The effects of certain angioneogenesis inhibitors in experimental endometriosis in rats

The pathogenetic theory of retrograde outflow of endometrial cells into the peritoneal cavity at endometriosis is gaining an increasing support. Adequate blood supply and angioneogenesis play an important role in successful implantation and occurrence of ectopic foci. VEGF plays an important role in this process. Determination of the pathways affecting the activity of this factor seems to be promising in terms of its effect produced on the early pathogenetic links of endometriosis.

to determine the effectiveness of dopamine agonist cabergoline and highly selective COX-2 inhibitor celecoxib in an experimental model of external genital endometriosis in rats. METHODS. 83 outbred white female rats of Rattus Norvegicus Wistar were involved in the experiment. Experimental induction of endometriosis was performed by surgery and implantation of the autologous uterine fragments. The type and volume of experimental endometriosis lesions on the peritoneum of experimental animals were evaluated macroscopically as well as their hystologic examination were performed. RESULTS. Administration of a dopamine receptor agonist as a VEGF inhibitor separately was found to produce a pronounced inhibitory effect on ectopic endometrioid formation. However, the use of a dopamine receptor agonist in combination with a highly selective COX-2 inhibitor does not lead to potentiation or summation of their effects. At the same time, the use of COX-2 inhibitor alone has shown significantly lower efficacy than using the dopamine receptor agonist as a VEGF inhibitor. CONCLUSION. At experimental endometriosis in rats, dopamine agonists and highly selective COX-2 inhibitors were found to be one of VEGF inhibitors available. KEY WORDS: experimental endometriosis; angioneogenesis; VEGF; dopamine agonist; COX-2 inhibitor; cabergoline; celecoxib ORIGINAL RESEARCH pro- and anti-inflammatory cytokines of the peritoneal fluid, plays a key role. The implantation of endometrium cells results from balance disorder and excretion of growth factors. Adequate blood supply and neoangio- genesis play an important role in successful implantation and occurrence of ectopic foci [10-12]. Considering the above, it can be suggested that one of the promising areas of conservative treatment of external genital endometriosis is the effect produced on one of the pathogenetic links, namely, inhibition of angiogenesis by the use of dopamine agonists and COX-2 inhibitors [13]. Angiogenesis is a complex process of formation of new blood vessels from the vessels existed before. This process plays a fundamental role in the reproduction, development and healing of wounds. In adults, endo- thelial proliferation is a strictly regulated process based on the balance between angiogenic and angiostatic factors which are activated in case www.transplantology.org 141 Cell and Organ Transplantology 2019 November; Vol. 7, No. 2 of necessity and subsequently completely inhibited when necessary [14, 15]. Cases of increased endothelial cell proliferation rate are often associ- ated with tumors and their development [16], which are known to be de- pendent on angiogenesis for growth and metastasis [17]. The survival of endometrioid implants in the abdominal cavity depends on the formation of blood supply to provide oxygen and nutrients to the developing lesions. Pathomorphological examination of endometriosis foci determines their dense vascularization [15, 18]. Similar to the process of tumor vascula- rization, endometriosis can utilize the mechanisms of angiogenesis and vasculogenesis to form its own vascular network to maintain viability. It should be supposed that endometrial fragments separated from the uterine endometrium may be the carriers of angiogenic potential. The hu- man endometrium, which consists of the functional and basal layer, is a unique organ that undergoes proliferation, differentiation and regeneration at each menstrual cycle under the regulation of steroid hormones of the ovaries – estrogens and progesterone. In addition to endometrial growth, the vascular bed of the endometrium undergoes proliferation and regen- eration at every cycle under the influence of ovarian steroids, especially estradiol (E2). Shifren et al. [19] identified the increased expression of vascular endothelial growth factor (VEGF) mRNA in the functional layer of the endometrium in the proliferative and secretory phase of the men- strual cycle, indicating the involvement of angiogenesis for proliferation and regeneration. The same study found that E2 is responsible for stimu- lating VEGF expression in isolated human endometrial cells, and E2 ad- ministration increased VEGF mRNA expression compared to endometrial cells without E2 stimulus. Endometriosis can be suggested to result from implantation of endometrial fragments into the abdominal cavity with sub- sequent activation of cellular aggression and proliferation mechanisms. VEGF appears to play an important role in supporting angiogenesis at endometriosis. It is a vasoactive substance which is involved