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Vascular endothelial cell dysfunction in septic shock
Abstract
Septic shock is reported as the most common cause of death in intensive care units. Worldwide data indicate a 30 to 60% mortality rate. This death rate has not really changed since the early 1970s despite improved antibiotic therapy, cardiovascular support, and various advances in the management and treatment of sepsis. Septic shock pathophysiology may be related to several altered blood vessel functions associated or not with obvious anatomical injury. Normally, endothelial cells act as a signaling sensor for blood messages to vascular smooth muscle cells. They play a pivotal role in the regulation of local vascular tone. Endothelial injury due to systemic inflammation and induction of the coagulation cascade has been well recognized in the pathophysiology of septic shock. A better understanding of endothelial cell abnormalities occurring during septic shock might prove to be a good way to optimize septic shock therapy.
... Abnormal endothelial-dependent vascular relaxation has been recognized in multiple sepsis conditions. Several investigations, including our own [17,60,61], have demonstrated attenuated acetylcholine-induced relaxation in vascular rings isolated from large arteries. Apart from anatomical injuries, such abnormalities observed in these vessels may result from several mechanisms: alteration in EC surface receptors; modified signal transduction pathways (receptor-ecNOS coupling); altered function and/or density of the ecNOS; changes in pathways that lead to release of NO; and/or changes in mechanisms that participate in subsequent degradation of NO. ...
... Taylor and coworkers concluded that sustained injury to the vascular endothelium secondary to reperfusion of the microvasculature occurred in those asymptomatic individuals. In our endotoxic model, we also demonstrated that at 5 days the rabbits had maximal monocyte TF expression, which coincided with maximal endothelial injury [6,17]. This, together with altered coagulation modulation properties, may ultimately result in intravascular microthrombosis. ...
... An important point is that EC injury is sustained over time. In an endotoxic rabbit model, we demonstrated that endothelium denudation is present at the level of the abdominal aorta as early as after several hours following injury and persisted for at least 5 days afterward [6,17]. After 21 days we observed that the endothelial surface had recovered. ...
During the past decade a unifying hypothesis has been developed to explain the vascular changes that occur in septic shock on the basis of the effect of inflammatory mediators on the vascular endothelium. The vascular endothelium plays a central role in the control of microvascular flow, and it has been proposed that widespread vascular endothelial activation, dysfunction and eventually injury occurs in septic shock, ultimately resulting in multiorgan failure. This has been characterized in various models of experimental septic shock. Now, direct and indirect evidence for endothelial cell alteration in humans during septic shock is emerging. The present review details recently published literature on this rapidly evolving topic.
The pathophysiology of acute illness and injury recognizes three main effectors: infection, trauma, and ischemia-reperfusion injury. Each of them can act by itself or in combination with the other two in developing a systemic inflammatory reaction syndrome (SIRS) that is a generalized reaction to the morbid event. The time course of SIRS is variable and influenced by the number and severity of subsequent insults (e.g., reparative surgery, acquired hospital infections). It occurs simultaneously with a complex of counter-regulatory mechanisms (compensatory anti-inflammatory response syndrome, CARS) that limit the aggressive effects of SIRS. In adjunct, a progressive dysfunction of the acquired (lymphocytes) immune system develops with increased risk for immunoparalysis and associated infectious complications. Both humoral and cellular effectors participate to the development of SIRS and CARS. The most important humoral mediators are pro-inflammatory (IL-1β, IL-6, IL-8, IL-12) and anti-inflammatory (IL-4, IL-10) cytokines and chemokines, complement, leukotrienes, and PAF. Effector cells include neutrophils, monocytes, macrophages, lymphocytes, and endothelial cells. The endothelium is a key factor for production of remote organ damage as it exerts potent chemo-attracting effects on inflammatory cells, allows for leukocyte trafficking into tissues and organs, and promotes further inflammation by cytokines release. Moreover, the loss of vasoregulatory properties and the increased permeability contribute to the development of hypotension and tissue edema. Finally, the disseminated activation of the coagulation cascade causes the widespread deposition of microthrombi with resulting maldistribution of capillary blood flow and ultimately hypoxic cellular damage. This mechanism together with increased vascular permeability and vasodilation is responsible for the development of the multiple organ dysfunction syndrome (MODS).
