**4. Transforming growth factor beta (TGF-ß)**

Transforming growth factor beta (TGF-β) is a protein that controls proliferation, cellular differentiation and other functions in most cells. TGF-β is a secreted protein that exists in at least five isoforms called TGF- ß 1, TGF- ß 2 and TGF- ß 3. It was also the original name for TGF- ß 1, which was the founding member of this family. TGF-beta acts as an antiproliferative factor in normal epithelial cells and at early stages of oncogenesis. Some cells that secrete TGF- ß also have receptors for TGF- ß. This is known as autocrine signalling. Cancerous cells increase their production of TGF-β, which also acts on surrounding cells.Oosterlynck et al. (Oosterlynck et al.,1994) found increased TGF-b activity in the peritoneal fluid of women with endometriosis. Transforming growth factor-b may be a cytokine that inhibits NK activity in the peritoneal fluid of women with endometriosis (Oosterlynck et al.,1994). It may play a major role in the biological processes leading to establishment and maintenance of endometriosis., in fact TGF- ß is implicated in the gene expression, cell motility, proliferation, apoptosis, differentiation, immune responses and tumorigenesis (Derynck et al., 2001). TGF-b is abundantly and differentially expressed in the endometrium and is secreted by endometrial cells and macrophages into the uterine fluid where interaction with the preimplantation embryo is suspected (Jones et al., 2006).

Endometriosis and Angiogenic Factors 195

**5. Plasminogen activator (uPA) and matrix metalloproteinase systems (MMS)** 

The PA system includes a wide cluster of proteolytic enzymes for plasmin generation. Plasminogen is activated to plasmin by two types of activators, urokinase-type PA (uPA) and tissue- type PA (tPA). Whereas tPA is involved in the role in the control of intravascular fibrin degradation, uPA is mainly implicated in cellular proteolysis and migration. The activity of the PAs is regulated by specific PA inhibitors (PAIs). (Kruithof et al., 1995;

The PA system and its specific plasminogen activator inhibitors (PAIs) exert physiological and pathophysiological functions such as fibrinolysis, tissue remodelling and tumor invasion, signal transduction, cell adherence and cell migration (Harbeck et al., 2001).

Fernández-Shaw et al., firstly reported high levels of urokinase and plasminogen in ectopic endometrium as a more invasive nature of the endometriotic implants in the peritoneal cavità (Fernández-Shawet al., 1995); afterwards, Sillem confirmed an altered activation of plasminogen in endometrium from women with endometriosis that could lead to a higher proteolytic potential of retrogradely menstruated endometrial fragments with consecutive

In situ hybridization studies performed by Bruse et al. showed that uPA mRNA seems to be up-regulated in both endometriotic glands and endometrial stroma from women with

Moreover, Lembessis and coworkers reported an increase in uPA mRNA expression in

Despite contrasting data in vitro culture model (Guan et al., 2002) ; recently, Cosin reported an increase in uPA antigenic levels in endometrium from women with endometriosis (Cosin

In relation to PA levels in eutopic endometrium from women with endometriosis, it has been suggested that a higher concentration of uPA in the endometrium might result in endometrial fragments with a higher potential to degrade the extracellular matrix after the implantation at ectopic sites (Spuijbroek et al., 1992; Bruse et al., 1998, 2004; Kobayashi,

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases involved in

Members of this family share high level of structural analogy and are secreted by several cell types as zymogens. In relation to substrate preference and protein-domain considerations, MMP family members have been categorized into subgroups that include gelatinases,

The activity of metalloproteinases is tightly regulated,as these molecules are potent proteolytic enzymes, at differente steps: transcriptional level (by cytokines, chemicals, and

stromelysins, collagenases, membrane-type (MT)-MMPs and 'other MMPs'.

endometriotic lesions compared to eutopic endometrium (Lembessis et al. (2003).

**5.1 Plasminogen activator system (PA)** 

Grancha et al., 1996; Heeb et al., 1987).

endometriosis (Bruse et al., 2005).

et al., 2010)

2000).

development of endometriotic foci (Sillem et al., 1997).

