Lena Holmdahl

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.

Increased plasma MMP-2 protein expression in lymph node-positive patients with colorectal cancer

BackgroundDegradation of the extracellular matrix plays an important part during the invasion of cancer cells into the surrounding tissue. The matrix metalloproteinases (MMPs) have a central role in this process as demonstrated in different malignancies. The aim of this study was to investigate the presence of several MMPs from tumour, adjacent tumour-free colon segment and from plasma, in order to evaluate how these factors might be used as predictors in colorectal malignancy.MethodsSeventy-two patients who underwent surgery because of a colorectal carcinoma were included. Biopsies from the tumour, macroscopically tumour-free bowel and plasma samples were analysed with enzyme-linked immunosorbent assay tests (ELISAs) quantifying protein expression of several MMPs.ResultsWe found highly elevated concentrations of MMP-1, MMP-2, MMP-3 and MMP-9 protein expression in tumour tissue compared with tumour-free tissue (p<0.0001). Elevated MMP-1 tumour levels were found in patients with Dukes’ C cancers (p=0.02). Lymph node status correlated with the expression of MMP-2 in plasma, which was significantly increased in patients with lymph node metastasis compared with those without (p=0.002). MMP-2 in plasma was higher in T3 and T2 tumours than in T4 tumours (p=0.0083).ConclusionThe MMPs we investigated were strongly elevated in tumour tissue compared with tumour-free bowel wall. Our results indicate that MMP-2 in plasma may possibly be used as a predictor in colorectal malignancy. The use of MMP-2 as a predicting tool in combination with different imaging techniques may give important preoperative information in patients with colorectal cancer.

Transforming growth factor beta isoforms production by human peritoneal mesothelial cells after exposure to hypoxia

PROBLEM: Although human mesothelial cells (HMC) line nearly the entire abdominal cavity, little is known about their role in adhesion formation. This study determines the effect of hypoxia and transforming growth factor (TGF)‐β1 on the ability of HMC to produce TGF‐β1‐3, which have been implicated as mediators of the healing process.
 METHOD OF STUDY: HMC were cultured under normal and hypoxic conditions, and treated with and without TGF‐β1 for 24 hr. RNA from each group was subjected to multiplex reverse transcription‐polymerase chain reaction to quantitate TGF‐β1‐3 mRNA levels.
 RESULTS: Hypoxia resulted in 2‐ and 3.3‐fold increase, while TGF‐β1 treatment resulted in 1.4‐ and 1.2‐fold increase (normoxia) and 0‐ and 4.8‐fold increase (hypoxia) in TGF‐β1 and TGF‐β2 mRNA levels, respectively. There was no detectable TGF‐β3 mRNA in HMC before or after treatments.
 CONCLUSION: TGF‐β1 treatment under hypoxia further extenuates endogenous TGF‐β2 but blocks TGF‐β1 production, thereby decreasing the TGF‐β1/TGF‐β2 ratio, which may result in the reduction of scarring and fibrosis.

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.

Role of fibrinolysis in the formation of postoperative adhesions

It has been hypothesized that peritoneal hypofibrinolysis is of importance in the formation of postoperative adhesions, but results from experiments with fibrinolytic modulators are conflicting. We tested this hypothesis in a controlled prospective study in rabbits, comparing the effects of fibrinolytic inhibition (tranexamic acid) to fibrinolysis enhancement by local instillation of gel containing tissue‐type plasminogen activator. Adhesion formation was measured after 1 week in a strictly standardized way and is presented as a percentage of an induced lesion that was covered by adhesions. Fibrinolytic inhibition significantly increased adhesion formation, both to the parietal peritoneum (34.2%± 3.2%) compared with untreated control (19.7%± 3.3%, p < 0.01) and to the bowel (76.3%± 5.8%) compared with untreated control (51.2%± 8.7%, p < 0.05). Control gel significantly increased adhesions to the parietal peritoneum (35.6%± 4.6%) versus untreated control (19.7%± 3.3%, p < 0.05), whereas gel containing tissue‐type plasminogen activator significantly reduced the amount of adhesions to the parietal peritoneum (4.9%± 1.7%) compared with untreated control (19.7%± 3.3%, p < 0.01) and abolished adhesion formation to the injured bowel. The fibrinolytic system thus seems to be intimately involved in the early formation of intraabdominal adhesions.

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.

