Fibrosis is a critical stage of many chronic diseases that can lead to organ dysfunction, illness and death. The burden associated with fibrosis is staggering with nearly half of all deaths in the United States attributed to fibrotic diseases including liver, lung, kidney, and heart disorders (Wynn, 2008; Rosenbloom et al., 2010). Unfortunately, the impact of fibrosis on mortality and morbidity rates has not been countered by effective treatments. Decades of research have identified many potential targets to combat fibrotic tissue damage, yet there currently are no effective therapies or FDA approved antifibrotic agents. This review highlights the potential of four agents that have emerged from basic science testing that show promise as translatable options for the treatment of fibrotic diseases in humans.

The development of antifibrotic therapies relies on the comprehensive understanding of profibrogenic mechanisms in multiple organ systems as well as disease-specific locations. The global and foremost mechanism involved in fibrosis is the activation of myofibroblasts resulting in the excessive and often continual production of extracellular matrix (ECM) components, the foundation of scar formation (McAnulty, 2007; Herzog and Bucala, 2010). Myofibroblast activation is initiated in chronic inflammatory diseases where healthy wound healing in response to injury is not controlled, resolved, or is repetitively stimulated by an inciting factor. As a result, differentiated fibroblasts are stimulated to be in a proliferative, activated state producing ECM components. In the last 25 years, heightened research has identified potential targetable pathways and related individual factors that are involved in the differentiation of quiescent fibroblasts and the persistent activation of myofibroblasts during fibrogenesis. These include an array of cytokines (e.g., interleukins, IL-1, IL-6, IL-25, IL-33; and tumor necrosis factor-α, TNF-α) that are produced as a consequence of inflammation and epithelial and endothelial tissue damage (Rosenbloom et al., 2013). The burst of inflammatory mediators can directly stimulate fibroblast activation as well as influence downstream adaptive and innate immune mechanisms (Wynn and Ramalingam, 2012). For example, a stimulated adaptive immune system can affect a complex array of T and B cell activities resulting in the resolution as well as promotion of fibrosis via interferon gamma (IFN-γ) signaling (Gurujeyalakshmi and Giri, 1995). Innate immune activation is similarly complex involving responses of multiple cell types (e.g., macrophages, neutrophils and mast cells) along with their associated cytokines and growth factors that drive a fibrotic signaling cascade (Barrientos et al., 2008; Lech and Anders, 2013). Among the factors found to be associated with innate immune cells, TNF-α and IL-1β are noted profibrotic mediators along with transforming growth factor-beta (TGF-β), which has long been identified as a key factor in fibrosis (Roberts et al., 1988). Altogether, inflammatory and immune cell factors ultimately stimulate myofibroblasts to produce smooth muscle actin and matrix components such as collagen and fibronectin. The goal to inhibit profibrogenic factors or pathways has been the focus of research for years. Unfortunately, successful translation of effective antifibrotic treatments to humans has been limited due to inefficient and off-target effects. However, recent preclinical assessments of several agents show promise in achieving an attainable inhibition of fibroblast activation and ECM expression through mechanisms involving the blockade of signaling receptors, TGF-β stimulation, or translation of fibrogenic genes (Friedman et al., 2013; Gourdie et al., 2016).

Of the various mechanisms that have been studied to inhibit fibroproliferative events, strategies to target the activation of the myofibroblasts are emerging with high potential. Recent advances have been noted in the understanding and importance of particular factors in the inhibition and/or resolution of fibrosis. Specifically, excitement has been noted for the antifibrotic potential of bone morphogenetic protein-7 (BMP7); micro-RNAs; peroxisome proliferator-activated receptor (PPAR) signaling pathways; and the hormone relaxin. A strong indication for the use of these factors has been shown in studies of atrial fibrosis, heart failure, renal disease and cirrhosis of the liver. Acknowledging the importance and emergent nature of cardiovascular disease, this review will discuss the antifibrotic potential of the above agents in a disease-specific manner with emphasis on cardiac injury, as well their applicability as a broad intervention.

