Olivier Huet

Alterations of mitochondrial function in sepsis and critical illness

Purpose of review Septic shock is the consequence of a conflict between a pathogenic agent and the immune system of the host. This conflict induces an immune-mediated cytokine storm, with a whole-body inflammatory response often leading to multiple organ failure. Although extensively studied, the pathophysiology of sepsis-associated multiorgan failure remains unknown. One postulated mechanism is changes in mitochondrial function with an inhibition of mitochondrial respiratory chain and a decrease of oxygen utilization. Recent findings Mitochondrion is a key organelle in supplying energy to the cell according to its metabolic need. Hypoxia and a number of the mediators implicated in sepsis and in the associated systemic inflammatory response have been demonstrated to directly impair mitochondrial function. A large body of evidence supports a key role of the peroxynitrite, which can react with most of the components of the electron transport chain, in the mitochondrial dysfunction. Summary A pivotal role is suggested for mitochondrial dysfunction during the occurrence of multiorgan failure. Understanding the precise effect of sepsis on the mitochondrial function and the involvement of mitochondria in the development of multiple organ failure is fundamental. More human studies are thus necessary to clarify the mitochondrial dysfunction in the various phases of sepsis (early and late phase) before testing therapeutic strategies targeting mitochondria.

Microcirculatory alterations in traumatic hemorrhagic shock

Objectives:Microcirculatory dysfunction has been well reported in clinical studies in septic shock. However, no clinical studies have investigated microcirculatory blood flow behavior in hemorrhagic shock. The main objective of this study was to assess the time course of sublingual microcirculation in traumatic hemorrhagic shock during the first 4 days after trauma. DesignProspective observational study. SettingICU. PatientsEighteen traumatic hemorrhagic shock patients. InterventionsThe sublingual microcirculation was estimated at the study inclusion after surgical or angiographic embolization to control bleeding (D1), and then three times at 24-hour intervals (D2, D3, and D4). Measurements and Main Results:Sublingual microcirculation was impaired for 72 hours despite restoration of the macrovascular circulation after control of bleeding in traumatic hemorrhagic shock patients. Furthermore, we found significantly higher decreases in the microvascular flow index and proportion of perfused vessels in high Sequential Organ Failure Assessment score patients at D4 (Sequential Organ Failure Assessment score ≥ 6) compared to low Sequential Organ Failure Assessment score patients at D4 (Sequential Organ Failure Assessment score < 6) without any differences in global hemodynamics between these two groups. Finally, the initial proportion of perfused vessels at D1 appears to be a good predictor of high Sequential Organ Failure Assessment score at D4. Conclusions:Alterations of microcirculation in traumatic hemorrhagic shock patients result from the interplay among hemorrhage-induced tissue hypoperfusion, trauma injuries, inflammatory response, and subsequent resuscitation interventions. Despite restoration of the macrocirculation, the sublingual microcirculation was impaired for at least 72 hours. The initial proportion of perfused vessels appears to be a good predictor of high Sequential Organ Failure Assessment score at D4. Further studies are required to firmly establish the link between microvascular alterations and organ dysfunction in traumatic hemorrhagic shock patients.

Plasma-induced endothelial oxidative stress is related to the severity of septic shock.

Objective:To estimate the capacity of plasma from septic shock patients to induce in vitro reactive oxygen species (ROS) production by endothelial cells and to analyze whether ROS production is related to the severity of the septic shock. Design:Prospective, observational study. Setting:Medical intensive care unit in a university hospital. Patients:Twenty-one patients with septic shock. Interventions:The in vitro capacity of plasma from septic shock patients to induce ROS production by naive human umbilical vein endothelial cells (HUVEC) was quantified by using a fluorescent probe (2′,7′-dichlorodihydrofluorescein diacetate). Measurements and Main Results:Blood samples were collected on day 1, day 3, and day 5 from 21 consecutive septic shock adult patients and from ten healthy volunteers. Patients mean age was 58 yrs old, mean Sequential Organ Failure Assessment (SOFA) score at admission was 12, mean severity illness assessed by Simplified Acute Physiology Score (SAPS) II was 53, and the mortality rate was 47%. In addition to assessment of in vitro ROS generation by HUVEC, oxidative stress in blood was evaluated by measuring lipid peroxidation products and enzymatic and nonenzymatic antioxidants. Septic shock was associated with oxidative stress and an imbalance in antioxidant status. As compared with controls, plasma-induced ROS production by naïve HUVEC was significantly higher in septic shock. Moreover ROS production was significantly correlated with SAPS II (p = .028) and SOFA values (p = .0012) and was higher in nonsurvivors than in survivors. In contrast, no correlation was found between the severity of the septic shock and any of the levels of lipid peroxidation products or enzymatic and nonenzymatic antioxidants. Conclusion:Plasma from septic shock patients induces ROS formation by naive HUVEC, and the extent of ROS formation correlates with mortality and with criteria of the severity of septic shock as SOFA score and SAPS II.

