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INFLAMMATION, IMMUNOPHARMACOLOGY, AND ASTHMA
Divisions of Biotechnology Discovery Research (M.A.R., A.G., D.T.B., B.G., G.R.S., M.S.C., J.G.H., B.W.G.), Diagnostic/Experimental Medicine (L.A.O.), and Pathology (S.S., E.J.G.), Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, Indiana
Received August 21, 2007; accepted January 2, 2008.
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Abstract |
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). Despite advances in the understanding of the pathophysiology of ALI/ARDS and improved supportive care with optimal ventilation and fluid balance, mortality rates range from 30 to 50% (Bastarache et al., 2006). ARDS is a systemic disease with the majority of patients dying from multiorgan dysfunction (Vincent and Zambon, 2006), and there remains a significant need for new therapeutic interventions.
Infection is a common cause of pulmonary injury, and in sepsis, the lung is the most often affected organ (Vincent et al., 2003; Bastarache et al., 2006). The lung is a major source of various inflammatory mediators that affect both local and systemic immune response (Ritter et al., 2005), and recent studies have identified specific markers associated with pulmonary dysfunction. For example, chemokines associated with leukocyte migration and activation such as MIP-1
and MIP-2 have been found in close association in pulmonary lesions (Sun et al., 2006) and associated with respiratory dysfunction (Bonville et al., 2006). In addition, the T-cell chemotactic factor TARC has been found to be elevated both in humans (Manabe et al., 2005) and models of lung inflammation (Ritter et al., 2005). The induction of nitric oxide (NO) through inducible NO synthase (iNOS) also plays a significant role in the inflammatory response, and its inhibition attenuates endotoxin-induced lung injury in rats (Su et al., 2007; Xia et al., 2007). Excessive production of angiotensin (Ang) II peptide, which is generated by elevated angiotensin-converting enzyme (ACE)-1 expression, has also been implicated in models of lung injury (for review, see Kuba et al., 2006). Recent studies also have begun to highlight the involvement of coagulation and fibrinolysis in the pathogenesis ALI/ARDS (Schultz et al., 2006) and the potential for therapies modulating microvascular coagulation (Hardaway, 2006). Therefore, multiple factors play a role in the balance of endothelial and leukocyte activation and dysfunction contributing to the pathogenesis of sepsis-induced ALI.
A key factor regulating the balance of endothelial and leukocyte function is activated protein C (APC) (O'Brien et al., 2006). Various studies have demonstrated cytoprotective and anti-inflammatory actions of APC that are mediated by its interaction with the endothelial protein C receptor (EPCR) (for review, see Mosnier et al., 2007). In addition, low PC levels are predictive of early death during sepsis in both human (Macias and Nelson, 2004) and rat model of polymicrobial sepsis (Heuer et al., 2004). Previous studies have demonstrated that patients with ALI/ARDS exhibit low PC levels relative to normal subjects regardless of the underlying etiology of lung injury (Ware et al., 2006), and this reduction in PC levels has been associated with adverse clinical outcomes. These studies signify the role of PC pathway in response to infection, along with the demonstrated efficacy of recombinant human APC in the treatment of severe sepsis (Bernard et al., 2001).
Previous studies have demonstrated that APC can attenuate ALI in lung injury models (for review, see Robriquet et al., 2006), and recombinant human APC decreased neutrophil migration into the airspaces and reduced local coagulation in volunteers given pulmonary endotoxin (Abraham, 2005; van der Poll et al., 2005). In a ewe model of sepsis, APC-treated animals had lower pulmonary arterial pressure and lung wet/dry ratio (Wang et al., 2007b). In the present study, we have explored the relationship between acquired PC deficiency and markers of lung injury in a rat cecal ligation and puncture (CLP) model of polymicrobial sepsis, a model known to be accompanied by acute lung failure (Martin et al., 2003). We demonstrate that acquired PC deficiency after sepsis is associated with increased tissue pathology and markers of lung inflammation but reduced pulmonary ACE-2 expression, a factor that functions as a negative regulator of AngII production (Donoghue et al., 2000). Moreover, treatment with APC results in suppression of lung pathology and the markers of injury and inflammation and restoration of ACE-2 levels and reducing AngII. The data presented here suggest that low endogenous PC levels during systemic inflammatory response may be pathophysiologically related to lung injury by reducing the ability to control the cascading inflammatory responses in the lung after infection.
