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Recently discovered interstitial cells “telocytes” as players in the pathogenesis of uterine leiomyomas
Medical Hypotheses 110 (2018) 64–67
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Recently discovered interstitial cells “telocytes” as players in the pathogenesis of uterine leiomyomas
MARK
⁎
Ivan Vargaa, , Martin Kleina, Ladislav Urbana,b, Ludovit Danihel Jr.c, Stefan Polaka, Ludovit Danihel Sr.d a
Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Spitalska Street 24, SK-81372 Bratislava, Slovakia Department of Gynecology and Obstetrics, ForLife General Hospital, Medercska Street 39, SK-945 75 Komarno, Slovakia c Third Department of Surgery, Faculty of Medicine, Comenius University and Hospital of The Brothers of Saint John of God, Bratislava, Namestie SNP 10, SK-814 65 Bratislava, Slovakia d Institute of Pathology, Faculty of Medicine, Comenius University in Bratislava, Spitalska Street 24, SK-81372 Bratislava, Slovakia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Telocytes Myometrium Uterine fibroid Immunohistochemistry
Uterine telocytes are interstitial cells characterized by a very long cytoplasmic prolongations, which form a 3D network, functionally integrating a wide variety of different cells. Leiomyomas (uterine fibroids) are benign tumors, which pose a huge threat concerning various health problems in women affected by this condition. The exact cause of leiomyomas development is, however, still largely unknown. Therefore, in an attempt to clarify their etiology, we performed an immunohistochemical characterization of telocytes in leiomyomas as well as in normal myometrium. Tissue samples of intramural leiomyomas from 26 women (age 46.26 ± 11.07) were immunohistochemically stained for the expression of c-kit (CD117) antigen, one of the markers of telocytes. C-kit (CD117) antigen is useful for a routine immunohistochemical identification of uterine telocytes in histological sections of myometrium. In normal, healthy myometrium the c-kit positive telocytes occupy approximately 2.2% of the area of a tissue slide, contrasting with no detectable c-kit positive cells within leiomyomas. As telocytes are thought to be key players in the regulation of tissue homoeostasis, our data suggest that uterine telocyte loss may have important implications in the pathogenesis of leiomyomas. In addition, we supposed to summarize three hypotheses on the association of the cells telocytes loss within the myometrium and formation of leiomyomas. These hypotheses include the loss of telocytes’ functions as “sex hormone sensors” and regulators of smooth muscle cells cycle; the role of telocytes as progenitor cells for the development of leiomyomas; and the hypothesis of decreased angiogenesis after telocytes’ loss with subsequent hypoxia (as a key factor for leiomyomas development).
Introduction There have been a huge body of scientific works published recently, concerning a novel population of interstitial cells termed telocytes. These cells are characterized by very long cytoplasmic prolongations, which form a 3D network, structurally and functionally integrating immunologically active cells, smooth muscle cells, epithelial cells, nerve fibers and blood vessels. Often these cells are called just ”connecting cells“. This arrangement has been repeatedly found in essentially every organ of the human body including organs of the female reproductive system [1,2]. Many studies describe the role of these cells not only in regulation of physiological functions, but also in plenty of pathological conditions, where the alteration of telocytes is regarded as one of the principal pathological changes. This disruption of normal
⁎
Corresponding author. E-mail address: [email protected] (I. Varga).
https://doi.org/10.1016/j.mehy.2017.11.003 Received 29 September 2017; Accepted 7 November 2017 0306-9877/ © 2017 Elsevier Ltd. All rights reserved.
telocytes physiology is commonly described in terms of their ultrastructure, quantitative changes and modification of their topographical relations with surrounding cells and extracellular components. Focusing on the female reproductive system, dysfunction of telocytes is associated with the pathogenesis of tubal infertility [3,4], endometriosis [5], premature ovarian failure [6], or preeclampsia [7,8]. Several works have been published recently, implying telocytes’ dysfunction as one of the key factors also in tumorigenesis [9–11]. Hence, the recent knowledge indicates, that telocytes represent a cardinal cell population with an irreplaceable role in tissue homeostasis and in cell proliferation, differentiation, and survival. Leiomyomas (uterine fibroids, myomas) are benign monoclonal tumors, which originate from the muscle layer of the uterus (myometrium). Apart from the uncontrolled proliferation of smooth muscle
Medical Hypotheses 110 (2018) 64–67
I. Varga et al.
Discussion
cells, leiomyomas are also characterized by an increased susceptibility to endocrine stimuli and typical changes in the morphology of extracellular matrix (ECM), with disorganized architecture and alterations in the structure and production of various ECM components [12,13]. Despite their benign nature, these tumors pose a huge threat concerning various health problems in women affected by this condition. The most severe complications associated with uterine fibroids are uterine bleeding, pelvic pain, infertility, miscarriage and a number of other pregnancy-associated complications [14–16]. Moreover, uterine leiomyomas are the most common tumors in women. According to Ahrendt et al. [17], in European population more than 40% of women over 30 years of age have suffered from leiomyomas and more than 50% of all women may develop leiomyomas at some time in their life. For all that, there is a high urgency to acquire a detailed insight into their etiology and pathogenesis. Intense research of leiomyomas in recent years introduced a number of risk factors and possible causes. The exact cause is, however, still largely unknown. Therefore, in attempt to clarify their etiology, we performed an immunohistochemical characterization of telocytes within leiomyomas as well as in normal myometrium. We searched for an answer about the possible role of these recently described interstitial cells in the pathogenesis of uterine leiomyomas development.
