Helder Mota-Filipe

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)

Agonists of Peroxisome-Proliferator Activated Receptor-Gamma Reduce Renal Ischemia/Reperfusion Injury

Background/Aims: Recent evidence indicates that peroxisome-proliferator activated receptor (PPAR) agonists protect against ischemia/reperfusion (I/R) injury. Here we investigate the effects of the PPAR-γ agonists, rosiglitazone and ciglitazone, on the renal dysfunction and injury caused by I/R of the rat kidney in vivo. Methods: Rosiglitazone or ciglitazone were administered to male Wistar rats prior to and during reperfusion. Biochemical indicators of renal dysfunction and injury were measured and histological scoring of kidney sections was used to assess renal injury. Expression of PPAR isoforms and intercellular adhesion molecule-1 during renal I/R were assessed using RT-PCR and Northern blot, respectively. Myeloperoxidase activity and activation of poly(ADP-ribose) polymerase (PARP) were used as indicators of polymorphonuclear (PMN) cell infiltration and oxidative stress, respectively. Results: Expression of PPAR-α, PPAR-β and PPAR-γ1 (but not PPAR-γ2) was observed in kidneys with down-regulation of PPAR-α expression during renal I/R. Rosiglitazone and ciglitazone significantly reduced biochemical and histological signs of renal dysfunction and injury. Renal expression of ICAM-1 caused by I/R was reduced by rosiglitazone and ciglitazone which was reflected by decreased PMN infiltration into reperfused renal tissues. Both rosiglitazone and ciglitazone reduced PARP activation indicating a reduction of oxidative stress. Conclusion: These results suggest that the PPAR-γ agonists rosiglitazone and ciglitazone reduce the renal dysfunction and injury associated with I/R of the kidney. We propose that one mechanism underlying the protective effects involves inhibition of the expression of ICAM-1, a reduction of PMN infiltration into renal tissues and subsequent reduction of oxidative stress.

Calpain inhibitor I reduces the activation of nuclear factor-kappaB and organ injury/dysfunction in hemorrhagic shock.

There is limited evidence that inhibition of the activity of the cytosolic cysteine protease calpain reduces ischemia/reperfusion injury. The multiple organ injury associated with hemorrhagic shock is due at least in part to ischemia (during hemorrhage) and reperfusion (during resuscitation) of target organs. Here we investigate the effects of calpain inhibitor I on the organ injury (kidney, liver, pancreas, lung, intestine) and dysfunction (kidney) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage and resuscitation with shed blood resulted in an increase in calpain activity (heart), activation of NF‐κB (kidney), expression of iNOS and COX‐2 (kidney), and the development of multiple organ injury and dysfunction, all of which were attenuated by calpain inhibitor I (10 mg/kg i.p.), administered 30 min prior to hemorrhage. Chymostatin, a serine protease inhibitor that does not prevent the activation of NF‐κB, had no effect on the organ injury/failure caused by hemorrhagic shock. Pretreatment (for 1 h) of murine macrophages or rat aortic smooth muscle cells (activated with endotoxin) with calpain inhibitor I attenuated the binding of activated NF‐κB to DNA and the degradation of IκBα, IKBβ, and IκBε. Selective inhibition of iNOS activity with L‐NIL reduced the circulatory failure and liver injury, while selective inhibition of COX‐2 activity with SC58635 reduced the renal dysfunction and liver injury caused by hemorrhagic shock. Thus, we provide evidence that the mechanisms by which calpain inhibitor I reduces the circulatory failure as well as the organ injury and dysfunction in hemorrhagic shock include 1) inhibition of calpain activity, 2) inhibition of the activation of NF‐κB and thus prevention of the expression of NFκB‐dependent genes, 3) prevention of the expression of iNOS, and 4) prevention of the expression of COX‐2. Inhibition of calpain activity may represent a novel therapeutic approach for the therapy of hemorrhagic shock.—McDonald, M. C., Mota‐Filipe, H., Paul, A., Cuzzocrea, S., Abdelrahman, M., Harwood, S., Plevin, R., Chatterjee, P. K., Yaqoob, M. M., Thiemermann, C. Calpain inhibitor I reduces the activation of nuclear factor‐κB and organ injury/dysfunction in hemorrhagic shock. FASEB J. 15, 171–186 (2001)

Recombinant human erythropoietin protects the liver from hepatic ischemia-reperfusion injury in the rat

Recently, erythropoietin was shown to have both hematopoietic as well as tissue‐protective properties. Erythropoietin (EPO) had a protective effect in animal models of cerebral ischemia, mechanical trauma of the nervous system, myocardial infarction, and ischemia‐reperfusion (I/R) injury of the kidney. It is not known whether EPO protects the liver against I/R injury. Using a rat model of liver I/R injury, we aimed to determine the effect of the administration of human recombinant erythropoietin (rhEPO) on liver injury. Rats were subjected to 30 min of liver ischemia followed by 2 h of reperfusion. When compared with the sham‐operated rats, I/R resulted in significant rises in the serum levels of aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, gamma‐glutamyl transferase, tissue lipid peroxidation, caspase‐3 activity and altered histology. Administration of rhEPO 5 min before ischemia was able to reduce the biochemical evidence of liver injury; however, this protection was not evident when rhEPO was administered 5 min before reperfusion. Mechanistically, early administration of rhEPO was able to reduce the oxidative stress and caspase‐3 activation, suggesting the subsequent reduction of apoptosis. This study provides the first evidence that rhEPO causes a substantial reduction of the liver injury induced by I/R in the rat.

