Differential expression of TGF-ß1 and TGF-ß3 in serosal tissues of human intraperitoneal organs and peritoneal adhesions*

Nasser Chegini1,5, Kristina Kotseos1, Yong Zhao1, Barbara Bennett1, Frederick W. McLean1, Michael P. Diamond2, Lina Holmdahl3, James Burns4 and The Peritoneal Healing and Adhesion Multi-University Study (PHAMUS) Group

1 Department of Obstetrics and Gynecology, University of Florida, Gainesville, 2 Wayne State University, Detroit, USA, 3 Department of Surgery, Gothenburg University, Sweden and 4 Genzyme Corporation, Cambridge, MA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Elevated local expression of transforming growth factor (TGF-ß) has been associated with increased incidence of peritoneal adhesion formation. In this study we determine whether differences in basal expression of TGF-ß in serosal tissue of peritoneal organs correlate with incidence of adhesion formation. Serosal tissue of parietal peritoneum, uterus, oviduct, ovary, omentum, large and small bowels as well as adhesions, skin, fascia, subcutaneous tissue, peritoneal fluid and serum were collected from 57 subjects with/without adhesions who were undergoing abdominal/pelvic surgery. To determine TGF-ß1 and TGF-ß3 mRNA and protein expression, total RNA and protein were isolated from these tissues and along with the fluids, subjected to quantitative RT–PCR and enzyme-linked immunosorbent assay (ELISA) respectively. Tissue sections were immunostained for TGF-ß1 and TGF-ß3 protein. We found that TGF-ß1 and TGF-ß3 mRNA and protein are expressed in these tissues and present in peritoneal fluids and serum, with considerable variations in level of their expression. Comparatively, there was more variation in TGF-ß1 than TGF-ß3 expression without age or gender relation. Adhesions express a significantly higher TGF-ß1 mRNA and have the highest TGF-ß1:TGF-ß3 ratio, with lowest concentrations and ratio detected in omentum, small and large bowels; in contrast uterus expresses higher TGF-ß3, with lowest concentrations detected in subcutaneous tissue and large bowels (P < 0.05). A similar trend was also observed for total (active + latent) TGF-ß1 protein expression, with low active TGF-ß1 that was not significantly different among the tissue extracts and fluids. However, the lowest active:total TGF-ß1 ratio was found in adhesions and ovary. In subjects with adhesions, the adhesions express significantly more TGF-ß1 compared to parietal peritoneum (P < 0.05). Immunoreactive TGF-ß1 and TGF-ß3 protein were present in various cell types in these tissues with intensity reflecting their mRNA and protein expression. In conclusion, we provided evidence that serosal tissue of various peritoneal organs and adhesions express TGF-ß1 and TGF-ß3. Since TGF-ß is expressed differently in these tissues and tissue injury often alters the expression of TGF-ß, we propose that tissues with a higher basal expression of TGF-ß may become predisposed to develop more adhesions compared to others.

Key words: fibrotic disorder/inflammatory immune response/peritoneal adhesion/TGF-ß1/TGF-ß3


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The molecular environment that regulates the outcome of peritoneal wound healing and adhesion formation is not well defined. Analysis of peritoneal fluid and adhesion tissue in humans, and in surgically induced adhesions in animal models, suggests that alteration in local expression of cytokines, growth factors and proteases is a key factor in adhesion formation (Hershlag et al., 1991Go; Williams et al., 1991Go; Chegini et al., 1994aGo, 1994bGo, 1999Go Chegini et al., 2000a, 2000b; Kaidi et al., 1995aGo, 1995bGo; Lucas et al., 1996Go; Saba et al., 1996Go, 1998Go; Chegini, 1997Go; Rong et al., 1997Go; Holschneider et al., 1997Go; Dou et al., 1997Go; Holmdahl, 1997Go; Ivarssen et al., 1998; Wiczyk et al., 1998Go; Lin et al., 1998Go; Krause et al., 1999Go; Holmdahl et al., 2000; Ghellai et al., 2000Go). Most noticeable among the cytokines are changes in the expression of interleukins (IL), tumour necrosis factor (TNF-{alpha}), transforming growth factor beta (TGF-ß), IL-10 and IFN-{gamma} that regulate the inflammatory and immune responses, enhance tissue fibrosis and anti-inflammatory and anti-fibrotic activities respectively. These cytokines also regulate the expression of endoproteases such as serine proteases, i.e. plasminogen activators (PA) and matrix metalloproteinases (MMP) as well as their physiological inhibitors, plasminogen activator inhibitors (PAI) and tissue inhibitor of MMP (TIMP), thus controlling fibrin deposition and degradation, chemotactic migration of inflammatory cells and fibroblasts, cell growth and differentiation, angiogenesis and deposition of extracellular matrix (Border and Noble, 1994Go; Roberts, 1995Go; Clark and Coker, 1998Go; Sang, 1998Go; Parks, 1999Go; Ma et al., 1999Go). Co-ordinate occurrence of these processes that are critical to normal tissue repair, are also regulated by differential expression of these cytokines, while their unregulated expression appears to account for impaired healing including tissue fibrosis.

