Vasoactive hormone adrenomedullin and its binding protein: anti-inflammatory effects by up-regulating peroxisome proliferator-activated receptor-gamma.

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-gamma (PPAR-gamma) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-gamma expression in vivo and in vitro and was associated with increased TNF-alpha production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-gamma levels in both models, resulting in TNF-alpha suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-alpha production in the absence of PPAR-gamma. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced TauNuF-alpha release, it did not alter PPAR-gamma expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-gamma and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-gamma.

Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve.

OBJECTIVE: To test the hypothesis that administration of ghrelin attenuates inflammatory responses in sepsis through vagal nerve stimulation. SUMMARY BACKGROUND DATA: Ghrelin has been demonstrated to possess multiple functions, including stimulation of the vagus nerve. Our recent study has shown that plasma levels of ghrelin were significantly reduced in sepsis; and ghrelin administration improved organ perfusion and function. However, it remained unknown whether ghrelin also decreases proinflammatory cytokines in sepsis and, if so, whether the down-regulatory effect of ghrelin is mediated by activation of the vagus nerve. METHODS: Male rats were subjected to sepsis by cecal ligation and puncture (CLP). At 5 hours after CLP, a bolus intravenous injection of 2 nmol ghrelin was followed by a continuous infusion of 12 nmol ghrelin via a primed 200-microL Alzet mini-pump for 15 hours. At 20 hours after CLP, plasma and peritoneal fluid levels of TNF-alpha and IL-6 were determined. The direct effect of ghrelin on cytokine production was studied using cultured normal rat Kupffer cells or peritoneal macrophages stimulated by lipopolysaccharide (LPS). In additional animals, vagotomy or sham vagotomy was performed in sham and septic animals immediately prior to ghrelin administration and cytokine levels were then measured. RESULTS: Ghrelin significantly reduced TNF-alpha and IL-6 levels in sepsis. In contrast, ghrelin did not inhibit TNF-alpha and IL-6 release from LPS-stimulated Kupffer cells or peritoneal macrophages. However, vagotomy, but not sham vagotomy, prevented ghrelin's down-regulatory effect on TNF-alpha and IL-6 production. CONCLUSIONS: Ghrelin down-regulates proinflammatory cytokines in sepsis through activation of the vagus nerve. Pharmacologic stimulation of the vagus nerve may offer a novel approach of anti-sepsis therapy.

Adrenomedullin and adrenomedullin-binding protein-1 downregulate inflammatory cytokines and attenuate tissue injury after gut ischemia-reperfusion.

BACKGROUND: Recent studies have shown that adrenomedullin (AM) and AM-binding protein-1 (AMBP-1) possess anti-inflammatory properties in sepsis. We hypothesized that administration of AM/AMBP-1 after gut ischemia-reperfusion (I/R) downregulates inflammatory cytokines and attenuates tissue injury. METHODS: Male Sprague-Dawley rats (275-325 g) were used. Gut ischemia was induced by placing a microvascular clip across the superior mesenteric artery (SMA) for 90 minutes. Upon release of the SMA clamp, the animals were treated by AM (12 microg per kilogram of body weight) and AMBP-1 (40 microg per kilogram of body weight) in combination, or vehicle (1 mL 0.9% NaCl) over 30 minutes via a femoral vein catheter. The animals undergoing sham operation or ischemia for 90 minutes only, did not receive AM/AMBP-1 treatment. At 60 minutes after the completion of the treatment (ie, 90 minutes after reperfusion), blood samples were collected. Plasma AM and AMBP-1 were measured by radioimmunoassay and Western blot analysis, respectively. Serum levels of TNF-alpha, interleukin (IL)-1beta, IL-6, IL-10, transaminases (ie, alanine aminotransaminase, aspartate aminotransaminase), lactate, and creatinine were determined with the use of enzyme-linked immunosorbent assay and other standard methods. In additional groups of animals, the 10-day survival rate was recorded after gut I/R. RESULTS: Ischemia alone was sufficient to downregulate both AM and AMBP-1. Unlike AMBP-1 that remained decreased, AM levels increased significantly after reperfusion. I/R but not ischemia alone significantly increased serum levels of inflammatory cytokines. Moreover, I/R-induced tissue injury was evidenced by increased levels of transaminases, lactate, and creatinine. Administration of AM/AMBP-1 after ischemia, however, markedly reduced cytokine levels, attenuated tissue injury, and improved survival. CONCLUSIONS: AM/AMBP-1 may be a novel treatment to attenuate the reperfusion injury after gut ischemia.