Bone morphogenetic protein-7 was discovered nearly 30 years ago as a critical factor in development and bone formation (Ozkaynak et al., 1990). Interestingly, BMP7 was also determined to be part of the TGF-β family. Considering the association of BMP7 with the fibrogenic factor TGF-β, the intervening years since its identification has produced a wealth of information related to mechanisms and the targetability of BMP7 pathways in fibrotic disease. To date, basic science discoveries have detailed the importance of BMP7 in organ homeostasis and specifically as an opposing mechanism to the profibrogenic actions of TGF-β. However, the translation of BMP7 as an antifibrotic agent remains in development. The following overview highlights the functional significance of BMP7 and related signaling mechanisms that may lead to translational successes and clinically relevant treatments for fibrosis.

BMP7 is structurally and functionally similar to members of the TGF-β superfamily (Weiskirchen et al., 2009). Structurally, BMP7 has a related modular form and sequence to TGF family members including a C-terminal biologically active region that is highly homologous to TGF-β. Also, similar to other family members, BMP7 engages with serine/threonine kinase receptors leading to the initiation of signal transduction cascades (Massague et al., 2005). Details of BMP7-mediated signaling have been eloquently characterized describing a variety of gene responses that are induced as a result of BMP7 activity (Weiskirchen et al., 2009). Outcomes of BMP7 signaling were shown to include the regulation of genes associated with embryonic development including kidney, eye and skeleton formation. Moreover, BMP7 signaling significantly influences organ homeostasis and importantly, the regulation of antifibrotic mechanisms. A summary of the major and most recent reports on the effectiveness of BMP7 treatment is presented in Table 1 and is discussed below.

In early studies using BMP7 deficient mice it was shown that a key factor in the antifibrotic effect of BMP7 involved the control of epithelial-to-mesenchymal transition (EMT) and TGF-β profibrogenic signaling in multiple organs (kidney, heart, liver, lung, and eye) (Luo et al., 1995; Zeisberg E.M. et al., 2007; Zeisberg M. et al., 2007; Myllarniemi et al., 2008). It is well known that TGF-β is a central inducer of myofibroblast activation and that TGF-β-mediated EMT is important in the transformation of fibroblasts involved in wound healing responses and fibrosis. Results from multiple fibrotic disease models demonstrated that BMP7 expression is downregulated during disease and that the restoration of BMP7 expression or treatment with recombinant protein resulted in the prevention or alleviation of fibrosis (Weiskirchen et al., 2009). The protective role of BMP7 was found to correlate with the inhibition of TGF-β-mediated profibrotic signaling. Despite the promising reports from various animal studies, the beneficial effects of exogenous BMP7 were found to be variable. This was evident from studies which failed to demonstrate an antifibrotic benefit of BMP7 as well as the indication that BMP7 expression in fact correlated with fibrotic disease (Ikeda et al., 2004; Tacke et al., 2007). However, the role of BMP7 as an opposing mechanism to the profibrogenic effects of TGF-β signaling remains clinically relevant, especially with the development of alternative strategies to alter BMP signaling through the use of small molecule inhibitors or agonists. Therefore, efforts continue to define the therapeutic effectiveness of BMP7 through more in-depth investigations including in vivo analyses, comparative studies and preliminary efficacy trials (Sugimoto et al., 2012; Lepparanta et al., 2013; Midgley et al., 2015; Lim et al., 2016).

