**5.1 Plasminogen activator system (PA)**

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

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

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 endometriosis (Bruse et al., 2005).

Moreover, Lembessis and coworkers reported an increase in uPA mRNA expression in endometriotic lesions compared to eutopic endometrium (Lembessis et al. (2003).

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 et al., 2010)

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, 2000).

#### **5.2 Matrix metalloproteinase systems (MMPs)**

Matrix metalloproteinases (MMPs) are a class of zinc-dependent endopeptidases involved in extracellular matrix remodelling (Matrisian, 1992).

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

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

Endometriosis and Angiogenic Factors 197

The catalytic domains of all MMPs share high amino acid similarity and their active sites are extensively conserved (Lauer-Fields et al., 2009). As a consequence, differenciate between different MMPs activities is extraordinarily difficult. However, some members of these

**Matrix metalloproteinase 7 (MMP7):** MMP7 is secreted mostly from the endometrial epithelium cells during the receptive phase localized to endometrial glandular and luminal

Moreover, MMP-7 has been shown to be the dominant metalloproteinase during the initial development of endometriosis in a baboon model (Fazleabas et al., 2002). In addition, a recent study clearly demonstrated that MMP-7 mRNA was identified in host peritoneal tissues during the development of endometriosis in a nude mouse model (Hull et al., 2008) MMP-7 protein expression in epithelial cells was significantly higher in red peritoneal lesions compared with that of deep infiltrating endometriosis, ovarian endometriosis and black peritoneal lesions, in all phases of the menstrual cycle. MMP-7 protein expression may be down-regulated during the evolution of peritoneal endometriotic implants, as active red

**Matrix metalloproteinase 5 (MMP5)**: The strongest MMP5 staining was seen in luminal

Both the gene chip expression analyses as well as PCR indicated strongly elevated transcript levels in most peritoneal endometriosis lesions. Moreover enhanced MT5-MMP expression has been detected in the eutopic endometrium from patients suffering from endometriosis.

**Matrix metalloproteinase 3 (MMP3)**: IMMP-3 is hormonally regulated during the menstrual cycle, with the highest levels of expression occurring during menses local

MMP-3 has not been well studied in endometriosis, however, studies suggest that retrogradely shed menstrual fragments, the putative precursors of endometriotic lesions,

Cox et al, demonstrated in a rat model that elevated MMP-3 expression by endometrial tissue leads to the establishment and progression of ectopic endometrial tissue growth. (KE.

Significant expression differences were obtained for MMP3 in the ovarian endometriomas. The deregulation of the different genes, included MMP3 genes, may be responsible for the

**Matrix metalloproteinase 2 (MMP):** Overexpression of stromal MMP-**2** may play a role in

**Matrix metalloproteinase 9 (MMP9):** Eutopic endometrium of women with endometriosis compared with normal women showed an increased release of MMP-9, and a decreased release of its natural inhibitor, TIMP-1, at both the protein and the mRNA levels (Chen *et al*.

epithelial cells, whereas endometrial glands frequently showed partial expression.

proteases showed a role in the pathogenesis of endometriosis.

lesions transition into inactive black lesions (Matsuzaki et al., 2010).

regulation that is absent in the in vitro cultures. (Hulboy et al., 1997;.)

loss of cellular homeostasis in endometriotic lesions (Meola et al., 2010)

express high levels of MMP- 3 (Koks et al., 2000).

the development of adenomyosis (Tokyol et al., 2009)

2004; Collette et al., 2004; Collette et al., 2006).

epithelium (Yanaihara et al., 2004; Zhang et al., 2005)

(Gaetje et al., 2007)

Cox, et al., 2001).

growth factors), post-translation modification and by a family of inhibitors: the tissue inhibitors of metalloproteinases or TIMPs (Matrisian, 1990).

Elevated cytokines may play a role in the establishment of ectopic endometrium in the peritoneal cavity by stimulating MMPs to remodel the mesothelial lining of the peritoneum thus allowing for tissue invasion.

MMPs are stimulated by cytokines and also by the protein Extracellular Matrix Metalloproteinase Inducer (EMMPRIN). Braundmeier et al., showed that IL-1beta stimulated MMP-1 protein secretion and mRNA levels in a time dependent manner (P < 0.05), MMP-2 mRNA in a time dependent manner and MMP-3 in a time and dose dependent manner. TNF-alpha stimulated MMP-1 and -3 protein secretion in a time dependent manner and stimulated MMP-1, -2 and -3 mRNA levels in a time dependent manner). Neither IL-1beta nor TNF-alpha treatment affected MMP-2 protein secretion. TGFbeta-1 inhibited MMP-1 and MMP-2 mRNAs at the highest treatment dose after 24 hr but there was no effect on protein secretion. TGF-beta-1 exerted no effect on MMP-3 mRNA or protein secretion (Braundmeier et al., 2010).