Adrenomedullin and adrenomedullin binding protein-1 prevent metabolic acidosis after uncontrolled hemorrhage in rats.

OBJECTIVE: Management of trauma victims with uncontrolled hemorrhage remains a major problem in combat casualty care at the far-forward battlefield setting. The neuroendocrine response to hemorrhage is to maintain perfusion to the heart and brain, often at the expense of other organ systems. Decreased organ perfusion after hemorrhagic shock is associated with metabolic acidosis, in which the up-regulated endothelin-1 plays an important role. We have recently shown that vascular responsiveness to adrenomedullin (AM), a newly discovered vasodilator peptide, is depressed after hemorrhage and resuscitation. Down-regulation of AM binding protein (AMBP-1) appears to be responsible for this hyporesponsiveness. We therefore hypothesized that administration of AM/AMBP-1 would prevent metabolic acidosis after uncontrolled hemorrhage via down-regulation of endothelin-1. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male Sprague-Dawley rats (275-325 g). INTERVENTIONS: A rat model of uncontrolled hemorrhage with an extremely low volume of fluid resuscitation was used to mimic the combat situation. MEASUREMENTS AND MAIN RESULTS: Both lumbar veins of male adult rats were isolated and severed at the junction to the vena cava. The abdomen was kept open but covered with a saline wet gauze for 45 mins and then closed in layers. The animals received 1 mL of normal saline (vehicle) with or without AM (12 microg/kg of body weight) and AMBP-1 (40 microg/kg of body weight) over 45 mins. Various variables were measured at 4 hrs after resuscitation. The bleed-out volumes in the vehicle group and the AM/ AMBP-1 treatment group were 6.78 +/- 0.19 and 6.81 +/- 0.25 mL/rat, respectively. The results indicate that AM/AMBP-1 administration prevented metabolic acidosis, mitigated organ injury, down-regulated preproendothelin-1 gene expression, and decreased plasma levels of endothelin-1 after hemorrhage. CONCLUSIONS: AM/AMBP-1 may provide a novel approach for the treatment of uncontrolled hemorrhage. The beneficial effect of AM/AMBP-1 is associated with down-regulation of endothelin-1.

Suppression of hepatocyte CYP1A2 expression by Kupffer cells via AhR pathway: the central role of proinflammatory cytokines.

The hepatic cytochrome P-450 (CYP) enzyme system provides a major aspect of liver function, yet alterations of CYP in sepsis remain largely unknown. Although we have recently shown that CYP1A2, one of the major isoforms of CYP in rats, is downregulated in sepsis, the underlying mechanism and possible therapeutic approaches warrant further investigation. The aim of this study was to determine whether Kupffer cells (KCs) play any role in suppressing CYP1A2 in the hepatocytes (HCs) and if so, how to modulate CYP1A2 expression in sepsis. To study this, primary KCs and HCs were cultured separately or together with or without transwells. Cells and supernatant samples were collected after various stimulations. Additionally, polymicrobial sepsis was induced in rats by cecal ligation and puncture (CLP) with or without curcumin pretreatment. Liver samples were harvested 20 h post-CLP. The results show that lipopolysaccharide (LPS) did not suppress CYP1A2 in HC or HC/KC coculture with transwells. However, LPS downregulated CYP1A2, aryl hydrocarbon receptor (AhR, a nuclear receptor) and AhR nuclear translocator (Arnt) in coculture without transwells. Anti-TNF-alpha and anti-IL-1beta antibodies attenuated this downregulation. Moreover, elevated hepatic levels of TNF-alpha and IL-1beta post-CLP were decreased by curcumin pre-treatment. This reduction was associated with increased expression of AhR and CYP1A2. These results indicate that KCs-derived proinflammatory cytokines may play an important role in downregulating CYP1A2 in sepsis. The reduction of AhR/Arnt may be the underlying mechanism for such downregulation. Inhibition of proinflammatory cytokines by curcumin may provide a novel approach to modulate the hepatic CYP function in sepsis.

The anti-inflammatory effect of curcumin in an experimental model of sepsis is mediated by up-regulation of peroxisome proliferator-activated receptor-gamma.