Research efforts have determined that a key component of the balance between pro and antifibrotic signaling is the opposing action of BMP7 on TGF-β/Smad signaling (Figure 1). It was determined that BMP7 induces the antifibrotic phosphorylation of Smad1/5/8 that opposes TGF-β mediated phosphorylation of Smad2/3 and fibrogenic gene expression (Derynck and Zhang, 2003). Further evaluations into the role of BMP7/Smad signaling in various fibrotic diseases have been performed. In liver disease studies, models of hepato-schistosomiasis and carbon tetrachloride-induced fibrosis have been used to demonstrate the effectiveness of either exogenous BMP7 or adenovirus treatment in reducing key parameters of injury including TGF-β/Smad signaling and hepatic stellate cell activation (Hao et al., 2012; Chen et al., 2013). Additionally, in efforts to define regulators of BMP7/Smad signaling, it was demonstrated that a herbal compound was effective in alleviating hepatic fibrosis via enhancements in p-Smad1/5/8 levels and BMP7 antifibrotic signaling (Hou et al., 2016). In the context of chronic kidney disease, the negative regulation of TGF-β/Smad signaling by BMP7 has been shown to be involved in various nephropathies (Wang et al., 2003; Zeisberg et al., 2003; Chan et al., 2008). Further investigations have detailed parameters involved in the therapeutic restoration mediated by BMP7 overexpression which opposes Smad 3 signaling and protects against TGF-β-induced renal damage (Meng et al., 2013). In other works, the beneficial role of BMP7/Smad signaling has been shown in fibrotic diseases of the lung and heart. Of note, a recent evaluation provided correlative evidence of BMP7 antifibrotic effects in humans compared to animal models of cardiac disease. Particularly, it was determined that left ventricular LV remodeling in patients with aortic stenosis as well as mice with aortic constriction involved impaired BMP/Smad 1/5/8 signaling and increased TGF/Smad2/3 pathways; and that exogenous supplementation with BMP7 reduced LV disease in the mice (Merino et al., 2016). In models of lung disease, similar opposing actions of BMP7 on TGF-β/Smad signaling were indicated by reduced p-Smad 2/3 levels and attenuation of silica-induced pulmonary fibrosis in animals treated with recombinant BMP7 (Yang et al., 2013; Liang et al., 2016). Overall, the importance of BMP7 as an antagonist to profibrogenic TGF-β/Smad signaling has been demonstrated in multiple organ model systems providing support for further investigations into the clinical efficacy of BMP7 treatment strategies.

In addition to affecting Smad-dependent pathways, the antifibrotic role of BMP7 has been linked to several other mechanisms that hold promise for translation into human treatments. For example, DNA methylation changes may be important in BMP7 activity since the prevention of kidney fibrosis was linked to the reversal of Rasal1 promoter hypermethylation (Tampe et al., 2014). In another study, changes in the expression of specific receptors on tubular epithelial cells (e.g., CD44v3) resulted in enhanced BMP7 synthesis and an associated reduction of renal fibrotic damage (Rampanelli et al., 2013). Also, the protective role of BMP7 was shown to be related to changes in the expression of miRNAs as the suppression of miR-21 in rat kidney cells was found to associate with BMP7-mediated inhibition of fibronectin secretion and apoptosis (Yu et al., 2016). Another novel mechanism identified to be associated with BMP7 activity is the production and degradation of hyaluronan in human lung fibroblasts (Midgley et al., 2015). The role of antagonists and competing ligands in the inhibition of BMP7 activity is also part of current investigations which may lead to future translational advancements (Lin et al., 2005; Yanagita et al., 2006; Tanaka et al., 2010). In particular, research is underway to define the role of Activin A, a member of the TGF-β family that acts as an antagonist to BMP7 antifibrotic signaling (Abe et al., 2004; Aykul and Martinez-Hackert, 2016). To date, preclinical studies have shown the potential benefit of inhibiting Activin A signaling by disrupting receptor kinase activity or by blocking the receptor binding site of the antagonistic ligand (Laping et al., 2002; Pearsall et al., 2008; Aykul and Martinez-Hackert, 2016). In addition to the use of antagonists, mechanisms are being studied to enhance antifibrotic signaling through the action of BMP agonists. Notably, a recently developed synthetic peptide (THR-123, Thrasos Therapeutics, Canada) to the BMP receptor activin-like kinase 3 (ALK3), was found to be effective in controlling renal fibrosis in mice (Sugimoto et al., 2012). The successful preclinical work with THR-123 has led to the development of a similar analog, THR-184, which has been tested in a phase 2 trial for the resolution of acute kidney injury in cardiac patients (ClinicalTrials.gov ID NCT01830920). Although it is anticipated that forthcoming results will be clinically revealing, there are limitations in the global use of BMP agonists since the ALK3 receptor is expressed predominantly in the kidney (Sugimoto et al., 2012). Thus, the organ-specificity of BMP agonists needs to be considered and requires further characterization.

