Background: Toll-like receptors (TLRs) have been implicated in myocardial ischemia/reperfusion (I/R) injury. The TLR9 ligand, CpG-ODN has been reported to improve cell survival. We examined effect of CpG-ODN on myocardial I/R injury. Methods: Male C57BL/6 mice were treated with either CpG-ODN, control-ODN, or inhibitory CpG-ODN (iCpG-ODN) 1. h prior to myocardial ischemia (60. min) followed by reperfusion. Untreated mice served as I/R control (n. =10/each group). Infarct size was determined by TTC straining. Cardiac function was examined by echocardiography before and after myocardial I/R up to 14. days. Results: CpG-ODN administration significantly decreased infarct size by 31.4% and improved cardiac function after myocardial I/R up to 14. days. Neither control-ODN nor iCpG-ODN altered I/R-induced myocardial infarction and cardiac dysfunction. CpG-ODN attenuated I/R-induced myocardial apoptosis and prevented I/R-induced decrease in Bcl2 and increase in Bax levels in the myocardium. CpG-ODN increased Akt and GSK-3β phosphorylation in the myocardium. In vitro data suggested that CpG-ODN treatment induced TLR9 tyrosine phosphorylation and promoted an association between TLR9 and the p85 subunit of PI3K. Importantly, PI3K/Akt inhibition and Akt kinase deficiency abolished CpG-ODN-induced cardioprotection. Conclusion: CpG-ODN, the TLR9 ligand, induces protection against myocardial I/R injury. The mechanisms involve activation of the PI3K/Akt signaling pathway.

Objective: Activation of PI3K/Akt signaling protects the myocardium from ischemia/reperfusion injury. MicroRNAs have been demonstrated to play an important role in the regulation of gene expression at the post-transcriptional level. In this study, we examined whether miR-130a will attenuate cardiac dysfunction and remodeling after myocardial infarction (MI) via PI3K/Akt dependent mechanism. Approaches and results: To determine the role of miR-130a in the proliferation and migration of endothelial cells, HUVECs were transfected with miR-130a mimics before the cells were subjected to scratch-induced wound injury. Transfection of miR-130a mimics stimulated the migration of endothelial cells into the wound area and increased phospho-Akt levels. To examine the effect of miR-130a on cardiac dysfunction and remodeling after MI, Lentivirus expressing miR-130a (LmiR-130a) was delivered into mouse hearts seven days before the mice were subjected to MI. Cardiac function was assessed by echocardiography before and for up to 21 days after MI. Ejection fraction (EF%) and fractional shortening (FS%) in the LmiR-130a transfected MI hearts were significantly greater than in LmiR-control and untransfected control MI groups. LmiR-130a transfection increased capillary number and VEGF expression, and decreased collagen deposition in the infarcted myocardium. Importantly, LmiR-130a transfection significantly suppressed PTEN expression and increased the levels of phosphorylated Akt in the myocardium. However, treatment of LmiR-130a-transfected mice with LY294002, a PI3K inhibitor, completely abolished miR-130a-induced attenuation of cardiac dysfunction after MI. Conclusions: miR-130a plays a critical role in attenuation of cardiac dysfunction and remodeling after MI. The mechanisms involve activation of PI3K/Akt signaling via suppression of PTEN expression.

