Abdominal adhesions are networks of fibrotic tissues that form between organs postoperatively. Current prophylactic strategies do not reproducibly prevent adhesive small bowel obstruction across the entire abdomen. Human placental-derived stem cells produce an anti-inflammatory secretome that has been applied to multiple fibrosing diseases. The purpose of this project is to test human placental stem cell (hPSC)-based therapies for prevention of abdominal adhesions in a clinically relevant rat model.

Fifty-four (n = 54, n = 6/group) male Sprague-Dawley rats (250-350 g) underwent model creation and treatment randomization under anesthesia. Experimental groups included human placental-derived stem cells (hPSC, 5 × 106 cells/10 mL Plasmalyte A), human placental-derived stem cells in a hyaluronic acid (HA-Mal-hPSC) hydrogel, the human placental-derived stem cell secretome from conditioned media in 10 mL Plasmalyte A, human placental-derived stem cells' conditioned media in a hyaluronic acid (HA-Mal-CM) hydrogel, Plasmalyte A (media alone, 10 mL), hyaluronic acid hydrogel alone (HA-Mal), Seprafilm (Baxter, Deerfield, IL), and the control groups, model with no treatment (MNT) and sham animals. Treatments were administered intraperitoneally, and the study period was 14 days postoperation. Adhesions were scored at necropsy and analyzed as the difference between means of an index statistic (Animal Index Score) versus MNT. Underlying molecular mechanisms were explored by functional genomic analysis and histology of peritoneal tissues.

Hyaluronic acid hydrogel alone, HA-Mal-CM hydrogel, and Seprafilm significantly reduced the overall appearance of abdominal adhesions by mean Animal Index Score at 14 days versus MNT. Human placental stem cell, HA-Mal-hPSC hydrogel, HA-Mal-CM hydrogel, HA-Mal hydrogel alone, and Seprafilm significantly reduced the collagen content of injured peritoneal tissues. Human placental stem cell and HA-Mal-hPSC hydrogel suppressed expression of the most profibrotic genes. Conditioned media, HA-Mal hydrogel alone, and media alone significantly altered the expression of proteins associated with peritoneal fibrotic pathways.

Human placental stem cell-based therapies reduce abdominal adhesions in a prospective randomized preclinical trial. This effect is supported by suppression of profibrotic genomic and proteomic pathways.

A hamster adhesion model was used to investigate the mechanism by which polyglycolic acid (PGA) sheets reinforce the surgical site through the acceleration of postoperative adhesion formation.

After receiving electrocautery burns on the inside of the abdominal wall, the hamsters were divided into the PGA group (a PGA sheet was placed on the burned area) and a non-treated group (a PGA sheet was not placed). The degree of adhesion was evaluated at 3, 14, 28, and 56 days after burn injury, and the mRNA levels of myeloperoxidase (MPO), tumor necrosis factor (TNF)-α, and transforming growth factor (TGF)-β1 at the surgical sites were measured.

Adhesion formation was observed 3 days after the burn injury in the non-treated group, but it decreased at 14, 28, and 56 days. On the other hand, a significant increase in adhesion formation was observed at 3 days in the PGA group relative to the non-treated group, with the increase continuing at 14 and 28 days. Significant increases in MPO, TNF-α, and TGF-β1 mRNA levels at the adhesion site were observed 3 days after the burn injury in both groups, with the increase continuing in the PGA group, but not in the non-treated group, at 14 and 28 days.

Acceleration of adhesion formation by PGA may be associated with upregulated MPO, TNF-α, and TGF-β1 mRNA levels.

Repeat cesarean deliveries (CDs) pose a risk in the development of intra-abdominal adhesions.

We aimed to examine the incidence and severity of adhesions in repeat CDs using a specific scoring system and assess the predictive power of the pre-operative value of transforming growth factor (TGF)-β and interleukin (IL)-6 with selected peripheral inflammatory biomarkers (PIBs) in the prediction of adhesion formation.

This prospective study enrolled 91 pregnant women at term, who had previously undergone at least one or more scheduled CDs. PIBs, namely C-reactive protein, white blood cell count, neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and systemic immune-inflammation index, TGF-β, and IL-6 were studied according to the presence and location of adhesions.

There was a significant difference only in the variables of the number of CDs, post-operative adhesion index (PAI) score, IL-6, and TGF-β on the presence of adhesion (p < 0.05). The linear regression model revealed that the effect of the number of CDs, PAI score, and IL-6 values on TGF-β was significant (p < 0.05). The effect of the PAI score on TGF-β was higher than that of IL-6. As a reciprocal relationship, the effect of the TGF-β value on the PAI score was also higher than that of IL-6.

In patients with a history of repeat CDs, the preoperative determination of TGF-β seems to be an important independent predictor of POA. The adverse events due to post-operative adhesion caused by repeat CDs can be overcome by detecting high-risk patients with a comprehensive assessment and individualized intervention integrated into overall patient management.

