Modulation of matrix metalloproteinase-9 and tissue inhibitor of matrix metalloproteinase-1 in sepsis.

Previous studies demonstrated that hepatic matrix metalloproteinase-9 (MMP-9) activity increased following cecal ligation and puncture (CLP) in rats, indicating liver injury in sepsis. The activity of MMP-9 in degrading extracellular matrix is controlled by activation of proenzymes and inhibition of tissue inhibitor of MMPs (TIMP-1). To further assess the proteolytic cascade imbalance in sepsis, hepatic MMP-9 and TIMP-1 expressions were examined in CLP rats. In this study, sepsis was induced in rats by CLP, and at 10 and 20 h after sepsis induction, liver samples were collected and MMP-2, MMP-9, and TIMP-1 gene and protein expressions were evaluated by real time PCR and Western blot analysis, respectively. Gene expression of MMP-9 was increased by 6.4-fold and 3.0-fold at 10 h and 20 h after CLP as compared to sham group, respectively. Likewise, MMP-9 protein expression was also significantly increased at both time points. In contrast, MMP-2 gene expression was not altered at 10 h and 20 h after CLP as compared to sham controls. Interestingly, TIMP-1 gene expression was elevated to 89-fold and 46-fold from sham levels at 10 h and 20 h after CLP, respectively. Similarly, TIMP-1 protein levels were also significantly increased at both time points. In addition, MMP-9/TIMP-1 protein ratio was lower at both 10 h and 20 h after CLP compared to sham rats. Results demonstrated an imbalance between MMP and TIMP, with a more evident role for MMP-9 than MMP-2, and high value of TIMP-1 was particularly evident in CLP rats. Our results indicate that MMP-9 and TIMP-1 expressions are increased and they may serve as useful markers to predict the outcome of sepsis.

Downregulation of protein disulfide isomerase in sepsis and its role in tumor necrosis factor-alpha release.

INTRODUCTION: Protein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an essential role in cell survival under various stress conditions. It has been reported that PDI can serve as a negative regulator of nuclear factor-kappa-B (NF-kappaB) and that it can inhibit lipopolysaccharide (LPS)-induced proinflammatory cytokine production in macrophages. Thus, PDI may be an intracellular anti-inflammatory molecule. Although we have previously shown that Kupffer cell-derived proinflammatory cytokines cause liver injury in sepsis, the effect of sepsis on PDI expression as well as the effect of PDI inhibition on cytokine production have not been investigated. We therefore hypothesized that sepsis downregulates PDI expression and that the inhibition of PDI promotes proinflammatory cytokine production. METHOD: Adult male rats were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia (continuous infusion of 1 microg/kg body weight LPS by an osmotic pump) for 20 hours. Hepatic tissues were collected and PDI gene expression was determined. In additional experiments, cells from a macrophage-like cell line, RAW 264.7, were treated with 100 ng/mL LPS for 4 hours and protein expressions were measured. RAW 264.7 cells were also treated with bacitracin, a specific PDI inhibitor, for 24 hours, and tumor necrosis factor-alpha (TNF-alpha) gene and protein expression as well as its release in the cell supernatant were determined. To further confirm the beneficial effect of PDI in sepsis, RAW 264.7 cells were transfected with PDI short interfering RNA (siRNA) and PDI gene expression and TNF-alpha release were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: PDI gene expression was significantly decreased by 28% and 69% at 20 hours after CLP or LPS infusion, respectively. LPS also decreased PDI protein expression by 33% in RAW 264.7 cells. Incubation of RAW 264.7 cells with bacitracin significantly increased TNF-alpha gene expression and TNF-alpha release as well as its cellular levels in a dose-dependent manner. Transfection of RAW 264.7 cells with PDI siRNA produced an average 36.8% inhibition of the PDI gene expression. This downregulation was correlated with a 3.19-fold increase in TNF-alpha release into the cell supernatant. CONCLUSION: Taken together, these results suggest that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production. Thus, prevention of PDI downregulation in sepsis may be a novel approach to attenuate hyperinflammation and to reduce tissue injury under such conditions.

The potential role of transcription factor aryl hydrocarbon receptor in downregulation of hepatic cytochrome P-450 during sepsis.

