The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

L’étude d’échantillons humains montre que les voies de signalisation RAS/MAPK et PI3K/AKT/mTOR sont fréquemment activées de manière aberrante dans les tumeurs de la prostate, d’autant plus dans les phases de résistance aux traitements. Ces deux voies de signalisation sont sensibles aux facteurs de croissances et impliquées dans la régulation de processus cellulaires fondamentaux tels que la prolifération, la croissance ou encore la différenciation cellulaire. Ces données suggèrent qu’elles ont un rôle essentiel dans la tumorigenèse prostatique. Cependant, le rôle respectif de chacune de ces voies dans la carcinogenèse prostatique, particulièrement dans les phases précoces, n’est pas clairement établit. L’objectif de ma thèse est donc de définir le rôle possible de ces deux voies dans l’initiation et la progression du cancer de la prostate, ainsi que les mécanismes impliqués dans leur co-dérégulation. Cette étude est réalisée dans un modèle in vivo alternatif, la drosophile, qui possèdent un équivalent fonctionnel de la prostate : les glandes accessoires. La première partie des travaux réalisés montre que seule la suractivation de la voie RAS/MAPK dans la glande accessoire conduit à un processus de tumorigenèse, avec la production de masses cellulaires récapitulant de nombreuses caractéristiques cancéreuses : croissance cellulaire et prolifération incontrôlée, expression de métalloprotéases, perte de l’expression de marqueurs épithéliaux et formation de nouvelles trachées. Cependant, les deux voies de signalisation sont nécessaires à la tumorigenèse, mais avec des rôles différents : la voie RAS/MAPK est activée précocement et est capable de recruter la voie PI3K/AKT/TOR grâce à la mise en place de deux boucles autocrines de régulation. La première dépend de spitz (dEGF) et du récepteur EGFR pour amplifier l’activation de la voie RAS/MAPK. La seconde dépend de l’activation d’ILP6 (dIGF1), produit suite à l’activation de la voie RAS/MAPK, et permet le recrutement de la voie PI3K/AKT/TOR par l’intermédiaire du récepteur à l’insuline InR. La deuxième partie des travaux réalisés montre que l’activation de la voie RAS/MAPK conduit à la production de MMP1 dans les cellules qui seront à l’origine des tumeurs avant leur extravasation hors de l’épithélium. Cette expression temporelle contrôlée correspond à une étape où une réorganisation du cytosquelette a lieu et où le microenvironnement est altéré. Ces données placent donc la dérégulation de la voie RAS/MAPK comme un évènement précoce de la tumorigenèse prostatique, capable de recruter la voie PI3K/AKT/TOR et d’entrainer la production de MMP1, pour in fine conduire à l’extravasation des cellules et à la formation de tumeurs. / Clinical studies have demonstrated that, in prostate cancer, RAS/MAPK and PI3K/AKT/TOR signaling pathways are often aberrantly co-activated in tumors, their activation levels increasing again in resistance phases. These pathways, that are regulated by growth factors, are implicated in fundamental cellular processes regulation such as proliferation, growth and cellular differentiation. These data suggest that they are likely implicated in prostate tumorigenesis. However, the relative implication of each of these two pathways during prostate tumorigenesis, especially during early phases, is not fully understood. Thus, the aim of my thesis is to define the possible implication of these pathways in prostate cancer initiation and progression and which molecular mechanisms are implicated in their co-deregulation. Therefore, we have developed an alternative in vivo model of prostate tumorigenesis in drosophila, where accessory glands are a functional equivalent of the human prostate. The first part of my work shows that only the hyperactivation of the RAS/MAPK pathway in accessory glands can promote tumorigenesis, with the formation of cell masses that recapitulate many cancer hallmarks including uncontrolled cell growth and proliferation, enhanced matrix metalloproteinases expression, loss of epithelial markers expression, neovascularization-like tracheogenesis. However, both pathways are necessary to tumorigenesis, even though they display different roles: the RAS/MAPK pathway is activated earlier and is able to recruit the PI3K/AKT/TOR pathway thanks to the formation of two feedback loops. The first depend on Spitz (dEGF) and EGFR receptor to amplify RAS/MAPK pathway activation. The second depends on ILP6 (dIGF1) activation, produced following RAS/MAPK pathway activation and allow PI3K/AKT/TOR pathway recruitment via insulin receptor InR. The second part of the work shows that RAS/MAPK pathway activation allows MMP1 production restricted to the cells that will be the origin of the tumors, before their actual extravasation. This temporally controlled step of MMP1 expression corresponds to a time window where the cells show strong cytoskeletal reorganization and where microenvironment is disturbed. These data place the RAS/MAPK pathway deregulation as an early event of prostate tumorigenesis, able to recruit the PI3K/AKT/TOR pathway and to induce MMP1 production to allow cell extravasation and tumor formation.

