Aleksandrovych, Veronika

Bereza, Tomasz

Depukat, Paweł

Gach-Kuniewicz, Barbara

Komnata, Kinga

Konarska, Monika

Lis, Maciej

Maleszka, Aleksandra

Paziewski, Mateusz

Sajewicz, Marek

Solewski, Bernard

Warchoł, Łukasz

Wrona, Anna Natalia

English

Jagiellonian Univeristy Repository

FOLIA MEDICA CRACOVIENSIAVol. LVI, 2, 2016: 37–43PL ISSN 0015-5616Angiogenesis and pro-angiogenic factors in uterine fi broids — facts and mythsMonika Konarska1, Anna Natalia Wrona2, Veronika Aleksandrovych3, Tomasz Bereza1, Marek Sajewicz4, Barbara Gach-Kuniewicz5, Maciej Lis1, Kinga Komnata1, Mateusz Paziewski1, Aleksandra Maleszka6, Paweł Depukat1, Bernard Solewski1, Łukasz Warchoł11Department of Anatomy, Jagiellonian University Medical Collegeul. Kopernika 12, 31-034 Kraków, Poland 2Szpital Specjalistyczny im. J. Śniadeckiego w Nowym Sączu, Oddział Ginekologiczno-Położniczyul. Młyńska 10, 33-300 Nowy Sącz, Poland3Department of Pathophysiology, Jagiellonian University Medical Collegeul. Czysta 18, 31-121 Kraków, Poland4Clinic of Obstetrics and Perinatology, Th e University Hospitalul. Kopernika 23, 31-501 Kraków, Poland5Szpital Specjalistyczny im. L. Rydygiera w Krakowie, Oddział Anestezjologii i Intensywnej Terapiios. Złotej Jesieni 1, 31-826 Kraków, Poland6Department of Diagnostics, Jagiellonian University Medical Collegeul. Kopernika 15A, 31-501 Kraków, PolandCorresponding author: Tomasz Bereza, MD, PhD; Department of AnatomyJagiellonian University Medical Collegeul. Kopernika 12, 31-034 Kraków, PolandPhone/Fax: +48 12 422 95 11; E-mail: t.bereza@wp.plAbstract: Uterine leiomyomata present major problem for females. Although they are benign tumors their frequency is associated with many symptoms like infertility, abdominal pain, menorrhagia. Authors based on their own morphological studies and review of the literature try to indicate main factors causing angiogenesis within leiomyomata and its infl uence on tumor growth. Th e strongest proangiogenic factor seems to be hypoxia, which stimulates up- and down-regulation of numerous genetically determined substances. Also mechanical pressure acting upon newly growing vessels is one of the factors which may determine formation of so called “vascular pseudocapsule” around the lesion.Key words: uterine leiomyomata, microvessel density, angiogenesis.38 Monika Konarska, Anna Natalia Wrona, et al.IntroductionAngiogenesis is currently considered to be a  very important factor controlling growth and ability to metastasize of malignant tumors [1]. Role of the angiogenesis in the growth of benign tumors still remains unclear. However if we consider the model of de-velopment of uterine fi broids based on adaptation of the vessels of normal vascular bed — their further regression and subsequent vascularization “de novo” — it may appear that angiogenesis plays key role also in the development of benign tumors. Formation of “vascular pseudocapsule” in the periphery of the fi broid requires undoubtedly involve-ment of pro-angiogenic factors. Although studies which consider angiogenic activity of uterine leiomyomata and surrounding myometrium, bring unanimous results by far. It was proved that uterine fi broids are source of the whole palette of growth factors, which seem to be probable or to be angiogenic factors, too. Family of factors associated with uterine leiomyomata consists of: vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), platelet derived endothelial cells growth factor (PD-ECGF), epidermal growth factor (EGF), transforming growth factor (TGF alpha and beta), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), adnenulomedullin (ADM) [2–12]. Some of mentioned factors, i.e. VEGF-A [6], bFGF [3, 11] and IGF-I [7] show increased expression in the uterine leiomyomata, comparing to the surrounding myometrium. Other factors, i.e. EGF [6], TGF-beta [13] show decreased expression in fi broids. It is generally assumed that VEGF and ADM show increased activity in uterine myomas [2]. From another hand however we have to cope with contrary reports. Lee and Novak described increased level of TGF-beta mRNA [10], while Vollenhoven et al. [14] did not show signifi cant changes in expression of EGF and TGF-beta of fi broids and surrounding myometrium. Similar results were achieved by