Zhao-Qin Cheng

Lysozyme: a mediator of myocardial depression and adrenergic dysfunction in septic shock in dogs

The objective of the present study was to identify the nature of a filterable cardiodepressant substance (FCS) that contributes to myocardial dysfunction in a canine model of Escherichia coli septic shock. In a previous study, it was found that FCS increased in plasma after 4 h of bacteremia (Am J Physiol 1993;264:H1402) in which FCS was identified by a bioassay that included a right ventricular trabecular (RVT) preparation. In that study, FCS was only partially identified by pore filtration techniques and was found to be a protein of molecular weight between 10 and 30 K. In the present study, FCS was further purified by size exclusion high-pressure liquid chromatography, until a single band was identified on one-dimensional gel electrophoresis. This band was then subjected to tandem mass spectrometry and protein-sequencing techniques and both techniques identified FCS as lysozyme c (Lzm-S), consistent with that originating from the canine spleen. Confirmatory tests showed that purified Lzm-S produced myocardial depression in the RVT preparation at concentrations achieved during sepsis in the in vivo preparation. In addition, Lzm-S inhibited the adrenergic response induced by field stimulation and the beta- agonist isoproterenol in in vitro preparations, these results suggesting that Lzm-S may inhibit the sympathetic response in sepsis. The present findings indicate that Lzm-S originating from disintegrating leukocytes from organs such as the spleen contributes to myocardial dysfunction in this model. The mechanism may relate to its binding or hydrolysis of a cardiac membrane glycoprotein thereby interfering with myocardial excitation-contraction coupling in sepsis.

N,n′,n′-triacetylglucosamine, an inhibitor of lysozyme, prevents myocardial depression in escherichia coli sepsis in dogs*

ObjectiveReversible myocardial depression in sepsis has been ascribed to the release of inflammatory mediators. We recently found that lysozyme c (Lzm-S), consistent with that originating from the spleen, was a mediator of myocardial depression in an Escherichia coli model of septic shock in dogs. We further showed in a right ventricular trabecular (RVT) preparation that Lzm-S’s depressant activity could be blocked by N,N′,N’ triacetylglucosamine (TAC), a competitive inhibitor of Lzm-S. We hypothesized that Lzm-S binds to or cleaves a cardiac membrane glycoprotein, thereby interfering with myocardial contraction in sepsis. In the present study, we examined whether TAC could prevent myocardial depression in an in vivo preparation and whether other related N-acetylglucosamine (NAG) structures could also inhibit Lzm-S’s effect in RVT. DesignRandomized experimental study. SettingUniversity laboratory. SubjectsAnesthetized, mechanically ventilated dogs. InterventionsWe produced sepsis by infusion of E. coli over an approximately 6-hr period. Measurements and Main ResultsWe examined the effect of TAC on stroke work, our primary index of myocardial function, when treatment was administered before sepsis (pretreatment) and after 1.5 hrs (early treatment study) and 3.5 hrs of sepsis (late treatment study; LTS). In the pretreatment study and early treatment study, myocardial depression would have not yet occurred but would have already been present in the late treatment study. In RVT, we assessed the effect of other NAG oligosaccharides and variants to the NAG structure on Lzm-S’s depressant activity. In pretreatment and the early treatment study, TAC prevented the reduction in stroke work observed in nontreated septic groups but did not reverse the reduction found in the late treatment study. In RVT, of the compounds tested, only N,N′-diacetylglucosamine showed an inhibitory effect. ConclusionsWe found that TAC, a competitive inhibitor of Lzm-S, prevented myocardial depression in experimental sepsis. Only specific NAG structures are inhibitory to Lzm-S’s depressant activity. TAC may be useful in attenuating cardiovascular collapse in sepsis.

Lysozyme, a mediator of sepsis that produces vasodilation by hydrogen peroxide signaling in an arterial preparation.