Background: Phosphatase and tensin homolog (PTEN) hamartoma tumor syndrome (PHTS) is caused by germ line mutations in the PTEN gene. Symptoms include cancer pre- disposition, immune deviations, and lipomas/lipomatosis. No causal standard therapy is available. We describe a therapeutic attempt with the mammalian target of rapamycin (mTOR) inhibitor sirolimus for a PHTS patient suffering from thymus hyperplasia and lipomatosis. We furthermore assessed the in vitro effects of sirolimus and other inhibitors on lipoma cells of the patient. Methods: The patient underwent clinical and blood examinations and whole-body magnetic resonance imaging to assess tumor sizes. Lipoma cells of the patient were incubated with inhibitors of the phosphoinositide3-kinase (PI3K)/AKT/ mTOR signaling pathway to analyze the effects on proliferation, adipocyte differentiation, and survival in vitro. Results: Sirolimus treatment improved somatic growth and reduced thymus volume. These effects diminished over the treatment period of 19 mo. Sirolimus decreased lipoma cell proliferation and adipocyte differentiation in vitro but did not cause apoptosis. PI3K and AKT inhibitors induced apoptosis significantly. Conclusion: Sirolimus treatment led to an improvement of the patient’s clinical status and a transient reduction of the thymus. Our in vitro findings point to PI3K and AKT inhibitors as potential treatment options for patients with severe forms of PHTS.