The vascular endothelium is a highly specialized tissue involved in modulating immune responses and vascular cell growth, and in regulating the level of hemostatic, inflammatory, and vasoactive agents in the blood (Table 1). Endothelial cells line vessels in every organ system and regulate the flow of nutrient substances, diverse biologically active molecules, and the blood cells themselves. This ‘gate-keeping’ role of the endothelium is effected through the presence of membrane-bound receptors for numerous molecules including proteins, lipid transporting particles, metabolites and hormones, as well as through specific junction proteins and receptors that govern cell-to-cell and cell-tomatrix interactions [1]. This feature explains why endothelium dysfunction and/or injury with subendothelium exposure facilitates leukocyte and platelet aggregation and aggravation of coagulopathy. Therefore, endothelial dysfunction and/or injury should favor impaired perfusion, tissue hypoxia and subsequent organ dysfunction.
The so-called immunopharmaconutrients are amino acids which display regulatory metabolic properties independently of the nitrogen they are bringing. Typically, glutamine, arginine and their common precursor, ornithine α-ketoglutarate, enter this category. These amino acids modulate protein turnover and are contributive to the gut trophicity and immune cell functionality. Besides a huge number of experimental and bioclinical studies, recent trials indicate that glutamine (in a free form or as dipeptides), given by parenteral route, and enterally administered arginine decrease the frequency of infection and the length of hospital stay in intensive care unit patients. Ornithine α-ketoglutarate exhibits a marked action on wound healing in burn patients and post-operative patients (head and neck cancer). The underlying mechanisms of action remain unclear. However, it is likely that the actions of these amino acids are mediated in part through their metabolism (into nitric oxide, glutathione, polyamines), through increased hormone secretions (insulin, growth hormone) and through cell swelling. This issue is complexified by the fact that glutamine, arginine and ornithine α-ketoglutarate are metabolically linked. The large splanchnic metabolism of glutamine explains the low efficacy of this amino acid when given enterally and perhaps when used in elderly patients. At the turn of the millenium, it makes clear that providing a glutamine-, arginine- or ornithine α-ketoglutarate-enriched diet is indicated in subgroups of catabolic patients. However, there are few data allowing to choose one of these molecules rather than the others. Don't forget that we are speaking of pharmacology: any random mixture of these molecules must be avoided, every new product must be carefully validated comparing its efficacy to the appropriate isonitrogenous control.
Un acide aminé à propriétés immuno- et pharmacomodulatrices est une molécule qui exerce des propriétésmétaboliques régulatrices indépendamment de l'apport azoté qu'elle réalise. Typiquement, il s'agit de la glutamine, de l'arginine et de leur précurseur commun l'α-cétogluta rate d'ornithine. Ces acides aminés modulent le turn over protéique, contribuent à la trophicité intestinale et à la fonctionnalité immunitaire. À côté de très nombreux travaux expérimentaux ou biocliniques, des études cliniques récentes indiquent que la glutamine (libre ou sous forme de dipeptide), par voie parentérale, et l'arginine, par voie entérale, diminuent la prévalence des infections et la durée d'hospitalisation chez le patient de réanimation. Pour sa part, l'α-cétoglutarate d'ornithine présente une action particulièrement marquée sur la cicatrisation des escarres, des brûlés et en chirurgie carcinologique ORL. Le mécanisme d'action des pharmaconutriments azotés est imparfaitement compris. II est cependant vraisemblable que leur action découle de leur métabolisme (monoxyde d'azote, glutathion, polyamines), d'effets secrétagogues (insuline, hormone de croissance) et de variations du volume cellulaire. Le problème est compliqué par le fait que la glutamine, l'arginine et l'α-cétoglutarate d'ornithine sont métaboliquement liés. L'importance du métabolisme splanchnique de la glutamine explique son peu d'efficacité lorsqu'elle est administrée par voie entérale et, peut-être, chez le sujet âgé. Il est maintenant clair que l'administration de nutrition enrichie en glutamine, en arginine ou en alphacétoglutarate d'ornithine est indiquée dans des groupes sélectionnés de patients. II n'existe cependant que très peu de données qui permette de préférer l'un de ces pharmaconutriments aux deux autres.
