Michael Tencati

PI3K-Akt Pathway Suppresses Coagulation and Inflammation in Endotoxemic Mice

Objective—In endotoxemia, lipopolysaccharide (LPS) induces a systemic inflammatory response and intravascular coagulation. Monocytes orchestrate the innate immune response to LPS by expressing a variety of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), and the procoagulant molecule, tissue factor (TF). In this study, we analyzed the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in the activation of coagulation and the innate immune response in a mouse model of endotoxemia. Methods and Results—Wortmannin and LY294002 were used to inhibit the PI3K-Akt pathway. We found that wortmannin inhibited LPS-induced Akt phosphorylation in blood cells. Inhibition of the PI3K-Akt pathway significantly increased TF mRNA expression in blood cells, TF antigen, and thrombin–antithrombin III levels in the plasma, and fibrin deposition in the liver of endotoxemic mice. Inhibition of the PI3K-Akt pathway also strongly enhanced LPS-induced cytokine expression and the levels of soluble E-selectin in the plasma, suggesting enhanced activation of both monocytes and endothelial cells. Wortmannin treatment also increased the number of macrophages in the liver and kidney of endotoxemic mice. Finally, wortmannin and LY294002 dramatically reduced the survival time of endotoxemic mice. Conclusions—These data suggest that the PI3K-Akt pathway suppresses LPS-induced inflammation and coagulation in endotoxemic mice.

Genetic Analysis of the Role of the PI3K-Akt Pathway in Lipopolysaccharide-Induced Cytokine and Tissue Factor Gene Expression in Monocytes/Macrophages

LPS stimulation of monocytes/macrophages induces the expression of genes encoding proinflammatory cytokines and the procoagulant protein, tissue factor. Induction of these genes is mediated by various signaling pathways, including mitogen-activated protein kinases, and several transcription factors, including Egr-1, AP-1, ATF-2, and NF-κB. We used a genetic approach to determine the role of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway in the regulation of LPS signaling and gene expression in isolated macrophages and in mice. The PI3K-Akt pathway is negatively regulated by the phosphatase and tensin homologue (PTEN). We used peritoneal exudate cells from Pik3r1-deficient mice, which lack the p85α regulatory subunit of PI3K and have reduced PI3K activity, and peritoneal macrophages from PTENflox/flox/LysMCre mice (PTEN−/−), which have increased Akt activity. Analysis of LPS signaling in Pik3r1−/− and PTEN−/− cells indicated that the PI3K-Akt pathway inhibited activation of the ERK1/2, JNK1/2, and p38 mitogen-activated protein kinases and reduced the levels of nuclear Egr-1 protein and phosphorylated ATF-2. Modulating the PI3K-Akt pathway did not affect LPS-induced degradation of IκBα or NF-κB nuclear translocation. LPS induction of TNF-α, IL-6, and tissue factor gene expression was increased in Pik3r1−/− peritoneal exudate cells and decreased in PTEN−/− peritoneal macrophages compared with wild-type (WT) cells. Furthermore, LPS-induced inflammation and coagulation were enhanced in WT mice containing Pik3r1−/− bone marrow compared with WT mice containing WT bone marrow and in mice lacking the p85α subunit in all cells. Taken together, our results indicate that the PI3K-Akt pathway negatively regulates LPS signaling and gene expression in monocytes/macrophages.

Protease-Activated Receptor-1 Contributes to Cardiac Remodeling and Hypertrophy

Background— Protease-activated receptor-1 (PAR-1) is the high-affinity receptor for the coagulation protease thrombin. It is expressed by a variety of cell types in the heart, including cardiomyocytes and cardiac fibroblasts. We have shown that tissue factor (TF) and thrombin contribute to infarct size after cardiac ischemia-reperfusion (I/R) injury. Moreover, in vitro studies have shown that PAR-1 signaling induces hypertrophy of cardiomyocytes and proliferation of cardiac fibroblasts. The purpose of the present study was to investigate the role of PAR-1 in infarction, cardiac remodeling, and hypertrophy after I/R injury. In addition, we analyzed the effect of overexpression of PAR-1 on cardiomyocytes. Methods and Results— We found that PAR-1 deficiency reduced dilation of the left ventricle and reduced impairment of left ventricular function 2 weeks after I/R injury. Activation of ERK1/2 was increased in injured PAR-1−/− mice compared with wild-type mice; however, PAR-1 deficiency did not affect infarct size. Cardiomyocyte-specific overexpression of PAR-1 in mice induced eccentric hypertrophy (increased left ventricular dimension and normal left ventricular wall thickness) and dilated cardiomyopathy. Deletion of the TF gene in cardiomyocytes reduced the eccentric hypertrophy in mice overexpressing PAR-1. Conclusions— Our results demonstrate that PAR-1 contributes to cardiac remodeling and hypertrophy. Moreover, overexpression of PAR-1 on cardiomyocytes induced eccentric hypertrophy. Inhibition of PAR-1 after myocardial infarction may represent a novel therapy to reduce hypertrophy and heart failure in humans.