Local anesthetics (LAs) are necessary for the regional anesthesia, spinal anesthesia, and pain management. However, the application of LAs may cause neurotoxicity and result in postoperative neurological complications. Lithium is a mood stabilizer for the treatment of bipolar disorder and may exert neuroprotective effects. In this study, we evaluated the effects of lithium on bupivacaine (a frequently used LAs)-induced injury in mouse neuroblastoma neuro 2a (N2a) cells. N2a cells were treated with bupivacaine in the presence or absence of lithium. After treatment, the cell injury was evaluated by examination of viability, morphology changes, and nuclear condensation. The levels of mitochondrial transmembrane potential (δψm) and activation of phosphatidylinositol-3-kinase (PI3K)/ threonine-serine protein kinase B (Akt) and extracellular signal-regulated kinase (ERK) were also examined. In a separate experiment, we investigated the effect of Akt and ERK inhibition on cell injury after bupivacaine and lithium treatment. Pretreatment of N2a cells with lithium significantly attenuated bupivacaine-induced cell injury. Lithium pretreatment completely reversed the suppression of PI3K/Akt and ERK signalings and significantly prevented the decline of δψ m in N2a cells after bupivacaine treatment. More importantly, pharmacological inhibition of Akt and ERK diminished the protective effect of lithium against bupivacaine-induced neuronal death. Our data suggest that lithium pretreatment provides a protective effect on bupivacaine-induced neuronal cell injury. This action of lithium is mediated through, at least in part, the activating of PI3K/Akt- and ERK-dependent mechanisms. Because lithium is a clinically proved safety drug for neurons, it is worthwhile to identify whether coadministration of LAs with lithium will decrease the risks of LAs-induced postoperative neurological complications in clinic practice.

Physical and psychological stress can alter the immune system in both humans and animals. Stress is a known risk factor for numerous human diseases, such as infectious and autoimmune diseases, and cancer. Toll-like receptors (TLRs) play a pivotal role in the induction of innate and adaptive immune response. Our previous studies have shown that TLR4 deficiency prevents stress-induced splenocyte reduction. However, the role of TLR2 in stress-mediated lymphocyte reduction is unknown. In this study, we investigated the effects of TLR2 ligands on stress-induced lymphocyte reduction. We also defined whether the phosphoinositide 3-kinases (PI3Ks)/Akt pathway contributes to TLR2-mediated lymphocyte numbers altered by stress. Our data have shown that stimulation of TLR2 by TLR2 ligands peptidoglycan (PGN) or Pam3CSK4 (Pam3) attenuates stress-induced reduction in lymphocyte numbers. However, TLR2 ligand-induced protection from stress-induced lymphocyte reduction is lost in TLR2 deficiency in mice. Furthermore, stimulation of TLR2 by PGN induces protection from stress-induced reduction in the number of splenocytes through PI3K. Moreover, PGN dramatically increases the level of phosphorylation of Akt through a PI3K-dependent manner. Moreover, we found that stimulation of TLR2 by PGN induced protection from stress-induced reduction in splenocyte numbers is abolished in β-arrestin 2 deficient mice. In addition, PGN-induced immune protection in stress-induced changes of cytokine levels appears to require -arrestin 2, a multifunctional adaptor and signal transducer. Collectively, our study thus demonstrates that stimulation of TLR2-mediated PI3K signaling attenuates splenocyte reduction induced by stress, and that β-arrestin 2 modulates TLR2-mediated immune response following stress.

Tocotrienols are members of the vitamin E family but, unlike tocopherols, possess an unsaturated isoprenoid side chain that confers superior anti-cancer properties. The ability of tocotrienols to selectively inhibit the HMG-CoA reductase pathway through posttranslational degradation and to suppress the activity of transcription factor NF-κB could be the basis for some of these properties. Our studies indicate that γ- and δ-tocotrienols have potent antiproliferative activity in pancreatic cancer cells (Panc-28, MIA PaCa-2, Panc-1, and BxPC-3). Indeed both tocotrienols induced cell death (> 50%) by the MTT cell viability assay in all four pancreatic cancer cell lines. We also examined the effects of the tocotrienols on the AKT and the Ras/Raf/MEK/ERK signaling pathways by Western blotting analysis. γ- and δ-tocotrienol treatment of cells reduced the activation of ERK MAP kinase and that of its downstream mediator RSK (ribosomal protein S6 kinase) in addition to suppressing the activation of protein kinase AKT. Suppression of activation of AKT by γ-tocotrienol led to downregulation of p-GSK-3β and upregulation accompanied by nuclear translocation of Foxo3. These effects were mediated by the downregulation of Her2/ErbB2 at the messenger level. Tocotrienols but not tocopherols were able to induce the observed effects. Our results suggest that the tocotrienol isoforms of vitamin E can induce apoptosis in pancreatic cancer cells through the suppression of vital cell survival and proliferative signaling pathways such as those mediated by the PI3-kinase/AKT and ERK/MAP kinases via downregulation of Her2/ErbB2 expression. The molecular components for this mechanism are not completely elucidated and need further investigation.

