- Review Article
- Published:
Angiogenic factors in peritoneal adhesion formation
Gynecological Surgery volume 3, pages 157–167 (2006)
Abstract
Abdominal surgery is considered as the leading cause of peritoneal adhesions and almost universally as adhesiogenic. Peritoneal injury at the time of surgery initiates an inflammatory reaction determining fibrin deposition on the wound surface. Depending on the balance between the different components of the plasminogen system, this fibrin can be either lysed, leading to normal peritoneal healing, or organised, serving as a scaffold for fibroblast ingrowth, extracellular matrix deposition and angiogenesis, leading to adhesion formation. The mechanism underlying the predisposition to form adhesions in some patients and in some specific anatomic sites and not in others after similar surgical procedures remains unknown. In spite of the many attempts proposed over the years for reducing the incidence of adhesion formation, peritoneal adhesions remain a major clinical problem, inducing intestinal obstruction, pelvic pain, female infertility and difficulties at the time of re-operation. The available evidence indicates that understanding the adhesion formation process at the molecular level is essential for developing successful strategies for preventing adhesions. Fortunately, the advancement in molecular biology during the last years has led to the identification of many molecules with the potential of regulating inflammatory and immune responses, tissue remodelling and angiogenesis, key events in peritoneal healing and adhesion formation. This review focuses on the role of angiogenesis and angiogenic factors in peritoneal adhesion formation.
Definition and aetiology of peritoneal adhesions
Adhesions are pathological bonds between surfaces within body cavities. These bonds can be a thin film of connective tissue, a thick fibrous bridge containing blood vessels and nerve tissue, or a direct contact between two organ surfaces [1]. Adhesions can be found in abdominal, pericardial, pleural, uterine and joint cavities, and in the chamber of the eyes. Adhesions in the abdominal cavity are also known as peritoneal adhesions because the peritoneum is always involved.
Peritoneal adhesions may be classified, according to the aetiology, as congenital or acquired, which in turn can be classified as postinflammatory or postoperative [2]. Abdominal surgery is the most common cause of adhesions, 70–85% of all adhesions being attributed to previous surgery. On the other hand, surgery has been documented as almost universally adhesiogenic, the reported incidence of adhesions in patients undergoing surgery being between 55 and 100% [3].
Among postoperative adhesions, different processes can be distinguished [4]:
-
Adhesions type 1 or de novo adhesion formation: adhesions formed at sites that did not have adhesions previously.
Type 1A: no previous operative procedures at the site of adhesions.
Type 1B: previous operative procedures at the site of adhesions.
-
Adhesions type 2 or adhesion reformation: adhesions formed at sites where adhesiolysis was performed.
Type 2A: no operative procedures at the site of adhesions besides adhesiolysis.
Type 2B: other operative procedures at the site of adhesions besides adhesiolysis.
Clinical significance of peritoneal adhesions
Depending on their location and structure, adhesions may remain silent or cause clinically important complications such as intestinal obstruction, chronic pelvic pain, female infertility and difficulties at the time of re-operation.
Intestinal obstruction is the most serious complication of peritoneal adhesions as it can be life threatening due to strangulation. Adhesions are the leading cause of intestinal obstruction in the Western world, accounting for more than 40% of all cases of intestinal obstruction and for 60–70% of those involving the small bowel [2].
Adhesion formation is a major cause of chronic pelvic pain and it has been reported as the primary cause in some 25% of patients with chronic pelvic pain. It was suggested that pelvic pain is a consequence of the restricted organ mobility imposed by adhesions. After adhesiolysis, a relief of symptoms has been consistently reported. From a clinical point of view, however, the relation between adhesions and chronic pelvic pain is unclear since their association does not necessarily imply a causal relationship. Indeed, it was demonstrated that a large number of infertility patients with adhesions do not experience pelvic pain [5].
Peritoneal adhesions are well recognised as a cause of female infertility. The proposed mechanism of infertility is that adhesions restrict the sweeping of the fimbria over the ovary. Periadnexal adhesions were found in some 20–30% of infertile women and marked increases in pregnancy rates were reported after adhesiolysis [6].
Adhesions increase the technical difficulty for surgeries, increasing the difficulty of accessing the abdomen and/or the operation site, the complication rates, the anaesthesia, operating and recovery time, the use of surgical materials and the need for blood transfusion. Therefore, the magnitude of adhesions related disorders (ARD) is larger than could be anticipated and is better illustrated by the reports showing that hospital readmission for ARD rival the number of hip replacements, heart bypass or appendix surgeries, that 35% of women having open gynaecologic surgery are readmitted 1.9 times in 10 years for operation due to adhesions or complicated by adhesions, and that the estimated annual cost for ARD in the USA is 1.3 billion US$ [7].
Pathogenesis of peritoneal adhesions
The peritoneum is one of the largest organs in humans with a surface of some 10.000 cm2. It serves to minimise friction and facilitate free movement of abdominal viscera, to resist and localise infections and to store fat, especially in the greater omentum. It forms a closed sac in males and an open sac in females, lining the abdominal walls (parietal peritoneum) and the viscera (visceral peritoneum). It is composed of a continuous layer of mesothelial cells and a layer of loose connective tissue [8].
Peritoneal mesothelial cells are highly differentiated, as are pleural and pericardial mesothelial cells, and their apical surface contain abundant long microvilli that increase the functional surface of the peritoneum for absorption and secretion. Mesothelial cells are connected to one another by desmosomes and very loosely attached to the underlining basement membrane. The connective tissue is composed of bundles of collagenous and elastic fibres oriented in different directions and a rich network of blood and lymphatic vessels. Interspersed among these fibres and vessels, there are poorly differentiated epithelioid-like cells, similar to fibroblasts, macrophages, mast cells and fat cells [8].
