Michelle C. McDonald

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)

Erythropoietin attenuates the tissue injury associated with hemorrhagic shock and myocardial ischemia.

Here we investigate the effects of erythropoietin (EPO) on the tissue/organ injury caused by hemorrhagic shock (HS), endotoxic shock, and regional myocardial ischemia and reperfusion in anesthetized rats. Male Wistar rats were anesthetized with thiopental sodium (85 mg/kg i.p.) and subjected to hemorrhagic shock (HS; i.e., mean arterial blood pressure reduced to 45 mmHg for 90 min, followed by resuscitation with shed blood for 4 h), endotoxemia (for 6 h), or left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h). HS and endotoxemia resulted in renal dysfunction and liver injury. Administration of EPO (300 IU/kg i.v., n = 10) before resuscitation abolished the renal dysfunction and liver injury in hemorrhagic, but not endotoxic, shock. HS also resulted in significant increases in the kidney of the activities of caspases 3, 8, and 9. This increase in caspase activity was not seen in HS rats treated with EPO. In cultured human proximal tubule cells, EPO concentration-dependently reduced the cell death and increase in caspase-3 activity caused by either ATP depletion (simulated ischemia) or hydrogen peroxide (oxidative stress). In the heart, administration of EPO (300 IU/kg i.v., n = 10) before reperfusion also caused a significant reduction in infarct size. In cultured rat cardiac myoblasts (H9C2 cells), EPO also reduced the increase in DNA fragmentation caused by either serum deprivation (simulated ischemia) or hydrogen peroxide (oxidative stress). We propose that the acute administration of EPO on reperfusion and/or resuscitation will reduce the tissue injury caused by ischemia-reperfusion of the heart (and other organs) and hemorrhagic shock.

The Production Of Hydrogen Sulfide Limits Myocardial Ischemia And Reperfusion Injury And Contributes To The Cardioprotective Effects Of Preconditioning With Endotoxin, But Not Ischemia In The Rat

ABSTRACT We investigated whether (endogenous) hydrogen sulfide (H2S) protects the heart against myocardial ischemia and reperfusion injury. Furthermore, we investigated whether endogenous H2S is involved in the protection afforded by (1) ischemic preconditioning and (2) the second window of protection caused by endotoxin. The involvement of one of the potential (end) effectors of the cardioprotection afforded by H2S was investigated using the mitochondrial KATP channel blocker, 5-hydroxydecanoate (5-HD; 5 mg/kg). Animals were subjected to 25 min regional myocardial ischemia followed by reperfusion (2 h) and were pretreated with the H2S donor, sodium hydrosulfide (3 mg/kg i.v.). Animals were also subjected to shorter periods of myocardial ischemia (15 min) and reperfusion (2 h) and pretreated with an irreversible inhibitor of cystathionine-&ggr;-lyase, dl-propargylglycine (PAG; 50 mg/kg i.v.). Animals were also pretreated with PAG (50 mg/kg) and subjected to either (1) ischemic preconditioning or (2) endotoxin (1 mg/kg i.p.) 16 h before myocardial ischemia. Myocardial infarct size was determined by p-nitroblue tetrazolium staining. Administration of sodium hydrosulfide significantly reduced myocardial infarct size, and this effect was abolished by 5-HD. Administration of PAG (50 mg/kg) or 5-HD significantly increased infarct size caused by 15 min of myocardial ischemia. The delayed cardioprotection afforded by endotoxin was abolished by 5-HD or PAG. In contrast, PAG (50 mg/kg) did not affect the cardioprotective effects of ischemic preconditioning. These findings suggest that (1) endogenous H2S is produced by myocardial ischemia in sufficient amounts to limit myocardial injury and (2) the synthesis or formation of H2S by cystathionine-&ggr;-lyase may contribute to the second window of protection caused by endotoxin.

Peptidoglycan and lipoteichoic acid in gram-positive bacterial sepsis: receptors, signal transduction, biological effects, and synergism.

