Lucas Liaudet

Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation

Diabetic patients frequently suffer from retinopathy, nephropathy, neuropathy and accelerated atherosclerosis. The loss of endothelial function precedes these vascular alterations. Here we report that activation of poly(ADP-ribose) polymerase (PARP) is an important factor in the pathogenesis of endothelial dysfunction in diabetes. Destruction of islet cells with streptozotocin in mice induced hyperglycemia, intravascular oxidant production, DNA strand breakage, PARP activation and a selective loss of endothelium-dependent vasodilation. Treatment with a novel potent PARP inhibitor, starting after the time of islet destruction, maintained normal vascular responsiveness, despite the persistence of severe hyperglycemia. Endothelial cells incubated in high glucose exhibited production of reactive nitrogen and oxygen species, consequent single-strand DNA breakage, PARP activation and associated metabolic and functional impairment. Basal and high-glucose-induced nuclear factor-κB activation were suppressed in the PARP-deficient cells. Our results indicate that PARP may be a novel drug target for the therapy of diabetic endothelial dysfunction.

Flagellin, a Novel Mediator of Salmonella-Induced Epithelial Activation and Systemic Inflammation: IκBα Degradation, Induction of Nitric Oxide Synthase, Induction of Proinflammatory Mediators, and Cardiovascular Dysfunction

Gram-negative sepsis is mediated by the actions of proinflammatory genes induced in response to microbes and their products. We report that flagellin, the monomeric subunit of flagella, is a potent proinflammatory species released by Salmonella. Flagellin (1 μg/ml) induces IκBα degradation, NF-κB nuclear translocation, and inducible NO synthase expression in cultured intestinal epithelial cells (IEC). Aflagellic Salmonella mutants do not induce NF-κB activation or NO production by cultured IEC. Antiserum to flagellin blocks NO production in IEC induced by medium conditioned by a variety of motile Gram-negative enteric pathogens (Escherichia coli, Salmonella muenchen, Serratia marcescens, Proteus mirabilis, and Proteus vulgaris). Flagellin, when injected systemically (∼10 μg/mouse), induces systemic inflammation characterized by the systemic expression of a range of proinflammatory cytokines and chemokines and of inducible NO synthase. At higher doses (∼300 μg/mouse), flagellin induces shock, characterized by hypotension, reduced vascular contractility in mice, and death. The effects of flagellin do not diminish in C3H/HeJ LPS-resistant mice, indicating that the Toll-like receptor-4 receptor is not involved in flagellin’s actions. In LPS-resistant mice, i.p. injection of S. dublin flagellin or medium conditioned by wild-type S. dublin induces serum IFN-γ and TNF-α, whereas medium conditioned by aflagellic mutants has no effect. Flagellin can be detected in the blood of rats with septic shock induced by live bacteria at approximately 1 μg/ml. We propose that flagellin released by Gram-negative pathogens may contribute to the inflammatory response by an LPS- and Toll-like receptor-4-independent pathway.

Potent Metalloporphyrin Peroxynitrite Decomposition Catalyst Protects Against the Development of Doxorubicin-Induced Cardiac Dysfunction

Background—Increased oxidative stress and dysregulation of nitric oxide have been implicated in the cardiotoxicity of doxorubicin (DOX), a commonly used antitumor agent. Peroxynitrite is a reactive oxidant produced from nitric oxide and superoxide in various forms of cardiac injury. Using a novel metalloporphyrinic peroxynitrite decomposition catalyst, FP15, and nitric oxide synthase inhibitors or knockout mice, we now delineate the pathogenetic role of peroxynitrite in rodent models of DOX-induced cardiac dysfunction. Methods and Results—Mice received a single injection of DOX (25 mg/kg IP). Five days after DOX administration, left ventricular performance was significantly depressed, and high mortality was noted. Treatment with FP15 and an inducible nitric oxide synthase inhibitor, aminoguanidine, reduced DOX-induced mortality and improved cardiac function. Genetic deletion of the inducible nitric oxide synthase gene was also accompanied by better preservation of cardiac performance. In contrast, inhibition of the endothelial isoform of nitric oxide synthase with N-nitro-l-arginine methyl ester increased DOX-induced mortality. FP15 reduced the DOX-induced increase in serum LDH and creatine kinase activities. Furthermore, FP15 prevented the DOX-induced increase in lipid peroxidation, nitrotyrosine formation, and metalloproteinase activation in the heart but not NAD(P)H-driven superoxide generation. Peroxynitrite neutralization did not interfere with the antitumor effect of DOX. FP15 also decreased ischemic injury in rats and improved cardiac function and survival of mice in a chronic model of DOX-induced cardiotoxicity. Conclusions—Thus, peroxynitrite plays a key role in the pathogenesis of DOX-induced cardiac failure. Targeting peroxynitrite formation may represent a new cardioprotective strategy after DOX exposure or in other conditions associated with peroxynitrite formation, including myocardial ischemia/reperfusion injury.

