Gentisic acid sodium salt, a phenolic compound, is superior to norepinephrine in reversing cardiovascular collapse, hepatic mitochondrial dysfunction and lactic acidemia in Pseudomonas aeruginosa septic shock in dogs.

BACKGROUND: The development of lactic acidemia (LA) in septic shock (SS) is associated with an ominous prognosis. We previously showed that the mechanism of LA in SS may relate to impaired hepatic uptake of lactate, but the mechanism was not clear. Uptake of lactate by the liver occurs by a membrane-associated, pH-dependent, antiport system known as the monocarboxylate transporter. In the hepatocyte, lactate can then be metabolized by oxidative phosphorylation or converted to glucose in the cytosol. In the present study, we examined (1) whether hepatic mitochondrial dysfunction accounted for decreased uptake of lactate in a canine model of Pseudomonas aeruginosa SS, (2) whether norepinephrine (NE) treatment by increasing mean arterial pressure (MAP) could improve mitochondrial dysfunction and LA in this model, and (3) whether gentisic acid sodium salt (GSS), a novel phenolic compound, was superior to NE in these effects. METHODS: In anesthetized/ventilated dogs, we infused the bacteria over ~10 h and measured hemodynamics in various treatment groups (see below). We then euthanized the animal and isolated the hepatic mitochondria. We measured hepatic mitochondrial oxygen consumption rates using the novel Seahorse XF24 analyzer under conditions that included: basal respiration, after the addition of adenosine- diphosphate to produce coupled respiration, and after the addition of a protonophore to produce maximal respiration. RESULTS: We found that in the septic control group, mean arterial pressure decreased over the course of the study, and that mitochondrial dysfunction developed in which there was a reduction in maximal respiration. Whereas both NE and GSS treatments reversed the reduction in mean arterial pressure and increased maximal respiration to similar extents in respective groups, only in the GSS group was there a reduction in LA. CONCLUSIONS: Hepatic mitochondrial dysfunction occurs in SS, but does not appear to be required for the development of LA in SS, since NE improved mitochondrial dysfunction without reversing LA. GSS, a phenolic compound restored mean arterial pressure, mitochondrial dysfunction, and LA in SS. This reduction in LA may be independent of its effect on improving hepatic mitochondrial function.

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

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.

Mechanisms of systemic vasodilation by lysozyme-c in septic shock.

In septic shock (SS), cardiovascular collapse is caused by the release of inflammatory mediators. We previously found that lysozyme-c (Lzm-S), released from leukocytes, contributed to systemic vasodilation in a canine model of SS. We then delineated the pathway by which this occurs in a canine carotid artery organ bath preparation (CAP). We showed that Lzm-S could intrinsically generate hydrogen peroxide (H(2)O(2)) and that H(2)O(2) subsequently reacted with endogenous catalase to form compound I, an oxidized form of catalase. In turn, compound I led to an increase in cyclic guanosine 3',5'-monophosphate to produce vasodilation. However, it was not clear from previous studies whether it is necessary for Lzm-S to bind to the vasculature to cause vasodilation or, alternatively, whether the generation of H(2)O(2) by Lzm-S in the surrounding medium is all that is required. We examined this question in the present study in which we used multiple preparations. In a partitioned CAP, we found that when we added Lzm-S to a partitioned space in which a semipermeable membrane prevented diffusion of Lzm-S to the carotid artery tissue, vasodilation still occurred because of diffusion of H(2)O(2). On the other hand, we found that Lzm-S could accumulate within the vascular smooth muscle layer (VSML) after 7 h of SS in a canine model. We also determined that when Lzm-S was located in close proximity to vascular smooth muscle cells, it could generate H(2)O(2) to produce lengthening in a human cell culture preparation. We conclude that there are two mechanisms by which Lzm-S can cause vasodilation in SS. In one instance, H(2)O(2) generated by Lzm-S in plasma diffuses to the VSML to cause vasodilation. In a second mechanism, Lzm-S directly binds to the VSML, where it generates H(2)O(2) to produce vasodilation.

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.

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-S's 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.

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.

N,N'-diacetylchitobiose, an inhibitor of lysozyme, reverses myocardial depression and lessens norepinephrine requirements in Escherichia coli sepsis in dogs.

