Extracellular matrix in uterine leiomyoma pathogenesis: a potential target for future therapeutics

Abstract

BACKGROUND

Uterine leiomyoma (also known as fibroid or myoma) is the most common benign tumor of the uterus found in women of reproductive age. It is not usually fatal but can produce serious clinical symptoms, including excessive uterine bleeding, pelvic pain or pressure, infertility and pregnancy complications. Due to lack of effective medical treatments surgery has been a definitive choice for the management of this tumor.

OBJECTIVE AND RATIONALE

Extracellular matrix (ECM) accumulation and remodeling are thought to be crucial for fibrotic diseases such as uterine leiomyoma. Indeed, ECM plays important role in forming the bulk structure of leiomyoma, and the ECM-rich rigid structure within these tumors is thought to be a cause of abnormal bleeding and pelvic pain. Therefore, a better understanding of ECM accumulation and remodeling is critical for developing new therapeutics for uterine leiomyoma.

SEARCH METHODS

PubMed and Google Scholar were searched for all original and review articles/book chapters related to ECM and medical treatments of uterine leiomyoma published in English until May 2017.

OUTCOMES

This review discusses the involvement of ECM in leiomyoma pathogenesis as well as current and future medical treatments that target ECM directly or indirectly. Uterine leiomyoma is characterized by elevated levels of collagens, fibronectin, laminins and proteoglycans. They can induce the mechanotransduction process, such as activation of the integrin-Rho/p38 MAPK/ERK pathway, resulting in cellular responses that are involved in pathogenesis and altered bidirectional signaling between leiomyoma cells and the ECM. ECM accumulation is affected by growth factors (TGF-β, activin-A and PDGF), cytokines (TNF-α), steroid hormones (estrogen and progesterone) and microRNAs (miR-29 family, miR-200c and miR-93/106b). Among these, TGF-βs (1 and 3) and activin-A have been suggested as key players in the accumulation of excessive ECM (fibrosis) in leiomyoma. The presence of elevated levels of ECM and myofibroblasts in leiomyoma supports the fibrotic character of these tumors. Interestingly, ECM may serve as a reservoir of profibrotic growth factors and enhance their activity by increasing their stability and extending their duration of signaling. At present, several classes of compounds, including gonadotropin-releasing hormone (GnRH) agonist (leuprolide acetate), GnRH antagonist (cetrorelix acetate), selective progesterone receptor modulators (ulipristate acetate and asoprisnil), antiprogestin (mifepristone) and natural compounds like vitamin D and resveratrol have been studied as medical treatments that target ECM in uterine leiomyoma.

WIDER IMPLICATIONS

Although several types of drugs (mostly antiproliferative agents) are available for leiomyoma treatment, none of them were introduced specifically as antifibrotic agents. In light of its critical role in the process of fibrosis in leiomyoma, we propose that ECM should be considered as a crucial target for future therapeutics. Thus, the introduction of drugs that are specifically antifibrotic could be a good solution to control abnormal leiomyoma growth and associated clinical symptoms. The antifibrotic compounds can be introduced based on their ability to regulate ECM components and their receptors, as well as growth factors, cytokines, steroid hormones and their corresponding receptors and intracellular signaling pathways, as well as microRNAs, involved in ECM production in leiomyoma.

uterine leiomyoma, fibrosis, extracellular matrix, growth factors, steroid hormones, mechanotransduction, inflammation, myofibroblast, medical treatment, ultrastructure

Introduction

Uterine leiomyoma (also known as fibroid or myoma), the most common benign tumor of the uterus, is found in women of reproductive age (Islam et al., 2013a). They may occur in more than 70% of women, and ~25% of women show clinically significant lesions (Buttram and Reiter, 1981; Cramer and Patel, 1990). Particularly, African-American women are reported to have a higher leiomyoma incidence (Marshall et al., 1997; Day Baird et al., 2003), with more severe clinical symptoms (Kjerulff et al., 1996) compared to Caucasians. Symptoms associated with leiomyoma include excessive uterine bleeding, pelvic pain or pressure, infertility and pregnancy complications (Buttram and Reiter, 1981). Due to lack of effective, non-invasive medical treatments (Islam et al., 2013b), surgery has been the primary choice for leiomyoma management. However, surgery is risky and expensive and negatively affects the quality of life of patients. In the USA, the annual economic burden of uterine leiomyoma is estimated to be between $5.9 and $34.4 billion (Cardozo et al., 2012).

The precise molecular and cellular changes that led to the development and growth of uterine leiomyoma are not well understood. However, one distinguishing characteristic of uterine leiomyoma is the excessive accumulation of extracellular matrix (ECM) components including collagens (Fig. 1), fibronectin, laminins and proteoglycans (Stewart et al., 1994; Arici and Sozen, 2000; Norian et al., 2009; Malik et al., 2012; Herndon et al., 2016). High levels of ECM proteins in uterine leiomyoma result in mechanotransduction, a process whereby increased tissue stiffness leads to bidirectional signaling via integrins and downstream mediators including Rho/p38 MAPK/ERK (Rogers et al., 2008; Malik et al., 2012; Chen et al., 2013; Thorne et al., 2015) (Fig. 2). Uterine leiomyoma also expresses proteolytic enzymes such as matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) that play key roles in ECM remodeling (Bogusiewicz et al., 2007; Malik et al., 2010). Furthermore, a number of studies indicate that ECM accumulation and function is regulated by growth factors (Joseph et al., 2010; Islam et al., 2014a), cytokines (Wang et al., 2015) and steroid hormones (Qiang et al., 2014). ECM binds and sequesters growth factors to promote their stability as well as restrict their activity. By degrading ECM components, MMPs and other proteolytic enzymes release growth factors and trigger activation of multiple signal transduction pathways (Fig. 3).

Figure 1

Hypothetical presentation of fibrosis in uterine leiomyoma. Fibrosis is initiated by inflammation in the presence or absence of genetic changes. Growth factors, cytokines and steroid hormones are produced at the site of injury and contribute to fibroblast activation and differentiation into myofibroblasts. Myofibroblasts produce ECM components to restore homeostasis and then should be eliminated by apoptosis. During chronic inflammation, myofibroblasts become resistant to elimination by apoptosis and produce excessive amounts of ECM components, leading to fibrotic transformation. Once established, fibroids can promote their own growth. Masson's trichrome stain highlighting collagen (green) and smooth muscle (red) in myometrium and leiomyoma was performed in our laboratory.

Figure 2

Mechanotransduction is caused by extracellular matrix in uterine leiomyoma cells. Mechanical force from the stiffness of ECM or osmotic stress activates mechanical signaling pathways through heterodimeric integrins (α and β) that function as transmembrane receptors for ECM components. Activation of FAK initiates actin polymerization and AKAP13 activation of RhoA, which in turn interacts with ROCK and activates the ERK/p38 MAPK-signaling cascade, resulting in changes in cell proliferation, decreased apoptosis and upregulation of genes involved in ECM composition and remodeling. Cells sense the mechanical force from newly generated altered ECM and further activate mechanical signaling that affects cell behavior and alters overall remodeling of the matrix. This process initiates cell-matrix interactions in a rapid, transient, and bidirectional manner, which is referred to as dynamic reciprocity. Compounds that can interrupt this signaling pathway are also listed (yellow square). FAK, focal adhesion kinase; AKAP13, A kinase anchor protein 13; RhoA, Ras homolog gene family, member A; ROCK, Rho kinase; ERK, extracellular signal–regulated kinase; MAPK, mitogen-activated protein kinase.

Figure 3

Regulation of extracellular matrix by growth factors, cytokines and steroid hormones in uterine leiomyoma. Extracellular matrix may act as both a reservoir of growth factors and a modulator of their actions. Compounds that can interrupt the signaling pathways are also listed (yellow squares). C, Curcumin; V, Vitamin D3; M, 2-Methoxyestradiol; L, Leuprolide acetate.

Uterine leiomyoma is thought to be a consequence of an improper inflammatory response (Wegienka, 2012; Leppert et al., 2013) with myofibroblasts possibly playing a key role in the development of fibrosis (Feng et al., 2016; Protic et al., 2016) (Fig. 1). In our recent study, we found α-smooth muscle actin (α-SMA) positive and desmin negative cells as well as a large amount of collagen in leiomyoma tissue, indicating the presence of myofibroblasts and their role in the ECM deposition (Protic et al., 2016). It is known that when women reach reproductive age, events such as ovulation, menstruation and implantation may create physiological injuries in the uterus. In addition to reproductive events, harmful stimuli, mechanical forces, hypoxia and oxidative stress may create a chronic inflammatory state in the uterus (Wegienka, 2012; Fletcher et al., 2013; Leppert et al., 2013; Santulli et al., 2013). In the inflammatory state associated with injury, myofibroblasts produce ECM to promote subsequent repair processes and tissue homeostasis (Wynn, 2007). However, during chronic inflammation, myofibroblasts continuously and excessively produce ECM resulting in pathological fibrosis (Fig. 1). It is hypothesized that growth factors including TGF-βs and activin-A are key players in driving myofibroblast differentiation during the process of fibrosis (Joseph et al., 2010; Islam et al., 2014a; Feng et al., 2016; Protic et al., 2016).

ECM accumulation is a critical event in producing the rigid structure of leiomyoma, and ECM stiffness is thought to be a cause of abnormal bleeding and pelvic pain or pressure. Therefore, the inhibition of further accumulation of ECM and associated fibrosis might be an option for the management of these tumors. In this review, we highlight the involvement of ECM components in leiomyoma pathogenesis. Particularly, we discuss the basis for the increase in abundance and regulation of ECM in leiomyoma as well as the role of the ECM as reservoir for growth factors and a modulator of their actions. We also present options for medical treatments that target ECM formation, which are currently used or may be attractive in the future.

Methods

We used multiple strategies to identify primary research publications and review articles/book chapters related to extracellular matrix and medical treatments of uterine leiomyoma published in English until May 2017. We conducted extensive searches in PubMed and/or Google Scholar using the following keywords either alone or in combination with ‘uterine leiomyoma and extracellular matrix’: sarcolemma, caveolae, myofilaments and intermediate filament, collagen, fibronectin, laminin, versican, proteoglycan, MMP, TIMP, mechanotransduction, mechanical stress, growth factor, transforming growth factor-β (TGF-β), activin-A, platelet-derived growth factor (PDGF), cytokines, chemokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-11, IL-13, granulocyte-macrophage colony-stimulating factor (GM-CSF), estrogen, progesterone, microRNA, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), inflammation, myofibroblast, fibrosis, medical treatment, pharmacological treatment, leuprolide acetate, cetrorelix acetate, asoprisnil, ulipristal acetate, mifepristone, raloxifene, CP8947, 2-methoxyestradiol, liarozole, all-trans retinoic acid, vitamin D, celecoxib, tranilast, pirfenidone, halofuginone, curcumin, resveratrol and collagenase Clostridium histolyticum. We also searched additional relevant articles in the bibliographies of downloaded articles. Overall, we reviewed most relevant articles and included them as appropriate.

Ultrastructural features of uterine leiomyoma

A number of studies have identified some ultrastructural features that may distinguish uterine leiomyoma from myometrium. These include sarcolemma (Richards et al., 1998), caveolae (Richards et al., 1998), extracellular matrix (Leppert et al., 2004; Wortham et al., 2006), myofilaments and intermediate filaments (Eyden et al., 1992; Wortham et al., 2006). Sarcolemma is the cell membrane of a striated muscle fiber cell and sarcolemmal dense bands were found to be significantly greater in length in fibromyomata and host myometria (non-neoplastic myometrial portion of fibromyomatous uteri) than in normal myometria (Richards et al., 1998). Caveolae are 50–100 nm invaginations of the plasma membrane with an omega (Ω) shape and the numbers of caveolae in host myometria and fibromyomata were found to be decreased in comparison to normal myometria (Richards et al., 1998). Collagens are central structural components of the ECM. In myometrium, collagen structure is ordered with tight bundles, whereas in leiomyoma, they are more loosely packed, with little common orientation (Leppert et al., 2004; Wortham et al., 2006). Myofibrils, composed of myofilaments, are rod-like units of a muscle cell. Eyden et al. reported an abundance of myofilaments with focal densities in both normal myometrium and leiomyomata with the organization of intermediate filaments being alike in both tissues (Eyden et al., 1992). However, a later study reported that myofilaments and intermediate filaments were ordered and aligned in the myometrium while disorganized structures were found in leiomyoma (Wortham et al., 2006).