Overall, it is well established that BMP7 is an opposing mechanism to TGF-β-mediated profibrogenic signaling and that BMP7 activity is downregulated in fibrotic tissue injury. The potential therapeutic restoration of BMP7 through overexpression or exogenous administration has been demonstrated in multiple animal and organ models of fibrotic disease. However, the effectiveness of these strategies could be limiting due to potential off-target effects and low bioavailability of exogenous BMP7. It is known that systemically administered BMP7 has a short half-life resulting in the need to use of high doses to reach pharmacological effects (Vukicevic et al., 1998). Consequently, the stimulation of BMP signaling can occur in unwanted organ systems due to the ubiquitous expression of BMP receptors throughout the body (Wang et al., 2014). Therefore, current research efforts are focusing on the development and testing of alternative strategies to more specifically target BMP7 including the use of antagonist inhibitors and BMP agonists. Such investigations will likely contribute to translational clinical trials and the advancement of BMP7 as an antifibrotic agent.

MicroRNAs are short non-coding RNA molecules that regulate target messenger RNAs through post-transcriptional or translational repression mechanisms (Ambros, 2004). It has been shown that miRNA expression levels can be altered in disease states compared to normal conditions, highlighting the potential use of miRNAs as diagnostic or treatment targets. Studies to date have identified unique miRNA profiles for disease states which are often found to be tissue and cellular specific. The dysregulation of miRNA expression during fibrotic disease can involve the aberrant overexpression as well as the downregulation of miRNAs (Bartel, 2009). Regardless of the change, altered miRNA levels can lead to the regulation of a multitude of protein coding genes and signaling mechanisms that promote disease. In fibrosis, miRNAs alterations can lead to disease-promoting changes in a variety of fibrotic mechanisms such as the action of signaling mediators such as TGF-β or the expression of tissue-remodeling ECM components (Jiang et al., 2010). Thus, because of the magnitude of their regulatory role, miRNAs have emerged as viable targets for therapeutic intervention of fibrotic pathways. The following discussion is a review of miRNAs that are promising candidates for the treatment of fibrotic disease.

