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
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Abstract |
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Key words: fibrotic disorder/inflammatory immune response/peritoneal adhesion/TGF-ß1/TGF-ß3
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Introduction |
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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, 1994, Roberts, 1995
). 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., 1999
; Holmdahl et al., 2000) and in surgically-induced adhesion formation in animal models (Williams et al., 1991
; Chegini et al., 1994a
; Lucas et al., 1996
; Rong et al., 1997
; Chegini 1997
; Ghellai et al., 2000
). 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., 1999
). Furthermore, post-operative peritoneal administration of TGF-ß1 has been shown to increase (Williams et al., 1991
), while neutralizing antibodies directed against TGF-ß reduced the incidence of adhesion formation (Lucas et al., 1996
).
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, 1997; Ellis et al., 1999
; Lower et al., 2000
). 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.
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Materials and methods |
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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, 1995). 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-RTPCR using an external cRNA standard as previously described (Dou et al., 1996). 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., 1999
).
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., 1999). 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., 1996). 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., 1996
). 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 KruskalWallis test using computer software program SigmaStat (Jandel Co., San Rafael, CA, USA). A probability level of P < 0.05 was considered significant.
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Results |
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Discussion |
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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., 1991; Hershlag et al., 1991
; Kaidi et al., 1995a
, b
; Lucas et al., 1996
; Rong et al., 1997
; Holschneider et al., 1997
; Dou et al., 1997
; Holmdahl, 1997
; Chegini, 1997
; Ivarssen et al., 1998; Saba et al., 1996
, 1998
; Wiczyk et al., 1998
; Lin et al., 1998
; Krause et al., 1999
; Chegini et al., 1999
Chegini et al., 2000a,b; Ma et al., 1999
; Saed et al., 1999
, 2000
; Ghellai et al., 2000
). 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., 1991
; Chegini et al., 1994a
, 1999
; Lucas et al., 1996
; Rong et al., 1997
; Dou et al., 1997
; Chegini, 1997
; Krause et al., 1999
; Ghellai et al., 2000
; Saed et al., 2000
; 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., 2000
). 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., 1994a
, 1999
; Osterlynek et al., 1994
; Chegini, 1997
; Chegini and Williams, 1997
; Rong et al., 1997
; Krause et al., 1999
; Ghellai et al., 2000
; Holmdahl et al., 2001).
Consistent with the role of TGF-ß in tissue fibrosis (Border and Noble, 1994; Roberts, 1995
; Chegini, 1997
), 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., 1991
; Chegini et al., 1994a
; Lucas et al., 1996
; Chegini, 1997
). Since cellular and tissue injury increases the expression of TGF-ß 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-ß 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., 1992
; Miyazono et al., 1993
; Clark and Coker, 1998
). 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., 1993
; Roberts, 1995
). These proteins are expressed by serosal tissue of the peritoneal organs and are present in the peritoneal fluid (Ramey and Archer, 1993
; Witz et al., 1998
; Saed et al., 1999
). 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, 1995; Clark and Coker, 1998
). 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., 1992
; Schultz-Cherry and Murphy-Ullrich, 1993
; Grainger et al., 1995
; Roberts, 1995
; Clark and Coker, 1998
). 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-ß gene activation (Falcone et al., 1993
; Grainger et al., 1995
; Rougier et al., 1998
). 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-ß 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., 1999
). 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., 2000
) 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., 1999). 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, 1995
; Lawrence, 1996
; Clark and Coker, 1998
; Rifkin et al., 1999
). 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., 1997
). 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., 2000
). 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, 1993
). 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., 1994), 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., 1995
). 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., 1999
). 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., 1994a
). 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., 1994a
). Furthermore, neutralization of both TGF-ß1 and TGF-ß3 equally resulted in reduced incidence of adhesion formation (Lucas et al., 1996
). 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., 1997
).
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, 1995; Clark and Coker, 1998
; Arora et al., 1999
). 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, 1995
; Parks, 1999
). These include fibrinolytic system, MMP and their inhibitors, PAI and TIMP (Roberts, 1995
; Clark and Coker, 1998
; Parks, 1999
). 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, 1994
; Roberts, 1995
; Parks, 1999
; Ma et al., 1999
; Saed et al., 1999
, 2000
; Flak et al., 2000
). 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, 1997
; Chegini et al., 1999
, 2001; Ghellai et al., 2000
). 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., 1998
). 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., 1999
), 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-ß 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.
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Acknowledgements |
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Notes |
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* This paper was presented in part at the 47th Annual Meeting of the Society for Gynecological Investigation, Chicago, IL, USA, 2000.
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Submitted on September 29, 2000; accepted on February 21, 2001.