Giovanna Piraino

Diverse cardioprotective signaling mechanisms of peroxisome proliferator-activated receptor-gamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone, in reperfusion injury: role of nuclear factor-kappaB, heat shock factor 1, and Akt.

Peroxisome proliferator-activated receptor-γ (PPAR-γ) is a nuclear receptor that regulates diverse biological functions including inflammation. The PPARγ ligands have been reported to exert cardioprotective effects and attenuate myocardial reperfusion injury. Here, we examined the molecular mechanisms of their anti-inflammatory effects. Male Wistar rats were subjected to myocardial ischemia and reperfusion and were treated with the PPAR-γ ligands, 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, or with vehicle only, in the absence or presence of the selective PPAR-γ antagonist GW-9662. In vehicle-treated rats, myocardial injury was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of creatine kinase and tumor necrosis factor-α. These events were preceded by activation of the nuclear factor-κB pathway. The PPAR-γ DNA binding was also increased in the heart after reperfusion. Treatment with ciglitazone or 15d-PGJ2 reduced myocardial damage and neutrophil infiltration and blunted creatine kinase levels and cytokine production. The beneficial effects of both ligands were associated with enhancement of PPAR-γ DNA binding and reduction of nuclear factor-κB activation. Treatment with 15d-PGJ2, but not ciglitazone, enhanced DNA binding of heat shock factor 1 and upregulated the expression of the cardioprotective heat shock protein 70. Treatment with 15d-PGJ2, but not ciglitazone, also induced a significant increase in nuclear phosphorylation of the prosurvival kinase Akt. The cardioprotection afforded by ciglitazone was attenuated by the PPAR-γ antagonist GW-9662. In contrast, GW-9662 did not affect the beneficial effects afforded by 15d-PGJ2. Thus, our data suggest that treatment with these chemically unrelated PPAR-γ ligands results in diverse anti-inflammatory mechanisms.

Activator protein-1 signalling pathway and apoptosis are modulated by poly(ADP-ribose) polymerase-1 in experimental colitis

Poly(ADP‐ribose) polymerase‐1 (PARP‐1) is activated in response to DNA injury in the nucleus of eukaryotic cells and has been implicated in intestinal barrier dysfunction during inflammatory bowel diseases. In this study we investigated whether PARP‐1 may regulate the inflammatory response of experimental colitis at the level of signal transduction mechanisms. Mice genetically deficient of PARP‐1 (PARP‐1–/–) and wild‐type littermates were subjected to rectal instillation of trinitrobenzene sulphonic acid (TNBS). Signs of inflammation were monitored for 14 days. In wild‐type mice, TNBS treatment resulted in colonic ulceration and marked apoptosis, which was associated with decreased colon content of the antiapoptotic protein Bcl‐2, whereas the proapoptotic Bax was unchanged. Elevated levels of plasma nitrate/nitrite, metabolites of nitric oxide (NO), were also found. These inflammatory events were associated with activation of c‐Jun‐NH2 terminal kinase (JNK), phosphorylation of c‐Jun and activation of the nuclear transcription factor activator protein‐1 (AP‐1) in the colon. In contrast, PARP‐1–/– mice exhibited a significant reduction of colon damage and apoptosis, which was associated with increased colonic expression of Bcl‐2 and lower levels of plasma nitrate/nitrite when compared to wild‐type mice. Amelioration of colon damage was associated with a significant reduction of the activation of JNK and reduction of the DNA binding of AP‐1. The data indicate that PARP‐1 exerts a pathological role in colitis possibly by regulating the early stress‐related transcriptional response through a positive modulation of the AP‐1 and JNK pathways.