Role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro

Doxorubicin (DOX) is a potent available antitumor agent; however, its clinical use is limited because of its cardiotoxicity. Cell death is a key component in DOX-induced cardiotoxicity, but its mechanisms are elusive. Here, we explore the role of superoxide, nitric oxide (NO), and peroxynitrite in DOX-induced cell death using both in vivo and in vitro models of cardiotoxicity. Western blot analysis, real-time PCR, immunohistochemistry, flow cytometry, fluorescent microscopy, and biochemical assays were used to determine the markers of apoptosis/necrosis and sources of NO and superoxide and their production. Left ventricular function was measured by a pressure-volume system. We demonstrated increases in myocardial apoptosis (caspase-3 cleavage/activity, cytochrome c release, and TUNEL), inducible NO synthase (iNOS) expression, mitochondrial superoxide generation, 3-nitrotyrosine (NT) formation, matrix metalloproteinase (MMP)-2/MMP-9 gene expression, poly(ADP-ribose) polymerase activation [without major changes in NAD(P)H oxidase isoform 1, NAD(P)H oxidase isoform 2, p22(phox), p40(phox), p47(phox), p67(phox), xanthine oxidase, endothelial NOS, and neuronal NOS expression] and decreases in myocardial contractility, catalase, and glutathione peroxidase activities 5 days after DOX treatment to mice. All these effects of DOX were markedly attenuated by peroxynitrite scavengers. Doxorubicin dose dependently increased mitochondrial superoxide and NT generation and apoptosis/necrosis in cardiac-derived H9c2 cells. DOX- or peroxynitrite-induced apoptosis/necrosis positively correlated with intracellular NT formation and could be abolished by peroxynitrite scavengers. DOX-induced cell death and NT formation were also attenuated by selective iNOS inhibitors or in iNOS knockout mice. Various NO donors when coadministered with DOX but not alone dramatically enhanced DOX-induced cell death with concomitant increased NT formation. DOX-induced cell death was also attenuated by cell-permeable SOD but not by cell-permeable catalase, the xanthine oxidase inhibitor allopurinol, or the NADPH oxidase inhibitors apocynine or diphenylene iodonium. Thus, peroxynitrite is a major trigger of DOX-induced cell death both in vivo and in vivo, and the modulation of the pathways leading to its generation or its effective neutralization can be of significant therapeutic benefit.

Predictors of awakening from postanoxic status epilepticus after therapeutic hypothermia