Surgery is performed to treat various diseases. During the process, the surgical site is healed through self-healing after surgery. Post-operative or tissue adhesion caused by unnecessary contact with the surgical site occurs during the normal healing process. In addition, it has been frequently found in patients who have undergone surgery, and severe adhesion can cause chronic pain and various complications. Therefore, anti-adhesion barriers have been developed using multiple biomaterials to prevent post-operative adhesion. Typically, anti-adhesion barriers are manufactured and sold in numerous forms, such as gels, solutions, and films, but there are no products that can completely prevent post-operative adhesion. These products are generally applied over the surgical site to physically block adhesion to other sites (organs). Many studies have recently been conducted to increase the anti-adhesion effects through various strategies. This article reviews recent research trends in anti-adhesion barriers.

Intrauterine adhesions (IUAs) can be dissected using hysteroscopic scissors (cold scissors) or other methods, but there is no consensus on which hysteroscopic method is preferable. There is also no consensus on the method of how to deal with the scar tissue on the surface of the intrauterine cavity.

From January 2016 to October 2017, 179 patients who had HA met the enrollment criteria (see the text below), and their data were analyzed retrospectively. In addition, all patients were divided into three groups according to the surgical techniques used. The groups were the ploughing group (PG) (using cold scissors to dissect the adhesion and cut the scar tissue using a ploughing technique) (n=81), the traditional group (TG) (using cold scissors to dissect the adhesion, but not deal with the scar tissue) (n=42), and the electrosurgical group (EG) (using a resectoscope to dissect the adhesion with an energy L-hook electrode, and not deal with the scar tissue) (n=56). Safety (surgical complications), feasibility (surgical technique replacement rate), and postoperative efficacy (reduction of AFS score, pregnancy, and live birth rate), were each evaluated between groups.

No statistically significant differences between the groups were observed in basic preoperative information (P>0.05), while there were significant differences between PG and TG, as well as PG and EG in postoperative AFS scores (PG vs. TG: P=0.007; PG vs. EG: P<0.001) and pregnancy outcome (PG vs. TG: P=0.039; PG vs. EG: P<0.001). No patients had surgical complications such as uterine perforations, moderate or severe fluid overload, heavy uterine bleeding, nor any surgical technique replacements (for example, transfer to use a resectoscope).

Cold scissors ploughing technique in HA is effective, feasible, and safe, and thus worthy of further study.

Postoperative peritoneal adhesions (PPAs) constitute a common complication of abdominal surgery with a high incidence. Bletilla striata (BS) is an important hemostatic drug used in China for nearly 2000 years. The purpose of this study was to investigate the effect of Bletilla striata on postoperative intestinal adhesion in rats. PPA was induced by cecal wall abrasion, and Bletilla striata was injected to observe its effect on adhesion in rats. The adhesion and inflammation score were assessed through visual observation and histopathologic evaluation. The levels of interleukin-1 (IL-1β), tumor necrosis factor (TNF-α), and interleukin-17F (IL-17F) in abdominal cavity and interleukin-6 (IL-6) in plasma were measured by enzyme-linked immunosorbent assay (ELISA) at 6 hours, 12 hours, 24 hours, and 1 week after operation. The tissue level of transforming growth factor beta-1 (TGF-β1) was also determined by ELISA on the seventh day after surgery. The expressions of collagen and TNF-α were, respectively, detected by Masson trichrome staining and immunohistochemical staining. The expression of TGF-β1 and alpha smooth muscle actin (α-SMA) was detected by Western blot. The result showed that Bletilla striata has obvious preventive effect on PPAs and celiac inflammation of PPAs. Bletilla striata could significantly reduce the level of IL-17F abdominal cavity and IL-6 in plasma. Masson trichrome staining and immunohistochemical staining results showed that Bletilla striata also decreased the expression of TNF-α and collagen. Western blot results showed that Bletilla striata decreased the expression of α-SMA and TGF-β1. Our results suggest that Bletilla striata decreased the development of abdominal adhesion in abrasion-induced model of rats and reduced the expression of the important substance which increased in PPAs. Bletilla striata can be further studied as a new and cheaper antiadhesive substance.

Adhesion formation that occurred after alkali-induced injury of the cecum was used as a novel adhesion model in rats, and it was compared with that of a common adhesion model after abrading the cecum. Using the novel adhesion model, inhibition of adhesion formation by a chymase inhibitor, Suc-Val-Pro-PheP(OPh)2, and by sodium hyaluronate/carboxymethylcellulose (Seprafilm) was evaluated, and their mechanisms were assessed. The degree of adhesion formation was more severe and more stable in the alkali-induced injury model than in the abrasion-induced injury model. Both the chymase inhibitor and Seprafilm showed significant attenuation of the degree of adhesion 14 days after alkali-induced injury. Chymase activity in the cecum was significantly increased after alkali-induced injury, but it was significantly attenuated by the chymase inhibitor and Seprafilm. Myeloperoxidase and transforming-growth factor (TGF)-β levels were significantly increased after alkali-induced injury, but they were attenuated by both the chymase inhibitor and Seprafilm. At the level of the adhesions, the numbers of both chymase-positive cells and TGF-β-positive cells were significantly increased, but their numbers were reduced by the chymase inhibitor and Seprafilm. In conclusion, a chymase inhibitor attenuated the degree of adhesions to the same degree as Seprafilm in a novel peritoneal adhesion model that was more severe and more stable than the common adhesion model, and not only the chymase inhibitor, but also Seprafilm reduced the chymase increase at the adhesions.

