Treatment of sepsis-induced acquired protein C deficiency reverses Angiotensin-converting enzyme-2 inhibition and decreases pulmonary inflammatory response.

The protein C (PC) pathway plays an important role in vascular and immune function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. However, the association of acquired PC deficiency with the pathophysiology of lung injury is unclear. We hypothesized that low PC induced by sepsis would associate with increased pulmonary injury and that replacement with activated protein C (APC) would reverse the activation of pathways associated with injury. Using a cecal ligation and puncture (CLP) model of polymicrobial sepsis, we examined the role of acquired PC deficiency on acute lung injury assessed by analyzing changes in pulmonary pathology, chemokine response, inducible nitric-oxide synthase (iNOS), and the angiotensin pathway. Acquired PC deficiency was strongly associated with an increase in lung inflammation and drivers of pulmonary injury, including angiotensin (Ang) II, thymus and activation-regulated chemokine, plasminogen activator inhibitor (PAI)-1, and iNOS. In contrast, the protective factor angiotensin-converting enzyme (ACE)-2 was significantly suppressed in animals with acquired PC deficiency. The endothelial protein C receptor, required for the cytoprotective signaling of APC, was significantly increased post-CLP, suggesting a compensatory up-regulation of the signaling receptor. Treatment of septic animals with APC reduced pulmonary pathology, suppressed the macrophage inflammatory protein family chemokine response, iNOS expression, and PAI-1 activity and up-regulated ACE-2 expression with concomitant reduction in AngII peptide. These data demonstrate a clear link between acquired PC deficiency and pulmonary inflammatory response in the rat sepsis model and provide support for the concept of APC as a replacement therapy in acute lung injury associated with acquired PC deficiency.

Activated protein C modulates chemokine response and tissue injury in experimental sepsis.

The protein C (PC) pathway plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality. We have explored the role of PC suppression in modulating early inflammatory events in a model of polymicrobial sepsis. We show that increased levels of organ damage and dysfunction are associated with decreased levels of endogenous PC. Notably, animals with low PC had correspondingly high levels of pulmonary iNOS expression, which correlated with chemokines KC/Gro and MIP2, previously shown to predict outcome in this model. Treatment with activated protein C (aPC) not only reduced the pathology score, leukocyte infiltration and markers of organ dysfunction, but also suppressed the induction of iNOS, and the chemokine response (including KC/Gro, MIP2, IP-10, RANTES, GCP-2 and lymphotactin), and increased apoA1. aPC treatment also suppressed the induction of VEGF, a marker recently suggested to play a pathophysiological role in sepsis. These data demonstrate a clear link between low protein C and degree of organ damage and dysfunction in sepsis, as well as the early reversal with aPC treatment. Moreover, our data show a direct role of aPC in broadly modulating monocyte and T-cell chemokines following systemic inflammatory response.

Role of protein C in renal dysfunction after polymicrobial sepsis.

Protein C (PC) plays an important role in vascular function, and acquired deficiency during sepsis is associated with increased mortality in both animal models and in clinical studies. This study explored the consequences of PC suppression on the kidney in a cecal ligation and puncture model of polymicrobial sepsis. This study shows that a rapid drop in PC after sepsis is strongly associated with an increase in blood urea nitrogen, renal pathology, and expression of known markers of renal injury, including neutrophil gelatinase-associated lipocalin, CXCL1, and CXCL2. The endothelial PC receptor, which is required for the anti-inflammatory and antiapoptotic activity of activated PC (APC), was significantly increased after cecal ligation and puncture as well as in the microvasculature of human kidneys after injury. Treatment of septic animals with APC reduced blood urea nitrogen, renal pathology, and chemokine expression and dramatically reduced the induction of inducible nitric oxide synthase and caspase-3 activation in the kidney. The data demonstrate a clear link between acquired PC deficiency and renal dysfunction in sepsis and suggest a compensatory upregulation of the signaling receptor. Moreover, these data suggest that APC treatment may be effective in reducing inflammatory and apoptotic insult during sepsis-induced acute renal failure.

FoxA2 involvement in suppression of protein C, an outcome predictor in experimental sepsis.

Low levels of protein C (PC) predict outcome as early as 10 h after insult in a rat polymicrobial sepsis model and were associated with suppression of PC mRNA, upstream transcription factor FoxA2, and cofactor hepatocyte nuclear factor 6 (HNF6). Small interfering RNA suppression of FoxA2 in isolated hepatocytes demonstrated regulation of both its cofactor HNF6 and PC. Our data suggest that reduced FoxA2 may be important in the suppression of PC and resulting poor outcome in sepsis.

Effects of hyperglycemia and insulin therapy on outcome in a hyperglycemic septic model of critical illness.

