining to determine the effect on apoptosis in the neonatal brain. Sections were counterstained with PI (red) and representative images are shown (B). Quantitation of TUNEL positive cells shows that KN93, but not KN92, attenuates the increase in apoptosis in the right hemisphere by HI (C). Values are presented as mean ± S.E from 6 animals per group. *p<0.05 vs. left hemisphere, †p<0.05 vs. HI+vehicle, ‡P<0.05 vs. HI+KN92.</p

KN93 attenuates NADPH oxidase activity and superoxide generation in the neonatal rat brain exposed to hypoxia-ischemia.

<p>P7 neonatal rats were pre-treated with KN93 (0.3 nmol), the inactive analog KN92 (0.3 nmol), or vehicle then exposed to HI. Two hours after HI, NADPH oxidase activity (A) and superoxide levels (B) were determined in the right hemisphere of the brain. There is a significant increase in both NADPH oxidase activity and superoxide levels in the right hemisphere of the neonatal brain that is attenuated by KN93, but not by KN92. Values are presented as mean ± S.E from 6 animals per group. *p<0.05 vs. sham, †p<0.05 vs. HI+vehicle, ‡P<0.05 vs. HI+KN92.</p

Decreased PPAR-γ signaling increases eNOS uncoupling in ovine pulmonary arterial endothelial cells.

<p>The levels of eNOS (A) and Hsp90 (B) protein levels were unchanged between scrambled and PPAR-γ siRNA transfected PAEC. The interaction of eNOS with Hsp90 was determined by immunoprecipitation (IP) using a specific antiserum raised against eNOS followed by Western blot (IB) analysis with an anti-Hsp90 antibody. The membrane was reprobed for eNOS to normalize for immunoprecipitation efficiency. There was a significant decrease in the association of eNOS with Hsp90 in PPAR-γ siRNA transfected cells (C). The effect of PPAR-γ inhibition on eNOS uncoupling was determined. PAEC were treated or not with eNOS inhibitor 2-ethyl-2-thiopseudourea (ETU). Superoxide and NO levels were then determined in the presence or absence of acute laminar shear stress (20dyn/cm², 15 min). PPAR-γ siRNA transfection significantly increased eNOS-derived superoxide levels (D) and decreased NO levels (E) but only under shear-stimulated conditions. Values are mean ± SE; n = 6. *P<0.05 <i>vs</i> scrambled siRNA, no shear; †P<0.05 <i>vs</i> scrambled siRNA, shear.</p

Decreased PPAR-γ signaling induces mitochondrial dysfunction in ovine pulmonary arterial endothelial cells.

<p>PAEC were transiently transfected with a PPAR-γ siRNA or a scrambled siRNA for 24 h then exposed or not to the PPAR-γ agonist, rosilglitazone (10 µM) for a further 24 h. The MitoSOX red mitochondrial ROS indicator was then added. Representative images after MitoSOX staining are shown (A, top). Images of 20 random fields were quantified to determine the mean fluorescence intensity of each sample. PPAR-γ inhibition significantly increased mitochondrial ROS levels and this was reversed by rosiglitazone (A). Mitochondrial membrane potential (MMP) was also determined using the DePsipher mitochondrial potential assay kit. Representative images after DePsipher staining are shown (B, top). PPAR-γ inhibition significantly decreased mitochondrial membrane potential and this was reversed by rosiglitazone (B). Total mitochondrial number was evaluated by fluorescent microscopy (C) and flow cytometry (D) in scrambled and PPAR-γ siRNA transfected PAEC stained with Mitotracker green. PPAR-γ gene silencing had no significant affect on mitochondrial number as evaluated by either method. There was also a significant reduction in ATP levels after PPAR-γ siRNA transfection (E). Values are mean ± SE; n = 7–12. *P<0.05 <i>vs</i> scrambled siRNA.</p