Extracellular matrix in uterine leiomyoma

The hallmark of uterine leiomyoma is the excessive deposition of ECM and key ECM components including collagens (Fig. 1), fibronectin, laminins and proteoglycans have been reported to be elevated in leiomyoma compared to myometrium (Stewart et al., 1994; Arici and Sozen, 2000; Norian et al., 2009; Malik et al., 2012).

Collagens

Collagen is an insoluble extracellular glycoprotein. It is a key structural component of ECM and maintains cellular morphology. In addition to their roles in wound healing and fibrosis, collagens are known to regulate cell migration, proliferation, differentiation and survival by signaling through cell surface receptors including integrins (Jones and Walker, 1999; Pickering, 2001). Leiomyoma cells have an increased expression of collagen subtypes and are organized by an abnormal collagen structure and orientation (Stewart et al., 1994; Leppert et al., 2004; Malik et al., 2010; Iwahashi and Muragaki, 2011). Stewart et al. reported overexpression of type I and III collagen mRNAs in leiomyoma compared to the adjacent myometrium (Stewart et al., 1994) while later studies confirmed an increase of type I and V collagen protein in leiomyoma compared to normal myometrium (Iwahashi et al., 2010; Iwahashi and Muragaki, 2011). Malik et al. measured levels of a series of collagen subtypes using microarray analysis and found that COL1A1, 4A2, 6A1, 6A2, 7A1 and 16A1 were each expressed to a greater extent in leiomyoma cells than in myometrial cells (Malik et al., 2010). Wolanska and co-scientists similarly reported that collagen concentration was higher in both small (defined as <10 g) and large (defined as >100 g) leiomyoma compared to normal myometrium (Wolanska et al., 1998). They also reported that small leiomyoma had a higher collagen concentration than large leiomyoma (Wolanska et al., 1998) suggesting that excessive accumulation of ECM components is an early event in the formation of these uterine leiomyoma.

Fibronectin

Like collagen, fibronectin is a secreted glycoprotein of the ECM and its role is to attach cells to a variety of ECM types (such as collagen, fibrin and heparin) through interacting with specific membrane receptors (such as integrins). Fibronectin is involved in cell migration, adhesion, growth and differentiation as well as fibrosis, tumor invasion and metastasis (Pankov and Yamada, 2002). Leiomyoma cells are reported to have an elevated level of fibronectin compared to myometrial cells (Arici and Sozen, 2000).

Laminins

Laminins are major glycoprotein components of ECM of the basal lamina (one of the layers of the basement membrane). They are composed of α-, β- and γ-subunits. Laminin has binding regions for collagen and fibronectin (Ockleford et al., 1993; Smith and Ockleford, 1994) and specific integrins also serve as cell surface receptors for laminins. Laminin plays an important role in cell migration, adhesion, growth and differentiation as well as assembly of ECM (Kleinman et al., 1985). Malik et al. demonstrated an increased expression of integrin β1 and integrin α6 subunit as well as laminin 5α, laminin 5β and laminin 5γ subunits in leiomyoma cells compared to myometrial cells (Malik et al., 2012). In addition, they plated leiomyoma cells in laminin coated culture plates, and found their alignment was parallel compared to plastic or collagen-1 coated plates (Malik et al., 2012).

Fibulin-3

Fibulins are calcium-binding glycoproteins that associate with basement membranes and elastic fibers in the ECM. Fibulins may interact with fibronectin, laminins, proteoglycans and tropoelastin, and play important roles in cell morphology, growth, adhesion and motility (Gallagher et al., 2005). Fibulin-3 is encoded by the gene EFEMP1 (EGF-containing fibulin-like ECM protein 1). Marsh et al. demonstrated reduced expression of EFEMP1 and fibulin-3 in leiomyoma compared to normal myometrial cells and tissues by using multiple approaches, including real-time PCR, western blotting, immunohistochemistry and immunofluorescence (Marsh et al., 2016a). Since fibulin-3 acts as an antagonist of angiogenesis (Albig et al., 2006), downregulation of EFEMP1 and the associated loss of fibulin-3 protein may be associated with increased leiomyoma angiogenesis.

Proteoglycans

Proteoglycans are glycosylated proteins consisting of a ‘core protein’ with covalently attached glycosaminoglycans (GAGs). GAGs are long, unbranched heteropolysaccharide chains composed of repeating disaccharide units. The common GAGs are chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, heparin and hyaluronic acid. Berto et al. reported that the amounts of chondroitin sulfate and dermatan sulfate (combined data) were increased in leiomyoma compared to normal myometrial tissue (Berto et al., 2001). In addition to the increases in chondroitin sulfate and dermatan sulfate (Berto et al., 2001; Mitropoulou et al., 2001), a rise in keratin sulfate was also found in leiomyoma relative to myometrial tissue (Wolanska et al., 1998). Wolanska et al. also reported elevations in heparan sulfate and heparin in leiomyoma relative to myometrial tissue (Wolanska et al., 1998). By contrast, however, a decrease in heparan sulfate concentration was reported in leiomyoma compared to myometrium in other studies (Berto et al., 2001; Mitropoulou et al., 2001). Hyaluronic acid is a large GAG and is unique in that it does not contain any sulfate and does not attach covalently to proteins. Available evidence suggests that expression levels of hyaluronic acid are not consistently higher or lower in leiomyoma than normal myometrium. For example, Wolanska et al. found no differences in the expression of hyaluronic acid in small leiomyoma compared to myometrial tissue (Wolanska et al., 1998), while another study found less hyaluronic acid in leiomyoma compared to normal myometrium (Mitropoulou et al., 2001). This apparent inconsistency may relate to the rather dynamic expression of proteoglycans in the uterus.

Versican is a large chondroitin sulfate proteoglycan that plays an important role in cell migration, adhesion, proliferation, tissue homeostasis and inflammation (Wight, 2002; Andersson-Sjöland et al., 2014). The expression of several versican subtypes was reported to be higher in leiomyoma tissue and primary cells compared to their healthy tissue counterparts by microarray analysis (Leppert et al., 2006; Malik and Catherino, 2007; Malik et al., 2010). Particularly, versican variant V0 was found to be dramatically elevated in leiomyoma (Malik et al., 2010). In addition, Carrino et al. reported higher amounts of versican in uterine leiomyoma and keloid scars compared to corresponding healthy tissues (Carrino et al., 2012), suggesting a molecular link in the ECM composition between these two fibrotic diseases.

Decorin is a small dermatan sulfate proteoglycan that regulates matrix assembly by binding to fibronectin and collagen via its core protein (Schmidt et al., 1991; Fiedler et al., 2008). Available evidence suggests that uterine leiomyoma contain less decorin than normal myometrial tissue (Carrino et al., 2012; Barker et al., 2015) and, since decorin may act as a antagonist of TGF-β signaling (Droguett et al., 2006), this may cause increased activation of TGF-β signaling that is important for fibrosis. Indeed, decorin treatment of several in vivo models of fibrosis was reported to ameliorate the fibrotic condition (Giri et al., 1997; Logan et al., 1999).

Fibromodulin is a small keratan sulfate proteoglycan that is widely expressed in many connective tissues. It has a close homology with decorin and biglycan. Fibromodulin binds to collagens, and influences the rate of fibrillogenesis (Hedbom and Heinegård, 1993; Font et al., 1998). Levens et al. reported that fibromodulin was highly expressed at mRNA and protein levels in leiomyoma compared to myometrial tissue (Levens et al., 2005).

MMPs and TIMPs

ECM remodeling is an essential process for development, wound healing and homeostasis as well as fibrosis. It is regulated by the combined action of MMPs and TIMPs. MMPs are a class of zinc-dependent endopeptidases that are responsible for the degradation of the ECM while TIMPs act as physiological regulators of the MMPs. Available evidence indicates that several MMPs and TIMPs are differentially expressed at both mRNA and protein level in leiomyoma compared to myometrium. Among the studied MMPs, the expression of MMP-1, -2, -3, -9, -11, -14, -16 and -24 is elevated, while the expression of MMP-7, -19 and -25 is decreased in leiomyoma (Palmer et al., 1998; Wolanska et al., 2004; Dimitrova et al., 2009; Malik et al., 2010; Tsigkou et al., 2015) (Table I). Particularly, the activity and circulating level of MMP-2 are elevated in leiomyoma compared to normal myometrium (Wolanska et al., 2004; Bogusiewicz et al., 2007; Korompelis et al., 2015), indicating a possible dominant role of this MMP in ECM remodeling in leiomyoma. The current research suggests that MMPs are not only ECM-degrading proteases; they participate in a wide range of physiological processes including cell migration, differentiation, growth, innate and adaptive immunity, inflammation, angiogenesis, apoptosis, bone remodeling and neurite growth (Nagase et al., 2006; Rodríguez et al., 2010; Löffek et al., 2011). Some MMPs are antifibrotic, whereas others can have profibrotic functions (Giannandrea and Parks, 2014) (Table 1). They can directly or indirectly affect the functions of various cytokines that play roles in inflammation and repair processes including interferon-β (Nelissen et al., 2003), VEGF (Bergers et al., 2000), EGF (Suzuki et al., 1997), FGF (Suzuki et al., 1997) and TGF-β1 (Shull et al., 1992). MMPs can regulate the bioavailability of angiogenic factors sequestered by the ECM. For instance, ECM-bound VEGF is mobilized by MMPs-1, -3, -7, -9, -16 and -19 cleavage of the VEGF-binding ECM proteins (Bergers et al., 2000; Colnot et al., 2003; Lee et al., 2005). MMPs also enable proteolytic release of different ECM-trapped growth factors such as FGFs or TGF-β that can show strong mitogenic properties (Rodríguez et al., 2010). MMPs can both activate or prevent apoptosis through proteolytic processing of particular signaling molecules. For example, the Fas/Fas ligand (FasL) signal transduction axis is a vital pro-apoptotic system affected by MMPs. MMP-7-mediated release of soluble FasL triggers apoptosis in epithelial cells while inhibiting cell death in tumors (Powell et al., 1999b; Mitsiades et al., 2001). MMP-7 also inhibits apoptosis in cancer cells by cleaving Fas (Strand et al., 2004). Regarding TIMPs, Bogusiewicz et al. reported that the levels of TIMP-1 and TIMP-2 were similar in both myometrial and leiomyoma tissues (Bogusiewicz et al., 2007) while circulating levels of TIMP-1 were reported to be significantly elevated in leiomyoma patients compared to controls (Korompelis et al., 2015). Overall, the available evidence suggests a dysregulation of MMPs and TIMPs that may play a critical role in the formation of a more fibrous ECM in leiomyoma.

Table I

Expression of MMPs and TIMPs in uterine leiomyoma compared to myometrium.