Peroxisome Proliferator-Activated Receptor δ Regulates Inflammation via NF-κB Signaling in Polymicrobial Sepsis

The nuclear peroxisome proliferator-activated receptor δ (PPARδ) is an important regulator of lipid metabolism. In contrast to its known effects on energy homeostasis, its biological role on inflammation is not well understood. We investigated the role of PPARδ in the modulation of the nuclear factor-κB (NF-κB)-driven inflammatory response to polymicrobial sepsis in vivo and in macrophages in vitro. We demonstrated that administration of GW0742, a specific PPARδ ligand, provided beneficial effects to rats subjected to cecal ligation and puncture, as shown by reduced systemic release of pro-inflammatory cytokines and neutrophil infiltration in lung, liver, and cecum, when compared with vehicle treatment. Molecular analysis revealed that treatment with GW0742 reduced NF-κB binding to DNA in lung and liver. In parallel experiments, heterozygous PPARδ-deficient mice suffered exaggerated lethality when subjected to cecal ligation and puncture and exhibited severe lung injury and higher levels of circulating tumor necrosis factor-α (TNFα) and keratinocyte-derived chemokine than wild-type mice. Furthermore, in lipopolysaccharide-stimulated J774.A1 macrophages, GW0742 reduced TNFα production by inhibiting NF-κB activation. RNA silencing of PPARδ abrogated the inhibitory effects of GW0742 on TNFα production. Chromatin immunoprecipitation assays revealed that PPARδ displaced the NF-κB p65 subunit from the κB elements of the TNFα promoter, while recruiting the co-repressor BCL6. These data suggest that PPARδ is a crucial anti-inflammatory regulator, providing a basis for novel sepsis therapies.

Ciglitazone ameliorates lung inflammation by modulating the inhibitor κB protein kinase/nuclear factor-κB pathway after hemorrhagic shock

Objective:Peroxisome proliferator-activated receptor-γ is a ligand-activated transcription factor. Ciglitazone, a peroxisome proliferator-activated receptor-γ ligand, has been shown to provide beneficial effects in experimental models of sepsis and ischemia/reperfusion injury. We investigated the effects of ciglitazone on lung inflammation after severe hemorrhage. Design:Prospective, laboratory study, rodent model of hemorrhagic shock. Setting:University hospital laboratory. Subjects:Male rats. Interventions:Hemorrhagic shock was induced by withdrawing blood to a mean arterial pressure of 50 mm Hg. At 3 hrs after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received ciglitazone (10 mg/kg) or vehicle intraperitoneally. Heart rate and mean arterial pressure were measured throughout the experiment. Plasma and lung tissue were collected for analysis up to 3 hrs after resuscitation. Measurements and Main Results:Ciglitazone treatment ameliorated mean arterial pressure, reduced lung injury, significantly blunted lung neutrophil infiltration, and lowered plasma interleukin-6, interleukin-10, and monocyte chemoattractant protein-1 levels. In a time course analysis, vehicle-treated rats had a significant increase in nuclear factor-&kgr;B DNA binding, which was preceded by increased inhibitor &kgr;B protein kinase activity and inhibitor &kgr;Bα degradation in the lung. Treatment with ciglitazone significantly reduced inhibitor &kgr;B protein kinase activity and inhibitor &kgr;Bα degradation and completely inhibited nuclear factor-&kgr;B DNA binding. This reduction of inhibitor &kgr;B protein kinase activity afforded by ciglitazone appeared to be a consequence of a physical interaction between peroxisome proliferator-activated receptor-γ and increased inhibitor &kgr;B protein kinase. Conclusion:Ciglitazone ameliorates the inflammatory response and may reduce lung injury after hemorrhagic shock. These protective effects appear to be mediated through inhibition of the inhibitor &kgr;B protein kinase/nuclear factor-&kgr;B pathway.

Proinsulin c-peptide exerts beneficial effects in endotoxic shock in mice.