Our study showed, that within the normal myometrium the c-kit positive telocytes occupy approximately 2% of the area of a tissue slide. The exact role of uterine telocytes is not clear, but most of the authors suppose their role in intercellular signaling, mechanical and chemical sensation (“hormone sensors”), bioelectrical signaling, modulation of myometrial contractility, or guidance of immune cells [18]. Based on our results, c-kit positive cells are not present within the uterine leiomyomas, only in normal myometrium surrounding the leiomyomas. According to our knowledge, this is the first report about the absence of c-kit positive cells, probably uterine telocytes, inside the uterine leiomyomas. Until now, only two articles have dealt with the association between telocytes and uterine leiomyomas. The first one is a short review article without own results and a valuable conclusion [19]. The second one is from an Egyptian research group, whose conclusions are completely different from ours. Results of Othman et al. [20] show significantly higher count of c-kit positive telocytes within the leiomyomas in comparison to normal myometrium. However, based on the figure captions of the aforementioned cited work, we beg to differ with the results of the authors’ research. Photomicrographs of Othman et al. [20] are somewhat blurred, making it impossible to verify the results – typical feature of telocytes is a c-kit positive cytoplasm, however, their study showed mainly c-kit positive cell nuclei, which is not characteristic of telocytes. The exact identification of spindle-shaped uterine telocytes in classically stained histological sections, which may be morphologically similar to connective tissue fibroblasts and smooth muscle cells, is impossible. Currently, with the aid of the methods of immunohistochemistry, the identification of telocytes is not so difficult. Various antigens are typical for telocytes, but at present, none of them are considered to be telocyte-specific only [21]. One of these antigens is the c-kit (CD117), which is considered to be the most specific marker of uterine telocytes [22]. However, within the uterus, c-kit is expressed also in mast cells [22], mesenchymal stem cells of endometrium [23] and endometrial cancer stem cells [24]. Nevertheless, development of antibodies against c-kit has allowed for routine identification of uterine telocytes in histological sections. The proto-oncogene c-kit, encoding a protein transmembrane protein kinase receptor, plays an important role in the signal transduction pathway that regulates cellular growth and repair. The c-kit is highly expressed in number of different neoplasms, e.g. leiomyosarcomas of the uterus [25,26] as well as other uterine sarcomas [27]. It seems, that c-kit may have a significant role in the oncogenesis of mesenchymal tumors of the uterus. In our study we demonstrated, that uterine leiomyomas are absolutely c-kit negative, so they do not contain c-kit positive telocytes. This is surprising because within leiomyomas of the digestive system, some researchers detected c-kit positive spindleshaped interstitial Cajal cells (note, that telocytes were firstly reported as interstitial Cajal-like cells) [28,29]. Based on the previously described functions of uterine telocytes, we propose three hypotheses of how can be the loss of telocytes within the myometrium involved in the pathogenesis of uterine leiomyomas (Table 1). It is well known, that leiomyoma tissue is more sensitive to estradiol or has more estrogen receptors in comparison to normal myometrium. Thus, leiomyomas can be considered, in general as estrogen-dependent tumors [30,31]. Therefore, increased levels of estrogens have the most significant impact on the development of uterine leiomyomas. On one hand, uterine telocytes act as the sensors of sex hormone levels, as there are progesterone and estrogen receptors present on their surface [32]. On the other hand, telocytes play a role in the regulation of proliferation and apoptosis of smooth muscle cells of the myometrium (the number of telocytes decreases during pregnancy, but it rises again after delivery, as the uterus undergoes involution). [33]. Therefore, the loss of telocytes can disrupt hormonally regulated processes of cellular proliferation and apoptosis within the
Patients and methods The specimens of uteruses were taken from 26 women (aged 19–69, mean age 46.26 ± 11.07) who underwent hysterectomy with diagnosis of leiomyomas. The study protocol was approved by the local ethical committee of the General Hospital in Komarno, Slovakia, and informed consent was obtained from every patient. All tissue samples were firstly examined by a pathologist and the diagnosis “intramural leiomyomas” was established. Five control samples of normal myometrium were obtained from the collection of tissue samples available at the Institute of Histology and Embryology, Comenius University in Bratislava, Slovakia. The areas of patients’ tissue samples devoid of leiomyomas were also considered normal for the purpose of our study. Tissue samples were fixed in formalin for 24 h, embedded in paraffin, and 5 μm thick sections were used for immunohistochemistry. Primary antibodies were used to label and detect the antigen of transmembrane tyrosine kinase receptor c-kit (CD 117) (DAKO, Denmark). Antibodies against c-kit are one of the most routinely used antibodies in the identification of telocytes. The primary antibodies were used according to the manufacturer’s protocols. For visualization, we used EnVisionTM FLEX Detection system (DAKO, Denmark) with diaminobenzidine as a chromogen. For better orientation within the slide, cell nuclei were stained with Mayer’s hematoxylin in dark blue. For visualization of histological sections by light microscopy, the LEICA DM2500 microscope was used and images were captured using the LEICA DFC290HD digital camera. The density of area with the positivity for ckit was calculated via a public domain open source software ImageJ. Results Among unstained smooth muscle cells of normal myometrium, telocytes’ cytoplasm showed strong c-kit (CD117) positivity, recognized by brown stain by using diaminobenzidine. The cell bodies of telocytes were spindle-shaped or star-shaped with visible beginnings (proximal portions) of their cytoplasmic projections (Fig. 1). Telocytes were present in all sublayers of myometrium and the average percentage of the positive area within the normal myometrium was 2.19 ± 0.51%. Surprisingly, inside leiomyomas we found no c-kit positive cells (Fig. 2). This negativity is not a methodical error. In tissue sections where we could saw both the normal myometrium and leiomyoma in one slide, c-kit positive cells were visible only in those parts of the slide, where the normal myometrium was localized (Fig. 3). 65
Medical Hypotheses 110 (2018) 64–67
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Fig. 1. a and b C-kit positive telocytes (brown color) within a normal myometrium (Magn. 200× and 400×).