MMMs and TIMPs	Expression in leiomyoma relative to myometrium	Matrix substrates/MMPs	Functions in different biological systems
MMP-1	↑	Collagen types I, II, III, VII and X as well as gelatin, entactin, aggrecan and tenascin (McCawley and Matrisian, 2001)	(I) Plays an antifibrotic role in liver fibrosis (Iimuro et al., 2003)
			(II) Possesses pro-apoptotic effects (Mannello et al., 2005)
MMP-2	↑	Fibronectin, laminin, gelatin, elastin, aggrecan and vitronectin as well as collagen types I, IV, V, VII, X and XI (McCawley and Matrisian, 2001)	(I) Plays an antifibrotic role in liver fibrosis (Onozuka et al., 2011)
			(II) Promotes angiogenesis (Webb et al., 2017)
			(III) Possesses both pro-apoptotic and anti-apoptotic effects (Mannello et al., 2005)
MMP-3	↑	Fibronectin, laminin, proteoglycans, gelatins, fibrinogen, entactin, tenascin and vitronectin as well as collagen types III, IV, V and IX (McCawley and Matrisian, 2001)	(I) Appears to be profibrotic in lung fibrosis (Yamashita et al., 2011)
			(II) Appears to play a role in angiogenesis (Anuar et al., 2016)
			(III) Possesses both pro-apoptotic and anti-apoptotic effects (Mannello et al., 2005)
MMP-7	↓	Collagen types III, IV, V, IX, X and XI as well as proteoglycans, laminin, fibronectin, gelatin, fibrinogen, entactin, tenascin and vitronectin (McCawley and Matrisian, 2001)	(I) Plays a profibrotic role in lung fibrosis (Manicone et al., 2009)
			(II) Induces angiogenesis (Nishizuka et al., 2001)
			(III) Possesses both pro-apoptotic and anti-apoptotic effects (Mannello et al., 2005)
MMP-9	↑	Elastin and gelatin as well as collagen types IV and V (Matrisian, 1992)	(I) Possesses both anti- and profibrotic functions in lung fibrosis (Lee et al., 2001; Cabrera et al., 2007)
			(II) Promotes angiogenesis (Webb et al., 2017)
			(III) Possesses both pro-apoptotic and anti-apoptotic effects (Mannello et al., 2005)
MMP­11	↑	Laminin, fibronectin and aggrecan (McCawley and Matrisian, 2001)	(I) Shows dual functions in tumourigenesis and cancer progression (Zhang et al., 2016)
			(II) Possesses both pro-apoptotic and anti-apoptotic effects (Mannello et al., 2005)
MMP-14	↑	Collagen, fibronectin, gelatin, vitronectin, tenascin, nidogen, aggrecan, fibrin, fibrinogen and laminin-5 (McCawley and Matrisian, 2001; Klein and Bischoff, 2011)	(I) Shows diverse and opposing functions in scarring or fibrosis (Rohani and Parks, 2015)
			(II) Promotes cell migration (Itoh, 2006)
			(III) Promotes tumor growth and angiogenesis (Sounni et al., 2002)
MMP-16	↑	Collagen type III, gelatin and fibronectin (Matsumoto et al., 1997)	(I) promotes cancer progression (Sounni and Noël, 2005)
MMP-19	↓	Fibronectin, tenascin-C, gelatin, nidogen and collagen type IV (Stracke et al., 2000)	(I) possesses both anti- and profibrotic functions in liver fibrosis (Wang et al., 2011; Jirouskova et al., 2012)
			(II) exhibits an anti-angiogenic effect on endothelial cells (Brauer et al., 2011)
MMP-24	↑	Fibronectin, gelatin, vitronectin, collagen and aggregan (McCawley and Matrisian, 2001)	(I) Plays an important role in ECM remodeling events in the brain and during embryonic development (Pei, 1999)
MMP-25	↓	Fibronectin, collagen type IV gelatin, fibrin and laminin-1 (McCawley and Matrisian, 2001)	() Appears to play a role in cancer progression (Velasco et al., 2000; Sun et al., 2007)
TIMP-1	~	Collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2, MMP-9), stromelysins (MMP-3, MMP-10, MMP-11 and MMP-18), matrilysins (MMP-7, MMP-26), membranous MMP (MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25), and others (MMP-11, MMP-12, MMP-19, MMP-20, MMP-23 and MMP-28) (Jakubowska et al., 2016)	(I) Promotes liver fibrosis (Yoshiji et al., 2000)
			(II) Inhibits apoptosis (Li et al., 1999)
			(III) Inhibits tumor growth and angiogenesis (Ikenaka et al., 2003)
TIMP-2	~	Collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2, MMP-9), stromelysins (MMP-3, MMP-10, MMP-11 and MMP-18), matrilysins (MMP-7, MMP-26), membranous MMP (MMP-14, MMP-15, MMP-16, MMP-17, MMP-24 and MMP-25), and others (MMP-11, MMP-12, MMP-19, MMP-20, MMP-23 and MMP-28) (Jakubowska et al., 2016)	(I) Promotes survival of hepatic stellate cells and endothelial cells (Murphy et al., 2002; Boulday et al., 2004)
			(II) Inhibits endothelial cell migration and angiogenesis (Seo et al., 2003; Oh et al., 2004)

Mechanotransduction of the extracellular matrix

Mechanotransduction is a process that allows cells to adapt to their changing physical surroundings by sensing their environment and translating mechanical stress into biochemical signals (Huang et al., 2004; Thorne et al., 2015). This process is promoted by the stiffness of the ECM or osmotic stress that initiates cell-matrix interactions in a rapid, transient and bidirectional manner which is referred to as ‘dynamic reciprocity’ (Bissell and Aggeler, 1986; Maniotis et al., 1997; Polacheck et al., 2014; Thorne et al., 2015) (Fig. 2). The activation of downstream mechanical signaling pathways can alter gene expression, leading to changes in ECM density, composition and organization that ultimately affect cell shape and contractility (Fig. 2). Cells sense the mechanical force from newly generated altered ECM and further activate mechanical signaling that result in altered cell behavior and overall remodeling of the matrix. This process appears to play a crucial role in cell adhesion, migration, proliferation and survival as well as inflammation and fibrosis (Seong et al., 2013; Duscher et al., 2014; Thorne et al., 2015). Mechanotransduction occurs at the sites of focal adhesion (Seong et al., 2013; Mui et al., 2016) where large protein complexes serve as a ‘molecular bridge’ between ECM and intracellular molecules/cytoskeleton (Geiger et al., 2001). At these sites, mechanical signals are transmitted from the ECM to the intracellular space through heterodimeric (α and β) transmembrane integrins. Upon ligation and clustering, integrins activate cytoplasmic tyrosine kinases including Src and focal adhesion kinase (FAK). Activation of FAK results in the recruitment of a number of SH2 (Src Homology 2) domain- and SH3 domain-containing proteins that mediate downstream signaling pathways (Parsons, 2003; Schlaepfer and Mitra, 2004) including ERK 1/2, p38 MAPK and JNK (c-Jun N-terminal kinase) (Ruwhof and van der Laarse, 2000; PaszeK et al., 2005). Rho is one of the targets of FAK (Hanks et al., 1992). The Rho family of GTPases, include Rac, Cdc42 and RhoA (Ras homolog gene family, member A). RhoA serves as a ‘molecular switch’ that mediates cycling between active GTP (guanosine triphosphate)-bound and inactive GDP (guanosine diphosphate)-bound states. It plays a crucial role in mediating several profibrotic responses in cardiac fibroblasts (Porter et al., 2004; Zhao et al., 2007). A kinase anchor protein 13 (AKAP13), a RhoA GTPase-specific guanine exchange factor (Rho-GEF), is known to activate RhoA from the inactivated form to the activated RhoA GTPase. The AKAP13/RhoA complex was recently reported to mediate the profibrotic effects in cardiac fibroblasts (Cavin et al., 2014). In addition, AKAP13 appears to play a key role in transmitting the osmotic stress or extracellular hyperosmolarity signal through nuclear factor of activated T cells 5 (NFAT5) (Kino et al., 2009). The cellular osmotic response results in fluid exchange between the cell and ECM that may affect cell shape and ECM composition and organization (Polacheck et al., 2014).

Uterine leiomyoma has been reported to be significantly stiffer than matched myometrium (Rogers et al., 2008), and appears to be under increased mechanical stress (Rogers et al., 2008). In leiomyomal cells, the levels of integrin α6 and integrin β1 were found to be overexpressed compared to normal counterparts (Malik et al., 2012; Chen et al., 2013). In addition, phosphorylation of FAK and ERK was increased in leiomyomal cells (Malik et al., 2012; Chen et al., 2013). The upstream target of ERK, RhoA was overexpressed in leiomyoma compared to myometrial cells (Norian et al., 2012). Malik et al. reported that inhibition of integrin-β1 led to a decrease in active RhoA in leiomyoma cells (Malik et al., 2012). Fasudil, an inhibitor of Rho kinase (ROCK, a downstream target of RhoA), was found to relax the contraction of leiomyoma cells in 3D collagen gels (Malik et al., 2014). The levels of AKAP13 (a Rho-GEF) also appeared higher in leiomyoma compared to myometrium (Rogers et al., 2008). Rogers and co-investigators further reported that p38 MAPK phosphorylation was greater in leiomyoma compared to matched myometrium (Rogers et al., 2008), and this increase was associated with upregulation of AKAP13 (Rogers et al., 2008).

NFAT5 is a transcription factor that induces the expression of genes involved in osmotic stress. Uterine leiomyoma cells demonstrated increased basal expression of NFAT5 compared to myometrial cells. The expression of NFAT5 as well as NFAT5-regulated genes, AR (aldose reductase) and SMIT (sodium myo-inositol transporter 1) was further increased in leiomyoma cells under hyperosmolar conditions (McCarthy-Keith et al., 2011).

A recent study suggested that collagen may play a role in smooth muscle cell proliferation in uterine leiomyoma (Koohestani et al., 2013). Using an in vitro model system of collagen, Koohestani et al. investigated the interaction of cultured leiomyoma smooth muscle cells with monomeric unpolymerized collagen films and fibrillar polymerized collagen gels, and found a significant increase in cell proliferation (Koohestani et al., 2013). The differences in cell proliferation were accompanied by changes in cell cycle progression and p21 (Koohestani et al., 2013). The overall result suggests a possible activation of ECM mediated integrin-FAK-Rho-ERK-p38 MAPK-signaling leading to cell proliferation. Indeed, Malik and co-investigators reported that inhibition of integrin β1 led to a decrease in phosphorylation of ERK as well as reduced proliferation of leiomyoma cells (Malik et al., 2012).

Regulation of extracellular matrix accumulation by growth factors and cytokines

As discussed below, several growth factors and cytokines regulate the production of ECM in leiomyoma cells and can contribute to fibrosis (Islam et al., 2013a, 2016) (Fig. 3).

Transforming growth factor-β

Transforming growth factor-β (TGF-β) is a secreted polypeptide with multifunctional properties. It has three isoforms, referred to as TGF-β1, TGF-β2 and TGF-β3. Myometrial and leiomyoma smooth muscle cells express mRNAs and proteins for each of the three TGF-β isoforms as well as for the TGF-β receptors, TGF-βR-I and TGF-βR-II (Dou et al., 1996; Tang et al., 1997). TGF-β3 appears to increase mRNA expression of ECM components, including collagen 1A1, connective tissue growth factor (CTGF) (Joseph et al., 2010), fibronectin (Arici and Sozen, 2000) and versican V0 (Norian et al., 2009) in both myometrial and leiomyoma cells. However, TGF-β3 was reported to decrease production of MMP-2 and MMP-11 in myometrial and leiomyoma cells (Joseph et al., 2010). In addition to TGF-β3, TGF-β1 has been reported to increase mRNA expression of CTGF (Luo et al., 2006), plasminogen activator inhibitor 1 (PAI-1) (Ding et al., 2004b) and fibromodulin (Levens et al., 2005) in myometrial and leiomyoma smooth muscle cells. The profibrotic role of TGF-β is mediated, at least in part, through activation of Smad 2/3 and ERK 2/3 signaling pathways (Xu et al., 2003; Ding et al., 2004b).

Activin-A

Activin-A, a member of the TGF-β superfamily, was originally isolated for its ability to stimulate secretion of FSH from the anterior pituitary gland but is now known to have wide-ranging physiological and pathophysiological roles in multiple tissues. The mRNA level of activin-A was found to be more highly expressed in leiomyoma compared to myometrium, whereas the levels of activin receptors (ALK4, ActRIIA and ActRIIB) were unchanged (Ciarmela et al., 2011a). The profibrotic role of activin-A in leiomyoma cells has recently been demonstrated by our group (Islam et al., 2014a). We found that activin-A can increase mRNA levels of several ECM components, including collagen 1A1, fibronectin and versican in human primary leiomyoma cells compared to untreated controls (Islam et al., 2014a). Furthermore, activin-A can induce phosphorylation of Smad2 and Smad3 in both myometrial and leiomyoma cells compared to untreated cells (Islam et al., 2014a), suggesting that the fibrotic role of activin-A is mediated, at least in part, by activation of Smad 2/3 signaling pathway.