OBJECTIVE: Although phytochemical curcumin has been shown to possess anti-inflammatory properties, it remains unknown whether this agent has any beneficial effects in sepsis. The purpose of this study was to demonstrate whether curcumin protects septic animals and, if so, whether activation of peroxisome proliferator-activated receptor (PPAR)-gamma, an anti-inflammatory nuclear receptor, plays any role. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: A bolus injection of 0.2 micromol of curcumin was given intravenously to male adult rats, followed by continuous infusion of curcumin (0.24 micromol/day) for 3 days via a primed 2-mL mini-pump. The rats were then subjected to sepsis by cecal ligation and puncture (CLP). MEASUREMENTS AND MAIN RESULTS: Serum levels of liver enzymes (alanine aminotransferase and aspartate aminotransferase), lactate, albumin, and tumor necrosis factor (TNF)-alpha were measured at 20 hrs after CLP (i.e., late stage of sepsis). In addition, a 10-day survival curve was conducted following CLP and cecal excision with or without curcumin treatment. Furthermore, macrophages cell line RAW 264.7 cells were treated with curcumin followed by stimulation with endotoxin. TNF-alpha and PPAR-gamma expression were then measured. The results indicate that intravenous administration of curcumin before the onset of sepsis attenuated tissue injury, reduced mortality, and decreased the expression of TNF-alpha in septic animals. Similar results were also found when curcumin was administered after the onset of sepsis. Moreover, the down-regulated PPAR-gamma in the liver at 20 hrs after CLP was significantly improved by curcumin treatment. Concurrent administration of curcumin and GW9662, a specific PPAR-gamma antagonist, completely abolished the beneficial effects of curcumin under such conditions. In cultured RAW 264.7 cells, curcumin inhibited endotoxin-induced increases in TNF-alpha expression and markedly up-regulated PPAR-gamma expression without affecting cell viability. Curcumin also prevented morphologic alterations in macrophages induced by endotoxin. CONCLUSIONS: The protective effect of curcumin makes it or its analogues strong candidates as a novel therapy for sepsis. The beneficial effect of curcumin appears to be mediated by up-regulation of nuclear receptor PPAR-gamma.

Proapoptotic effect of curcumin on human neutrophils: activation of the p38 mitogen-activated protein kinase pathway.

OBJECTIVE: Despite advances in the management of sepsis and acute respiratory distress syndrome, the mortality rate remains high. Delayed apoptosis of neutrophils is associated with multiple organ failure under those conditions. Thus, development of nontoxic neutrophil apoptosis regulating molecules may provide a novel therapeutic strategy. Curcumin is a promising dietary supplement for cancer prevention. However, the effect of curcumin on human neutrophil apoptosis remains unknown. We therefore hypothesized that curcumin would produce a proapoptotic effect on neutrophils. DESIGN: Prospective, controlled, and randomized in vitro study. SETTING: Research institute laboratory. SUBJECTS: Human peripheral neutrophils obtained from normal subjects. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: In the presence or absence of curcumin, both spontaneous neutrophil apoptosis and apoptosis of neutrophils following transmigration across a human lung endothelium-epithelium bilayer were studied by morphology and terminal dUTP nucleotide end labeling analyses, respectively. Myeloperoxidase activity and migration assays were performed to determine the impact of curcumin on neutrophil function. To elucidate the potential mechanism, the p38 mitogen-activated protein kinase pathway and caspase-3 activity were examined by Western blotting and enzymatic analyses. The data demonstrate that curcumin increased constitutive neutrophil apoptosis and abrogated the transbilayer migration-induced delay in neutrophil apoptosis. Neutrophil activation was reduced by curcumin treatment as evidenced by a decrease in migration and myeloperoxidase release. A marked increase in p38 phosphorylation and caspase-3 activity was observed following curcumin exposure. In addition, inhibition of p38 mitogen-activated protein kinase with SB203580 suppressed apoptosis and caspase-3 activation induced by curcumin. Thus, activation of p38 mitogen-activated protein kinase or an increase in caspase-3 activity appears to contribute to the proapoptotic effect of human neutrophil apoptosis by curcumin. CONCLUSION: The characteristics of curcumin, including its proapoptotic effect and antidegranulation effect, make it a potential candidate for the therapy of neutrophil-induced lung injury and sepsis.

Mechanisms responsible for vascular hyporesponsiveness to adrenomedullin after hemorrhage: the central role of adrenomedullin binding protein-1.