We recently demonstrated that the hepatic cytochrome P-450 (CYP) isoform 1A2 is downregulated in sepsis, which appears to play an important role in the inflammatory response and liver injury. However, the mechanism responsible for the decreased CYP1A2 remains unknown. Since the transcription factor aryl hydrocarbon receptor (AhR) regulates the expression of CYP1A2 and the disruption of the AhR gene causes liver injuries, we hypothesized that downregulation of AhR plays an important role in the reduced hepatic CYP1A2 during sepsis. Adult male rats were subjected to sepsis by cecal ligation and puncture (CLP). Hepatic tissues were collected at 5, 10, and 20 h after CLP or sham-operation. The gene expression of AhR was assessed by RT-PCR technique. Its protein was determined by Western blot analysis. In addition, subcellular localization of AhR was examined by immunohistochemical staining. The results indicate that hepatic AhR gene expression decreased at 5 h and remained downregulated at 10-20 h after CLP. AhR protein levels were significantly reduced at 10-20 h after CLP. Immunohistochemical examination showed that AhR was mainly located in hepatocyte cytoplasm in sham animals. The translocation of AhR from the cytoplasm to the nucleus was observed in septic animals. The downregulation of hepatic AhR and CYP1A2 observed in septic animals does not appear to be due to the elevated endotoxin levels since administration of polymyxin B (an endotoxin-binding agent) did not affect AhR and CYP1A2 gene expression. However, proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta decreased AhR and CYP1A2 expression. As AhR activates the specific gene expression by binding to the target genes, the translocation of AhR to the nucleus in sepsis would suggest that alterations at AhR binding sites may also contribute to the downregulated CYP1A2 expression in sepsis. Since AhR gene expression decreased earlier than the occurrence of depression of CYP1A2 (CYP1A2 decreased at 10-20 h post CLP), the decreased AhR may play an important role in downregulating hepatic CYP1A2 during the progression of sepsis.

Adrenomedullin and adrenomedullin binding protein-1 protect endothelium-dependent vascular relaxation in sepsis.

Downregulation of vascular endothelial constitutive nitric oxide synthase (ecNOS) contributes to the vascular hyporesponsiveness in sepsis. Although coadministration of the potent vasodilatory peptide adrenomedulin (AM) and the newly discovered AM binding protein (AMBP-1) maintains cardiovascular stability and reduces mortality in sepsis, it remains unknown whether AM/AMBP-1 prevents endothelial cell dysfunction. To investigate this possibility, we subjected adult male rats to sepsis by cecal ligation and puncture (CLP), with or without subsequent intravenous administration of the combination of AM (12 microg/kg) and AMBP-1 (40 microg/kg). Thoracic aortae were harvested 20 h after CLP (i.e., the late stage of sepsis) and endothelium-dependent vascular relaxation was determined by the addition of acetylcholine (ACh) in an organ bath system. In addition, ecNOS gene and protein expression was assessed by RT-PCR and immunohistochemistry, respectively. The results indicate that ACh-induced (i.e., endothelium-dependent) vascular relaxation was significantly reduced 20 h after CLP. Administration of AM/AMBP-1 prevented the reduction of vascular relaxation. In addition, ecNOS gene expression in aortic and pulmonary tissues was downregulated 20 h after CLP and AM/AMBP-1 attenuated such a reduction. Moreover, the decreased ecNOS staining in thoracic aortae of septic animals was prevented by the treatment with AM/AMBP-1. These results, taken together, indicate that AM/AMBP-1 preserves ecNOS and prevents reduced endothelium-dependent vascular relaxation (i.e., endothelial cell dysfunction) in sepsis. In light of our recent finding that AM/AMBP-1 improves organ function and reduces mortality in sepsis, it is most likely that the protective effect of these compounds on ecNOS is a mechanism responsible for the salutary effect of AM/AMBP-1 in sepsis.

Inhibition of matrix metalloproteinase on hepatic transforming growth factor beta1 and caspase-3 activation in hemorrhage.