Fig. 2. a and b Leiomyomas are absolutely c-kit negative (Magn. 200×).
Fig. 3. a and b Boundary between normal myometrium (M) with c-kit positive telocytes (arrows) and leiomyomas (L) without c-kit positive cells (Magn. 200×).
Table 1 Review of possible influences of telocytes in the development of uterine leiomyomas. Physiological role of uterine telocytes
What can happen after the loss of uterine telocytes
2.
Sensors of sex hormone levels and regulators of the proliferation and apoptosis of smooth muscle cells Mesenchymal stem cells for tissue reparation/regeneration
3.
Regulation of new blood vessels formation, angiogenesis
Increased density of estrogen receptors on the surface of smooth muscle cells and dysregulation of their cell cycle Telocytes as progenitor cells are the source of increased number of leiomyoma-like phenotype cells within myometrium Local hypoxia results in the decrease of apoptosis of leiomyoma cells and causes the acquisition of leiomyoma-like phenotype within the smooth muscle cells of myometrium
1.
66
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myometrium. In the state of decreased number of telocytes and their receptors for sex hormones, this receptor function is taken over by smooth muscle cells of the myometrium itself and without the telocytes’ regulatory function, smooth muscle cells can proliferate uncontrollably. The second hypothesis comes from the assumption, that telocytes are not an individual cell population. According to several authors, telocytes are just non-differentiated mesenchymal stem cells, important for tissue reparation/regeneration. For example, Díaz-Flores et al. [34] described telocytes as progenitor cells of fibroblasts and myofibroblasts in the processes of repair through granulation tissue and fibrosis. An increasing evidence supports the hypothesis that uterine leiomyomas originate from the myometrial stem cell population [35]. It is possible, that c-kit positive telocytes are these stem cells which, after differentiation, play a role in myometrial regeneration or growth (under normal conditions), but they may also be the source of uterine leiomyomas development (under pathological conditions). Still, very little is known about the initial steps of the uterine leiomyomas tumorigenesis. The third hypothesis is based on the knowledge, that telocytes play an important role during angiogenesis, e.g. in myocardium after myocardial infarction [36] or during regular physical exercise [37]. The loss of uterine telocytes may be responsible for decreased vessel formation within the myometrium and subsequent shifting of aerobic to anaerobic metabolism of smooth muscle cells. According to Fletcher et al. [38], this change in metabolism of myometrium results in a decrease in apoptosis of leiomyoma cells and causes the normal smooth muscle cells to acquire the leiomyoma-like phenotype. Decreased density of blood vessels or decreased angiogenesis within the uterine leiomyomas were described also by Sajewicz et al. [39] and Uluer et al. [40]. These three hypotheses based on our results further underscore the importance of the basic research of telocytes, which has also a huge impact in terms of clinical applications. To give an example, it was observed lately, that near-infrared low-level laser stimulation is a useful tool for uterine telocytes growth and development [41], what suggests a possible application of this type of laser-therapy in uterine regenerative medicine, and perhaps in the future also in the therapy of uterine leiomyomas.
impairment during preeclampsia. Med Hypotheses 2015;84:72–7. [8] Bosco CB, Díaz EG, Gutierrez RR, et al. Placental hypoxia developed during preeclampsia induces telocytes apoptosis in chorionic villi affecting the maternal-fetus metabolic exchange. Curr Stem Cell Res Ther 2016;11:420–5. [9] Mirancea N, Moroşanu AM, Mirancea GV, et al. Infrastructure of the telocytes from tumor stroma in the skin basal and squamous cell carcinomas. Rom J Morphol Embryol 2013;54:1025–37. [10] Mou Y, Wang Y, Li J, et al. Immunohistochemical characterization and functional identification of mammary gland telocytes in the self-assembly of reconstituted breast cancer tissue in vitro. J Cell Mol Med 2013;17:65–75. [11] Mihalcea CE, Moroşanu AM, Murăraşu D, et al. Particular molecular and ultrastructural aspects in invasive mammary carcinoma. Rom J Morphol Embryol 2015;56:1371–81. [12] Bulun SE. Uterine fibroids. N Engl J Med 2013;369(14):1344–55. [13] Aleksandrovych