OBJECTIVE: Irreversible hypovolemia remains a major clinical problem. Preliminary studies indicate that administration of adrenomedullin and adrenomedullin binding protein-1 in combination (AM/AMBP-1) after hemorrhage, improves cardiovascular function despite the increased levels of AM. Our aim was to determine whether vascular responsiveness to AM is reduced after hemorrhage and, if so, to elucidate the possible mechanism responsible for such hyporesponsiveness. METHODS: Male rats were bled to and maintained at a mean arterial pressure of 40 mm Hg for 90 minutes. The animals were then resuscitated with 4 times the volume of shed blood with lactated Ringer's solution over 60 minutes. At 1.5 hours postresuscitation, vascular responses to AM and AMBP-1, plasma levels of AM and AMBP-1, AMBP-1 and AM receptor gene expression were measured. In additional animals, AM and AMBP-1 were administered intravenously at 15 minutes after resuscitation over 45 minutes. Serum levels of liver enzymes, lactate, creatinine, TNF-alpha, IL-6, and IL-10 were measured at 1.5 hours postresuscitation. RESULTS: AM-induced vascular relaxation decreased significantly after hemorrhage and resuscitation, which was markedly improved by AMBP-1. However, AM receptor gene expression did not change under such conditions. Hemorrhage-induced AM hyporesponsiveness was accompanied by the decreased expression and release of AMBP-1. Moreover, AM/AMBP-1 treatment down-regulated TNF-alpha and IL-6, up-regulated IL-10, and attenuated organ injury. CONCLUSIONS: The decreased AMBP-1 levels rather than alterations in AM receptors are responsible for producing AM hyporesponsiveness after hemorrhage. Thus, administration of AMBP-1 in combination with AM can be useful to reduce organ injury after severe hypovolemia.

Gut-derived norepinephrine plays an important role in up-regulating IL-1beta and IL-10.

Previous studies have shown that the gut is a major source of norepinephrine (NE) released in early sepsis and that gut-derived NE plays an important role in up-regulating TNF-alpha expression in Kupffer cells (KC) via an alpha(2)-adrenoceptor (alpha(2)-AR) pathway. However, it remains unknown whether NE affects the release of other inflammatory cytokines such as IL-1beta and IL-10 and, if so, whether alpha(2)-AR is also involved in such a process. To study this, a branch of the portal vein in normal adult male rats was cannulated under anesthesia. NE (20 muM in ascorbate saline), NE plus yohimbine (YHB, a specific alpha(2)-AR antagonist, 1 mM) or vehicle (0.1% ascorbate saline) was infused at a rate of 13 mul/min for 2 h. The above rate of NE infusion was used to increase the portal level of NE to approximately 20 nM, similar to that observed in sepsis. Blood samples were then collected and serum levels of IL-1beta and IL-10 were measured. In addition, the KC was isolated from normal rats and stimulated with either NE (20 nM) or NE plus YHB (1 muM). The gene expression of IL-1beta and IL-10 in KC and their supernatant levels were assessed. The results indicate that serum levels of IL-1beta and IL-10 increased significantly after the intraportal infusion of NE. Co-administration of NE and YHB, however, significantly attenuated IL-1beta and IL-10 production. Similarly, IL-1beta and IL-10 gene expression and release from KC were up-regulated by NE stimulation, whereas YHB attenuated both cytokines. Thus, gut-derived NE up-regulates IL-1beta and IL-10 expression and release in the liver through an alpha(2)-AR pathway. Since adenylate cyclase activator forskolin prevents the increase in NE-induced IL-1beta and IL-10, the up-regulatory effect of NE on those cytokines appears to be mediated, at least in part, by inhibition of adenylate cyclase and reduction in intracellular cyclic AMP levels.

Divergent roles of murine neutrophil chemokines in hemorrhage induced priming for acute lung injury.

Neutrophil associated lung injury is identified with a variety of local and systemic priming insults. In vitro studies have shown that TNF-alpha mediated suppression of neutrophil apoptosis is due to the secretion of interleukin-8 (IL-8), a human chemokine shown to alter neutrophil chemotaxis. Our initial in vitro antibody neutralization studies with neutrophil chemotactic proteins, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2alpha (MIP-2alpha), mouse IL-8 homologues, indicate that MIP-2alpha but not KC appears to mediate TNF-alpha suppression of mouse neutrophil apoptosis. Therefore, we hypothesized that in vivo neutralization of KC or MIP-2alpha during an initial priming insult would produce differential effects on the extent of lung injury by restoring normal neutrophil apoptotic function. To assess this, mice were hemorrhaged followed with septic challenge at 24 h. Antibody against KC or MIP-2alpha or a nonspecific IgG was given during resuscitation immediately following hemorrhage. Anti-MIP-2alpha treatment resulted in a significant reduction in lung tissue IL-6 and myeloperoxidase levels. Percentage of neutrophil apoptosis increased significantly in the anti-KC group. Tissue and plasma KC and MIP-2alpha were reduced in their respective treatment groups. These data suggest that KC and MIP-2alpha differ in their mediation of neutrophil function (apoptosis and chemotaxis) and contribution to the pathogenesis of lung injury following hemorrhage subsequent to sepsis.

Adrenomedullin and its binding protein attenuate the proinflammatory response after hemorrhage.