For instance, over-expression of TGF-ß1 has been implicated in fibrotic disorders at various sites throughout the body such as pulmonary fibrosis, glomerulonephritis, cirrhosis of the liver, and dermal scarring (Border and Noble, 1994Go, Roberts, 1995Go). Evidence that implicates TGF-ß in peritoneal adhesion formation comes from experiments showing elevated concentrations of TGF-ß in adhesion tissues and peritoneal fluid of patients with adhesions (Chegini et al., 1999Go; Holmdahl et al., 2000) and in surgically-induced adhesion formation in animal models (Williams et al., 1991Go; Chegini et al., 1994aGo; Lucas et al., 1996Go; Rong et al., 1997Go; Chegini 1997Go; Ghellai et al., 2000Go). Mice heterozygous for TGF-ß1 (+/-) have been shown to have significantly lower adhesions and expressed at least two-fold lower TGF-ß1 protein in their peritoneal fluids compared with wild type (+/+) animals as early as 2 h post-injury (Krause et al., 1999Go). Furthermore, post-operative peritoneal administration of TGF-ß1 has been shown to increase (Williams et al., 1991Go), while neutralizing antibodies directed against TGF-ß reduced the incidence of adhesion formation (Lucas et al., 1996Go).

Clinical observations indicate that peritoneal adhesions develop in the vast majority of subjects with more frequent occurrence with individual organ, subjects and surgical procedures as opposed to others (Ginsburg and Diamond, 1997Go; Ellis et al., 1999Go; Lower et al., 2000Go). The molecular basis for such a predisposition is not known. Since TGF-ß is a key regulator of tissue fibrosis, including peritoneal adhesions, we hypothesized that serosal tissue of the organs that develop adhesions more frequently express a higher basal level of TGF-ß, compared to others that develop less or no adhesions. To test this hypothesis we determined the expression of TGF-ß1 and TGF-ß3 mRNA and protein expression in serosal tissue of parietal peritoneum, uterus, oviduct, ovary, omentum, large and small bowels, and adhesions, and compared their expression in fascia, subcutaneous tissue and skin in subjects with and without adhesions who were undergoing abdominal/pelvic surgery.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
All the materials for isolation of cellular RNA, reverse transcription polymerase chain reaction (RT–PCR), enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry were purchased from commercial sources as previously described (Dou et al., 1996Go; Chegini et al., 1999Go). Human specific TGF-ß1 ELISA kit with limit of detection of 2 pg/ml was purchased from Promega Inc. (Cambridge, MA, USA). Portions of serosal tissue of parietal peritoneum (n = 33), uterus (n = 22), Fallopian tube (n = 7), ovary (n = 5), large and small bowels (n = 14), omentum (10) and adhesions (n = 16) as well as skin (n = 34), fascia (n = 8) and subcutaneous tissue (12) were collected from 57 patients scheduled to undergo abdominal/pelvic surgery. Of the 57 subjects, 12 were male and 45 were female, ranging in age from 24–83 years. Of the female subjects 32 were premenopausal and 13 postmenopausal, of whom 23 had previous invasive or non-invasive pelvic surgical procedures including Caesarean section, bilateral tubal intervention, appendectomy, ovarian cystectomy, hysterectomy and/or treatment for endometriosis. Based on their last menstrual period and endometrial histology nine of the premenopausal women were from the proliferative phase and 23 from the secretory phase of the menstrual cycle. Male patients were undergoing various gastrointestinal surgical procedures.

Prior to and following the initiation of surgical procedures, 10 ml of blood and peritoneal fluids were collected respectively. Peritoneal fluids were excluded if grossely contaminated, thus peritoneal fluids from 15 subjects were included in the analysis. Peritoneal fluids were also collected from six women, with normal pelvic anatomy, who were scheduled to undergo tubal ligation. The patient's pelvic findings at surgery were used to assess the type of adhesions and classified based on their severity as previously described (The Myomectomy Adhesion Multicenter Study Group, 1995Go). The specimens used in this study were collected at the University of Florida as well as Wayne State and Gothenburg Universities following standardization for handling and shipment, and after obtaining approval from the Institutional Review Board of the above universities prior to initiation of the study. All patients gave informed written consent prior to tissue collection.

