Alex Dyson

Animal models of sepsis: why does preclinical efficacy fail to translate to the clinical setting?

Objective:To postulate reasons as to why the benefits seen with novel therapies in animal models of sepsis fail to translate to the clinical setting. Data Source:MEDLINE searches and relevant book chapters. Data Summary:Thousands of preclinical trials performed over more than five decades have failed to find more than a handful of drugs and techniques that significantly improve outcomes in clinical sepsis. We review current concepts surrounding the variety of animal models used today, ranging from simple models of acute toxemia to more complex models of abdominal sepsis. Differences between animal and human populations are also examined including species, age, comorbidity, and the use of supportive therapies. Finally, we examine differences between preclinical and clinical trial design, and the potential for experimental and publication bias. Conclusions:Animal models of sepsis are still too heterogeneous with regard to type of insult, duration, and supportive therapy to be regarded as representative of the human condition. Using standardized animal models may eliminate some of the differences between animal and human studies, allowing a greater degree of translation.

Key bioactive reaction products of the NO/H2S interaction are S/N-hybrid species, polysulfides, and nitroxyl

Significance Reactions of sulfur-centered nucleophiles with nitrogenous species have been studied independently for more than a century for synthetic/industrial purposes; to understand geochemical, atmospheric, and biological processes; and to explain the origins of life. Various products and reaction mechanisms were proposed. We here identify a singular process comprising a network of cascading chemical reactions that form three main bioactive products at physiological pH: nitrosopersulfide, polysulfides, and dinitrososulfite. These anionic products scavenge, transport, and release NO/HNO or sulfide/sulfane sulfur, each displaying distinct chemistries and bioactivities. Our observations provide a chemical foundation for the cross-talk between the NO and H2S signaling pathways in biology and suggest that the biological actions of these entities can be neither considered nor studied in isolation. Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO−), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO− is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO− synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.

Cardioprotection, attenuated systemic inflammation, and survival benefit of β1-adrenoceptor blockade in severe sepsis in rats*

Objective: To explore the hypothesis that beta-1 adrenoreceptor blockade may be protective through the attenuation of sympathetic hyperactivity and catecholaminergic inflammatory effects on cardiac and hepatic function. Design: Prospective, randomized, controlled study. Setting: Animal laboratory in a university medical center. Subjects: Male adult Wistar rats. Interventions: Peripheral &bgr;1-adrenoceptor blockade through daily intraperitoneal injection (metoprolol, 100 mg·kg−1; atenolol, 6 mg·kg−1) or central nervous system &bgr;1-adrenoceptor blockade (intracerebroventricular metoprolol, 25 &mgr;g) to achieve ∼20% heart rate reduction in rats for 2 days before or after the induction of lethal endotoxemia, cecal ligation and puncture, or fecal peritonitis. Measurements and Main Results: Peripheral &bgr;1-adrenoceptor blockade established for 2 days before lethal endotoxemia markedly improved survival in both metoprolol-treated (n = 16; log rank test, p = .002) and atenolol-treated (n = 15; p = .03) rats. Overall mortality in cecal ligation and puncture was similar between metoprolol (40%; n = 10) and saline (50%; n = 10) pretreatment (p = .56), but the median time to death was increased by 33 hrs in metoprolol-treated rats (p = .03). Metoprolol pretreatment reduced hepatic expression of proinflammatory cytokines and lowered plasma interleukin-6 (both p < .05). Myocardial protein expression of interleukin-18 and monocyte chemoattractant protein-1, key mediators of cardiac dysfunction in sepsis, were also reduced (p < .05). Peripheral &bgr;1-adrenoceptor blockade commenced 6 hrs after lethal endotoxemia or fecal peritonitis did not improve survival. However, arterial blood pressure was preserved and left ventricular contractility restored similar to that found in nonseptic controls. Central nervous system &bgr;1-adrenoceptor blockade (metoprolol) did not reduce plasma cytokines or mortality, despite enhancing parasympathetic tone. Conclusions: Peripheral &bgr;1-adrenoceptor blockade offers anti-inflammatory and cardioprotective effects, with mortality reduction if commenced before a septic insult. Its role in sepsis should be explored further.

Liver Dysfunction and Phosphatidylinositol-3-Kinase Signalling in Early Sepsis: Experimental Studies in Rodent Models of Peritonitis

Experimental studies in a rat model of fecal peritonitis conducted by Michael Bauer and colleagues show that in this model, changes in liver function occur early in the development of sepsis, with potential implications for prognosis and development of new therapeutic approaches.

