RESUMEN
BACKGROUND: Despite many animal studies and clinical trials, mortality in sepsis remains high. This may be due to the fact that most experimental studies of sepsis employ young animals, whereas the majority of septic patients are elderly (60 - 70 years). The objective of the present study was to examine the sepsis-induced inflammatory and pro-coagulant responses in aged mice. Since running exercise protects against a variety of diseases, we also examined the effect of voluntary running on septic responses in aged mice. METHODS: Male C57BL/6 mice were housed in our institute from 2-3 to 22 months (an age mimicking that of the elderly). Mice were prevented from becoming obese by food restriction (given 70-90% of ad libitum consumption amount). Between 20 and 22 months, a subgroup of mice ran voluntarily on wheels, alternating 1-3 days of running with 1-2 days of rest. At 22 months, mice were intraperitoneally injected with sterile saline (control) or 3.75 g/kg fecal slurry (septic). At 7 h post injection, we examined (1) neutrophil influx in the lung and liver by measuring myeloperoxidase and/or neutrophil elastase in the tissue homogenates by spectrophotometry, (2) interleukin 6 (IL6) and KC in the lung lavage by ELISA, (3) pulmonary surfactant function by measuring percentage of large aggregates, (4) capillary plugging (pro-coagulant response) in skeletal muscle by intravital microscopy, (5) endothelial nitric oxide synthase (eNOS) protein in skeletal muscle (eNOS-derived NO is putative inhibitor of capillary plugging) by immunoblotting, and (6) systemic blood platelet counts by hemocytometry. RESULTS: Sepsis caused high levels of pulmonary myeloperoxidase, elastase, IL6, KC, liver myeloperoxidase, and capillary plugging. Sepsis also caused low levels of surfactant function and platelet counts. Running exercise increased eNOS protein and attenuated the septic responses. CONCLUSIONS: Voluntary running protects against exacerbated sepsis-induced inflammatory and pro-coagulant responses in aged mice. Protection against pro-coagulant responses may involve eNOS upregulation. The present discovery in aged mice calls for clinical investigation into potential beneficial effects of exercise on septic outcomes in the elderly.
Asunto(s)
Carrera/fisiología , Sepsis/fisiopatología , Envejecimiento/metabolismo , Envejecimiento/fisiología , Análisis de Varianza , Animales , Ensayo de Inmunoadsorción Enzimática/métodos , Interleucina-6/análisis , Interleucina-6/sangre , Elastasa de Leucocito/análisis , Elastasa de Leucocito/sangre , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/análisis , Peroxidasa/sangreRESUMEN
BACKGROUND: We discovered that lipopolysaccharide (LPS, an initiating factor in sepsis) and hypoxia-reoxygenation (H/R, a confounding factor) reduce electrical coupling between microvascular endothelial cells from wild-type (WT) but not Cx40-/- mice. Because Cx40 knockout could result in nonspecific effects, this discovery may not establish the causal relationship between Cx40 and reduced coupling. Using the same cell culture model, we aimed to address this uncertainty by using the rescue-of-function approach. METHODS/RESULTS: Electrical coupling between endothelial cells (hind-limb muscle origin) was determined by electrophysiology. LPS, H/R and concurrent LPS + H/R reduced coupling between WT but not Cx40-/- cells. The defect in Cx40-/- cells was rescued by ectopic expression of Cx40, after infecting the cells with adenovirus encoding Cx40. Cx40-/- cells were also engineered to express mutant Cx40 that lacked the carboxyl terminal domain beginning at residue 236 (Cx40x0394;237-358) or 344 (Cx40x0394;345-358). No response to inflammatory stimuli was observed in cells expressing either of these 2 mutants. CONCLUSION: Our data establish the causal relationship between Cx40 and reduced coupling and suggest that the 345-358 amino acid motif of the Cx40 carboxyl terminal is required for reduced coupling. Cx40 may participate in compromised conducted response in the microvasculature during sepsis.