Effects of 5-aminoisoquinolinone, a water-soluble, potent inhibitor of the activity of poly (ADP-ribose) polymerase on the organ injury and dysfunction caused by haemorrhagic shock

Poly (ADP‐ribose) synthetase (PARP) is a nuclear enzyme activated by strand breaks in DNA, which are caused inter alia by reactive oxygen species (ROS). Here we report on (i) a new synthesis of a water‐soluble and potent PARP inhibitor, 5‐aminoisoquinolinone (5‐AIQ) and (ii) investigate the effects of 5‐AIQ on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat. Exposure of human cardiac myoblasts (Girardi cells) to hydrogen peroxide (H2O2, 3 mM for 1 h, n=9) caused a substantial increase in PARP activity. Pre‐treatment of these cells with 5‐AIQ (1 μM–1 mM, 10 min prior to H2O2) caused a concentration‐dependent inhibition of PARP activity (IC50: ∼0.01 mM, n=6). Haemorrhage and resuscitation resulted (within 4 h after resuscitation) in a delayed fall in blood pressure (circulatory failure) as well as in rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma‐glutamyl‐transferase (γ‐GT) (liver injury and dysfunction), (iii) lipase (pancreatic injury) and (iv) creatine kinase (CK) (neuromuscular injury) (n=10). Administration (5 min prior to resuscitation of 5‐AIQ) (0.03 mg kg−1 i.v., n=8, or 0.3 mg kg−1 i.v., n=10) reduced (in a dose‐related fashion) the multiple organ injury and dysfunction, but did not affect the circulatory failure, associated with haemorrhagic shock. Thus, 5‐AIQ abolishes the multiple organ injury caused by severe haemorrhage and resuscitation.

A membrane-permeable radical scavenger reduces the organ injury in hemorrhagic shock.

Reactive oxygen species (ROS) contribute to the multiple organ failure (MOF) in hemorrhagic shock. Here we investigate the effects of a membrane-permeable radical scavenger (tempol) on the circulatory failure and the organ injury and dysfunction (kidney, liver, lung, intestine) associated with hemorrhagic shock in the anesthetized rat. Hemorrhage (sufficient to lower mean arterial blood pressure to 500 mmHg for 90 min) and subsequent resuscitation with shed blood resulted (within 4 h after resuscitation) in a delayed fall in blood pressure, renal and liver injury and dysfunction as well as lung and gut injury. In all organs, hemorrhage and resuscitation resulted in the nitrosylation of proteins (determined by immunohistochemistry for nitrotyrosine) suggesting the formation of peroxynitrite and/or reactive oxygen species. Treatment of rats upon resuscitation with the membrane-permeable radical scavenger tempol (30 mg/kg bolus injection followed by an infusion of 30 mg/kg/h i.v.) attenuated the delayed circulatory failure as well as the multiple organ injury and dysfunction associated with hemorrhagic shock. Thus, we propose that an enhanced formation of ROS and/or peroxynitrite importantly contributes to the MOF in hemorrhagic shock, and that membrane-permeable radical scavengers, such as tempol, may represent a novel therapeutic approach for the therapy of hemorrhagic shock.

High density lipoproteins reduce organ injury and organ dysfunction in a rat model of hemorrhagic shock

High density lipoproteins (HDLs) inhibit the cytokine‐induced expression of endothelial cell adhesion molecules both in vitro and in vivo. We examined the ability of HDLs to mediate a functional anti‐inflammatory effect by measuring their ability to prevent neutrophil adhesion and transmigration in vitro. Treatment of human endothelial cell cultures with physiologic concentrations of HDLs inhibited neutrophil binding by 68 ± 5.9% (mean and SE, n=6, P<0.05) and neutrophil transmigration by 48.7 ± 6.7% (n=8, P<0.05). We then examined the effect of HDLs on inflammatory infiltration and subsequent multiple organ dysfunction syndrome (MODS), associated with trauma in a rat model of hemorrhagic shock. Rats given human HDLs (80 mg apo A‐I/kg, i.v.) 90 min after hemorrhage (which reduced mean arterial pressure to 50 mmHg) and 1 min before resuscitation showed attenuation of the increases in the serum levels of markers of MODS normally observed in this model. Severe disruption of the architecture of tissues and the extensive cellular infiltration into those tissues were also largely inhibited in animals that received HDLs. Human HDLs attenuate the MODS associated with ischemia and reperfusion injury after hemorrhagic shock in rats.