Differential regulation of mesothelial cell fibrinolysis by transforming growth factor beta 1

Inflammation and tissue trauma during the surgical procedure reduce the peritoneal fibrinolytic capacity. These conditions promote adhesion formation, and are associated with increased expression of transforming growth factor beta 1 (TGF-beta1). The objective of the present study was to investigate whether TGF-beta1 regulates the expression of fibrinolytic components in peritoneal mesothelial cells. Human peritoneal mesothelial cells (HPMC) were cultured and treated with various concentrations of human recombinant TGF-beta1 (0.1, 1.0 and 10 ng/mL) for 24 h. Levels of tissue- and urokinase plasminogen activator (t-PA and uPA), plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) mRNA and protein were assessed by quantitative reverse transcriptase polymerase chain reaction (Q-RT-PCR) and ELISA, respectively. HPMC expressed these components at the gene and protein level. TGF-beta1 downregulated, dose-dependently t-PA mRNA and protein to about 50% of control values (p = 0.0010), and doubled PAI-1 protein production (p = 0.0008) compared to untreated controls. Although uPA gene expression increased in cells exposed to TGF-beta1, the corresponding protein concentration in conditioned media did not. PAI-2 was not affected, either at the gene or protein level. In conclusion, the results indicate that fibrinolytic capacity of mesothelial cells is reduced by TGF-beta1, suggesting that peritoneal adhesion formation induced by TGF-beta1 may be mediated, in part, through reduction in fibrin degradation capacity at an early stage of peritoneal tissue repair.

Expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP in serosal tissue of intraperitoneal organs and adhesions

OBJECTIVE To compare expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP (TIMP-1) in serosal tissue of intraperitoneal organs and adhesions. DESIGN Prospective and cross-sectional study. SETTING Academic research centers. PATIENT(S) Patients undergoing abdominal or pelvic surgery. INTERVENTION(S) MMP-1 and TIMP-1 expression. MAIN OUTCOME MEASURE(S) Expression of messenger ribonucleic acid (mRNA) and protein was measured by using quantitative reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay. RESULT(S) Serosal tissue of intraperitoneal organs and adhesions express MMP-1 and TIMP-1 mRNA and protein at levels that are consistently varied with 10- to 10,000-fold and 2- to 10-fold higher TIMP, mRNA and protein, respectively. Parietal peritoneum, fallopian tubes and ovaries express higher MMP-1 mRNA levels compared with uterus and adhesions; the lowest expression is found in small and large bowels, subcutaneous tissue. and omentum. Expression of TIMP-1 mRNA was less variable; the highest level was found in the uterus and the lowest in subcutaneous tissue and small bowels. There was less variability in MMP-1 and TIMP-1 protein content than mRNA expression; ovaries and adhesions contained the highest MMP-1 and TIMP-1 levels, respectively, and peritoneum contained the lowest. The MMP-1 and TIMP-1 content and ratios further indicate limited MMP-1 proteolytic activity. Although tissues from premenopausal women express more MMP-1 and TIMP-1, expression did not differ by sex or age. CONCLUSION(S) Because MMP-1 and TIMP-1 expression varies consistently among the serosal tissues of peritoneal organs and adhesions, and because tissue injury alters their expression, site-specific variations in expression of these substances may predispose a particular organ to develop more adhesions.

Peritoneal and systemic pH during pneumoperitoneum with CO2 and helium in a pig model

BackgroundLocal peritoneal effects of laparoscopic gases might be important in peritoneal biology during and after laparoscopic surgery. The most commonly used gas, CO2, is known to be well tolerated, but also causes changes in acid-base balance. Helium is an alternative gas for laparoscopy. Although safe, it is not widely used. In this study a method for monitoring peritoneal pH during laparoscopy was evaluated and peritoneal pH during CO2 and helium pneumoperitoneum was studied as well as its systemic reflection in arterial pH.MethodsFor these experiments 20 pigs were used, with ten exposed to pneumoperitoneum with CO2, and ten to helium. Peritoneal and sub-peritoneal pH were continuously measured before and during gas insufflation, during a 30-minute period with a pneumoperitoneum and during a 30-minute recovery period. Arterial blood-gases were collected immediately before gas insufflation, at its completion, at 30 minutes of pneumoperitoneum and after the recovery period.ResultsPeritoneal pH before gas insufflation was in all animals 7.4. An immediate local drop in pH (6.6) occurred in the peritoneum with CO2 insufflation. During pneumoperitoneum pH declined further, stabilising at 6.4, but was restored after the recovery period (7.3). With helium, tissue pH increased slightly (7.5) during insufflation, followed by a continuous decrease during pneumoperitoneum and recovery, reaching 7.2. Systemic pH decreased significantly with CO2 insufflation, and increased slightly during helium insufflation. Systemic pH showed co-variation with intra-peritoneal pH at the the end of insufflation and after 30 minutes of pneumoperitoneum.ConclusionsInsufflation of CO2 into the peritoneal cavity seemed to result in an immediate decrease in peritoneal pH, a response that might influence biological events. This peritoneal effect also seems to influence systemic acid-base balance, probably due to trans-peritoneal absorption.