Research into the role of miRNAs as an antifibrotic therapy in cardiovascular disease is a leading area of investigation. As in other organs, the transformation of cardiac tissue after injury can involve TGF-β signaling and fibroblast activation leading to detrimental scar formation (Khan and Sheppard, 2006). Studies have identified miRNAs in cardiac cells that contribute to the fibrotic cascade in various models of heart disease including atrial fibrosis, cardiac infarction and heart failure as summarized in Table 2. Several miRNAs have been noted to be upregulated in animal and human hearts including miR-214, miR-223, and miR-21 (Thum et al., 2008; van Rooij et al., 2008; Roy et al., 2009). Of note, the enhancement of miR-21 led to functional changes in cardiac cells through MAP kinase signaling, fibroblast growth factor 2 expression and the survival of cardiac fibroblasts. Further, a direct link to profibrotic mechanisms was confirmed when the inhibition of miR-21 via antogomir-21 treatments resulted in recovery of heart function in mice subjected to transverse aortic constriction (Thum et al., 2008). In other works, the downregulation of miRNAs was also observed in cardiac disease models including reductions in miR-133, miR-590, miR-30, miR155, miR-22, miR-29, and miR101 (van Rooij et al., 2008; Duisters et al., 2009; Shan et al., 2009; Pan et al., 2012; Kishore et al., 2013; Hong et al., 2016). In recent preclinical assessments, the functional significance of many of the downregulated miRNAs has been described using several models of cardiac fibrotic diseases. In a model of ischemia/reperfusion injury, the dysregulation of miR-29-30-133 were linked to the activation of TGF-β signaling which could be reversed by triiodothyronine (T3) treatment (Nicolini et al., 2015). Particularly, T3 treatment was found to be effective in countering the injury-related downregulation of miR-29c, miR-30c, and miR-133a resulting in the reduction of profibrogenic matrix metalloproteinase (MMP)-2 and CTGF expressions (Nicolini et al., 2015). In models of fibrosis induced by myocardial infarction (MI), the reduction of miRNAs has been shown to regulate many profibrotic proteins and signaling mediators. For example, the targeting of miR-22 shows clinical potential as this miRNA was found to be a negative regulator of cardiac fibrosis through the suppression of TGF-βR1 (Hong et al., 2016). The miR-29 and miR-101 families have also been identified as regulators of antifibrotic mechanisms and have been evaluated for their potential as therapeutic agents. In the case of the miR-29 family, miR-29a, miR-29b, and miR-29c were found to be reduced after MI and associated with the gene expression of ECM proteins and TGF-β signaling (van Rooij et al., 2008; Kriegel et al., 2012). Further, the synthetic overexpression of miR-29 reduced collagen production and fibrosis following MI (van Rooij et al., 2008). Interestingly, the antifibrotic effect of the cardioprotective drug, Tanshinone IIA, was found to involve the upregulation of miR-29b (Yang et al., 2015). Studies evaluating the miR-101 family have similarly demonstrated a protective role in healthy tissue and the importance of designing strategies to increase their expression following cardiac injury. As such, decreased levels of miR-101a/b after coronary artery ligation have been associated with the induction of profibrogenic signaling mediated by c-Fos and TGF (Pan et al., 2012). Importantly, the overexpression of miR-101a inhibited the signaling pathways and alleviated fibrosis in the injured heart. Overall, there is strong preclinical evidence in various injury models that miR-based therapy may be an effective antifibrotic strategy, and especially for the treatment of MI-related cardiac fibrosis. As discussed, the successful modulation of miRNAs by the use of inhibitors, genetic manipulation or pharmacological agents indicates the high therapeutic value of targeting miRNAs. Although the characterization of potential off-target effects of systemically delivered miRNA modulators remains to be determined, the future is bright for the effective and tissue-specific delivery of miRNA targeting treatments.

In addition to cardiac disease, the potential of targeting miRNAs in other fibrotic diseases is also of current clinical importance. In the last 10 years, research efforts have increased in the study of miRNAs in hepatic, renal, and pulmonary fibrosis. In liver fibrosis research, the role of hepatic stellate cells (HSCs) in the production of ECM components and fibrotic injury has been well-characterized (Iredale et al., 2013; Lemoinne et al., 2013). As with other fibrotic conditions, several miRNAs were found to be either upregulated or reduced with the altered expression level associating with HSC activation and liver fibrogenesis (Guo et al., 2009). The correlation of miRNA changes to functional outcomes has been shown for several of the miRNAs highlighting their translational potential (Schon et al., 2016). Examples include a noted reduction in HSC-produced collagens in response to the overexpression of miR-133a (Roderburg et al., 2013). In another study, the activation of HSCs was inhibited by treatment with salvianolic acid B and the induced expression of miR-152 (Yu et al., 2015). And as observed in cardiac disease, the downregulation of miR-29 is strongly associated with hepatic fibrosis. Notably, miR-29 mimics or overexpression has been shown to control ECM production by HSCs demonstrating therapeutic potential (Kwiecinski et al., 2011).

The role of miRNAs in renal fibrosis has also been associated with the regulation of signaling pathways that induce ECM factors such as collagens and fibronectin (Wang et al., 2008; Kato et al., 2009). Multiple works have defined the role of miRNAs in renal fibrosis including the effects of miR-148b and members of the miR-29 and miR-let7 families (Fang et al., 2013; Nagai et al., 2014; Szeto and Li, 2014; Srivastava et al., 2016). Additionally, recent studies have demonstrated functional advancements as protection from fibrosis was gained from anti-miR-214 treatment (Denby et al., 2014). Further, the delivery of miR-let7c-expressing mesenchymal stem cells was found to be an effective method to target miRNAs in the damaged kidney (Wang B. et al., 2016). Altogether, research continues to define the therapeutic potential of miRNAs in renal fibrosis.

