**2.1 Vascular Endothelial Growth Factor(VEGF)**

#### **2.1.1 VEGF family and its receptors**

Vascular endothelial growth factors are important signaling proteins involved in both vasculogenesis (the *de novo* formation of the embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). VEGF family comprises seven members: VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and PlGF. All members have a common VEGF homology domain. This core region is composed of a cystine knot motif, with eight invariant cysteine residues involved in inter- and intramolecular disulfide bonds at one end of a conserved central four-stranded-sheet within each monomer, which dimerize in an antiparallel, side-by-side orientation (Neufeld G, wt al 1999).

The human *VEGF* gene (*VEGFA*, OMIM 192240) is located on chromosome 6p12 (Zhao Z, et al 2008).

Endometriosis and Angiogenic Factors 185

soluble factors released from cells. Many factors are involved in this complex mechanism, including FGF-a, FGF- b, PD-ECGF and VEGF, stimulate vascular endothelial cell growth *in* 

The development and maintenance of endometriosis is dependent on the recruitment of blood vessels to the endometriotic lesions from pre-existing ones to guarantee oxygen and essential nutrient supply. It has been shown that those endometriotic lesions recruit blood vessels by inducing angiogenesis. In a rat model, analyses of the assessed microvessel density demonstrated that angiogenesis is higher in ectopic endometriotic lesions compared

Promoting angiogenesis, VEGF is involved in the etiology but also in the maintenance of peritoneal endometriosis. The endometrial tissue that has been migrated into the peritoneal space develops different angiogenic properties and invasive patterns, which could have a role in the implantation in the peritoneum of the pelvic cavity. It is likely that the action of VEGF in the implant of endometrial ectopic cells is to promote the differentiation of mesodermic cells into endothelial cells and to regulate the tubular capillary formation (Di

VEGF-A is localized predominantly in the glandular epithelium of endometriosis lesions. Peritoneal fluid concentrations of VEGF have been demonstrated to be significantly higher in women with endometriosis than in the control patient and a positive correlation between the severity of endometriosis and the concentrations of VEGF in peritoneal fluid has been observed. Peritoneal fluid itself induced higher expression of the protein participating in the

In patients with endometriosis, high concentrations of VEGF in cystic and peritoneal fluids may be ascribed to a state of inflammation, where macrophages, which are the main source

It has been shown that cytokines released from immune cells play an important role in the pathogenesis of endometriosis, and many of these cytokines possess angiogenic activity. Pelvic implants and peritoneal fluid macrophages are the most likely source of VEGF-A in peritoneal fluid. McLaren (McLaren J, et al 2006) showed that peritoneal fluid macrophages express receptors for steroid hormones and secrete VEGF-A in response to ovarian steroids. The VEGF receptors VEGFR-1 and VEGFR-2 were also detected, suggesting an autocrine regulation. Peritoneal macrophages and activated lymphocytes seem to play an integral role in the secretion of proinflammatory/proangiogenic cytokines resulting in upregulation of

Ovarian endometriotic cysts also over express VEGF in their cystic fluid with respect to follicular and serous cysts and to a similar degree as ovarian cystadenocarcinoma and a negative or positive correlation of VEGF expression has been reported with cyst diameter. In ovary the process of angiogenesis is characterized by the existence of complex interrelations between the cell components of the ovarian cyst: diffuse VEGF expression in epithelial cells was associated with larger cysts; high VEGF expression in capsular fibroblast was associated with bilateral cysts; and expression of VEGF was found to be related in epithelial cells, capsular fibroblast and vessels, suggesting that neoangiogenesis might especially affect the outer cell cyst wall, thus contributing to cyst growth.( Goteri G. et al,

establishment and persistence of peritoneal endometriosis (Fasciani G. Et al, 2000).

VEGF from infiltrating neutrophils and macrophages (Machado DE et al, 2010).

*vitro* and angiogenesis *in vivo* (Gordon J. Et al, 1995).

with the eutopic endometrium (Machado DE et al, 2010).

Carlo C. Et al 2009).

of this growth factor, play a central role.

Vascular endothelial growth factor (VEGF) is one of the most potent and specific angiogenic factors. When VEGF binds to its targeted receptor, the VEGF receptor activation leads to a rapid increase in intracellular Ca2+ and inositol triphosphate concentrations in endothelial cells. The basic physiological function of VEGF is to induce angiogenesis, which allows the endometrium to repair itself following menstruation. It also modulates the characteristics of the newly formed vessels by controlling the microvascular permeability and permitting the formation of a fibrin matrix for endothelial cell migration and proliferation. This modulation may be responsible for local endometrial edema, which helps prepare the endometrium for embryo implantation. In endometriosis patients, VEGF is localized in the epithelium of endometriotic implants, particularly in hemorrhagic red implants. Moreover, the concentration of VEGF is increased in the PF of endometriosis patients. The exact cellular sources of VEGF in PF have not yet been precisely defined. Although evidence suggests that endometriotic lesions themselves produce this factor, activated peritoneal macrophages also can synthesize and secrete VEGF.

