**2.1.3 Genetic polymorphisms of VEGF genes**

An important aspect of the correlation between endometriosis and VEGF, also for possible future therapeutic application is that the polymorphisms in vascular endothelial growth factor gene are associated with the risk of familial endometriosis. The human *VEGF* gene (*VEGFA*, OMIM 192240) is located on chromosome 6p12 (Zhao Z, et al 2008). *VEGF* messenger ribonucleic acid and protein were significantly higher in women with endometriosis, which supported a key role for *VEGF* in the pathological angiogenesis in endometriosis (Gilabert-Estelles J, 2007). In particular several transcription factor-binding sites are found in the VEGF 5' –untranslated region and variation within the region increases the transcriptional activity. A single family in two generations with four members who have histologically proven endometriosis showed that the circulating levels of *VEGF* were higher than the healthy control group, indicating a role for *VEGF* in disease susceptibility (Simpson JL et al 2003). In Chinese patients, the T allele of the *VEGF* gene −60 T/C (rs833061) polymorphism was associated with a higher risk of endometriosis. Study of the *VEGF* +405 G/C (rs2010963) polymorphism in a Korean population showed that the SNP was associated with the risk of advanced stage endometriosis. The analysis of both SNPs in an Indian population identified a haplotype associated with endometriosis In addition, the analysis of *VEGF* −460 T/C (rs833061), +405 G/C (rs2010963) and +936 C/T

2004). The inner layers of cysts are characterized by high microvessel density but low expression of VEGF, whereas in the outer fibrosclerotic capsule, the vessels were less abundant, but Had a higher expression of VEGF and survive, thus activated to proliferate and protected from programmed cells death. Angiogenesis mediated by VEGF in the outer capsule contributed to the cyst growth and to the fibrosing process of adhesion (Goteri G. et al 2010). Antiangiogenetic drugs could act on the capsular vasculature and block the growth of ovarian cyst. The high VEGF levels could provoke an increase in the subperitoneal vascular network and facilitate implantation and viability of endometrial cells in the retroperitoneal space. Concerning sVEGFR-1, the highest levels of this protein were found in peritoneal fluids and cystic fluids of endometriosis patients with respect to both benign and malignant serous cysts. The soluble form of VEGFR-1, as already stated, should function as a modulator of VEGF's angiogenic activity. For this reason, it seems that sVEGFR-1 is secreted in proportionate amounts as VEGF itself. In benign cyst and peritoneal fluids, VEGF and sVEGFR-1 concentrations are proportionately low, while in endometriosis cyst and peritoneal fluids, VEGF and its soluble receptor are both expressed in much higher concentrations. Endothelial sVEGFR-1 is also known to be up-regulated by its ligand, VEGF-A, and the high levels of VEGF-A found in endometriomata compared with cystadenomas are likely to further contribute to up-regulation of sVEGFR-1. On the other hand, in patients affected by cystadenocarcinomas, there was discordance between the levels of VEGF and the levels of sVEGFR-1 in both cyst and peritoneal fluids. In fact, in malignant processes there seems to be an imbalance between pro-angiogenetic factors, represented by VEGF, and antiangiogenetic factors, represented by sVEGFR-1, leading to a disordered and exaggerated

The microenvironment of endometriosis is a locale of important secretion of angiogenic factors that play a key role in the establishment and maintenance of endometriotic lesions, and suggest that the balance of these local pro-antiangiogenic factors and cytokines may

An important aspect of the correlation between endometriosis and VEGF, also for possible future therapeutic application is that the polymorphisms in vascular endothelial growth factor gene are associated with the risk of familial endometriosis. The human *VEGF* gene (*VEGFA*, OMIM 192240) is located on chromosome 6p12 (Zhao Z, et al 2008). *VEGF* messenger ribonucleic acid and protein were significantly higher in women with endometriosis, which supported a key role for *VEGF* in the pathological angiogenesis in endometriosis (Gilabert-Estelles J, 2007). In particular several transcription factor-binding sites are found in the VEGF 5' –untranslated region and variation within the region increases the transcriptional activity. A single family in two generations with four members who have histologically proven endometriosis showed that the circulating levels of *VEGF* were higher than the healthy control group, indicating a role for *VEGF* in disease susceptibility (Simpson JL et al 2003). In Chinese patients, the T allele of the *VEGF* gene −60 T/C (rs833061) polymorphism was associated with a higher risk of endometriosis. Study of the *VEGF* +405 G/C (rs2010963) polymorphism in a Korean population showed that the SNP was associated with the risk of advanced stage endometriosis. The analysis of both SNPs in an Indian population identified a haplotype associated with endometriosis In addition, the analysis of *VEGF* −460 T/C (rs833061), +405 G/C (rs2010963) and +936 C/T

formation of blood vessels (Artini PG et al, 2008).