And lastly, investigation into the role of miRNAs in pulmonary fibrosis is emerging with potential targetable miRNAs identified including miR-29 (Cushing et al., 2015), miR-155 (Pottier et al., 2009), miR-21 (Zhou et al., 2015b; Liu et al., 2016), miR-26a (Liang et al., 2014), and miR-326 (Das et al., 2014). Interestingly, preclinical support of translational effects was indicated by the demonstration of pulmonary fibrosis that is induced as a result of miR-26a inhibition (Liang et al., 2014); and that pulmonary fibrosis could be effectively attenuated following the intranasal deliver of miR-326 mimics (Das et al., 2014). Overall, the increasing and emerging research in this field exemplify the power of targeting miRNAs for the treatment of fibrotic diseases and support the need of future evaluations.

The PPARs are nuclear transcription factors that form obligate heterodimers with retinoid-X receptors to modulate transcription of target genes. Three PPAR subtypes have been identified, known as PPARα, PPARβ/δ, and PPARγ. Due to its role in the insulin-sensitizing effects of the thiazolidinedione (TZD) drugs, PPARγ is by far the most widely studied PPAR isoform. However, preclinical studies have implicated all three PPARs as potential targets for antifibrotic therapy (summarized in Figure 2). Recent studies supporting the use of PPAR agonists for this purpose are described below, along with discussion of recently completed or ongoing clinical trials.

Relaxin is a polypeptide of the insulin/relaxin superfamily. It is produced by the corpus luteum of the ovary during pregnancy (Sherwood, 2004; Bathgate et al., 2013). Its roles during pregnancy involve widespread hemodynamic changes such as vasodilation, decreased systemic vascular resistance, and increased renal plasma flow and glomerular filtration rate (Conrad and Davison, 2014). Its roles outside of pregnancy were less clear until the generation of the relaxin knockout mouse, which developed widespread fibrosis with aging (Samuel et al., 2016a). Importantly, the male mice developed fibrosis as well, but the source of relaxin in males and non-pregnant females is unclear. Relaxin protects against fibrosis both by decreasing collagen production, and promoting collagen degradation by increasing the levels and activity of MMPs (Samuel et al., 2016b; Bennett, 2009). Furthermore, many of relaxin’s effects involve antagonism of the effects of TGFβ. The relatively recent discovery of its receptor (RXFP1) has led to rapid development of new discoveries for the use of relaxin as an antifibrotic agent (Bathgate et al., 2013). Some of these findings are summarized in Figure 3.

The earliest human studies using relaxin to target fibrotic diseases began in the late 1950s, using partially purified porcine relaxin to treat scleroderma, with variable success (Samuel et al., 2016a). Preclinical animal studies have supported the use of relaxin for dermal fibrosis, (Unemori et al., 1993), and the relaxin knockout mouse spontaneously develops age-related skin fibrosis (Samuel et al., 2005). A peptide activator of RXFP1 prevented bleomycin-induced dermal fibrosis in mice (Pini et al., 2010). Finally, the development of recombinant human relaxin led to more recent clinical scleroderma trials. In a phase 2 study, beneficial effects of relaxin were observed in patients with moderate to severe diffuse systemic sclerosis after 24 weeks infusion with low relaxin (25 μg/kg/day), but not at a higher dose (100 μg/kg/day) (Seibold et al., 2000). However, in a subsequent phase 3 trial, relaxin had no clinically significant effects (Khanna et al., 2009). Part of the lack of success of this study may have been due to the relatively advanced degree of scleroderma in the patient population, a notion supported by the finding that relaxin treatment failed to reverse advanced scleroderma in the relaxin knockout mouse (Samuel et al., 2005), or the uncertainty in clinically meaningful outcomes measures in human systemic sclerosis trials (Seibold, 2002).