**5.2 Matrix metalloproteinase systems (MMPs)** 

extracellular matrix remodelling (Matrisian, 1992).

Secretion of TGF-b into peritoneal fluid of women suffering from endometriosis suggests that they may be crucial in establishment and/or maintenance of endometriosis. Omwandho et al., showed that all TGF- ß and their high-affinity receptors were stagespecifically expressed in the human endometrium with highest levels around menstruation . Many researchers have reported staining of TGF-b1 and 3 in stromal and glandular cells (Chegini et al., 1994); (Gold et al., 1994) ; (Johnson et al., 2005) ; (Komiyama et al., 2007); (Gaide Chevronnay et al., 2008) and for TGF-b1 also in nerve fibres (Tamburro et al., 2003) and inflammatory cells specially in macrophages (Chegini et al., 1994); (Tamura et al., 1999); (Komiyama et al., 2007). TGF- ß 2 is more strongly expressed in stromal compared with glandular cells (Gold et al., 1994); (Bruner et al., 1995); (Gaide Chevronnay et al., 2008), although opposite staining intensity has been reported (Chegini et al., 1994). Localization of TbRII and RI was observed in both cellular compartments of the endometrium (Chegini et al., 1994; (Gaide Chevronnay et al., 2008) with stronger expression of TbRII than TbRI (Gaide Chevronnay et al., 2008) suggesting that TbRI might be a limiting factor for signal transduction in the endometrium or during endometriosis. TGF-b1 was found in the stromal cells (Johnson et al., 2005) and expression increased in the epithelial cells of endometriotic cysts (Tamura et al., 1999) and endometriotic nerve fibers (Tamburro et al., 2003). The TGF-b signal transducers Smad3, pSmad3, Smad4 (SMADs are intracellular proteins that transduce extracellular signals from TGF beta ligands to the nucleus where they activate downstream TGF- ß gene transcription) and the inhibitory Smad7 proteins were also observed in the endometrial stromal and epithelial cells (Luo et al., 2003). These observation suggest a role of the TGF- ß s in the normal function of the human endometrium. In fact is well known that TGF- ß sprevent the breakdown of the endometrial tissue (Tabibzadeh, 2002). This assumption is based on the observation that Lefty-2/EBAF (endometrial bleeding associated factor), a member of the TGF-b family, is dramatically up-regulated during endometriosis (Kothapalli et al., 1997) and antagonized TGF- ß signaling by inhibiting phosphorylation of Smad2 downstream of the TbRI (Ulloa et al., 2001). That Lefty-2 was noticeably more abundant in patients with endometriosis who did not conceive compared with those who became pregnant, suggested a role in implantation (Tabibzadeh et al., 2000). More, it has been shown that TGF- ß 1also induces contractions of decidual stromal cells (Kimatrai et al., 2003) and inhibits motility of stromal endometrial cells (Nasu et al., 2005).It is important to show how TGF- ß 1stimulated DNA synthesis in epithelial cells at low concentrations, but inhibited DNA synthesis at higher concentrations in women with and without endometriosis (Meresman et al., 2003). Additional evidence showed that TGF- ß 1induces expression of FasL mRNA and protein in endometrial stromal cells (Garci-Velasco et al., 1999), possibly preventing apoptosis during transit to the peritoneal cavity. Another very important analysis showed that TGF- ß 1represses the immune system (Shull et al., 1992) and the escape from immune surveillance is also important for adhesion of endometriotic cells in the peritoneum. Finally TGF- ßs participate in the initiation of menstruation via vasoconstriction, in menstrual tissue repair and in endometriosis. In a classic experiment, Luo et al. (2003b) demonstrated that the pretreatment with a GnRH antaonist resulted in further suppression of Smad3 in endometrial stromal cells but co-treatment with GnRH and TGF- ß 1or pretreatment with TbRII antisense partially inhibited TGF- ß 1-activated Smad3. Taken collectively, these observations suggest that GnRH may prevent endometriosis by altering expression and activation of Smads and interrupting TGF- ß receptor signaling.