**2.1.3 Genetic polymorphisms of VEGF genes** 

determine whether endometriotic lesions develop and grow.

(rs3025039) polymorphisms in 147 endometriosis cases and 181 controls found a positive association between stages III–IV disease and the *VEGF* +936 T allele in a Japanese population. (Zhao Z, et al 2008). The first reported study in a Caucasian population of +405 G/C (rs2010963) in 203 Italian women affected with endometriosis and 140 controls reported a weak association of the C allele with endometriosis (Gentilini D et al, 2008).

#### **2.1.4 Therapeutical approach**

Therapy of endometriosis consists of surgical removal of implants or medical treatment such as analogues of Gonadotrophin releasing hormone or oral contraceptive or progestins. This therapeutic approach has been shown to be of limited benefit so new approaches need to be developed. Considering the importance of angiogenesis in developing and maintaining disease, and the role of vascular endothelial growth factor, anti-angiogenetic drugs could be very important.

Romidepsin, the Histone deacetylase (HDC) inhibitor, modulates the expression of a variety of genes by alteriting gormatin structure. It has been recently shown to inhibit proliferation and activate apoptosis in human epithelial endometriotic cells. In particle Imesch demonstrated that his epigenetically acting drug inhibits VEGF transcription at low nanomolar concentration with high efficency. It works at the transcriptional level down regulated VEGF expression. Romidepsin reduced the level of HIF-α protein, indicating that VEGF mRNA expression may be related to the reduction of HIF-α protein levels. The issue of whether VEGF transcription is the primary target of romidepsin or if it acts preventing deacytalation of HIF-α, must still be solved. However Romidepsin, acting at a transcriptional level, could be more effective than other angiogenetic drugs, which inhibit the VEGF active form in targeting angiogenesis, and it can be considered a novel therapeutic candidate to counter endometriosis (Imesch P et al, 2011).

Gonadotrophin- releasing hormone agonist (GnHRa I) have been applied with success, in the treatment of endometriosis combined with the laparoscopic surgery. It leads to a reduction of ovarian hormone levels, to atrophy of endometriotic implants and it has antiproliferation and apoptotic effects. It reduce VEGF expression and it has been seen that after GnRHa treatment the concentration in peritoneal fluid of VEGF are significantly lower New molecule GnRHa II has been studied. Fengying et al demonstarted that this molecule can dose-dependently reduce VEGF protein secreted by ectopic and eutopic endometrial stromal cells cultured in vitro, and the inhibition effect is stronger than that of GnRHa I. GnRH II may reduce the secretion also of immune factors such as interleukin-8 and cyclooxigenase 2 relating to the incidence of endometriosis, suggesting for a antiproliferation and anti-inflammatory effect on endometrial cells (Fengying, H et al 2010).

Yilmaz showed (Yilmaz B et al, 2010.) the effect of metformin of endometriosis implants for his antioxidant characteristics and the beneficial effects on VEGF, and matrix metalloproteinases. In particular it reduces endometriotic implants in rats reducing VEGF levels.

Molecular therapies have been proposed as a treatment alternative for recurrent endometriosis. The use of concitionally replicative adenovirus (CRADs) has been explored for the therapy of disease. In particular Adenovirus constructed with the VEGF promoter controlling the expression of a marker gene have been evaluated in vitro culture of endometriotic cells. AdVEGFE1 replicates in a short-term culture of purified ectopic

