Karen L. Reed

Statins (HMG-CoA reductase inhibitors) decrease postoperative adhesions by increasing peritoneal fibrinolytic activity.

Objectives:The aims of this study were to determine if statins reduce adhesion formation in vivo and to identify the mechanism of action in vitro. Background:Intraperitoneal adhesions develop in up to 95% of patients following laparotomy. Adhesions are reduced by mechanisms that up-regulate fibrinolysis within the peritoneum. Statins promote fibrinolysis in the cardiovascular system and may play a role in the prevention of adhesions. Methods:Adhesions were induced in rats (n = 102) using our previously described ischemic button model. Rats received vehicle (controls), lovastatin (30 mg/kg), or atorvastatin (30 mg/kg) as a single intraperitoneal dose at the time of laparotomy. Animals were killed and adhesions were quantified at day 7. Peritoneal fluid and tissue were collected at day 1 to measure tissue plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1) by real-time PCR and ELISA. To assess the effects of statins on wound healing, burst pressures were measured in anastomoses of the colon. The effects of lovastatin on tPA and PAI-1 production were measured in vitro in human mesothelial cells (HMC) in the presence or absence of mevalonate (MVA), geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), all intermediates in the cholesterol pathway downstream of HMG-CoA. The effect of a Rho protein inhibitor, exoenzyme C3 transferase, on tPA production was also determined. Results:Lovastatin and atorvastatin reduced adhesion formation by 26% and 58%, respectively (P < 0.05), without affecting anastomotic burst pressure. At 24 hours, tPA mRNA levels in peritoneal tissue and tPA activity in peritoneal fluid from lovastatin-treated animals were increased by 57% and 379%, respectively (P < 0.05), while PAI-1 levels were unchanged. HMC incubated with either lovastatin or atorvastatin showed concentration-dependent increases in tPA production and decreases in PAI-1 production (P < 0.05). These lovastatin-induced changes in tPA and PAI-1 production were significantly reversed by the addition of MVA, GGPP, and FPP. The Rho protein inhibitor increased tPA production and rescued tPA production from the inhibitory effect of GGPP. Conclusion:These data suggest that statins administered within the peritoneum can up-regulate local fibrinolysis, while the in vitro studies show that this effect may be mediated, in part, by intermediates of the cholesterol biosynthetic pathway that regulate Rho protein signaling.

NF-κB Activation Precedes Increases in mRNA Encoding Neurokinin-1 Receptor, Proinflammatory Cytokines, and Adhesion Molecules in Dextran Sulfate Sodium–Induced Colitis in Rats

Nuclear factor kappa B (NF-κ B) plays a key role in initiating inflammation associated with colitis. A systematic study was conducted in the rat DSS colitis model to determine the temporal relationship between NF-κ B activation and expression of substance P (SP), neurokinin-1 receptor (NK-1R), proinflammatory cytokines, and adhesion molecules. Rats were given 5% DSS in their water and sacrificed daily for 6 days. Colon tissue was collected for assessment of histological changes, NF-κ B activation, myeloperoxidase (MPO) activity, and expression of NK-1R, SP, TNFα, IL-1β, VCAM-1, ICAM-1, E-selectin, CINC-1, MIP-1α, and iNOS. NF-κ B activation increased, biphasically, on Day 1 and again on Days 4–6. The mRNA levels for ICAM-1, CINC-1, IL-1β, TNFα, VCAM-1, and NK-1R rose significantly (P< 0.05) by 2–4 days. Increased iNOS mRNA levels, MPO activity, and mucosal damage occurred on Day 6. These data demonstrate that NF-κ B activation substantially precedes the onset of physical disease signs and active inflammation.