The intact peritoneal cavity contains 3–50 ml of fluid with a pH of 7.5–8.0 and with a significant buffering capacity. The peritoneal fluid (PF) contains plasma proteins, including a large amount of fibrinogen, and a variety of free-floating cells, including macrophages, lymphocytes, eosinophils, mast cells and desquamated mesothelial cells [8].
Peritoneal injury, due to surgery, infection or irritation, initiates an inflammatory reaction that increases all components of the PF, i.e. proteins and cells, generating a fibrinous exudate and the formation of fibrin [9]. Fibrin formation is the result of the activation of the coagulation cascade, which includes two pathways, i.e. the contact factor or intrinsic pathway and the tissue factor or extrinsic pathway. Activation of these pathways transforms prothrombin (Factor II) into thrombin (Factor IIa) via the common pathway. Thrombin then triggers the conversion of fibrinogen into monomers of fibrin, which interact with each other and polymerise. The initially soluble polymer becomes insoluble by some coagulation factors such as Factor XIIIa and is deposited on the wound surface [9]. Within this fibrinous exudate, polymorphonuclears (PMN), macrophages, fibroblasts and mesothelial cells migrate, proliferate and/or differentiate. During the first two postoperative days, a large number of PMN enter and, in the absence of infection, depart within 3–4 days. Macrophages increase in number and change their functions, becoming the most important component of the leukocyte population after day 5. They phagocyte more accurately, have greater respiratory burst activity and secrete a variety of substances including cytokines and growth factors that recruit new mesothelial cells onto the injury surface. Mesothelial cells migrate, form islands throughout the injured area and proliferate in order to cover the denuded area. This healing process is different from that occurring in the skin because the entire surface becomes epithelialised simultaneously from the islands of mesothelial cells and not gradually from the borders. Therefore, it is irrespective of the size of the injury and is complete in 5–7 days [8].
These cells release a variety of substances including plasminogen system components, arachidonic acid metabolites, reactive oxygen species (ROS), cytokines and growth factors such as interleukins (IL), tumour necrosis factor α (TNF-α), transforming growth factors α and β (TGF-α and TGF-ß), which modulate the process of peritoneal healing and adhesion formation at different stages [9, 10].
This fibrinous exudate and fibrin deposition is an essential part of normal tissue repair, but its complete resolution is required to restore the preoperative conditions. The degradation of fibrin is regulated by the plasminogen system. In this system, the inactive proenzyme plasminogen is converted into active plasmin by plasminogen activators (PAs), which are inhibited by plasminogen activator inhibitors (PAIs) [11]. Plasminogen is a glycoprotein synthesised in the liver that is abundant in almost all tissues. It is the inactive precursor of plasmin, a serine protease that is highly effective in the degradation of fibrin into fibrin degradation products (FDP) and that has a role in other stages of tissue repair such as extracellular matrix (ECM) degradation, [12] activation of proenzymes of the matrix metalloprotease (MMP) family [13] and activation of growth factors [14]. The principal activator of plasminogen is the serine protease tissue-type PA (tPA), which is expressed in endothelial cells, mesothelial cells and macrophages. tPA has a high affinity for fibrin and binds to a specific receptor, which exposes a strong plasminogen-binding site on the surface of the fibrin molecule. Therefore, in the presence of fibrin the activation rate of plasminogen is strikingly enhanced, whereas in the absence of fibrin, tPA is a poor activator of plasminogen [15, 16]. This results in higher plasminogen activation on the sites where it is required, whereas systemic activation is prevented. The other activator of plasminogen is the serine protease urokinase-type PA (uPA). The properties of uPA differ from those of tPA as it lacks high-affinity binding for fibrin and thus the increased activity in the presence of fibrin. Therefore, uPA is limited in its capacity to activate plasminogen [17].
The action of the PAs is counteracted by PAI-1 and PAI-2 through the formation of inactive complexes. The most potent inhibitor of tPA and uPA is the glycoprotein PAI-1, which is expressed in endothelial cells, mesothelial cells, macrophages, platelets and fibroblasts. The glycoprotein PAI-2 is a relatively poor inhibitor of tPA and uPA and is expressed in mesothelial cells, macrophages and epithelial cells. The role of other PAIs, i.e. PAI-3 and protease nexin 1, and plasmin inhibitors, i.e. α2-macroglobulin, α2-antiplasmin and α1-antitrypsin, in peritoneal fibrinolysis remains unknown.
The balance between fibrin deposition and degradation is critical in determining normal peritoneal healing or adhesion formation. If fibrin is completely degraded, normal peritoneal healing will occur. In contrast, if fibrin is not completely degraded, it will serve as a scaffold for fibroblasts and capillary ingrowth. Indeed, fibroblast will invade the fibrin matrix and ECM will be produced and deposited. The ECM can be completely degraded by MMPs, leading to normal healing. However, if this process is inhibited by tissue inhibitors of MMPs (TIMPs), peritoneal adhesions will be formed.
Angiogenesis, angiogenic factors and adhesion formation
The formation of new blood vessels on peritoneal adhesions has been universally claimed to be important and supported by animal data demonstrating increasing vascularisation over days [18]. The details of the angiogenesis process in peritoneal adhesion formation remains, however, largely unexplored.