Myeloid differentiation protein-88 (MyD88) is a crucial adaptor protein in the innate immune response. A protective role for MyD88 in normal cardiac function has been proposed in a surgical hypertrophic model. To assess the in vivo role of MyD88 in cardiac remodeling, we generated transgenic mice with cardiac-restricted expression of a dominant negative mutant of MyD88 (dnMyD88). Surprisingly, dnMyD88 transgenic mice displayed characteristic features of heart failure; including heart weight increase, cardiomyocytes enlargement, interstitial fibrosis, and re-expression of "fetal" genes. Echocardiographic examination of dnMyD88 hearts revealed dilated chamber volume and reduced cardiac contractility. DnMyD88 mice died from heart failure before they were 7 months old, as shown by Kaplan-Meier analysis. Additionally, the heart failure phenotype of dnMyD88 mice was associated with abnormal activation of the Akt/GSK-3β signaling pathway. These data provide the first evidence that normal MyD88 signaling is crucial for maintaining the physiological function of the adult heart.

Cardiac dysfunction is a major consequence of septic shock and may be responsible for the high mortality of sepsis. We have reported that transgenic mice with cardiac-specific overexpression of heat shock protein 27 (Hsp27 Tg) exhibited the protection against doxorubicin-induced cardiac dysfunction. We hypothesized that overexpression of Hsp27 will attenuate cardiac dysfunction during endotoxemia. Hsp27 Tg and age-matched wild-type (WT) mice were injected with LPS. Cardiac function was evaluated by echocardiography, survival rate was carefully monitored, and activities of signaling pathways were determined by immunoblot. LPS administration significantly decreased cardiac function in WT mice. In Hsp27 Tg mice, LPS-induced cardiac dysfunction was significantly attenuated as evidenced by increased ejection fraction (27.3%) and fractional shortening (37.1%), respectively, compared with LPS-treated WT mice. Heat shock protein 27 Tg mice were more resistant to LPS-induced mortality than WT. The levels of phospho-Akt and phospho-glycogen synthase kinase 3β (phospho-GSK-3β) in the myocardium were significantly increased in Hsp27 Tg mice compared with WT after LPS administration. Nuclear factor κB-binding activity was significantly decreased in Hsp27 Tg mice compared with WT mice after LPS challenge. Similar results were observed in in vitro studies using Hsp27-transfected rat cardiomyoblasts. Importantly, phosphoinositide 3-kinase inhibition abolished the protective effect of Hsp27 in LPS-induced cardiac dysfunction and mortality of endotoxemia. Our results suggest that Hsp27 plays an important role in attenuation of cardiac dysfunction and mortality in endotoxemia and that the mechanisms of the protection may involve activation of the PI3K/Akt signaling pathway.