Objective:Insulin connecting peptide (c-peptide) aids the folding of proinsulin and has been considered to have little biological activity. Recently, c-peptide has been shown to improve diabetic neuropathy and nephropathy as well as vascular inflammation. In vitro studies have reported that c-peptide may activate peroxisome proliferator-activated receptor-&ggr;, a nuclear transcription factor that plays a regulatory role in inflammation. This study was designed to investigate the biological effects of c-peptide during endotoxemia. Design:Prospective, randomized laboratory investigation that used an established murine model of endotoxic shock. Setting:University hospital laboratory. Subjects:Mice were subjected to endotoxic shock by intraperitoneal administration of Escherichia coli lipopolysaccharide. Interventions:Mice received vehicle or c-peptide (70–140 nmol/kg) intraperitoneally at 3 hrs and 6 hrs after lipopolysaccharide. Mortality was monitored for 96 hrs. In a separate experiment, mice were killed at 4, 7, and 18 hrs after lipopolysaccharide administration. Lungs and plasma were collected for biochemical assays. Measurements and Main Results:In vehicle-treated mice, endotoxic shock resulted in lung injury and was associated with a 41% survival rate and elevation in plasma tumor necrosis factor-&agr;, macrophage inflammatory protein-1&agr;, monocyte chemoattractant protein-1, and keratinocyte-derived chemokine levels. Lung nuclear levels of phosphorylated extracellular signal-regulated kinases 1 and 2 were significantly increased in vehicle-treated mice. On the other hand, lung nuclear expression and DNA binding of proliferator-activated receptor-&ggr; were decreased in comparison to control animals. Treatment with c-peptide (140 nmol/kg) improved survival rate (68%) and reduced plasma levels of tumor necrosis factor-&agr;, macrophage inflammatory protein-1&agr;, and monocyte chemoattractant protein-1, but it did not exert hypoglycemic effects. Treatment with c-peptide also up-regulated lung nuclear expression and DNA binding of proliferator-activated receptor-&ggr; and reduced phosphorylation of extracellular signal-regulated kinases 1 and 2 in comparison to vehicle-treated mice. Conclusions:Our data show that c-peptide has beneficial effects in endotoxic shock, and this therapeutic effect is associated with activation of proliferator-activated receptor-&ggr;.

Synergistic effect of peroxisome proliferator activated receptor-gamma and liver X receptor-alpha in the regulation of inflammation in macrophages.

ABSTRACT Peroxisome proliferator-activated receptor-&ggr; (PPAR&ggr;) and liver X receptor-&agr; (LXR&agr;) are nuclear ligand-activated transcription factors, which regulate lipid metabolism and inflammation. Murine J774.2 macrophages were stimulated with Escherichia coli lipopolysaccharide (concentration, 10 &mgr;g/mL) with or without the PPAR&ggr; ligand, 15-deoxy-&Dgr;12,14 prostaglandin J2 (15d-PGJ2), or the LXR&agr; ligands, 22(R)-hydroxycholesterol and T0901317 (concentration range, 0.01-10 &mgr;mol/L), alone or in combination. Nitric oxide (NO) metabolites and tumor necrosis factor &agr; production, inducible NO synthase expression, and mitochondrial respiration were measured. When added to the cells as single agents, 15d-PGJ2, 22(R)-hydroxycholesterol, or T0901317 reduced the lipopolysaccharide-induced NO and tumor necrosis factor &agr; production and the inducible NO synthase expression, and partially maintained mitochondrial respiration in a concentration-dependent manner. When added to the cells in combination at suboptimal concentrations, 15d-PGJ2 with 22(R)-hydroxycholesterol, or 15d-PGJ2 with T0901317, exerted anti-inflammatory effects similar to much higher concentrations (10,000-fold to 100,000-fold) of each ligand alone. The anti-inflammatory effects of these ligands, alone or in combination, were associated with reduction of nuclear factor-&kgr;B activation and with enhancement of PPAR&ggr; DNA binding. LXR&agr; expression was upregulated in response to 15d-PGJ2 and to the LXR&agr; ligands when added alone or in combination. Immunoprecipitation experiments revealed that PPAR&ggr; interacted with LXR&agr;. Our data demonstrate that the PPAR&ggr; ligand, 15d-PGJ2, and the LXR&agr; ligands, 22(R)-hydroxycholesterol and T0901317, although binding to different nuclear receptors (i.e., PPAR&ggr; and LXR&agr;, respectively), affect mediator production through common cell signaling events and exert a synergistic potentiation in a combined treatment at suboptimal concentrations. Thus, our data suggest that PPAR&ggr; and LXR&agr; may interact in controlling the inflammatory response in macrophages.