After collection tissue pieces (serosal tissue of parietal peritoneum, uterus, Fallopian tubes, ovaries, large and small bowels) were divided into multiple portions, and several portions were snap frozen and stored in liquid nitrogen. The concentration of TGF-ß1 and TGF-ß3 mRNA expression was determined using total RNA isolated from each individual tissue and subjected to competitive Q-RT–PCR using an external cRNA standard as previously described (Dou et al., 1996Go). The cDNA was synthesized in a series of standard reactions each containing 2 µg of total RNA isolated from each tissue and several dilutions of cRNA standard (102 to 108 copies/reaction). The PCR products were separated on 2% agarose gels containing ethidium bromide, and images were captured using a Kodak 120 digital camera. The band intensities were determined and following normalization for their molecular weight, the ratio of the cRNA/sample band intensity was plotted against the copy number of cRNA and final mRNA quantity was determined where the ratio was equal to 1. The data were analysed by equation of best fit lines and reported as mean ± SEM of copies mRNA/µg of total RNA (Dou et al., 1999Go).

The concentration of TGF-ß1 protein content in tissue extracts, peritoneal fluids and serum was determined by ELISA specific for human TGF-ß1. Individual tissue pieces were homogenized in a homogenizing buffer and centrifuged at 10 000 g as previously described (Chegini et al., 2001). Peritoneal fluids and blood were centrifuged at 4000 g for 15 min. Total protein content of the tissue extracts and peritoneal fluid supernatants and serum was determined using standard protein assay kit (Bio-Rad, Hercules, CA, USA); aliquots were made and stored at –80°C until assayed. An equal volume of total protein from each preparation in duplicate was subjected to ELISA (Chegini et al., 1999Go). The results are reported as mean ± SEM of ng of TGF-ß1/mg of total protein.

For immunohistochemistry a portion of the tissues was immediately fixed in Bouins solution and paraffin embedded as previously described (Dou et al., 1996Go). Tissue sections 5 µm thick were prepared and immunostained for TGF-ß1 and TGF-ß3 proteins using monoclonal antibodies generated against human recombinant TGF-ß1 and TGF-ß3 (Oncogene Research Products, Boston, MA, USA) as previously described (Dou et al., 1996Go). Tissue sections immunostained with pre-absorbed antibodies or non-immune normal serum instead of the primary antibodies were used as control.

Where appropriate the data were expressed as the mean ± SEM. Parametric data were analysed using the unpaired Student's t-test and non-parametric data by Kruskal–Wallis test using computer software program SigmaStat (Jandel Co., San Rafael, CA, USA). A probability level of P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Standard RT–PCR indicates that all tissues examined in our study express TGF-ß1 and TGF-ß3 mRNA. For quantitative analysis of TGF-ß1 and TGF-ß3 mRNA expression we performed Q–RT–PCR, and a representative example is shown in Figure 1Go. Analysis of Q-RT–PCR results indicate that levels of TGF-ß1 and TGF-ß3 mRNA expression vary significantly among these tissues ranging from 10- to 1000-fold (Figure 2Go). The level of TGF-ß1 mRNA expression in serosal tissue of uterus, Fallopian tubes, ovaries and parietal peritoneum were similar, but significantly higher compared with omentum and small and large bowels that expressed the lowest level of TGF-ß1 mRNA (P < 0.001; Figure 2Go). In contrast, the serosal tissue of these organs express similar levels of TGF-ß3 mRNA with highest level detected in the uterus (P < 0.05; Figure 2BGo). Adhesions express a significantly higher level of TGF-ß1, but not TGF-ß3 mRNA compared with parietal peritoneum from all the subjects with or without adhesions (Figure 2A, BGo). In comparison, skin and subcutaneous tissues express a similar level of TGF-ß1 and TGF-ß3 mRNA to that found in parietal peritoneum. The ratio of TGF-ß1:TGF-ß3 mRNA is shown in Figure 2CGo indicating that adhesions and serosal tissue of the ovary and Fallopian tubes, as well as subcutaneous tissues, express more TGF-ß1 mRNA compared to omentum and small and large bowels that express more TGF-ß3 mRNA (P < 0.05).



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Figure 1. Competitive quantitative RT–PCR analysis of TGF-ß1 and TGF-ß3 mRNA using total cellular RNA isolated from parietal peritoneum. The upper bands are the PCR products generated from the specific message in cellular RNA and the lower bands are from the standard cRNA (shown from left to right at dilutions corresponding to 103 to 108 copies/reaction). The far left lanes are the DNA markers.

