Marie-Louise Ivarsson

The role of cytokines, coagulation, and fibrinolysis in peritoneal tissue repair.

Peritoneal tissue repair is a distinct entity. Regardless of the type of injury, a common series of events follows, culminating in inflammation and restoration. Molecular actors interact in a series of events in which the balance of fibrin deposition and degradation is vital. Although the complexity of the repair is illustrated by the multitude of effects and the overlap of molecular mediators involved, a framework is emerging. In this context, the overall role of cytokines is to shift the balance of fibrin deposition and degradation in favour of fibrin residues. Coagulation, as well as generating fibrin, is probably of importance in stimulating remesothelialisation, and fibrinolysis is instrumental in the degradation of fibrin deposits. As far as wound healing in concerned, we propose that the ultimate goal may not be to prevent adhesions, but rather to control their formation. To attain this, site-specific modulation of the repair process is essential. The new insights in mediators and modulators reviewed in this paper may provide means for site-specific modulation of peritoneal tissue repair as well as constituting molecular markers of the repair process.

Plasminogen activators and inhibitors in peritoneal tissue

Serosal trauma elicits an inflammatory response which leads to the deposition of fibrin at injured sites, the residuals of which appear to be essential in excessive tissue repair and formation of intraabdominal adhesions. Local plasminogen activity may modulate this early phase of tissue repair. The present study was undertaken to investigate the distribution and cellular expression of plasminogen activators and their inhibitors in human peritoneal normal and inflamed tissue. Tissue‐type plasminogen activator (t‐PA) was expressed in subserosal capillary walls, and in normal mesothelium, but not in inflammation. Immunoreactivity for the plasminogen activator inhibitor type 1 (PAI‐1) was present in normal mesothelium, and substantially increased in inflammation, where, in addition, immunoreactivity was found throughout the submesothelial tissue. This PAI‐1 was partly co‐localized with macrophages, as was the urokinase plasminogen activator (u‐PA), suggesting an involvement of these cells in peritoneal tissue fibrinolysis. Inflammation or abrasion of the mesothelium during surgery is likely to cause a depletion of the local t‐PA source and expose the potentially PAI‐1‐containing submesothelial tissue, thus promoting persistence of fibrin and formation of adhesions.

Characterization and fibrinolytic properties of mesothelial cells isolated from peritoneal lavage

Human peritoneal mesothelial cells were harvested from patients undergoing open or laparoscopic surgery for non-septic conditions using three different approaches: (1) from a peritoneal biopsy, (2) from peritoneal fluid, and (3) from lavage fluid collected from peritoneal cavity. When these different methods were compared, cells derived from peritoneal fluid or lavage were more likely to result in established cultures than those obtained from biopsies. The cells displayed morphological, immunohistochemical and ultrastructural characteristics of mesothelial cells. The cultured mesothelial cells produced tissue type plasminogen activator (t-PA), urokinase plasminogen activator (uPA), and plasminogen activator inhibitor type-1 and type-2 (PAI-1 and PAI-2) during unstimulated conditions. Treatment with the proinflammatory mediators LPS and TNF-alpha resulted in an overall decreased fibrinolytic capacity with a decrease in the release of t-PA and an increase in plasminogen activator inhibitors PAI-1 and PAI-2. TNF-alpha had a more profound effect than LPS, especially on the release of t-PA. This may be an important mechanism by which inflammatory mediators disrupt the fibrin degradation. In conclusion, peritoneal lavage is a convenient and reproducible source of mesothelial cells for culture.

Increased TGF‐Beta1 protein expression in patients with advanced colorectal cancer

There is evidence that TGF‐β1 plays a role as a tumor suppressor in early disease and has pro‐oncogenic effects in advanced tumor stage. The aim of the study was to correlate TGF‐β1 in plasma and tissue to clinical and pathological parameters in patients with various stages of disease progression.

Expression and kinetics of fibrinolytic components in plasma and peritoneum during abdominal surgery