Angiogenesis, the formation of new blood vessels extending from existing vessels, occurs when the distance between cells and the nearest capillary exceeds an efficient diffusion range for maintaining an adequate supply of oxygen and nutrients to cells. This process is regulated by cellular hypoxia through the modulation of angiogenic factors and their inhibitors.
Angiogenesis is a self-limited and strictly controlled process that occurs in a sequential manner, involving degradation of the vascular basement membrane and interstitial matrix, migration and proliferation of endothelial cells and finally tubologenesis and formation of capillary loops. The proteolytic enzymes production such as MMPs and PAs, in response to angiogenic factors is fundamental for all stages of angiogenesis, i.e. degradation of perivascular matrix and tissue stroma, migration and proliferation of endothelial cells. Since these proteases are produced in inactive forms and must become activated to initiate their actions, their activities are regulated by naturally occurring physiological inhibitors, i.e. TIMPs and PAIs. Cytokines and growth factors such as IL 1, IL 8, TNF-α, vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), TGF-α, TGF-β, plateled-derived growth factor (PDGF) are considered as angiogenic factors due to their ability to regulate the expression of MMPs, PAs and their inhibitors and to modulate endothelial cell migration and proliferation [19].
VEGF, the most potent known angiogenic factor, is a family that includes VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental growth factor (PlGF). These factors are transcribed from single genes and processed by alternative splicing into different isoforms. VEGF-A, also known as VEGF, is processed into four isoforms in humans (VEGF-A121, VEGF-A165, VEGF-A189 and VEGF-A206) and three in mice (VEGF-A120, VEGF-A164 and VEGF-A188). VEGF-B is processed into two isoforms in humans and two in mice (VEGF-B167 and VEGF-B186), whereas PlGF is processed into three isoforms in humans (PlGF-1, PlGF-2 and PlGF-3) and one in mice (PlGF-2). These factors bind to two high-affinity transmembrane tyrosine kinase receptors with 7 immunoglobulin-like extracellular domains and a kinase intracellular domain, i.e. VEGFR-1/Flt-1 (for VEGF-A, VEGF-B and PlGF) and VEGFR-2/Flk-1 (for VEGF-A). These receptors are selectively but not exclusively expressed on endothelial cells. A truncated soluble form of VEGFR-1, resulting from alternative splicing and retaining its binding activity, is present in serum. VEGFR-1 is, unlike VEGFR-2, also expressed on inflammatory cells. Therefore, VEGF-A, VEGF-B and PlGF can stimulate inflammation in addition to angiogenesis (Fig. 1) [20–23].
Because of the presence of VEGF in endothelial cells of blood vessels supplying pelvic adhesions, a key role for VEGF in angiogenesis during adhesion formation has been suggested [24]. This observation was supported by studies in rats demonstrating up-regulation of VEGF188 and VEGF120 during early stages of peritoneal healing and down-regulation of VEGF164 24–48 h following open surgery [25], suggesting a compensatory mechanism to regulated angiogenesis in order to provide nutrients and oxygen to the injured tissues. The role of VEGF is also supported by the reduction of adhesion formation after treatment with antibodies against VEGF in an open surgery mouse model [26].
The role of VEGF-A, VEGF-B and PlGF in adhesion formation after laparoscopic surgery has been addressed in studies using wild type mice (i.e. VEGF-A+/+, VEGF-B+/+, PlGF+/+), transgenic mice and monoclonal antibodies. Adhesions were induced during laparoscopy and scored after 7 days during laparotomy. Since adhesions increase with the duration of the pneumoperitoneum and the insufflation pressure [27, 28], the CO2 pneumoperitoneum was maintained at 14 mmHg for the minimum time needed to induce the lesions (10 min) or for a longer period (60 min) to evaluate “basal adhesions” and “pneumoperitoneum-enhanced adhesions”, respectively [29]. In all control groups, 60 min of pneumoperitoneum increased adhesion formation. In transgenic mice for VEGF-A, (i.e. deficient for VEGF-A120 and VEGF-A188 and expressing exclusively VEGF-A164: VEGF-A164/164), basal adhesions were higher than in VEGF-A+/+ mice, the pneumoperitoneum slightly increased adhesions, and “pneumoperitoneum-enhanced adhesions” were higher than in VEGF-A+/+ mice (Fig. 2) [30]. In mice deficient for VEGF-B (VEGF-B−/−), “basal adhesions” were similar than in VEGF-B+/+ mice and the pneumoperitoneum did not increase adhesions, “pneumoperitoneum-enhanced adhesions” being therefore lower than in VEGF-B+/+ mice (Fig. 3) [30]. In mice deficient for PlGF (PlGF−/−), basal adhesions were slightly lower than in PlGF+/+ mice and the pneumoperitoneum did not increase adhesions, pneumoperitoneum-enhanced adhesions being therefore lower than in PlGF+/+ (Fig. 4) [30]. The role of PlGF was confirmed by using monoclonal antibodies with different neutralising capacities of the binding of PlGF to its receptor. In mice treated with neutralising antibodies, basal adhesions were lower than in the control groups and the pneumoperitoneum did not increase adhesions, pneumoperitoneum-enhanced adhesions being therefore lower than in the control groups [30] (Fig. 5).
The role of the common receptor of VEGF-A, VEGF-B and PlGF, i.e. VEGF-R1, was evaluated by using monoclonal antibodies against VEGFR-1. In the control group, i.e. IgG-treated mice, pneumoperitoneum increased adhesions. In VEGFR-1 antibodies-treated mice, basal adhesions were similar than in IgG-treated mice and the pneumoperitoneum did not increase adhesions, pneumoperitoneum-enhanced adhesions being therefore lower than in IgG-treated mice (Fig. 6) [31].
