Barbara Pisano

Ligands of the peroxisome proliferator-activated receptors (PPAR-γ and PPAR-α) reduce myocardial infarct size

This study was designed to investigate the effects of various chemically distinct activators of PPAR‐γ and PPAR‐α in a rat model of acute myocardial infarction. Using Northern blot analysis and RT‐PCR in samples of rat heart, we document the expression of the mRNA for PPAR‐γ (isoform 1 but not isoform 2) as well as PPAR‐β and PPAR‐α in freshly isolated cardiac myocytes and cardiac fibroblasts and in the left and right ventricles of the heart. Using a rat model of regional myocardial ischemia and reperfusion (in vivo), we have discovered that various chemically distinct ligands of PPAR‐γ (including the TZDs rosiglitazone, ciglitazone, and pioglitazone, as wel as the cyclopentanone prostaglandins 15D‐PGJ2 and PGA1) cause a substantial reduction of myocardial infarct size in the rat. We demonstrate that two distinct ligands of PPAR‐α (including clofibrate and WY 14643) also cause a substantial reduction of myocardial infarct size in the rat. The most pronounced reduction in infarct size was observed with the endogenous PPAR‐γ ligand, 15deoxyΔ12,14‐prostagalndin J2 (15D‐PGJ2). The mechanisms of the cardioprotective effects of 15D‐PGJ2 may include 1) activation of PPAR‐α, 2) activation of PPAR‐γ, 3) expression of HO‐1, and 4) inhibition of the activation of NF‐KB in the ischemic‐reperfused heart. Inhibition by 15D‐PGJ2 of the activation of NF‐κB in turn results in a reduction of the 1) expression of inducible nitric oxide synthase and the nitration of proteins by peroxynitrite, 2) formation of the chemokine MCP‐1, and 3) expression of the adhesion molecule ICAM‐1. We speculate that ligands of PPAR‐γ and PPAR‐α may be useful in the therapy of conditions associated with ischemia‐reperfusion of the heart and other organs. Our findings also imply that TZDs and fibrates may help protect the heart against ischemiareperfusion injury. This beneficial effect of 15D‐PGJ2 was associated with a reduction in the expression of the 1) adhesion molecules ICAM‐1 and P‐selectin, 2) chemokine macrophage chemotactic protein 1, and 3) inducible isoform of nitric oxide synthase. 15D‐PGJ2 reduced the nitration of proteins (immunohistological analysis of nitrotyrosine formation) caused by ischemiareperfusion, likely due to the generation of peroxynitrite. Not all of the effects of 15D‐PGJ2, however, are due to the activation of PPAR‐γ. For instance, exposure of rat cardiac myocytes to 15D‐PGJ2, but not to rosiglitazone, results in an up‐regulation of the expression of the mRNA for heme‐oxygenase‐1 (HO‐1). Taken together, these results provide convincing evidence that several, chemically distinct ligands of PPAR‐γ reduce the tissue necrosis associated with acute myocardial infarction.—Wayman, N. S., Hattori, Y., McDonald, M. C., Mota‐Filipe, H., Cuzzocrea, S., Pisano, B., Chatterjee, P. K., Thiemermann, C. Ligands of the peroxisome proliferator‐activated receptors (PPAR‐γ and PPAR‐α) reduce myocardial infarct size. FASEB J. 16, 1027–1040 (2002)

Rosiglitazone and 15-deoxy-Δ12,14-prostaglandin J2, ligands of the peroxisome proliferator-activated receptor-γ (PPAR-γ), reduce ischaemia/reperfusion injury of the gut