V, Bereza T, Sajewicz M, et al. Uterine fibroid: common features of widespread tumor (Review article). Folia Med Cracov 2015;55:61–75. [14] Segars JH, Parrott EC, Nagel JD, et al. Proceedings from the Third National Institutes of Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary and future recommendations. Hum Reprod Update 2014;20:309–33. [15] Huang YT, Wu RC, Chao A, Chao AS. Spontaneous rupture of infarcted leiomyoma into endometrial cavity in a perimenopausal woman. Arch Gynecol Obstet 2017. http://dx.doi.org/10.1007/s00404-017-4454-1. (in press). [16] Sagi-Dain L, Ojha K, Bider D, et al. Pregnancy outcomes in oocyte recipients with fibroids not impinging uterine cavity. Arch Gynecol Obstet 2017;295:497–502. [17] Ahrendt HJ, Tylkoski H, Rabe T, et al. Prevalence of uterine myomas in women in Germany: data of an epidemiological study. Arch Gynecol Obstet 2016;293:1243–53. [18] Roatesi I, Radu BM, Cretoiu D, Cretoiu SM. Uterine telocytes: a review of current knowledge. Biol Reprod 2015;93:10. [19] Aleksandrovych V. Uterine leiomyoma and telocytes: impact on pathogenesis? EC Gynaecology 2017;ECO.01:16–7. [20] Othman ER, Elgamal DA, Refaiy AM, et al. Identification and potential role of telocytes in human uterine leiomyoma. Contraception Reprod Med 2016;1:12. [21] Varga I, Urban L, Kajanová M, Polák Š. Functional histology and possible clinical significance of recently discovered telocytes inside the female reproductive system. Arch Gynecol Obstet 2016;294:417–22. [22] Cretoiu SM, Cretoiu D, Marin A, et al. Telocytes: ultrastructural, immunohistochemical and electrophysiological characteristics in human myometrium. Reproduction 2013;145:357–70. [23] Lara E, Rivera N, Rojas D, et al. Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle. Reprod Domest Anim 2017. http://dx.doi.org/10.1111/rda.12969. (in press). [24] Kyo S, Kato K. Endometrial cancer stem cell as a potential therapeutic target. Semin Reprod Med 2015;33:341–9. [25] Wang L, Felix JC, Lee JL, et al. The proto-oncogene c-kit is expressed in leiomyosarcomas of the uterus. Gynecol Oncol 2003;90:402–6. [26] Sah SP, McCluggage WG. DOG1 immunoreactivity in uterine leiomyosarcomas. J Clin Pathol 2013;66:40–3. [27] Erdogan G, Bassorgun CI, Pestereli HE, et al. C-kit protein expression in uterine and ovarian mesenchymal tumours. APMIS 2007;115:204–9. [28] Janevska V, Qerimi A, Basheska N, et al. Superficial leiomyomas of the gastrointestinal tract with interstitial cells of Cajal. Int J Clin Exp Pathol 2015;8:15977–84. [29] Ye JX, Liu Y, Qin Y, et al. Mast cells or not? - CD117 positive cells in esophageal leiomyoma. Histol Histopathol 2015;30:581–8. [30] McWilliams MM, Chennathukuzhi VM. Recent advances in uterine fibroid etiology. Semin Reprod Med 2017;35:181–9. [31] Dvorská D, Braný D, Danková Z, et al. Molecular and clinical attributes of uterine leiomyomas. Tumour Biol 2017;39. 1010428317710226. [32] Cretoiu D, Ciontea SM, Popescu LM, et al. Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach. J Cell Mol Med 2006;10:789–95. [33] Salama N. Immunohistochemical characterization of telocytes in rat uterus in different reproductive states. Egypt J Histol 2013;36:185–94. [34] Díaz-Flores L, Gutiérrez R, Pino García M, et al. Telocytes as a source of progenitor cells in regeneration and repair through granulation tissue. Curr Stem Cell Res Ther 2016;11:395–403. [35] Mas A, Stone L, O'Connor PM, et al. Developmental exposure to endocrine disruptors expands murine myometrial stem cell compartment as a prerequisite to leiomyoma tumorigenesis. Stem Cells 2017;35:666–78. [36] Manole CG, Cismaşiu V, Gherghiceanu M, Popescu LM. Experimental acute myocardial infarction: telocytes involvement in neo-angiogenesis. Cell Mol Med 2011;15:2284–96. [37] Xiao J, Chen P, Qu Y, et al. Telocytes in exercise-induced cardiac growth. J Cell Mol Med 2016;20:973–9. [38] Fletcher NM, Abusamaan MS, Memaj I, et al. Oxidative stress: a key regulator of leiomyoma cell survival. Fertil Steril 2017;107:1387–94. [39] Sajewicz M, Konarska M, Wrona AN, et al. Vascular density, angiogenesis and proangiogenic factors in uterine fibroids. Folia Med Cracov 2016;56:27–32. [40] Uluer ET, Inan S, Ozbilgin K, et al. The role of hypoxia related angiogenesis in uterine smooth muscle tumors. Biotech Histochem 2015;90:102–10. [41] Campeanu RA, Radu BM, Cretoiu SM, et al. Near-infrared low-level laser stimulation of telocytes from human myometrium. Lasers Med Sci 2014;29:1867–74.