in vari- ous normal physiological processes, including wound healing and endo- metrial revascularization, mediating endothelial proliferation and migration. In oncogenesis VEGF concentration usually correlates with increased blood supply in various tissue types that are associated with the tumor [20]. In normal endometrium, elevation of VEGF mRNA levels and expression of the corresponding proteins can be caused by hypoxia [21]. No wonder that the peritoneal fluid of women with the last stages of endometriosis con- tains higher VEGF concentrations than that of women with initial stages of endometriosis or healthy women [22]. Various sources of VEGF have been identified, including endometrioid lesions [23] and peritoneal fluid macro- phages that increase VEGF expression when treated with ovarian steroids such as E2 and progesterone [24], transforming the notion that VEGF is involved in angiogenesis associated with endometrial lesions [25]. VEGF, also known as VEGF-A, is a secretory cytokine structurally related to platelet-derived growth factor (PDGF), which mediates physio- logical and tumor angiogenesis [26-29]. In mice a complete damage of this factor is lethal and causes severe cardiovascular abnormalities [30, 31]. In vitro it promotes endothelial cell proliferation, migration, and vas- cular tube formation [32]. In various in vivo models it induces a strong angiogenic response [33]. At adulthood VEGF is involved in wound hea- ling, menstruation, pregnancy and blood pressure support [29]. During physiological angiogenesis, it is produced by various cell types, including neutrophils, platelets, and macrophages [29]. Currently there are the following ways of VEGF inhibition: (1) neutra- lizing monoclonal antibodies against VEGF and VEGFR; (2) small-mole- cule VEGF receptor tyrosine kinase inhibitors; (3) soluble VEGF receptors (VEGF-Trap) and (4) ribozymes [34]. In addition to the above mentioned VEGF inhibition pathways which are widely discussed in clinical onco- logy, there is the evidence of possibile inhibition of VEGF-induced an- giogenesis by using dopamine neurotransmitter [35]. Bacic et al [36] reported that dopaminergic receptors are linked to adenylyl cyclase in the human cerebral microvascular endothelium. Dopamine is a catecholamic neurotransmitter involved in pathogenesis of both Parkinson’s disease and development of schizophrenia [37-40]. Dopamine and its derivative molecules have shown inhibitory potential in several types of malignant tumors in mice, and its effects have been explained by inhibition of tumor cell proliferation, stimulation of immunity etc. [41-44]. Recent studies demonstrate the presence of D2 dopamine receptors on endothelial cells [45, 46], which may affect angiogenesis. It is this mechanism that is likely to be important in inhibiting tumorigenesis [47, 48], as VEGF is the most important angiogenic cytokine in tumors and in other types of pathologi- cal angiogenesis [49, 50]. VEGF is believed to induce angiogenesis by involving VEGF receptor type 2 (VEGFR-2), which leads to its phosphorylation and downstream signaling events series [51]. 1 μM dopamine was found to inhibit VEGF- induced phosphorylation of VEGFR-2 in vitro. D2 bromocriptine and quinpyrrole agonists similarly inhibit VEGF-induced phosphorylation of VEGFR-2 [52-54]. Dopamine is indicated to stimulate internalization of VEGFR-2 surface, probably by endocytosis, leaving less VEGFR-2 on the surface to interact with VEGF [52-54]. Thus, the inhibitory effect of do- pamine on VEGF-induced angiogenesis is explained by its action at the early signaling stage. It is important to note that dopamine inhibits an- giogenesis mediated by VEGF, but probably does not affect angiogenesis mediated by other mechanisms [54]. VEGF is not the only way to activate angiogenesis. There are alterna- tive activation pathways, such as through COX-2. COX-2 converts arachi- donic acid into prostaglandin H2, which can be enzymatically converted into five major prostanoids: prostaglandin E2, prostaglandin D2, prosta- glandin I2 (prostacyclin), prostaglandin F2a and thromboxane. COX-2 and prostaglandin E2 have been previously demonstrated to induce angioge- nesis, but most studies indicate an indirect role by means of stimulating other proangiogenic growth factors [55]. In addition to indirect effects, hypothetically, the COX-2 / PGE2 pathway may play a direct role in stimu- lating angiogenesis through VEGF-independent paracrine mechanisms that directly affect endothelial cells [56]. Certain studies have shown that COX-2 regulates VEGF-induced angiogenesis, and also that VEGF con- trols COX-2-induced angiogenesis, indicating reciprocity between these pathways [57, 58]. The ability of COX-2/PGE2 pathway to induce VEGF- independent angiogenesis suggests that this pathway may promote an- giogenesis when VEGF pathway is blocked. Moreover, scientific literature contains certain information concer- ning suppression of VEGF-induced phosphorylation of type 2 VEGF re- ceptor by dopamine [59-61], occurring due to phosphorylation of tyrosine phosphatase 2 [62], and therefore, its inactivation.