The left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas.

Regulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3β, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas.

In clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3β showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3β, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3β resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3β suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively.

Upregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2.

Over the past several years, there has been increasing recognition that pathogenesis of adhesion development includes significant contributions of hypoxia induced at the site of surgery, the resulting oxidative stress, and the subsequent free radical production. Mitochondrial dysfunction generated by surgically induced tissue hypoxia and inflammation can lead to the production of reactive oxygen and nitrogen species as well as antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase which when optimal have the potential to abrogate mitochondrial dysfunction and oxidative stress, preventing the cascade of events leading to the development of adhesions in injured peritoneum. There is a significant cross talk between the several processes leading to whether or not adhesions would eventually develop. Several of these processes present avenues for the development of measures that can help in abrogating adhesion formation or reformation after intraabdominal surgery.

To evaluate inflammatory/angiogenic cytokines-interleukin-1β (IL-1β), IL-6, IL-8, IL-12, interferon-γ (IFN-γ), tumor necrosis factor (TNF), and vascular endothelial growth factor A (VEGF-A)-in the peritoneal fluid of patients with endometriosis in relation to the occurrence and severity of pelvic adhesions and in control women without pelvic pathology.

Case-control study.

University research institution and hospital.

Sixty-five women with laparoscopically and histopathologically confirmed endometriosis, including 40 women with pelvic adhesions, and 37 control women without pelvic pathology.

Peritoneal fluid aspirated during routine diagnostic laparoscopic examination.

Cytokines evaluated in the peritoneal fluid via specific enzyme-linked immunosorbent assays.

Endometriosis and the revised American Fertility Society score of this disease were associated with statistically significantly increased levels of peritoneal IL-6 and IL-8 whereas the incidence and score of endometriosis-related pelvic adhesions were negatively associated with increased levels of VEGF-A. Notably, the concentration of VEGF-A predicted adhesion development and severity after adjustment for endometriosis severity. The adhesion score also correlated with increased levels of IL-6; however, after adjustment for endometriosis severity, the effect of this cytokine was no longer statistically significant.

Increased levels of VEGF-A may be associated with a decreased rate of pelvic adhesion formation in the course of endometriosis.

Adhesions are the most frequent complication of abdominopelvic surgery, yet the extent of the problem, and its serious consequences, has not been adequately recognized. Adhesions evolved as a life-saving mechanism to limit the spread of intraperitoneal inflammatory conditions. Three different pathophysiological mechanisms can independently trigger adhesion formation. Mesothelial cell injury and loss during operations, tissue hypoxia and inflammation each promotes adhesion formation separately, and potentiate the effect of each other. Studies have repeatedly demonstrated that interruption of a single pathway does not completely prevent adhesion formation. This review summarizes the pathogenesis of adhesion formation and the results of single gene therapy interventions. It explores the promising role of combinatorial gene therapy and vector modifications for the prevention of adhesion formation in order to stimulate new ideas and encourage rapid advancements in this field.

To compare the expression profiles of transforming growth factor-beta 1 (TGF-β1) and its receptors in occluded tubes of infertile women with those of control patients and to evaluate the potential correlation with postsurgical pregnancy outcome.

The expression profiles of TGF-β1, TGF-β1R1, and TGF-β1R2 in occluded fallopian tubes were compared using immunohistochemistry between 60 infertile patients with adhered tubes and 60 control patients with normal tubes; potential correlations with postsurgical fertility were evaluated at 2-year follow up.

Immunostainings of TGF-β1, TGF-β1R1, and TGF-β1R2 were all significantly elevated in patients with adhered tubes compared with normal specimens (P<0.001). In adhered specimens, correlation analyses showed positive correlations between TGF-β1 and TGF-β1R1 (P=0.008), and TGF-β1 and TGF-β1R2 (P=0.035). At 2-year follow up, 32 of the 60 infertile women had achieved normal pregnancies, 5 had had ectopic pregnancies, and 23 remained infertile. Correlation analysis showed that TGF-β1 expression level was negatively correlated with pregnancy outcome (r=-0.445, P<0.001), independent of adhesion severity or patient age.

TGF-β1 expression was independently correlated with the postsurgical pregnancy outcome among infertile women.

Postoperative intra-abdominal adhesion is associated with high morbidity and mortality. Smad7, a protein that occupies a strategic position in fibrogenesis, inhibits the transforming growth factor (TGF) beta/Smad signalling pathway. In this study the therapeutic potential of exogenous Smad7 in preventing fibrogenesis in postoperative intra-abdominal adhesion was investigated.

Intra-abdominal adhesion was induced in a rodent model by peritoneal abrasion. Smad7 was delivered into the peritoneal cavity by a non-viral ultrasound-microbubble-mediated naked gene transfection system. The effect of Smad7 transgene on adhesion formation was studied by measuring changes in TGF-beta, fibrogenic factors, alpha-SMA and Smad2/3 activation in the anterior abdominal wall.