Liver X receptor α activation with the synthetic ligand T0901317 reduces lung injury and inflammation after hemorrhage and resuscitation via inhibition of the nuclear factor κB pathway.

Liver X receptor &agr; (LXR&agr;) is a nuclear transcription factor that regulates lipid metabolism. Recently, it has been shown that activation of LXR&agr; with synthetic ligands has anti-inflammatory effects in atherosclerosis and chemical-induced dermatitis. We investigated the effect of the LXR&agr; agonist, T0901317, on lung inflammation in a rodent model of hemorrhagic shock. Hemorrhagic shock was induced in male rats by withdrawing blood to a goal mean arterial blood pressure of 50 mmHg. Blood pressure was maintained at this level for 3 h, at which point rats were rapidly resuscitated with shed blood. Animals were then treated with T0901317 (50 mg · kg−1) or vehicle i.p. and sacrificed at 1, 2, and 3 h after resuscitation. Treatment with T0901317 significantly improved the cardiac and stroke volume indices as well as the heart rate of rats during the resuscitation period as compared with vehicle-treated rats. The T0901317-treated animals showed significant improvement in the plasma level of lactate, whereas base deficit and bicarbonate levels both trended toward improvement. The T0901317-treated animals also showed lower levels of plasma cytokines and chemokines monocyte chemoattractant protein 1, macrophage inflammatory protein 1&agr;, TNF-&agr;, KC, and IL-6. Lung injury and neutrophil infiltration were reduced by treatment with T0901317, as evaluated by histology and myeloperoxidase assay. At molecular analysis, treatment with T0901317 increased nuclear LXR&agr; expression and DNA binding while also inhibiting activation of nuclear factor &kgr;B, a proinflammatory transcription factor, in the lung. Thus, our data suggest that LXR&agr; is an important modulator of the inflammatory response and lung injury after severe hemorrhagic shock, likely through the inhibition of the nuclear factor &kgr;B pathway.

C-peptide ameliorates kidney injury following hemorrhagic shock.

Reperfusion injury following hemorrhagic shock is accompanied by the development of a systemic inflammatory state that may lead to organ failure. Insulin connecting peptide (C-peptide) has been shown to exert anti-inflammatory effects in sepsis and myocardial ischemia-reperfusion injury and to ameliorate renal dysfunction in diabetic animals. Hence, we investigated the effect of C-peptide on kidney injury after hemorrhagic shock. We hypothesized that C-peptide would exert renoprotective effects by blunting inflammation. Hemorrhagic shock was induced in male rats (3-4 months old) by withdrawing blood from the femoral artery to a mean arterial pressure of 50 mmHg. Animals were kept in shock for 3 h, at which time they were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, one group of animals received C-peptide (280 nmol/kg), whereas another group received vehicle. Hemorrhagic shock resulted in significant rise in plasma levels of creatinine and elevated kidney neutrophil infiltration as evaluated by myeloperoxidase activity in vehicle-treated rats in comparison with sham rats, thus suggesting kidney injury. Treatment with C-peptide significantly attenuated the rise in creatinine and kidney myeloperoxidase activity when compared with vehicle group. At a molecular level, these effects of C-peptide were associated with reduced expression of the c-Fos subunit and reduced activation of the proinflammatory kinases, extracellular signal-regulated kinase 1/2 (ERK 1/2), and c-Jun N-terminal kinase and subsequently reduced DNA binding of activator protein 1 in the kidney. Thus, our data suggest that C-peptide may exert renoprotective effects after hemorrhagic shock by modulating activator protein 1 signaling.