The focus of this dissertation is to understand the physicochemical factors that affect the spatiotemporal control of phosphoinositide signaling events. Despite their low abundance in cellular membranes ( ~ 1% of total lipids) phosphoinositides are assuming major roles in the spatiotemporal regulation of cellular signaling, therefore making this group of lipids an attractive area of study, especially for identifying drug targets. The main phosphoinositide studied in this dissertation is phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2], which regulates various intracellular signaling pathways, notably the PI3K/AKT pathway. The PI3K/AKT pathway plays a critical role in regulating diverse cellular functions including metabolism, growth, proliferation, and survival. Thus, dysregulation of the PI3K/AKT pathway is implicated in a number of human diseases including cancer, diabetes, cardiovascular disease and neurological diseases. PI(4,5)P2 regulates phosphoinositide signaling in the PI3K/AKT pathway through interaction of its highly anionic headgroup with polybasic proteins. The highly specific manner that allows hundreds of structurally diverse proteins to interact with lipid species found in such low supply may require the local formation of PI(4,5)P2 clusters (domains). Although a significant amount of evidence has accumulated over the past decade that supports the notion of PI(4,5)P2-rich clusters, our understanding regarding the structural determinants required for cluster formation remains limited. Studies have shown that PI(4,5)P2 clustering is induced by cellular cations interacting with PI(4,5)P2 via electrostatic interactions, suggesting that non-clustering/clustering transitions are particularly sensitive to ionic conditions. However, why some ions are more effectively cluster PI(4,5)P2 than others remains to be understood. For our first research aim, we investigated the effects of divalent (Ca2+) and monovalent cations (Na+, K+ ) on PI(4,5)P2 clustering to understand the ionic environment required for electrostatic PI(4,5)P2 cluster formation. We used monolayers at the air/water interface (Langmuir films) to monitor PI(4,5)P2 molecular packing in the presence of each cation. Our results indicated that Ca2+ individually and Ca2+ along with K+ had a greater effects on PI(4,5)P2 cluster formation than Na+ and K+, individually and combined. We hypothesize that the cations shield the negatively charged headgroups, allowing adjacent PI(4,5)P2 molecules to interact via H- bonding networks. The analysis of the electrostatic environment required for stable PI(4,5)P2 clustering will help us understand important aspects of PI(4,5)P2 mediated signaling events, such as the temporal control of protein binding to PI(4,5)P2 clusters to enhance their function. Another important spatiotemporal modulator that affects the local concentration of PI(4,5)P2 clusters is cholesterol, a steroid present in large quantities (30-40 mole%) in the plasma membrane. Cholesterol has been shown to induce the formation of liquid-ordered domains when interacting with an otherwise gel phase forming lipid, however, the interaction of cholesterol with an inner leaflet lipid species that favors more of a disordered environment to form clusters is poorly understood. We hypothesize that cations along with cholesterol work synergistically to induce PI(4,5)P2 clustering. Thus, our second research aim was to investigate the role of cholesterol on PI(4,5)P2 clustering by monitoring the molecular packing of PI(4,5)P2 in the presence of both cholesterol and cations. This aim was investigated similarly to the first aim with Langmuir trough monolayer film experiments. Our results showed that cholesterol in the presence of Ca2+ had an additive effect leading to the strongest condensation of the monolayer (increase in PI(4,5)P2 packing). Our hypothesis is that Ca2+ significantly reduces the negative electron density of the phosphate groups, allowing the cholesterol hydroxyl group to interact with PI(4,5)P2 headgroup through hydrogen-bond formation. To confirm our hypothesis, we collaborated with a computational group at the NIH that performed all-atom molecular dynamics (MD) simulations that closely agreed with our experimental data. Thus we were able to determine that the cholesterol hydroxyl group directly interacts via hydrogen-bonding with the phosphodiester group as well as the PI(4,5)P2 hydroxyl groups in the 2- and 6-position. The insight into the structural positioning of cholesterol moving closer to the PI(4,5)P2 headgroup region suggests this unique interaction is important for PI(4,5)P2 cluster formation. Other anionic lipid species are suspected to interact with PI(4,5)P2 and strengthen PI(4,5)P2 clustering. We were particularly interested in the interaction of PI(4,5)P2 with phosphatidylinositol (PI) and phosphatidylserine (PS) because both are abundant in the plasma membrane, ~6-10% and ~10-20% respectively, and both electrostatically bind to peripheral proteins. Therefore, the third research aim analyzed the capacity of PI and PS to form stable clusters with PI(4,5)P2. We hypothesize that a mixed PI/PI(4,5)P2 or PS/PI(4,5)P2 domains are ideal for protein binding, since in combination PI or PS with PI(4,5)P2 would provide the necessary negative electrostatic environment, while PI(4,5)P2 would provide the high specificity and additional electrostatics for protein binding. Langmuir trough monolayer films were used to investigate the stabilization of PI/PI(4,5)P2 and PS/PI(4,5)P2 monolayers in the presence of Ca2+. Our results showed a condensation of the monolayer for both PI/PI(4,5)P2 and PS/PI(4,5)P2 with an increase in Ca2+concentrations, which suggests that Ca2+ shields the highly negatively charged phosphomonoester groups of PI(4,5)P2 allowing PI and PS to participate in PI(4,5)P2’s hydrogen-bond network. Interestingly, both PI and PS equally stabilized PI(4,5)P2 cluster formation, therefore it is highly likely that these lipids interact in vivo to form large stable electrostatic domains required for protein binding. The first three aims provided us with information about the physiological relevant environments required for PI(4,5)P2 cluster formation, while the last aim was geared towards understanding the temporal control of protein association with phosphoinositides in the plasma membrane. Specifically, we analyzed the plasma membrane association of PTEN-L, a translation variant protein of PTEN, that has the ability to exit and enter back into cells, unlike classical PTEN. The ability of PTEN-L to facilitate entry across the anionic and hydrophobic layers of the plasma membrane (in the case of direct transport of PTEN-L across the membrane) or into phospholipid transport vesicles (in the case of vesicular transport of PTEN-L across cells) is likely due to the addition of the 173 N-terminal amino acids, the alternative translated region (ATR-domain). Thus, our fourth research aim focused on the biophysical role of the ATR-domain to associate with inner leaflet plasma membrane lipids. Using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy to monitor secondary structural changes of the ATR-domain upon lipid binding, it was revealed that both PS and PI(4,5)P2 induced conformational change towards a slight increase in β-sheet content in an otherwise unstructured domain suggesting these lipids are required for ATR-domain interaction with the PM. Further studies revealed that the ATR-domain affects the integrity of PS lipid vesicles, further indicating the presence of PS is required to drive ATR-domain across the membrane. This aim provides information on ATR-domain lipid binding preferences aiding in our understanding of the biological and functional role of PTEN-L as a deliverable tumor suppressor protein. The overall goal of the research in this dissertation is to understand factors that fine-tune PI(4,5)P2 cluster formation in space and time. Our first three research aims were designed to understand the synergistic effects of spatiotemporal modulators (cations, cholesterol, and anionic lipids) on local concentration of PI(4,5)P2 clusters. Our results indicate that Ca2+, cholesterol, and the presence of anionic lipids PI and PS all induce stable domains, thus it is highly likely this is part of the biological environment required in vivo for cationic proteins to bind. The last aim, the association of the ATR-domain with phospholipids in the plasma membrane, provided evidence that PS is likely required to drive the ATR-domain across the plasma membrane. This dissertation unifies nearly two decades worth of research by shedding light on synergistic modulators of PI(4,5)P2 cluster formation (Figure 1). Thus, this work has potentially far reaching consequences for understanding temporal control of the spatially resolved protein activity.

No description available.

Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6 h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.