A new transcription factor that regulates TNF-α gene expression, LITAF, is increased in intestinal tissues from patients with CD and UC

Background: The proinflammatory cytokine tumor necrosis factor‐&agr; (TNF‐&agr;) plays a key role in the pathogenesis of Crohns disease (CD) and ulcerative colitis (UC). Recently, a new transcription factor termed LITAF (lipopolysaccharide‐induced TNF‐&agr; factor) was shown to mediate TNF‐&agr; expression in human macrophages by direct binding to specific sequences in the promoter region of the TNF‐&agr; gene. Methods: In this report, we identified LITAF in resected ileal and colonic tissues from patients with CD and UC by immunohistochemistry, real‐time polymerase chain reaction, and Western blot analysis. LITAF expression in inflamed and noninflamed areas of the tissues was compared. Results: This is the first demonstration of LITAF, a newly discovered transcription factor that regulates TNF‐&agr; gene transcription in ileal and colonic tissues from patients with either CD or UC. LITAF immunostaining was localized to lamina propria macrophages and was markedly increased relative to tissues from controls without inflammatory bowel disease. In patients with CD, a 5‐fold increase in LITAF mRNA was measurable in noninflamed colonic tissues compared with controls without inflammatory bowel disease. LITAF mRNA in tissues from inflamed areas of the colon was increased by an additional 60% compared with noninflamed tissues. In patients with UC, LITAF mRNA levels in colonic tissues resected from noninflamed areas were elevated 15‐fold above nondisease controls, but they were not different in tissues resected from inflamed areas. Western blot analysis showed that in patients with CD, there was a marked increase in LITAF protein in inflamed areas compared with noninflamed areas. LITAF protein levels were not different between noninflamed and inflamed tissues obtained from patients with UC. TNF‐&agr; mRNA and protein levels paralleled LITAF. Similarly, in inflamed ileal tissues from patients with CD, LITAF is also localized to lamina propria macrophages. LITAF mRNA and LITAF protein were significantly increased in inflamed ileal tissues compared with noninflamed areas. Conclusions: LITAF is readily detectable in ileal and colonic tissues from patients with either CD or UC, is significantly elevated above controls, and is localized to macrophages, a major source of TNF‐&agr;. These data provide strong evidence of a role for LITAF in the pathophysiological regulation of the TNF‐&agr; gene and underscore the potential value of anti‐LITAF strategies in the clinical management of these diseases.

N-acetyl-l-cysteine decreases intra-abdominal adhesion formation through the upregulation of peritoneal fibrinolytic activity and antioxidant defenses.

BACKGROUND Intraperitoneal adhesions occur in more than 94% of patients after abdominal surgery. Mechanisms that decrease oxidative stress and upregulate peritoneal fibrinolysis reduce adhesions. N-acetyl-l-cysteine (NAC) is a clinically relevant antioxidant whose effect on peritoneal fibrinolysis and ability to decrease adhesions has not been established. The aims of this study were to determine if NAC reduces adhesions and to characterize its potential mechanism(s) of action. METHODS Male Wistar rats (n = 92) received 0.9% saline (OP Control), intraperitoneal NAC (150 mg/kg, OP + NAC), or oral NAC (1200 mg/kg) twice daily on preoperative day 1, day of operation, and postoperative day 1. Adhesions were induced on the day of operation using our previously described ischemic button model. Animals were killed on postoperative day 7 for adhesion scoring. Peritoneal tissue and fluid from the intraperitoneal NAC group were measured at 24 hours for fibrinolytic activity, tissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1), total glutathione, and 8-isoprostane (8-IP). The effect of NAC on tPA and PAI-1 production was tested in vitro in human mesothelial cells. The effect of NAC on intestinal wound healing was measured using colonic anastomotic burst pressures. RESULTS Intraperitoneal NAC reduced adhesions by 53% (P < .001) compared to OP Controls without affecting anastomotic wound healing. NAC increased the tPA/PAI-1 protein ratio and peritoneal fibrinolytic activity by 69% and 127%, respectively, compared to OP Controls (P < .05). NAC did not restore total glutathione levels in peritoneal adhesion tissue but decreased 8-IP by 46% and 65% (P < .05) in peritoneal tissue and fluid, respectively, compared to OP Controls. Human mesothelial cells incubated with NAC exhibited a concentration-dependent increase in the tPA/PAI-1 ratio, which supported in vivo observations (P < .05). Oral NAC did not decrease adhesions. CONCLUSION NAC administered intraperitoneally decreased adhesion formation while upregulating peritoneal fibrinolytic activity and antioxidant defenses without affecting normal anastomotic wound healing. These data suggest a potential new therapeutic use for NAC in adhesion prevention.