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Materials and Methods |
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). In brief, Sprague-Dawley rats (245–265 g) were purchased from Harlan (Indianapolis, IN) and allowed to acclimate a minimum of 6 days before surgery. Animals were anesthetized with 3% isoflurane (1:1.5 with O2), and polyethylene catheters (Strategic Applications, Inc., Libertyville, IL) were implanted surgically into the femoral vein. Immediately after femoral catheterization, CLP was performed with a single puncture with a 16-gauge needle, and care was taken to ligate the same length of cecum (1 cm measured by a ruler on the scalpel). Sham rats received identical surgery (except for CLP). After surgery, the rats were given ketoprofen (2 mg/kg) i.m. for pain relief, received injections s.c. with 5 ml of prewarmed saline, and then were continuously infused with 5% dextrose in 0.9% saline (Abbott Laboratories, North Chicago, IL) at a rate of 2 ml/h via the femoral catheter until endpoint of the study at 22 h. Treatment with rat APC, prepared as described previously (Gerlitz and Grinnell, 1996), was infused at 200 µg/kg/h starting 10 h post-CLP. This recombinant rat APC has been shown to be effective in preventing mortality in the rat CLP model (T. Huynh, personal communication). Each animal was sacrificed at the 22-h time point for collection of lung tissue for pathology, protein, and mRNA analysis. The 22-h time point was selected base on the Kaplin-Meyer survival curve for the rat CLP model as described previously (Heuer et al., 2004). At this time point, the level of PC was highly predictive of mortality outcome in this model, where early death was associated with low protein C levels. Saline vehicle was used because previous studies have demonstrated that active site-inhibited APC has no in vivo activity (Kerschen et al., 2007). All experimental methods were approved by the Institutional Animal Care and Use Committee and were in accordance with Institute of Laboratory Animal Resources (1996).
Plasma and Tissue Measurements. Whole blood (90 µl) was collected from the retro-orbital sinus into a tube containing 10 µl of 3.8% sodium citrate and 500 mM benzamidine-HCl, and plasma was collected and stored frozen until analysis. The enzyme-linked immunosorbent assay for measurement of PC levels was performed as described previously (Heuer et al., 2004). Measurements of MIP-2, MIP-1, and MIP-1β were done by immunoassay using the Rodent Multi-Analyte Profile (Rules Based Medicine, Austin, TX). PAI-1 activity was assayed using a functional PAI-1 activity assay HPAIKT following the manufacturer's recommendations (Molecular Innovations, Inc., Southfield, MI). Total RNA was purified from lung tissue samples that had been preserved in RNA-later (Ambion, Austin, TX) using the RNeasy kit (QIAGEN, Valencia, CA) with RNA integrity determined by agarose gel electrophoresis. DNase-treated total RNA was used for first-strand cDNA synthesis primed with random hexamers using the Superscript II cDNA synthesis system (Invitrogen, Carlsbad, CA), and parallel control reactions (–RT) were performed in the absence of reverse transcriptase. TaqMan Gene Expression Assays for TARC, iNOS, ACE-1, and ACE-2 were purchased from Applied Biosystems (Foster City, CA).
Determination of Concentration of AngII in Rat Lung Tissue. Ang II levels were measured using the kit supplied by Phoenix Pharmaceuticals (Belmont, CA) following the manufacturer's instructions. To determine lung AngII levels, tissue samples were homogenized in lysis buffer (10 mM Tris, pH 7.5). The homogenate was centrifuged at 1600g for 15 min at 4°C. Tissue AngII concentration was measured after extraction through the Sep-Pak C-18 column supplied by the manufacturer (Phoenix Pharmaceuticals). The peptide was eluted very slowly with 3 ml of 60% acetonitrile in 1% trifluoroacetic acid, and the fluent was collected in a polypropylene tube. The eluent was lyophilized, and the residue was then dissolved in 250 µl of radioimmunoassay buffer and assayed for AngII according to manufacturer's specifications. Results were normalized to the amount of protein per sample, as determined by bicinchoninic acid assay (Pierce, Rockford, IL). The rat AngII assay has no cross-reactivity with endothelin-1, substance P, [Arg8]-vasopressin, and Ang1 peptides.