Role of cardiac myocyte tissue factor in heart hemostasis.

Summary.  Background: The tissue‐specific pattern of tissue factor (TF) expression suggests that it plays a major role in the hemostatic protection of specific organs, such as the heart and lung. In support of this notion, we found that mice expressing very low levels of TF exhibit hemostatic defects in the heart and lung. Hemosiderosis and fibrosis are observed in the hearts of all low TF mice as early as 3 months of age. In contrast, TF+/– mice expressing ∼50% of wild‐type levels of TF had no detectable hemostatic defects. Objective and methods: The objective of this study was to determine the threshold of TF that is required to maintain hemostasis under normal and pathologic conditions, and to investigate the specific role of cardiac myocyte TF in heart hemostasis using mice with altered levels of TF expression in cardiac myocytes. Results: First, we found that mice with 20% of wild‐type levels of TF activity in their hearts had hemosiderosis and fibrosis by 6 months of age. Secondly, mice with a selective deletion of the TF gene in cardiac myocytes had a mild hemostatic defect under normal conditions but exhibited a significant increase in hemosiderosis and fibrosis after challenge with isoproterenol. Finally, we showed that cardiac myocyte‐specific overexpression of TF abolished hemosiderin deposition and fibrosis in the hearts of low TF mice. Conclusions: Taken together, our results indicate that TF expression by cardiac myocytes is important to maintain heart hemostasis under normal and pathologic conditions.

Insulin Activation of the Phosphatidylinositol 3-Kinase/Protein Kinase B (Akt) Pathway Reduces Lipopolysaccharide-Induced Inflammation in Mice

Insulin is used to control pro-inflammatory hyperglycemia in critically ill patients. However, recent studies suggest that insulin-induced hypoglycemia may negate its beneficial effects in these patients. It is noteworthy that recent evidence indicates that insulin has anti-inflammatory effects that are independent of controlling hyperglycemia. To date, the mechanism by which insulin directly reduces inflammation has not been elucidated. It is well established that insulin activates phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling in many cell types. We and others have shown that this pathway negatively regulates LPS-induced signaling and pro-inflammatory cytokine production in monocytic cells. We hypothesized that insulin inhibits inflammation during endotoxemia by activation of the PI3K/Akt pathway. We used a nonhyperglycemic mouse model of endotoxemia to determine the effect of continuous administration of a low dose of human insulin on inflammation and survival. It is noteworthy that insulin treatment induced phosphorylation of Akt in muscle and adipose tissues but did not exacerbate lipopolysaccharide (LPS)-induced hypoglycemia. Insulin decreased plasma levels of interleukin-6, tumor necrosis factor-α, monocyte chemotactic protein 1 (MCP1)/JE, and keratinocyte chemoattractant, and decreased mortality. The PI3K inhibitor wortmannin abolished the insulin-mediated activation of Akt and the reduction of chemokine and interleukin-6 levels. We conclude that insulin reduces LPS-induced inflammation in mice in a PI3K/Akt-dependent manner without affecting blood glucose levels.

A Novel Class of Antioxidants Inhibit LPS Induction of Tissue Factor by Selective Inhibition of the Activation of ASK1 and MAP Kinases