Endothelial cells can be the prime target for an infection and infected endothelial cells may serve as an initiating system for a systemic response as these cells are able to secrete many mediators known to be of paramount importance. Endothelial cell functions in turn are regulated by these circulating mediators. Cellular interactions with leukocytes revealed protective and destructive functions. Single cell and animal studies indicate that endothelial permeability is increased and apart from clinical obvious edema formation in septic patients, the endothelial component remains unknown. Endothelial coagulation activation has been shown in vitro, however human data supporting an endothelial procoagulatory state are lacking. Defects in endothelium dependent vasoregulation in animal models are well known and again human studies are largely missing. An imbalanced production of reactive oxygen species including nitric oxide has been found to be involved in all endothelial functions and may provide a common link which at present can be supported only in animal studies.
The so-called immunopharmaconutrients are amino acids which display regulatory metabolic properties independently of the nitrogen they are bringing. Typically, glutamine, arginine and their common precursor, ornithine α-ketoglutarate enter this category. These amino acids modulate protein turn over and are contributive to the gut trophicity and immune cell functionality. Besides a huge number of experimental and bioclinical studies, recent trials indicate that glutamine (in a free form or as dipeptides), given by parenteral route, and enterally administered arginine, decrease the frequency of infection and the stay at the hospital in intensive care unit patients. Ornithine αketoglutarate exhibits a marked action on wound healing in burn patients and post-operative patients (head and neck cancer). The underlying mechanisms of action remain unclear. However, it is likely that the actions of these amino acids are mediated in part through their metabolism (into nitric oxide, glutathione, polyamines) through increased hormone secretions (insulin, growth hormone) and through cell swelling. This issue is complexified by the fact that glutamine, arginine and ornithine α-ketoglutarate are metabolically linked. The large splanchnic metabolism of glutamine explains the low efficacy of this amino acid when given enterally and also when used in elderly patients. At the turn of the millenium, it makes clear that providing a glutamine-, arginine- or ornithine α-ketoglutarate-enriched diets is indicated in subgroups of catabolic patients. However, there are few data allowing to choose one of these molecules rather than the others. Don't forget that we are speaking pharmacology: avoiding any mixture of these molecules at random, every new product must be carefully validated comparing its efficacy to the appropriate isonitrogenous control.
Vascular endothelial cells influence microvessel diameters in vivo and in vitro and participate in host-defense mechanisms during sepsis. We examined whether small arteriole dilation in skeletal muscle during high cardiac output bacteremia (HOB) and low cardiac output live Escherichia coli sepsis (LOS) is mediated by an endothelium-derived relaxing factor (EDRF). Local chemical blockade of EDRF by hydroquinone (HQ) substantially blunted acetylcholine-induced dilation of small arterioles. HQ also prevented large arteriole (55-135 microns) constriction and small arteriole (6-22 microns) dilation in the cremaster muscle of rats during HOB. In LOS, small arteriole dilation was also prevented by HQ but only during the early period when blood pressure was unchanged from baseline. HQ did not alter large arteriole constriction during LOS. We conclude that small arteriole vasodilation in skeletal muscle is mediated at least in part by EDRF during bacteremia. Because EDRF cannot mediate large arteriole constriction and because HQ blunted large arteriole constriction during HOB, we now suspect that HQ also interferes at least in part with some large arteriole vasoconstrictor mechanism, possibly leukotrienes or an endothelium-derived constricting factor, which mediates large arteriole constriction during HOB. Our data also suggest that large arteriole constriction during LOS is partly mediated by factors that are unaffected by HQ. The endothelium appears to play an important role in the microcirculatory responses of skeletal muscle to live E. coli sepsis through more than one mechanism.
The effect of arginine (Arg) was studied on norepinephrine- (1 microM) precontracted small mesenteric arteries removed from rats treated with E. coli lipopolysaccharide (LPS). The addition of L- (but not D-) Arg (1 mM) relaxed, within 3 min, the small mesenteric arteries from LPS-treated but not from control rats, the maximal relaxation (65.3 +/- 11%) being reached with less than 100 microM L-Arg. NG-Nitro-L-arginine methyl ester (1 mM) and methylene blue (10 microM) restored contractions to the level reached before addition of L-Arg. These results show that LPS induces the production of an L-Arg-derived, nitric oxide-like, relaxing factor in small mesenteric arteries.