Postresuscitation syndrome: Potential role of hydroxyl radical-induced endothelial cell damage

Objective:After out of hospital cardiac arrest, it has been reported that endothelium dysfunction may occur during the postresuscitation syndrome. However, the consequences of the reperfusion phase on endothelial reactive oxygen species production and redox homeostasis have not been explored in out of hospital cardiac arrest patients. Design:Prospective, observational study. Setting:Medical intensive care unit in a university hospital. Patients:Twenty successfully resuscitated out of hospital cardiac arrest patients, seven septic shock patients, and ten healthy volunteers. Intervention:Plasma was collected from patients at admission and 12, 24, 36, 48, and 72 hrs after cardiac arrest. We studied the production of reactive oxygen species and cell survival during plasma perfusion using perfused endothelial cells (human umbilical vein endothelial cells) as a model. Cell antioxidant response was studied by measuring superoxide dismutase, glutathione peroxidase, and glutathione reductase activities and reduced and oxidized glutathione levels. Mitochondrial respiratory chain activity was assessed by measuring complex I, II, III, and IV activities and anaerobic glycolysis by measuring glucose-6-phosphate dehydrogenase activity. Measurements and Main Results:Using perfused endothelial cells as a model, we demonstrate that plasma from out of hospital cardiac arrest patients induced on naive human umbilical vein endothelial cells a significant and massive cell death compared to plasma from septic shock patients and healthy volunteers. An increase of reactive oxygen species production with a decrease in antioxidant defenses (superoxide dismutase, glutathione peroxidase, and glutathione reductase activities, reduced and oxidized glutathione levels) was observed. The metabolic consequence of plasma exposure showed that mitochondrial respiratory chain activity was significantly impaired and anaerobic glycolysis was significantly increased. Inhibiting hydroxyl radical production significantly decreased cell death, suggesting that plasma from out of hospital cardiac arrest induced significant cell death by triggering the Fenton reaction. Conclusion:Plasma from out of hospital cardiac arrest induces major endothelial toxicity with an acute pro-oxidant state in the cells and impairment of mitochondrial respiratory chain activity. This toxicity could be due to hydroxyl radical production by activation of the Fenton reaction.

Dicarbonyl stress in the absence of hyperglycemia increases endothelial inflammation and atherogenesis similar to that observed in diabetes.

The deleterious effects of high glucose levels and enhanced metabolic flux on the vasculature are thought to be mediated by the generation of toxic metabolites, including reactive dicarbonyls like methylglyoxal (MG). In this article, we demonstrate that increasing plasma MG to levels observed in diabetic mice either using an exogenous source (1% in drinking water) or generated following inhibition, its primary clearance enzyme, glyoxalase-1 (with 50 mg/kg IP bromobenzyl-glutathione cyclopentyl diester every second day), was able to increase vascular adhesion and augment atherogenesis in euglycemic apolipoprotein E knockout mice to a similar magnitude as that observed in hyperglycemic mice with diabetes. The effects of MG appear partly mediated by activation of the receptor for advanced glycation end products (RAGE), as deletion of RAGE was able to reduce inflammation and atherogenesis associated with MG exposure. However, RAGE deletion did not completely prevent inflammation or vascular damage, possibly because the induction of mitochondrial oxidative stress by dicarbonyls also contributes to inflammation and atherogenesis. Such data would suggest that a synergistic combination of RAGE antagonism and antioxidants may offer the greatest utility for the prevention and management of diabetic vascular complications.

Activation of the Renin-Angiotensin System Mediates the Effects of Dietary Salt Intake on Atherogenesis in the Apolipoprotein E Knockout Mouse

Dietary salt intake is a major determinant of the activation state of renin-angiotensin-aldosterone system. Given the important role of the renin-angiotensin-aldosterone system in plaque accumulation, we investigated its role in the development of atherogenesis associated with sodium intake in apolipoprotein E knockout mice. Six-weeks of a low-salt diet (containing 0.03% sodium) resulted in a 4-fold increase in plaque accumulation in apolipoprotein E knockout mice when compared with mice receiving normal chow (containing 0.30% sodium). This was associated with activation of the renin-angiotensin-aldosterone system, increased vascular expression of adhesion molecules and inflammatory cytokines, and increased adhesion of labeled leukocytes across the whole aorta on a dynamic flow assay. These changes were blocked with the angiotensin-converting enzyme inhibitor perindopril (2 mg/kg per day). A high-salt diet (containing 3% sodium) attenuated vascular inflammation and atherogenesis, associated with suppression of the renin-angiotensin-aldosterone system, although systolic blood pressure levels were modestly increased (5±1 mmHg). Constitutive activation of the renin-angiotensin-aldosterone system in angiotensin-converting enzyme 2 apolipoprotein E knockout mice was also associated with increased atherosclerosis and vascular adhesion, and this was attenuated by a high-salt diet associated with suppression of the renin-angiotensin-aldosterone system. By contrast, a low-salt diet failed to further activate the renin-angiotensin-aldosterone system or to increase atherosclerosis in angiotensin-converting enzyme 2 apolipoprotein E knockout mice. Together, these data validate a relationship between salt-mediated renin-angiotensin-aldosterone system activation and atherogenesis, which may partly explain the inconclusive or paradoxical findings of recent observational studies, despite clear effects on blood pressure.