Relaxin has been extensively studied as a treatment for several preclinical fibrosis models (summarized in Table 4), including cardiac fibrosis. The relaxin knockout mouse serves as a model of age-related cardiac fibrosis and associated left ventricular dysfunction, but interestingly, only in the male mice, due to additional detrimental effects of testosterone (Du et al., 2003; Hewitson et al., 2012). The increased left ventricular collagen deposition was reversed by treatment with relaxin, by a mechanism that appeared to involve inhibition of cardiac myofibroblast differentiation (Samuel et al., 2004a). In preclinical animal models, relaxin treatment, or relaxin delivered by adenovirus, effectively reduced cardiac fibrosis induced by β-adrenergic stimulation in rodents (Samuel et al., 2004a; Zhang et al., 2005; Bathgate et al., 2008). More recently, relaxin was more effective than the angiotensin converting enzyme inhibitor enalapril in treating isoproterenol-induced cardiac injury, while the combination of relaxin and enalapril was more efficacious than either treatment alone, in both prevention and treatment approaches (Samuel et al., 2014). Furthermore, the effect of relaxin treatment on isoproterenol-induced cardiac fibrosis may involve inhibition of endothelial to mesenchymal transition (Zhou et al., 2015a). Relaxin was also effective in reversing cardiac fibrosis in the spontaneously hypertensive rat model (Lekgabe et al., 2005). Furthermore, relaxin reduced both fibrosis and atrial fibrillation in spontaneously hypertensive rats (Parikh et al., 2013). Relaxin reduced collagen content in a rat model of angiotensin II-induced fibrosis, and furthermore, berberine was used to induce relaxin expression, with similar results (Gu et al., 2012). Relaxin was also successful in reducing collagen in a diabetic cardiomyopathy model (Samuel et al., 2008). Conversely, in a chronic pressure overload model, relaxin knockout mice had no more collagen deposition in the heart than wild-type mice (Xu et al., 2008), suggesting that the effectiveness of relaxin may depend on the nature of cardiac injury.

In rats subjected to MI, relaxin reduced cardiac fibrosis, inhibited cardiac myofibroblast differentiation, and promoted induction of MMPs (Bonacchi et al., 2009; Samuel et al., 2011). Furthermore, a recent study suggested that in addition to reduced post-MI-induced fibrosis, relaxin reduced tachyarrhythmia and cardiac dysfunction in rats (Wang D. et al., 2016). Recombinant human relaxin has been used in clinical studies of acute heart failure, with some promising results in regard to dyspnea and hemodynamic properties, as well as cardiovascular death and all-cause mortality in a phase III study (Teerlink et al., 2009). A second phase III trial is currently underway to study the effect of relaxin on acute heart failure (ClinicalTrials.gov ID#NCT02064868).

The fibrotic lung is also a therapeutic target of relaxin. The relaxin knockout mouse developed age-related pulmonary fibrosis, and as with cardiac fibrosis, the effect was more pronounced in male mice, and was reversed after relaxin treatment (Samuel et al., 2003). Similarly, the relaxin knockout mouse developed more severe fibrosis in studies using ovalbumin-induced allergic airway disease (Mookerjee et al., 2006; Samuel et al., 2007). In rodent models of pulmonary fibrosis induced by bleomycin or ovalbumin-induced allergic airway disease, lung collagen content and alveolar thickness were reduced with relaxin (Unemori et al., 1996; Royce et al., 2009). Recent studies also showed efficacy of intranasal relaxin administration in allergic airway disease and cigarette smoke induced lung damage (Royce et al., 2014; Pini et al., 2016). In an exciting development, relaxin and bone marrow-derived mesenchymal stem cells or amnionic epithelial stem cells were found to be synergistic in the treatment of established airway disease (Royce et al., 2015, 2016). The mechanism for this effect is currently under investigation, but appears to be related to the matrix remodeling and anti-TGFβ effects of relaxin promoting a favorable environment for the establishment of stem cell residency in the damaged tissue (Samuel et al., 2016b).