Four weeks after surgical abrasion, all rats developed significant peritoneal adhesion with enhanced TGF-beta expression, increased levels of extracellular matrix components and activated myofibroblasts, accompanied by decreased Smad7 expression and increased Smad2/3 activation. In rats treated with the Smad7 transgene, the incidence and severity of peritoneal adhesion were significantly reduced, with biochemical downregulation of fibrogenic factors and inhibition of Smad2/3 activation. Serial quantitation using magnetic resonance imaging revealed a significant reduction in adhesion areas from day 14 onwards.

Ultrasound-microbubble-mediated gene transfection provides timely targeted gene delivery for the treatment of postoperative peritoneal adhesions.

The peritoneum is a serous membrane, which has a protective function for the contents of the abdominal cavity. It maintains homeostasis by allowing exchange of molecules and production of peritoneal fluid, thus providing an environment in which intra-abdominal organs can function properly. When traumatized, whether by surgery or due to inflammatory processes, a series of responses come into action to regenerate the injured part of the peritoneum. The inflammatory reaction causes influx of inflammatory cells but also activates resident mesothelial cells, ultimately leading to a fibrinous exudate. Depending on the severity of the trauma this exudate is transient due to fibrinolysis, or becomes more dense as a result of fibroblasts persisting, leading to fibrinous adhesions. A pivotal role is taken by the enzyme plasmin and its promotors and inhibitors; it is mainly the tissue-type plasminogen activator/plasminogen activator inhibitor ratio which determines the rate of fibrinolysis and therefore the rate of adhesion formation. The rate of injury determines the rate and extent of the inflammatory response to that injury; in its turn the inflammatory reaction determines the extent of adhesion formation. One should realize this when performing intra-abdominal surgery, which is in fact operating inside the peritoneal organ.

Peritoneal injury is often associated with alterations of the mesothelium, resulting in peritoneal healing and adhesion formation. We analyzed the effects of pro-inflammatory cytokines on cell morphology and proliferation of human peritoneal mesothelial cells (HPMC). After 48 hours, HPMC formed a confluent layer with cell volumes of 2,662+/-111 fL. Treatment of HPMC with interleukin-1beta and tumor necrosis factor-alpha (TNF-alpha) induced mesothelial disintegration and alterations in mesothelial cell morphology, which were associated with an interleukin-1beta-triggered increase in cell volume (3,028+/-118 fL; p<0.05) and exfoliation of cells into the supernatants of cell cultures (p<0.05). Whereas TNF-alpha arrested HPMC in the G0/G1 phase (p<0.05), interleukin-1beta caused an increase of cells into the S phase of the cell cycle. In addition, interleukin-1beta and interferon-gamma exerted a proliferative effect on HPMC. These changes were independent from mesothelial Na+/H+ antiporter-1 expression. Our data indicate that the response of HPMC to inflammatory injury is regulated by interleukin-1beta and TNF-alpha reflecting their putative role in peritoneal wound healing and adhesion formation.

To determine the response of adhesion and normal peritoneal fibroblasts to interferon-gamma (IFN-gamma) under normal and hypoxic conditions.

Prospective experimental study.

University medical center.

Primary cultures of fibroblasts established from peritoneal and adhesion tissue of the same patients.

Hypoxia and IFN-gamma treatment of the primary cultured fibroblasts.

Primary cultures of fibroblasts were established from peritoneal and adhesion tissues of the same patients (n = 5). The expression of extracellular matrix components (type I collagen and fibronectin) in adhesion and peritoneal fibroblasts under normal (20% O2) and hypoxic (2% O2) conditions was evaluated by multiplex reverse-transcription polymerase chain reaction analysis.

Adhesion fibroblasts (ADF) have increased basal levels of type I collagen as compared with normal peritoneal fibroblasts (NF). Interferon-gamma treatment resulted in a dose-response decrease in type I collagen and fibronectin mRNA levels in both ADF and NF. Hypoxia treatment resulted in a time-response increase in type I collagen and fibronectin mRNA levels in NF and ADF. Hypoxia had no effect on type I collagen and fibronectin mRNA levels in the presence of increasing dose of IFN-gamma in both NF and ADF. Interferon-gamma can block the stimulating effects of hypoxia on type I collagen expression, supporting the antifibrogenic nature of this cytokine.

Understanding the mechanism by which IFN-gamma exerts its effect will be important in the utilization of this cytokine as a therapy for postoperative adhesion and tissue fibrosis.

Transforming growth factor-beta (TGF-beta), a multifunctional growth factor, represents three mammalian isoforms, TGF-beta1, TGF-beta2, and TGF-beta3. In cutaneous wound healing, combined neutralization of TGF-beta1 and -beta2 or addition of TGF-beta3 reduces scar formation. Here, we investigated whether experimental manipulation of TGF-beta isoforms reduced adhesion formation after injury to the peritoneum. Adhesions were produced in mice by surgical abrasion of adjacent serosa followed by close apposition. In the first part of this study, a detailed analysis of TGF-beta isoform distribution was performed through immunolocalization. TGF-beta isoforms clearly showed a unique temporal and spatial pattern of expression after peritoneal wounding. Based on this pharmacokinetic data, we next administered neutralizing antibodies to TGF-beta1 and -beta2 or exogenous TGF-beta3 peptide by local application and intraperitoneal injection at various times before and after surgery. At day 7 after surgery, addition of neutralizing antibodies to both TGF-beta1 and -beta2 significantly reduced the number and size of adhesions (P < 0.05) compared with the vehicle control. By contrast, exogenous addition of TGF-beta3 either had no effect or increased adhesion formation compared to the vehicle control. In conclusion, these results show that by blocking both TGF-beta1 and TGF-beta2 using neutralizing antibodies, it is possible to prevent abdominal adhesion formation.