Platelet-derived growth factor

PDGF is a family of growth factors consisting of four homodimers, namely PDGF-AA, PDGF-BB, PDGF-CC and PDGF-DD, and one heterodimer, PDGF-AB. The protein levels of PDGF-AA, PDGF-BB and PDGF-CC and their receptors were found to be highly expressed in leiomyoma compared to myometrial cells and/or tissues (Liang et al., 2006; Hwu et al., 2008; Yu et al., 2008; Suo et al., 2009). PDGF exerts a profibrotic effect in myometrial and leiomyoma cells through increasing collagen α1 (I) expression (Liang et al., 2006). Additionally, PDGF can stimulate myometrial and leiomyoma cell proliferation (Liang et al., 2006; Mesquita et al., 2010) by regulating of VEGF production (Taniguchi et al., 2001) and activating the ERK 1/2 signaling pathway (Mesquita et al., 2010).

Tumor necrosis factor-α

Tumor necrosis factor (TNF)-α is a pleiotropic cytokine that is primarily secreted by activated macrophages. It plays an important role in controlling inflammation, immunity, cell growth and differentiation, and apoptosis. The level of TNF-α protein expression was found to be higher in leiomyoma compared to normal myometrial cells and tissues (Kurachi et al., 2001; Plewka et al., 2013). Wang et al. reported that TNF-α can significantly upregulate the protein and mRNA levels of MMP-2 in cultured leiomyoma smooth muscle cells but not in matched myometrial smooth muscle cells (Wang et al., 2015). Recently, we found that TNF-α can increase activin-A mRNA expression in both myometrial and leiomyoma cells (Islam et al., 2013b). The increased expression of activin-A in leiomyoma (Ciarmela et al., 2011a), and the ability of activin-A is to increase production of collagen 1A1, fibronectin and versican in leiomyoma smooth muscle cells (Islam et al., 2014a), supports the possible role of TNF-α and activin-A in the process of fibrosis following leiomyoma growth. Additionally, Nair and Al-Hendy demonstrated the possible of role of TNF-α in human uterine leiomyoma cell proliferation (Nair and Al-Hendy, 2011). The proliferative and profibrotic effect of TNF-α in leiomyoma cells was mediated, at least in part, by activating ERK 1/2 signaling pathway (Wang et al., 2015).

Other cytokines

Several cytokines, including interleukin (IL)-1β, IL-11, IL-13 and granulocyte-macrophage colony-stimulating factor (GM-CSF) have been reported to be highly expressed in leiomyoma (Chegini et al., 1999; Ding et al., 2004a; Luo et al., 2005; Syssoev et al., 2008; Plewka et al., 2013). Since these cytokines are known to play important roles in the development of fibrosis in different cellular systems (Xing et al., 1997; Chakir et al., 2003; Fichtner-Feigl et al., 2005; Guo et al., 2013), they may have fibrotic roles in the pathogenesis of leiomyoma.

Hormonal regulation of extracellular matrix accumulation

It is well accepted that estrogen and progesterone play prominent roles as regulators of uterine leiomyoma growth (Maruo et al., 2004; Ciarmela et al., 2011b; Islam et al., 2013a). However, the underlying mechanisms are not completely understood. A number of studies suggest that the stimulatory effects of estrogen and progesterone on leiomyoma growth are mediated, at least in part, by regulation of ECM proteins, growth factors and their signaling pathways (Barbarisi et al., 2001; Chegini et al., 2002; Hoekstra et al., 2009; Nierth-Simpson et al., 2009; Qiang et al., 2014; Barker et al., 2015) (Fig. 3).

Estrogen

Estrogens exert multiple effects on their target cells through binding to estrogen receptors (ERα and ERβ) (Nilsson et al., 2001). Uterine leiomyoma exhibits higher mRNA and protein expression levels of ERα (more highly expressed) and ERβ than normal myometrium (Benassayag et al., 1999; Kovacs et al., 2001). Particularly, ER-α and ER-β are more highly expressed at both mRNA and protein levels in leiomyoma fibroblasts compared to smooth muscle fibroblasts (Feng et al., 2016). Using primary cultures, Zbucka et al. found that collagen biosynthesis was strongly stimulated by low doses of estrogen (5 nM) in leiomyoma cells while no changes occurred in myometrial cells (Zbucka et al., 2008). In contrast, collagen production was inhibited by 10 nM estrogen in both leiomyoma as well as control cells (Zbucka et al., 2008). In uterine leiomyoma xenografts, 17β-estradiol was found to upregulate mRNAs for multiple collagens through downregulation of miR-29b (Qiang et al., 2014). Recently, higher expression of collagen I, fibronectin and laminin, were found in estrogen-treated leiomyoma and smooth muscle fibroblasts compared to untreated control groups (Feng et al., 2016). However, Zbucka et al. found that estrogen can inhibit MMP-2 in leiomyoma and myometrial cells (Zbucka et al., 2008) while transfection of a dominant-negative estrogen receptor resulted in significant augmentation of MMP-1 protein expression in primary and immortalized human leiomyoma cultures (Hassan et al., 2007). Chegini and co-investigators demonstrated that estradiol stimulated both total and active TGF-β1 production in both leiomyoma and myometrial smooth muscle cells (Chegini et al., 2002). The secretion of TGF-β and IGF-1 was also found to be significantly increased in response to estrogen treatment in both leiomyoma and smooth muscle fibroblasts compared to untreated controls (Feng et al., 2016). Recently, we found that estrogen can downregulate activin-A mRNA expression in human myometrial explants (Ciarmela et al., 2011a). Barbarisi et al. reported that estrogen can upregulate PDGF expression in cultured leiomyoma cells (Barbarisi et al., 2001). The ability of estrogen to induce PDGF expression was further confirmed by the observation by Barbarisi et al. that an antiestrogenic compound ICI 182 780 prevented estrogen-induced PDGF expression in cultured leiomyoma cells (Barbarisi et al., 2001). They further reported rapid and transient activation of the MAPK pathway in estrogen-treated leiomyoma smooth muscle cells compared to untreated groups (Barbarisi et al., 2001). Treatment of leiomyoma smooth muscle cells with estrogen was also reported to increase phosphorylation of several downstream intracellular proteins, such as GAP (GTPase-activating protein), PI3K, PLCγ (phospholipase-Cγ) and ERK 1/2 (Barbarisi et al., 2001; Nierth-Simpson et al., 2009). A recent study reported that estrogen can also promote phosphorylation of MEK, ERK1/2 and AKT (also known as protein kinase B or PKB) in both leiomyoma and smooth muscle fibroblasts compared to untreated controls (Feng et al., 2016).

Progesterone

Progesterone exerts physiologic actions on its target cells by interacting with progesterone receptors (PR-A and PR-B) (Kastner et al., 1990). Progesterone seems to be the dominant steroidal influence on uterine leiomyoma as indicated by the increased mitotic rates of leiomyoma cells during the secretory phase of the menstrual cycle (Kawaguchi et al., 1989). Recently, Barker et al. tested the effects of progesterone on decorin expression in human uterine leiomyoma compared with normal myometrial tissue from different stages of the menstrual cycle (Barker et al., 2015). This group found lower expression of decorin at both the mRNA and protein levels in leiomyoma than in adjacent myometrium (Barker et al., 2015). They also found reduced level of decorin mRNA expression in leiomyoma tissue from the secretory phase compared to the proliferative phase (Barker et al., 2015), suggesting an inhibitory effect of progesterone on decorin expression in leiomyoma. As expected, progesterone was found to decrease mRNA expression of decorin in uterine leiomyoma cells compared to controls (Barker et al., 2015). Since decorin inhibits the activity of TGF-β (Yamaguchi et al., 1990), its reduced level in leiomyoma may enhance ECM deposition through TGF-β activity. Chegini et al. demonstrated that treatment of primary myometrial and leiomyoma cell cultures with medroxyprogesterone acetate, a synthetic variant of the steroid hormone progesterone, stimulated both total and active TGF-β1 production (Chegini et al., 2002). Consistently, the highest level of TGF-β3 mRNA was observed in leiomyoma from the mid-secretory phase of menstrual cycle (Arici and Sozen, 2000). Hoekstra et al. reported that progestins can rapidly increase the levels of phosphorylated-AKT and its downstream effectors, pGSK3β and pFOXO1 in leiomyoma cells (Hoekstra et al., 2009). Recently, ECM regulation by progesterone via miRNA was reported by Qiang and co-workers (Qiang et al., 2014). This group found a lower level of miR-29b in uterine leiomyoma compared to myometrial tissues as well as primary cells (Qiang et al., 2014). The restoration of miR-29b in the uterine leiomyoma cells reduced the COL1A1 level in cultured uterine leiomyoma cells. In uterine leiomyoma xenografts, restoring miR-29b also inhibited the accumulation of several collagen subtypes and the development of solid tumors (Qiang et al., 2014). This group demonstrated that progesterone can upregulate mRNA expression of collagens via downregulating miR-29b in a leiomyoma xenograft model (Qiang et al., 2014).

Regulation of extracellular matrix accumulation by microRNA

MicroRNAs (or miRNAs) are small non-coding RNAs (∼22-nt long) that regulate post-transcriptional gene expression. By binding to the 3'-UTR (untranslated region) of target mRNAs, miRNAs prevent protein production by inducing mRNA degradation and/or directly repress translation. A significant number of studies have suggested that miRNAs play an important role in the regulation of ECM accumulation during leiomyoma pathogenesis (Wang et al., 2007; Luo and Chegini, 2008; Chuang et al., 2012a, 2012b; Chuang and Khorram, 2014; Qiang et al., 2014; Chuang and Khorram, 2016; Marsh et al., 2016b).

miR-29 family

miR-29a

Uterine leiomyoma has been demonstrated to have decreased levels of miR-29a in leiomyoma relative to myometrium (Marsh et al., 2016b). Marsh's group used Ambion miRNA precursors to successfully overexpress miRNA-29 in cell culture, and demonstrate its role in ECM production. They found that overexpression of miRNA-29a causes a decrease in the production of collagen subtypes (types II and III) in leiomyoma cells compared to control cells (Marsh et al., 2016b). However, the knockdown of miRNA-29 failed to impact collagen expression in leiomyoma cells compared to control cells (Marsh et al., 2016b).

miR-29b

Uterine leiomyoma expressed a lower level of miR-29b than myometrium (Wang et al., 2007; Qiang et al., 2014; Marsh et al., 2016b) and forced expression of miR-29b reduced the COL1A1, COL2A1 and COL3A1 protein level in primary leiomyoma cells (Qiang et al., 2014; Marsh et al., 2016b). In leiomyoma xenografts, overexpression of miR-29b also inhibited the accumulation of collagens (COL1A1 and COL1A3) and the development of solid tumors (Qiang et al., 2014). However, knockdown of miR-29b in myometrium xenografts increased the expression of collagens (COL1A1, COL1A2, COL3A1, COL5A1, COL5A3 and COL7A1) without transformation into leiomyoma (Qiang et al., 2014). This result suggests that the downregulation of miR-29b is essential but not sufficient for leiomyoma tumourigenesis. Qiang's group reported that estrogen and progesterone can downregulate miR-29b, and upregulate collagen expression in leiomyoma xenografts (Qiang et al., 2014), indicating that steroid hormones promote leiomyoma growth, at least in part, through increasing ECM production via downregulation of the tumor suppressor miR-29b.

miR-29c

Similar to miR-29a and miR-29b, miR-29c is also expressed at low levels in leiomyoma compared to myometrium (Luo and Chegini, 2008; Chuang and Khorram, 2016; Marsh et al., 2016b). Levels of miR-29c were inversely associated with expression of its target, COL3A1 (Chuang and Khorram, 2016) and gain of function of miR-29c inhibited the protein and mRNA expression of COL3A1, and reduced secreted COL3A1, and the rate of cell proliferation (Chuang and Khorram, 2016). Marsh et al. also reported that overexpression of miRNA-29c decreased protein expression of the major collagen subtypes (COL1A1, COL2A1 and COL3A1) in primary leiomyoma cells compared to control cells (Marsh et al., 2016b). The suppression of miR-29c in leiomyoma smooth muscle cells was primarily mediated by steroid hormones, NF-κB and SP1transcription factors, and DNA methylation (Chuang and Khorram, 2016).

miR-200c

Uterine leiomyoma expresses very low levels of miR-200c relative to myometrium (Chuang et al., 2012b; Chuang and Khorram, 2014). Gain-of-function of miR-200c repressed the expression of ZEB1 and ZEB2, TIMP2 and FBLN5 (fibulin 5) at the mRNA and protein levels in leiomyoma and/or smooth muscle cells (Chuang et al., 2012b). Gain-of-function of miR-200c in leiomyoma smooth muscle cells also reduced mRNA and protein levels of IL8 (an inflammatory mediator) (Chuang and Khorram, 2014). In contrast, knockdown of miR-200c produced the opposite effect with a significant increase in IL8 mRNA in these cells (Chuang and Khorram, 2014). This regulation was mediated by suppressing the NF-κB pathway through targeting IKBKB (Chuang and Khorram, 2014).

miR-93/106b

Uterine leiomyoma expresses significantly lower levels of miR-93, but not miR-106b, as compared with myometrium (Chuang et al., 2012a). The gain of function of miR-93 and miR-106b in leiomyoma smooth muscle cells and myometrial smooth muscle cells repressed mRNA and protein levels of tissue factor (F3) and IL8 at through direct interactions with their respective mRNA3'-UTRs (Chuang et al., 2012a) and indirectly inhibited IL8, CTGF and PAI-1 expression through F3 repression (Chuang et al., 2012a).