Conflict of interest We declare that, we have no conflict of interest. Ethical approval This study was approved by the local ethical committee of the General Hospital in Komárno, Slovakia. The patients provided written informed consent prior to participating in the study. Acknowledgement The authors thank Mrs. Gabriela Fujerikova for technical assistance during histological preparation. References [1] Aleksandrovych V, Walocha JA, Gil K. Telocytes in female reproductive system (human and animal). J Cell Mol Med 2016;20:994–1000. [2] Cretoiu D, Cretoiu SM. Telocytes in the reproductive organs: current understanding and future challenges. Sem Cell Dev Biol 2016;55:44–9. [3] Aleksandrovych V, Sajewicz M, Walocha JA, Gil K. Tubal telocytes: factor infertility reason? Folia Med Cracov 2016;56:17–23. [4] Dixon RE, Ramsey KH, Schripsema JH, et al. Time-dependent disruption of oviduct pacemaker cells by Chlamydia infection in mice. Biol Reprod 2010;8:244–53. [5] Yang XJ, Xu JY, Shen ZJ, Zhao J. Immunohistochemical alterations of Cajal-like type of tubal interstitial cells in women with endometriosis and tubal ectopic pregnancy. Arch Gynecol Obstet 2013;288:1295–300. [6] Liu T, Wang S, Li Q, et al. Telocytes as potential targets in a cyclophosphamideinduced animal model of premature ovarian failure. Mol Med Rep 2016;14:2415–22. [7] Bosco C, Diaz E, Gutierrez R, et al. A putative role for telocytes in placental barrier
67
Contents lists available at ScienceDirect
Medical Hypotheses journal homepage: www.elsevier.com/locate/mehy
Recently discovered interstitial cells “telocytes” as players in the pathogenesis of uterine leiomyomas
MARK
⁎
Ivan Vargaa, , Martin Kleina, Ladislav Urbana,b, Ludovit Danihel Jr.c, Stefan Polaka, Ludovit Danihel Sr.d a
Institute of Histology and Embryology, Faculty of Medicine, Comenius University in Bratislava, Spitalska Street 24, SK-81372 Bratislava, Slovakia Department of Gynecology and Obstetrics, ForLife General Hospital, Medercska Street 39, SK-945 75 Komarno, Slovakia c Third Department of Surgery, Faculty of Medicine, Comenius University and Hospital of The Brothers of Saint John of God, Bratislava, Namestie SNP 10, SK-814 65 Bratislava, Slovakia d Institute of Pathology, Faculty of Medicine, Comenius University in Bratislava, Spitalska Street 24, SK-81372 Bratislava, Slovakia b
A R T I C L E I N F O
A B S T R A C T
Keywords: Telocytes Myometrium Uterine fibroid Immunohistochemistry
Uterine telocytes are interstitial cells characterized by a very long cytoplasmic prolongations, which form a 3D network, functionally integrating a wide variety of different cells. Leiomyomas (uterine fibroids) are benign tumors, which pose a huge threat concerning various health problems in women affected by this condition. The exact cause of leiomyomas development is, however, still largely unknown. Therefore, in an attempt to clarify their etiology, we performed an immunohistochemical characterization of telocytes in leiomyomas as well as in normal myometrium. Tissue samples of intramural leiomyomas from 26 women (age 46.26 ± 11.07) were immunohistochemically stained for the expression of c-kit (CD117) antigen, one of the markers of telocytes. C-kit (CD117) antigen is useful for a routine immunohistochemical identification of uterine telocytes in histological sections of myometrium. In normal, healthy myometrium the c-kit positive telocytes occupy approximately 2.2% of the area of a tissue slide, contrasting with no detectable c-kit positive cells within leiomyomas. As telocytes are thought to be key players in the regulation of tissue homoeostasis, our data suggest that uterine telocyte loss may have important implications in the pathogenesis of leiomyomas. In addition, we supposed to summarize three hypotheses on the association of the cells telocytes loss within the myometrium and formation of leiomyomas. These hypotheses include the loss of telocytes’ functions as “sex hormone sensors” and regulators of smooth muscle cells cycle; the role of telocytes as progenitor cells for the development of leiomyomas; and the hypothesis of decreased angiogenesis after telocytes’ loss with subsequent hypoxia (as a key factor for leiomyomas development).
Introduction There have been a huge body of scientific works published recently, concerning a novel population of interstitial cells termed telocytes. These cells are characterized by very long cytoplasmic prolongations, which form a 3D network, structurally and functionally integrating immunologically active cells, smooth muscle cells, epithelial cells, nerve fibers and blood vessels. Often these cells are called just ”connecting cells“. This arrangement has been repeatedly found in essentially every organ of the human body including organs of the female reproductive system [1,2]. Many studies describe the role of these cells not only in regulation of physiological functions, but also in plenty of pathological conditions, where the alteration of telocytes is regarded as one of the principal pathological changes. This disruption of normal
⁎
Corresponding author. E-mail address: [email protected] (I. Varga).
https://doi.org/10.1016/j.mehy.2017.11.003 Received 29 September 2017; Accepted 7 November 2017 0306-9877/ © 2017 Elsevier Ltd. All rights reserved.
telocytes physiology is commonly described in terms of their ultrastructure, quantitative changes and modification of their topographical relations with surrounding cells and extracellular components. Focusing on the female reproductive system, dysfunction of telocytes is associated with the pathogenesis of tubal infertility [3,4], endometriosis [5], premature ovarian failure [6], or preeclampsia [7,8]. Several works have been published recently, implying telocytes’ dysfunction as one of the key factors also in tumorigenesis [9–11]. Hence, the recent knowledge indicates, that telocytes represent a cardinal cell population with an irreplaceable role in tissue homeostasis and in cell proliferation, differentiation, and survival. Leiomyomas (uterine fibroids, myomas) are benign monoclonal tumors, which originate from the muscle layer of the uterus (myometrium). Apart from the uncontrolled proliferation of smooth muscle
Medical Hypotheses 110 (2018) 64–67
I. Varga et al.
Discussion
cells, leiomyomas are also characterized by an increased susceptibility to endocrine stimuli and typical changes in the morphology of extracellular matrix (ECM), with disorganized architecture and alterations in the structure and production of various ECM components [12,13]. Despite their benign nature, these tumors pose a huge threat concerning various health problems in women affected by this condition. The most severe complications associated with uterine fibroids are uterine bleeding, pelvic pain, infertility, miscarriage and a number of other pregnancy-associated complications [14–16]. Moreover, uterine leiomyomas are the most common tumors in women. According to Ahrendt et al. [17], in European population more than 40% of women over 30 years of age have suffered from leiomyomas and more than 50% of all women may develop leiomyomas at some time in their life. For all that, there is a high urgency to acquire a detailed insight into their etiology and pathogenesis. Intense research of leiomyomas in recent years introduced a number of risk factors and possible causes. The exact cause is, however, still largely unknown. Therefore, in attempt to clarify their etiology, we performed an immunohistochemical characterization of telocytes within leiomyomas as well as in normal myometrium. We searched for an answer about the possible role of these recently described interstitial cells in the pathogenesis of uterine leiomyomas development.