OBJECTIVE: The neuroendocrine response to hemorrhage is to maintain perfusion to the heart and brain, often at the expense of other organ systems. Systemic inflammation and tissue injury are important components of pathophysiologic consequences of hemorrhage. We have recently shown that administration of adrenomedullin (AM, a potent vasodilator peptide) and adrenomedullin binding protein-1 (AMBP-1) prevented the transition from the hyperdynamic to the hypodynamic stage in the progression of sepsis. However, the effect of AM/AMBP-1 on the inflammatory response after hemorrhage remains unknown. We therefore hypothesized that administration of AM/AMBP-1 during fluid resuscitation in hemorrhaged animals (i.e., posttreatment) attenuates tissue injury and the proinflammatory response. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: Male adult rats. INTERVENTIONS: Rats were bled, and then a mean arterial pressure was maintained at 40 mm Hg for 90 mins. They were then resuscitated by infusion of four times the volume of shed blood using Ringer's lactate solution for 60 mins. MEASUREMENTS AND MAIN RESULTS: Fifteen minutes after the beginning of resuscitation, AM (12 microg/kg of body weight) in combination with AMBP-1 (40 microg/kg of body weight) was administered via a femoral venous catheter for 45 mins. Blood samples were collected 4 hrs postresuscitation and assayed for levels of liver enzymes (i.e., alanine aminotransferase and aspartate aminotransferase), lactate, creatinine, proinflammatory cytokines tumor necrosis factor and high mobility group box 1, and anti-inflammatory cytokine interleukin-10. The results indicate that levels of alanine aminotransferase, aspartate aminotransferase, creatinine, lactate, tumor necrosis factor, and high mobility group box 1 markedly elevated after hemorrhage and resuscitation, and AM/AMBP-1 treatment significantly attenuated these increases. In contrast, the serum concentration of anti-inflammatory cytokine interleukin-10 was increased by the treatment of AM/AMBP-1. Moreover, AM/AMBP-1 treatment significantly improved the survival rate from 35% in vehicle-treated animals to 73% in AM/AMBP-1-treated animals in a low-volume resuscitation model of hemorrhage. CONCLUSION: The combined administration of AM and AMBP-1 effectively suppresses hemorrhage-elicited organ injury and reduces hemorrhage-induced mortality, partly through down-regulation of proinflammatory cytokines (tumor necrosis factor and high mobility group box 1) and up-regulation of the anti-inflammatory cytokine interleukin-10.

A novel approach to maintaining cardiovascular stability after hemorrhagic shock: beneficial effects of adrenomedullin and its binding protein.

BACKGROUND: Vascular responsiveness to adrenomedullin (AM), a recently discovered vasodilator peptide, is depressed after hemorrhage and resuscitation. Downregulation of AM binding protein-1 (ie, AMBP-1) appears to be responsible for this hyporesponsiveness. Therefore, we hypothesize that administration of AM/AMBP-1 improves cardiovascular responses after hemorrhagic shock and resuscitation. METHODS: Male rats were bled to and maintained at a mean blood pressure of 40 mm Hg for 90 minutes. The animals were then resuscitated with 4 times the volume of shed blood with Ringer's lactate over 60 minutes. At 15 minutes after the beginning of resuscitation in hemorrhaged animals, AM alone, AMBP-1 alone, AM in combination with AMBP-1, or vehicle (phosphate-buffered saline solution) was administered intravenously over 45 minutes. At 4-hour postresuscitation, in vivo left ventricular contractility parameters, maximal rates of ventricular pressure increase (+dP/dt max ) and decrease (-dP/dt max ), were determined. Cardiac output and organ blood flow were measured with the use of radioactive microspheres. In an additional group of animals, cardiac tumor necrosis factor-alpha (TNF-alpha) levels were measured by an enzyme-linked immunosorbent assay. RESULTS: Four hours after resuscitation, +dP/dt max , -dP/dt max , cardiac output, and organ blood flow in the liver, small intestine, and kidneys were decreased while treatment with AM/AMBP-1 increased these parameters ( P < .05). Moreover, cardiac TNF-alpha levels were elevated at 4 hours after hemorrhage and resuscitation, while AM/AMBP-1 treatment reduced them to sham levels ( P < .05). CONCLUSIONS: Administration of AM/AMBP-1 appears to be a useful approach for restoring and maintaining cardiovascular stability after severe hemorrhagic shock and crystalloid resuscitation.

Regulation of polymorphonuclear leukocyte apoptosis: role of lung endothelium-epithelium bilayer transmigration.