Extracellular matrix acts as a reservoir for growth factors and modulator of their actions

An elegant experimental in vitro co-culture model by Moore and co-workers has demonstrated that leiomyoma-derived fibroblasts enhance collagen type I production, activate receptor tyrosine kinases (RTKs) and TGF-β signaling and stimulate leiomyoma cell proliferation (Moore et al., 2010). They also found an increased secretion of several growth factors, including IGF, PDGF, TGF-β, EGF and FGF in the ECM of cultured uterine leiomyoma cells (Moore et al., 2010), suggesting an important link among ECM, growth factors, their receptors and signaling pathways in the pathogenesis of leiomyoma.

Uterine leiomyoma is reported to express several GAG components of proteoglycans, such as heparan sulfate, heparin and keratan sulfate (Wolanska et al., 1998; Berto et al., 2001; Mitropoulou et al., 2001). Among these, heparin or heparan sulfate is known to bind to many growth factors. For example, heparan sulfate can bind to bFGF as a cofactor to facilitate the formation and signaling of bFGF-bFGF receptor complexes (Rapraeger et al., 1991; Yayon et al., 1991). Endothelial cell-derived heparan sulfate can also bind to bFGF and protect it from proteolytic degradation (Saksela et al., 1988), effectively serving as a storage depot for this growth factor. bFGF is commonly known as an inducer of angiogenesis and uterine leiomyoma has an increased expression of bFGF in leiomyoma compared to myometrium (Mangrulkar et al., 1995; Wolanska and Bankowski, 2006). FGFR-1 and FGFR-2 receptor expression levels were also found to be elevated in leiomyoma compared to myometrium (Wolanska and Bankowski, 2006; Yu et al., 2008). bFGF was found to be primarily bound to the ECM of myometrium and leiomyoma (Mangrulkar et al., 1995; Dixon et al., 2000). Particularly, leiomyoma showed stronger staining for bFGF because of large areas of ECM in these tumors (Mangrulkar et al., 1995; Dixon et al., 2000). ECM of uterine leiomyoma may therefore serve as a reservoir for bFGF.

A direct physical interaction between VEGF and heparan sulfate has been reported (Ortéga et al., 1998) and VEGF can bind to the heparin-II domain of fibronectin (Wijelath et al., 2006). The activation of VEGFR-2 can induce tyrosine phosphorylation of integrin β3 (Mahabeleshwar et al., 2007). Thus, the relationship between VEGFR-2 and integrin β3 seems to be synergistic. Uterine leiomyoma and myometrium were both reported to express VEGFR-1 and VEGFR-2 (Harrison-Woolrych et al., 1995; Brown et al., 1997; Gentry et al., 2001; Sanci et al., 2011). However, VEGF-A protein levels were found to be higher in leiomyoma than in adjacent myometrium (Gentry et al., 2001). Hassan et al. demonstrated that VEGF was required for continuous growth of tumors or leiomyoma in a xenograft mouse model (Hassan et al., 2008).

Growth factors can also bind to heparan sulfate indirectly through accessory proteins. For example, an activin-binding protein, follistatin, associates with heparan sulfate in follicular granulosa cells (Nakamura et al., 1991). Similarly, TGF-β binds to heparan sulfate via a 60 kDa protein (Bützow et al., 1993). In certain growth factors, such as PDGF-A and VEGF, the major heparin-binding site is localized to a linear 20 amino acid stretch rich in basic residues. The presence of heparan sulfate binding sequences in PDGF-A and VEGF can be modulated by alternative splicing (Raines and Ross, 1992; Park et al., 1993).

The interaction between TGF-β and the core protein of the proteoglycan, decorin has been reported (Border et al., 1990; Yamaguchi et al., 1990). TGF-β1 can also induce the synthesis of decorin by mesangial cells (Border et al., 1990). Decorin, in turn, binds to TGF-β and acts a ligand trap that blocks its ability to bind its receptors on target cells (Yamaguchi et al., 1990), suggesting a negative feedback loop. Uterine leiomyoma tissue contains less decorin protein than normal myometrial tissues (Carrino et al., 2012), suggesting a mechanism for increased activation of TGF-β signaling. Collagen type II contains a chordin-like VWC domain which can also bind to TGF-β1 (Abreu et al., 2002) and act as a negative regulator for this growth factor. By contrast, a membrane-anchored proteoglycan, betaglycan (TGF-β type III receptor) was reported to facilitate the formation of TGF-β receptors complexes (Shi and Massagué, 2003). TGF-β binds first to betaglycan and that binding and ‘presentation’ play key roles in mediating TGF-β binding to the type II receptor (Shi and Massagué, 2003).

A hallmark of the integrins is the ability to recognize multiple ligands. They can activate growth factor mediated signaling pathways independently (Assoian and Schwartz, 2001) or synergistically with other receptors (Alam et al., 2007). These include TGF-β receptors (Scaffidi et al., 2004), VEGF receptors (Mahabeleshwar et al., 2007), PDGF-β receptors (Schneller et al., 1997), insulin-like growth factor receptors (IGFR) (Doerr and Jones, 1996), and EGF receptors (Bill et al., 2004). In addition to integrins, several ECM proteins, including laminin, fibrillin, tenascin and thrombospondin contain EGF-like domains, which can directly bind to EGF receptors and modulate their signaling (Schenk et al., 2003).

Fibrosis in uterine leiomyoma: role of myofibroblasts and inflammation

Fibrosis is caused by an excessive accumulation of ECM proteins resulting from an imbalance in wound healing and repair processes during chronic tissue injury and/or inflammation (Wynn, 2007). Myofibroblasts are specialized cell type, activated by tissue injury, inflammation, hypoxia and oxidative stress, that play critical roles in wound healing and tissue homeostasis as well as fibrosis (Poli, 2000; Higgins et al., 2007; Wynn, 2007; Toullec et al., 2010) (Fig. 1). A biological function of myofibroblasts is to produce ECM proteins in order to heal wounds and maintain functional integrity of organs/tissues after injury. After finishing this role in tissue repair and homeostasis, myofibroblasts disappear via apoptosis (Desmouliere et al., 1995). When this process fails to proceed normally, persistence of cells with myofibroblastic phenotype can lead to excessive ECM production and development of fibrosis (Powell et al., 1999a; Tomasek et al., 2002) (Fig. 1).

Myofibroblast cells are characterized by α-SMA expression and a high level of collagen synthesis (Wynn, 2008). The fibronectin domain ED-A is also crucial for the myofibroblastic phenotype (Serini et al., 1998). Several cell types, including fibroblasts, fibrocytes, stem cells, smooth muscle cells and endothelial cells have been reported to acquire the myofibroblast phenotype in large variety of organs (Hinz et al., 2007) and a number of studies suggest that myofibroblastic transformation could occur from fibroblasts, stem cells and smooth muscle cells in the myometrium (Ono et al., 2007; Chang et al., 2010; Moore et al., 2010; Holdsworth-Carson et al., 2014; Zheng et al., 2014; Mas et al., 2015; Yin et al., 2015; Feng et al., 2016; Protic et al., 2016) (Fig. 1). CD90, a fibroblast specific marker, was found to be expressed in both smooth muscle leiomyoma fibroblasts but not in smooth muscle cells themselves (Luo et al., 2014). In addition, FAP (fibroblast activation protein), a marker of active fibroblasts, was highly expressed at the protein level in fibroblasts from uterine leiomyoma compared to those from normal muscle tissues (Luo et al., 2014). Recently, we identified numerous CD34+ fibroblasts in myometrium and leiomyoma, which are known to give origin to myofibroblasts when they lose CD34 expression (Protic et al., 2016). In addition, Yin et al. identified human uterine leiomyoma stem/progenitor cells expressing CD34 and CD49b, which are able to initiate tumors in vivo (Yin et al., 2015). In our recent study, we reported the presence of α-SMA positive and desmin negative cells as well as large amount of collagen inside the leiomyoma tissue that supports the existence of myofibroblast and fibrotic characters of this tumor (Protic et al., 2016).

Myofibroblast differentiation depends on environmental cues, including tension in the matrix and a variety of soluble mediators, such as growth factors, steroid hormones, cytokines and chemokines (Tomasek et al., 2002; Gabbiani, 2003; Wipff et al., 2007; Wynn, 2007, 2008; Luo et al., 2014; Feng et al., 2016). The central player of myofibroblast differentiation is TGF-β. It is induced by mechanical tension, induces α-SMA expression (Desmoulière et al., 1993), and enhances the synthesis of both collagens (Lindahl et al., 2002) and ED-A fibronectin (Serini et al., 1998). The cross-linking of the ECM may further activate resident cells to myofibroblastic transition (Desmouliere et al., 2005; Ho et al., 2014). In addition to TGF-β, activin-A has been demonstrated to play an important role in tissue repair as well as fibrosis (Werner et al., 1999). Both TGF-β and activin-A are highly expressed in uterine leiomyoma (Dou et al., 1996; Tang et al., 1997; Ciarmela et al., 2011a) where their abilities to enhance synthesis of ECM components, such as collagen 1A1, CTGF, fibronectin, versican and PAI-1 (Arici and Sozen, 2000; Ding et al., 2004b; Joseph et al., 2010; Islam et al., 2014a), supports their roles in driving myofibroblast differentiation. Recently, Feng et al. reported that after a biological insult of serum starvation and serum add-back, myometrial cells undergo a transition to a myofibroblast-like phenotype that was related to an increased activation of TGF-β signaling via the Smad 2/3 pathway (Feng et al., 2016). These authors further showed that in response to TGF-β3 treatment during the biological insult, myometrial cells migrated into nodules containing collagen and fibronectin (Feng et al., 2016). Using transmission electron microscopy, they also found myofibroblast-like cells and fibril-like structures in the extracellular spaces of the nodules (Feng et al., 2016).

In addition to TGF-β and activin-A, TNF-α can be considered as an important regulator of myofibroblast differentiation. Indeed, TNF-α increases the expression of activin-A in both myometrial and leiomyoma cells (Islam et al., 2013b) and thereby promotes the fibrotic role of activin-A in the process of myofibroblast differentiation following fibrosis.

Estrogen and progesterone have been reported to increase the proliferation of both leiomyoma and smooth muscle fibroblasts compared to untreated groups (Luo et al., 2014). Estrogen-mediated fibroblast proliferation was mediated, at least in part, by increased secretion of TGF-β and IGF-1 as well as activation of MEK/ERK1/2 and AKT pathways (Luo et al., 2014). The effect of estrogen on fibroblast activation was documented by the observation that expression of FAP as well as collagen I, fibronectin and laminin proteins was increased in estrogen-treated leiomyoma and smooth muscle fibroblasts compared to untreated controls (Luo et al., 2014). FAP knockdown was reported to attenuate the estrogen-mediated proliferation of fibroblasts, as well as phosphorylation levels of MEK, ERK, AKT and protein levels of c-fos (Luo et al., 2014). The decreased protein expression of ECM components (collagen I, fibronectin and laminin) was also observed in leiomyoma fibroblasts compared to untreated controls in response to suppression of FAP expression (Luo et al., 2014). These results suggest that the effect of estrogen on fibroblast cell activation is mediated partially through the FAP pathway.