An FDA Approved Neurokinin-1 Receptor Antagonist is Effective in Reducing Intraabdominal Adhesions when Administered Intraperitoneally, But Not Orally

R Lim, J Morrill, S G Prushik, K L Reed, A C Gower, S E Leeman, A F Stucchi, J M Becker (2008) An FDA Approved Neurokinin-1 Receptor Antagonist is Effective in Reducing Intraabdominal Adhesions when Administered Intraperitoneally, But Not Orally. Journal of Gastrointestinal Surgery 12:1754–1761. DOI:10.1007/s11605-008-0634-4. This article was incorrectly published as a 2008 SSAT Plenary Presentation. The Discussion published with it does not belong with that article, but instead belongs to the article by Lim, et al (DOI:10.1007/s11605-008-0724-3) appearing in this issue. J Gastrointest Surg (2009) 13:43 DOI 10.1007/s11605-008-0767-5

Inhibitory Effects of a Neurokinin‐1 Receptor Antagonist on Postoperative Peritoneal Adhesion Formation

Intra‐abdominal adhesions are a costly, long‐term sequela of abdominal surgeries. They occur in up to 94% of patients following abdominal operation and cause significant postoperative morbidity including difficult reoperative surgeries, small bowel obstructions, and infertility. The pathophysiology of adhesion formation remains poorly defined, and a uniformly effective method of adhesion prevention does not exist. Research focused on understanding the mechanisms underlying adhesion formation is essential for the development of safe and effective therapeutic approaches to adhesion prevention. The proinflammatory peptide substance P (SP), known to participate in inflammatory and wound‐healing events, may contribute to the early processes of adhesion formation. SP is the most widely studied ligand of the neurokinin‐1 receptor (NK‐1R), and we have determined in a rat model that intraoperative administration of an NK‐1R antagonist, CJ‐12–255 (Pfizer), that blocks ligand binding to the NK‐1R, significantly reduces adhesion formation. It also has been determined that animals administered the NK‐1R antagonist intraperitoneally have increased peritoneal fibrinolytic and matrix metalloproteinase activities, and reduced levels of oxidative stress postoperatively, all of which may contribute to the observed reduction in adhesion formation. Studies suggest that intra‐abdominal adhesion formation begins within hours of surgery and that the regulation of fibrin deposition, and degradation is of key importance. A pharmacologic agent, such as an NK‐1R antagonist, administered at the time of surgery that could augment postoperative peritoneal fibrinolytic activity without compromising wound healing, would be a beneficial tool in the prevention of postoperative adhesions.

The efficacy of a hyaluronate-carboxymethylcellulose bioresorbable membrane that reduces postoperative adhesions is increased by the intra-operative co-administration of a neurokinin 1 receptor antagonist in a rat model

BACKGROUND Bioresorbable membranes composed of hyaluronic acid and carboxymethylcellulose (HA/CMC) are the most effective method to prevent intra-abdominal adhesions; however, their efficacy may be limited to the site of application. Previous studies in our laboratory have shown that the intraperitoneal administration of a neurokinin-1 receptor antagonist (NK-1RA) reduces adhesions; however, the co-administration of HA/CMC plus an NK-1RA has not been studied. METHODS Adhesions were induced in rats by creating ischemic buttons on the peritoneum. Rats received NK-1RA, HA/CMC, HA/CMC+NK-1RA or saline intraperitoneally at surgery. The HA/CMC was applied either bilaterally over all ischemic buttons or unilaterally over half the ischemic buttons. Animals were sacrificed and adhesions quantified at 7 days. Peritoneal fluid was collected at 24 hours to measure peritoneal tissue plasminogen activator (tPA) activity using a bioassay. RESULTS The bilateral placement of HA/CMC alone reduced adhesions by 62% (P < .05) while the NK-1RA when administered alone reduced adhesions by 45% (P < .05), both groups compared with saline controls. The bilateral placement of HA/CMC+ NK-1RA decreased adhesions by 86% (P < .05) compared with saline controls and by 70% (P < .05) compared with either HA/CMC or NK-1RA alone. Unilateral application of HA/CMC resulted in a 41% decrease (P < .05) in adhesions where placed compared with the distal unprotected buttons in the same animal. However, the unilateral placement of HA/CMC+NK-1RA reduced adhesions by nearly 75% (P < .05) at the site of HA/CMC application compared with HA/CMC + saline, and by 45% (P < .05) at the distal unprotected buttons compared with saline controls. HA/CMC and the NK-1RA alone as well as HA/CMC+NK-1RA increased peritoneal tPA activity by 124%, 432%, and 192%, respectively (P < .05) compared with saline controls. CONCLUSION The co-administration of HA/CMC plus NK-1RA not only increases the efficacy of the membrane at the site of application, but significantly reduces adhesions formation at distal unprotected sites. This combination may represent an emerging concept in more effective adhesion prevention throughout the peritoneum.