Asunto(s)
Conexinas/metabolismo , Células Endoteliales/efectos de los fármacos , Inflamación/metabolismo , Lipopolisacáridos/farmacología , Microvasos/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Oxígeno/metabolismo , Sepsis/metabolismo , Animales , Hipoxia de la Célula , Forma de la Célula/efectos de los fármacos , Conexinas/deficiencia , Conexinas/genética , Conductividad Eléctrica , Células Endoteliales/metabolismo , Células Endoteliales/patología , Genotipo , Células HEK293 , Miembro Posterior , Humanos , Inflamación/genética , Inflamación/patología , Ratones Noqueados , Microvasos/metabolismo , Microvasos/patología , Mutación , Fenotipo , Dominios Proteicos , Sepsis/genética , Sepsis/patología , Transducción Genética , Transfección , Proteína alfa-5 de Unión ComunicanteRESUMEN
BACKGROUND: Sepsis, a potential risk associated with surgery, leads to a systemic inflammatory response including the plugging of capillary beds. This plugging may precipitate organ failure and subsequent death. We have shown that capillary plugging can be reversed rapidly within 1 h by intravenous injection of ascorbate in mouse skeletal muscle. It is unknown whether, in parallel with this effect, ascorbate negatively affects the protective responses to sepsis involving the fibrinolytic and immune systems. We hypothesized that treatment with ascorbate for 1 h does not alter bacterial content, plasminogen activator inhibitor 1 (PAI-1), and neutrophil infiltration in lung, kidney, spleen, and liver (organs with high immune response) of septic mice. MATERIALS AND METHODS: Sepsis was induced by feces injection into the peritoneum. Mice were injected intravenously with ascorbate at 6 h (10 mg/kg), and samples of peritoneal fluid, arterial blood, and organs collected at 7 h were subjected to analyses of bacterial content, PAI-1 messenger RNA and enzymatic activity, and myeloperoxidase (MPO) (a measure of neutrophil infiltration). RESULTS: Sepsis increased bacterial content in all fluids and organs and increased PAI-1 messenger RNA and enzymatic activity in the lung and liver. Sepsis increased the myeloperoxidase level in the lung and liver, and lowered it in the spleen. Except for decreasing the bacterial content in blood, these responses to sepsis were not altered by ascorbate. CONCLUSIONS: The rapid effect of ascorbate against capillary plugging in the septic mouse skeletal muscle is not accompanied by alterations in PAI-1 or myeloperoxidase responses in the organs with high immune response.
Asunto(s)
Ácido Ascórbico/farmacología , Bacterias/aislamiento & purificación , Peroxidasa/metabolismo , Inhibidor 1 de Activador Plasminogénico/genética , Sepsis/tratamiento farmacológico , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Infiltración Neutrófila , ARN Mensajero/análisis , Sepsis/inmunología , Sepsis/microbiologíaRESUMEN
OBJECTIVE: Compromised perfusion of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous injection of ascorbate inhibits platelet adhesion and plugging in septic capillaries. In this study, we hypothesized that ascorbate reduces aggregation of platelets and their surface expression of P-selectin (a key adhesion molecule) in mice. METHODS: Platelets were isolated from control mice and subjected to agents known to be released into the bloodstream during sepsis (thrombin, ADP or U46619, thromboxane A2 analog). Platelet aggregation was analyzed by aggregometry and P-selectin expression by flow cytometry. RESULTS: Platelet-activating agents increased aggregation and P-selectin expression. Ascorbate inhibited these increases. This inhibitory effect was NOS-independent (LNAME had no effect). In contrast to the platelet-activating agents, direct stimuli lipopolysaccharide, TNFα, or plasma from septic mice did not increase aggregation/expression, a finding consistent with the literature. The results suggest a complex mechanism of platelet aggregation and P-selectin expression in sepsis, where generation of platelet-activating stimuli is required first, before platelet aggregation and adhesion in capillaries occur. CONCLUSION: The ability of ascorbate to reduce platelet aggregation and P-selectin expression could be an important mechanism by which ascorbate inhibits capillary plugging in sepsis.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Modelos Biológicos , Selectina-P/biosíntesis , Agregación Plaquetaria/efectos de los fármacos , Sepsis/metabolismo , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Mediadores de Inflamación/metabolismo , Ratones , Óxido Nítrico/metabolismoRESUMEN