Objective—Oxidative stress contributes to the pathogenesis of many diseases, including atherosclerosis and sepsis. We have previously described a novel class of therapeutic compounds with antioxidant and antiinflammatory properties. However, at present, the intracellular targets of these compounds have not been identified. The purpose of this study was to elucidate the mechanism by which 2 structurally-related antioxidants (AGI-1067 and AGI-1095) inhibit LPS induction of tissue factor (TF) expression in human monocytic cells and endothelial cells. Methods and Results—We found that succinobucol (AGI-1067) and AGI-1095 inhibited LPS induction of TF expression in both monocytic cells and endothelial cells. These compounds also reduced LPS induction of nuclear AP-1 and expression of Egr-1 without affecting nuclear translocation of NF-&kgr;B. Importantly, these antioxidants inhibited LPS activation of the redox-sensitive kinase, apoptosis signal-regulating kinase-1 (ASK1) and the mitogen-activated protein kinases (MAPKs) p38, ERK1/2, and JNK1/2. Conclusions—AGI-1067 and AGI-1095 inhibit TF gene expression in both monocytic cells and endothelial cells through a mechanism that involves the inhibition of the redox-sensitive MAP3K, ASK1. These compounds selectively reduce the activation/induction of MAPK, AP-1, and Egr-1 without affecting NF-&kgr;B nuclear translocation.

The synthetic pentasaccharide fondaparinux reduces coagulation, inflammation and neutrophil accumulation in kidney ischemia–reperfusion injury

Summary.  Ischemia–reperfusion (I/R) injury is associated with activation of coagulation and inflammation. Interestingly, various anticoagulants have been shown to reduce both coagulation and inflammation in animal models of kidney I/R injury. Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in the coagulation cascade. The aim of this study was to investigate the effect of fondaparinux in a lethal murine model of kidney I/R injury. A murine model of kidney I/R was established. In this model, we measured activation of the coagulation cascade and induction of inflammation. Administration of fondaparinux to I/R‐injured mice reduced fibrin deposition in the kidney, reduced serum creatinine levels and increased survival from 0 to 44% compared with saline‐treated control mice. Fondaparinux also reduced interleukin‐6 and macrophage inflammatory protein‐2 expression and decreased neutrophil accumulation in the injured kidneys. Finally, we showed that fondaparinux reduced thioglycollate‐induced recruitment of neutrophils into the peritoneum and inhibited the binding of U937 cells to P‐selectin in vitro. Our data suggest that fondaparinux reduces kidney I/R injury primarily by inhibiting the recruitment of neutrophils.

Dexamethasone enhances LPS induction of tissue factor expression in human monocytic cells by increasing tissue factor mRNA stability

Glucocorticoids, such as dexamethasone (Dex), are used clinically in the treatment of various inflammatory diseases. Dex acts by inhibiting the expression of inflammatory mediators, such as tumor necrosis factor α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1). It is surprising that Dex enhances bacterial lipopolysaccharide (LPS) induction of tissue factor (TF) expression in human monocytic cells. TF is a transmembrane glycoprotein that activates the coagulation protease cascade. In this study, we analyze the mechanism by which Dex enhances LPS‐induced TF expression in human monocytic cells. We found that Dex reduced LPS‐induced TF gene transcription but increased the stability of TF mRNA. Dex decreased the stability of MCP‐1 mRNA and did not affect TNF‐α mRNA stability. Finally, we showed that Dex increased the stability of a transcript consisting of the final 297 nucleotides of the TF mRNA in in vitro decay assays. This region contains AU‐rich elements that regulate mRNA stability and may mediate the Dex response. Therefore, despite an inhibition of TF gene transcription, Dex enhances TF expression in human monocytic cells by increasing the stability of TF mRNA.

A non-anticoagulant synthetic pentasaccharide reduces inflammation in a murine model of kidney ischemia-reperfusion injury

Fondaparinux is a synthetic pentasaccharide that selectively inhibits factor Xa (FXa) in an antithrombin-dependent fashion. This newly developed anticoagulant is used in the prevention and treatment of venous thromboembolism. Recently, we showed that fondaparinux reduces inflammation and protects the kidney from ischemia-reperfusion (I/R) injury. However, the relative contributions of the anticoagulant and anti-inflammatory activities of fondaparinux to the observed protection is unknown. To address this, we chemically modified fondaparinux to abolish its affinity for antithrombin and analyzed the effect of this non-anticoagulant (NAC)-pentasaccharide on binding of U937 cells to P-selectin in vitro and on inflammation in a murine model of kidney I/R injury. NAC-pentasaccharide was as effective as fondaparinux at inhibiting the binding of U937 cells to P-selectin. In addition, NAC-pentasaccharide significantly reduced IL-6 and MIP-2 expression and injury in the kidney I/R model. These findings indicate that the anti-inflammatory activity of fondaparinux can be dissociated from its anticoagulant activity and that NAC-pentasaccharide is protective in kidney I/R injury.