In sepsis and multiple organ dysfunction syndrome (MODS) caused by gram-negative bacteria, lipopolysaccharide (LPS) initiates the early signaling events leading to the deleterious inflammatory response. However, it has become clear that LPS can not reproduce all of the clinical features of sepsis, which emphasize the roles of other contributing factors. Gram-positive bacteria, which lack LPS, are today responsible for a substantial part of the incidents of sepsis with MODS. The major wall components of gram-positive bacteria, peptidoglycan and lipoteichoic acid, are thought to contribute to the development of sepsis and MODS. In this review, the literature underlying our current understanding of how peptidoglycan and lipoteichoic acid activate inflammatory responses will be presented, with a focus on recent advances in this field.

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.

Tempol reduces infarct size in rodent models of regional myocardial ischemia and reperfusion.

Reactive oxygen species (ROS) contribute to ischemia-reperfusion injury of the heart. This study investigates the effects of tempol, a membrane-permeable radical scavenger on (i) the infarct size caused by regional myocardial ischemia and reperfusion of the heart in vivo (rat, rabbit) and in vitro (rat), and (ii) the cell injury caused by hydrogen peroxide (H2O2) in rat cardiac myoblasts (H9c2 cells). In the anesthetized rat, tempol reduced the infarct size caused by regional myocardial ischemia (25 min) and reperfusion (2 h) from 60 +/- 3% (control, n = 8) to 24 +/- 5% (n = 6, p < .05). In the anesthetized rabbit, tempol also attenuated the infarct size caused by myocardial ischemia (45 min) and reperfusion (2 h) from 59 +/- 3% (control, n = 6) to 39 +/- 5% (n = 5, p < .05). Regional ischemia (35 min) and reperfusion (2 h) of the isolated, buffer-perfused heart of the rat resulted in an infarct size of 54 +/- 4% (control n = 7). Reperfusion of hearts with buffer containing tempol (n = 6) caused a 37% reduction in infarct size (n = 6, p < .05). Pretreatment of rat cardiac myoblasts with tempol attenuated the impairment in mitochondrial respiration caused by H2O2 (1 mM for 4 h). Thus, the membrane-permeable radical scavenger tempol reduces myocardial infarct size in rodents.

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.

Effects of tempol, a membrane-permeable radical scavenger, in a gerbil model of brain injury.

There is evidence that the excessive generation of reactive-oxygen radicals contributes to the brain injury associated with transient, cerebral ischemia. This study investigates the effects of tempol, a small, water-soluble molecule, that crosses biological membranes, on the brain injury caused by bilateral occlusion and reperfusion of both common carotid arteries in the gerbil (BCO). Treatment of gerbils with tempol (30 mg/kg i.p. at 30 min prior to reperfusion and at 1 and 6 h after the onset of reperfusion) reduced the formation of post-ischemic brain oedema. Tempol also attenuated the increase in the cerebral levels of malondialdehyde (MDA) and the hippocampal levels of myeloperoxidase (MPO) caused by cerebral ischemia and reperfusion. The immunohistochemical analysis of the hippocampal region of brains subjected to ischemia-reperfusion exhibited positive staining for nitrotyrosine (an indicator of the generation of peroxynitrite) and poly(ADP-ribose) synthetase (PARS) (an indicator of the activation of this nuclear enzyme secondary to single strand breaks in DNA). In gerbils subjected to BCO, which were treated with tempol, the degree of staining for nitrotyrosine and PARS was markedly reduced. Tempol increased survival and reduced the hyperactivity (secondary to the ischemia-induced neurodegeneration) caused by cerebral ischemia and reperfusion. The loss of neurons from the pyramidal layer of the CA1 region caused by ischemia and reperfusion was also attenuated by treatment of gerbils with tempol. This is the first evidence that the membrane-permeable, radical scavenger tempol reduces the cerebral injury caused by transient, cerebral ischemia in vivo.

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.