MMPs have been implicated in the endometrial remodelling during the menstrual cycle with higher levels during menstrual and proliferative phases and decreased levels during the secretory phase (Salamonsen and Woolley, 1996);

Montly, in the absence of pregnancy, degradation of the ECM is a critical step in the initiation of tissue breakdown that leads to menstruation (Marbaix *et al*., 1996; Salamonsen and Woolley, 1996).

During the proliferative phase of the natural cycle, MMP-1, MMP-3 and MMP-9 are downregulated in the stroma (Hulboy *et al*., 1997), presumably to allow endometrial stable growth. The expression of MMPs then decline in the early secretory phase and then increase during the late secretory phase in anticipation of the next proliferative phase. These modification are related to serum progesterone levels, which has led to the suggestion that endometrial expression of MMPs is under gonadal steroid hormone control. Critically, MMP-9 expression is highest in the menstrual phase endometrium when tissue breakdown occurs.

Moreover, several repots suggest that these proteases are also involved in the ectopic invasion of endometriotic cells associated with endometriosis (Cox et al., 2001).

Deregulation of peritoneal fluid cytokines levels of women with endometriosis show indicate that an altered immune system may play an important role in the pathogenesis of endometriosis. The invasion of ectopic endometrium into peritoneal mesothelium, in association of different angiogenic factors, requires matrix metalloproteinases (MMPs) for tissue remodeling. Several MMPs are differentially expressed in human uterine endometrium with menstrual endometrium showing the highest level of expression. (Braundmeier et al., 2010)

MMP systems closely interact with PA system, because plasmin is an active enzyme, which degrades a variety of extracellular matrix proteins and activates MMPs and growth factors (Murphy et al., 2000).

growth factors), post-translation modification and by a family of inhibitors: the tissue

Elevated cytokines may play a role in the establishment of ectopic endometrium in the peritoneal cavity by stimulating MMPs to remodel the mesothelial lining of the peritoneum

MMPs are stimulated by cytokines and also by the protein Extracellular Matrix Metalloproteinase Inducer (EMMPRIN). Braundmeier et al., showed that IL-1beta stimulated MMP-1 protein secretion and mRNA levels in a time dependent manner (P < 0.05), MMP-2 mRNA in a time dependent manner and MMP-3 in a time and dose dependent manner. TNF-alpha stimulated MMP-1 and -3 protein secretion in a time dependent manner and stimulated MMP-1, -2 and -3 mRNA levels in a time dependent manner). Neither IL-1beta nor TNF-alpha treatment affected MMP-2 protein secretion. TGFbeta-1 inhibited MMP-1 and MMP-2 mRNAs at the highest treatment dose after 24 hr but there was no effect on protein secretion. TGF-beta-1 exerted no effect on MMP-3 mRNA or

MMPs have been implicated in the endometrial remodelling during the menstrual cycle with higher levels during menstrual and proliferative phases and decreased levels during

Montly, in the absence of pregnancy, degradation of the ECM is a critical step in the initiation of tissue breakdown that leads to menstruation (Marbaix *et al*., 1996; Salamonsen

During the proliferative phase of the natural cycle, MMP-1, MMP-3 and MMP-9 are downregulated in the stroma (Hulboy *et al*., 1997), presumably to allow endometrial stable growth. The expression of MMPs then decline in the early secretory phase and then increase during the late secretory phase in anticipation of the next proliferative phase. These modification are related to serum progesterone levels, which has led to the suggestion that endometrial expression of MMPs is under gonadal steroid hormone control. Critically, MMP-9 expression is highest in the menstrual phase endometrium when tissue breakdown

Moreover, several repots suggest that these proteases are also involved in the ectopic

Deregulation of peritoneal fluid cytokines levels of women with endometriosis show indicate that an altered immune system may play an important role in the pathogenesis of endometriosis. The invasion of ectopic endometrium into peritoneal mesothelium, in association of different angiogenic factors, requires matrix metalloproteinases (MMPs) for tissue remodeling. Several MMPs are differentially expressed in human uterine endometrium with menstrual endometrium showing the highest level of expression.

MMP systems closely interact with PA system, because plasmin is an active enzyme, which degrades a variety of extracellular matrix proteins and activates MMPs and growth factors

invasion of endometriotic cells associated with endometriosis (Cox et al., 2001).

inhibitors of metalloproteinases or TIMPs (Matrisian, 1990).

thus allowing for tissue invasion.

protein secretion (Braundmeier et al., 2010).

and Woolley, 1996).

(Braundmeier et al., 2010)

(Murphy et al., 2000).

occurs.

the secretory phase (Salamonsen and Woolley, 1996);

The catalytic domains of all MMPs share high amino acid similarity and their active sites are extensively conserved (Lauer-Fields et al., 2009). As a consequence, differenciate between different MMPs activities is extraordinarily difficult. However, some members of these proteases showed a role in the pathogenesis of endometriosis.