OF THE STUDY: to determine the effectiveness of dopa- mine agonist cabergoline and highly selective COX-2 inhibitor celecoxib in an experimental model of external genital endometriosis in rats. The experiment involved 83 outbred, sexually mature, nulliparous white Rattus Norvegicus Wistar rats weighing 170-200 g, 14-17 weeks of age. All the manipulations on animals were carried out in accordance with the recommendations of the UNESCO Intergovernmental Committee on Bioethics (IGBC). All the procedures and experiments of this study respect the ethical standards in the Helsinki Declaration of 1975, as revised in 2008 [5], as well as the national law. All institutional and national guide- lines for the care and use of laboratory animals were followed. At the end of the adaptation period, starting from the first day of the experiment, rats were injected subcutaneously in the back with 0.06 mg/kg body weight of estradiol valerate. Estradiol valerate injection at the dose of 0.06 mg/kg was repeated on the third day of the experiment. On the fourth day of the experiment, experimental induction of en- dometriosis was performed by means of surgery and implantation of an autologous uterine fragment using the method described by A. Golan et al. [63] and T. Hirata et al. [64]. After awakening from ketamine-xylazine intraperitoneal anesthesia, rats were randomly and evenly divided into groups. Rats of all the groups were treated with estrogenic hormonal

142 Cell and Organ Transplantology 2019 November; Vol. 7, No. 2 support in the form of daily subcutaneous injections of 0.03 mg/kg body weight of estradiol valerate until the last day of the experiment. In the first experimental group comprising 21 rats, dopamine recep- tors agonist cabergoline (ATC: G02CB03; Dostinex ® , Pfizer Italia S.r.l.) was used as a subcutaneous injection in the dose of 0.075 mg/kg body weight daily. In the second experimental group including 19 rats, a do- pamine receptor agonist and a COX-2 inhibitor were administered as a single subcutaneous injection of cabergoline in the dose of 0.075 mg/kg of animal body weight and celecoxib (АТХ: L01XX33, M01AH01; Cele- brex ® , Pfizer Inc.) in the dose of 30 mg/kg of animal body weight daily. In the third experimental group of 20 rats, the COX-2 inhibitor celecoxib was administered as a subcutaneous injection in the dose of 30 mg/kg of ani- mal body weight daily. In the fourth, the control group, which comprised 20 rats, nothing more than estrogen support has been applied. In the first, second and third experimental groups, drugs were admi- nistered from the 12 th day of the experiment (from the 8 th day after sur- gery) to the end of the experiment (the 25 th day of the experiment, the 21 st day after surgery). On the 26 th day of the experiment (22 nd day after surgery) euthanasia was performed by decapitation using thiopental an- esthesia in the dose of 5.7 mg/kg of the body weight. The presence and type of lesions were evaluated macroscopically: cysts filled with dark fluid, cysts filled with light fluid, lesions in the form of solid tissue, or the absence of macroscopic signs of lesions. Using the office transparent millimeter ruler the smallest and largest diameters of lesions were measured. The following formula was used to determine the volume of lesions: where d 1 and d 2 – are the smallest and largest diameters of lesion respectively. ORIGINAL RESEARCH Sampling of the material for morphological studies was performed according to standard requirements for making histological preparations. The tissue fragments after washing in distilled water were fixed in 10-12 % solution of neutral formalin, after which they were transferred to 3-5 % solution of neutral formalin, where they were stored. The preparations were dehydrated by sequentially running the objects through ethanol of increasing concentration. Subsequently, paraffin embedded blocks were made and 3 to 5 μm thick histologic sections were perfomed using a microtome. To obtain differentiated polychromy, the tissue was stained with hematoxylin-eosin. Histological sections were examined, analyzed and photographed using a computer-based image analysis system consisting of an CX-21 light mi- croscope (Olympus, Japan) and C450 digital camera (Olympus, Japan). Estimation of optical density and the area