In women, physiological neoangiogensis is presented during the female reproductive cycle. VEGF-A is important in luteal angiogenesis. VEGF-A mRNA or protein is detectable in the granulosa cells of primordial and primary follicles, as they progressively become localized to the granulosa surrounding the oocyte and theca cells of the preovulatory follicle. After ovulation, VEGF-A mRNA and protein expression are observed in granulosa-derived luteal cells. VEGF-A expression in the corpus luteum appears highest early in the luteal phase and declines after the mid-luteal phase, with little or no expression in the late corpus luteum (Fraser HM, et al 2000). Gonadotrophic hormones, particularly luteinizing hormone (LH), appear to be major regulators of angiogenesis in the ovary (Hyder SM. Et al, 1999). The LHstimulated luteinization of granulosa cells at the time of ovulation is associated with enhanced VEGF-A expression.

When VEGF is overexpressed, it can contribute to disease. Three VEGF tyrosine kinase receptors have been identified: The fms-like tyrosine kinase Flt-1 (VEGFR-1/Flt-1), the kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3). Each receptor has seven immunoglobulinlike domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain (Ortega N et al, 1999). Binding its receptors R1, VEGF regulates development of tubular capillaries; binding R2 receptor, it promotes mesodermic cells differentiation into endothelial cells. In vivo and in vitro experiments indicate that steroid hormones, hypoxia and nitric oxide are potent inducers of vascular endothelial growth factor gene expression by enhancing hypoxia inducible factor 1-α activity and by the activation of the AKT/protein kinase B pathway (Kimura H, et al 2006).

### **2.1.2 VEGF role in endometriosis**

Although the aetiology of endometriosis is unknown, it is generally accepted that the condition is a result of the implantation of exfoliated endometrium, deposited in the peritoneal cavity following retrograde menstruation (Sampson J 1927). When the exfoliated endometrium enters the peritoneal cavity and becomes attached to the mesothelial layer through attachment proteins like the cadherins, a process of angiogenesis is essential for further implantation and the development of peritoneal endometriosis (Nisolle M et al 1993). Angiogenesis is a fundamental process by which new blood vessels are formed and is considered as a major process in the pathogenesis of endometriosis. It is dependent on

Vascular endothelial growth factor (VEGF) is one of the most potent and specific angiogenic factors. When VEGF binds to its targeted receptor, the VEGF receptor activation leads to a rapid increase in intracellular Ca2+ and inositol triphosphate concentrations in endothelial cells. The basic physiological function of VEGF is to induce angiogenesis, which allows the endometrium to repair itself following menstruation. It also modulates the characteristics of the newly formed vessels by controlling the microvascular permeability and permitting the formation of a fibrin matrix for endothelial cell migration and proliferation. This modulation may be responsible for local endometrial edema, which helps prepare the endometrium for embryo implantation. In endometriosis patients, VEGF is localized in the epithelium of endometriotic implants, particularly in hemorrhagic red implants. Moreover, the concentration of VEGF is increased in the PF of endometriosis patients. The exact cellular sources of VEGF in PF have not yet been precisely defined. Although evidence suggests that endometriotic lesions themselves produce this factor, activated peritoneal macrophages also

In women, physiological neoangiogensis is presented during the female reproductive cycle. VEGF-A is important in luteal angiogenesis. VEGF-A mRNA or protein is detectable in the granulosa cells of primordial and primary follicles, as they progressively become localized to the granulosa surrounding the oocyte and theca cells of the preovulatory follicle. After ovulation, VEGF-A mRNA and protein expression are observed in granulosa-derived luteal cells. VEGF-A expression in the corpus luteum appears highest early in the luteal phase and declines after the mid-luteal phase, with little or no expression in the late corpus luteum (Fraser HM, et al 2000). Gonadotrophic hormones, particularly luteinizing hormone (LH), appear to be major regulators of angiogenesis in the ovary (Hyder SM. Et al, 1999). The LHstimulated luteinization of granulosa cells at the time of ovulation is associated with