This study was to evaluate the feasibility of using an acellular bovine pericardium fixed with genipin (AGP) to repair an abdominal wall defect created in a rat model.

The glutaraldehyde-fixed acellular pericardium (AGA), the genipin-fixed cellular pericardium (GP), and a commercially available polypropylene mesh were used as controls.

Gross examination at 3-month post-operatively revealed that dense adhesions to the visceral organs were observed for the polypropylene mesh and the AGA patch, while a filmy to dense adhesion was seen for the GP patch. In contrast, no adhesion to the visceral organs was observed for the AGP patch. Histologically, inflammatory cells were found mainly surrounding the GP patch. In contrast, host cells (inflammatory cells, fibroblasts, and neo-capillaries) were able to infiltrate into the AGA and AGP patches. Unlike the AGA patch, the AGP patch retrieved at 1-month post-operatively became well integrated with the host tissue near the suture line. Additionally, there were some mesothelial cells, identified by the van Gieson stain, observed on the AGP patch. At 3-month post-operatively, a neo-peritoneum was observed on the AGP patch. The neo-peritoneum consisted of organized vascularized connective tissues covered by an intact layer of mesothelial cells. The calcium contents of the polypropylene mesh and the AGA patch increased significantly at 3-month post-operatively, while those of the GP and AGP patches stayed minimal throughout the entire course of the study.

The results obtained in the study revealed that the AGP patch effectively repaired abdominal wall defects in rats and successfully prevented the formation of post-surgical abdominal adhesions.

To quantify the expression of transforming growth factor beta1 in nerve fibers in endometriotic lesions and to correlate it with dysmenorrhea and appearance of endometriotic implants.

Prospective comparative study.

University hospital.

Peritoneal endometriotic specimens obtained from 35 patients diagnosed with endometriosis were compared with biopsies of normal peritoneum from 10 patients without endometriosis.

Endometriosis-associated dysmenorrhea for each patient was evaluated before surgery using a 10-point visual analog scale, which was followed by a laparoscopic staging of the patient's endometriosis.

Immunohistochemical analysis of the peritoneal endometriotic specimens evaluated the maximal intensity of staining (INTMMAX) of TGFbeta1, defined as higher staining intensity found within a selected structure.

When the nerve fibers of endometriotic lesions were compared with those of normal peritoneum, statistically significant differences were found in the INTMMAX of TGFbeta1. Greater TGFbeta1 INTMMAX was found in red lesions and deep endometriotic foci than in black lesions and normal peritoneum. A statistically significant relationship was found between the TGFbeta1 INTMMAX score and dysmenorrhea; a relationship also was found to the color of the lesions.

The physical appearance of endometriotic implants and the severity of dysmenorrhea appear to be related to the expression of TGFbeta1 in nerve fibers.

To examine the role of aerobic metabolism in fibroblasts from normal peritoneum and adhesions in the differential expression of extracellular matrix (ECM) and transforming growth factor-beta (TGF-beta), an inflammatory cytokine that regulates ECM expression.

Cell culture under normoxic and hypoxic conditions.

University research laboratory.

Human fibroblasts cultures from normal peritoneum and adhesions.

Exposure to dichloroacetic acid (DCA), which activates pyruvate dehydrogenase, for 24 hours under normal and hypoxic (2% O(2)) conditions.

Multiplex reverse transcriptase polymerase chain reaction (RT/PCR) of type III collagen, fibronectin, TGF-beta1, and beta-actin was performed, with analysis of PCR-amplified products performed by densimetric analysis of gel bands using the National Institutes of Health Image analysis program.

DCA inhibited human peritoneal fibroblast and adhesion fibroblast TGF-beta1 mRNA expression under normoxic and hypoxic conditions. DCA also markedly reduced fibronectin and type III collagen expression under hypoxic conditions in fibroblasts from normal peritoneum and adhesions.

These observations provide further support for the suggestion that regulation of metabolic activity of peritoneal cells may provide a target for interventions designed to reduce postoperative adhesions.