The peroxisome proliferator‐activated receptor‐γ (PPAR‐γ) is a member of the nuclear receptor superfamily of ligand‐dependent transcription factors related to retinoid, steroid and thyroid hormone receptors. The thiazolidinedione rosiglitazone and the endogenous cyclopentenone prostaglandin (PG)D2 metabolite, 15‐deoxy‐Δ12,14‐PGJ2 (15d‐PGJ2), are two PPAR‐γ ligands, which modulate the transcription of target genes. The aim of this study was to investigate the effect of rosiglitazone and 15d‐PGJ2 on the tissue injury caused by ischaemia/reperfusion (I/R) of the gut. I/R injury of the intestine was caused by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp allowing reperfusion for 2 or 4 h. This procedure results in splanchnic artery occlusion (SAO) shock. Rats subjected to SAO developed a significant fall in mean arterial blood pressure, and only 10% of the animals survived for the entire 4 h reperfusion period. Surviving animals were killed for histological examination and biochemical studies. Rats subjected to SAO displayed a significant increase in tissue myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels, significant increases in plasma tumour necrosis factor (TNF)‐α and interleukin (IL)‐1β levels and marked injury to the distal ileum. Increased immunoreactivity to nitrotyrosine was observed in the ileum of rats subjected to SAO. Staining of sections of the ileum obtained from SAO rats with anti‐intercellular adhesion molecule (ICAM‐1) antibody resulted in diffuse staining. Administration at 30 min prior to the onset of gut ischaemia of the two PPAR‐γ agonists (rosiglitazone (0.3 mg kg−1 i.v.) and 15d‐PGJ2 (0.3 mg kg−1 i.v.)) significantly reduced the (i) fall in mean arterial blood pressure, (ii) mortality rate, (iii) infiltration of the reperfused intestine with polymorphonuclear neutrophils (MPO activity), (iv) lipid peroxidation (MDA levels), (v) production of proinflammatory cytokines (TNF‐α and IL‐1β) and (vi) histological evidence of gut injury. Administration of rosiglitazone and 15d‐PGJ2 also markedly reduced the nitrotyrosine formation and the upregulation of ICAM‐1 during reperfusion. In order to elucidate whether the protective effects of rosiglitazone and 15d‐PGJ2 are related to the activation of the PPAR‐γ receptor, we also investigated the effect of a PPAR‐γ antagonist, bisphenol A diglycidyl ether (BADGE), on the protective effects of rosiglitazone and 15d‐PGJ2. BADGE (1 mg kg−1 administered i.v. 30 min prior to the treatment of rosiglitazone or 15d‐PGJ2) significantly antagonised the effect of the two PPAR‐γ agonists and thus abolished the protective effect against gut I/R. These results demonstrate that the two PPAR‐γ agonists, rosiglitazone and 15d‐PGJ2, significantly reduce I/R injury of the intestine.

The cyclopentenone prostaglandin 15-deoxy-Δ12,14-PGJ2 attenuates the development of colon injury caused by dinitrobenzene sulphonic acid in the rat

Inflammatory bowel disease (IBD) is characterized by oxidative and nitrosative stress, leukocyte infiltration, and increased expression of the adhesion molecules intercellular adhesion molecule 1 (ICAM‐1) in the colon. Recent evidence also suggests that the cyclopentenone prostaglandin (PG) 15‐deoxy‐Δ12,14‐PGJ2 (15d‐ PGJ2) functions as an early anti‐inflammatory signal. The aim of the present paper is to investigate the effects of 15d‐PGJ2 in rats subjected to experimental colitis. Colitis was induced in rats by intra‐colonic instillation of dinitrobenzene sulphonic acid (DNBS). 15d‐PGJ2 was administered daily as intraperitoneal injection (20 or 40 μg kg−1). On day 4, animals were sacrificed and tissues were taken for histological and biochemical analysis. 15d‐PGJ2 significantly reduced the degree of haemorrhagic diarrhoea and weight loss caused by administration of DNBS. 15d‐PGJ2 also caused a substantial reduction of (i) the degree of colonic injury, (ii) the rise in myeloperoxidase (MPO) activity (mucosa), (iii) the increase in the tissue levels of malondialdehyde (MDA) and (iv) of the pro‐inflammatory cytokines tumour necrosis factor‐alpha (TNF‐α) and interleukin‐1β (IL‐1β). Furthermore, 15d‐PGJ2 reduced the increase in immunohistochemical staining for (i) inducible nitric oxide synthase (iNOS), (ii) nitrotyrosine and (iii) poly (ADP‐ribose) polymerase (PARP), as well as (iv) the increased expression of ICAM‐1 caused by DNBS in the colon. Electrophoresis mobility shift assay (EMSA) of inflamed colon revealed that 15d‐ PGJ2 also caused a substantial reduction of the activation of nuclear factor‐kappaB (NF‐κB). Furthermore, 15d‐PGJ2 stimulates the activation of heat shock protein 72 (hsp72) in the inflamed colon, as assessed by Western blot analysis. In conclusion, 15d‐PGJ2 reduces the development of experimental colitis.