Plugging of the capillary bed can lead to organ failure and mortality in sepsis. We have reported that intravenous ascorbate injection reduces platelet adhesion to the capillary wall and capillary plugging in septic mice. Both platelet adhesion and capillary plugging require P-selectin, a key adhesion molecule. To elucidate the beneficial effect of ascorbate, we hypothesized that ascorbate reduces platelet-endothelial adhesion by reducing P-selectin surface expression in endothelial cells. We used mouse platelets, and monolayers of cultured microvascular endothelial cells (mouse skeletal muscle origin) stimulated with lipopolysaccharide, to examine platelet-endothelial adhesion. P-selectin mRNA expression in endothelial cells was determined by real-time PCR and P-selectin protein expression at the surface of these cells by immunofluorescence. Secretion of von Willebrand factor from cells into the supernatant (a measure of P-selectin-containing granule exocytosis) was determined by ELISA. Lipopolysaccharide (10âµg/ml, 1âh) increased platelet-endothelial adhesion. P-selectin-blocking antibody inhibited this adhesion. Lipopolysaccharide also increased P-selectin mRNA in endothelial cells, P-selectin expression at the endothelial surface, and von Willebrand factor secretion. Ascorbate pretreatment (100âµmol/l, 4âh) inhibited the increased platelet adhesion, surface expression of P-selectin, and von Willebrand factor secretion, but not the increase in P-selectin mRNA. The lipopolysaccharide-induced increase in platelet-endothelial adhesion requires P-selectin presence at the endothelial surface. Ascorbate's ability to reduce this presence could be important in reducing both platelet adhesion to the capillary wall and capillary plugging in sepsis.
Asunto(s)
Ácido Ascórbico/farmacología , Células Endoteliales/metabolismo , Selectina-P/metabolismo , Sepsis/metabolismo , Animales , Humanos , Ratones , Ratones Endogámicos C57BL , Adhesividad PlaquetariaRESUMEN
The microcirculation during sepsis fails due to capillary plugging involving microthrombosis. We demonstrated that intravenous injection of ascorbate reduces this plugging, but the mechanism of this beneficial effect remains unclear. We hypothesize that ascorbate inhibits the release of the antifibrinolytic plasminogen activator inhibitor-1 (PAI-1) from endothelial cells and platelets during sepsis. Microvascular endothelial cells and platelets were isolated from mice. Cells were cultured and stimulated with lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFα), or thrombin (agents of sepsis), with/without ascorbate for 1-24âh. PAI-1 mRNA was determined by quantitative PCR. PAI-1 protein release into the culture medium was measured by ELISA. In platelets, PAI-1 release was measured after LPS, TNFα, or thrombin stimulation, with/without ascorbate. In endothelial cells, LPS and TNFα increased PAI-1 mRNA after 6-24âh, but no increase in PAI-1 release was observed; ascorbate did not affect these responses. In platelets, thrombin, but not LPS or TNFα, increased PAI-1 release; ascorbate inhibited this increase at low extracellular pH. In unstimulated endothelial cells and platelets, PAI-1 is released into the extracellular space. Thrombin increases this release from platelets; ascorbate inhibits it pH-dependently. The data suggest that ascorbate promotes fibrinolysis in the microvasculature under acidotic conditions in sepsis.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Plaquetas/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Inhibidor 1 de Activador Plasminogénico/genética , Sepsis/tratamiento farmacológico , Animales , Plaquetas/inmunología , Plaquetas/metabolismo , Células Cultivadas , Células Endoteliales/inmunología , Células Endoteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Inhibidor 1 de Activador Plasminogénico/inmunología , ARN Mensajero/genética , Sepsis/genética , Sepsis/inmunologíaRESUMEN