Bowel perforation can lead to significant bacterial spillage, which may then cause septic peritonitis, characterized by a systemic inflammatory response and organ dysfunction. There are several reports that have shown that the development of peritoneal adhesions is dependent on inflammatory cytokine levels and that these adhesions can reduce bacterial spread, possibly by sealing off the cecum in the cecal ligation and puncture (CLP) model of septic peritonitis. There have not, however, been any studies that have utilized a strategy to accelerate tissue repair in order to seal off the injured cecum and reduce bacterial spread as well as ameliorate systemic inflammation. In the present study, we demonstrate that the administration of anti-gamma interferon (IFN-gamma) antibody (1.2 mg/kg of body weight, intravenously) accelerated tissue repair via increased fibrin deposition 12 and 24 h after CLP in rats. This increase in fibrin deposition was associated with peritoneal adhesion 24 h after CLP and a reduction in bacterial load compared to the bacterial load of rats given irrelevant antibody. Plasma fibrin levels, however, were not altered after IFN-gamma antibody administration, suggesting that the inhibition of IFN-gamma activity specifically increased fibrin deposition to the site of injury. Furthermore, plasma interleukin-6, used as a marker of systemic inflammatory response, was reduced in CLP rats given IFN-gamma antibody compared to that found in those given irrelevant antibody. These results suggest that the early inhibition of IFN-gamma activity in the CLP model is beneficial by accelerating fibrin deposition in cecal tissue to prevent bacterial spread and reduce the systemic inflammatory response. Importantly, increased fibrin deposition in the ceca was not associated with increased plasma fibrin whereas the latter may have detrimental effects associated with coagulation disorders.

To establish whether the concentration of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta (TGF-beta) is influenced by the presence or absence of adhesions, and whether the concentration of these mediators vary throughout the menstrual cycle.

Prospective case-control study.

Women undergoing laparoscopy in a university hospital in the United Kingdom.

Women undergoing laparoscopy for benign gynecological conditions.

Samples of peritoneal fluid were collected at diagnostic laparoscopy in one group, and at laparoscopy and serially during the first 48 hours after laparoscopic adhesiolysis in a second group. We correlated the concentrations of mediators in serially sampled peritoneal fluid during the 48 hours following laparoscopic adhesiolysis to the adhesion formation and reformation found during second-look laparoscopy.

The concentrations of MMP-9, TIMP-1, and TGF-beta in peritoneal fluid.

MMP-9 concentration was lower in the follicular phase than the luteal phase of the menstrual cycle. MMP-9 concentration was significantly lower in women with pelvic adhesions than in women with a normal pelvis. The MMP-9/TIMP-1 ratio is significantly higher in women with significant adhesions at second-look laparoscopy compared to women with minimal or no adhesions.

The components of extracellular matrix remodeling may play an important part in the adhesion formation/reformation process.

Interferon-inducible protein 10 (IP-10) is a key regulator of neutrophils, monocytes, and lymphocytes, cells infiltration whose secretory products play a key role in peritoneal wound healing. The objective of the present study was to determine whether IP-10 is expressed by parietal peritoneum and whether its temporal and spatial expression is altered following surgically induced peritoneal injury during healing. Peritoneal sidewall injuries were induced in mice (N = 60), and the severity of adhesions were graded at 12 hours and 1, 2, 4, and 7 days postsurgery. After collection of peritoneal washes, the injured peritoneum with associated adhesion and intact parietal peritoneum were collected to determine IP-10 mRNA and protein expression using quantitative reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, and immunohistochemistry. Peritoneal injury resulted in adhesion formation with increased severity by day 7 postsurgery. The intact parietal peritoneum expressed IP-10 mRNA, whose level of expression significantly increased following peritoneal injury and reached a maximum at day 4 (p = 0.001), declining to the uninjured control levels by day 7 post-injury. The level of IP-10 in peritoneal washes also increased as a result of peritoneal injury. Immunohistochemically, IP-10 was localized to various inflammatory and immune cells, adhesion fibroblasts, and mesothelial cells, and its intensity increased during the course of wound healing. In conclusion, we showed that parietal peritoneum expresses IP-10 and peritoneal tissue injury results in an elevated level of its expression throughout the early phase of wound healing. The results suggest that IP-10 and its elevated expression may play a role in peritoneal cellular activities that influence the early phases of tissue repair and, possibly, the development of peritoneal adhesions.

Interleukins (IL) 13 and 15 are key regulators of inflammatory and immune responses, processes that are central to endometriosis and associated abnormalities. The present study examined (1) whether ectopic endometrial tissue expresses IL-13 and IL-15 (2) if their expression differs compared with matched eutopic endometrium and control endometrium from normal fertile women, and (3) if peritoneal fluids (PF) content of these cytokines reflects the disease compared with PF from women with peritoneal adhesions unrelated to endometriosis and those without pelvic pathology.

The expression of IL-13 and IL-15 mRNA and protein was determined using quantitative RT-PCR, ELISA and immunohistochemistry.

Ectopic endometrium expresses IL-13 and IL-15 mRNA and protein with elevated levels compared with eutopic and control endometrium, irrespective of the phases of the menstrual cycle, with predominance in IL-13 expression. Endometrial epithelial cells were found to be the primary site of IL-13 and IL-15 expression. The PF content of IL-13 and IL-15 show a trend toward higher concentrations in women with adhesion and endometriosis, respectively, compared with fertile control without pelvic pathology.

Interleukins 13 and 15 are expressed in ectopic endometrium and present in PF of women with endometriosis and their elevated expression in ectopic endometrium suggests that these cytokines play a key role in local inflammatory/immune responses that are critical in endometriosis-associated abnormalities.