In septic shock, systemic vasodilation and myocardial depression contribute to the systemic hypotension observed. Both components can be attributed to the effects of mediators that are released as part of the inflammatory response. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression that develops in a canine model of septic shock. Lzm-S binds to the endocardial endothelium, resulting in the production of nitric oxide (NO), which, in turn, activates the myocardial soluble guanylate cyclase (sGC) pathway. In the present study, we determined whether Lzm-S might also play a role in the systemic vasodilation that occurs in septic shock. In a phenylephrine-contracted canine carotid artery ring preparation, we found that both canine and human Lzm-S, at concentrations similar to those found in sepsis, produced vasorelaxation. This decrease in force could not be prevented by inhibitors of NO synthase, prostaglandin synthesis, or potassium channel inhibitors and was not dependent on the presence of the vascular endothelium. However, inhibitors of the sGC pathway prevented the vasodilatory activity of Lzm-S. In addition, Aspergillus niger catalase, which breaks down H(2)O(2), as well as hydroxyl radical scavengers, which included hydroquinone and mannitol, prevented the effect of Lzm-S. Electrochemical sensors corroborated that Lzm-S caused H(2)O(2) release from the carotid artery preparation. In conclusion, these results support the notion that when Lzm-S interacts with the arterial vasculature, this interaction results in the formation of H(2)O(2), which, in turn, activates the sGC pathway to cause relaxation. Lzm-S may contribute to the vasodilation that occurs in septic shock.

Ethyl gallate, a scavenger of hydrogen peroxide that inhibits lysozyme-induced hydrogen peroxide signaling in vitro, reverses hypotension in canine septic shock

Although hydrogen peroxide (H2O2) is a well-described reactive oxygen species that is known for its cytotoxic effects and associated tissue injury, H2O2 has recently been established as an important signaling molecule. We previously demonstrated that lysozyme (Lzm-S), a mediator of sepsis that is released from leukocytes, could produce vasodilation in a phenylephrine-constricted carotid artery preparation by H2O2 signaling. We found that Lzm-S could intrinsically generate H2O2 and that this generation activated H2O2-dependent pathways. In the present study, we used this carotid artery preparation as a bioassay to define those antioxidants that could inhibit Lzm-Ss vasodilatory effect. We then determined whether this antioxidant could reverse the hypotension that developed in an Escherichia coli bacteremic model. Of the many antioxidants tested, we found that ethyl gallate (EG), a nonflavonoid phenolic compound, was favorable in inhibiting Lzm-S-induced vasodilation. In our E. coli model, we found that EG reversed the hypotension that developed in this model and attenuated end-organ dysfunction. By fluorometric H2O2 assay and electrochemical probe techniques, we showed that EG could scavenge H2O2 and that it could reduce H2O2 production in model systems. These results show that EG, an antioxidant that was found to scavenge H2O2 in vitro, was able to attenuate cardiovascular dysfunction in a canine in vivo preparation. Antioxidants such as EG may be useful in the treatment of hemodynamic deterioration in septic shock.

Lysozyme, a mediator of sepsis that intrinsically generates hydrogen peroxide to cause cardiovascular dysfunction

In septic shock, cardiovascular collapse is caused by the release of inflammatory mediators. We previously found that lysozyme (Lzm-S), released from leukocytes, contributed to the myocardial depression and arterial vasodilation that develop in canine models of septic shock. To cause vasodilation, Lzm-S generates hydrogen peroxide (H(2)O(2)) that activates the smooth muscle soluble guanylate cyclase (sGC) pathway, although the mechanism of H(2)O(2) generation is not known. To cause myocardial depression, Lzm-S binds to the endocardial endothelium, resulting in the formation of nitric oxide (NO) and subsequent activation of myocardial sGC, although the initial signaling event is not clear. In this study, we examined whether the myocardial depression produced by Lzm-S was also caused by the generation of H(2)O(2) and whether Lzm-S could intrinsically generate H(2)O(2) as has been described for other protein types. In a canine ventricular trabecular preparation, we found that the peroxidizing agent Aspergillus niger catalase, that would breakdown H(2)O(2), prevented Lzm-S- induced decrease in contraction. We also found that compound I, a species of catalase formed during H(2)O(2) metabolism, could contribute to the NO generation caused by Lzm-S. In tissue-free experiments, we used a fluorometric assay (Ultra Amplex red H(2)O(2) assay) and electrochemical sensor techniques, respectively, to measure H(2)O(2) generation. We found that Lzm-S could generate H(2)O(2) and, furthermore, that this generation could be attenuated by the singlet oxygen quencher sodium azide. This study shows that Lzm-S, a mediator of sepsis, is able to intrinsically generate H(2)O(2). Moreover, this generation may activate H(2)O(2)-dependent pathways leading to cardiovascular collapse in septic shock.