When VEGF is overexpressed, it can contribute to disease. Three VEGF tyrosine kinase receptors have been identified: The fms-like tyrosine kinase Flt-1 (VEGFR-1/Flt-1), the kinase domain region, also referred to as fetal liver kinase (VEGFR-2/KDR/Flk-1), and Flt-4 (VEGFR-3). Each receptor has seven immunoglobulinlike domains in the extracellular domain, a single transmembrane region, and a consensus tyrosine kinase sequence interrupted by a kinase insert domain (Ortega N et al, 1999). Binding its receptors R1, VEGF regulates development of tubular capillaries; binding R2 receptor, it promotes mesodermic cells differentiation into endothelial cells. In vivo and in vitro experiments indicate that steroid hormones, hypoxia and nitric oxide are potent inducers of vascular endothelial growth factor gene expression by enhancing hypoxia inducible factor 1-α activity and by the

Although the aetiology of endometriosis is unknown, it is generally accepted that the condition is a result of the implantation of exfoliated endometrium, deposited in the peritoneal cavity following retrograde menstruation (Sampson J 1927). When the exfoliated endometrium enters the peritoneal cavity and becomes attached to the mesothelial layer through attachment proteins like the cadherins, a process of angiogenesis is essential for further implantation and the development of peritoneal endometriosis (Nisolle M et al 1993). Angiogenesis is a fundamental process by which new blood vessels are formed and is considered as a major process in the pathogenesis of endometriosis. It is dependent on

activation of the AKT/protein kinase B pathway (Kimura H, et al 2006).

can synthesize and secrete VEGF.

enhanced VEGF-A expression.

**2.1.2 VEGF role in endometriosis** 

soluble factors released from cells. Many factors are involved in this complex mechanism, including FGF-a, FGF- b, PD-ECGF and VEGF, stimulate vascular endothelial cell growth *in vitro* and angiogenesis *in vivo* (Gordon J. Et al, 1995).

The development and maintenance of endometriosis is dependent on the recruitment of blood vessels to the endometriotic lesions from pre-existing ones to guarantee oxygen and essential nutrient supply. It has been shown that those endometriotic lesions recruit blood vessels by inducing angiogenesis. In a rat model, analyses of the assessed microvessel density demonstrated that angiogenesis is higher in ectopic endometriotic lesions compared with the eutopic endometrium (Machado DE et al, 2010).

Promoting angiogenesis, VEGF is involved in the etiology but also in the maintenance of peritoneal endometriosis. The endometrial tissue that has been migrated into the peritoneal space develops different angiogenic properties and invasive patterns, which could have a role in the implantation in the peritoneum of the pelvic cavity. It is likely that the action of VEGF in the implant of endometrial ectopic cells is to promote the differentiation of mesodermic cells into endothelial cells and to regulate the tubular capillary formation (Di Carlo C. Et al 2009).

VEGF-A is localized predominantly in the glandular epithelium of endometriosis lesions. Peritoneal fluid concentrations of VEGF have been demonstrated to be significantly higher in women with endometriosis than in the control patient and a positive correlation between the severity of endometriosis and the concentrations of VEGF in peritoneal fluid has been observed. Peritoneal fluid itself induced higher expression of the protein participating in the establishment and persistence of peritoneal endometriosis (Fasciani G. Et al, 2000).

In patients with endometriosis, high concentrations of VEGF in cystic and peritoneal fluids may be ascribed to a state of inflammation, where macrophages, which are the main source of this growth factor, play a central role.

It has been shown that cytokines released from immune cells play an important role in the pathogenesis of endometriosis, and many of these cytokines possess angiogenic activity. Pelvic implants and peritoneal fluid macrophages are the most likely source of VEGF-A in peritoneal fluid. McLaren (McLaren J, et al 2006) showed that peritoneal fluid macrophages express receptors for steroid hormones and secrete VEGF-A in response to ovarian steroids. The VEGF receptors VEGFR-1 and VEGFR-2 were also detected, suggesting an autocrine regulation. Peritoneal macrophages and activated lymphocytes seem to play an integral role in the secretion of proinflammatory/proangiogenic cytokines resulting in upregulation of VEGF from infiltrating neutrophils and macrophages (Machado DE et al, 2010).

Ovarian endometriotic cysts also over express VEGF in their cystic fluid with respect to follicular and serous cysts and to a similar degree as ovarian cystadenocarcinoma and a negative or positive correlation of VEGF expression has been reported with cyst diameter.

In ovary the process of angiogenesis is characterized by the existence of complex interrelations between the cell components of the ovarian cyst: diffuse VEGF expression in epithelial cells was associated with larger cysts; high VEGF expression in capsular fibroblast was associated with bilateral cysts; and expression of VEGF was found to be related in epithelial cells, capsular fibroblast and vessels, suggesting that neoangiogenesis might especially affect the outer cell cyst wall, thus contributing to cyst growth.( Goteri G. et al,

Endometriosis and Angiogenic Factors 187

(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).

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

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

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

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

**2.1.4 Therapeutical approach** 

candidate to counter endometriosis (Imesch P et al, 2011).

very important.

levels.

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

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 determine whether endometriotic lesions develop and grow.