Humanin (HN) is an anti-apoptotic peptide that suppresses neuronal cell death induced by Alzheimer's disease, prion protein fragments, and serum deprivation. Recently, we demonstrated that Gly14-HN (HNG), a variant of HN in which the 14th amino acid serine is replaced with glycine, can decrease apoptotic neuronal death and reduce infarct volume in a focal cerebral ischemia/reperfusion mouse model. In this study, we postulate that the mechanism of HNG's neuroprotective effect is mediated by the PI3K/Akt pathway. Oxygen-glucose deprivation (OGD) was performed in cultured mouse primary cortical neurons for 60 min. The effect of HNG and PI3K/Akt inhibitors on OGD-induced cell death was examined at 24 h after reperfusion. HNG increased cell viability after OGD in primary cortical neurons, whereas the PI3K/Akt inhibitors wortmannin and Akti-1/2 attenuated the protective effect of HNG. HNG rapidly increased Akt phosphorylation, an effect that was inhibited by wortmannin and Akti-1/2. Mouse brains were injected intraventricularly with HNG before being subjected to middle cerebral artery occlusion (MCAO). HNG treatment significantly elevated p-Akt levels after cerebral I/R injury and decreased infarct volume. The protective effect of HNG on infarct size was attenuated by wortmannin and Akti-1/2. Taken as a whole, these results suggest that PI3K/Akt activation mediates HNG's protective effect against hypoxia/ischemia reperfusion injury.

Aims: The ability of lipopolysaccharide (LPS) pre-treatment to induce cardioprotection following ischaemia/reperfusion (I/R) has been well documented; however, the mechanisms have not been fully elucidated. LPS is a Toll-like receptor 4 (TLR4) ligand. Recent evidence indicates that there is cross-talk between the TLR and phosphoinositide 3-kinase/Akt (PI3K/Akt) signalling pathways. We hypothesized that activation of PI3K/Akt signalling plays a critical role in LPS-induced cardioprotection. Methods and results: To evaluate this hypothesis, we pre-treated mice with LPS 24 h before the hearts were subjected to ischaemia (45 min) and reperfusion (4 h). We examined activation of the PI3K/Akt/GSK-3β signalling pathway. The effect of PI3K/Akt inhibition on LPS-induced cardioprotection was also evaluated. LPS pre-treatment significantly reduced infarct size (71.25%) compared with the untreated group (9.3 ± 1.58 vs. 32.3 ± 2.92%, P < 0.01). Cardiac myocyte apoptosis and caspase-3 activity in LPS-pre-treated mice were significantly reduced following I/R. LPS pre-treatment significantly increased the levels of phospho-Akt, phospho-GSK-3β, and heat shock protein 27 in the myocardium. Pharmacological inhibition of PI3K by LY294002 or genetic modulation employing kinase-defective Akt transgenic mice abolished the cardioprotection induced by LPS. Conclusion: These results indicate that LPS-induced cardioprotection in I/R injury is mediated through a PI3K/Akt-dependent mechanism.

A zebrafish transcript dubbed GA2692 was initially identified via a whole-mount in situ hybridization screen for vessel specific transcripts. Its mRNA expression during embryonic development was detected in ventral hematopoietic and vasculogenic mesoderm and later throughout the vasculature up to 48 hours post fertilization. Morpholino-mediated knockdown of GA2692 in embryos resulted in multiple defects in vasculature, particularly, at sites undergoing active capillary sprouting: the intersegmental vessels, sub-intestinal vessels and the capillary sprouts of the pectoral fin vessel. During the course of these studies, a homology search indicated that GA2692 is the zebrafish orthologue of mammalian HspA12B, a distant member of the heat shock protein 70 (Hsp70) family. By a combination of northern blot and realtime PCR analysis, we showed that HspA12B is highly expressed in human endothelial cells in vitro. Knockdown of HspA12B by small interfering RNAs (siRNAs) in human umbilical vein endothelial cells blocked wound healing, migration and tube formation, whereas overexpression of HspA12B enhanced migration and accelerated wound healing - data that are consistent with the in vivo fish phenotype obtained in the morpholino-knockdown studies. Phosphorylation of Akt was consistently reduced by siRNAs against HspA12B. Overexpression of a constitutively active form of Akt rescued the inhibitory effects of knockdown of HspA12B on migration of human umbilical vein endothelial cells. Collectively, our data suggests that HspA12B is a highly endothelial-cell-specific distant member of the Hsp70 family and plays a significant role in endothelial cells during development and angiogenesis in vitro, partially attributable to modulation of Akt phosphorylation.