Delayed polymorphonuclear leukocyte (PMN) apoptosis exacerbates acute lung injury. To reach the alveolar spaces, PMNs must migrate across both pulmonary endothelial and epithelial cell layers. We hypothesized that transmigration across the endothelium-epithelium bilayer suppresses PMN apoptosis and sought to elucidate the underlying mechanisms. PMNs freshly isolated from normal volunteers were allowed to migrate across polycarbonate membranes alone or membranes coated with a bilayer of human lung endothelial and epithelial cells. After migration toward different chemoattractants (IL-8, formyl-Met-Leu-Phe, or leukotriene B(4)), PMN apoptosis and caspase activities were assessed by annexin V, histology, and enzymatic assays, respectively. Messenger RNA and specific protein expression in three receptor ligand-mediated, apoptosis-inducing pathways (Fas, TNF-alpha, and TNF-related apoptosis-inducing ligand) were further examined by gene array, RT-PCR, flow cytometry, and Western blot analyses. The data demonstrated that transbilayer migration suppressed PMN apoptosis, and this effect was not chemoattractant type specific. Kinetic analyses further showed that the delay of apoptosis was sustained to at least 18 h. Transbilayer migration caused significant decreases in caspase (-3, -8, and -9) activities. The changes in apoptosis-related gene expression support the survival role of transbilayer migration. Furthermore, the reduced apoptosis was correlated with downregulation of Fas ligand and TNF receptor 1 expression. Our data reveal that migration across a lung endothelium-epithelium bilayer suppresses PMN apoptosis. The decreased activity and/or expression of proapoptotic proteins may provide possible targets for the regulation of inappropriate delay in PMN apoptosis during lung inflammation and injury.

NF-kappaB protects lung epithelium against hyperoxia-induced nonapoptotic cell death-oncosis.

Prolonged exposure to hyperoxia induces pulmonary epithelial cell death and acute lung injury. Although both apoptotic and nonapoptotic morphologies are observed in hyperoxic animal lungs, nonapoptotic cell death had only been recorded in transformed lung epithelium cultured in hyperoxia. To test whether the nonapoptotic characteristics in hyperoxic animal lungs are direct effects of hyperoxia, the mode of cell death was determined both morphologically and biochemically in human primary lung epithelium exposed to 95% O(2). In contrast to characteristics observed in apoptotic cells, hyperoxia induced swelling of nuclei and an increase in cell size, with no evidence for any augmentation in the levels of either caspase-3 activity or annexin V incorporation. These data suggest that hyperoxia can directly induce nonapoptotic cell death in primary lung epithelium. Although hyperoxia-induced nonapoptotic cell death was associated with NF-kappaB activation, it is unknown whether NF-kappaB activation plays any causal role in nonapoptotic cell death. This study shows that inhibition of NF-kappaB activation can accelerate hyperoxia-induced epithelial cell death in both primary and transformed lung epithelium. Corresponding to the reduced cell survival in hyperoxia, the levels of MnSOD were also low in NF-kappaB-deficient cells. These results demonstrate that NF-kappaB protects lung epithelial cells from hyperoxia-induced nonapoptotic cell death.

Delayed administration of human inter-alpha inhibitor proteins reduces mortality in sepsis.

OBJECTIVE: We have recently shown that administration of human inter-alpha inhibitor proteins (IalphaIp) very early after the onset of sepsis maintains cardiovascular stability and reduced mortality. However, it remains unknown whether injection of IalphaIp at later time points of sepsis has any beneficial effects. We therefore hypothesized that IalphaIp and its active component bikunin are reduced in sepsis and that the delayed administration of IalphaIp also improves survival rate. DESIGN: : Prospective, controlled, and randomized animal study. SETTING: A research institute laboratory. SUBJECTS: : Male adult Sprague-Dawley rats. INTERVENTIONS: Rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). MEASUREMENTS AND MAIN RESULTS: : Bikunin gene expression in the liver was measured by reverse transcription polymerase chain reaction. Plasma concentrations of IalphaIp were determined by Western blot at 5 and 20 hrs after CLP. IalphaIp clearance was assessed by injecting radioactive IalphaIp at 12 hrs post-CLP, and the half-life was determined. In addition, IalphaIp (30 mg/kg of body weight) or vehicle was administered at 1, 5, or 10 hrs (single treatment) or at both 10 and 20 hrs (double treatment) post-CLP. The necrotic cecum was excised at 20 hrs post-CLP, and 10-day survival was recorded. The results indicate that bikunin gene expression decreased significantly at 20 hrs post-CLP. Moreover, IalphaIp concentrations decreased significantly at 5 and 20 hrs post-CLP, and its half-life increased from 5.6 +/- 0.3 hrs to 11.8 +/- 2.7 hrs (p <.05), suggesting down-regulation of IalphaIp in sepsis despite the decreased clearance. Administration of IalphaIp at 1 hr post-CLP improved the survival rate from 50% to 92% (p <.05), whereas there was no significant improvement when IalphaIp was administrated at 5 or 10 hrs post-CLP. However, double injection of IalphaIp at 10 and 20 hrs post-CLP (i.e., severe sepsis) increased the survival rate from 44% to 81% (p <.05). CONCLUSION: Since delayed but repeated administration of human IalphaIp improves survival after CLP, this compound appears to be a useful agent for the treatment of severe sepsis.