EPCR Western Analysis. Protein lysates were prepared for Western analysis using the T-PER reagent (Pierce) containing complete protease inhibitor cocktail (Roche Diagnostics GmbH, Mannheim, Germany) from lung tissues that had been preserved in RNA-later (Ambion). The protein lysates were quantified by bicinchoninic acid assay (Pierce) and equal concentrations of each lysate were loaded for SDS-polyacrylamide gel electrophoresis and electroblotting. EPCR was detected using anti-EPCR antibody (1:1000; Zymed Laboratories, San Francisco, CA). The blots were stripped and reprobed using a monoclonal antibody to β-actin (Sigma-Aldrich, St. Louis, MO) for normalization. Levels of EPCR and β-actin were quantified by analyzing the pixel density of each band from scanned autoradiograms using UnScanIt software (Silk Scientific Corporation, Orem, UT).
Tissue Pathology and Immunohistochemistry. For pathology and immunohistochemistry, tissues were fixed, sectioned, and stained as described previously (Gupta et al., 2007). For EPCR staining, 10 µg/ml of the anti-EPCR antibody (or irrelevant control antibody) followed by a biotinylated secondary antibody plus streptavidin-horseradish peroxidase kit (Dako LSAB2; Dako North America, Inc., Carpinteria, CA) was used along with a DAB Chromagen and peroxide substrate to detect the bound antibody complexes. For iNOS, lungs were fixed in 4% paraformaldehyde and embedded in paraffin. Five-micrometer sections were immunostained for iNOS using the automated Ventana Discovery XT staining module (Ventana Medical Systems, Tucson, AZ). The sections were incubated with rabbit anti-mouse iNOS (5 µg/ml; BD Biosciences Transduction Laboratories, Lexington, KY) for 60 min followed by biotinylated goat anti-rabbit IgG (1:200; Dako North America, Inc.) for 20 min. Detection was performed using Ventana's DAPMap kit, and sections were taken offline for routine counterstaining with hematoxylin. MIP-1 staining was performed similarly on the Ventana using a rabbit anti-rat MIP-1 (catalog number 500-P77; PeproTech, Rocky Hill, NJ) for 1 h followed by the secondary Dako anti-rabbit. The slides were reviewed using light microscopy to evaluate the intensity and localization of the staining.
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). The rapid drop in PC to a level of <60% baseline demonstrated a 100% sensitivity and specificity for early death by ROC analysis (Berg et al., 2006). We were interested in determining a possible relationship between pulmonary dysfunction and low PC level. Shown in Fig. 1A is the distribution of plasma PC levels at 22 h post-CLP compared with surgical sham animals as a control population. Approximately one half (14 of 25) of the animals had a significant reduction in PC levels, using the previously defined cut-off of <60% of baseline (Berg et al., 2006). As indicated in the introduction, the activations of the MIP chemokines and TARC have been characterized as markers of pulmonary dysfunction, so we analyzed levels of these markers with respect to the PC deficiency. As shown in Fig. 1B, animals with low PC had a significant elevation in pulmonary TARC expression (3.5 ± 0.3-fold), compared with no change in TARC in those animals that maintained PC levels within the normal range. Likewise, the MIP chemokines were significantly elevated in the low-PC animals (Fig. 1C) but not in the normal-PC animals. Moreover, there were significant negative correlations between plasma PC and TARC (r2 =–0.62, p < 0.001), MIP-2 (r2 =–0.61, p < 0.005), MIP-1 (r2 =–0.63, p < 0.002), and MIP-1β (r2 =–0.70, p < 0.0005). Unlike these chemokines, the cytokines IL-1, TNF, and IL-6 were not associated with PC deficiency at 22 h, probably because they peak earlier that the chemokines, which continue to increase from 10 h to the 22-h time point (Supplemental Table S1).
Acute lung injury results in increased coagulation and locally suppressed fibrinolysis as a result of strongly elevated levels of PAI-1 (Schultz et al., 2006). As shown in Fig. 1D, animals with PC deficiency had significantly elevated levels of pulmonary PAI-1 activity compared both with sham animals and animals whose PC level had not dropped. Although IL-6 levels in the plasma were not significantly different in low-PC animals, we assessed levels directly in the lung because IL-6 is strongly induced by local thrombin generation (Shin et al., 1999). As shown in Fig. 2A, there was a significantly higher level of pulmonary IL-6 in the low-PC animals, with normal-PC animals no different from shams. We also examined the level of MCP1 as another inflammatory activator induced by thrombin (Wang et al., 2007a) and observed significant elevation in the low-PC animals. Moreover, the correlation between the level of pulmonary IL-6 and MCP1 was r = 0.93 (p < 0.00001, n = 25).