Inflammation is a key component of the wound healing process. However, local chronic inflammation makes a suitable microenvironment for development of fibrosis. Chronic inflammation is characterized by infiltration of mononuclear immune cells (macrophages, monocytes, lymphocytes and plasma cells). Of note, inflammatory cells, in particular macrophages, are widely accepted as important regulators of cytokines and growth factors during the wound healing process (Leibovich and Ross, 1975). Recently, we found higher numbers of macrophages present inside and in the vicinity of leiomyoma compared to the more distant surrounding myometrium (Protic et al., 2016).

Chronic inflammatory reactions are induced by a variety of stimuli, including chemical insults, radiation, persistent infections, tissue injury, autoimmune reactions and allergic responses (Wynn, 2008). In the uterus, reproductive events, including ovulation, menstruation and implantation, may trigger inflammatory reactions. In addition, many factors have been hypothesized to increase inflammation in uterus, such as infection, injury, talc use, an intrauterine device, cesarean section, male reproductive proteins and stress (work, home or perceived racism) (Wegienka, 2012). Repeated stimulation by reproductive events, mechanical forces, injury, hypoxia and oxidative stress may also create a chronic inflammatory state in the uterus (Wegienka, 2012; Fletcher et al., 2013; Leppert et al., 2013; Santulli et al., 2013). Regardless of the cause of chronic uterine inflammation, it is under this condition that myofibroblasts cells produce ECM in an unregulated manner and fail to undergo normal apoptosis leading to fibrosis.

Although most women experience causes of uterine inflammation, e.g. reproductive events, they all do not have leiomyoma. This observation suggests that initiation of uterine leiomyoma does not solely depend on inflammation. There are other factors that may influence the risk of developing leiomyoma under the chronic inflammatory condition. Accumulated evidence suggests that genetic and epigenetic factors may influence the risk for developing leiomyoma (Gallagher and Morton, 2016; Yang et al., 2016). In addition, black race, heredity, nulliparity, obesity, polycystic ovary syndrome, diabetes and hypertension are associated with increased risk of this tumor (Okolo, 2008).

Extracellular matrix as a therapeutic target of current and future medical treatments

Several classes of compounds including GnRH agonizts, GnRH antagonists, selective progesterone receptor modulators, antiprogestin and natural compounds have been studied as medical treatments that target ECM in uterine leiomyoma (Table II).

Table II

Current and potential treatments for leiomyoma that target the extracellular matrix.

Treatment modalities	Fibrosis-related molecular targets	Effects on leiomyoma and related symptoms	Safety and side effects
Current treatments
Leuprolide  acetate	↓TGF-β1, ↓TGF-β3, ↓TGF-βR type I, ↓TGF-βR type II, ↓Smad4, ↓pSmad3, ↑Smad7, ↓ collagen type I, ↓↑fibronectin, ↓versican, ↓fibromodulin, ↓TIMP-1, ↑MMP-1, ↑MMP-2, ↑MMP-3, and ↑MMP-9	(I) Reduces uterine and leiomyoma volume (Stovall et al., 1995)	(I) Prolonged therapy (>6 months) is not recommended
		(II) Alleviates bleeding and increase hemoglobin levels (Stovall et al., 1991)	(II) Side effects-hot flashes, vaginitis and bone loss (Stovall et al., 1995)
Cetrorelix  acetate	↓COL1A1, ↓fibronectin, and ↓versican variant V0	(I) Decreases uterine and leiomyoma volume (Gonzalez-Barcena et al., 1997; Felberbaum et al., 2001; Engel et al., 2007)	(I) Side effects-amenorrhea and hot flashes (Gonzalez-Barcena et al., 1997)
		(II) Abolishes symptoms of menorrhagia and reduce uterine pain (Engel et al., 2007)
Asoprisnil	↓TGF-β3, ↓TGF-βR type II, ↑EMMPRIN, ↑MMP-1, ↑MT1-MMP, ↓TIMP-1, ↓TIMP-2, ↓ collagen type I and ↓collagen type III	(I) Suppress leiomyoma and uterine volume (Chwalisz et al., 2007)	(I) Abnormal endometrial changes (Williams et al., 2007)
		(II) Reduces uterine bleeding as well as bloating and pelvic pressure (Chwalisz et al., 2007)	(II) Side effects-hot flashes or night sweats (Chwalisz et al., 2007)
Ulipristal  acetate	↑MMP-1, ↑MMP-2, ↑MMP-3, ↑MMP-8, ↑MMP-9, ↑EMMPRIN, ↓TIMP-1, ↓TIMP-2, ↓ collagen type I, ↓collagen type III and ↓fibronectin	(I) Reduces leiomyoma and uterine volume (Donnez et al., 2012a, 2012b., 2015)	(I) Quite well tolerated (Donnez et al., 2015).
		(II) Controls bleeding and pain (Donnez et al., 2015)	(II) Side effects-hot flashes and headaches (Donnez et al., 2015)
		(III) Improves quality of life (Donnez et al., 2012a, 2012b., 2015)
Mifepristone	↓COL1A1, ↓fibronectin, ↓versican and ↓dermatopontin	(I) Reduces uterine and leiomyoma volume (Esteve et al., 2012)	(I) No endometrial hyperplasia or cellular atypia(Yerushalmi et al., 2014)
		(II) Alleviates hypermenorrhea, menstrual blood loss, pelvic painandpressure, anemia and dysmenorrhea (Shen et al., 2013; Yerushalmi et al., 2014)	(II) Side effects-hot flashes, nausea, weakness, abdominal pain and vaginal discharge (Yerushalmi et al., 2014)
		(III) Reduces leiomyoma cell viability and proliferation (Chung et al., 2014)
Raloxifene	↓Collagen and ↓MMP-2	(I) prevents progression of uterine leiomyoma (Jirecek et al., 2004)	(I) quite well tolerated (Jirecek et al., 2004)
			(II) side effects-hot flashes and leg cramps (Martino et al., 2005), and risk of venous thromboembolism (Ettinger et al., 1999)
Investigational compounds
CP8947	↓COL1A1 and ↓COL7A1	(I) Inhibits the leiomyoma cell proliferation (Catherino et al., 2010)	Limited
		(II) No effect on myometrial cell proliferation (Catherino et al., 2010)
2-methoxyestradiol	↓Collagen type I, ↓Collagen type III, ↓PAI-1, ↓CTGF, ↓α-SMA, ↓pSmad2/3 and ↓PI3K/Akt/mTOR	(I) Inhibits cell proliferation in rat and human leiomyoma cells (Salama et al., 2006)	(I) Well tolerated (Harrison et al., 2011)
		(II) induces apoptosis in rat and human leiomyoma cells(Salama et al., 2006)	(II) Side effects-hot flashes, muscle cramps, headache, fatigue or weakness, nausea, vomiting, diarrhea, gastrointestinal hemorrhage and hyponatremia (Rajkumar et al., 2007)
Liarozole	↓COL1A1, ↓COL4A2, ↓versican, ↓fibromodulin, ↓fibronectin and ↓TGF-β3	(I) Inhibits proliferation of both myometrial and leiomyoma cells (Gilden et al., 2012)	(I) Well tolerated (Denis et al., 1998)
			(II) Side effects-skin disorders and dryness of mouth/eyes/lips (Goss et al., 2000)
All-trans retinoic  acid	↓Collagen 1, ↓collagen 4, ↓fibronectin, ↓versican and ↓TGF-β3	(I) Downregulates immortalized leiomyoma cell proliferation (Malik et al., 2008)	(I) Quite well tolerated (Kurzrock et al., 1993; Böcher et al., 2008)
			(II) Side effects-transient headache, dry skin and mucosa, nausea and vomiting, myalgias or muscle pain, dyspnea (shortness of breath), and sensorineural hearing loss (Kurzrock et al., 1993; Böcher et al., 2008)
Vitamin D	↓fibronectin, ↓collagen type 1, ↓PAI-1, ↓pSmad2, ↓Wnt4, ↓β-catenin, ↓mTOR, ↓fibromodulin, ↓biglycan and ↓versican	(I) Shrinks uterine leiomyoma tumors in Eker rat model (Halder et al., 2012)	Limited
Celecoxib	↓collagen A, ↓fibronectin, ↓PDGF, and ↓TGF-β	(I) Inhibits leiomyoma cell proliferation (Ke et al., 2013)	(I) Increases the risk of myocardial infarction, stroke or heart failure (Solomon et al., 2005)
		(II) No effect on myometrial cell proliferation (Ke et al., 2013)
Tranilast	↓COL1A1, ↓fibronectin ↓versican and ↓activin-A	(I) Inhibits proliferation of myometrial and leiomyoma cells (Shime et al., 2002; Islam et al., 2012)	(I) Well tolerated with low toxicity (Konneh, 1998)
Pirfenidone	↓collagen type I, ↓collagen type III	(I) Inhibits proliferation of myometrial and leiomyoma cells (Lee et al., 1998)	(I) Rather well tolerated by patients with idiopathic pulmonary fibrosis (Chaudhuri et al., 2014)
			(II) Gastrointestinal adverse effects (Chaudhuri et al., 2014)
Halofuginone	↓collagen type I (a1), ↓collagen type III (a1) and ↓TGF-β1	(I) Reduces uterine leiomyoma volume in a mouse xenograft model (Koohestani et al., 2016)	(I) Appears to be safe and well tolerated (De Jonge et al., 2006)
		(II) Inhibits both myometrial and leiomyoma cell proliferation (Grudzien et al., 2010)	(II) No clinically adverse events (De Jonge et al., 2006)
		(III) Induces apoptosis (Grudzien et al., 2010)
Curcumin	↓fibronectin	(I) Inhibits leiomyoma cell proliferation (Malik et al., 2009)	(I) Appears to be safe and well tolerated by patients (Gupta et al., 2013)
			(II) Side effects-nausea, diarrhea, headache, rash and yellow stool (Gupta et al., 2013)
Resveratrol	↓fibronectin, ↓collagen types I, ↓collagen III, ↓fibromodulin. ↓biglycan, and ↓MMP-9, ↑TIMP2	(I) Inhibits proliferation of human uterine leiomyoma cells (Catherino et al., 2011)	(I) Appears safe and well tolerated by patients (Turner et al., 2015)
			(II) Adverse events-nausea, diarrhea and weight loss (Turner et al., 2015)
Collagenase C.  histolyticum	↓ collagen	(I) Reduces fibrosis (collagen) and tissue stiffness in uterine leiomyoma (Jayes et al., 2016)	Limited

Current treatments

Leuprolide acetate

Leuprolide acetate is a synthetic analog of GnRH. GnRH is produced in the hypothalamus of the brain and after its release travels to the pituitary gland where it stimulates the production of LH and follicle stimulating hormone (FSH). Through peripheral circulation, LH and FSH travel to the ovaries where they stimulate the production of estrogen. Estrogen maintains its own levels within an appropriate range by acting as a negative feedback regulator of GnRH, LH and FSH production. Leuprolide acetate acts by producing an initial stimulation of FSH and LH as well as estrogen but after a few weeks, levels of LH and FSH drop because the pituitary gland stops responding to GnRH and leuprolide. This induces a state of hypoestrogenism, which has been used for the treatment of leiomyoma (Stovall et al., 1991). Leuprolide acetate is able to alleviate bleeding and increase hemoglobin levels (Stovall et al., 1991) as well as reduce uterine and leiomyoma volume (Stovall et al., 1995). However, this treatment is associated with some side effect related to hypoestrogenism, such as hot flashes, vaginitis and bone loss, which constitute the major limitation of long-term use (Stovall et al., 1995). The therapeutic effects of leuprolide acetate on leiomyoma are mediated by regulation of TGF-β receptor signaling and substantial tissue remodeling (Dou et al., 1997; Chegini et al., 2003; Ding et al., 2004b). Leuprolide acetate was reported to inhibit TGF-β1, TGF-β3, TGF-βR type I and type II as well as Smad4 and pSmad3 levels while increasing Smad7 expression in both myometrium and leiomyoma relative to untreated controls (Dou et al., 1996; Chegini et al., 2003). Leuprolide acetate also inhibited fibronectin mRNA expression in myometrial cells with a moderate increase in its expression in leiomyoma cells while also inhibiting type I collagen expression in both myometrial and leiomyoma cells (Ding et al., 2004b). A recent study reported that 3D leiomyoma cultures exposed to estrogen and progesterone demonstrated an increased expression of collagen-1, fibronectin and versican and this effect was inhibited by leuprolide acetate (Malik et al., 2016). TGF-β1 increased the expression of fibromodulin in myometrial cells, whereas leuprolide acetate inhibited this effect in both myometrial and leiomyoma cells (Levens et al., 2005). Dou et al. reported that leuprolide acetate treatment also induced a significant decrease in TIMP-1, and an increase in MMP-1, MMP-2, MMP-3 and MMP-9 mRNA expression in both leiomyoma and myometrium compared with untreated groups (Dou et al., 1997).