 



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Figure 2. The bar graphs show the levels of TGF-ß1 and TGF-ß3 mRNA expression in serosal tissue of parietal peritoneum (PERT), ovaries, uterus, Fallopian tubes (F. Tube), large (LBW) and small (SBW) bowels, omentum (OMNT), subcutaneous tissue (SC), adhesions (ADHS) and skin. The values were calculated from the band densities where the ratio of cRNA/sample RNA is equal to 1 and presented as mean ± SEM of copies of mRNA/µg total RNA. (A) c is different from a, b, e, f, g, h, j and k (P < 0.05) and h, j and k differ from others (P < 0.001). (B) d is different from a, b, c, f, g and k (P < 0.05). (C) shows the ratio of TGF-ß1:TGF-ß3 mRNA with adhesions and the serosal tissue of the ovary and Fallopian tubes, as well as subcutaneous tissues expressing more TGF-ß1 mRNA compared to omentum and small and large bowels that express more TGF-ß3 mRNA (P < 0.05).

 
The concentration of total TGF-ß1 protein in the tissue extracts was also variable, ranging from 2–10 ng/mg of protein with similar concentration of active TGF-ß1 (Figure 3Go). Ovarian serosal tissue contains the highest TGF-ß1 concentration, with the lowest concentration detected in the omentum and large bowels (P = 0.03, 0.02, 0.008; Figure 3AGo). Although adhesions contain higher TGF-ß1 content, these concentrations were not significantly different from the mean values found in intact parietal peritoneum from all subjects with or without adhesions (P = 0.06; Figure 3AGo). Analysis of the ratio between active:total TGF-ß1 in serosal tissue of interaperitoneal organs from all patients showed that the lowest ratio is present in the ovary and adhesion (P = 0.02, 0.01, 0.03; Figure 3BGo). The concentration of active and total TGF-ß1 (Figure 4Go) and active:total TGF-ß1 ratio (Figure 4DGo) in parietal peritoneum, adhesions and skin of subjects with adhesions showed a significant variations among and between the tissues and subjects (Figure 4A, BGo). However, comparing their mean values, we found a significantly higher total TGF-ß1 (Figure 4CGo) and lower active:total TGF-ß1 ratio (Figure 4DGo) in adhesions compared with parietal peritoneum and skin from the same subjects with adhesions (P < 0.05).



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Figure 3. Bar graph shows the concentration of active and total (active + latent) TGF-ß1 in extracts from serosal tissue of parietal peritoneum (Perit), ovaries, uterus, Fallopian tubes (F. Tube), large bowels (LBwl), omentum (Oment), skin, subcutaneous tissue (Subc), fascia and adhesions (Adhs), shown as mean ± SEM of TGF-ß1/mg of total protein (A). (B) shows the ratios between active/total TGF-ß1 in tissue extracts from parietal peritoneum (Perit), ovaries, uterus, Fallopian tubes (F. Tube), large bowels (LBwl), omentum (Oment), skin, subcutaneous tissue (Subc), fascia and adhesions (Adhs). In (A), c differs from a (P = 0.03), i (P = 0.02) and j (P = 0.008) and b differs from a, i and j (P < 0.05). In (B), * differs from **: adhesion, uterus and F. Tube (P = 0.02), ovary (P = 0.01) and oment (P = 0.03), adhesion versus peritoneum (P = 0.06) and skin (P = 0.05).

 


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Figure 4. Bar graphs show the concentrations of total (active + latent) and active TGF-ß1 content in adhesions, intact parietal peritoneum and skin presented individually for 10 subjects with adhesions (A and B), with mean ± SEM given in (C). (D) represents the ratio between active and total TGF-ß1 in these tissues. In (C), * is significantly higher compared with ** and in (D), ** is lower than skin and peritoneum (P < 0.05).

 
The concentration of total, but not active, or active:total TGF-ß1 was significantly lower in peritoneal fluid compared with serum of all subjects with or without adhesions (P = 00007; Figure 5A, BGo). In subjects with adhesions, the peritoneal fluid content of total TGF-ß1 was higher, but it did not reach statistically significant (P = 0.09). The serum concentration of TGF-ß1 in subjects with extensive, moderate or mild adhesions also was not significantly different; however, these concentrations were significantly higher compared with serum of subjects without adhesions (P = 0.0001, 0.001 and 0.003 respectively; Figure 6AGo). Furthermore, the ratio between active:total TGF-ß1 was significantly lower in serum of subjects with mild adhesions compared with those who had extensive and moderate adhesions or without adhesions (Figure 6BGo; P = 0.03). We did not find any correlation between TGF-ß1 and TGF-ß3 expression with age, gender, or menopausal status.