Benefits of ethyl gallate versus norepinephrine in the treatment of cardiovascular collapse in Pseudomonas aeruginosa septic shock in dogs.

Interventions: Vasopressor therapy is required in septic shock to maintain tissue perfusion in the face of hypotension. Unfortunately, there are significant side effects of current vasopressors, and newer agents need to be developed. We recently discovered that ethyl gallate, a nonflavonoid phenolic antioxidant found in food substances, could reverse low mean arterial pressure found in an experimental model of septic shock due to inhibition of hydrogen peroxide signaling. In the present study, we compared the hemodynamic and biochemical effects of ethyl gallate vs. those of the commonly used vasopressor, norepinephrine, in a bacteremic canine model of Pseudomonas aeruginosa sepsis in two protocols. Measurements and Main Results: We performed these studies in anesthetized and mechanically ventilated dogs. In the early treatment protocol, we infused P. aeruginosa until mean arterial pressure first decreased to ∼60 mm Hg (about 2–3 hrs), after which we stopped the infusion and randomly administered ethyl gallate or norepinephrine in respective groups. In the late treatment protocol, we administered ethyl gallate or norepinephrine after a sustained ∼5-hr decrease in mean arterial pressure to 60 mm Hg and continued the infusion for the duration of the experiment. We followed parameters for over 10 hrs after the initiation of P. aeruginosa in both groups. We measured stroke work, urine output, serum creatinine, among other parameters, and used serum troponin T as an index of myocardial injury. We found that in both protocols, ethyl gallate and norepinephrine improved mean arterial pressure and stroke work to similar extents over the duration of the study. Particularly in the late treatment protocol, ethyl gallate resulted in a lower heart rate, a lower troponin T, and a greater urine output as compared with norepinephrine (p < .05). Conclusions: These results suggest that phenolic antioxidants, such as ethyl gallate, that inhibit hydrogen peroxide signaling, may represent an alternative class of vasopressors for use in septic shock.

Lysozyme, a mediator of sepsis that deposits in the systemic vasculature and kidney as a possible mechanism of acute organ dysfunction.

ABSTRACT In septic shock (SS), dysfunction of many organ systems develops during the course of the illness, although the mechanisms are not clear. In earlier studies, we reported that lysozyme-c (Lzm-S), a protein that is released from leukocytes and macrophages, was a mediator of the myocardial depression and vasodilation that develop in a canine model of Pseudomonas aeruginosa SS. Whereas both of these effects of Lzm-S are dependent on its ability to intrinsically generate hydrogen peroxide, we subsequently showed that Lzm-S can also deposit within the vascular smooth muscle layer of the systemic arteries in this model. In the present study, we extend our previous findings. We used a canine carotid artery organ bath preparation to study the time course and dose dependence of Lzm-S deposition within the vascular smooth muscle layer. We used a human aortic vascular smooth muscle cell preparation to determine whether Lzm-S can persistently inhibit contraction in this preparation. We also used a canine P. aeruginosa model to determine whether Lzm-S deposition might occur in other organs such as the kidney, liver, and small intestine. The results showed that, in the carotid artery organ bath preparation, Lzm-S deposition occurred within minutes of instillation and there was a dose-response effect. In the human aortic vascular smooth muscle cell preparation, Lzm-S inhibited contraction during a 4-day period. In the in vivo model, Lzm-S accumulated in the kidney and the superior mesenteric artery. In a canine renal epithelial preparation, we further showed that Lzm-S can be taken up by the renal tubules to activate inflammatory pathways. We conclude that Lzm-S can deposit in the systemic vasculature and kidneys in SS, where this deposition could lead to acute organ dysfunction.