calculations of microscopic structures was performed using free-licensed software ImageJ, ver. 1.50b (NIH, USA). The optical density of cytoplasm and nuclei of secretory epi- thelial cells was estimated using 0-255 RGB gradation (0 – black color, 255 – white color). The results were statistically processed by MedCalc 15.8 software (MedCalc Software bvba, Belgium) using Mann-Whitney U-test and de- scriptive statistics analysis. Data were presented as mean +/- standard deviation (SD). Graph data were presented as clustered bar chart and box and whisker charts. Fig. 1. Macroscopic view of ectopic uterine tissue on the peritoneum when using cabergoline only ( A), celecoxib (B) or combination of cabergoline and celecoxib (C) compared to control group (D). Arrows indicate implant growth sites. D B C А www.transplantology.org 143 Cell and Organ Transplantology 2019 November; Vol. 7, No. 2 Most current theories of endometriosis are based on the theory of Sampson’s retrograde menstruation. However, an open question remains why retrograde menstruation occurs in 90 % of women of reproductive age, while the incidence of endometriosis is several times less. Among the contributing factors for the implantation of eutopic endometrium to the peritoneum, we have identified VEGF as one of the major contributors to the growth of heterotopias. Dopamine agonists and highly selective COX-2 inhibitors have been identified as one of the available VEGF inhibi- tors. In order to determine the effectiveness of their use, we created an experimental model of endometriosis in rats. Macroscopic examination of endometrioid foci was performed imme- diately after euthanasia, after opening the abdominal cavity of a rat using U-shaped incision. Considering the fact that the only reliable method of diagnosis of endometriosis is visual identification of foci during laparo- scopy with subsequent histological verification, it is advisable to visualize and perform morphometry in an experimental model of endometriosis. Based on morphometry, data the volume of lesions was determined, which is another parameter of objectification when conducting statistical research. Due to the fact that two flaps of their own uterine horn were transplanted to each animal, the number of observed cases was doubled in relation to the number of animals in this group. The morphological data we have obtained coincide with those of other researchers who used a similar technique. Thus, Elgamal et al. [65] describes ectopic endometrioid lesions macroscopically in the form of cysts filled with fluid, and the histological picture obtained by us com- pletely corresponds to the described one by Rezende et al. [66], Neto et al. [67] and Amaral et al. [68] (Fig. 1). While comparing the types of lesions (Fig. 2), we determined that the least cystic endometrioid formation occurred when cabergoline was used. The use of celecoxib alone and in combination with cabergoline showed less effectiveness concerning this parameter. While comparing the volume of lesions (Fig. 3), we found that the best

were obtained when cabergoline was used separately. The volume of lesions after cabergoline administration is almost 9 times smaller than that in the control group. It should be noted that the volume of lesions after administration of cabergoline + celecoxib combination is almost 3.5 times smaller than that in the control. When celecoxib is used separately, there is also a significant difference in the volume of lesions (celecoxib reduces the size of lesions), but less pronounced (slightly more than 2 times). One of the indirect signs of vascular and capillary proliferation, as well as a sign of successful implantation of ectopic endometrium, is the growth of the glandular epithelium and its secretory activity. During microscopic examination of endometrioid lesions, we ob- served glandular secreting epithelium oriented by the secretory pole to- wards the cystic cavity. The stroma of the glands is found lower, which respectively lie on the myometrial basis (which was preserved during transplantation). We observed the viability and functional activity of the glandular epithelium of ectopic transplants (Fig. 4). Several morphometric parameters were used to objectify microscop- ic examination and functional activity of the cells. They include determi- nation of the