A neurokinin-1 receptor antagonist that reduces intraabdominal adhesion formation increases peritoneal matrix metalloproteinase activity

Adhesions remain a significant complication of abdominal surgery. There is a growing body of evidence suggesting that remodeling of peritoneal extracellular matrix by matrix metalloproteinases (MMPs) is involved in adhesion formation. We have shown that administration of a specific neurokinin‐1 receptor (NK‐1R) antagonist (CJ‐12,255, Pfizer) to rats within 5 hours of surgery reduces intraabdominal adhesion formation. Because substance P (SP), the primary NK‐1R ligand, is known to augment tissue fibrosis, the aim of this study was to determine the effects of NK‐1R antagonist administration on peritoneal MMP expression and activity 24 hours after surgery in a rat adhesion model. Following laparotomy, four ischemic buttons were created on the peritoneum of rats that received either an intraperitoneal NK‐1R antagonist or a vehicle at surgery. Adhesion formation was assessed 7 days later. Peritoneal fluid and tissue were collected at 24 hours to assess total MMP activity, as well as MMP‐2, MMP‐8, and MMP‐9 activity. Specific MMP and tissue inhibitors of MMP mRNAs were measured, and the effects of SP on MMP‐3 expression were determined in Met‐5A cells, a human peritoneal mesothelial cell line. NK‐1R antagonist administration reduced adhesion formation by 47% (p<0.05) at 7 days and significantly increased the total MMP activity in peritoneal fluid at 24 hours. There was an accompanying increase (p<0.05) in MMP‐8 and MMP‐9 mRNA expression and activity in peritoneal tissue and fluid, respectively. MMP‐3 mRNA was also increased in the 24‐hour peritoneal tissue, and exposure of Met‐5A cells to SP reduced MMP‐3 expression and activity. These data support a role for MMPs, specifically MMP‐3, MMP‐8, and MMP‐9, in intraabdominal adhesion formation and suggest that the NK‐1R antagonist may reduce adhesions, in part, by increasing MMP activity in the peritoneum by 24 hours after surgery.

LITAF Mediation of Increased TNF-α Secretion from Inflamed Colonic Lamina Propria Macrophages

Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions.

Pharmacologic Inhibition of Adhesion Formation and Peritoneal Tissue-Type Plasminogen Activator Activity

Intraperitoneal adhesions remain a costly, long-term sequela of abdominal surgery. They cause significant postoperative morbidity and difficult reoperative surgery. Although adhesions have been recognized for more than 250 years, a uniformly effective method of adhesion prevention does not exist. In recent years, research has become more focused on understanding the biochemical and cellular processes involved in adhesion formation--a necessary step in the development of safe and effective means of adhesion prevention. Studies suggest that events critical to adhesion outcome begin within hours of an abdominal operation with the balance between fibrin deposition and degradation being of central importance. A pharmacologic agent administered at the time of surgery that could tip the fibrinolytic balance in favor of fibrin degradation without interfering with postoperative wound healing would be an ideal candidate in the prevention of adhesion formation. Further research into the molecular and cellular events that underlie adhesion formation is essential and undoubtedly will lead to successful adhesion prevention.