To comparatively analyse the expression of matrix metalloproteinase (MMP-3) and tissue inhibitor of MMP (TIMP-2) in serosal tissue of intraperitoneal organs and adhesions, as well as peritoneal fluid and serum of subjects with and without adhesions.

Cross sectional study.

Academic research centres.

Patients undergoing abdominal/pelvic surgery.

Messenger ribonucleic acid (mRNA) and protein expression using quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), ELISA and immunohistochemistry.

All the tissues examined express MMP-3 and TIMP-2 mRNA and protein at consistently varying levels ranging from 100- to 1000-fold and 2- to 10-fold mRNA and protein, respectively. Serosa of uterine, fallopian tube, ovary and partial peritoneum express a higher MMP-3 mRNA compared with small and large bowels and omentum, while TIMP-2 expression was less variable with the highest level found in uterine serosa (P < 0.05). The expression of MMP-3 and TIMP-2 mRNA in adhesions was not significantly different compared with parietal peritoneum. MMP-3 and TIMP-2 protein content in tissue extracts, peritoneal fluids and serum also varied with higher TIMP-2 than MMP-3 (P < 0.05). TIMP-2 levels were lower in serum of subjects with moderate/extensive adhesions compared with subjects without adhesions. Immunoreactive MMP-3 and TIMP-2 proteins were detected in various cell types in these tissues. There was no correlation between MMP-3 and TIMP-2 expression, and age, gender or menstrual status of subjects with or without adhesions.

MMP-3 and TIMP-2 are expressed at varying levels in serosal tissues of peritoneal organs and adhesions, with higher TIMP-2 than MMP-3. Based on the knowledge that tissue injury alters the expression of MMPs and TIMPs, such variations may predispose an organ to develop more adhesions than others. In addition, the results suggest that serum level of TIMP-2 may represent a marker for subjects who will form adhesions with greater severity.

To assess the presence of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP (TIMP-1) in peritoneal fluid and serum of subjects with and without adhesions.

Cross-sectional study.

Academic research centers.

Sixty-three patients who underwent abdominal/pelvic surgery.

MMP-1, TIMP-1, and MMP-1-TIMP-1 complex content.

ELISA.

Peritoneal fluids (PF) and sera of subjects with and without peritoneal adhesions contain MMP-1, TIMP-1, and MMP-1-TIMP-1 complex at varying levels with 10- to 100-fold higher TIMP-1 than MMP-1. Compared with serum, PF contains a lower level of MMP-1 in subjects with mild adhesions and without adhesions, higher TIMP-1 in subjects with extensive adhesions, and lower MMP-1-TIMP-1 complex in subjects with moderate adhesions. However, the serum and PF content of MMP-1, TIMP-1, and MMP-1-TIMP-1 complex was not statistically different among subjects with or without adhesions, with the exception of TIMP-1 in PF of subjects with extensive adhesions. MMP1-TIMP-1 ratio indicates that a major portion of MMP-1 is in complex with TIMP-1. There was no age- or gender-dependent difference in MMP-1 and TIMP-1 content in serum or PF.

Despite differences in MMP-1 and TIMP-1 levels in serum and PF of subjects with extensive and moderate adhesions, there is no correlation between MMP-1 and TIMP-1, with the exception of higher TIMP-1 in PF of subjects with extensive adhesions.

To compare expression of matrix metalloproteinase (MMP-1) and tissue inhibitor of MMP (TIMP-1) in serosal tissue of intraperitoneal organs and adhesions.

Prospective and cross-sectional study.

Academic research centers.

Patients undergoing abdominal or pelvic surgery.

MMP-1 and TIMP-1 expression.

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.

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.

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.

To assess the expression of integrin alpha v and beta 3 in the serosal tissue of intraperitoneal organs and adhesions in persons with and without adhesions.

Prospective study.

Academic research centers.

Fifty-seven patients undergoing abdominal or pelvic surgery.

Integrin alpha v and beta 3 messenger RNA (mRNA) expression was evaluated by quantitative reverse transcription polymerase chain reaction.

The serosal tissue of the parietal peritoneum, uterus, fallopian tubes, ovary, and the large and small bowel, as well as peritoneal adhesions, skin, fascia, subcutaneous tissue, and omentum, expresses integrin alpha v and beta 3 mRNA. The level of alpha v and beta 3 mRNA expression varied among these tissues; expression of the former substance was highest in uterine serosa and lowest in the skin and small bowel, and expression of the latter substance was highest in the fallopian tubes and skin and lowest in the uterine serosa. Parietal peritoneum and adhesions express equal levels of integrin alpha v; however, integrin beta 3 expression was >100-fold lower in adhesions than in peritoneum. The level of integrin beta 3 expression in omentum, small and large bowels, and subcutaneous tissue was 100-fold to 10,000-fold lower than in other tissues.

Serosal tissue of peritoneal organs and adhesions express variable levels of integrin alpha v and beta 3 mRNA. On the basis of such variation and the knowledge that tissue injury alters local integrin expression, integrins may play a key role in adhesion development, particularly in tissue with higher integrin expression.

To determine the response of adhesion and peritoneal fibroblasts to hypoxia.

Prospective experimental study.

University medical center.

Primary cultures of fibroblasts established from the peritoneal and adhesion tissues of the same patients (n = 2) to minimize genetic variations.