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Figure 5. Notched box plot (A) showing the concentration of TGF-ß1 in serum (n = 38) and peritoneal fluid (n = 15) of all the subjects with adhesions. The vertical line within the box boundaries represents the distribution of the middle 50%, the thin horizontal lines within the boxes are the median and the thick lines are the arithmetic mean of the factors assayed. The notches show the 95th and the error bars 90th and 10th percentiles. In (A), a is significantly different from b (P = 0.00007). (B) shows the ratio of active:total TGF-ß1 in serum and peritoneal fluids in these subjects.

 


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Figure 6. Notched box plot (A) showing the concentration of active and latent TGF-ß1 is serum of subjects with extensive (n = 13), moderate (n = 10) and mild (n = 15) adhesions and without adhesion (n = 14). B shows the ratio between active and total TGF-ß1 presented as the mean ± SEM. In A total TGF-ß1 in serum of subjects with extensive, moderate and mild adhesions (*) are significantly higher compared with those with no adhesion (**)(P = 0.0001, 0.001 and 0.003 respectively). In (B) the serum of subjects with mild adhesion (**) had a lower TGF-ß1 active:total ratio compared with those with extensive adhesions (P = 0.03), but not with other groups.

 
TGF-ß1 and TGF-ß3 were immunolocalized in the serosal cell layer of the parietal peritoneum, uterus, ovary, Fallopian tubes, large and small bowels, keratinocyte cell layer and subcutaneous fibroblasts in skin, and fibroblasts in adhesions and omentum (Figure 7Go). Representative micrographs show specific immunoreactive TGF-ß1 (Figure 7A, C, EGo) and TGF-ß3 (Figure 7BGo, D, F), in association with various cell types in skin (A, B), parietal peritoneum (C, D), and adhesion (E, F). Preabsorption or replacement of the primary antibodies with non-immune normal serum resulted in considerable reduction in immunostaining intensity associated with these cells (Figure 7GGo, H).



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Figure 7. Immunohistochemical localization of TGF-ß1 (A, C and E) and TGF-ß3 (B, D and F) in intact parietal peritoneum (A and B), adhesions (C and D) and skin (E and F). Note the presence of immunoreactive TGF-ß in association with the mesothelial cells (arrows), fibroblasts in sub-mesothelial tissue (A and B) and in adhesions (C and D) and keratinocyte cell layer (E and F). Pre-absorption of the primary antibodies with respective proteins or replacement with non-immune normal serum resulted in a considerable reduction in immunostaining over the immunostained area shown in skin as an example (G and H). Magnification x82. Bar = 100 µm.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Peritoneal mesothelial cells that line the serosal surface of the peritoneal cavity provide a natural protective barrier that prevents the organs from adhering to adjacent opposing surfaces. However, cellular/tissue injury induced following surgical procedures, infection or inflammation compromises the integrity of the mesothelial cells, which results in local biological response with the intention of repairing the defected surface. If cellular or tissue injury is relatively extensive, leading to excess migration and proliferation of various wound cells, i.e. fibroblasts, this response initiates a cascade of events that often results in the development of peritoneal adhesions. These adhesions are known to be the major cause of bowel obstruction, pain and infertility and hospital readmission (Ginsburg and Diamond, 1997Go; Ellis et al., 1999Go; Lower et al., 2000Go).

An array of molecules whose expression following cell/tissue injury and during the course of healing is altered has been considered to influence the outcome of peritoneal adhesions (Williams et al., 1991Go; Hershlag et al., 1991Go; Kaidi et al., 1995aGo, bGo; Lucas et al., 1996Go; Rong et al., 1997Go; Holschneider et al., 1997Go; Dou et al., 1997Go; Holmdahl, 1997Go; Chegini, 1997Go; Ivarssen et al., 1998; Saba et al., 1996Go, 1998Go; Wiczyk et al., 1998Go; Lin et al., 1998Go; Krause et al., 1999Go; Chegini et al., 1999Go Chegini et al., 2000a,b; Ma et al., 1999Go; Saed et al., 1999Go, 2000Go; Ghellai et al., 2000Go). Experimental data from peritoneal adhesions in human and animal models of surgically induced adhesions suggest that among these factors, the local over-expression of TGF-ß is a key to development of adhesions (Williams et al., 1991Go; Chegini et al., 1994aGo, 1999Go; Lucas et al., 1996Go; Rong et al., 1997Go; Dou et al., 1997Go; Chegini, 1997Go; Krause et al., 1999Go; Ghellai et al., 2000Go; Saed et al., 2000Go; Holmdahl et al., 2001). In the present study, which is the first comparative analysis of the expression of TGF-ß1 and TGF-ß3 in serosal tissue of intraperitoneal organs and adhesions, we demonstrated that TGF-ß1 and TGF-ß3 mRNA expression in these tissues varied considerably ranging from 10- to 1000-fold. Comparatively, adhesions express a higher concentration of TGF-ß1, with large bowel and omentum expressing the lowest concentrations, while TGF-ß3 expression was relatively less variable than TGF-ß1 with uterine serosa expressing the highest concentration. Although the effect of length of time since these adhesions were formed is not known, the results provide further evidence supporting the involvement of TGF-ß in development of adhesions. Consistent with these observations we have recently reported an increased concentration of TGF-ß1 expression in adhesions by as much as 15-fold within 4 days following the induction of peritonitis in the rat (Ghellai et al., 2000Go). Such an elevated level of TGF-ß1 expression has also been demonstrated in adhesions and peritoneal fluids of subjects with peritoneal inflammation, endometriosis and in surgically-induced peritoneal adhesions in animals (Chegini et al., 1994aGo, 1999Go; Osterlynek et al., 1994Go; Chegini, 1997Go; Chegini and Williams, 1997Go; Rong et al., 1997Go; Krause et al., 1999Go; Ghellai et al., 2000Go; Holmdahl et al., 2001).