PREDICTORS OF AWAKENING FROM POSTANOXIC STATUS EPILEPTICUS AFTER THERAPEUTIC HYPOTHERMIA To the Editor: We read with interest the article by Rossetti et al.,1 who studied 6 cardiac arrest (CA) cases with postanoxic status epilepticus (PSE) treated with therapeutic hypothermia. The authors highlight important observations about EEG in the age of therapeutic hypothermia. The study raises the question: If specific EEG patterns emerge after CA, would these patterns now represent a treatable CA with potential for better outcomes? However, the authors apparently did not perform EEG during the hypothermic period in which vecuronium was used. Nonetheless, the authors’ data showed half of their cases with clinically evident seizures at the bedside confirmed by EEG while the other half had PSE detected only on EEG in coma. The authors’ observations demonstrate that not all clinically evident post CA myoclonic jerking represents myoclonus status epilepticus, which is a poor prognostic sign. Recently published American Academy of Neurology guidelines emphasized the utility of EEG after CA.2 It has been our observation in 2 years of using hypothermia after CA that myoclonic jerks after CA are an ominous sign especially in concert with absent or partially absent brainstem reflexes. We find the authors’ study important for clinicians, since all myoclonic jerking seen at the bedside after CA should not be interpreted as indicative of myoclonus status epilepticus without EEG confirmation. To do so could lead to a self-fulfilling prophecy of poor outcome and untreated status epilepticus. Additionally, the majority of the authors’ cases with PSE on EEG had background reactivity, intact brainstem reflex testing, and intact N20 responses on somatosensory evoked potential testing. This group of patients might be expected to do better than most. For example, we treated 26 patients with hypothermia after cardiac arrest over 2 years (mixed in and out of hospital arrest, ventricular fibrillation, pulseless electrical activity, and asystole) and observed 3 cases (11%) with severe myoclonic jerking after hypothermia, sedation, and muscle relaxation was lifted. In these patients, there was typically partial loss of brainstem reflexes prehypothermia and posthypothermia, and in 1 case absent N20 potentials. In 2 of our cases, the EEG indicated a myoclonus status epilepticus pattern, in which the background is typically suppressed, unreactive, and alternating bursts of activity correlated with myoclonic jerks.3 Whether the hypothermia improves the EEG patterns relative to clinical outcomes during the period of therapeutic hypothermia is still unclear.

Novel phenanthridinone inhibitors of poly(adenosine 5'-diphosphate-ribose) synthetase: Potent cytoprotective and antishock agents

ObjectiveTo synthesize novel inhibitors of the nuclear enzyme poly(adenosine 5′-diphosphate [ADP]-ribose) synthetase (PARS), also known as poly(ADP-ribose) polymerase (PARP), and to test them in in vitro models of oxidant-induced cytotoxicity and in endotoxin and splanchnic occlusion-reperfusion-induced shock. DesignRandomized, prospective laboratory study. SettingResearch laboratory. SubjectsMurine macrophages, thymocytes, and endothelial cells; Balb/c mice and Wistar rats. InterventionsMacrophages and endothelial cells were treated with peroxynitrite and bleomycin to induce PARS activation, and thymocytes were treated with peroxynitrite to induce cell necrosis. Novel PARS inhibitors were synthesized and used to reduce PARS activation and to reverse cytotoxicity. Balb/c mice were subjected to splanchnic occlusion and reperfusion and were pretreated with various doses (1–10 mg/kg intraperitoneally) of PJ34, a selected, potent, water-soluble PARS inhibitor. The passage of fluorescein isothiocyanate-conjugated dextran (4 kDa) was analyzed in everted gut ileal sacs incubated ex vivo as an index of gut permeability. Wistar rats were subjected to Escherichia coli bacterial lipopolysaccharide (40 mg/kg intraperitoneally). PJ34 was also used at 10 mg/kg intraperitoneally, 1 hr before lipopolysaccharide or at 25 mg/kg intraperitoneally 1 hr after lipopolysaccharide treatment. Serum concentrations of indicators or multiple organ injury, concentrations of various proinflammatory mediators, and tissue concentrations of myeloperoxidase and malondialdehyde were measured. In addition, survival rates and vascular contractile and relaxant responses were recorded. Measurements and Main ResultsAppropriate modifications of the phenanthridinone core structure yielded significant increases in the potency of the compounds, both as PARS inhibitors and as cytoprotective agents. The compound N-(6-oxo-5,6-dihydro-phenanthridin-2-yl) -N,N-dimethylacetamide (designated as PJ34) was one of the potent PARS inhibitors of the series, and it dose-dependently protected against thymocyte necrosis, with a half-maximal restoration of cell viability of 35 nM and complete protection at 200 nM. PARS activation also was visualized by immunohistochemistry and was dose-dependently suppressed by PJ34. The effect of PJ34 was dose-dependently reversed by excess nicotinamide adenine dinucleotide (oxidized). The PARS inhibitors dose-dependently suppressed proinflammatory cytokine and chemokine production and restored viability in immunostimulated macrophages. PJ34 was selected for the subsequent in vivo studies. PJ34 significantly protected against splanchnic reperfusion-induced intestinal hyperpermeability in the mouse. PJ34 reduced peak plasma concentrations of tumor necrosis factor-&agr;, interleukin-1&bgr;, and nitrite/nitrate in the plasma of lipopolysaccharide-treated rats. PJ34 ameliorated the lipopolysaccharide-induced increases in indexes of liver and kidney failure and concentrations of myeloperoxidase and malondialdehyde in the lung and gut. Lipopolysaccharide elicited vascular dysfunction, which was normalized by PJ34. Lipopolysaccharide-induced mortality was reduced by PJ34 (both pre- and posttreatment). ConclusionsThe novel series of phenanthridinone PARS inhibitors have potent cytoprotective effects in vitro and significant protective effects in shock and reperfusion injury in rodent models in vivo.