Role of cyclopentenone prostaglandins in rat carrageenin pleurisy.

In this study, using rat carrageenin‐induced pleurisy, we found that treatment of rats with either indomethacin or NS‐398 suppressed the pleurisy at 2 h but significantly exacerbated this reaction at 48 h. Exacerbated inflammation was associated with reduced prostaglandin D2 levels, decreased heat shock factor 1 (HSF1) activation, reduced hsp72 expression and increased activation of nuclear factor κB (NF‐κB). Replacement of cyclopentenone prostaglandins by treating rats with either prostaglandin J2 or prostaglandin D2 reversed the exacerbating effects of cyclooxygenase inhibitors leading to the resolution of the reaction. In conclusion, we demonstrate that cyclopentenone prostaglandins may act as anti‐inflammatory mediators by inducing in inflammatory cells HSF1‐dependent hsp72 expression and NF‐κB inhibition, two crucial events for the remission of inflammation.

HSF1/hsp72 pathway as an endogenous anti-inflammatory system

We investigated the occurrence and the role of HSF1 activation and inducible hsp72 expression in the carrageenin pleurisy in the rat. Molecular analysis performed on pleural cells collected from rat pleural cavity after carrageenin challenge revealed increased HSF1 activation and hsp72 expression. Moreover, local injection of a double‐stranded oligodeoxynucleotide (ODN) containing the heat shock element sequence, acting as transcription factor decoy, exacerbated the inflammatory reaction. The exacerbation, induced by wild‐type, but not by mutant ODN decoy, was associated to both inhibition of HSF1/DNA binding activity and reduction of hsp72 expression. In conclusion, this study shows that HSF1 activation and hsp72 expression both actually occur in acute inflammation and that the remission of the inflammatory reaction is tightly associated to the HSF1‐dependent hsp72 expression, suggesting a a relevant role for the HSF1/hsp72 pathway as an endogenous anti‐inflammatory system.

Tempol reduces the activation of nuclear factor-κB in acute inflammation

Recent studies have demonstrated that Tempol, a membrane-permeable radical scavenger, exerts protective effects in various models of inflammation and shock. Since nuclear factor-kappaB (NF-kappaB) is a transcription factor, which plays a pivotal role in the induction of genes involved in physiological processes as well as in the response to inflammation, we have investigated the effect of Tempol on NF-kappaB activation in a model of acute inflammation in mice. Injection of carrageenan into the pleural cavity of mice induced an acute inflammatory response characterized by fluid accumulation in the pleural cavity, which contained a large number of neutrophils, as well as an increased production of tumor necrosis factor-alpha, (TNF-alpha) and interleukin-1alpha (IL-1alpha). Tempol (100mg/kg i.p 30min prior to carrageenan administration) significantly attenuated the degree of pleuritis caused by carageeenan (all parameters measured). Administration of carageeenan into the chest cavity (pleuritis) was associated with the activation of NF-kappaB in the lung. In particular, the appearance of IkappaB-alpha in homogenates of lung tissue was investigated by immunoblot analysis at 4h after carrageenan administration. IkappaB-alpha levels were substantially reduced in the lung tissue from carrageenan-treated mice in comparison with sham-treated mice. Furthermore, to detect any effects of Tempol on NF-kappaB/DNA binding, lung extracts were analyzed by EMSA. The DNA binding activity significantly increased in extracts obtained from lungs of vehicle-treated mice at 4h after carrageenan administration. Treatment of mice with Tempol caused a significant inhibition of carrageenan-induced IkappaB-alpha degradation and NF-kappaB/DNA binding activity. These data confirm that Tempol exerts potent anti-inflammatory properties and clearly demonstrates for the first time that Tempol reduces the activation of NF-kappaB in vivo.