glandular epithelium cellular height, determination of the height of the secretory part of glandular epithelium cells, determination of the nucleus area, carrying out densitometry of the nucleus and cells in general. Statistical processing and analysis of morphometric examination of histological specimens resulted in the following. The height of secretory endometrioid epitheliocytes was significantly lower (p < 0.0001) only when cabergoline was used. No significant difference was found in case of caber- goline and celecoxib combination, and with celecoxib alone (Fig. 5). The following regularity was found after examination of a secretory part of epitheliocytes: a reliable decrease was observed only with the use of cabergoline. No significant difference was found in the use of cele-

AND DISCUSSION Fig. 3. Average volume of experimental endometriosis lesions on the peritoneum in experimental groups, mm 3 . Note: * – p < 0.0001 compared to control group; ▼▲ – extreme outliers. Fig. 2. Types of experimental endometriosis lesions on the peritoneum in experimental groups. 700 600 500 400 300 200 100 0 Cabergolin* Cabergolin+Celecoxib Celecoxib Control group No cystic structures Cysts with dark contentsCysts with light contents Types of lesions 2 20 20 20 29 30 40 10 10 10 7 8 12 4 0 18 18 Cabergolin Celecoxib* Cabergolin & Celecoxib Control Cabergolin* Celecoxib* Cabergolin & Celecoxib Control 90 80 70 60 50 40 30 0 10 20 Cabergolin* Celecoxib* Cabergolin & Celecoxib Control 140 120 60 60 100 40 0 20 700 600 500 400 300 200 100 0 Cabergolin* Cabergolin+Celecoxib Celecoxib Control group відсутність кіст кісти з темним вмістомкісти з світлим вмістом Типи уражень 2 20 20 20 29 30 40 10 10 10 7 8 12 4 0 18 18 Cabergolin Celecoxib* Cabergolin & Celecoxib* Control Cabergolin* Celecoxib* Cabergolin & Celecoxib* Control 90 80 70 60 50 40 30 0 10 20 Cabergolin* Celecoxib*** Cabergolin & Celecoxib*** Control 140 120 60 60 100 40 0 20 coxib, and in case of cabergoline and celecoxib combination, a significant increase in the secretory portion of epitheliocytes was detected. The determined area of the epitheliocyte nucleus (Fig. 6) was sig- nificantly smaller in all the experimental groups (without a significant difference between the experimental groups) compared to the control. Morphometry of samples from the control group of animals revealed a smaller nucleus compared to that of the eutopic endometrium. A relative densitometric density of epitheliocyte nuclei was signifi- cantly (p < 0.05) higher (indicating less compacting of nuclei) in all the experimental groups compared to that of the control one. However, in fact, it was equal to a relative nucleus density of eutopic epitheliocytes. Determination of the relative density of epitheliocytes did not reveal a significant difference (p > 0.05) between the experimental groups and the control group. According to current literature data, we suggest that the effectiveness (according to the above mentioned indices) of cabergoline administration in experimental endometriosis compared with that of the control group is caused by its direct effect on dopamine D2 receptors. In its turn, it activates phosphorylation of VEGF receptors and their internalization via the adenylate cyclase pathway, which directly blocks the action of VEGF further leading to inhibition of angiogenesis. Due to inability to form new vessels, with nutrient and oxygen deficiency, the ectopic endometrioid 144 Cell and Organ Transplantology 2019 November; Vol. 7, No. 2 ORIGINAL RESEARCH Fig. 4. Microscopic views of ectopic uterine tissue on the peritoneum in rats when using cabergoline ( A), celecoxib (B) or combination of cabergoline and celecoxib (C) compared to control group (D). Arrow indicates secretory epithelium. Hematoxylin and eosin stain. Ob. 10х (A, C) and 40х (B, D), Oc. 10х. D B C А Fig. 5. Height of epitheliocytes in ectopic endometrium on experimental endometriosis, μm. Notes: * – p 0.05 compared to control group; ▲ – extreme outliers. Fig. 6. Nucleus area of epitheliocytes in ectopic endometrium on experimental endometriosis, μm 2 . Note: * – p < 0.0001 compared to control group; ▲ – extreme outliers 700 600 500 400 300 200 100 0 Cabergolin* Cabergolin+Celecoxib Celecoxib Control group відсутність кіст кісти з темним вмістомкісти з світлим вмістом Типи