BACKGROUND: The effects of hyperglycemia and insulin therapy on septic outcome have not been well studied. METHODS: Septic hyperglycemia was induced by infusion of TPN (254 kcal/kg x d) immediately following cecal ligation and puncture (CLP) surgery in rats. Animals (N = 109) were monitored for blood glucose and followed for survival for 4 days. Separate cohorts (N = 36) were sacrificed at 22 hours post-CLP and analyzed for cytokines/chemokines, hormones, and organ damage markers. The effects of insulin treatment on 4 day survival were also examined (N = 60). RESULTS: Hyperglycemic septic animals had significantly higher blood glucose (p < 0.0001), plasma proinflammatory cytokine levels, serum organ damage markers (p < 0.05) and reduced mean survival time (p < 0.001). Insulin treatment (2 IU/kg/hr) resulted in significantly lower blood glucose (p < 0.01) and improved 4 day survival (p < 0.03). CONCLUSIONS: Hyperglycemia is associated with greater morbidity and mortality in sepsis. Insulin therapy significantly improved survival suggesting that management of hyperglycemia with insulin may improve outcome in septic patients.

Cecal ligation and puncture with total parenteral nutrition: a clinically relevant model of the metabolic, hormonal, and inflammatory dysfunction associated with critical illness.

BACKGROUND: Standard rat cecal ligation and puncture (CLP) results in only transient hyperglycemia, making an examination of the effects of glucoregulatory agents, such as insulin, on the morbidity and mortality of CLP problematic. Accordingly, we sought to develop a model of rat CLP with prolonged hyperglycemia through continuous infusion of total parenteral nutrition (TPN) post CLP. MATERIALS AND METHODS: Polyethylene catheters were implanted into the femoral vein of female Sprague Dawley rats (245-265 g) which were subsequently subjected to CLP. TPN was initiated at different intervals following CLP, and mortality, bacteremia, blood glucose, hormonal, and inflammatory responses were monitored. RESULTS: Without TPN, CLP resulted in significantly lower blood glucose at 22 h post CLP. In contrast, CLP rats receiving TPN exhibited significant prolonged hyperglycemia that was responsive to insulin treatment. Mortality and hyperglycemia tended to increase with puncture size in CLP TPN rats, with early initiation of TPN leading to poorer outcome. There were time-dependent differences in bacteremia and mortality based on time of TPN initiation. Levels of insulin, leptin, and glucagon were significantly elevated in CLP TPN rats, as were many inflammatory markers. Organ damage was evident as early as 12 h post CLP and blood cell kinetics indicated significantly depressed neutrophil and lymphocyte counts. CONCLUSIONS: Our results indicate that addition of TPN to CLP provides a clinically relevant animal model of critical illness with associated hyperglycemia that may provide utility for the testing of glucoregulatory and other therapeutic modalities.

Evaluation of protein C and other biomarkers as predictors of mortality in a rat cecal ligation and puncture model of sepsis.

OBJECTIVE: To evaluate protein C and other factors associated with the septic response as predictors of mortality in a clinically relevant animal model of sepsis. DESIGN: Laboratory investigation. SETTING: Eli Lilly and Company discovery research laboratory. SUBJECTS: Forty female Sprague Dawley rats weighing 245-265 g. INTERVENTIONS: Polyethylene catheters were surgically implanted into the femoral vein and sepsis was induced by cecal ligation and puncture (CLP). A solution of 5% dextrose in 0.9 % saline was continuously infused via femoral catheters immediately following surgery. Blood sampling was done before surgery and at 6 and 20 hrs after surgery. Rats were then monitored for survival out to 4 days. MEASUREMENTS AND MAIN RESULTS: Blood collections were used to measure blood glucose, bacteremia, plasma protein C, D-dimer, hormones, chemokines, cytokines, and myoglobin (as a marker of organ damage). Mortality was categorized into three groups: early death (before 30 hrs post-CLP), late death (after 30 hrs post-CLP), and survivors (96 hrs post-CLP). Compared with survivors, early death rats had statistically significant differences in 30 variables indicative of severe inflammation, coagulopathy, and muscle damage including less bacterial clearance, hypoglycemia, lower plasma protein C, higher plasma D dimer, higher plasma cytokine/ chemokines, and higher plasma myoglobin concentrations. Twenty variables had a moderate to strong correlation with time of death. Receiver operator characteristic curves generated from a simple logistic regression model indicated that KC and macrophage inflammatory protein-2, rodent homologues of the human growth related oncogene CXC chemokine family, and protein C were the best predictors of mortality in this model. CONCLUSIONS: The data from this study indicate that an early decrease in protein C concentration predicts poor outcome in a rat sepsis model. The data further indicate that increases in the CXC chemokines macrophage inflammatory protein-2 and KC precede poor outcome.