Cetrorelix acetate

Cetrorelix acetate, a GnRH antagonist, has been reported to decrease mean uterine and leiomyoma volume in pre-menopausal women (Gonzalez-Barcena et al., 1997; Felberbaum et al., 2001; Engel et al., 2007). This compound can abolish symptoms of menorrhagia and reduce uterine pain (Engel et al., 2007). However, cetrorelix acetate is associated with amenorrhea and hot flashes (Gonzalez-Barcena et al., 1997) and its therapeutic use is also limited by its prohibitive cost and requirement for daily injections (Kashani et al., 2016). Britten et al. investigated the effect of cetrorelix acetate on ECM production in human uterine leiomyoma and patient-matched myometrial cells (Britten et al., 2012). They found that cetrorelix decreased mRNA and protein expression of COL1A1, fibronectin and versican variant V0 in a time dependent manner in leiomyoma cells compared to untreated cells (Britten et al., 2012). In 3D leiomyoma cultures, cetrorelix also decreased estrogen- and progesterone-induced expression of collagen-1, fibronectin and versican (Malik et al., 2016).

Asoprisnil

Asoprisnil is a selective progesterone receptor modulator that shows a high degree of receptor and tissue selectivity. It has high-binding affinity for progesterone receptors, no binding affinity for estrogen or mineralocorticoid receptors, moderate affinity for glucocorticoid receptors and low affinity for androgen receptors. Asoprisnil has been reported to suppress leiomyoma and uterine volume as well as uterine bleeding and was associated with significant reduction in bloating and pelvic pressure (Chwalisz et al., 2007). The safety profile indicates that asoprisnil treatment is associated with endometrial changes including abnormal vascular growth (Williams et al., 2007). The adverse events, such as hot flashes or night sweats, were also detected but they were mild or moderate in severity, because asoprisnil does not induce a hypoestrogenic state (Chwalisz et al., 2007; Kashani et al., 2016). Asoprisnil exerts therapeutic effects on leiomyoma by regulating growth factor expression as well as ECM turnover and tissue remodeling (Wang et al., 2006; Morikawa et al., 2008). Treatment of leiomyoma cells with asoprisnil caused a decrease in the levels of TGF-β3 mRNA and protein and phosphorylated TGF-βR type II receptor compared to untreated controls (Wang et al., 2006). Morikawa and co-investigators reported that asoprisnil treatment also significantly increased levels of EMMPRIN, MMP-1 and MT1-MMP (membrane type 1-MMP) while decreasing levels of TIMP-1, TIMP-2, collagen type I and type III in cultured leiomyoma cells compared to untreated control cells (Morikawa et al., 2008). This group also reported that asoprisnil had no effect on protein contents of ECM and ECM-remodeling enzymes in myometrial cells (Morikawa et al., 2008).

Ulipristal acetate

Ulipristal acetate (also known as CDB-2914) is a selective progesterone receptor modulator that binds to progesterone receptors A and B with high affinity. Use of this drug has been approved in Europe and Canada for preoperative leiomyoma treatment (Melis et al., 2012). Ulipristal acetate is able to reduce leiomyoma and uterine volume and improves leiomyoma-related symptoms such as bleeding and pain as well as quality of life (Donnez et al., 2012a, 2012b, 2015). While this treatment is associated with some adverse events, such as hot flashes and headaches, and breast tenderness, these occurred in ≤10% of patients as circulating estradiol levels are maintained in the mid-follicular range throughout the treatment duration (Kashani et al., 2016). Moreover, it may be associated with physiologic endometrial changes (PAECs) that consist of benign cystic glandular dilation but these have not been associated to date with an increased risk of endometrial hyperplasia or malignancy (Donnez et al., 2015). Ulipristal acetate exerts therapeutic effects on leiomyoma growth by regulating the fibrotic process and its use results in lower ECM volume and higher MMP-2 expression in women with symptomatic leiomyoma compared to untreated leiomyoma (Courtoy et al., 2015). In cultured leiomyoma cells, ulipristal acetate significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents as well as MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, while decreasing mRNA and protein levels of TIMP-1 and TIMP-2 as well as collagen types I and III content, without comparable effects on cultured normal myometrial cells (Xu et al., 2008). Recently, we also demonstrated that ulipristal acetate can block activin-A induction of fibronectin mRNA expression in myometrial and leiomyoma cultured cells (Ciarmela et al., 2014).

Mifepristone

Mifepristone (also known as RU-486, mifegyne, mifeprex) is a synthetic steroid with antiprogesterone and antiglucocorticoid activity. It binds progesterone receptors and competitively antagonizes progesterone binding and signaling. Several clinical studies have demonstrated that mifepristone can reduce uterine and leiomyoma volume as well as alleviate leiomyoma-related symptoms including hypermenorrhea, menstrual blood loss, pelvic pain or pressure, anemia and dysmenorrhea (Esteve et al., 2012, 2013; Shen et al., 2013; Yerushalmi et al., 2014). A recent study reported that no endometrial hyperplasia or cellular atypia was observed after vaginal mifepristone treatment (Yerushalmi et al., 2014). However, some side effects, including hot flashes (10.3%), nausea (6.9%), weakness (6.9%), abdominal pain (24.1%) and vaginal discharge (20.7%), were observed at some point during the course of the 3 month study (Yerushalmi et al., 2014). Moreover, the increased risk of developing endometrial hyperplasia (28%) (10/36) during treatment with mifepristone raised safety concerns about its use in the management of leiomyoma (Steinauer et al., 2004; Guo and Segars, 2012). Antifibrotic effects of mifepristone have recently been demonstrated by Patel et al. (2016). This group reported that the progesterone agonist R5020 directly stimulated production of several ECM components including COL1A1, fibronectin, versican and dermatopontin in human leiomyoma cells compared to untreated cells and this effect was inhibited by mifepristone (Patel et al., 2016). Additionally, mifepristone can significantly reduce leiomyoma cell viability and proliferation (Chung et al., 2014) at least partly by downregulation of LAT2 (L-type amino acid transporter 2) mRNA expression (Luo et al., 2009). Furthermore, Yin et al. reported that mifepristone robustly up-regulated mRNA and protein levels of the known tumor suppressor KLF11 in leiomyoma smooth muscle cells (Yin et al., 2010).

Raloxifene

Raloxifene is a selective estrogen receptor modulator that interacts with estrogen receptors to elicit tissue-specific responses. It inhibits the growth of uterine leiomyoma in pre-menopausal women (Jirecek et al., 2004). Raloxifene is quite well tolerated (Jirecek et al., 2004) but has been associated with the risk of venous thromboembolism (Ettinger et al., 1999) as well as hot flashes and leg cramps (Martino et al., 2005). Raloxifene appears to inhibit collagen biosynthesis in leiomyoma cells while only slightly affecting collagen biosynthesis in control myometrial cells (Zbucka et al., 2008). Raloxifene was also found to inhibit MMP-2 in leiomyoma as well as in control myometrial cells (Zbucka et al., 2008).

Investigational compounds

CP8947

CP8947 is a novel non-steroidal selective progesterone receptor modulator derived from Penicillium oblatum. It has high selectivity for progesterone receptors and lacks affinity for estrogen receptor-α, androgen receptor and glucocorticoid receptor. Catherino et al. investigated the effect of CP8947 on cell proliferation and ECM components in leiomyoma cells (Catherino et al., 2010). They found that CP8947 was effective in inhibiting leiomyoma cell proliferation without disrupting myometrial cell proliferation and that it also decreased mRNA expression of COL1A1 and COL7A1 (Catherino et al., 2010).

2-Methoxyestradiol

2-Methoxyestradiol is a naturally occurring estradiol metabolite with low affinity for estrogen receptors. 2-Methoxyestradiol is currently being evaluated in ongoing advanced phases of clinical trials in patients with multiple myeloma, glioblastoma, ovarian cancer, metastatic renal cell carcinoma and prostate cancer. It is well tolerated by patients but associated with some adverse events such as hot flashes, muscle cramps, headache, fatigue or weakness, nausea, vomiting, diarrhea, gastrointestinal hemorrhage and hyponatremia (Rajkumar et al., 2007; Harrison et al., 2011). 2-Methoxyestradiol has been reported to induce apoptosis as well as inhibit cell proliferation and collagen production in rat and human leiomyoma cells (Salama et al., 2006). Salama et al. investigated the antifibrotic effect of 2­-methoxyestradiol on TGF-β3 mediated fibrosis-related factors in leiomyoma cells (Salama et al., 2012). They found that 2-methoxyestradiol abrogated TGF-β3-induced expression of collagen types I and III, PAI-1, CTGF and α-SMA in immortalized human uterine leiomyoma smooth muscle cells compared to untreated controls (Salama et al., 2012). 2-Methoxyestradiol also inhibited TGF-β3-induced activation of the PI3K/AKT/mTOR pathway as well as ameliorated TGF-β3-induced phosphorylation and nuclear translocation of Smad2/3 in this cell type (Salama et al., 2012). It has been shown that relatively high concentrations of 2-methoxyestradiol can also induce spindle aberrations in oocytes (Eichenlaub-Ritter et al., 2007).

Liarozole

Liarozole inhibits the cytochrome P450 (CYP)-dependent catabolism of retinoic acid and thereby increases intracellular retinoic acid levels. Liarozole is well tolerated in clinical studies and has been studied as treatment for prostate cancer (Denis et al., 1998) and breast cancer (Goss et al., 2000). The adverse events were predominantly dermatological including skin disorders (88%) and dryness of mouth/eyes/lips (69%) (Goss et al., 2000). The antifibrotic effects of liarozole have been studied in leiomyoma cells (Gilden et al., 2012; Levy et al., 2014). Gilden and co-investigators reported that liarozole can inhibit proliferation of both myometrial and leiomyoma cells at suprapharmacologic concentrations and also decrease mRNA and protein expression of COL1A1, COL4A2, versican, fibromodulin and fibronectin in a dose-dependent manner in leiomyoma cells compared with myometrial cells (Gilden et al., 2012). Interestingly, they found no statistically significant alteration in ECM regulation in liarozole treated myometrial cells (Gilden et al., 2012). A recent study reported that liarozole can inhibit TGF-β3 and TGF-β3 induction of ECM components, including versican, COL1A1 and fibronectin in human three-dimensional leiomyoma cultures (Levy et al., 2014).

All-trans retinoic acid

All-trans retinoic acid (also known as tretinoin) is a derivative of vitamin A that functions as a ligand for the retinoic acid receptor (RAR). It is quite well tolerated by patients but has been associated with common side effects including transient headache, dry skin and mucosa, nausea and vomiting, myalgias or muscle pain, dyspnea (shortness of breath) and sensorineural hearing loss (Kurzrock et al., 1993; Böcher et al., 2008). Human uterine smooth muscle cells express retinoic acid receptors RAR α, β and γ and retinoid X receptors RXR α and β as well as all-trans retinoic acid (Boettger-Tong et al., 1997). Treatment of immortalized leiomyoma cells with all-trans retinoic acid was reported to downregulate cell proliferation as well as protein production of ECM components including collagen 1, collagen 4, fibronectin and versican as well as mRNA expression of TGF-β3 compared to untreated controls (Malik et al., 2008).