Cardiovascular dysfunction in septic shock (SS) is ascribed to the release of inflammatory mediators. Norepinephrine (NE) is often administered to treat low MAP in SS. We recently found that lysozyme c (Lzm-S) released from leukocytes was a mediator of myocardial depression in an Escherichia coil model of SS in dogs. This effect can be blocked in an in vitro preparation by chitobiose, a competitive inhibitor of Lzm-S. In the present study, we examined whether chitobiose treatment can reverse myocardial depression and obviate NE requirements in two respective canine E. coli preparations. In a 6-h study, we administered chitobiose after 3.5 h of E. coli bacteremia and compared stroke work (SW) and MAP at 6 h with a sepsis control group. In a 12-h study, we determined whether chitobiose treatment can reduce the need for NE requirements during 12 h of bacteremia. In the latter study, either chitobiose or NE was given when MAP decreased approximately 20% from the presepsis value in respective groups. In anesthetized, mechanically ventilated dogs, we monitored hemodynamic parameters during continuous E. coli infusion. In the 6-h study, chitobiose improved SW and MAP at the 6-h period as compared with the nontreated sepsis group. In the 12-h study, SW and MAP increased after chitobiose without the necessity of NE administration. These results suggest that inhibitors of Lzm-S such as chitobiose may improve myocardial depression and reduce the need for NE requirements in SS.

Lysozyme, a mediator of sepsis, impairs the cardiac neural adrenergic response by nonendothelial release of NO and inhibitory G protein signaling.

We previously showed that lysozyme (Lzm-S), derived from leukocytes, caused myocardial depression in canine sepsis by binding to the endocardial endothelium to release nitric oxide (NO). NO then diffuses to adjacent myocytes to activate the cGMP pathway. In a canine right ventricular trabecular (RVT) preparation, Lzm-S also decreased the inotropic response to field stimulation (FSR) during which the sympathetic and parasympathetic nerves were simulated to measure the adrenergic response. In the present study, we determined whether the pathway by which Lzm-S decreased FSR was different from the pathway by which Lzm-S reduced steady-state (SS) contraction. Furthermore, we determined whether the decrease in FSR was due to a decrease in sympathetic stimulation or enhanced parasympathetic signaling. In the RVT preparation, we found that the inhibitory effect of Lzm-S on FSR was prevented by NO synthase (NOS) inhibitors. A cGMP inhibitor also blocked the depressant activity of Lzm-S. However, in contrast to the Lzm-S-induced decline in SS contraction, chemical removal of the endocardial endothelium by Triton X-100 to eliminate endothelial NO release did not prevent the decrease in FSR. An inhibitory G protein was involved in the effect of Lzm-S, since FSR could be restored by treatment with pertussis toxin. Atropine prevented the Lzm-S-induced decline in FSR, whereas beta(1)- and beta(2)-adrenoceptor function was not impaired by Lzm-S. These results indicate that the Lzm-S-induced decrease in FSR results from a nonendothelial release of NO. NO then acts through inhibitory G protein to enhance parasympathetic signaling.

Volume reduction surgery impairs immediate postoperative pulmonary function in canine emphysema.

RATIONALE: In severe pulmonary emphysema, lung volume reduction surgery (LVRS) improves pulmonary function over a 2-yr period in selected patients. However, the changes in lung function and maximal flow (Vmax) occurring immediately postoperatively are not clear and may contribute to the high morbidity observed. In the present study, we used a chronic canine model of upper lobe emphysema to address this question. METHODS: Bilateral upper lobe emphysema was produced by the intrabronchial administration of papain. Measurements were made before and immediately after LVRS was performed. A vacuum-assisted surgical system (VALR Surgical System; Spiration, Redmond, WA) that deploys a compression sleeve over portions of the disease tissue was used to produce LVRS. Changes in Vmax were interpreted in terms of the wave-speed theory of flow limitation in which a pressure sensor was placed into the airway to determine the site of limitation and intrabronchial pressures. RESULTS: In the emphysema group, total lung capacity postemphysema increased to approximately 20% above the preemphysema value, whereas Vmax was reduced as compared with a control group. After LVRS, tidal respiratory compliance and Vmax decreased, whereas lung elastic recoil and frictional resistance increased in both the emphysema and control groups as compared with presurgery. CONCLUSION: The acute effect of LVRS leads to an impairment in lung mechanical properties. These changes could contribute to ventilatory complications, including the difficulty of weaning patients from mechanical ventilation and the mortality observed from this procedure.