Phosphorylation of extracellular signal-regulated kinase (ERK)-1/2 Is associated with the downregulation of peroxisome proliferator-activated receptor (PPAR)-γ during polymicrobial sepsis.

Peroxisome proliferator-activated receptor (PPAR)-γ is a ligand-activated transcription factor and regulates inflammation. Posttranslational modifications regulate the function of PPARγ, potentially affecting inflammation. PPARγ contains a mitogen-activated protein kinase (MAPK) site, and phosphorylation by extracellular signal-regulated kinase (ERK)-1/2 leads to inhibition of PPARγ. This study investigated the kinetics of PPARγ expression and activation in parenchymal and immune cells in sepsis using the MAPK/ERK kinase (MEK)-1 inhibitor, an upstream kinase of ERK1/2. Adult male Sprague Dawley rats were subjected to polymicrobial sepsis by cecal ligation and puncture. Rats received intraperitoneal injection of vehicle or the MEK1 inhibitor PD98059 (5 mg/kg) 30 min before cecal ligation and puncture. Rats were euthanized at 0, 1, 3, 6 and 18 h after cecal ligation and puncture. Control animals used were animals at time 0 h. Lung, plasma and peripheral blood mononuclear cells (PBMCs) were collected for biochemical assays. In vehicle-treated rats, polymicrobial sepsis resulted in significant lung injury. In the lung and PBMCs, nuclear levels of PPARγ were decreased and associated with an increase in phosphorylated PPARγ and phosphorylated ERK1/2 levels. Treatment with the MEK1 inhibitor increased the antiinflammatory plasma adipokine adiponectin, restored PPARγ expression in PBMCs and lung, and decreased lung injury. The inflammatory effects of sepsis cause changes in PPARγ expression and activation, in part, because of phosphorylation of PPARγ by ERK1/2. This phosphorylation can be reversed by ERK1/2 inhibition, thereby improving lung injury.

C-peptide, a Novel Inhibitor of Lung Inflammation following Hemorrhagic Shock

C-peptide is a 31-amino acid peptide cleaved from proinsulin during insulin synthesis. Initially thought to be inert, C-peptide may modulate the inflammatory response in the setting of endotoxemia and ischemia reperfusion. However, the spectrum of its biological effects is unclear. We hypothesized that exogenous administration of C-peptide would modulate pro- and anti-inflammatory signaling pathways and thereby attenuate lung inflammation in an in vivo model of hemorrhagic shock. Hemorrhagic shock was induced in male Wistar rats (aged 3-4 mo) by withdrawing blood to a mean arterial pressure of 50 mmHg. At 3 h after hemorrhage, rats were rapidly resuscitated by returning their shed blood. At the time of resuscitation and every hour thereafter, animals received C-peptide (280 nmol/kg) or vehicle parenterally. Animals were euthanized at 1 and 3 h after resuscitation. C-peptide administration at resuscitation following hemorrhagic shock ameliorated hypotension and blunted the systemic inflammatory response by reducing plasma levels of IL-1, IL-6, macrophage inflammatory protein-1α, and cytokine-induced neutrophil chemoattractant-1. This was associated with a reduction in lung neutrophil infiltration and plasma levels of receptor for advanced glycation end products. Mechanistically, C-peptide treatment was associated with reduced expression of proinflammatory transcription factors activator protein-1 and NF-κB and activation of the anti-inflammatory transcription factor peroxisome proliferator-activated receptor-γ. Our data suggest that C-peptide ameliorates the inflammatory response and lung inflammation following hemorrhagic shock. These effects may be modulated by altering the balance between pro- and anti-inflammatory signaling in the lung.