Adrenomedullin and adrenomedullin binding protein-1 attenuate vascular endothelial cell apoptosis in sepsis.

OBJECTIVE: To determine whether vascular endothelial cell apoptosis occurs in the late stage of sepsis and, if so, whether administration of a potent vasodilatory peptide adrenomedullin and its newly reported specific binding protein (AM/AMBP-1) prevents sepsis-induced endothelial cell apoptosis. SUMMARY BACKGROUND DATA: Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Our recent studies have shown that administration of AM/AMBP-1 delays or even prevents the transition from the hyperdynamic phase to the hypodynamic phase of sepsis, attenuates tissue injury, and decreases sepsis-induced mortality. However, the mechanisms responsible for the beneficial effects of AM/AMBP-1 in sepsis remain unknown. METHODS: Polymicrobial sepsis was induced by cecal ligation and puncture in adult male rats. Human AMBP-1 (40 microg/kg body weight) was infused intravenously at the beginning of sepsis for 20 minutes and synthetic AM (12 microg/kg body weight) was continuously administered for the entire study period using an Alzert micro-osmotic pump, beginning 3 hours prior to the induction of sepsis. The thoracic aorta and pulmonary tissues were harvested at 20 hours after cecal ligation and puncture (ie, the late stage of sepsis). Apoptosis was determined using TUNEL assay, M30 Cytodeath immunostaining, and electromicroscopy. In addition, anti-apoptotic Bcl-2 and pro-apoptotic Bax gene expression and protein levels were assessed by RT-PCR and Western blot analysis, respectively. RESULTS: Vascular endothelial cells underwent apoptosis formation at 20 hours after cecal ligation and puncture as determined by three different methods. Moreover, partial detached endothelial cell in the aorta was observed. Bcl-2 mRNA and protein levels decreased significantly at 20 hours after the onset of sepsis while Bax was not altered. Administration of AM/AMBP-1 early after sepsis, however, significantly reduced the number of apoptotic endothelial cells. This was associated with significantly increased Bcl-2 protein levels and decreased Bax gene expression in the aortic and pulmonary tissues. CONCLUSION: The above results suggest that vascular endothelial cell apoptosis occurs in late sepsis and the anti-apoptotic effects of AM/AMBP-1 appear to be in part responsible for their beneficial effects observed under such conditions.

Differential expression of cytochrome P450 isoforms in the lungs of septic animals.

OBJECTIVE: Sepsis is characterized by an early, hyperdynamic phase and a late, hypodynamic phase. Although studies have shown that cytochrome P450 (CYP) plays an important role in the regulation of vascular reactivity, alterations of vascular CYP isoforms in sepsis remain unknown. Since CYP2C11 and CYP2J4 convert arachidonic acid to vasodilative epoxyeicosatrienoic acids, and CYP4A3 metabolizes arachidonic acid to both epoxyeicosatrienoic acids and vasoconstrictive 19,20-hydroxyeicosatetraenoic acid, the aim of this study was to examine the expression of these isoforms in sepsis and their association with hemodynamic changes. DESIGN: Prospective, controlled, and randomized animal study. SETTING: An institute research laboratory. SUBJECTS: Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). INTERVENTIONS: At 5 hrs (early sepsis) or 20 hrs (late sepsis) after cecal ligation and puncture, blood vessel-rich tissues (i.e., lungs) were harvested. The expression of CYP isoforms at both messenger RNA and protein levels was determined by reverse transcription polymerase chain reaction and Western blot analysis (CYP2C11), respectively. Hemodynamic variables were measured by radioactive microspheres. MAIN RESULTS: The results indicate that the gene expression of CYP2C11 and CYP2J4 was significantly down-regulated at 20 hrs after cecal ligation and puncture, whereas the expression of CYP4A3 was markedly up-regulated at 5 hrs. The protein concentrations of CYP2C11 also decreased significantly at 20 hrs after cecal ligation and puncture. Although total peripheral resistance markedly increased, mean arterial pressure did not change significantly at 20 hrs after the onset of sepsis. In contrast, cardiac output and pulmonary perfusion markedly decreased in late sepsis. CONCLUSIONS: Since the up-regulated CYP4A3 is associated with the early, hyperdynamic phase of sepsis and the down-regulated CYP2C11 and CYP2J4 are associated with the late, hypodynamic phase, vascular CYP isoforms that metabolize arachidonic acid may be involved in regulating the cardiovascular response during the progression of sepsis.