Background and purpose: HSPA12B is a newly discovered member of the Hsp70 family proteins. This study investigated the effects of HSPA12B on focal cerebral ischemia/reperfusion (I/R) injury in mice. Methods: Transgenic mice overexpressing human HSPA12B (Tg) and wild-type littermates (WT) were subjected to 60. min of middle cerebral artery occlusion to induce ischemia and followed by reperfusion (I/R). Neurological deficits, infarct volumes and neuronal death were examined at 6 and 24. hrs after reperfusion. Blood-brain-barrier (BBB) integrity and activated cellular signaling were examined at 3. hrs after reperfusion. Results: After cerebral I/R, Tg mice exhibited improvement in neurological deficits and decrease in infarct volumes, when compared with WT I/R mice. BBB integrity was significantly preserved in Tg mice following cerebral I/R. Tg mice also showed significant decreases in cell injury and apoptosis in the ischemic hemispheres. We observed that overexpression of HSPA12B activated PI3K/Akt signaling and suppressed JNK and p38 activation following cerebral I/R. Importantly, pharmacological inhibition of PI3K/Akt signaling abrogated the protection against cerebral I/R injury in Tg mice. Conclusions: The results demonstrate that HSPA12B protects the brains from focal cerebral I/R injury. The protective effect of HSPA12B is mediated though a PI3K/Akt-dependent mechanism. Our results suggest that HSPA12B may have a therapeutic potential against ischemic stroke.

Background. Toll-like receptors (TLRs) play a role in the pathophysiology of sepsis and multiple organ failure. This study examined the effect of CpG oligodeoxynucleotide (CpG-ODN), the TLR9 ligand, on polymicrobial sepsis-induced cardiac dysfunction.Methods. Male C57BL/6 mice were treated with CpG-ODN, control CpG-ODN (control-ODN), or inhibitory CpG-ODN (iCpG-ODN) 1 hour prior to cecal ligation and puncture (CLP)-induced sepsis. Mice that underwent sham surgery served as sham controls. Cardiac function was examined by echocardiography before and 6 hours after CLP.Results. Cardiac function was significantly decreased 6 hours after CLP. CpG-ODN prevented CLP-induced cardiac dysfunction, as evidenced by maintenance of the ejection fraction and fractional shortening. Control-ODN or iCpG-ODN did not alter CLP-induced cardiac dysfunction. CpG-ODN significantly attenuated CLP-induced myocardial apoptosis and increased myocardial Akt and extracellular-signal-related kinase (ERK) phosphorylation levels following CLP. In vitro experiments demonstrated that CpG-ODN promotes an association between TLR9 and Ras, resulting in Akt and ERK phosphorylation. Inhibition of phosphoinositide 3-kinase (PI3K) by Ly294002 or inhibition of ERK by U0126 in vivo abolished CpG-ODN attenuation of CLP-induced cardiac dysfunction.Conclusions. CpG-ODN prevents CLP-induced cardiac dysfunction, in part through activation of PI3K/Akt and ERK signaling. Modulation of TLR9 could be an effective approach for treatment of cardiovascular dysfunction in patients with sepsis or septic shock.

Myocardial dysfunction is an important manifestation of sepsis/septic shock. Activation of Phosphatidylinositol 3-kinase(PI3K)/protein kinase B (Akt) signaling pathway has been shown to improve cardiac performance during sepsis/septic shock. We have reported previously that α-lipoic acid (LA) activates PI3K/Akt pathway in neuronal cells. It is possible, therefore, that treatment with LA will attenuate cardiac dysfunction during sepsis/septic shock through a PI3K/Akt-dependent mechanism. To test this possibility, we treated mice with LA prior to lipopolysaccharide (LPS) challenge. Cardiac function was analyzed by echocardiography 6 h after LPS challenge. LPS significantly suppressed cardiac function as evidenced by decreases in EF% and FS% in mice. However, LA pretreatment significantly attenuated cardiac dysfunction following LPS challenge. LA pretreatment also improved survival in LPS-challenged mice. Furthermore, LA markedly attenuated the LPS-induced inflammatory response in myocardium, as evidenced by decreases in the upregulation of VCAM-1, ICAM-1 and iNOS, as well as myocardial leucocytes infiltration. Moreover, LPS challenge significantly decreased the phosphorylation levels of Akt and Gsk-3β, which was prevented by LA pretreatment. More importantly, inhibition of PI3K/Akt signaling by Wortmannin (WM) completely abrogated the LA-induced protection in cardiac dysfunction following LPS challenge. Collectively, our results demonstrated that LA improved cardiac function during endotoxemia. The mechanism was through, at least in part, preserved activation of the PI3K/Akt signaling.