For basal adhesions the data clearly demonstrate a role for VEGF-A164, whereas for pneumoperitoneum-enhanced adhesions, the data indicate that the pneumoperitoneum increases adhesions through VEGF-B and PlGF up-regulation and probably also through VEGF-A164 up-regulation. Indeed, pneumoperitoneum-enhanced adhesions is absent in VEGF-B−/− and PlGF−/− mice because the pneumoperitoneum cannot up-regulate these nonexistent factors. This is fully consistent with the observations in mice treated with PlGF antibodies. The only slight increase in adhesions following 60 min of pneumoperitoneum in VEGF-A164/164 mice does not rule out VEGF-A164 up-regulation because adhesion formation could already be near maximal due to the over-expression of this factor.
Since PlGF, VEGF-A and VEGF-B have a common receptor, i.e. VEGFR-1, and since antibodies against VEGFR-1 prevent pneumoperitoneum-enhanced adhesions, the data indicate that the effects of the VEGF family are mediated to a large extent by this receptor. This is supported by the recently reported reduction of peritoneal fibrosis after soluble VEGFR-1 gene transfer in mice [32], since this isoform, by retaining its binding capacity, reduces the binding of the ligands to the functional cellular receptors. As VEGFR-1 is expressed on endothelial cells and on inflammatory cells, it remains unclear whether these effects are mainly related to stimulation of angiogenesis and/or inflammation.
Several mechanisms have been proposed for VEGF-driven angiogenesis [21, 33]. VEGF-A induces angiogenesis by activating VEGFR-2, while VEGFR-1 might function as an inert “decoy” regulating the availability of VEGF-A to activate VEGFR-2. PlGF stimulates angiogenesis by several mechanisms. First, PlGF displaces VEGF-A from VEGFR-1, increasing the fraction of VEGF-A available to activate VEGFR-2. Second, PlGF up-regulates the expression of VEGF-A. Third, PlGF transmits its own intracellular angiogenic signals through VEGFR-1. Fourth, PlGF activates receptor cross-talk between VEGFR-1 and VEGFR-2, enhancing VEGFR-2-driven angiogenesis. Fifth, PlGF forms heterodimers with VEGF-A. On the other hand, VEGF-driven inflammation is mediated by VEGFR-1 by increasing mobilisation of bone marrow-derived myeloid progenitors into peripheral blood, by increasing myeloid cell differentiation, mobilisation and activation, and by increasing cytokines production by macrophages [21, 36].
Regardless of the main mechanism of action of VEGF, the available data point to peritoneal hypoxia as the trigger factor. The hypoxic response is not restricted to specific specialised cell types and a general similar mechanism might act in a variety of cell types. Most mammalian cells can respond to alterations in oxygen levels by increasing or decreasing the expression of specific genes [34, 35]. The hypoxic regulation of many of these genes takes place at both transcriptional and post-transcriptional levels. The transcriptional regulation is mediated by transcription factors known as hypoxia inducible factors (HIFs) [36–38]. Since VEGF is up-regulated by hypoxia through HIFs and since HIFs have a well-known role in angiogenesis [39], a role for these factors in adhesion formation can be postulated.
HIFs are nuclear proteins that bind to hypoxia response elements (HRE) in the promoter or enhancer regions of hypoxia inducible genes, activating gene transcription in response to hypoxia [43]. HIFs are members of the basic helix-loop-helix (bHLH) periodic (Per) aryl hydrocarbon receptor nuclear translocator (ARNT) single-minded (Sim) (PAS) domain protein family. Several proteins have been identified in this bHLH-PAS family that belong to the α or β classes. Each member of the α class form a stable heterodimer with a member of the β class. Whereas β class members are constitutively expressed in a ubiquitous or a tissue-specific way, α class members are often inducible by environmental stimuli such as light or hypoxia [40]. HIF-1 is composed of HIF-1α and HIF-1β subunits [41–43], whereas HIF-2 is composed of HIF-2α and HIF-1β subunits [44]. HIF-1α and HIF-2α, the specific hypoxia-regulated subunits, are structurally very similar and share the same heterodimerisation partner. Therefore, both HIF-1 and HIF-2 have a high similarity in structure and regulatory domains and are able to bind to the same HRE of target genes (Fig. 7).
The specific role of HIFs in adhesion formation was evaluated in mice partially deficient for HIF-1α or HIF-2α using the model previously described. While, in the control groups, 60 min of pneumoperitoneum increased adhesions, in the transgenic mice this effect was not observed, pneumoperitoneum-enhanced adhesions being therefore nonexistent (Figs. 8 and 9) [45]. These observations are consistent with the effects of the VEGF family [30, 34] and are also supported by the absence of pneumoperitoneum-enhanced adhesions in mice deficient for PAI-1 [29], since PAI-1 is up-regulated by hypoxia through HIF-1α [46].
Angiogenesis not only depends on the angiogenic factors but also on the availability of their inhibitors. Among the angiogenic suppressors are TGF-β, TNF-α, interferons, collagen synthesis modifiers, protamine, cyclosporine, hyaluronic acid, thrombospondin, angiostatin and endogenous oestrogen metabolites [19]. Since peritoneal vascular endothelial cells contain receptors for ovarian steroids [60], these steroids can potentially regulate peritoneal healing and adhesion formation related angiogenesis.