Vitamin D

Vitamin D is a fat-soluble vitamin found in many foods including fish, eggs, fortified milk and cod liver oil. Its major physiologically relevant forms are D2 (ergocalciferol) and D3 (cholecalciferol). Several recent in vitro and in vivo studies implicate vitamin D insufficiency as an important contributor to the development of uterine leiomyoma (Halder et al., 2012; Baird et al., 2013; Paffoni et al., 2013). Women with sufficient vitamin D have been reported to have an estimated 32% lower odds of leiomyoma compared to women with vitamin D insufficiency (Baird et al., 2013). Similarly, Paffoniand et al. reported that women with a vitamin D deficiency experienced 2.4 times more leiomyoma compared to women with an adequate level of vitamin D (Paffoni et al., 2013). Using the Eker rat model, Halder and co-investigators found that vitamin D3 [1,25(OH)2D3] treatment was able to shrink uterine leiomyoma tumors (Halder et al., 2012). The therapeutic effect of vitamin D3 on leiomyoma was mediated, at least in part, by regulation of TGF-β responsive genes (Halder et al., 2011, 2013), as well as Wnt/β-catenin and mTOR signaling pathways (Al-Hendy et al., 2016). Vitamin D3 reduced TGF-β3-induced expression of fibronectin, collagen type 1 and PAI-1 protein in human uterine leiomyoma cells (Halder et al., 2011). Vitamin D3 also reduced TGF-β3-induced Smad2 phosphorylation as well as Smad2 and Smad3 nuclear translocation in human uterine leiomyoma cells (Halder et al., 2011). Al-Hendy et al. reported that vitamin D3 can reduce the levels of Wnt4 and β-catenin as well as mTOR in both immortalized and primary human uterine leiomyoma cells (Al-Hendy et al., 2016). Furthermore, vitamin D3 was reported to reduce mRNA and protein levels of fibromodulin, biglycan and versican in human uterine leiomyoma cells (Halder et al., 2013). The ability of vitamin D3 to downregulate ECM (fibronectin and collagen type 1) expression as well as Wnt4/β-catenin and cell proliferation was further confirmed by the observation that silencing expression of the vitamin D receptor (VDR) gene in normal myometrial cells increased ECM production as well as Wnt4/β-catenin and cell proliferation (Al-Hendy et al., 2016). Initial results regarding the opportunity for Vitamin D3 supplementation in women with leiomyoma are encouraging, but they must be confirmed by further studies (Ciavattini et al., 2016).

Celecoxib

Celecoxib is an inhibitor of cyclooxygenase 2 (COX-2) that is commonly used to manage pain or inflammation. The function of COX-2 is to convert arachidonic acid into prostaglandin H2, a common substrate for specific prostaglandin synthases. Uterine leiomyoma cells have higher levels of COX-2 mRNA and protein than myometrial cells (Ke et al., 2013). A recent study reported that celecoxib can significantly inhibit uterine leiomyoma cell proliferation without affecting proliferation of healthy myometrial smooth muscle cells (Ke et al., 2013). Celcoxib can also reduce expression of collagen A, fibronectin, PDGF and TGF-β at the mRNA level in uterine leiomyoma cells compared to untreated controls (Park et al., 2014). Although initial results are encouraging, the potential health risks may hinder the use of celecoxib for the treatment uterine leiomyoma. Clinical evidence indicates that celecoxib use is associated with increased risk of cardiovascular events, myocardial infarction, stroke and heart failure (Solomon et al., 2005).

Tranilast

Tranilast is an orally administered synthetic drug of low toxicity used for the treatment of allergic disorders in Japan and South Korea. It is well tolerated by patients (Konneh, 1998) supporting the possibility for its use to treat uterine leiomyoma. The antifibrotic effects of tranilast have been reported in myometrial and leiomyoma cells (Islam et al., 2014b). Recently, we demonstrated that tranilast can decrease expression of COL1A1 and fibronectin at mRNA and protein levels, as well as versican and activin-A at mRNA levels in myometrial and leiomyoma cells (Islam et al., 2014b). Additionally, tranilast can also inhibit the proliferation of uterine myometrial and leiomyoma cells (Shime et al., 2002; Islam et al., 2012).

Pirfenidone

Pirfenidone is a synthetic pyridone compound used for the treatment of idiopathic pulmonary fibrosis. It is rather well tolerated by patients, with adverse effects that are predominantly gastrointestinal (Chaudhuri et al., 2014). The antifibrotic potential of pirfenidone has been studied in leiomyoma cells by Lee et al. (1998). They reported that pirfenidone was effective in regulating proliferation of myometrial and leiomyoma cells in vitro as well as in reducing the mRNA level of collagen types I and III in a dose-dependent manner (Lee et al., 1998).

Halofuginone

Halofuginone, an analog of febrifugine, is a small alkaloid isolated from the plant Dichroa febrifuga. It has been used as a coccidiostat (an antiprotozoal agent) in chickens since the 1960s. Phase I/II clinical trials of halofuginone have been completed in patients with progressive advanced solid tumors and HIV-related Kaposi's sarcoma. Halofuginone at dose 0.5 mg/d appears to be safe and well tolerated, with no clinically adverse events (De Jonge et al., 2006). The antifibrotic effect of halofuginone on uterine leiomyoma has been reported (Grudzien et al., 2010). Grudzien et al. reported that halofuginone can significantly reduce collagen type I (a1), collagen type III (a1) and TGF-β1 mRNA levels in leiomyoma and myometrial cells compared to corresponding untreated cells (Grudzien et al., 2010). Halofuginone can also inhibit both myometrial and leiomyoma cell proliferation by inducing apoptosis and inhibiting DNA synthesis in a dose-dependent manner (Grudzien et al., 2010). A recent study by Koohestani et al. reported that halofuginone was effective in reducing uterine leiomyoma volume in a mouse xenograft model (Koohestani et al., 2016). This group found a 35–40% reduction in leiomyoma/tumor volume in mice carrying human uterine leiomyoma xenografts after treatment with halofuginone compared to control groups. The halofuginone-induced reduction of tumor volume was accompanied by decreased cell proliferation and increased apoptosis (Koohestani et al., 2016). However, these authors found no significant difference in the content of collagen between helofuginone-treated mice carrying human uterine leiomyoma xenografts and control groups (Koohestani et al., 2016).

Curcumin

Curcumin is a polyphenol derived from rhizome of turmeric (Curcuma longa). Turmeric is commonly used in Asian foods. It has shown beneficial effects in a plethora of human diseases. Extensive clinical trials indicate that curcumin is safe and well tolerated by patients. However, this treatment is associated with some undesired adverse effects such as nausea, diarrhea, headache, rash and yellow stool (Gupta et al., 2013). Antifibrotic effects of curcumin on human uterine leiomyoma cells have been demonstrated by Malik et al. (2009). This group reported that curcumin can inhibit mRNA expression of fibronectin in leiomyoma cells compared to untreated controls (Malik et al., 2009). They also found that curcumin can inhibit proliferation of leiomyoma cells without affecting patient-matched myometrial cells (Malik et al., 2009). These effects were mediated by inducing caspase-3 and caspase-9 protein expression as well as inhibiting ERK 1/2 and NF-κB protein expression (Malik et al., 2009).

Resveratrol

Resveratrol is a polyphenolic compound found in peanuts, grapes and some berries and is produced by plants in response to environmental stress, pathogen infection and ultraviolet radiation. Resveratrol is safe and well tolerated by patients, with common adverse events including nausea, diarrhea and weight loss (Turner et al., 2015). The ability of resveratrol to interfere with ECM formation and deposition in multiple diseases has recently been discussed by Agarwal and Agarwal (2017). Resveratrol appears to reduce expression of fibronectin, collagen types I and III as well as fibromodulin and biglycan at mRNA and/or protein levels in ELT-3 cells and/or human uterine leiomyoma smooth muscle cells compared to corresponding untreated cells (Catherino et al., 2011; Wu et al., 2016). Resveratrol also reduced MMP-9 protein expression while increasing TIMP2 protein expression in ELT-3 cells and healthy uterine smooth muscle cells compared to corresponding untreated cells (Wu et al., 2016). Furthermore, resveratrol was reported to inhibit proliferation and induce apoptosis and cell cycle arrest in human uterine leiomyoma cells compared to untreated controls (Catherino et al., 2011).

Collagenase C. histolyticum

Collagenase C.histolyticum (CCH) is a bacterial enzyme that breaks down collagen. CCH has been approved by the US FDA for the treatment of Dupuytren's contracture (a thickening of the fibrous tissue layer underneath the skin of the palm and fingers) and Peyronie's disease (a connective tissue disorder of the penis). A recent study by Jayes et al. investigated whether CCH was effective in the digestion of interstitial collagen in uterine leiomyoma (Jayes et al., 2016). They found 37–77% fibrosis in untreated leiomyoma, indicating the collagen-rich nature of these tumors (Jayes et al., 2016). A reduced amount of fibrosis ranging from 5.3 to 2.4% was recorded after treatment with CCH. Furthermore, complete digestion of collagen fibrils was confirmed by transmission electron microscopy. Tissue stiffness was also reduced with CCH treatment (Jayes et al., 2016). The above results suggest that CCH may reduce leiomyoma size and bulk symptoms possibly through collagen digestion and by modulating mechanotransduction process. Clinical trials are necessary to evaluate the safety and efficacy of CCH.

Conclusions and future perspectives

Uterine leiomyoma expresses a wide variety of ECM components, including collagens, fibronectin, laminins, proteoglycans and integrins as well as MMPs and TIMPs. ECM proteins can induce mechanotransduction, thus activating pleiotropic intracellular signaling cascades such as the integrin-Rho/p38 MAPK/ERK pathways.

ECM accumulation is regulated by growth factors, cytokines and steroid hormones. Among growth factors and cytokines, TGF-β, activin-A, PDGF and TNF-α are able to increase the synthesis of ECM components through activation of multiple signaling pathways (e.g. Smad 2/3 and MEK/ERK). In addition, estrogen and progesterone increase ECM production by regulating the expression and activity of growth factors (TGF-β and IGF), again leading to stimulation of several signaling pathways (MEK/ERK, AKT and PLCγ).

ECM may act as a reservoir of growth factors and protect them from degradation in the ECM microenvironment. ECM proteins and their receptors (integrins) can interact with growth factors independently or synergistically. MMPs can degrade ECM proteins and GAGs of proteoglycans, thereby inducing local release of soluble growth factors from their insoluble state.

Uterine leiomyoma is thought to be an inflammatory/fibrotic disorder, and possibly myofibroblasts are thought to play key role in the process of fibrosis. Reproductive events (mainly menstruation), infection, mechanical stress, injury, oxidative stress or hypoxia may act as the initiator of inflammation in the uterus. In response to inflammation, myofibroblast cells produce ECM to promote necessary repair and tissue homeostasis, but deregulation of normal myofibroblast function may lead to fibrosis. The differentiation of myofibroblasts is regulated by TGF-β and activin-A and estrogen.

The above findings support the fact that ECM is a major molecular switch that can be considered to be a crucial therapeutic target to control abnormal growth of leiomyoma. Currently, several types of drugs are available for leiomyoma treatment but none of them has been introduced specifically as antifibrotic agents (Table II). Therefore, the introduction of effective antifibrotic drugs which target ECM components directly or interfere with their expression and deposition has the potential to be an effective solution for the management of uterine leiomyoma. The antifibrotic compounds can be introduced based on their ability to regulate ECM components and ECM receptors as well as growth factors, cytokines, steroid hormones and their corresponding receptors and intracellular signaling pathways, as well as miRNAs, involved in ECM production in leiomyoma. Since ECM is involved in both pathological and physiological processes, therefore, it will be an important challenge to identify compounds that specifically target unwanted fibrosis and optimize their use in interrupting the fibrotic process without disturbing the normal physiological environment.

Authors' roles

M.S.I. and P.C. conceived and designed the study. M.S.I. conducted the review of the literature and wrote the first draft of the manuscript. A.C., F.P., M.C. and P.C. supervised the work and corrected the manuscript. All authors contributed to analysis and interpretation of the data, critically revised and approved the manuscript.

Funding

The ‘Fondazione Cassa di Risparmio di Fabriano e Cupramontana’ (to M.C. and P.C.).

Conflict of interest

The authors have nothing to declare.