Beneficial effects of caffeic acid phenethyl ester in a rat model of vascular injury

The aim of this study was to evaluate whether caffeic acid phenethyl ester (CAPE), an active component of propolis, was able to reduce neointimal formation in a model of endothelial injury of rat carotid artery (balloon angioplasty). Furthermore, we investigated the relationship between neointima formation and nuclear factor‐κB (NF‐κB) activation and we correlated NF‐κB activation to the expression of inducible isoform of cyclo‐oxygenase (COX‐2) in injured carotids. In control group a significant proliferation of neointima was observed 14 days after balloon angioplasty, which was correlated to an increase of NF‐κB/DNA binding activity as well as p50/p65 nuclear levels compared to those observed in the carotids from sham‐operated rats. Furthermore, NF‐κB activation was correlated to increased COX‐2, but not β‐actin, protein expression. Treatment of rats for 14 days with CAPE (3, 10, 30 mg kg−1) caused a significant inhibition of all the parameters assayed, except β‐actin protein expression. These results indicate that treatment with CAPE may lead to a reduction of neointima formation by inhibiting NF‐κB activation and suggest that this agent may have therapeutic relevance for the prevention of human restenosis.

Superoxide-Related Signaling Cascade Mediates Nuclear Factor-κB Activation in Acute Inflammation

The nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays a pivotal role in the induction of genes involved in physiological processes, as well as in the response to inflammation. In this study, we used a selective nonpeptidyl superoxide dismutase mimetic, M40403, to investigate the role of superoxide anion in NF-kappaB activation during acute inflammation in mice. Injection of carrageenan into the pleural cavity of mice induced an acute inflammatory response characterized by fluid accumulation in the pleural cavity that contained a large number of neutrophils, as well as an increased production of tumor necrosis factor-alpha and interleukin-1beta. All parameters of inflammation were attenuated by M40403 (10 mg/kg i. p., 30 min prior to carrageenan administration). These inflammatory events were associated with the activation of NF-kappaB in the lung. In particular, the appearance of inhibitory protein kappaB-alpha (IkappaB-alpha) in homogenates of lung tissues was investigated by immunoblot analysis at 4 h after carrageenan administration. IkappaB-alpha levels were substantially reduced in the lung tissue from carrageenan-treated mice in comparison with sham-treated mice. Furthermore, to detect NF-kappaB/DNA binding activity, whole extracts from lung tissue of each mouse were analyzed by electrophoretic mobility-shift assay. The DNA binding activity significantly increased in whole extracts obtained from lung tissues of vehicle-treated mice 4 h after carrageenan administration. Treatment of mice with M40403 caused a significant inhibition of carrageenan-induced IkappaB-alpha degradation and NF-kappaB/DNA binding activity. These data confirm that M40403 exerts a potent antiinflammatory activity and clearly demonstrate that the reduction of the inflammatory process is associated with modification of the activation of signal transduction pathways.

Plakortethers A−G: A New Class of Cytotoxic Plakortin−Derived Metabolites

Plakortethers A−G (4−10) represent a new class of plakortin-related metabolites containing a trisubstituted tetrahydrofuran ring. They have been isolated from the Caribbean sponge Plakortis simplex and their stereostructures fully characterized by a combination of spectroscopic data and chemical evidence, based on a three-step conversion of the cycloperoxide plakortin into plakortether B (5). Plakortethers A (4), B (5), D (7), and E (8) exhibited selective cytotoxicity against the RAW 264-7 cell line (murine macrophage).

New Glycosphingolipids from the Marine Sponge Aplysinella rhax and Their Potential as Nitric Oxide Release Inhibitors

Several new glycosphingolipids were isolated by means of reversed-phase HPLC from the less-polar fraction of the methanol extract of the sponge Aplysinella rhax. Their structures, showing very close similarity, were elucidated by extensive NMR experiments including 1H,1H (COSY, TOCSY and ROESY) and 1H,13C (HMQC and HMBC) spectroscopy and chemical evidence. The molecular masses of the glycosphingolipids were determined by negative-ion fast-atom bombardment mass spectrometry, while HREIMS and HRFABMS provided important information for the identification of the fatty acids and sphingoid bases. The mixture of glycosphingolipids exhibited a good inhibitory activity on LPS-induced NO2− release by J774 A.1 macrophages.