Our study showed, that within the normal myometrium the c-kit positive telocytes occupy approximately 2% of the area of a tissue slide. The exact role of uterine telocytes is not clear, but most of the authors suppose their role in intercellular signaling, mechanical and chemical sensation (“hormone sensors”), bioelectrical signaling, modulation of myometrial contractility, or guidance of immune cells [18]. Based on our results, c-kit positive cells are not present within the uterine leiomyomas, only in normal myometrium surrounding the leiomyomas. According to our knowledge, this is the first report about the absence of c-kit positive cells, probably uterine telocytes, inside the uterine leiomyomas. Until now, only two articles have dealt with the association between telocytes and uterine leiomyomas. The first one is a short review article without own results and a valuable conclusion [19]. The second one is from an Egyptian research group, whose conclusions are completely different from ours. Results of Othman et al. [20] show significantly higher count of c-kit positive telocytes within the leiomyomas in comparison to normal myometrium. However, based on the figure captions of the aforementioned cited work, we beg to differ with the results of the authors’ research. Photomicrographs of Othman et al. [20] are somewhat blurred, making it impossible to verify the results – typical feature of telocytes is a c-kit positive cytoplasm, however, their study showed mainly c-kit positive cell nuclei, which is not characteristic of telocytes. The exact identification of spindle-shaped uterine telocytes in classically stained histological sections, which may be morphologically similar to connective tissue fibroblasts and smooth muscle cells, is impossible. Currently, with the aid of the methods of immunohistochemistry, the identification of telocytes is not so difficult. Various antigens are typical for telocytes, but at present, none of them are considered to be telocyte-specific only [21]. One of these antigens is the c-kit (CD117), which is considered to be the most specific marker of uterine telocytes [22]. However, within the uterus, c-kit is expressed also in mast cells [22], mesenchymal stem cells of endometrium [23] and endometrial cancer stem cells [24]. Nevertheless, development of antibodies against c-kit has allowed for routine identification of uterine telocytes in histological sections. The proto-oncogene c-kit, encoding a protein transmembrane protein kinase receptor, plays an important role in the signal transduction pathway that regulates cellular growth and repair. The c-kit is highly expressed in number of different neoplasms, e.g. leiomyosarcomas of the uterus [25,26] as well as other uterine sarcomas [27]. It seems, that c-kit may have a significant role in the oncogenesis of mesenchymal tumors of the uterus. In our study we demonstrated, that uterine leiomyomas are absolutely c-kit negative, so they do not contain c-kit positive telocytes. This is surprising because within leiomyomas of the digestive system, some researchers detected c-kit positive spindleshaped interstitial Cajal cells (note, that telocytes were firstly reported as interstitial Cajal-like cells) [28,29]. Based on the previously described functions of uterine telocytes, we propose three hypotheses of how can be the loss of telocytes within the myometrium involved in the pathogenesis of uterine leiomyomas (Table 1). It is well known, that leiomyoma tissue is more sensitive to estradiol or has more estrogen receptors in comparison to normal myometrium. Thus, leiomyomas can be considered, in general as estrogen-dependent tumors [30,31]. Therefore, increased levels of estrogens have the most significant impact on the development of uterine leiomyomas. On one hand, uterine telocytes act as the sensors of sex hormone levels, as there are progesterone and estrogen receptors present on their surface [32]. On the other hand, telocytes play a role in the regulation of proliferation and apoptosis of smooth muscle cells of the myometrium (the number of telocytes decreases during pregnancy, but it rises again after delivery, as the uterus undergoes involution). [33]. Therefore, the loss of telocytes can disrupt hormonally regulated processes of cellular proliferation and apoptosis within the
Patients and methods The specimens of uteruses were taken from 26 women (aged 19–69, mean age 46.26 ± 11.07) who underwent hysterectomy with diagnosis of leiomyomas. The study protocol was approved by the local ethical committee of the General Hospital in Komarno, Slovakia, and informed consent was obtained from every patient. All tissue samples were firstly examined by a pathologist and the diagnosis “intramural leiomyomas” was established. Five control samples of normal myometrium were obtained from the collection of tissue samples available at the Institute of Histology and Embryology, Comenius University in Bratislava, Slovakia. The areas of patients’ tissue samples devoid of leiomyomas were also considered normal for the purpose of our study. Tissue samples were fixed in formalin for 24 h, embedded in paraffin, and 5 μm thick sections were used for immunohistochemistry. Primary antibodies were used to label and detect the antigen of transmembrane tyrosine kinase receptor c-kit (CD 117) (DAKO, Denmark). Antibodies against c-kit are one of the most routinely used antibodies in the identification of telocytes. The primary antibodies were used according to the manufacturer’s protocols. For visualization, we used EnVisionTM FLEX Detection system (DAKO, Denmark) with diaminobenzidine as a chromogen. For better orientation within the slide, cell nuclei were stained with Mayer’s hematoxylin in dark blue. For visualization of histological sections by light microscopy, the LEICA DM2500 microscope was used and images were captured using the LEICA DFC290HD digital camera. The density of area with the positivity for ckit was calculated via a public domain open source software ImageJ. Results Among unstained smooth muscle cells of normal myometrium, telocytes’ cytoplasm showed strong c-kit (CD117) positivity, recognized by brown stain by using diaminobenzidine. The cell bodies of telocytes were spindle-shaped or star-shaped with visible beginnings (proximal portions) of their cytoplasmic projections (Fig. 1). Telocytes were present in all sublayers of myometrium and the average percentage of the positive area within the normal myometrium was 2.19 ± 0.51%. Surprisingly, inside leiomyomas we found no c-kit positive cells (Fig. 2). This negativity is not a methodical error. In tissue sections where we could saw both the normal myometrium and leiomyoma in one slide, c-kit positive cells were visible only in those parts of the slide, where the normal myometrium was localized (Fig. 3). 65
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Fig. 1. a and b C-kit positive telocytes (brown color) within a normal myometrium (Magn. 200× and 400×).