Reconstituted High-density Lipoprotein Attenuates Organ Injury and Adhesion Molecule Expression in a Rodent Model of Endotoxic Shock

The salutary effects of high-density lipoproteins (HDLs) in animal and human models of endotoxic shock have in the past been attributed to the ability of this lipoprotein to bind to lipopolysaccharide. However, the precise mechanisms for the protective effect of HDL are unclear. The first objective of this study was to determine the effects of HDLs on the organ injury and dysfunction associated with acute severe endotoxemia. Second, to gain insight into the mechanism of action of HDL, we also investigated the effect of HDLs on 1) the expression of P-selectin and intercellular adhesion molecule-1 in the kidneys of rats treated with endotoxin and 2) the rise in the plasma levels of tumor necrosis factor-&agr; (TNF-&agr;). Rats were given Escherichia coli lipopolysaccharide (6 mg/kg i.v.), pretreated with either vehicle (n = 9) or reconstituted HDL (rHDL; apolipoprotein A-I/phosphatidylcholine proteoliposomes, n = 10), and were monitored for 6 h. Here we report that rHDL attenuates the renal injury and dysfunction caused by endotoxin in the rat. In addition, rHDL reduced the degree of histological tissue injury in the lung, liver and intestine and attenuated the expression of P-selectin and intercellular adhesion molecule-1 in the renal glomerulus. Interestingly, pretreatment of rats with rHDL did not prevent the hypotension nor the rise in plasma levels of TNF-&agr; (at 90 min) caused by endotoxin. Thus, rHDL reduces the organ injury/dysfunction, but does not affect the circulatory failure, nor the rise in plasma levels of TNF-&agr; caused by endotoxin in the rat. We propose that the mechanisms of these beneficial effects of HDL may be related to direct inhibition of adhesion molecule expression.

High density lipoprotein (HDL) reduces renal ischemia/reperfusion injury

High-density lipoproteins (HDL) have been shown to reduce organ injury and mortality in animal models of shock via modulation of the expression of adhesion molecules and pro-inflammatory enzymes. As renal inflammation plays an important role in the development of ischemia/reperfusion (I/R) injury of the kidney, the aim of this study was to investigate the ability of HDL to alleviate renal dysfunction and injury in a rat model of renal I/R. HDL (80 mg/kg, intravenous) was administered to male Wistar rats 30 min before bilateral renal ischemia for 45 min followed by reperfusion for up to 48 h. After 6-h reperfusion, HDL significantly reduced (1) renal and tubular dysfunction, (2) tubular and reperfusion-injury, and (3) histologic evidence of renal injury. HDL also improved renal function (after 24-h and 48-h reperfusion) and reduced histologic signs of renal injury (after 48-h reperfusion). Administration of HDL significantly reduced the numbers of polymorphonuclear leukocytes (PMN) infiltrating into renal tissues during reperfusion, which was reflected by an attenuation of the increase in renal myeloperoxidase activity caused by I/R. Furthermore, HDL markedly reduced expression of the adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), and P-selectin during reperfusion. The increase in renal malondialdehyde levels caused by renal I/R was also significantly reduced by HDL, suggesting attenuation of lipid peroxidation subsequent to oxidative stress. These results demonstrate that HDL significantly reduces renal I/R injury and severity of ischemic acute renal failure. It is proposed that the mechanism of protection involves reduction of the expression of adhesion molecules, resulting in reduction of PMN infiltration and oxidative stress.

Anti-inflammatory effect of lycopene on carrageenan-induced paw oedema and hepatic ischaemia–reperfusion in the rat

The regular intake of tomatoes or its products has been associated with a reduced risk of chronic diseases and these effects have been mainly attributed to lycopene. Here, we evaluated the anti-inflammatory properties of lycopene and its protective effects on organ injury in two experimental models of inflammation. In order to study the effects of lycopene in local inflammation, a carrageenan-induced paw oedema model in rats was performed. Lycopene was administered as an acute (1, 10, 25 or 50 mg/kg, intraperitoneally, 15 min before carrageenan injection) and chronic treatment (25 or 50 mg/kg per d, 14 d). Inflammation was assessed by the measurement of paw volume increase after 6 h. Lycopene significantly inhibited paw oedema formation at two doses (25 and 50 mg/kg) in both acute and repeated administration. The effect of lycopene on liver inflammation was evaluated in a liver ischaemia-reperfusion (I/R) model. Rats were subjected to 45 min of ischaemia of three-quarters of the liver followed by 2 h of reperfusion. In this model, lycopene was administered daily at two doses (25 and 50 mg/kg) during the 14 d that preceded the experiments. Repeated administration of lycopene reduced liver injury induced by I/R, as demonstrated by the reduction of the increase in liver injury markers (aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase and gamma-glutamyl transferase) and attenuation of liver tissue lipoperoxidation was evidenced by a decrease in malondialdehyde production. The present results show that lycopene exhibited local anti-inflammatory activity and also attenuated liver injury induced by I/R. We speculate that lycopene administration might be useful in the pharmacological modulation of inflammatory events.