Treatment of experimental renal fibrosis has also supported relaxin as a novel antifibrotic agent. As with the heart, skin and lungs, the relaxin knockout mouse develops renal fibrosis with age, which can be reversed with relaxin treatment (Samuel et al., 2004b). In several animal models of renal fibrosis, relaxin was effective in reducing collagen deposition (Garber et al., 2001, 2003; McDonald et al., 2003; Lekgabe et al., 2005; Danielson et al., 2006; Yoshida et al., 2011; Yoshida et al., 2014). Relaxin reduced unilateral uteretic obstruction-induced renal disease by antagonizing the effect of TGF-β, and that this effect was enhanced by coadministration of mesenchymal stem cells (Hewitson et al., 2010; Huuskes et al., 2015). On the other hand, relaxin was not effective in a diabetes-related renal disease model (Wong et al., 2013; Dschietzig et al., 2015). However, unlike the unilateral uteretic obstruction models, TGFβ was not increased in the diabetic model. Therefore, given relaxin’s well-known role in opposing the profibrotic effects of TGFβ, the lack of effect in the diabetes model is perhaps not surprising.

There is also evidence that relaxin can treat liver fibrosis. In prevention models, relaxin reduced collagen production and promoted MMP in CCl₄-induced hepatic fibrosis (Williams et al., 2001; Bennett et al., 2009). In a more clinically relevant model, relaxin treatment also reduced established liver fibrosis (Bennett et al., 2014). Finally, relaxin reduced portal hypertension in animal models of liver disease (Fallowfield et al., 2014), and a phase 2 trial is currently underway to study the effect of relaxin on portal hypertension in cirrhotic patients (ClinicalTrials.gov ID# NCT02669875).

There is evidence of interplay between relaxin and other signaling pathways (Figure 4). It was recently shown that the relaxin receptor RXFP1 can form heterodimers with the angiotensin II type 2 (AT2R), and that renal-protective effects of relaxin required the presence and activity of AT2R (Chow et al., 2014). One major mechanism for relaxin’s antifibrotic effects is by antagonism of TGFβ signaling (Samuel et al., 2016a,b). Relaxin binding to RXFP1 results in activation of the protein kinase ERK1, with downstream activation of endothelial nitric oxide synthase (eNOS, or NOS1) and increased nitric oxide production. This in turn activates soluble guanylyl cyclase and cGMP production. This pathway has been shown to inhibit the phosphorylation of Smad2/3, resulting in decreased TGFβ signaling (Chow et al., 2012). An additional RXFP1 signaling pathway results from coupling to Gαs to promote activation of adenylyl cyclase, cAMP production and activation of protein kinase A (PKA). It was recently shown that this pathway, through phosphorylation of the transcription factor cAMP response element binding protein (CREB), promotes expression of the coactivator protein PPARγ coactivator 1α (PGC1α), which serves to increase PPARγ activity, providing potential cross-talk between these two antifibrotic pathways (Singh et al., 2015; Singh and Bennett, 2010). Relaxin activation of PPARγ activity has also been detected in brain arterioles (Chan and Cipolla, 2011; Chan et al., 2013). In support of this concept, a recent study showed that relaxin enhanced the response of other airway dilators, including the PPARγ agonist rosiglitazone (Lam et al., 2016).

The current options for the treatment of fibrotic diseases are extremely limited, and to date no effective drug has emerged that successfully targets established fibrosis. The four avenues of potential treatments discussed here show considerable progress, but to date have not translated to clinical treatment. Useful antifibrotic therapies must be effective against not only against reducing the excess collagen accumulation, but also accommodate collagen degradation. Ideally, the agents should have oral bioavailability. This presents challenges to peptide-based treatments, such as BMP7 or relaxin, and novel small molecule receptor agonists may hold the key to future therapeutics in these areas. In addition, and perhaps most importantly, off-target effects should be minimized, which might be overcome with nanoformulated preparations of the drugs. The novel agents provided in this review show promise as potential future treatments for fibroses, but more work is needed to determine if they will be ultimately translated to human disease.

All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.