Antiangiogenic therapy for peritoneal adhesion prevention
Although antiangiogenic therapy is widely used in other fields such as cancer, to the best of our knowledge, only recently have antiangiogenic agents been used for evaluating their efficacy for peritoneal adhesion prevention. The angiogenesis inhibitor TNP-4 [34], an analogue of fumagillin secreted by the fungus Aspergillus fumigatus, has been shown to reduce peritoneal adhesions and to delay vascular ingrowth in a laparotomy mouse model [48]. However, side effects of the drug such as neurotoxicity and delayed wound healing, precluded further investigations of TNP-470.
Enzymes involved in the transformation of the arachidonic acid as the first step in the prostaglandin synthesis pathway, cyclooxygenase-1 and 2 (COX-1 and COX-2), were also evaluated for adhesion prevention. In contrast to COX-1, which is expressed on endothelial cells of normal blood vessels, COX-2 is present on new angiogenic endothelial cells [49], as well as in fibroblasts associated with surgical adhesions [50]. Therefore, COX-2 inhibitors have the potential of reducing angiogenesis and adhesion formation. In a laparotomy mouse model, in which adhesions were created by rubbing the cecum and by a silicone patch attached to the abdominal wall, animals were treated with the selective COX-2 agents, celecoxib or rofecoxib, and the nonspecific COX inhibitors, aspirin, naproxen, ibuprofen or indomethacin. Animals treated with selective and nonselective COX-2 inhibitors, except aspirin, had significantly fewer adhesions than control animals. Celecoxib produced a maximal reduction in adhesion formation compared with rofecoxib and the nonselective COX-2 inhibitors. Adhesions from mice treated with celecoxib had reduced microvessel density, suggesting inhibition of peritoneal adhesions through an antiangiogenic mechanism [51]. These observations were further supported by the reduced human fibroblast expression of VEGF after in vitro treatment with another COX-2 inhibitor, i.e. NS-358, and by stimulation of aerobic metabolism with dichloroacetic acid [52].
Although it was withdrawn from the market for the teratogenic side effects, the antiangiogenesis inhibitor thalidomide was shown to reduce adhesions formation after colonic anastomosis in rabbits [53]. The antiangiogenenic agent tamoxifen, however, did not show any beneficial effect for reducing adhesion formation in an ileo-ileal anastomosis rat model, although no adverse effects on wound or anastomotic healing were reported [54]. As mentioned before, mice data clearly demonstrate a reduction in adhesion formation after treatment with antibodies again VEGF [55], PlGF [30] or VEGFR-1 [31] opening new alternatives for adhesion prevention in humans.
Conclusions
Peritoneal adhesions, induced by infection, inflammation or surgery, are a leading cause of pelvic pain, intestinal obstruction and female infertility and cause increasing difficulties at the time of re-operation as well as increasing medical costs. It remains unknown why peritoneal wounds heal without adhesions in some patients, whereas in others, severe adhesions are formed from seemingly equal procedures, and why adhesions can develop in one surgical site and not in others in the same patient.
These observations, together with the failure of the many strategies developed over the years to prevent or at least to reduce peritoneal adhesions, clearly highlight the importance of understanding adhesion formation at the molecular level. Fortunately, we have witnessed, during the past decade, extraordinary advances in molecular biology that have led to the identification of many molecules, e.g. cytokines, growth factors, chemokines and proteases, with the potential of regulating inflammatory response, tissue remodelling and angiogenesis—events that are central to normal wound healing and to tissue fibrosis. However, the roles of all these molecules, specifically in the peritoneal biology and in the adhesion formation process, remain speculative to a large extent. Recently a specific adhesion phenotype has been reported, describing the substantial differences between the adhesion peritoneum and the apparently normal adjacent peritoneum [57], an observation that may be crucial for prevention of adhesion reformation (type 2 adhesions).
In addition to the cellular players, molecules and processes involved in postoperative adhesion formation, we have reported the importance of taking into account the potential effect of the local environment, i.e. CO2 pneumoperitoneum for laparoscopy and air for laparotomy, to fully understand the intrinsic mechanisms involved. These observations should not be underestimated since environment-related factors such as hypoxia [28–57], hyperoxia [58, 59], desiccation and hypothermia [60, 61] could modulate every stage of the adhesion formation process in different ways. Indeed, we have initially postulated that the CO2 pneumoperitoneum induces peritoneal hypoxia by compressing the capillary flow at the time of insufflation, which could enhance the formation of adhesions. This hypothesis was supported by the increase in adhesion formation with the duration of the pneumoperitoneum and the insufflation pressure using both CO2 and helium pneumoperitoneum and by the decrease in adhesion formation observed after adding 2–4% of oxygen to any of the two insufflation gases [27, 28]. It was also confirmed by the absence of pneumoperitoneum-enhanced adhesions in mice deficient for the genes encoding for factors regulated by hypoxia such as HIFs [54], VEGF [30] and PAI-1 [29]. The important role of hypoxia in adhesion formation was further supported by a series of in vitro studies demonstrating increased expression of many adhesiogenic factors produced by fibroblasts cultured under hypoxic conditions [62–79].
Since angiogenesis is one of the essential steps in adhesion formation, and since it is mainly regulated by hypoxia, all these data together indicate that antiangiogenic measurements, either by preventing hypoxia and thus angiogenesis or by using antiangiogenic drugs, could be an alternative for prevention of peritoneal adhesions.