Early predictors of outcome in comatose survivors of ventricular fibrillation and non-ventricular fibrillation cardiac arrest treated with hypothermia: a prospective study.

Objectives:Current indications for therapeutic hypothermia (TH) are restricted to comatose patients with cardiac arrest (CA) due to ventricular fibrillation (VF) and without circulatory shock. Additional studies are needed to evaluate the benefit of this treatment in more heterogeneous groups of patients, including those with non-VF rhythms and/or shock and to identify early predictors of outcome in this setting. Design:Prospective study, from December 2004 to October 2006. Setting:32-bed medico-surgical intensive care unit, university hospital. Patients:Comatose patients with out-of-hospital CA. Interventions:TH to 33 ± 1°C (external cooling, 24 hrs) was administered to patients resuscitated from CA due to VF and non-VF (including asystole or pulseless electrical activity), independently from the presence of shock. Measurements and Main Results:We hypothesized that simple clinical criteria available on hospital admission (initial arrest rhythm, duration of CA, and presence of shock) might help to identify patients who eventually survive and might most benefit from TH. For this purpose, outcome was related to these predefined variables. Seventy-four patients (VF 38, non-VF 36) were included; 46% had circulatory shock. Median duration of CA (time from collapse to return of spontaneous circulation [ROSC]) was 25 mins. Overall survival was 39.2%. However, only 3.1% of patients with time to ROSC >25 mins survived, as compared to 65.7% with time to ROSC ≤25 mins. Using a logistic regression analysis, time from collapse to ROSC, but not initial arrest rhythm or presence of shock, independently predicted survival at hospital discharge. Conclusions:Time from collapse to ROSC is strongly associated with outcome following VF and non-VF cardiac arrest treated with therapeutic hypothermia and could therefore be helpful to identify patients who benefit most from active induced cooling.

Hypertension: A Disease of the Microcirculation?

In the last decade, the pathophysiology of microcirculation has become an actively developing field of hypertension research, which we have felt it useful to review. An exact definition of microcirculation is elusive. It is often taken morphologically, to encompass all of the blood vessels with a diameter <150 μm, that is, some small arteries, arterioles, capillaries, and venules,1 with the morphological distinction between small arteries and arterioles not entirely clear, because some2 but not all authors3 limit the concept of arteriole to vessels containing a single layer of smooth muscle cells. Functionally, it is usually accepted that the arterial side of the microcirculation composes most of the resistance vessels, meaning that the largest part of the pressure drop between large conduit arteries and veins takes place in this segment.2,4 The “resistance” property of small arteries and arterioles is intimately, although not exclusively, related to the prevalence of myogenic tone in these vessels.5,6 Myogenic tone is an intrinsic property of vascular smooth muscle, which contracts in response to stretching, independent of any nerve or humoral mediation.5 All arteries have myogenic tone and, therefore, contract in response to an increase in blood pressure. Myogenic tone gains in importance with decreasing vessel caliber,7,8 and only in small arteries and arterioles (diameter: 15 to 300 μm, depending on species and organ) can it provoke substantial luminal narrowing (or even closure) in reaction to an increase in transmural pressure.8,9 In the hamster cheek pouch, for example, control of arterial/arteriolar luminal size by myogenic tone seems minimal in the feeding saccular arteries (diameter 135 μm) but progressively more intense in the A1 (80 μm), A2 (40 μm), and A3 arterioles (28 μm).8 Myogenic tone serves a simple purpose: to protect the distal capillaries against deleterious local hypertension. …

Cannabidiol attenuates cardiac dysfunction, oxidative stress, fibrosis, and inflammatory and cell death signaling pathways in diabetic cardiomyopathy.

