Timothy J. Burroughs

15-Deoxy-delta(12,14)-prostaglandin J(2) (15D-PGJ(2)), a peroxisome proliferator activated receptor gamma ligand, reduces tissue leukosequestration and mortality in endotoxic shock.

Peroxisome proliferator-activated receptor-γ (PPARγ) is a nuclear receptor that requires ligand activation for transcription. Experimental studies have shown that 15-deoxy-Δ12,14-PGJ2 (15d-PGJ2) is a natural PPARγ ligand which has potent anti-inflammatory properties. This study was designed to examine the effect and the molecular mechanisms of 15d-PGJ2 on tissue neutrophil infiltration and survival in endotoxic shock. Male Swiss albino mice were subjected to intraperitoneal injection of Escherichia coli lipopolysaccharide (LPS, 25 mg/kg). Three hours after LPS mice received vehicle or 15d-PGJ2 (1 mg/kg) and continued treatment every 12 hours. Survival was monitored for 72 hours. In a separate experiment, mice were sacrificed 6 hours after LPS and tissue examined. In vehicle-treated mice, LPS injection resulted in a survival rate of 9%. Marked lung injury was characterized by hemorrhage, infiltration of inflammatory cells and reduction of alveolar space. Elevated levels of myeloperoxidase activity in lung and small intestine were indicative of infiltration of neutrophils. Increased expression of intercellular adhesion molecule-1, vascular cellular adhesion molecule-1 and E-selectin were observed in the lung and small intestine. These inflammatory events were associated with reduced expression of PPARγ and with activation of nuclear factor-κB (NF-κB) in the lung. Treatment with 15d-PGJ2 improved survival rate to 55%, downregulated expression of adhesion molecules and reduced neutrophil infiltration in tissues. These beneficial effects were associated with reduced activation of NF-κB DNA binding, whereas expression and DNA binding of PPARγ and expression of the cytoprotective heat shock protein (HSP) 70 were increased in the lung. Our data demonstrate that 15d-PGJ2 ameliorates endotoxic shock most likely through repressing the proinflammatory pathway of NF-κB and enhancement of the cytoprotective heat shock response.

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.

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.

Lung injury after hemorrhage is age-dependent: role of peroxisome proliferator activated receptor γ

Objective:The incidence of multiple organ failure in pediatric trauma victims is lower than in the adult population. However, the molecular mechanisms are not yet defined. We investigated whether the pathophysiologic characteristics of hemorrhage-induced lung injury may be age dependent and may be regulated by the peroxisome proliferator-activated receptor &ggr; (PPAR&ggr;). Design:Prospective, laboratory investigation that used an established rodent model of hemorrhagic shock. Setting:University hospital laboratory. Subjects:Young (n = 67; 3–5 months old) and mature (n = 66; 11–13 months old) male rats. Interventions:Hemorrhagic shock was induced in young and mature rats by withdrawing blood to a mean arterial blood pressure of 50 mm Hg. After 3 hours, rats were rapidly resuscitated by infusing the shed blood and killed 3 hours thereafter. Measurements and Main Results:In young rats, lung injury was characterized by accumulation of red cells and neutrophils at the end of the resuscitation period; on Western blot analysis, lung expression of intercellular adhesion molecule-1 was increased. In contrast, the severity of lung injury was more pronounced in mature rats. Lung myeloperoxidase activity and expression of constitutive and inducible intercellular adhesion molecule-1 was significantly higher in mature rats compared with young rats. Mature rats also had higher plasma levels of cytokines and chemokines compared with young rats. This heightened inflammation was associated with higher degree of activation of nuclear factor-&kgr;B and down-regulation of PPAR&ggr; and heat shock factor-1 in the lung of mature rats compared with young rats. Treatment with the PPAR&ggr; ligand, the cyclopentenone prostaglandin 15-deoxy-&Dgr;12,14-prostaglandin J2, ameliorated lung injury in young, but not in mature animals. Conclusions:Lung injury after severe hemorrhage is age dependent and may be secondary to a diverse regulation of PPAR&ggr;.