The coronary vasoconstriction induced by the thromboxane mimetic U46619 (9, 11 dideoxy methanoepoxy 9 alpha, 11 alpha prostaglandin F2 alpha, 3-30 nM) was significantly attenuated in hearts obtained from rabbits treated with endotoxin (lipopolysaccharide, LPS, 200 micrograms kg-1, i.v.) 4 h before isolation of the heart. Under these conditions the vasoconstriction induced by two inhibitors of nitric oxide (NO) synthase, NG-monomethyl-L-arginine (L-NMMA) and N-iminoethyl-L-ornithine (L-NIO) (1-100 microM for each) was significantly enhanced when compared to that induced in hearts from control animals. Both the decreased response to U46619 and the increased response to inhibitors of NO synthase were significantly attenuated by administration of dexamethasone (4 mg kg-1, i.v.) 90 min before treatment with LPS. These data are consistent with the induction, by LPS, of an NO synthase, and the inhibition of this induction by dexamethasone. The enhanced NO synthesis contributes to the haemodynamic changes known to occur in endotoxin shock.
Endotoxic shock is characterized by a variety of hemodynamic disturbances which result in tissue hypoperfusion. There is some evidence for endothelial damage caused by endotoxin. The present study addressed the hypothesis that vascular responsiveness to endothelial-dependent vasodilators is altered in endotoxic shock. Dose-response relationships for an endothelial-dependent vasodilator, acetylcholine, and an endothelial-independent vasodilator, adenosine, were determined in guinea pig aortic rings. Rings were examined from either control (untreated) animals or from animals given Escherichia coli endotoxin (4 mg/kg, i.p.) 16 hr prior to functional studies. Dose-response relationships to adenosine were similar in aortic rings from control and shocked animals. However, response to acetylcholine were attenuated by 30% (P less than .05) in the shocked group. To distinguish between a direct, acute effect of endotoxin versus effects produced by systemic changes that occur during shock, rings were isolated from untreated animals and incubated with endotoxin in vitro for 30 min prior to and during dose-response measurements. Incubation with endotoxin caused no change in aortic responses to adenosine or acetylcholine. Electron microscopy revealed a separation of the endothelium from the internal elastic lamina and an increase in inter-endothelial gaps in rings isolated from shocked animals. These structural changes were not observed in rings from untreated animals or in rings incubated with endotoxin in vitro. We conclude that endothelial-dependent vasodilation is attenuated during endotoxic shock. The functional changes are correlated with ultrastructural alterations of the endothelium.
The aortic endothelium from control and Escherichia coli (E. coli) endotoxin-treated rats and rabbits was examined by transmission and scanning electron microscopy. Following the intravenous injection of endotoxin, the animals were sacrificed at intervals ranging from 1 min to 4 hr. As early as 1 min after endotoxin, there was a widening of the subendothelial space (SES) and an increase in tortuosity of the internal elastic lamina (IEL). At 5 min, the tortuosity of the IEL increased to a peak value, and the SES showed an increase in the amount of smooth muscle cells (SMC). Initial endothelial damage occurred 5 min after endotoxin: SEM showed some spindle-shaped endothelial cells starting to peel from the underlying SES, and TEM showed some endothelial cells protruding or arching into the lumen. The new findings in this study are that endotoxin injection a) has a very rapid (less than 15 min) effect on rat and rabbit aortic endothelium, including localized endothelial injuries in the intima, and b) induces ultrastructural alterations also in the SES, IEL and portions of the tunica media. These effects were largely reversed within 1 hr after endotoxin administration, thus indicating that the endothelium and other components of the arterial wall can recover with great speeds.