Upregulation of cardiovascular ghrelin receptor occurs in the hyperdynamic phase of sepsis.

Ghrelin, a newly identified endogenous ligand for growth hormone secretagogue receptor 1a (GHSR-1a, i.e., ghrelin receptor), was recently demonstrated to be a potent vasoactive peptide. Although sepsis is characterized by an early, hyperdynamic phase, it remains unknown whether ghrelin or GHSR-1a plays a role in the cardiovascular response to sepsis. To determine this, polymicrobial sepsis was induced by cecal ligation and puncture in male adult rats. At 5 h (i.e., early sepsis) or 20 h (i.e., late sepsis) after cecal ligation and puncture, blood and tissue samples were collected. Ghrelin levels and ghrelin and GHSR-1a mRNA expression were assessed by RIA and RT-PCR, respectively. In addition, GHSR-1a protein levels in aorta, heart, and small intestine were determined by Western blotting. The vascular response to ghrelin was determined by using an isolated gut preparation. A primary rat aortic smooth muscle cell culture was used to determine the effects of LPS on GHSR-1a expression. The results indicate that although ghrelin levels decreased at early and late sepsis, its receptor was markedly elevated in early sepsis. Moreover, ghrelin-induced relaxation in resistance blood vessels of the isolated small intestine increased significantly during early sepsis but was not altered in late sepsis. Furthermore, GHSR-1a expression in smooth muscle cells was significantly increased at mRNA and protein levels with stimulation by LPS at 10 ng/ml. These results demonstrate that GHSR-1a expression is upregulated and vascular sensitivity to ghrelin stimulation is increased in the hyperdynamic phase of sepsis.

Down-regulation of hepatic CYP1A2 plays an important role in inflammatory responses in sepsis.

OBJECTIVE: Although hepatic cytochrome P-450 protein concentrations are altered following endotoxin shock, changes in P-450 isoforms in sepsis have not been fully investigated. The aim of this study was to determine whether the major P-450 isoform in rat liver (i.e., CYP1A2) is down-regulated during the progression of sepsis and, if so, whether reduction of P-450 enzyme system plays an important role in the inflammatory response. DESIGN: Prospective, controlled, and randomized animal study. SETTING: A university/institute research laboratory. SUBJECTS: Male adult Sprague-Dawley rats were subjected either to polymicrobial sepsis by cecal ligation and puncture (CLP) or to sham operation followed by the administration of normal saline solution (i.e., fluid resuscitation). INTERVENTIONS: P-450 isoforms in the liver (i.e., CYP1A2 and 4A1) were determined using reverse transcription polymerase chain reaction and Western blot analysis at various time points after CLP. MEASUREMENTS AND MAIN RESULTS: The results indicate that CYP1A2 messenger RNA expression decreased significantly at 10 and 20 hrs whereas its protein concentrations decreased at 20 hrs after the induction of sepsis. In contrast, CYP4A1 messenger RNA and protein concentrations were not altered even at 20 hrs after CLP. In an additional experiment, all P-450 isoforms were inhibited by pretreatment with 1-aminobenzotriazole to determine the effect of cytochrome P-450 blockade on inflammatory responses by assessing proinflammatory cytokines. The results show further increases in serum concentrations of tumor necrosis factor-alpha, interleukin-1beta, and interleukin-6 in aminobenzotriazole-treated animals at 10 hrs after CLP, which was associated with elevated concentrations of circulating lactate and severe morphologic alterations in the liver. These results suggest that the integrity of the cytochrome P-450 enzyme system plays an important role in septic inflammatory response. CONCLUSION: The major hepatic P-450 isoform CYP1A2 is down-regulated and inhibition of P-450 enzyme system is associated with an exacerbated inflammatory response in sepsis. Treatment with pharmaceutical agents that regulate or are metabolized by P-450 enzymes might be approached cautiously in the septic patient if this holds true in a clinical setting.

Standardizing vascular surgery residency: merger of two programs to create one model.

The practice of vascular surgery has been transformed in the last decade. It is now necessary to change the way we train vascular surgeons, to keep pace with this rapidly evolving specialty.