Consistent with the role of TGF-ß in tissue fibrosis (Border and Noble, 1994Go; Roberts, 1995Go; Chegini, 1997Go), post-operative intraperitoneal administration of TGF-ß has been shown to increase, while neutralization of TGF-ß reduces the incidence of adhesion formation (Williams et al., 1991Go; Chegini et al., 1994aGo; Lucas et al., 1996Go; Chegini, 1997Go). Since cellular and tissue injury increases the expression of TGF we propose that the serosal surface of peritoneal organs that expresses a higher basal concentration of TGF-ß may become more predisposed to form adhesions than others following injury. Moreover, our data suggest that TGF-ß1 may play a more specific role influencing the outcome of adhesion formation compared to TGF-ß3. This is due to higher expression and more variation in the concentration of TGF-ß1 mRNA in peritoneal serosal tissues and adhesions compared to TGF-ß3. Beside transcriptional regulation of TGF in local tissue fibrosis, regulation of their protein expression and activation is critical in this process. Although we did not measure TGF-ß3 protein content in the tissue extracts and fluids, the concentration of TGF-ß1 protein in the tissue extracts varied substantially as seen with TGF-ß1 mRNA expression. This suggests that TGF-ß1 expression in these tissues may be regulated differently at the translational level. Interestingly, TGF-ß is synthesized and secreted by cells as latent or biologically inactive forms that upon secretion can become associated with extracellular matrix (ECM) or with the cell membrane, acting as an additional level of control for TGF-ß local action (Noble et al., 1992Go; Miyazono et al., 1993Go; Clark and Coker, 1998Go). A variety of matrix proteins including biglycan, decorin, type IV collagen, fibronectin, and thrombospondin can associate with TGF-ß sequestered into the ECM, in addition to covalent and non-covalent complex formation with ß2-macroglobulin (Miyazono et al., 1993Go; Roberts, 1995Go). These proteins are expressed by serosal tissue of the peritoneal organs and are present in the peritoneal fluid (Ramey and Archer, 1993Go; Witz et al., 1998Go; Saed et al., 1999Go). The assay used in our study does not allow differentiation between the matrix and cell-associated TGF-ß in the tissue extract; however, immunohistochemical observations revealed that all cell types in these tissues contain immunoreactive TGF-ß1 and TGF-ß3.

Matrix-associated TGF-ß can become activated through a mechanism that includes proteolytic processes, low pH, and binding to thrombospondin (Roberts, 1995Go; Clark and Coker, 1998Go). Endoproteases such as sialadases and serine proteases that activate TGF-ß are secreted by inflammatory cells, which infiltrate the tissue following ischaemia and injury (Harpel et al., 1992Go; Schultz-Cherry and Murphy-Ullrich, 1993Go; Grainger et al., 1995Go; Roberts, 1995Go; Clark and Coker, 1998Go). Such a proteolytically active environment, characteristic of the early stage of wound healing, may serve to release the matrix-associated TGF-ß for immediate local action before initiation of TGF gene activation (Falcone et al., 1993Go; Grainger et al., 1995Go; Rougier et al., 1998Go). Interestingly, TGF-ß can up-regulate its own expression; thus, activated TGF-ß released from sequestrated local environment may be critical in the early stage of wound healing and the outcome of adhesion formation. Although alteration in local tissue expression of TGF is a critical factor in the outcome of tissue fibrosis, we have previously reported that peritoneal fluids of women with adhesion, particularly those who had extensive adhesions and endometriosis, contain significantly higher concentration of TGF-ß1 compared with those without adhesions (Chegini et al., 1999Go). The result of our present study indicates that the peritoneal fluid content of TGF-ß1 in subjects with adhesions is similar to those we have previously reported; however, its relation to severity of adhesions could not be determined due to inadequate number of samples for each category. Human peritoneal mesothelial cells have been shown to express several growth factors and cytokines including TGF-ß (Saed et al., 2000Go) that may serve as a source of TGF-ß in peritoneal fluid. In addition, TGF-ß in peritoneal fluid can derive from serum; however, the concentration of TGF-ß in the serum was significantly higher than in peritoneal fluids. Interestingly, we found that regardless of severity of peritoneal adhesions, the serum concentration of TGF-ß1 is significantly higher in subjects with adhesions compared to those who had no adhesions. These observations suggest that alterations in TGF-ß expression at both local and systemic levels may influence the outcome of peritoneal adhesion. Therefore, circulating TGF-ß may serve as an indicator of peritoneal adhesions.