Hypoxia treatment of the primary cultured fibroblast.

Analyze the expression of extracellular matrix (ECM) components, metalloproteinases and their tissue inhibitors, growth factors, and cytokines in adhesion and peritoneal fibroblasts under normal and hypoxic conditions by reverse transcriptase/polymerase chain reaction analysis.

Compared to peritoneal fibroblasts, adhesion fibroblasts had a significant increase in the basal mRNA levels for collagen I, fibronectin, MMP-1, TIMP-1, TGF-beta 1, TGF-beta 2, and IL-10. Hypoxia resulted in a further increase in collagen 1, fibronectin, TIMP-1, TGF-beta 1, TGF-beta 2, IL-10, and IFN-gamma mRNA levels in both peritoneal and adhesion fibroblasts. The increase was more profound in adhesion fibroblasts.

Hypoxia induces molecular changes in both peritoneal and adhesion fibroblasts, creating a milieu that favors adhesion development. The effect of hypoxia was more profound on adhesion fibroblasts.

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.

Peritonitis is a major cause of intra-abdominal adhesion formation. The overexpression of transforming growth factor beta-1 (TGF-Beta1), a potent mitogen, chemoattractant, and stimulant for collagen synthesis by fibroblasts, has been linked to tissue fibrosis at various sites throughout the body including peritoneal adhesion formation. Hence we hypothesized that the mechanism(s) involved in peritonitis-induced adhesion formation may be mediated through the upregulation of TGF-Beta1 expression. Peritonitis was induced in rats by cecal ligation and puncture, while a control group underwent sham operation. Adhesions were scored and harvested from both groups at 0, 6 and 12 hours and at 1, 2, 4, 7, and 28 days. Tissue expression of TGF-Beta1 mRNA was determined by quantitative reverse transcription-polymerase chain reaction and TGF-Beta1 protein was localized by immunohistochemical analysis. Serum and peritoneal fluid TGF-Beta1 concentrations were quantified by enzyme-linked immunosorbent assay. Compared with sham operation, peritonitis was associated with a significantly greater incidence of abdominal adhesions and a significant increase in the levels of TGF-Beta1 mRNA expression at days 2, 4, and 7. Immunostaining intensity of TGF-Beta1 in adhesions from the peritonitis group also steadily rose through day 7. In peritoneal fluid, the ratio of active:total TGF-Beta1 was significantly increased in the peritonitis group on days 1, 2, and 4 compared with the sham group. These results suggest that peritonitis is associated with the upregulation of TGF-Beta1, a mechanism that may exacerbate adhesion formation.

To determine the temporal and spatial expression of granulocyte macrophage-colony stimulating factor (GM-CSF) and GM-CSF alpha and beta receptor mRNA and protein in human endometrium.

The endometrial expression of GM-CSF and GM-CSF receptor mRNA and protein was determined using competitive quantitative reverse transcription polymerase chain reaction (Q-RT-PCR), in situ hybridization, and immunohistochemistry.

Endometrium expresses GM-CSF and GM-CSF alpha receptor mRNA with maximal expression occurring during the mid-secretory phase (21.1 +/- 4.2 and 32.2 +/- 7.7 x 10(6) mRNA copies/microg total RNA) compared to the proliferative phase (1.46 +/- 0.4 and 7.5 +/- 0.5 x 10(6) copies) of the menstrual cycle, with a significant reduction (0.67 +/- 0.1 and 1.7 +/- 0.2 x 10(6) mRNA copies) during the post-menopausal period (P < 0.05). The endometrium expresses a significantly lower level of GM-CSF beta receptor mRNA (approximately 0.01 x 10(5) mRNA copies). Endometrial luminal and glandular epithelial cells are the primary site of GM-CSF mRNA and protein expression, while arteriole endothelial, stromal, and inflammatory cells are the primary site of GM-CSF alpha receptor protein. GM-CSF beta receptor protein has a similar cellular distribution as GM-CSF.

Temporal and spatial expression of GM-CSF and GM-CSF receptors in human endometrium during the menstrual cycle suggests that epithelial-derived GM-CSF in an autocrine/paracrine manner may influence various endometrial biological activities, local inflammatory response, and macrophage survival.

Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor-beta1, a key regulator of tissue fibrosis. For this purpose we used Met-5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U-937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor-beta1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP-1 (collagenase-1), MMP-3 (stromelysin-1), TIMP-1 and TIMP-2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP-1 than MMP-3 (p < 0.001). The mesothelial cells express significantly less MMP-1, higher MMP- 3 and similar levels of TIMP mRNA compared to macrophages. In a time-dependent manner, treatment of the mesothelial cells with transforming growth factor-beta1 resulted in a significant decrease in the expression of MMP-1, while increasing the expression of TIMP-1 mRNA (p = 0.05). In contrast, MMP-3 and TIMP-2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP-1 and TIMP-2 by mesothelial cells following transforming growth factor-beta1 treatments in a time-dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP-1 in the culture conditioned media of both cell types was found in complex with TIMP-1. The ratios of MMP-1/TIMPs production were significantly higher than MMP-3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor-beta1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor-beta1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.