References
Diamond MP, Freeman ML (2001) Clinical implications of postsurgical adhesions. Hum Reprod Update 7:567–576
Ellis H (1997) The clinical significance of adhesions: focus on intestinal obstruction. Eur J Surg (Suppl):5–9
Weibel MA, Majno G (1973) Peritoneal adhesions and their relation to abdominal surgery: a postmortem study. Am J Surg 126:345–353
Diamond MP, Nezhat F (1993) Adhesions after resection of ovarian endometriomas. Fertil Steril 59:934–935
Duffy DM, diZerega GS (1996) Adhesion controversies: pelvic pain as a cause of adhesions, crystalloids in preventing them. J Reprod Med 41:19–26
Marana R, Muzii L (2006) Infertility and adhesions. In: diZerega GS (ed) Peritoneal surgery. Springer, Berlin Heidelberg New York, pp 329–333
Ray NF, Denton WG, Thamer M, Henderson SC, Perry S (1998) Abdominal adhesiolysis: inpatient care and expenditures in the United States in 1994. J Am Coll Surg 186:1–9
diZerega GS (2006) Peritoneum, peritoneal healing and adhesion formation. In: diZerega GS (ed) Perotoneal surgery. Springer, Berlin Heidelberg New York, pp 3–38
Holmdahl L (1997) The role of fibrinolysis in adhesion formation. Eur J Surg Suppl 24–31
Chegini N (1997) The role of growth factors in peritoneal healing: transforming growth factor beta (TGF-beta). Eur J Surg Suppl 17–23
Holmdahl L, Falkenberg M, Ivarsson ML, Risberg B (1997) Plasminogen activators and inhibitors in peritoneal tissue. APMIS 105:25–30
Wong AP, Cortez SL, Baricos WH (1992) Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells. Am J Physiol 263:F1112–F1118
Murphy G, Atkinson S, Ward R, Gavrilovic J, Reynolds JJ (1992) The role of plasminogen activators in the regulation of connective tissue metalloproteinases. Ann N Y Acad Sci 667:1–12
Saksela O, Rifkin DB (1990) Release of basic fibroblast growth factor-heparan sulfate complexes from endothelial cells by plasminogen activator-mediated proteolytic activity. J Cell Biol 110:767–775
Ichinose A, Takio K, Fujikawa K (1986) Localization of the binding site of tissue-type plasminogen activator to fibrin. J Clin Invest 78:163–169
Norrman B, Wallen P, Ranby M (1985) Fibrinolysis mediated by tissue plasminogen activator: disclosure of a kinetic transition. Eur J Biochem 149:193–200
Lu HR, Wu Z, Pauwels P, Lijnen HR, Collen D (1992) Comparative thrombolytic properties of tissue-type plasminogen activator (t-PA), single-chain urokinase-type plasminogen activator (u-PA) and K1K2Pu (a t-PA/u-PA chimera) in a combined arterial and venous thrombosis model in the dog. J Am Coll Cardiol 19:1350–1359
Bigatti G, Boeckx W, Gruft L, Segers N, Brosens I (1995) Experimental model for neoangiogenesis in adhesion formation. Hum Reprod 10:2290–2294
Chegini N (2002) Peritoneal molecular environment, adhesion formation and clinical implication. Front Biosci 7:e91–e115
Luttun A, Tjwa M, Carmeliet P (2002) Placental growth factor (PlGF) and its receptor Flt-1 (VEGFR-1): novel therapeutic targets for angiogenic disorders. Ann N Y Acad Sci 979:80–93
Luttun A, Tjwa M, Moons L, Wu Y, Angelillo-Scherrer A, Liao F, Nagy JA, Hooper A, Priller J, De Klerck B, Compernolle V, Daci E, Bohlen P, Dewerchin M, Herbert JM, Fava R, Matthys P, Carmeliet G, Collen D, Dvorak HF, Hicklin DJ, Carmeliet P (2002) Revascularization of ischemic tissues by PlGF treatment, and inhibition of tumor angiogenesis, arthritis and atherosclerosis by anti-Flt1. Nat Med 8:831–840
Clauss M (2000) Molecular biology of the VEGF and the VEGF receptor family. Semin Thromb Hemost 26:561–569
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z (1999) Vascular endothelial growth factor (VEGF) and its receptors. FASEB J 13:9–22
Wiczyk HP, Grow DR, Adams LA, O’Shea DL, Reece MT (1998) Pelvic adhesions contain sex steroid receptors and produce angiogenesis growth factors. Fertil Steril 69:511–516
Rout UK, Oommen K, Diamond MP (2000) Altered expressions of VEGF mRNA splice variants during progression of uterine-peritoneal adhesions in the rat. Am J Reprod Immunol 43:299–304
Saltzman AK, Olson TA, Mohanraj D, Carson LF, Ramakrishnan S (1996) Prevention of postoperative adhesions by an antibody to vascular permeability factor/vascular endothelial growth factor in a murine model. Am J Obstet Gynecol 174:1502–1506
Molinas CR, Mynbaev O, Pauwels A, Novak P, Koninckx PR (2001) Peritoneal mesothelial hypoxia during pneumoperitoneum is a cofactor in adhesion formation in a laparoscopic mouse model. Fertil Steril 76:560–567
Molinas CR, Koninckx PR (2000) Hypoxaemia induced by CO(2) or helium pneumoperitoneum is a co-factor in adhesion formation in rabbits. Hum Reprod 15:1758–1763
Molinas CR, Elkelani O, Campo R, Luttun A, Carmeliet P, Koninckx PR (2003) Role of the plasminogen system in basal adhesion formation and carbon dioxide pneumoperitoneum-enhanced adhesion formation after laparoscopic surgery in transgenic mice. Fertil Steril 80:184–192