Secretion of TGF-b into peritoneal fluid of women suffering from endometriosis suggests that they may be crucial in establishment and/or maintenance of endometriosis. Omwandho et al., showed that all TGF- ß and their high-affinity receptors were stagespecifically expressed in the human endometrium with highest levels around menstruation . Many researchers have reported staining of TGF-b1 and 3 in stromal and glandular cells (Chegini et al., 1994); (Gold et al., 1994) ; (Johnson et al., 2005) ; (Komiyama et al., 2007); (Gaide Chevronnay et al., 2008) and for TGF-b1 also in nerve fibres (Tamburro et al., 2003) and inflammatory cells specially in macrophages (Chegini et al., 1994); (Tamura et al., 1999); (Komiyama et al., 2007). TGF- ß 2 is more strongly expressed in stromal compared with glandular cells (Gold et al., 1994); (Bruner et al., 1995); (Gaide Chevronnay et al., 2008), although opposite staining intensity has been reported (Chegini et al., 1994). Localization of TbRII and RI was observed in both cellular compartments of the endometrium (Chegini et al., 1994; (Gaide Chevronnay et al., 2008) with stronger expression of TbRII than TbRI (Gaide Chevronnay et al., 2008) suggesting that TbRI might be a limiting factor for signal transduction in the endometrium or during endometriosis. TGF-b1 was found in the stromal cells (Johnson et al., 2005) and expression increased in the epithelial cells of endometriotic cysts (Tamura et al., 1999) and endometriotic nerve fibers (Tamburro et al., 2003). The TGF-b signal transducers Smad3, pSmad3, Smad4 (SMADs are intracellular proteins that transduce extracellular signals from TGF beta ligands to the nucleus where they activate downstream TGF- ß gene transcription) and the inhibitory Smad7 proteins were also observed in the endometrial stromal and epithelial cells (Luo et al., 2003). These observation suggest a role of the TGF- ß s in the normal function of the human endometrium. In fact is well known that TGF- ß sprevent the breakdown of the endometrial tissue (Tabibzadeh, 2002). This assumption is based on the observation that Lefty-2/EBAF (endometrial bleeding associated factor), a member of the TGF-b family, is dramatically up-regulated during endometriosis (Kothapalli et al., 1997) and antagonized TGF- ß signaling by inhibiting phosphorylation of Smad2 downstream of the TbRI (Ulloa et al., 2001). That Lefty-2 was noticeably more abundant in patients with endometriosis who did not conceive compared with those who became pregnant, suggested a role in implantation (Tabibzadeh et al., 2000). More, it has been shown that TGF- ß 1also induces contractions of decidual stromal cells (Kimatrai et al., 2003) and inhibits motility of stromal endometrial cells (Nasu et al., 2005).It is important to show how TGF- ß 1stimulated DNA synthesis in epithelial cells at low concentrations, but inhibited DNA synthesis at higher concentrations in women with and without endometriosis (Meresman et al., 2003). Additional evidence showed that TGF- ß 1induces expression of FasL mRNA and protein in endometrial stromal cells (Garci-Velasco et al., 1999), possibly preventing apoptosis during transit to the peritoneal cavity. Another very important analysis showed that TGF- ß 1represses the immune system (Shull et al., 1992) and the escape from immune surveillance is also important for adhesion of endometriotic cells in the peritoneum. Finally TGF- ßs participate in the initiation of menstruation via vasoconstriction, in menstrual tissue repair and in endometriosis. In a classic experiment, Luo et al. (2003b) demonstrated that the pretreatment with a GnRH antaonist resulted in further suppression of Smad3 in endometrial stromal cells but co-treatment with GnRH and TGF- ß 1or pretreatment with TbRII antisense partially inhibited TGF- ß 1-activated Smad3. Taken collectively, these observations suggest that GnRH may prevent endometriosis by altering expression and activation of Smads and interrupting TGF- ß receptor signaling.