Fig. 2. a and b Leiomyomas are absolutely c-kit negative (Magn. 200×).
Fig. 3. a and b Boundary between normal myometrium (M) with c-kit positive telocytes (arrows) and leiomyomas (L) without c-kit positive cells (Magn. 200×).
Table 1 Review of possible influences of telocytes in the development of uterine leiomyomas. Physiological role of uterine telocytes
What can happen after the loss of uterine telocytes
2.
Sensors of sex hormone levels and regulators of the proliferation and apoptosis of smooth muscle cells Mesenchymal stem cells for tissue reparation/regeneration
3.
Regulation of new blood vessels formation, angiogenesis
Increased density of estrogen receptors on the surface of smooth muscle cells and dysregulation of their cell cycle Telocytes as progenitor cells are the source of increased number of leiomyoma-like phenotype cells within myometrium Local hypoxia results in the decrease of apoptosis of leiomyoma cells and causes the acquisition of leiomyoma-like phenotype within the smooth muscle cells of myometrium
1.
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myometrium. In the state of decreased number of telocytes and their receptors for sex hormones, this receptor function is taken over by smooth muscle cells of the myometrium itself and without the telocytes’ regulatory function, smooth muscle cells can proliferate uncontrollably. The second hypothesis comes from the assumption, that telocytes are not an individual cell population. According to several authors, telocytes are just non-differentiated mesenchymal stem cells, important for tissue reparation/regeneration. For example, Díaz-Flores et al. [34] described telocytes as progenitor cells of fibroblasts and myofibroblasts in the processes of repair through granulation tissue and fibrosis. An increasing evidence supports the hypothesis that uterine leiomyomas originate from the myometrial stem cell population [35]. It is possible, that c-kit positive telocytes are these stem cells which, after differentiation, play a role in myometrial regeneration or growth (under normal conditions), but they may also be the source of uterine leiomyomas development (under pathological conditions). Still, very little is known about the initial steps of the uterine leiomyomas tumorigenesis. The third hypothesis is based on the knowledge, that telocytes play an important role during angiogenesis, e.g. in myocardium after myocardial infarction [36] or during regular physical exercise [37]. The loss of uterine telocytes may be responsible for decreased vessel formation within the myometrium and subsequent shifting of aerobic to anaerobic metabolism of smooth muscle cells. According to Fletcher et al. [38], this change in metabolism of myometrium results in a decrease in apoptosis of leiomyoma cells and causes the normal smooth muscle cells to acquire the leiomyoma-like phenotype. Decreased density of blood vessels or decreased angiogenesis within the uterine leiomyomas were described also by Sajewicz et al. [39] and Uluer et al. [40]. These three hypotheses based on our results further underscore the importance of the basic research of telocytes, which has also a huge impact in terms of clinical applications. To give an example, it was observed lately, that near-infrared low-level laser stimulation is a useful tool for uterine telocytes growth and development [41], what suggests a possible application of this type of laser-therapy in uterine regenerative medicine, and perhaps in the future also in the therapy of uterine leiomyomas.
impairment during preeclampsia. Med Hypotheses 2015;84:72–7. [8] Bosco CB, Díaz EG, Gutierrez RR, et al. Placental hypoxia developed during preeclampsia induces telocytes apoptosis in chorionic villi affecting the maternal-fetus metabolic exchange. Curr Stem Cell Res Ther 2016;11:420–5. [9] Mirancea N, Moroşanu AM, Mirancea GV, et al. Infrastructure of the telocytes from tumor stroma in the skin basal and squamous cell carcinomas. Rom J Morphol Embryol 2013;54:1025–37. [10] Mou Y, Wang Y, Li J, et al. Immunohistochemical characterization and functional identification of mammary gland telocytes in the self-assembly of reconstituted breast cancer tissue in vitro. J Cell Mol Med 2013;17:65–75. [11] Mihalcea CE, Moroşanu AM, Murăraşu D, et al. Particular molecular and ultrastructural aspects in invasive mammary carcinoma. Rom J Morphol Embryol 2015;56:1371–81. [12] Bulun SE. Uterine fibroids. N Engl J Med 2013;369(14):1344–55. [13] Aleksandrovych V, Bereza T, Sajewicz M, et al. Uterine fibroid: common features of widespread tumor (Review article). Folia Med Cracov 2015;55:61–75. [14] Segars JH, Parrott EC, Nagel JD, et al. Proceedings from the Third National Institutes of Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary and future recommendations. Hum Reprod Update 2014;20:309–33. [15] Huang YT, Wu RC, Chao A, Chao AS. Spontaneous rupture of infarcted leiomyoma into endometrial cavity in a perimenopausal woman. Arch Gynecol Obstet 2017. http://dx.doi.org/10.1007/s00404-017-4454-1. (in press). [16] Sagi-Dain L, Ojha K, Bider D, et al. Pregnancy outcomes in oocyte recipients with fibroids not impinging uterine cavity. Arch Gynecol Obstet 2017;295:497–502. [17] Ahrendt HJ, Tylkoski