Summary Objective: A reduced peritoneal tissue fibrinolytic capacity is thought to be pathogenetic in intra-abdominal adhesion formation. Therefore, the aim of this study was to characterize the expression of fibrinolytic components in peritoneal tissue and peripheral blood at the beginning and end of abdominal surgery. Design: Peripheral blood and peritoneal tissue were sampled simultaneously from consecutive patients operated for non-septic causes (Group 1, n = 27) or for intra-abdominal infections (Group 2, n = 10). Setting: Surgical department, University hospital. Subjects: Thirty-seven surgical patients. Interventions: Abdominal surgery. Main outcome measures: Tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1) and the inactive complex formation between t-PA and PAI-1 were assayed in plasma and peritoneal tissue extracts sampled at the beginning and end of surgery. Results: The expression of fibrinolytic components in plasma was similar in the two groups when surgery commenced. However, the intraoperative response was partly different. In Group 1, significant increases in t-PA antigen, PAI-1 antigen and t-PA/PAI complex were observed during surgery as opposed to Group 2. When surgery started, Group 2 displayed a reduced fibrinolytic capacity in peritoneal tissue compared with Group 1. In the latter, there was a rapid decline in t-PA activity, followed by an increase in uPA, PAI-1 and t-PA-PAI complex. The changes in Group 2 were similar, but attenuated. There was no correlation in concentrations of fibrinolytic parameters between peritoneal and blood samples. Conclusion: These observations provide novel insights into the early systemic and tissue response to surgery and may lead to new therapeutic strategies.

Increased concentration of tissue-degrading matrix metalloproteinases and their inhibitor in complicated diverticular disease

Objective. Complicated diverticular disease is associated with extensive structural changes of the colonic wall. Turnover of extracellular matrix (ECM) plays a pivotal role in this process. Proteolytic enzymes, including matrix metalloproteinases (MMPs), are capable of degrading most components of ECM. Their activity is regulated by inhibitors, tissue inhibitors of metalloproteinases (TIMPs). Disturbances of the MMP-TIMP balance can cause tissue degradation or fibrosis. The aim of this study was to assess the concentration and distribution of MMPs and TIMPs in colonic biopsies. Material andmethods. Twenty-seven patients who had undergone sigmoid colectomy were included in the study. Full-thickness biopsies from affected and non-affected parts of each resected specimen were collected. Expressions of the proteins MMP-1, -2, -3, -9, TIMP-1 and TIMP-2 were quantified by ELISA and localized by immunohistochemistry. Results. The concentrations of MMP-1, MMP-2 and TIMP-1 were significantly higher in affected tissue than concentrations in non-affected tissue (MMP-1 p=0.005, MMP-2 p=0.0003 and TIMP-1 p<0.0001). In affected segments in general, there was an increased expression in the entire bowel wall, predominantly for MMP-2, MMP-3 and TIMP-1. Conclusions. Concentrations of MMP-1, MMP-2 and TIMP-1 were increased in intestinal segments affected by complicated diverticular disease and distributed throughout the entire bowel wall, which may explain the structural changes.

uPA and PAI-1 in rectal cancer--relationship to radiotherapy and clinical outcome.

BACKGROUND It is well known that the fibrinolytic system is of importance in inflammation, wound healing, and fibrosis development. However, it is also important in the process of tumor invasion and metastasis. We have investigated protein levels of urokinase plasminogen activator (uPA) and plasminogen activator inhibitor-1 (PAI-1) in rectal cancer and effects of radiotherapy, links to clinical outcome, and potential use as prognostic factors. MATERIALS AND METHODS Ninety-one patients with rectal cancer were studied. Blood samples and biopsies were taken during surgery and assayed with enzyme-linked immunosorbent assay for uPA and PAI-1, and patients were followed prospectively (0-96 mo). RESULTS Higher levels of uPA (P < 0.0001) and PAI-1 (P < 0.0001) were found in tumor compared with mucosa. Mucosa exposed to radiotherapy had higher levels of uPA (P < 0.0001) and of PAI-1 (P < 0.0001). Irradiated tumor tissue had higher levels of PAI-1 (P < 0.001). PAI-1 in tumor was correlated with T stage (P < 0.001) and N stage (P < 0.01). PAI-1 in plasma was higher in patients with synchronous distant metastases (P < 0.001). Cox regression was used to identify high levels of PAI-1 in tumor as an independent factor related to short disease-free survival (P < 0.01) and the ratio of uPA/PAI-1 to development of metastases (P < 0.01). CONCLUSIONS There is a relationship between PAI-1 in plasma and rectal cancer metastases. PAI-1 in tumor tissue is correlated to histopathological data and to outcome of rectal cancer. If these findings can be confirmed in larger trials, there will be a possibility to use PAI-1 as a prognostic factor.