уражень 2 20 20 20 29 30 40 10 10 10 7 8 12 4 0 18 18 Cabergolin Celecoxib* Cabergolin & Celecoxib* Control Cabergolin* Celecoxib* Cabergolin & Celecoxib* Control 90 80 70 60 50 40 30 0 10 20 Cabergolin* Celecoxib*** Cabergolin & Celecoxib*** Control 140 120 60 60 100 40 0 20 700 600 500 400 300 200 100 0 Cabergolin* Cabergolin+Celecoxib Celecoxib Control group відсутність кіст кісти з темним вмістомкісти з світлим вмістом Типи уражень 2 20 20 20 29 30 40 10 10 10 7 8 12 4 0 18 18 Cabergolin Celecoxib* Cabergolin & Celecoxib* Control Cabergolin* Celecoxib* Cabergolin & Celecoxib* Control 90 80 70 60 50 40 30 0 10 20 Cabergolin* Celecoxib*** Cabergolin & Celecoxib*** Control 140 120 60 60 100 40 0 20 www.transplantology.org 145 Cell and Organ Transplantology 2019 November; Vol. 7, No. 2 implant has no potential for further development and secretory activity. This is confirmed by the data indicating decrease in epitheliocytes and their secretory poles in size, as well as the compacting of the epitheliocyte nuclei. According to the theoretical data reported in the relevant publications, COX-2 inhibitors potentially produce an inhibitory effect on angiogenesis by deactivating PGE2 formation, which, in the opinion of many authors, is responsible for an alternative (non-VEGF) pathway of angiogenesis. However, as indicated by our study, the use of celecoxib in experimen- tal endometriosis in rats did not produce the expected efficacy. Despite a significant reduction of lesions in size (although less significant than after cabergoline use), the size of epitheliocytes, their secretory part re- mained unchanged. Moreover, endometrioid lesions predominate in this group in the form of cystic structures, including almost 1/3 of them with a dark content confirming their activity. Apparently blocking non-VEGF pathways of angiogenesis is insufficient to suppress the growth of endo- metrioid lesions in the experiment. A similar statement is found in Xu et al. [69], where the authors did not obtain the expected results when using highly selective COX-2 inhibitors to inhibit tumor angiogenesis. Obvious- ly, a possible role and time-related effects of the use of highly selective COX-2 inhibitors in endometriosis should be investigated. A complex administration of cabergoline, a dopamine receptor ago- nist, and celecoxib, a highly selective COX-2 inhibitor, similar to that of the previous group (using celecoxib alone) did not produce the expected efficacy, despite a noticeable efficacy of cabergoline alone. Although an average volume of lesions is significantly smaller than that in the control group, the size of epitheliocytes did not decrease significantly compared to the control, and their secretory part is even larger than in the control group. After administration of both cabergoline in combination with ce- lecoxib, and celecoxib alone, vacuolation of epitheliocytes was observed, as it is evidenced by an increased rate of relative cell density. At the same time, compaction of epitheliocyte nuclei was observed. These data in- dicate insufficient inhibition of endometrial cell functional activity and, therefore, insufficient regression of endometrioid ectopic foci. Despite a theoretically expected synergistic effect of cabergoline and celecoxib, the results of our study are indicative of its lack. Similar conclu- sions were also drawn by Xu et al. (2015) [69]. In this study, bevacizumab (a direct specific VEGF blocker) in combination with celecoxib was shown to have much less effectiveness in blocking tumor angiogenesis than in a single administration. At the same time, in a separate administration, celecoxib showed less efficacy in blocking angiogenesis in tumors than bevacizumab. Therefore, the use of a dopamine receptor agonist as a VEGF inhibi- tor in combination with a highly selective COX-2 inhibitor does not lead to potentiation or summation of their effects. At the same time, the use of the COX-2 inhibitor alone showed significantly lower efficacy than using the dopamine D2 receptor agonist as a VEGF inhibitor. On the basis of our study, the use of dopamine receptor agonists with the purpose to inhibit angiogenesis in endometriosis has real prospects for further study, including other animal models and the clinical studies.

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