Despite the differences, most of the TGF-ß1 detected in the tissue extracts, peritoneal fluids and serum in subjects with and without adhesions was in latent form that requires activation before binding to TGF-ß1 receptors to exert its specific biological activity. Although the concentration of active TGF-ß1 in tissue extracts, peritoneal fluids and serum was low, we observed considerable variation in its levels in adhesions, peritoneum and skin of subjects with adhesions, expressing more TGF-ß1 and having a lower active:total TGF-ß1 ratio. Additionally, we have previously reported a higher concentration of active TGF-ß1 in peritoneal fluid of women with adhesions compared to women with normal pelvic anatomy (Chegini et al., 1999Go). Such low concentrations of active TGF-ß1 may be due to lack of availability rather than production, because active TGF-ß1 can immediately bind to its receptors which are present in all the cell types, or sequestrates by TGF-ß1 type III receptors in the ECM, a mechanism which controls TGF-ß action (Roberts, 1995Go; Lawrence, 1996Go; Clark and Coker, 1998Go; Rifkin et al., 1999Go). Latent TGF-ß1 has a considerably longer plasma half-life compared with active that may explain a higher concentration of total TGF-ß1 and reduction in active:total ratio, particularly in peritoneal serosal tissues compared with skin, subcutaneous tissue and fascia. We have previously demonstrated that the peritoneal fluid concentrations of total and active TGF-ß1 significantly increased during the first week post-injury in surgically-induced peritoneal wounds in mice (Rong et al., 1997Go). However, following induction of peritonitis in rats the concentrations of total or active TGF-ß1 in the peritoneal fluid did not significantly change, while the active:total ratio increased during the first 4 days, possibly due to increased activation of the latent TGF-ß under this environment (Ghellai et al., 2000Go). A higher influx of inflammatory cells during peritonitis, the major source of the proteases that activate the latent TGF-ß, may account for the differences. Unregulated balance between the active and latent TGF-ß1 which results in excessive activity has been demonstrated in fibroblasts obtained from normal and hypertrophic scar (Messadi and Bertolami, 1993Go). Our results indicate that adhesions had the lowest active:total TGF-ß1 ratio compared to serosal tissue of interaperitoneal organs.

Although the involvement of TGF-ß3 in peritoneal adhesion formation awaits detailed investigation, TGF-ß1 is reported to constitute more than 85% of the total TGF-ß detected in adult skin wound fluid (Longaker et al., 1994Go), making it a key isoform in peritoneal adhesion formation. Furthermore, it has been reported that neutralization of TGF-ß1 and TGF-ß2 or exogenous addition of TGF-ß3 to cutaneous rat wounds reduces the incidence of scarring (Shah et al., 1995Go). Similar wounds induced in transgenic TGF-ß1 over-expressing mice healed with reduced scarring, accompanied by an increase in immunostaining for TGF-ß3 and TGF-ß type II receptor and a decrease in TGF-ß1 compared with wounds in control mice (Shah et al., 1999Go). We have previously reported that intact rat parietal peritoneum contains lower concentrations of immunoreactive TGF-ß3 compared with TGF-ß1 and TGF-ß2 (Chegini et al., 1994aGo). However, the immunostaining intensity of all TGF-ß isoforms increased following surgically-induced adhesion, suggesting a lack of specificity for the action of TGF-ß isoforms in peritoneal adhesion formation (Chegini et al., 1994aGo). Furthermore, neutralization of both TGF-ß1 and TGF-ß3 equally resulted in reduced incidence of adhesion formation (Lucas et al., 1996Go). Interestingly, we found that the ratio of TGF-ß1:TGF-ß3 mRNA is the highest in peritoneal adhesions and the serosal tissue of the ovary and Fallopian tubes, as well as in subcutaneous tissues, while the lowest was found in omentum and small and large bowels. Although such a difference in TGF-ß1:TGF-ß3 ratios among these tissues appears to support the concept that an increase in TGF-ß3 and a decrease in TGF-ß1 expression may result in less tissue scarring, we can not rely solely on mRNA expression; an accurate and quantitative protein assay is required to support this hypothesis. Interestingly, it has been reported that TGF-ß3 accelerates skin wound healing without alteration of scar prominence (Wu et al., 1997Go).