Harrisson-Woolrych et al. in EGF mRNA [9].Material and methodsTh e study was carried out on 51 human uteri of females aged between 32–66 years, which died because of diseases not associated with the internal female genital tracts, obtained during autopsies. Th e study was approved by local Ethical Committee. Th e whole material was collected upon 12–22 hrs from the moment of decease. Th e pro-tocol used was described in details in [14, 15]. Additionally 5 specimens were injected with water solution of acrylic emulsion (Liquitex R, Binney and Smith, USA) [16]. Th us obtained material was cut into slides, which aft er dehydration in rising concen-trations of ethyl alcohol and immersed in methyl benzoesane were paraffi  nized and dyed with hematoxylin and eosin. Th e material was studied under light microscope (magnifi cation 5–40x). Some of tissue blocs were deparaffi  nized and hydrated to per-form immunohistochemical reaction for von Willebrandt factor.  Angiogenesis and pro-angiogenic factors in uterine fi broids  — facts and myths 39Results and discussionMost of proangiogenic factors are located in cytoplasm of smooth muscle cells — of both myometrium and cells of blood vessels. TGF-alpha, EGF and FGF-2 were ob-served also within endothelial cells, and IGF-1 in the fi broblasts of connective tissue [6]. It is interesting that bFGF is located mainly within intercellular matrix, and its relatively high level was associated with its condensation [11].Growth factors present in fi broids may infl uent all its factors: smooth muscle cells, vascular endothelial cells and connective tissue fi broblasts. Th eir specifi c participation in angiogenesis hasn’t been suffi  ciently elicited yet. It is interesting that so far any relationship between increased vascular density and level of VEGF expression hasn’t been proved, and VEGF is considered to be the main factor which stimulates angiogenesis [17, 18]. Th at kind of positive correlation was observed according to PD-ECGF, TGF-alpha and ADM. Expression of this last factor correlates positively also with the endothelial cells proliferation index [2]. ADM, opposite to VEGF has limited mitogenic action, limited almost to endothelial cells only. Furthermore, it shows relatively wide infl uence on proliferation and growth of tumor cells, both malignant and benign, and may play key role in angiogenesis of uterine leiomyomata. Regulation of angiogenic factors probably follows few complicated mechanisms. Genetic aberrations of chromosomes 6, 7, 12, and 14 may cause increased tolerance considering angiogenic growth factors or decreased ability of production or response to angiogenic factors with the growth of the tumor. Genetic failures leading to increased sensitivity to angiogenic inhibitors, i.e. angiostatin [19], may be responsible for clinical picture of necrosis within certain leiomyomata.Factors which stimulate angiogenesis, i.e. VEGF and substances from group of FGF, prove frequently increased activity in hypoxia, tissue damage and growth of new tissues. ADM and VEGF prove certain similarities, because both these strong angiogenic factors are induced by hypoxia, and both increase vascular permeability. It has been well documented that on induction both these factors require hypoxia stadium [2]. It is postulated that similar to most of the neoplasms, uterine leiomyomata show decreased level of oxygen.In our studies one could observe relatively dense network of vessels penetrating the lesions, and in the specimens injected with Liquitex R we could see compressed blood vessels — even not fi lled with dye which were visible by staining using von Willebrandt factor. Th ese compressed by growing tumor masses vessels were useless — and such condition led to signifi cant hypoxia, a strong proangiogenic condition.It is obvious that angiogenesis in the uterine fi broids may be infl uenced by ovarian steroids. Estrogens regulate expression of VEGF in the uterus at the transcription level [20]. In physiology they promote endometrial angiogenesis through regulation of VEGF expression in glandular cells and stroma [21]. Regulation of bFGF and its 40 Monika Konarska, Anna Natalia Wrona, et al.receptors seems to be also dependent on the level of ovarian hormones [12]. PDGF and bFGF