Molinas CR, Campo R, Dewerchin M, Eriksson U, Carmeliet P, Koninckx PR (2003) Role of vascular endothelial growth factor and placental growth factor in basal adhesion formation and in carbon dioxide pneumoperitoneum-enhanced adhesion formation after laparoscopic surgery in transgenic mice. Fertil Steril 80(Suppl 2):803–811
Molinas CR, Merces BM, Carmeliet P, Koninckx PR (2004) Role of vascular endothelial growth factor receptor 1 in basal adhesion formation and in carbon dioxide pneumoperitoneum-enhanced adhesion formation after laparoscopic surgery in mice. Fertil Steril 82(Suppl 3):1149–1153
Motomura Y, Kanbayashi H, Khan WI, Deng Y, Blennerhassett PA, Margetts PJ, Gauldie J, Egashira K, Collins SM (2005) The gene transfer of soluble VEGF type I receptor (Flt-1) attenuates peritoneal fibrosis formation in mice but not soluble TGF-beta type II receptor gene transfer. Am J Physiol Gastrointest Liver Physiol 288:G143–G150
Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, Scholz D, Acker T, DiPalma T, Dewerchin M, Noel A, Stalmans I, Barra A, Blacher S, Vandendriessche T, Ponten A, Eriksson U, Plate KH, Foidart JM, Schaper W, Charnock-Jones DS, Hicklin DJ, Herbert JM, Collen D, Persico MG (2001) Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 7:575–583
Semenza GL (1999) Perspectives on oxygen sensing. Cell 98:281–284
Gleadle JM, Ratcliffe PJ (1998) Hypoxia and the regulation of gene expression. Mol Med Today 4:122–129
Semenza GL (2000) HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J Appl Physiol 88:1474–1480
Semenza GL (1999) Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1. Annu Rev Cell Dev Biol 15:551–578
Semenza GL (1998) Hypoxia-inducible factor 1: master regulator of O2 homeostasis. Curr Opin Genet Dev 8:588–594
Hirota K, Semenza GL (2006) Regulation of angiogenesis by hypoxia-inducible factor 1. Crit Rev Oncol Hematol
Taylor BL, Zhulin IB (1999) PAS domains: internal sensors of oxygen, redox potential, and light. Microbiol Mol Biol Rev 63:479–506
Semenza GL, Wang GL (1992) A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol 12:5447–5454
Wang GL, Jiang BH, Rue EA, Semenza GL (1995) Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc Natl Acad Sci USA 92:5510–5514
Wang GL, Semenza GL (1995) Purification and characterization of hypoxia-inducible factor 1. J Biol Chem 270:1230–1237
Ema M, Taya S, Yokotani N, Sogawa K, Matsuda Y, Fujii-Kuriyama Y (1997) A novel bHLH-PAS factor with close sequence similarity to hypoxia-inducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc Natl Acad Sci USA 94:4273–4278
Molinas CR, Campo R, Elkelani OA, Binda MM, Carmeliet P, Koninckx PR (2003) Role of hypoxia inducible factors 1alpha and 2alpha in basal adhesion formation and in carbon dioxide pneumoperitoneum-enhanced adhesion formation after laparoscopic surgery in transgenic mice. Fertil Steril 80(Suppl 2):795–802
Uchiyama T, Kurabayashi M, Ohyama Y, Utsugi T, Akuzawa N, Sato M, Tomono S, Kawazu S, Nagai R (2000) Hypoxia induces transcription of the plasminogen activator inhibitor-1 gene through genistein-sensitive tyrosine kinase pathways in vascular endothelial cells. Arterioscler Thromb Vasc Biol 20:1155–1161
Wiczyk HP, Grow DR, Adams LA, O’Shea DL, Reece MT (1998) Pelvic adhesions contain sex steroid receptors and produce angiogenesis growth factors. Fertil Steril 69:511–516
Chiang SC, Cheng CH, Moulton KS, Kasznica JM, Moulton SL (2000) TNP-470 inhibits intraabdominal adhesion formation. J Pediatr Surg 35:189–196
Masferrer JL, Koki A, Seibert K (1999) COX-2 inhibitors. A new class of antiangiogenic agents. Ann N Y Acad Sci 889:84–86
Saed GM, Munkarah AR, Diamond MP (2003) Cyclooxygenase-2 is expressed in human fibroblasts isolated from intraperitoneal adhesions but not from normal peritoneal tissues. Fertil Steril 79:1404–1408
Greene AK, Alwayn IP, Nose V, Flynn E, Sampson D, Zurakowski D, Folkman J, Puder M (2005) Prevention of intra-abdominal adhesions using the antiangiogenic COX-2 inhibitor celecoxib. Ann Surg 242:140–146
Diamond MP, El Hammady E, Munkarah A, Bieber EJ, Saed G (2005) Modulation of the expression of vascular endothelial growth factor in human fibroblasts. Fertil Steril 83:405–409
Mall JW, Schwenk W, Philipp AW, Muller JM, Pollmann C (2002) Thalidomide given intraperitoneally reduces the number of postoperative adhesions after large bowel resection in rabbits. Eur J Surg 168:641–645
McNamara DA, Walsh TN, Kay E, Bouchier-Hayes DJ (2003) Neoadjuvant antiangiogenic therapy with tamoxifen does not impair gastrointestinal anastomotic repair in the rat. Colorectal Dis 5:335–341
Saltzman AK, Olson TA, Mohanraj D, Carson LF, Ramakrishnan S (1996) Prevention of postoperative adhesions by an antibody to vascular permeability factor/vascular endothelial growth factor in a murine model. Am J Obstet Gynecol 174:1502–1506