Design, Characterization, and First-In-Human Study of the Vascular Actions of a Novel Biased Apelin Receptor Agonist

[Pyr1]apelin-13 is an endogenous vasodilator and inotrope but is downregulated in pulmonary hypertension and heart failure, making the apelin receptor an attractive therapeutic target. Agonists acting at the same G-protein–coupled receptor can be engineered to stabilize different conformational states and function as biased ligands, selectively stimulating either G-protein or &bgr;-arrestin pathways. We used molecular dynamics simulations of apelin/receptor interactions to design cyclic analogues and identified MM07 as a biased agonist. In &bgr;-arrestin and internalization assays (G-protein–independent), MM07 was 2 orders of magnitude less potent than [Pyr1]apelin-13. In a G-protein–dependent saphenous vein contraction assay, both peptides had comparable potency (pD2:[Pyr1]apelin-13 9.93±0.24; MM07 9.54±0.42) and maximum responses with a resulting bias for MM07 of ≈350- to 1300-fold for the G-protein pathway. In rats, systemic infusions of MM07 (10-100nmol) caused a dose-dependent increase in cardiac output that was significantly greater than the response to [Pyr1]apelin-13. Similarly, in human volunteers, MM07 produced a significant dose-dependent increase in forearm blood flow with a maximum dilatation double that is seen with [Pyr1]apelin-13. Additionally, repeated doses of MM07 produced reproducible increases in forearm blood flow. These responses are consistent with a more efficacious action of the biased agonist. In human hand vein, both peptides reversed an established norepinephrine constrictor response and significantly increased venous flow. Our results suggest that MM07 acting as a biased agonist at the apelin receptor can preferentially stimulate the G-protein pathway, which could translate to improved efficacy in the clinic by selectively stimulating vasodilatation and inotropic actions but avoiding activating detrimental &bgr;-arrestin–dependent pathways.

The Key Role of Nitric Oxide in Hypoxia: Hypoxic Vasodilation and Energy Supply–Demand Matching

SIGNIFICANCE A mismatch between energy supply and demand induces tissue hypoxia with the potential to cause cell death and organ failure. Whenever arterial oxygen concentration is reduced, increases in blood flow--hypoxic vasodilation--occur in an attempt to restore oxygen supply. Nitric oxide (NO) is a major signaling and effector molecule mediating the bodys response to hypoxia, given its unique characteristics of vasodilation (improving blood flow and oxygen supply) and modulation of energetic metabolism (reducing oxygen consumption and promoting utilization of alternative pathways). RECENT ADVANCES This review covers the role of oxygen in metabolism and responses to hypoxia, the hemodynamic and metabolic effects of NO, and mechanisms underlying the involvement of NO in hypoxic vasodilation. Recent insights into NO metabolism will be discussed, including the role for dietary intake of nitrate, endogenous nitrite (NO₂⁻) reductases, and release of NO from storage pools. The processes through which NO levels are elevated during hypoxia are presented, namely, (i) increased synthesis from NO synthases, increased reduction of NO₂⁻ to NO by heme- or pterin-based enzymes and increased release from NO stores, and (ii) reduced deactivation by mitochondrial cytochrome c oxidase. CRITICAL ISSUES Several reviews covered modulation of energetic metabolism by NO, while here we highlight the crucial role NO plays in achieving cardiocirculatory homeostasis during acute hypoxia through both vasodilation and metabolic suppression. FUTURE DIRECTIONS We identify a key position for NO in the bodys adaptation to an acute energy supply-demand mismatch.

MICROVASCULAR AND INTERSTITIAL OXYGEN TENSION IN THE RENAL CORTEX AND MEDULLA STUDIED IN A 4-H RAT MODEL OF LPS-INDUCED ENDOTOXEMIA

The pathophysiology of sepsis-induced acute kidney injury remains poorly understood. As changes in renal perfusion and oxygenation have been shown, we aimed to study the short-term effects of endotoxemia on microvascular and interstitial oxygenation in the cortex and medulla, in conjunction with global and renal hemodynamics. In a 4-h rat model of endotoxemia, we simultaneously assessed renal artery blood flow and microvascular and interstitial oxygen tensions in the renal cortex and medulla using ultrasonic flowmetry, dual wavelength phosphorimetry, and tissue oxygen tension monitoring, respectively. Whereas medullary microvascular and interstitial oxygen tensions decreased promptly in line with macrovascular blood flow, changes in cortical oxygenation were only seen later on. During the entire experimental protocol, the gradient between microvascular PO2 and tissue oxygen tension remained unchanged in both cortex and outer medulla. At study end, urine output was significantly decreased despite a maintained oxygen consumption rate. In this 4-h rat model of endotoxemia, total renal oxygen consumption and the gradient between microvascular PO2 and tissue oxygen tension remained unaltered, despite falls in renal perfusion and oxygen delivery and urine output. Taken in conjunction with the decrease in urine output, our results could represent either a functional renal impairment or an adaptive response.