OBJECTIVES In this study, we have investigated the effects of cannabidiol (CBD) on myocardial dysfunction, inflammation, oxidative/nitrative stress, cell death, and interrelated signaling pathways, using a mouse model of type I diabetic cardiomyopathy and primary human cardiomyocytes exposed to high glucose. BACKGROUND Cannabidiol, the most abundant nonpsychoactive constituent of Cannabis sativa (marijuana) plant, exerts anti-inflammatory effects in various disease models and alleviates pain and spasticity associated with multiple sclerosis in humans. METHODS Left ventricular function was measured by the pressure-volume system. Oxidative stress, cell death, and fibrosis markers were evaluated by molecular biology/biochemical techniques, electron spin resonance spectroscopy, and flow cytometry. RESULTS Diabetic cardiomyopathy was characterized by declined diastolic and systolic myocardial performance associated with increased oxidative-nitrative stress, nuclear factor-κB and mitogen-activated protein kinase (c-Jun N-terminal kinase, p-38, p38α) activation, enhanced expression of adhesion molecules (intercellular adhesion molecule-1, vascular cell adhesion molecule-1), tumor necrosis factor-α, markers of fibrosis (transforming growth factor-β, connective tissue growth factor, fibronectin, collagen-1, matrix metalloproteinase-2 and -9), enhanced cell death (caspase 3/7 and poly[adenosine diphosphate-ribose] polymerase activity, chromatin fragmentation, and terminal deoxynucleotidyl transferase dUTP nick end labeling), and diminished Akt phosphorylation. Remarkably, CBD attenuated myocardial dysfunction, cardiac fibrosis, oxidative/nitrative stress, inflammation, cell death, and interrelated signaling pathways. Furthermore, CBD also attenuated the high glucose-induced increased reactive oxygen species generation, nuclear factor-κB activation, and cell death in primary human cardiomyocytes. CONCLUSIONS Collectively, these results coupled with the excellent safety and tolerability profile of CBD in humans, strongly suggest that it may have great therapeutic potential in the treatment of diabetic complications, and perhaps other cardiovascular disorders, by attenuating oxidative/nitrative stress, inflammation, cell death and fibrosis.

Resistance to acute septic peritonitis in poly(ADP-ribose) polymerase-1-deficient mice

Sepsis is associated with a widespread production of proinflammatory cytokines and various oxidant species. Activation of the enzyme poly(ADP-ribose) polymerase (PARP) has been shown to contribute to cell necrosis and organ failure in various diseases associated with inflammation and reperfusion injury. The aim of the current study was to elucidate the role of PARP activation in the multiple organ dysfunction complicating sepsis in a murine model of polymicrobial sepsis induced by cecal ligation and puncture (CLP). Mice genetically deficient in PARP (PARP−/−) and their wild-type littermates (PARP+/+) were subjected to CLP. After 12 and 24 h, the proinflammatory cytokines TNF-&agr; and IL-6, as well as the anti-inflammatory cytokine IL-10, and nitrite/nitrate were measured in plasma samples. Organs were harvested for the measurement of myeloperoxidase (MPO) and malondialdehyde (MDA) levels, and immunohistochemical staining for nitrotyrosine and poly(ADP ribose) was performed in gut sections. PARP−/− mice, and their wild-type littermate showed a similar time-dependent increase in plasma nitrite/nitrate and in gut and lung MDA content, as well as the presence of nitrotyrosine in the gut. In contrast to wild-type mice showing a PARP activation in the gut, PARP−/− mice had no staining for poly(ADP ribose). PARP−/− mice had significantly lower plasma levels of TNF-&agr;, IL-6, and IL-10, and they exhibited a reduced degree of organ inflammation, indicated by decreased MPO activity in the gut and lung. These effects were associated with a significant improvement in the survival of CLP in PARP−/− mice. Thus, PARP activation has an important role in systemic inflammation and organ damage in the present model of polymicrobial septic shock.