levels decrease in patients treated with agonists of gonadotropin releasing hormone (GnRH) [22, 23]. From another hand however in these patients nobody observed specifi c changes of microvessel density nor VEGF expression in fi broids [8, 24]. However the endothelial cells proliferation index of fi broids, myometrium and endometrium did not diff er in particular phases of the menstruation cycle [2]. Xu et al. postulated that progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells [25]. Also Catherino et al [26] indicate new potential targets for genetic researches.In this context diff erences in vascular density observed between leiomyomata and the normal tissue may at least partially be result of diff erent level of estrogen receptors (ER), which was shown in fi broids, comparing to the level of ER in regular myometrium [25, 27, 28]. Th e level of ER alpha RNA and immunoreactivity ER alpha are higher in leiomyomata than in surrounding myometrial tissue. Myometrial cells and in vitro cultured fibroid cells show increased expression of ER alpha receptors, and do not show changes in expression of ER beta. On the other hand endothelium of microvessels shows significant changes in expression of ER beta receptors [29]. Estrogens play vital role in survival of endothelial cells [30], so on this path they can stimulate changes which support or/and cause angiogenic response of microvessel endothelial cells, thus promoting growth of the new vessels within a tumor.Despite expression of numerous proangiogenic factors within uterine leiomyomata, angiogenesis seems not to be specially outstanding during tumorigenesis, what is confi rmed by relatively low vascular density (Fig. 1).Th is hypothesis was lastly confi rmed by studies of Weston et al., which proved in the leiomyomata decreased expression of two factors promoting angiogenesis: connective tissue growth factor (CTGF) and cysteine rich factor inducing angiogenesis (CYR61), and also decreased expression of collagen 4 alpha2 (COL4A2), precursor of angiogenic inhibitor — canstatine. Th is is why fi broids comparing to the surrounding myometrium show antiangiogenic profi le of gene expression.For contrast, the most intense angiogenesis seems to occur at the border leiomyoma/myometrium, which leads to formation of vascular rich “pseudocapsule”, visible especially in greater uterine leiomyomata. It might be a  result of release of proangiogenic factors through stimulated by fi broid angiogenesis within surrounding myometrium. As a matter of fact we do not know, if the arrangement of the vessels in the central region of the lesion is resulted by physical pressure of the new vessels ‘invading’ the tumor or is it result of activity of the factors which inhibit angiogenesis in these parts of the tumor? Th is problem remains in the sphere of speculations, by far. Angiogenesis and pro-angiogenic factors in uterine fi broids  — facts and myths 41Fig. 1. Uterus of 43 years old female. Subserous leiomyoma injected through the arteries with water solu-tion of Acrylic emulsion Liquitex R (Binney and Smith) (*). Immunostaining for von Willebrandt fac-tor. Centrally visible numerous minute, not fi lled with emulsion vessels (arterioles and capillaries) (↑). Bar = 500 μm.Confl ict of interestNone declared.References 1. Folkman J.: Role of angiogenesis in tumor growth and metastasis. Semin Oncol. 2002; 29, Suppl. 16: 15–18. 2. Hague S., Zhang L., Oehler M.K., Manek S., MacKenzie I.Z., Bicknell R., Rees M.C.P.: Expression of hypoxically regulated angiogenic factor adrenomedullin correlates with uterine leiomyoma vascular density. Clin Cancer Res. 2000; 6: 2808–2814. 3. Anania C.A., Stewart E.A., Quade B.J., Hill J.A., Nowak R.A.: Expression of the fi broblast growth factor receptor in women with leiomyomas and abnormal uterine bleeding. Mol Hum Reprod. 1997; 3: 685–691. 4. Anderson J.: Factors in fi broid growth. Bailliére’s Clin Obstet Gynecol. 1998; 12: 225–243. 5. 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Angiogenesis and pro-angiogenic factors in uterine fi broids : facts and myths

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Angiogenesis and pro-angiogenic factors in uterine fi broids : facts and myths

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