Early functional and transcriptomic changes in the myocardium predict outcome in a long-term rat model of sepsis.

Myocardial function is depressed in sepsis and is an important prognosticator in the human condition. Using echocardiography in a long-term fluid-resuscitated Wistar rat model of faecal peritonitis we investigated whether depressed myocardial function could be detected at an early stage of sepsis and, if so, whether the degree of depression could predict eventual outcome. At 6 h post-insult, a stroke volume <0.17 ml prognosticated 3-day mortality with positive and negative predictive values of 93 and 80%, respectively. Subsequent fluid loading studies demonstrated intrinsic myocardial depression with poor-prognosis animals tolerating less fluid than either good-prognosis or sham-operated animals. Cardiac gene expression analysis at 6 h detected 527 transcripts significantly up- or down-regulated by the septic process, including genes related to inflammatory and cell cycle pathways. Predicted mortality was associated with significant differences in transcripts of genes expressing proteins related to the TLR2/MyD88 (Toll-like receptor 2/myeloid differentiation factor 88) and JAK/STAT (Janus kinase/signal transducer and activator of transcription) inflammatory pathways, β-adrenergic signalling and intracellular calcium cycling. Our findings highlight the presence of myocardial depression in early sepsis and its prognostic significance. Transcriptomic analysis in heart tissue identified changes in signalling pathways that correlated with clinical dysfunction. These pathways merit further study to both better understand and potentially modify the disease process.

Is MOF an outcome parameter or a transient, adaptive state in critical illness?

Purpose of reviewThe term ‘multiorgan failure’ (MOF) carries the negative connotation of major homeostatic breakdown and severe malfunction. However, this traditional paradigm may not be necessarily accurate. This review will investigate the rationale for no longer considering MOF to be simply a ‘failed’ pathophysiological state. Recent findingsMultiorgan failure is characterized by a hypometabolic, immunodepressed state with clinical and biochemical evidence of decreased functioning of the bodys organ systems. Notwithstanding these findings, evidence for cell death is scarce and organ recovery is frequently the rule in surviving patients without pre-existing organ disease. Decreased mitochondrial activity appears to play a key role in the processes underlying MOF, both as a victim and a player. Reduced ATP production will compromise normal metabolic functioning. To protect itself from dying, the cell may adapt by decreasing its metabolic rate, and this is clinically manifest as organ dysfunction. Mitochondrial modulation may thus represent an important therapeutic target. SummaryThe concept of MOF could be revisited as a transient state of metabolic shutdown analogous to hibernation. Avoiding the detrimental effects of inappropriate and counter-adaptive iatrogenic interventions is an important cornerstone of therapeutic management.

An integrated approach to assessing nitroso-redox balance in systemic inflammation

Most studies examining the metabolic fate of NO during systemic inflammation have focused on measuring the quantitatively predominating, stable anions nitrite and nitrate within the circulation. However, these are not necessarily the NO-related products that govern NO metabolism and signaling in tissues. We assessed all major NO derivatives temporally in blood and vital organs during inflammation and explored their relationship to insult severity and redox status. Male rats receiving intraperitoneal endotoxin or vehicle were sacrificed for organ and blood sampling between 0 and 24 h. Endotoxin induced transient and organ-specific changes in a variety of NO metabolites. Nitrite and nitrate increased, peaking at 8 and 12 h, respectively. S- and N-nitrosation and heme-nitrosylation products also peaked at 8 h; these posttranslational protein modifications were associated with decreased myocardial function (echocardiography). Evidence of oxidative stress and systemic inflammation was also obtained. The rise in most NO derivatives was proportional to insult severity. All metabolite levels normalized within 24 h, despite evidence of persisting myocardial dysfunction and clinical unwellness. Our findings point to a complex interplay between NO production, antioxidant defense, and redox status. Although the precise (patho)physiologic roles of specific NO derivatives and their diagnostic/prognostic utility await further investigation, nitroso species in erythrocytes are the most sensitive markers of NO in systemic inflammation, detectable before clinical symptoms manifest.