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chemokine expression and as shown in Fig. 3C observed significant margination of MIP-1-positive cells in the low-PC animals but not in those with normal PC. As would be suggested by the ED1 staining, the majority of the MIP-1-positive cells were mononuclear (Supplemental Fig. S1).
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staining.
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Low PC and ACE-2. ACE-2 is a negative regulator of AngII production, and its deficiency has been shown to be associated with worsening of lung function and leukocyte accumulation in acute lung injury (Imai et al., 2005). Therefore, we sought to determine whether there was any relationship between the PC pathway and this protective factor. As shown in Fig. 5A, animals with low PC had a significantly lower level of ACE-2 expression in the lung compared with animals whose PC level had not dropped. In contrast, the level of ACE-1 expression in the lung was increased by 
50% (p < 0.001) in the CLP animals in both the low- and normal-PC animals (data not shown). Because an increase in ACE-1 should drive higher levels of AngII, whereas ACE-2 should counter-regulate the induction, we measured the level of AngII peptide in the lung. As shown in Fig. 5B, there was an 8-fold increase in the AngII level in the low-PC animals but significantly lower levels in the normal-PC animals, consistent with the reduced ACE-2 in the low-PC group. Taken together, the above data suggest a strong association between acquired PC deficiency following polymicrobial sepsis and the degree of pulmonary injury and inflammation.
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); however, little is known regarding its regulation during pulmonary injury. We examined the levels of EPCR in the lungs after CLP and found a 2.5-fold increase in mRNA, which was significantly higher in animals with low PC (Fig. 6A), and a corresponding increase in the level of protein expression by Western blot analysis (Fig. 6B). We also assessed EPCR by immunohistochemistry, and as shown in Fig. 6C, we observed staining for EPCR in the small vessels, consistent with previous reports (Gupta et al., 2007). As was observed with the mRNA and protein analyses, the level of EPCR by immunohistochemistry was elevated in the low-PC animals as shown by increased staining in the vasculature as well as in the infiltrating leukocytes. Thus, injury to the lung seems to result in an increase in the key vascular factor mediating the anti-inflammatory signaling of APC.
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Treatment with APC Reduces Pulmonary Pathology, iNOS, and Inflammation. The low level of circulating PC probably compromises the ability to naturally generate APC, thereby resulting in a reduction in the endogenous mechanism to protect from pulmonary injury. To test this hypothesis, we examined the effect of infusing rat APC on pulmonary pathology, level of iNOS expression, leukocyte margination, and chemokine inflammation markers. Animals were subjected to CLP and, 10 h later, infused with vehicle or APC for 12 h before sacrifice. As shown in Fig. 7A, treatment of animals with APC starting at 10 h post-CLP resulted in a significant decrease in the level of iNOS mRNA expression in the lung. Because the level of iNOS mRNA was highly related both to the degree of leukocyte margination and tissue pathology in the untreated animals above, we assessed the effect of APC treatment on pulmonary iNOS expression by immunohistochemistry. As shown in Fig. 7B, after APC treatment, we did not observe the high degree of iNOS-positive cells marginating to the vasculature that was seen in untreated animals, as shown in Fig. 4C.
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Because the MIP chemokines were significantly associated with lung injury and low PC and were highly correlated with the level of iNOS (r2 = 0.84, p < 0.001), we assessed the effect of APC treatment on level of MIP chemokine family members. As shown in Fig. 8, MIP-2, MIP-1, and MIP-1β were significantly reduced by the APC treatment. These data suggest that APC down-regulates chemokine-mediated responses during CLP that may account for the observed improvement in lung pathology. As was observed with iNOS, we found very few MIP-1-positive cells by immunohistochemistry after APC treatment (data not shown). Although we observed a significant suppression of leukocyte margination in the lung by APC treatment, we observed no significant effect on peripheral leukocyte populations (data not shown).