Ensuring animal welfare while meeting scientific aims using a murine pneumonia model of septic shock.

ABSTRACT With animal models, death as an intentional end point is ethically unacceptable. However, in the study of septic shock, death is still considered the only relevant end point. We defined eight humane end points into four stages of severity (from healthy to moribund) and used to design a clinically relevant scoring tool, termed “the mouse clinical assessment score for sepsis” (M-CASS). The M-CASS was used to enable a consistent approach to the assessment of disease severity. This allowed an ethical and objective assessment of disease after which euthanasia was performed, instead of worsening suffering. The M-CASS displayed a high internal consistency (Cronbach &agr; = 0.97) with a high level of agreement and an intraclass correlation coefficient equal to 0.91. The plasma levels of cytokines and markers of oxidative stress were all associated with the M-CASS score (Kruskal-Wallis test, P < 0.05). The M-CASS allows tracking of disease progression and animal welfare requirements.

Pivotal role of glutathione depletion in plasma-induced endothelial oxidative stress during sepsis.

Objective:Plasma from septic shock patients can induce production of reactive oxygen species (ROS) by human umbilical vein endothelial cells (HUVEC) in vitro. How endothelial cells defend themselves against ROS under increased oxidative stress has not yet been examined. This study investigates the antioxidant defenses of HUVEC exposed to plasma obtained from either septic shock patients or healthy volunteers. Design:Prospective, observational study. Setting:Medical intensive care unit in a university hospital. Patients:Twenty-five patients with septic shock and 10 healthy volunteers. Interventions:Blood samples were collected within the first 24 hrs of septic shock. In vitro HUVEC production of ROS was studied by spectrofluorimetry using 2′,7′-dichlorodihydrofluorescein diacetate fluorescent dye. Reactive nitrogen species were also assessed. Intracellular reduced glutathione (GSH) levels were measured using monochlorobimane fluorescent dye. Activity of catalase and superoxide dismutase in HUVEC were also measured. Cell death was assessed using YOPRO fluorescent dye and the MTT assay. Measurements and Results:On admission, the septic shock populations mean age was 55 yrs old, the mean Sequential Organ Failure Assessment score was 12, mean simplified acute physiology score was 50, and intensive care unit mortality rate was 45%. Evaluation of HUVEC antioxidant defenses showed a significantly decreased GSH level, increased catalase activity, and unchanged superoxide dismutase activity. ROS levels and cell death were significantly reduced when cells were pretreated with N-acetylcysteine or GSH, but no changes in reactive nitrogen species were observed. Conclusion:This study demonstrates that plasma-induced ROS production by HUVEC is associated with an intracellular decrease in reduced GSH. Both ROS levels and cell death decreased when N-acetylcysteine or GSH were added before exposing the cells to plasma. These data suggest a pivotal role of alterations in GSH in damage caused by sepsis-generated ROS in endothelial cell.

Direct Endothelial Nitric Oxide Synthase Activation Provides Atheroprotection in Diabetes-Accelerated Atherosclerosis

Patients with diabetes have an increased risk of developing atherosclerosis. Endothelial dysfunction, characterized by the lowered bioavailability of endothelial NO synthase (eNOS)–derived NO, is a critical inducer of atherosclerosis. However, the protective aspect of eNOS in diabetes-associated atherosclerosis remains controversial, a likely consequence of its capacity to release both protective NO or deleterious oxygen radicals in normal and disease settings, respectively. Harnessing the atheroprotective activity of eNOS in diabetic settings remains elusive, in part due to the lack of endogenous eNOS-specific NO release activators. We have recently shown in vitro that eNOS-derived NO release can be increased by blocking its binding to Caveolin-1, the main coat protein of caveolae, using a highly specific peptide, CavNOxin. However, whether targeting eNOS using this peptide can attenuate diabetes-associated atherosclerosis is unknown. In this study, we show that CavNOxin can attenuate atherosclerotic burden by ∼84% in vivo. In contrast, mice lacking eNOS show resistance to CavNOxin treatment, indicating eNOS specificity. Mechanistically, CavNOxin lowered oxidative stress markers, inhibited the expression of proatherogenic mediators, and blocked leukocyte-endothelial interactions. These data are the first to show that endogenous eNOS activation can provide atheroprotection in diabetes and suggest that CavNOxin is a viable strategy for the development of antiatherosclerotic compounds.

Compound 21, a selective agonist of angiotensin AT2 receptors, prevents endothelial inflammation and leukocyte adhesion in vitro and in vivo

Angiotensin AT2 receptors are upregulated in disease states such as atherosclerosis and blockade of the AT2 receptors exacerbates plaque formation. Direct stimulation of these receptors is anti‐atherogenic but the mechanisms and pathways involved remain unknown. We examined the effect of direct AT2 receptor stimulation with Compound 21 (C21) on the leukocyte adhesion cascade in vitro, right through to plaque formation in vivo.