H, Rabe T, et al. Prevalence of uterine myomas in women in Germany: data of an epidemiological study. Arch Gynecol Obstet 2016;293:1243–53. [18] Roatesi I, Radu BM, Cretoiu D, Cretoiu SM. Uterine telocytes: a review of current knowledge. Biol Reprod 2015;93:10. [19] Aleksandrovych V. Uterine leiomyoma and telocytes: impact on pathogenesis? EC Gynaecology 2017;ECO.01:16–7. [20] Othman ER, Elgamal DA, Refaiy AM, et al. Identification and potential role of telocytes in human uterine leiomyoma. Contraception Reprod Med 2016;1:12. [21] Varga I, Urban L, Kajanová M, Polák Š. Functional histology and possible clinical significance of recently discovered telocytes inside the female reproductive system. Arch Gynecol Obstet 2016;294:417–22. [22] Cretoiu SM, Cretoiu D, Marin A, et al. Telocytes: ultrastructural, immunohistochemical and electrophysiological characteristics in human myometrium. Reproduction 2013;145:357–70. [23] Lara E, Rivera N, Rojas D, et al. Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle. Reprod Domest Anim 2017. http://dx.doi.org/10.1111/rda.12969. (in press). [24] Kyo S, Kato K. Endometrial cancer stem cell as a potential therapeutic target. Semin Reprod Med 2015;33:341–9. [25] Wang L, Felix JC, Lee JL, et al. The proto-oncogene c-kit is expressed in leiomyosarcomas of the uterus. Gynecol Oncol 2003;90:402–6. [26] Sah SP, McCluggage WG. DOG1 immunoreactivity in uterine leiomyosarcomas. J Clin Pathol 2013;66:40–3. [27] Erdogan G, Bassorgun CI, Pestereli HE, et al. C-kit protein expression in uterine and ovarian mesenchymal tumours. APMIS 2007;115:204–9. [28] Janevska V, Qerimi A, Basheska N, et al. Superficial leiomyomas of the gastrointestinal tract with interstitial cells of Cajal. Int J Clin Exp Pathol 2015;8:15977–84. [29] Ye JX, Liu Y, Qin Y, et al. Mast cells or not? - CD117 positive cells in esophageal leiomyoma. Histol Histopathol 2015;30:581–8. [30] McWilliams MM, Chennathukuzhi VM. Recent advances in uterine fibroid etiology. Semin Reprod Med 2017;35:181–9. [31] Dvorská D, Braný D, Danková Z, et al. Molecular and clinical attributes of uterine leiomyomas. Tumour Biol 2017;39. 1010428317710226. [32] Cretoiu D, Ciontea SM, Popescu LM, et al. Interstitial Cajal-like cells (ICLC) as steroid hormone sensors in human myometrium: immunocytochemical approach. J Cell Mol Med 2006;10:789–95. [33] Salama N. Immunohistochemical characterization of telocytes in rat uterus in different reproductive states. Egypt J Histol 2013;36:185–94. [34] Díaz-Flores L, Gutiérrez R, Pino García M, et al. Telocytes as a source of progenitor cells in regeneration and repair through granulation tissue. Curr Stem Cell Res Ther 2016;11:395–403. [35] Mas A, Stone L, O'Connor PM, et al. Developmental exposure to endocrine disruptors expands murine myometrial stem cell compartment as a prerequisite to leiomyoma tumorigenesis. Stem Cells 2017;35:666–78. [36] Manole CG, Cismaşiu V, Gherghiceanu M, Popescu LM. Experimental acute myocardial infarction: telocytes involvement in neo-angiogenesis. Cell Mol Med 2011;15:2284–96. [37] Xiao J, Chen P, Qu Y, et al. Telocytes in exercise-induced cardiac growth. J Cell Mol Med 2016;20:973–9. [38] Fletcher NM, Abusamaan MS, Memaj I, et al. Oxidative stress: a key regulator of leiomyoma cell survival. Fertil Steril 2017;107:1387–94. [39] Sajewicz M, Konarska M, Wrona AN, et al. Vascular density, angiogenesis and proangiogenic factors in uterine fibroids. Folia Med Cracov 2016;56:27–32. [40] Uluer ET, Inan S, Ozbilgin K, et al. The role of hypoxia related angiogenesis in uterine smooth muscle tumors. Biotech Histochem 2015;90:102–10. [41] Campeanu RA, Radu BM, Cretoiu SM, et al. Near-infrared low-level laser stimulation of telocytes from human myometrium. Lasers Med Sci 2014;29:1867–74.
Conflict of interest We declare that, we have no conflict of interest. Ethical approval This study was approved by the local ethical committee of the General Hospital in Komárno, Slovakia. The patients provided written informed consent prior to participating in the study. Acknowledgement The authors thank Mrs. Gabriela Fujerikova for technical assistance during histological preparation. References [1] Aleksandrovych V, Walocha JA, Gil K. Telocytes in female reproductive system (human and animal). J Cell Mol Med 2016;20:994–1000. [2] Cretoiu D, Cretoiu SM. Telocytes in the reproductive organs: current understanding and future challenges. Sem Cell Dev Biol 2016;55:44–9. [3] Aleksandrovych V, Sajewicz M, Walocha JA, Gil K. Tubal telocytes: factor infertility reason? Folia Med Cracov 2016;56:17–23. [4] Dixon RE, Ramsey KH, Schripsema JH, et al. Time-dependent disruption of oviduct pacemaker cells by Chlamydia infection in mice. Biol Reprod 2010;8:244–53. [5] Yang XJ, Xu JY, Shen ZJ, Zhao J. Immunohistochemical alterations of Cajal-like type of tubal interstitial cells in women with endometriosis and tubal ectopic pregnancy. Arch Gynecol Obstet 2013;288:1295–300. [6] Liu T, Wang S, Li Q, et al. Telocytes as potential targets in a cyclophosphamideinduced animal model of premature ovarian failure. Mol Med Rep 2016;14:2415–22. [7] Bosco C, Diaz E, Gutierrez R, et al. A putative role for telocytes in placental barrier
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