Disseminated intravascular coagulation is a frequent finding in critically ill patients, and may be diagnosed in the majority of patients with Gram-negative sepsis. Tissue damage may result from intravascular thrombosis, and disseminated intravascular coagulation is an underestimated causal factor in the pathogenesis of organ failure in sepsis. The diagnosis of disseminated intravascular coagulation is difficult, as the initial coagulation process that leads to thrombosis is counteracted by fibrinolytic responses, that in the context of ongoing consumption of clotting factors may result in an overwhelming bleeding tendency. Hence, depending on underlying disease, and the time at which the patient is evaluated, different laboratory results may be obtained. The treatment of disseminated intravascular coagulation is even more challenging than its diagnosis. No well-designed, controlled clinical trials exist that form a basis for rational treatment decisions. Treatment frequently needs to be individualized, and rapid adjustments may be necessitated by the course of the disease. Nonetheless, we believe that recent insights in the pathophysiology of disseminated intravascular coagulation, in particular concerning the role of the extrinsic coagulation pathway, provide ground for some optimism concerning future therapeutic options.
Although vascular endothelial cell function (i.e., the release of endothelium-derived nitric oxide) decreases and plasma tumor necrosis factor (TNF) increases during sepsis, it is not known whether the elevated TNF is responsible for the depression of endothelial cell function under such conditions. The aim of this study, therefore, was to determine if inhibition of TNF biologic activity by polyethylene glycol dimerized conjugate of the recombinant human form of the p55 soluble TNF receptor (PEG-(rsTNF-R1)2) maintains endothelial function during sepsis.
Rats were subjected to sepsis by cecal ligation and puncture (CLP). Immediately before the onset of sepsis, 600 microgram/rat PEG-(rsTNF-R1)2 or an equal volume of saline was infused intravenously. At 10 hours after CLP (i.e., hyperdynamic sepsis), the thoracic aorta was isolated, cut into rings, and placed in organ chambers. Dose responses for an endothelium-dependent vasodilator, acetylcholine (ACh), and an endothelium-independent vasodilator, nitroglycerine (NTG), were determined. Endothelial cell structure was examined by transmission electron microscopy.
Endothelium-dependent vascular relaxation was depressed at 10 hours after the onset of sepsis. Administration of PEG-(rsTNF-R1)2 before CLP, however, maintained ACh-induced relaxation. In contrast, no significant difference in NTG-induced relaxation was seen, irrespective of administration of PEG-(rsTNF-R1)2 Furthermore, the deterioration in endothelial structure during sepsis was prevented by PEG-(rsTNF-R1)2 pretreatment.
Since administration of PEG-(rsTNF-R1)2 maintains vascular endothelial cell structure and function, it can be concluded that TNF plays a pivotal role in producing endothelial dysfunction during sepsis. Thus, pharmacologic agents that inhibit TNF biologic activity and/or its production may be useful for protecting endothelial cells during sepsis.
Angiotensin-converting enzyme (ACE) inhibitors reduce intimal hyperplasia after balloon injury. A role for nitric oxide (NO) has been suggested in this effect. Because recent data suggest that NO may modulate some features of endothelial cells and because endothelial cells are involved in the control of intimal hyperplasia, we investigated the role of NO synthesis in the effect of an ACE inhibitor, perindopril, on neoendothelial dysfunction and intimal hyperplasia in a rabbit model of unilateral iliac balloon injury. New Zealand White male rabbits received placebo, perindopril, or cotreatment with perindopril and NG-nitro-L-arginine methyl ester (L-NAME) and were evaluated 4 wk after the injury. Fifteen rabbits (5 in each group) were used to assess in vitro vasoreactivity and twenty-four (8 in each group) for morphometric analysis. In injured vessels, neoendothelium-dependent relaxation in ACE inhibitor-treated animals was improved compared with placebo (P < 0.05) and restored to the level of noninjured vessels (NS). The improvement observed with ACE inhibitor was abolished by cotreatment with L-NAME (P < 0.05). In the same vessels, no effect was observed on neoendothelium-independent vasoreactivity. The improved neoendothelial dysfunction with ACE inhibitor was associated with a 66% reduction in intimal thickening (P < 0.01). The effect was also reversed by cotreatment with L-NAME (P < 0.01). In noninjured vessels, treatment did not alter vasoreactivity or morphology of the vessel wall. These results suggest that NO synthesis may play a key role in the improvement of vascular function seen with ACE inhibitor in balloon-injured vessels.