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We examined the effect of APC treatment on both IL-6 and MCP1 in the lung, and as shown in Fig. 9, both of these thrombin-activated cytokines were significantly suppressed by APC treatment. The reduction in local IL-6 in the lung, but not in the plasma, by APC treatment suggests the effect of APC may be directly on lung. However, we also examined the effect of APC on the systemic inflammatory response, using multiplex profiling as described previously (Heuer et al., 2004). As shown in Supplemental Table S2, which lists the significant fold changes in plasma level after treatment, APC broadly reduced inflammatory markers associated with leukocyte activation and infiltration, suggesting a significant effect on the systemic inflammatory component in this model. However, as also shown, APC reduced tissue factor, increased fibrinogen levels, and decreased d-dimer. Thus, APC treatment does result in significant changes consistent with an anti-inflammatory role but also reduces systemic coagulopathy as would be expected. Further studies will be required to better define whether the effect of APC on the lung is local or the result of suppression of systemic inflammatory and coagulopathic responses.
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), we examined the effect of APC on active caspase levels in the lung. At the 22-h time point, we observed very few apoptotic cells in the lung (20–30 caspase-positive cells per section), making it unlikely that apoptosis is playing a significant role in the degree of lung inflammation and thus in the protection with APC treatment. Treatment with APC Increases Lung ACE-2 and Decreases AngII and PAI-1. As indicated above, low PC was significantly associated with a reduction in the ACE-2 and increased AngII. Therefore, we examined the effect of infusing rat APC on both of these factors. As shown in Fig. 10A, treatment of animals at 10 h post-CLP with APC resulted in a significant increase in the level of ACE-2, while at the same time resulting in a significant decrease in the level of pulmonary AngII peptide (Fig. 10B).
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Suppression of ACE-2 in Lung Endothelium by Inflammatory Mediators and Protection with APC. We examined whether or not ACE-2 expression could be inhibited in vitro, using human cultured pulmonary endothelial cells. We found that a cytokine mix (IL-1, TNF, interferon 
) significantly reduced ACE-2 expression (Fig. 11). Moreover, treatment of the pulmonary endothelial cells with MIP-1 dramatically reduced ACE-2 protein levels. As also shown, treatment of cells with APC in the absence of the inflammatory mediators had no effect on expression of ACE-2 protein; however, it significantly blocked the down-reduction by the inflammatory mediators.
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Discussion |
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Acquired PC deficiency was associated with a significant elevation of the MIP-family chemokine response, which could by reversed by APC treatment. The chemokine response seems to be a very important driver of pulmonary injury and is critical for amplification of the host response during infection. Sun et al. (2006) demonstrated that MIP-1β and associated T cell responses were found to be in close association in pulmonary lesions. Using microarray studies, Bonville et al. (2006) identified expression profiles of the proinflammatory mediators MIP-1 and MIP-2 that correlated with persistent respiratory dysfunction in a murine model of pneumonia. In our studies, the increase in MIP chemokines was negatively correlated with PC level but highly correlated with the level of iNOS. Kim et al. (2003) have suggested that iNOS may regulate chemokine response during injury, which correlates with the degree of tissue inflammatory response (for review, see Gupta et al., 2007).
Several reports have demonstrated an increase in pulmonary NO production in response to sepsis. Fujii et al. (1998) have shown an enhanced production of pulmonary NO in septic animals, which was contributed by macrophage-mediated iNOS expression. In addition, iNOS-deficient mice exhibit reduced pulmonary albumin vascular leakage during endotoxemia (Wang et al., 2002). These findings suggest that iNOS plays a detrimental role in the pathogenesis of sepsis-induced lung dysfunction. In the present study, we observed increased iNOS expression in lungs of PC-deficient animals, largely the result of increased margination of iNOS-positive leukocytes. Although there were MPO-stained cells in the lung, most of the marginating leukocytes appeared to be mononuclear in origin, as indicated by the ED-1 and MIP-1 staining and by histopathological determination, and were substantially reduced after APC treatment. Nick et al. (2004) have shown reduction in neutrophil accumulation into the airways in a localized pulmonary inflammation model induced by bacterial pneumonia after APC treatment, and MPO staining was reduced in our study (data not shown). Thus, it seems that infiltration of both neutrophil and iNOS-positive mononuclear cell is suppressed by APC in the lung. In terms of other cell types in the lung, recent studies have demonstrated the important role of mast cells and IL-15 in the CLP model (Orinska et al., 2007); however, we found no effect of APC on either IL-15 or the number of T-blue staining cells in the lung, suggesting that the protective effect does not involve mast cells.