The active form of TGF-ß is a potent chemotactic agent for fibroblasts and inflammatory cells such as macrophages, promotes angiogenesis, cell proliferation and differentiation, and stimulates the expression and deposition of extracellular matrix and integrin in fibroblasts, markedly altering their migration into the wound (Roberts, 1995Go; Clark and Coker, 1998Go; Arora et al., 1999Go). TGF-ß also regulates the expression of proteases that are critical not only in cellular migration, but also angiogenesis, cell proliferation, ECM expression, deposition and turnover (Roberts, 1995Go; Parks, 1999Go). These include fibrinolytic system, MMP and their inhibitors, PAI and TIMP (Roberts, 1995Go; Clark and Coker, 1998Go; Parks, 1999Go). In fibroblasts and peritoneal mesothelial cells, TGF-ß inhibits MMP-1, stimulates TIMP-1 expression and prevents plasmin generation by increasing the expression of PAI-1, allowing unopposed deposition of ECM (Border and Noble, 1994Go; Roberts, 1995Go; Parks, 1999Go; Ma et al., 1999Go; Saed et al., 1999Go, 2000Go; Flak et al., 2000Go). Parietal peritoneum, serosal surface of peritoneal organs, adhesions and peritoneal fluid express TGF-ß1, MMP, TIMP, tPA and PAI-1 in human and surgically-induced adhesions in animal models during the early stages of wound repair (Holmdahl, 1997Go; Chegini et al., 1999Go, 2001; Ghellai et al., 2000Go). Following cellular or tissue injury, excess production of TGF-ß can sustain the expression of ECM, PAI-1 and TIMP thus promoting matrix deposition leading to further impairment of normal peritoneal healing (Rougier et al., 1998Go). Even though peritoneal mesothelial cells are critical to peritoneal healing, it has been shown that these cells can undergo a reversible change into a fibroblastic phenotype, with increased adhesion and migration on type I collagen during wound healing (Leavesley et al., 1999Go), that can potentially become detrimental in promoting adhesion formation.

In conclusion, evidence is provided that TGF-ß is differentially expressed in serosal tissue of peritoneal organs and adhesions, with higher expression in adhesions and serum of subjects who had developed peritoneal scars. Since tissue injury and increased expression of TGF-ß is critical in peritoneal adhesion formation, it is proposed that the serosal surface of peritoneal organs with higher basal TGF expression may be more predisposed to form adhesions following injury. It is further proposed that TGF-ß1 may be more critical in altering the outcome of peritoneal adhesion formation; however, the potential involvement of TGF-ß3 remains to be further investigated.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The members of Peritoneal Healing and Adhesion Multi University Study (PHAMUS) Group are: University of Florida, Department of Obstetrics/Gynecology: Nasser Chegini, Barbara Bennett, Fredrick W.McLean, R.Stan Williams, Yong Zhao, Chunfeng Ma, Kristina Ketoses and Alpa Patel; Biomaterials Center, Department of Material Sciences and Engineering: Eugene Goldberg and Lynn Peck; Wayne State University: Michael P.Diamond, Ghassen Saed, Richard Leach, Karen Collins, Frank Yealin, David Svinvich and Yoram Serokin; University of Gothenburg, Sweden: Lena E.Holmdahl, Peter Falk, Marie-Louise Ivarsson, Maria Hedgren, Maria Bergstrom and Ingrid Palmgren; Genzyme Co: Jim Burns, Kevin Skinner and Cindy Nickerson. This work was supported by Genzyme Co., Cambridge, MA, USA. The author would like to acknowledge the crucial contributions of residents and nurses toward the collection of the tissues for this study, Dr Hossin Yarandi, College of Nursing, University of Florida for statistical analysis, Ruth Ann Klockowski and Landis Young, University of Florida, Department of Obstetrics/Gynecology for editorial assistance.


    Notes
 
5 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, University of Florida, College of Medicine, Box 100294, Gainesville, FL 32610–0294, USA. E-mail: cheginin{at}obgyn.ufl.edu Back

* This paper was presented in part at the 47th Annual Meeting of the Society for Gynecological Investigation, Chicago, IL, USA, 2000. Back


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 Introduction
 Materials and methods
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 Acknowledgements
 References
 
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Submitted on September 29, 2000; accepted on February 21, 2001.





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