OBJECTIVES: Hemorrhage initiates an inflammatory response that induces the systemic release of cytokines and sequestration of polymorphonuclear neutrophils. Sequestered polymorphonuclear neutrophils release proteases, including matrix metalloproteinases (MMPs) that degrade elements of the extracellular matrix, contributing to the morbidity and mortality seen from hemorrhage. Activation of MMPs may be associated with changes in transforming growth factor beta1 (TGF-beta1) and caspase-3 signaling pathways. In this study, the authors examined hemorrhage-induced changes in the expression of rat hepatic MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-l), TGF-beta1, and caspase-3 activities in the presence and absence of the MMP inhibitor hydroxamate. METHODS: Hemorrhagic shock was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure level of 40 mm Hg, maintained for 90 minutes by withdrawal and infusion of blood, followed by a resuscitation period of lactated Ringer's infusion. Rats received either hydroxamate (25 mg/kg) or vehicle by gavage before hemorrhage. Twenty-four hours after resuscitation, plasma and liver samples were collected. Liver MMP-9, TGF-beta1, and caspase-3 levels were quantified by Western immunoblotting. Plasma glutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvic transaminase (GPT) were determined enzymatically. RESULTS: Plasma GOT, plasma GPT, and liver MMP-9, TGF-beta1, and caspase-3 levels were all significantly elevated at 24 hours postresuscitation when compared with the control values. Hepatic TIMP-1, an in vivo inhibitor of MMP-9, was unaltered at 24 hours. Hydroxamate treatment reduced GOT, GPT, MMP-9, TGF-beta1, and caspase-3 levels at 24 hours. The mortality of hemorrhaged untreated rats was 29% after 24 hours, and pretreatment with hydroxamate reduced mortality to 0%. CONCLUSIONS: These results indicate the beneficial effects of MMP inhibitor in preventing an increase in GOT, GPT, MMP-9, TGF-beta1, and caspase-3 activity with the potential for improvement of hepatic injury due to hemorrhage.

Effect of chemically modified tetracycline on transforming growth factor-beta1 and caspase-3 activation in liver of septic rats.

OBJECTIVES: We have previously demonstrated that hepatic matrixmetalloproteinase (MMP)-9 and gelatinase activity increased significantly after sepsis, and pretreatment with chemically modified tetracycline (CMT-3) inhibited these expressions and improved survivability. It has been established that MMP-9 release from hepatic nonparenchymal cells activates transforming growth factor (TGF)-beta1, which in turn catalyzes the conversion of procaspase-8 into active caspase-8. Caspase-8 activates caspase-3, which in turn degrades fibronectin and focal adhesion kinase and leads to disruption of hepatic architecture and integrity. We have been interested in investigating the role of posttreatment with CMT-3 on hepatic MMP-9, TGF-beta1, and caspase-3 activity following sepsis. DESIGN: Laboratory experiment. SETTING: University laboratory. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: In this study, sepsis was induced in rats by cecal ligation and puncture (CLP), and 2 hrs later, half of the rats received CMT-3 (25 mg/kg), whereas the other half received vehicle by gavage. Twenty-four and 48 hrs after sepsis induction, blood and liver samples were collected. MEASUREMENTS AND MAIN RESULTS: Plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) levels were determined by enzymatic method, and the activation states of hepatic MMP-9, MMP-2, tissue inhibitor of metalloproteinase (TIMP)-1, TGF-beta1, and caspase-3 were determined by Western immunoblotting. Plasma GOT, GPT, and hepatic MMP-9 activity increased 2.5-fold, and TFG-beta1 and caspase-3 activity increased 1.5- to 2-fold at 24 hrs and 48 hrs post-CLP; CMT-3 treatment blocked these increases. Furthermore, CMT-3 treatment also led to increased TIMP-1 level, an in vivo inhibitor of MMP-9. MMP-2 level was unaffected by CLP. The 24-hr and 48-hr mortality rates for CLP rats were 29% and 50%, whereas posttreatment with CMT-3 resulted in 0% mortality. CONCLUSIONS: Our results are consistent with an MMP-9-induced caspase-3 activation in response to CLP. CMT-3 posttreatment increased TIMP-1 level and thereby inhibited MMP-9, which in turn decreased TGF-beta1 and caspase-3 signaling pathways and improved survivability in septic rats.

Role of chemically modified tetracycline on TNF-alpha and mitogen-activated protein kinases in sepsis.

Chemically modified tetracyclines are orally active inhibitors of multiple proteases and cytokines. In this study, we focused on the regulation of tumor necrosis factor (TNF)-alpha and mitogen-activated protein kinases (MAPKs) in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracycline-3 (CMT-3), which retains their antiinflammatory activity. Sepsis was induced in rats by cecal ligation and puncture (CLP). At 24 h and 1 h before CLP, treated rats received CMT-3 (25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. TNF-alpha was determined by ELISA, and MAPKs were determined by Western blot analysis. A significant activation of p38 MAPK was observed after 0.5 h and 1.5 h of sepsis that appeared to coincide with the increased circulating TNF-alpha level. The activation of p42/44 was increased after 24 h of sepsis, whereas that of SAPK/JNK was unaltered throughout the course of sepsis. CMT-3 pretreatment inhibited the TNF-alpha level as well as p38 MAPK activation seen after 0.5 and 1.5 h of CLP and also suppressed the activation of p42/44 after 24 h post-CLP. These results indicate increased activity of TNF-alpha and MAPK following sepsis and demonstrate the beneficial effect of CMT-3 in preventing the increase in TNF-alpha, p38 MAPK, p42/44 MAPK, and the progression of septic shock.