Endometriosis and Angiogenic Factors 189

Studies have shown the role of some cytokines in the implantation of ectopic endometrial tissue, and its progression and infiltration. In the implantation and growth of ectopic tissue, a primary role was attributed to several cytokines contained in the peritoneal fluid including interleukin IL-1,IL-6, IL-8, IL-12 and tumor necrosis factor-a (TNF-a) (Arici et al.,1996); (Iwabe et al., 1998); (Ho et al.,1997) Normally, peritoneal fluid contains leukocytes in concentrations of 0.5 to 2.0 3 106/mL, of which approximately 85% are macrophages (Syrop et al., 1987). Halme et al., (Halme et al.,1984) postulated that peritoneal macrophage activation may be a central contributor to the pathogenesis of endometriosis and activated macrophages in the peritoneal cavity of women with endometriosis (Vinatier et al., 1996) are potent producers of cytokines (Halme, 1989) ;( Fakih et al., 1987); (Rana et al., 1996). Thus, peritoneal fluid contains a rich cocktail of cytokines. Cytokines play a major role in the initiation, propagation, and regulation of immune and inflammatory responses. Immune cell activation results in a burst and cascade of inflammatory cytokines. These cytokines have pleiotropic and redundant activities that culminate in recruitment of numerous cell types to the site of inflammation (Harada et al, 2001). More, cytokines may regulate the actions of leukocytes in the peritoneal fluid or may act directly on ectopic endometrium, where they may play various roles in the pathogenesis and pathophysiology of endometriosis. Increased levels of cytokines in the peritoneal fluid of women with endometriosis may reflect increased synthesis of cytokines by peritoneal macrophages, lymphocytes, ectopic endometrial implants, or mesothelial cells of the peritoneum, all of which can produce cytokines (Tabibzadeh et al., 1989) ; (Betjes et al., 1993). The main source of cytokines is thought to be the macrophages, which originate in bone marrow, circulate as monocytes, and migrate to various body cavities. It seems that the cytokines playing the

most important role in the endometriosis are : IL-1, IL-6, IL-8,IL-12, TNF- α.

**• Interleukin-1 (IL1)** is one of the major proinflammatory cytokine found in the peritoneal fluid of women with endometriosis (Mori et al., 1991); (Taketani et al., 1992); (Fakih et al., 1987). This multifunctional cytokine was shown to stimulate the production of angiogenic factors by ectopic endometrial cells and therefore play a role in ectopic endometrial cell growth (Lebovic et al., 2000). Other studies pointed to a possible role for IL1 in endometriosis-associated infertility (Fakih et al.,1987); (Sueldo et al.,1990). Both ectopic and eutopic endometrial cells of women with endometriosis display an increased sensitivity to IL1, which results in an enhanced production of angiogenic, growth, and proinflammatory factors (Lebovic et al., 2000); (Akoum et al., 1995); (Akoum et al.,2002).Some previous studies showed that the increased endometrial and endometriotic cell responsiveness to IL1 may in part be due to a deficiency in the expression of interleukin-1 receptor type II (IL1R2) revealed in eutopic and ectopic endometrial tissues (Akoum et al., 2001);(Kharfi et al.,2002). The soluble IL1R2 levels were found to be reduced in the peripheral blood of women with endometriosis, which may account for the activation of peripheral blood monocytes in them (Kharfi et al., 2002). More, the IL1R2 has no signaling properties in contrast to the functional signaling IL1R1, which mediates cell activation by IL1 (Bossu et al., 1995); (Dinarello 2004); (Colotta et al.,1993). However, the membrane form of this receptor and the soluble form, which is shed by proteolysis from the cell surface (Cui et al.,2003); (Orlando et al.,1997), bind to IL1 and with higher affinity to IL1b, which is the circulating and the preferential ligand for IL1R2, in particular for its soluble form (Bossu et al., 1995). This inhibits the interaction of

**3.1 Cytokines and pathogenesis of endometriosis** 

endometriosis cells. The virus induces apoptosis in endometriotic cells in vitro (Rein DT. Et al, 2010). Ad VEGFE1 allowed specific replication and efficient killing of endometriotic cells.

Another approach, which has been recently been published (Essam –Eldin R et al, 2008) is the transfection of endometriotic cells by dominant negative estrogen receptor gene via Ad vector. Dominant negative mutants of the estrogen receptor are altered estrogen receptors forms that are unable to activate transcription of estrogen-responsive genes when estradiol binds them, resulting in decreased cell proliferation and increased apoptosis.