**Matrix metalloproteinase 7 (MMP7):** MMP7 is secreted mostly from the endometrial epithelium cells during the receptive phase localized to endometrial glandular and luminal epithelium (Yanaihara et al., 2004; Zhang et al., 2005)

Moreover, MMP-7 has been shown to be the dominant metalloproteinase during the initial development of endometriosis in a baboon model (Fazleabas et al., 2002). In addition, a recent study clearly demonstrated that MMP-7 mRNA was identified in host peritoneal tissues during the development of endometriosis in a nude mouse model (Hull et al., 2008)

MMP-7 protein expression in epithelial cells was significantly higher in red peritoneal lesions compared with that of deep infiltrating endometriosis, ovarian endometriosis and black peritoneal lesions, in all phases of the menstrual cycle. MMP-7 protein expression may be down-regulated during the evolution of peritoneal endometriotic implants, as active red lesions transition into inactive black lesions (Matsuzaki et al., 2010).

**Matrix metalloproteinase 5 (MMP5)**: The strongest MMP5 staining was seen in luminal epithelial cells, whereas endometrial glands frequently showed partial expression.

Both the gene chip expression analyses as well as PCR indicated strongly elevated transcript levels in most peritoneal endometriosis lesions. Moreover enhanced MT5-MMP expression has been detected in the eutopic endometrium from patients suffering from endometriosis. (Gaetje et al., 2007)

**Matrix metalloproteinase 3 (MMP3)**: IMMP-3 is hormonally regulated during the menstrual cycle, with the highest levels of expression occurring during menses local regulation that is absent in the in vitro cultures. (Hulboy et al., 1997;.)

MMP-3 has not been well studied in endometriosis, however, studies suggest that retrogradely shed menstrual fragments, the putative precursors of endometriotic lesions, express high levels of MMP- 3 (Koks et al., 2000).

Cox et al, demonstrated in a rat model that elevated MMP-3 expression by endometrial tissue leads to the establishment and progression of ectopic endometrial tissue growth. (KE. Cox, et al., 2001).

Significant expression differences were obtained for MMP3 in the ovarian endometriomas. The deregulation of the different genes, included MMP3 genes, may be responsible for the loss of cellular homeostasis in endometriotic lesions (Meola et al., 2010)

**Matrix metalloproteinase 2 (MMP):** Overexpression of stromal MMP-**2** may play a role in the development of adenomyosis (Tokyol et al., 2009)

**Matrix metalloproteinase 9 (MMP9):** Eutopic endometrium of women with endometriosis compared with normal women showed an increased release of MMP-9, and a decreased release of its natural inhibitor, TIMP-1, at both the protein and the mRNA levels (Chen *et al*. 2004; Collette et al., 2004; Collette et al., 2006).

Endometriosis and Angiogenic Factors 199

(Salata et al., 2008; De Sanctis et al., 2010). On the other hand a prospective, blinded, longitudinal study show that MMP-2, MMP-9, and MMP-9/neutrophil gelatinase-associated lipocalin were significantly more likely to be detected in the urine of patients with

Bruner-Tran et al. described progesterone treatment inhibits expression of MMP-3 and -7 in human endometrium and prevents the establishment of ectopic lesions in a nude mouse

A study concerning human endometrium intraperitoneally transplanted into nude mice, demonstrated a significant suppression of MMP-2 transcription by all progestins tested, and a significant down-regulation of MMP-3 by dydrogesterone (Mönckedieck et al., 2009).

In conclusion, angiogenesis is proposed as an important mechanism for the pathogenesis of endometriosis. Different evidences support the hypothesis that the endometrium of women with endometriosis has an increased capacity to proliferate, implant and grow in the peritoneal cavity. Further studies are needed to better understand critical steps of the pathogenesis of endometriosis; nevertheless excessive endometrial angiogenesis suggests

Aeby TC, Huang T, Nakayama RT. The effect of peritoneal fluid from patients with

Akoum A, Jolicoeur C, Boucher A. Estradiol amplifies interleukin-1 induced monocyte

Akoum A, Jolicoeur C, Kharfi A, Aube M. Decreased expression of the decoy interleukin-1 receptor type II in human endometriosis. Am J Pathol 2001;158:481–9. Akoum A, Lemay A, Brunet C, Hebert J. Cytokine-induced secretion of monocyte

d'Investigation en Gynecologie. Am J Obstet Gynecol 1995;172:594–600. Akoum A, Lemay A, Maheux R. Estradiol and interleukin-1beta exert a synergistic

Akoum A, Lemay A, Paradis L, Rheault N, Maheux R. Secretion of interleukine-6 by human

Akoum A, Mahera Al-Akoum, Andre Lemay, Rodolphe Maheux,Mathieu Leboeuf.

American Society for Reproductive Medicine. Revised American Society for Reproductive Medicine classification of endometriosis. Fertil Steril 1997; 67: 817–821.

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**6. References** 