The lung possesses its own renin-angiotensin system that acts independently of the circulating system in terms of pulmonary function. A key element of the system is the enzyme ACE-2, which hydrolyzes AngII to the vasodilator Ang (1–7), thereby regulating the net level of AngII and facilitating the mitigation of the biological actions of AngII (Ferrario et al., 2005). Increasing ACE-2 levels by administration of recombinant protein has been shown to improve ALI associated with acid aspiration and CLP-induced sepsis (Imai et al., 2005). In the present study, PC deficiency was associated with a marked down-regulation of ACE-2 expression coupled with elevated AngII levels and worsened lung pathology. Moreover, administration of APC restored ACE-2 expression and reduced AngII peptide. Because AngII has been shown to induce neutrophil accumulation in vivo (Nabah et al., 2004), reduction of pulmonary AngII levels by APC may contribute to its ability to suppress leukocyte adhesion in the present study. In addition, reduction in AngII has been shown to improve the fibrinolytic balance by reducing plasma PAI-1 levels (Arndt et al., 2006), and local suppression of fibrinolysis as a result of elevated PAI-1 levels has been demonstrated in ALI, pneumonia, and sepsis (for review, see Schultz et al., 2006). Therefore, the reduction in AngII by APC treatment may have contributed to the observed reduction in PAI-1 levels by APC, although the ability of APC to directly inhibit PAI-1 is also likely a contributing factor. Taken together, these data suggest a role for APC in modulating pulmonary function by altering ACE-2 expression, which may also contribute to reduction in PAI-1 and, along with the inhibition of chemokine activation, play a role in the suppression of iNOS-positive leukocyte infiltration in the lung. In support of this last point, we observed a highly significant negative correlation (r2 =–0.8, p < 0.0001) between the level of ACE-2 and iNOS in the lung.
EPCR mediates the receptor-associated anti-inflammatory and cytoprotective effects of APC (Mosnier et al., 2007), and we now show higher level of EPCR expression in the lung postsepsis. Several studies have suggested that EPCR is suppressed during tissue injury (Dahlbäck and Villoutreix, 2005), although EPCR has been shown to be increased after renal injury (Gupta et al., 2007). The increase in pulmonary EPCR may be a compensatory mechanism to allow for increased protective signaling via APC. The even higher levels of EPCR in animals with low PC shown in Fig. 5, i.e., with a reduced ability to generate endogenous PC, would be consistent with this hypothesis.
Overall, our studies suggest that acquired PC deficiency in sepsis may be pathophysiologically related to compromised pulmonary function, probably due to the inability to generate sufficient APC to limit inflammation and tissue injury. In acute lung injury associated with acquired PC deficiency, APC treatment may provide a means of replacing the loss of a natural protective mechanism.
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Acknowledgements |
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Footnotes |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: ALI, acute lung injury; ARDS, acute respiratory distress syndrome; MIP, macrophage inflammatory protein; TARC, thymus and activation-regulated chemokine; NO, nitric oxide; iNOS, inducible nitric-oxide synthase; Ang, angiotensin; ACE, angiotensin-converting enzyme; APC, activated protein C; EPCR, endothelial protein C receptor; PC, protein C; CLP, cecal ligation and puncture; PAI, plasminogen activator inhibitor; ROC, receiver operator characteristic; IL, interleukin; TNF, tumor necrosis factor; MPO, myeloperoxidase.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Brian W. Grinnell, Biotechnology Discovery Research, Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285. E-mail: bgrinnell{at}lilly.com
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References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abraham E (2005) Effects of recombinant human activated protein C in human models of endotoxin administration. Proc Am Thorac Soc 2: 243–247.
Arndt P, Young S, Poch K, Nick J, Falk S, Schrier R, and Worthen G (2006) Systemic inhibition of the angiotensin-converting enzyme limits lipopolysaccharide-induced lung neutrophil recruitment through both bradykinin and angiotensin II-regulated pathways. J Immunol 177: 7233–7241.
Bastarache J, Ware L, and Bernard G (2006) The role of the coagulation cascade in the continuum of sepsis and acute lung injury and acute respiratory distress syndrome. Semin Respir Crit Care Med 27: 365–376.[CrossRef][Medline]
Berg D, Gerlitz B, Sharma G, Richardson M, Stephens E, Grubbs R, Holmes K, Montani D, Zhang T, Cramer M, et al. (2006) FoxA2 involvement in suppression of protein C, an outcome predictor in experimental sepsis. Clinical Vaccine Immunol 13: 426–432.[CrossRef]
Bernard G, Vincent J, Laterre P, LaRosa S, Dhainaut J, Lopez-Rodriguez A, Steingrub J, Garber G, Helterbrand J, Ely E, et al. (2001) Efficacy and safety of recombinant human activated protein C for severe sepsis. New Engl J Med 344: 699–705.