Differential Prognostic Impact of uPA and PAI-1 in Colon and Rectal Cancer

Background and Objectives: Degradation of extracellular matrix is important for tumour growth and invasion, which in part is regulated by the plasminogen activation system. The aim of the study was to evaluate the protein expression of urokinase plasminogen activator (uPA) and plasminogen-activating inhibitor-1 (PAI-1) in plasma, tumour-free mucosa and tumour tissue regarding their prognostic value in colon and rectal cancer. Methods: Patients (n = 221) undergoing surgery for colorectal cancer were prospectively included. Samples were assayed by ELISA technique. Results: PAI-1 in tumour tissue (p = 0.006), plasma (<0.0001) and uPA in tumour-free mucosa (p = 0.006) were associated with survival in rectal cancer in univariate analysis. An uPA expression level below 1.1 ng/mg (log rank test, p < 0.0001) in tumour-free mucosa was associated with poor survival in rectal cancer. This was true also for patients without disseminated disease (M₀, p = 0.02). PAI-1 in plasma correlated with metastatic disease (p < 0.0001). uPA and PAI-1 were not associated with survival in either tumour tissue, mucosa or plasma in patients with colon cancer. Conclusions: uPA and PAI-1 have a differential prognostic impact in colon and rectal cancer. Preoperative mucosal uPA and plasma PAI-1 protein expression could possibly be used as prognostic factors in rectal cancer.

A local imbalance between MMP and TIMP may have an implication on the severity and course of appendicitis

BackgroundMatrix metalloproteinases (MMPs) and the tissue inhibitors of MMPs (TIMPs) have been demonstrated to be involved in inflammatory conditions in the intestine. The purpose of this study was to investigate whether the alterations of the MMP/TIMP balance might reflect the course of the inflammatory process in acute appendicitis and if the expression and localisation of MMPs and TIMP is variable in the various clinical manifestations of appendicitis.Materials and methodsThe study comprises 40 patients (26 men and 14 women) having emergency appendectomy and a control group constituting of 10 patients (5 men and 5 women) having a hemicolectomy for other reasons. MMP and TIMP expressions were assessed and compared in tissue specimens from phlegmonous (n = 15), gangrenous (n = 7), perforated appendicitis (n = 11) and controls with noninflamed appendices (n = 10) by means of enzyme-linked immunosorbent assay technique. Localisation of the enzymes was performed by immunohistochemistry.ResultsMMP-1 was significantly higher in gangrenous and perforated appendicitis compared with phlegmonous appendicitis and controls (p < 0.05) while MMP-2 was significantly lower in gangrenous appendicitis compared with phlegmonous appendicitis and controls. MMP-2 was also lower in perforated appendicitis when compared with controls (p < 0.01). Elevated expression of MMP-9 was demonstrated in all groups of appendicitis compared with the controls (p < 0.001).ConclusionsMMP-9 is the most abundantly expressed MMP of those investigated in inflamed appendix. We postulate that a local imbalance between MMP-9 and TIMP-1 may trigger a perforation. These results suggest that MMPs might be useful as biomarkers of appendices prone to perforation.

Studies of TGF-β1-3 in Serosal Fluid During Abdominal Surgery and Their Effect on In Vitro Human Mesothelial Cell Proliferation

BACKGROUND Increased transforming growth factor-beta (TGF-beta) levels are associated with fibrosis, affected cell proliferation, and postsurgical adhesion development, but the knowledge regarding TGF-beta response to the surgical trauma is limited. This study investigated TGF-beta(1-3) isoforms and fibrinolytical factors in peritoneal serosal fluid during abdominal surgery, together with the in vitro effect of TGF-beta(1-3) on human mesothelial cell proliferation. MATERIALS AND METHODS Total as well as biologically active TGF-beta(1-3) and fibrinolytic factors: t-PA, uPA, and PAI-1 were measured in serosal fluid and plasma from 23 patients undergoing colorectal cancer surgery. In vitro proliferation of human primary mesothelial cell cultures upon TGF-beta(1-3) stimulation was also investigated. RESULTS Total TGF-beta1 and TGF-beta2 levels were similar in serosal fluid and plasma while active fractions were increased in serosal fluid. In contrast, total fraction of TGF-beta3 was higher in serosal fluid compared with plasma, while levels of active fractions did not differ. Plasminogen activators (t-PA, uPA) were elevated while the inhibitor (PAI-1) was decreased in serosal fluid compared with plasma. The in vitro mesothelial cell proliferation studies revealed that high TGF-beta(1-3) concentrations decreased cell proliferation, while lower concentrations of TGF-beta1 increased mesothelial cell proliferation. CONCLUSIONS This human study shows increased active TGF-beta levels in peritoneal serosal fluid, compared with plasma, during abdominal surgery and that TGF-beta1 at physiological concentrations increased human mesothelial cell proliferation in vitro. TGF-beta cytokines may be involved in postsurgical adhesion formation.