Plugging of the capillary bed in tissues correlates with organ failure during sepsis. In septic mouse skeletal muscle, we showed that blood in capillaries becomes hypercoagulable and that ascorbate injection inhibits capillary plugging. In the present study, we hypothesized that ascorbate promotes fibrinolysis, reversing this plugging. Sepsis in mice was induced by fecal injection into peritoneum. Mice were injected intravenously with a bolus of streptokinase (fibrinolytic agent) or ascorbate at 5-6âh. Both agents reversed capillary plugging in muscle at 7âh. Sepsis increased mRNA expression of urokinase plasminogen activator (u-PA) (profibrinolytic) and plasminogen activator inhibitor 1 (PAI-1) (antifibrinolytic) in muscle and liver homogenates at 7âh. Ascorbate did not affect u-PA mRNA in either tissue, but it inhibited PAI-1 mRNA in muscle, suggesting enhanced fibrinolysis in this tissue. However, ascorbate did not affect increased PAI-1 mRNA in the liver (dominant source of soluble PAI-1 in systemic blood). Consistently, ascorbate affected neither elevated PAI-1âprotein/enzymatic activity in septic liver nor lowered plasmin antiplasmin level in septic blood. Furthermore, hypocoagulability of septic blood revealed by thrombelastography and thrombin-induced PAI-1 release from isolated platelets (ex-vivo model of sepsis) were not affected by ascorbate. Based on the PAI-1 protein data, the present study does not support the hypothesis that ascorbate promotes fibrinolysis in sepsis.
Asunto(s)
Ácido Ascórbico/farmacología , Fibrinólisis/efectos de los fármacos , Músculo Esquelético/irrigación sanguínea , Músculo Esquelético/efectos de los fármacos , Sepsis/sangre , Animales , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Sepsis/patología , Serpina E2/genética , Serpina E2/metabolismo , Tromboelastografía/métodos , Activador de Tejido Plasminógeno/genética , Activador de Tejido Plasminógeno/metabolismo , Activador de Plasminógeno de Tipo Uroquinasa/genética , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
PURPOSE: Impaired microvascular perfusion in sepsis is not treated effectively because its mechanism is unknown. Since inflammatory and coagulation pathways cross-activate, we tested if stoppage of blood flow in septic capillaries is due to oxidant-dependent adhesion of platelets in these microvessels. METHODS: Sepsis was induced in wild type, eNOS(-/-), iNOS(-/-), and gp91phox(-/-) mice (n = 14-199) by injection of feces into the peritoneum. Platelet adhesion, fibrin deposition, and blood flow stoppage in capillaries of hindlimb skeletal muscle were assessed by intravital microscopy. Prophylactic treatments at the onset of sepsis were intravenous injection of platelet-depleting antibody, P-selectin blocking antibody, ascorbate, or antithrombin. Therapeutic treatments (delayed until 6 h) were injection of ascorbate or the glycoprotein IIb/IIIa inhibitor eptifibatide, or local superfusion of the muscle with NOS cofactor tetrahydrobiopterin or NO donor S-nitroso-N-acetylpenicillamine (SNAP). RESULTS: Sepsis at 6-7 h markedly increased the number of stopped-flow capillaries and the occurrence of platelet adhesion and fibrin deposition in these capillaries. Platelet depletion, iNOS and gp91phox deficiencies, P-selectin blockade, antithrombin, or prophylactic ascorbate prevented, whereas delayed ascorbate, eptifibatide, tetrahydrobiopterin, or SNAP reversed, septic platelet adhesion and/or flow stoppage. The reversals by ascorbate and tetrahydrobiopterin were absent in eNOS(-/-) mice. Platelet adhesion predicted 90% of capillary flow stoppage. CONCLUSION: Impaired perfusion and/or platelet adhesion in septic capillaries requires NADPH oxidase, iNOS, P-selectin, and activated coagulation, and is inhibited by intravenous administration of ascorbate and by local superfusion of tetrahydrobiopterin and NO. Reversal of flow stoppage by ascorbate and tetrahydrobiopterin may depend on local eNOS-derived NO which dislodges platelets from the capillary wall.