Bonville C, Bennett N, Koehnlein M, Haines D, Ellis J, DelVecchio A, Rosenberg H, and Domachowske J (2006) Respiratory dysfunction and proinflammatory chemokines in the pneumonia virus of mice (PVM) model of viral bronchiolitis. Virology 349: 87–95.[CrossRef][Medline]
Dahlbäck B and Villoutreix B (2005) The anticoagulant protein C pathway. FEBS Lett 579: 3310–3316.[CrossRef][Medline]
Donoghue M, Hsieh F, Baronas E, Godbout K, Gosselin M, Stagliano N, Donovan M, Woolf B, Robison K, Jeyaseelan R, et al. (2000) A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1–9. Circ Res 87: E1–E9.[Medline]
Ferrario CM, Trask AJ, and Jessup JA (2005) Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1–7) in regulation of cardiovascular function. Am J Physiol Heart Circ Physiol 289: H2281–H2290.
Fujii Y, Goldberg P, and Hussain SN (1998) Contribution of macrophages to pulmonary nitric oxide production in septic shock. Am J Respir Crit Care Med 157: 1645–1651.[Medline]
Gerlitz B and Grinnell BW (1996) Mutation of protease domain residues Lys37–39 in human protein C inhibits activation by the thrombomodulin-thrombin complex without affecting activation by free thrombin. J Biol Chem 271: 22285–22288.
Gupta A, Berg D, Gerlitz B, Sharma G, Syed S, Richardson M, Sandusky G, Heuer J, Galbreath E, and Grinnell B (2007) Role of protein C in renal dysfunction after polymicrobial sepsis. J Am Soc Nephrol 18: 860–867.
Hardaway R (2006) A brief overview of acute respiratory distress syndrome. World J Surg 30: 1829–1834.[CrossRef][Medline]
Heuer JG, Sharma GR, Gerlitz B, Zhang T, Bailey DL, Ding C, Berg DT, Perkins D, Stephens EJ, Holmes KC, et al. (2004) Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis. Crit Care Med 32: 1570–1578.[CrossRef][Medline]
Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, Yang P, Sarao R, Wada T, Leong-Poi H, et al. (2005) Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 436: 112–116.[CrossRef][Medline]
Institute of Laboratory Animal Resources (1996) Guide for the Care and Use of Laboratory Animals 7th ed. Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council, Washington DC.
Kerschen E, Fernandez J, Cooley B, Yang X, Sood R, Mosnier L, Castellino F, Mackman N, Griffin J, and Weiler H (2007) Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med 204: 2439–2448.
Kim JY, Kim D, Lee EM, Choi I, Park CG, Kim KS, Ha J, Kim SJ, Yang J, Kim YS, et al. (2003) Inducible nitric oxide synthase inhibitors prolonged the survival of skin xenografts through selective down-regulation of pro-inflammatory cytokine and CC-chemokine expressions. Transpl Immunol 12: 63–72.[CrossRef][Medline]
Kuba K, Imai Y, and Penninger J (2006) Angiotensin-converting enzyme 2 in lung diseases. Curr Opin Pharmacol 6: 271–276.[CrossRef][Medline]
Macias WL and Nelson DR (2004) Severe protein C deficiency predicts early death in severe sepsis. Crit Care Med 32: S223–S228.[CrossRef][Medline]
Manabe K, Nishioka Y, Kishi J, Inayama M, Aono Y, Nakamura Y, Ogushi F, Bando H, Tani K, and Sone S (2005) Elevation of macrophage-derived chemokine in eosinophilic pneumonia: a role of alveolar macrophages. J Med Invest 52: 85–92.[CrossRef][Medline]
Martin EL, Moyer BZ, Pape MC, Starcher B, Leco KJ, and Veldhuizen RA (2003) Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis. Am J Physiol Lung Cell Mol Physiol 285: L1222–L1232.
Mosnier L, Zlokovic B, and Griffin J (2007) The cytoprotective protein C pathway. Blood 109: 3161–3172.
Nabah YN, Mateo T, Estelles R, Mata M, Zagorski J, Sarau H, Cortijo J, Morcillo EJ, Jose PJ, and Sanz MJ (2004) Angiotensin II induces neutrophil accumulation in vivo through generation and release of CXC chemokines. Circulation 110: 3581–3586.