Saed GM, Diamond MP (2004) Molecular characterization of postoperative adhesions: the adhesion phenotype. J Am Assoc Gynecol Laparosc 11:307–314
Elkelani OA, Binda MM, Molinas CR, Koninckx PR (2004) Effect of adding more than 3% oxygen to carbon dioxide pneumoperitoneum on adhesion formation in a laparoscopic mouse model. Fertil Steril 82:1616–1622
Binda MM, Molinas CR, Koninckx PR (2003) Reactive oxygen species and adhesion formation: clinical implications in adhesion prevention. Hum Reprod 18:2503–2507
Elkelani OA, Binda MM, Molinas CR, Koninckx PR (2004) Effect of adding more than 3% oxygen to carbon dioxide pneumoperitoneum on adhesion formation in a laparoscopic mouse model. Fertil Steril 82:1616–1622
Binda MM, Molinas CR, Mailova K, Koninckx PR (2004) Effect of temperature upon adhesion formation in a laparoscopic mouse model. Hum Reprod 19:2626–2632
Binda MM, Molinas CR, Hansen P, Koninckx PR (2006) Effect of desiccation and temperature during laparoscopy on adhesion formation in mice. Fertil Steril
Diamond MP, El Hammady E, Wang R, Kruger M, Saed G (2004) Regulation of expression of tissue plasminogen activator and plasminogen activator inhibitor-1 by dichloroacetic acid in human fibroblasts from normal peritoneum and adhesions. Am J Obstet Gynecol 190:926–934
Diamond MP, El Hammady E, Wang R, Saed G (2004) Regulation of matrix metalloproteinase-1 and tissue inhibitor of matrix metalloproteinase-1 by dichloroacetic acid in human fibroblasts from normal peritoneum and adhesions. Fertil Steril 81:185–190
Diamond MP, El Hammady E, Wang R, Saed G (2003) Regulation of transforming growth factor-beta, type III collagen, and fibronectin by dichloroacetic acid in human fibroblasts from normal peritoneum and adhesions. Fertil Steril 79: 1161–1167
Diamond MP, El Hammady E, Wang R, Saed G (2002) Metabolic regulation of collagen I in fibroblasts isolated from normal peritoneum and adhesions by dichloroacetic acid. Am J Obstet Gynecol 187:1456–1460
Rout UK, Saed GM, Diamond MP (2005) Expression pattern and regulation of genes differ between fibroblasts of adhesion and normal human peritoneum. Reprod Biol Endocrinol 3:1
Rout UK, Saed GM, Diamond MP (2002) Transforming growth factor-beta1 modulates expression of adhesion and cytoskeletal proteins in human peritoneal fibroblasts. Fertil Steril 78:154–161
Saed GM, Munkarah AR, Abu-Soud HM, Diamond MP (2005) Hypoxia upregulates cyclooxygenase-2 and prostaglandin E(2) levels in human peritoneal fibroblasts. Fertil Steril 83(Suppl 1):1216–1219
Saed GM, Abu-Soud HM, Diamond MP (2004) Role of nitric oxide in apoptosis of human peritoneal and adhesion fibroblasts after hypoxia. Fertil Steril 82(Suppl 3):1198–1205
Saed GM, Diamond MP (2004) Differential expression of alpha smooth muscle cell actin in human fibroblasts isolated from intraperitoneal adhesions and normal peritoneal tissues. Fertil Steril 82(Suppl 3):1188–1192
Saed GM, Kruger M, Diamond MP (2004) Expression of transforming growth factor-beta and extracellular matrix by human peritoneal mesothelial cells and by fibroblasts from normal peritoneum and adhesions: effect of Tisseel. Wound Repair Regen 12:557–564
Saed GM, Collins KL, Diamond MP (2002) Transforming growth factors beta1, beta2 and beta3 and their receptors are differentially expressed in human peritoneal fibroblasts in response to hypoxia. Am J Reprod Immunol 48:387–393
Saed GM, Diamond MP (2003) Modulation of the expression of tissue plasminogen activator and its inhibitor by hypoxia in human peritoneal and adhesion fibroblasts. Fertil Steril 79:164–168
Saed GM, Diamond MP (2002) Hypoxia-induced irreversible up-regulation of type I collagen and transforming growth factor-beta1 in human peritoneal fibroblasts. Fertil Steril 78:144–147
Saed GM, Diamond MP (2002) Apoptosis and proliferation of human peritoneal fibroblasts in response to hypoxia. Fertil Steril 78: 137–143
Saed GM, Zhang W, Diamond MP (2001) Molecular characterization of fibroblasts isolated from human peritoneum and adhesions. Fertil Steril 75:763–768
Saed GM, Zhang W, Diamond MP (2000) Effect of hypoxia on stimulatory effect of TGF-beta 1 on MMP-2 and MMP-9 activities in mouse fibroblasts. J Soc Gynecol Investig 7:348–354
Saed GM, Zhang W, Chegini N, Holmdahl L, Diamond MP (2000) Transforming growth factor beta isoforms production by human peritoneal mesothelial cells after exposure to hypoxia. Am J Reprod Immunol 43: 285–291
Saed GM, Zhang W, Chegini N, Holmdahl L, Diamond MP (1999) Alteration of type I and III collagen expression in human peritoneal mesothelial cells in response to hypoxia and transforming growth factor-beta1. Wound Repair Regen 7:504–510
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Molinas, C.R., Binda, M.M. & Koninckx, P.R. Angiogenic factors in peritoneal adhesion formation. Gynecol Surg 3, 157–167 (2006). https://doi.org/10.1007/s10397-006-0236-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10397-006-0236-7