Inhibition of matrix metalloproteinases by chemically modified tetracyclines in sepsis.

Sepsis precipitates a systemic inflammatory stimulus that causes systemic release of cytokines and sequestration of polymorphonuclear neutrophils, resulting in degranulation of matrix metalloproteinases (MMPs), which causes extracellular matrix basement membrane degradation. One of the important anti-inflammatory properties of tetracyclines is their ability to inhibit MMPs. In this study, we focused on the regulation of MMPs in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracyclines (CMTs), which retain their anti-inflammatory activity. Sepsis was induced by cecal ligation and puncture (CLP) method. At 24 h and 1 h before CLP, some rats received CMT-3 (25 mg/kg), another group of rats received hydroxamate (H; an inhibitor of MMP; 25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. Plasma and liver MMP-9 by zymography and Western immunoblotting, plasma nitric oxide by measuring nitrate level, plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) by enzymatic method, and liver gelatinase by radiolabeled gelatin lysis assay and 24 h mortality were determined. Plasma MMP-9 (92 kDa), nitrate, and GOT and GPT levels were elevated compared with the time 0 level and reached peak at 1.5 h CLP and remained high for 24 h. Both CMT-3 and H treatment reduced GOT,GPT, 92-kDa gelatinase, and nitrate levels throughout the 24 h. CMT-3 and H are equally effective in sepsis treatment. The 24-h mortality for CLP rats was 30%, whereas pretreatment with CMT-3 and H resulted in 0% mortality. Hepatic MMP-9 and gelatinase activity increased significantly after CLP, and pretreatment with CMT-3 and H inhibited these expressions. These results indicate the beneficial effect of CMT-3 in preventing the increase in GOT, GPT, NO, MMP-9, gelatinase activity, and the ensuing septic shock.

Modulations of signal transduction pathways during sepsis and the effects of insulin and mifepristone.

OBJECTIVES: The phosphorylation states and the inferred activation of specific members of the mitogen-activated protein kinase (MAPK) pathways (p42/44, p38, and SAPK/JNK) were quantitated in the livers of rats by Western blot analysis during the progression of sepsis. In addition, the authors examined the effects of insulin and mifepristone (RU-486) administration on these signal transduction pathways during sepsis. METHODS: Sepsis was brought about by the cecal ligation and puncture (CLP) method. The control group underwent sham operation. One experimental group was injected with insulin (0.5 U/kg, IV); prior to the CLP or sham procedure, the second group was injected with RU-486, a glucocorticoid antagonist (mifepristone, 20 mg/kg, IP), and the third group was injected with saline (control). RESULTS: The activities of SAPK/JNK were unaltered throughout the course of sepsis, while those of p42/44 MAPK increased four-fold after 24 hours of sepsis. The activation of p38 MAPK was also increased after 0.5 and 1.5 hours of sepsis, but returned to normal during late sepsis (24 hours). Rats that had received RU-486 before CLP showed no change in p42/44 MAPK compared with the untreated group, but the drug appeared to suppress the activation after 24 hours post-CLP. SAPK/JNK activation, on the other hand, was slightly enhanced by RU-486 pretreatment after 1.5 and 24 hours of CLP compared with the untreated CLP and the treated sham-operated rats. Insulin, given preoperatively, had no effect on the activation of SAPK/JNK, although p42/44 MAPK pathways were activated by the hormone at 0.5 hours and 1.5 hours post-CLP only, but not in the sham-operated controls or at 24 hours post-CLP. The p38 MAPK activation seen after 0.5 and 1.5 hours of CLP was suppressed by RU-486. Insulin pretreatment appears to suppress the phosphorylation of p38 in both the CLP and sham-operated groups. CONCLUSIONS: The data suggest that sepsis induces two phases of MAPK activation, an early phase in which p38 is active, followed by a second, more pronounced phase of p42/44 MAPK activation that occurs only late in sepsis. Hormonal administration results suggest that RU-486 suppresses the activation of p38 seen in early sepsis, and the activation of p42/44 MAPK seen in late sepsis.