Nick JA, Coldren CD, Geraci MW, Poch KR, Fouty BW, O'Brien J, Gruber M, Zarini S, Murphy RC, Kuhn K, et al. (2004) Recombinant human activated protein C reduces human endotoxin-induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 104: 3878–3885.
O'Brien L, Gupta A, and Grinnell B (2006) Activated protein C and sepsis. Front Biosci 11: 676–698.[CrossRef][Medline]
Orinska Z, Maurer M, Mirghomizadeh F, Bulanova E, Metz M, Nashkevich N, Schiemann F, Schulmistrat J, Budagian V, Giron-Michel J, et al. (2007) IL-15 constrains mast cell-dependent antibacterial defenses by suppressing chymase activities. Nat Med 13: 927–934.[CrossRef][Medline]
Ritter M, Mennerich D, Weith A, and Seither P (2005) Characterization of Toll-like receptors in primary lung epithelial cells: strong impact of the TLR3 ligand poly(I:C) on the regulation of Toll-like receptors, adaptor proteins and inflammatory response. J Inflamm (Lond) 29: 16.
Robriquet L, Collet F, Tournoys A, Prangere T, Neviere R, Fourrier F, and Guery BP (2006) Intravenous administration of activated protein C in Pseudomonas-induced lung injury: impact on lung fluid balance and the inflammatory response. Respir Res 7: 41.[CrossRef][Medline]
Schultz M, Haitsma J, Zhang H, and Slutsky A (2006) Pulmonary coagulopathy as a new target in therapeutic studies of acute lung injury or pneumonia: a review. Crit Care Med 34: 871–877.[Medline]
Shin H, Kitajima I, Nakajima T, Shao Q, Tokioka T, Takasaki I, Hanyu N, Kubo T, and Maruyama I (1999) Thrombin receptor mediated signals induce expressions of interleukin 6 and granulocyte colony stimulating factor via NF-kappa B activation in synovial fibroblasts. Ann Rheum Dis 58: 55–60.
Su C, Yang F, and Chen H (2007) Inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats. Clin Exp Pharmacol Physiol 34: 339–346.[Medline]
Sun X, Jones H, Hodge L, and Simecka J (2006) Cytokine and chemokine transcription profile during Mycoplasma pulmonis infection in susceptible and resistant strains of mice: macrophage inflammatory protein 1beta (CCL4) and monocyte chemoattractant protein 2 (CCL8) and accumulation of CCR5+ Th cells. Infect Immun 74: 5943–5954.
van der Poll T, Levi M, Nick JA, and Abraham E (2005) Activated protein C inhibits local coagulation after intrapulmonary delivery of endotoxin in humans. Am J Respir Crit Care Med 171: 1125–1128.
Vincent J and Zambon M (2006) Why do patients who have acute lung injury/acute respiratory distress syndrome die from multiple organ dysfunction syndrome? Implications for management. Clin Chest Med 27: 725–731.[CrossRef][Medline]
Vincent JL, Sakr Y, and Ranieri VM (2003) Epidemiology and outcome of acute respiratory failure in intensive care unit patients. Crit Care Med 31: S296–S299.[CrossRef][Medline]
Wang H, Yi T, Zheng Y, and He S (2007a) Induction of monocyte chemoattractant protein-1 release from A549 cells by agonists of protease-activated receptor-1 and -2. Eur J Cell Biol 86: 233–242.[CrossRef][Medline]
Wang LF, Patel M, Razavi H, Weicker, Joseph M, McCormack D, and Mehta S (2002) Role of inducible nitric oxide synthase in pulmonary microvascular protein leak in murine sepsis. Am J Respir Crit Care Med 165: 1634–1639.
Wang Z, Su F, Rogiers P, and Vincent J (2007b) Beneficial effects of recombinant human activated protein C in a ewe model of septic shock. Crit Care Med Sep 35: 2594–2600.
Ware LB, Camerer E, Welty-Wolf K, Schultz MJ, and Matthay MA (2006) Bench to bedside: targeting coagulation and fibrinolysis in acute lung injury. Am J Physiol Lung Cell Mol Physiol 291: L307–L311.
Xia Z, Wang X, Chen X, and Xia Z (2007) Effect of NO donor sodium nitroprusside on lipopolysaccharide induced acute lung injury in rats. Injury 38: 53–59.[Medline]
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