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British Journal of
Pharmacology
British Journal of Pharmacology (2017) 174 962–976
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Themed Section: Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors
REVIEW ARTICLE
Anti-fibrotic actions of relaxin
Correspondence Associate Professor Chrishan S. Samuel, PhD, Cardiovascular Disease Program, Biomedicine Discovery Institute and
Department of Pharmacology, Monash University, Clayton, Vic. 3800, Australia; and Professor Robert G. Bennett, PhD, Research Service
151 VA Nebraska-Western Iowa Health Care System, Omaha, NE 68105, USA. E-mail: chrishan.samuel@monash.edu; rgbennet@unmc.edu
Received 7 March 2016; Revised 19 May 2016; Accepted 23 May 2016
C S Samuel1, S G Royce1, T D Hewitson3, K M Denton2, T E Cooney4 and R G Bennett5,6
1
Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Pharmacology, Monash University, Melbourne, Vic., Australia,
2
Cardiovascular Disease Program, Biomedicine Discovery Institute and Department of Physiology, Monash University, Melbourne, Vic., Australia,
Department of Nephrology, Royal Melbourne Hospital, Melbourne, Vic., Australia, 4University of Pittsburgh Medical Centre (UPMC) Hamot, Erie, PA,
3
USA, 5Research Service 151, VA Nebraska-Western Iowa Health Care System, Omaha, NE, USA, and 6Department of Internal Medicine, University of
Nebraska Medical Center, Omaha, NE, USA
Fibrosis refers to the hardening or scarring of tissues that usually results from aberrant wound healing in response to organ injury,
and its manifestations in various organs have collectively been estimated to contribute to around 45–50% of deaths in the
Western world. Despite this, there is currently no effective cure for the tissue structural and functional damage induced by fibrosisrelated disorders. Relaxin meets several criteria of an effective anti-fibrotic based on its specific ability to inhibit pro-fibrotic
cytokine and/or growth factor-mediated, but not normal/unstimulated, fibroblast proliferation, differentiation and matrix
production. Furthermore, relaxin augments matrix degradation through its ability to up-regulate the release and activation of
various matrix-degrading matrix metalloproteinases and/or being able to down-regulate tissue inhibitor of metalloproteinase
activity. Relaxin can also indirectly suppress fibrosis through its other well-known (anti-inflammatory, antioxidant, antihypertrophic, anti-apoptotic, angiogenic, wound healing and vasodilator) properties. This review will outline the organ-specific
and general anti-fibrotic significance of exogenously administered relaxin and its mechanisms of action that have been documented in various non-reproductive organs such as the cardiovascular system, kidney, lung, liver, skin and tendons. In addition, it
will outline the influence of sex on relaxin’s anti-fibrotic actions, highlighting its potential as an emerging anti-fibrotic therapeutic.
LINKED ARTICLES
This article is part of a themed section on Recent Progress in the Understanding of Relaxin Family Peptides and their Receptors.
To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.10/issuetoc
Abbreviations
α-SMA, α-smooth muscle actin; AT2 receptor, angiotensin type 2 receptor; CCl4, carbon tetrachloride; ECM, extracellular matrix; HSC, hepatic stellate cell; INSL, insulin-like; KO, knockout; MSC, mesenchymal stem cell; PMA, phorbol
12-myristate 13-acetate; RLN, relaxin gene; rhRLX, synthetically or recombinantly produced drug form of relaxin;
RXFP1, relaxin family peptide receptor 1; TIMP, tissue inhibitor of metalloproteinase
DOI:10.1111/bph.13529
© 2016 The British Pharmacological Society
Anti-fibrotic actions of relaxin
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Tables of Links
TARGETS
Other protein targets
LIGANDS
a
Enzymes
VEGF
GPCRs
b
d
Angiotensin II
Relaxin
Akt (PKB)
cAMP
TGF-β1
MMP-1
Enalapril
TIMP1
TIMP2
β2-adrenoceptor
MMP-2
IFN-γ
AT2 receptor
MMP-9
Methylprednisolone
RXFP1 receptor
MMP-13
RXFP2 receptor
Nuclear hormone receptors
nNOS
c
PKA
PPARγ
These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org,
the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide
a,b,c,d
Alexander et al., 2015a,b,c,d).
to PHARMACOLOGY 2015/16 (
Introduction
The tissue response to stress depends on a number of intrinsic
and extrinsic factors including the length and extent of injury.
When injury or disease is mild or transient, then the tissue response leads to remodelling or regeneration of the organ parenchyma. However, if the injury is either severe or prolonged, the
process is characterized by ongoing inflammation and extracellular matrix (ECM) production. Fibrosis is therefore a failure of
the wound healing process, where ECM synthesis is ongoing
(Wynn, 2008; Hewitson, 2009). This maladaptive response is
particularly dependent on paracrine and autocrine production
of TGF-β1 and the consequent recruitment of activated fibroblasts (myofibroblasts). Over-abundant ECM production and
failure to resolve lead to significant organ damage and dysfunction, which depends not only on the quantity of matrix produced (fibrogenesis) but also the degree of its cross-linking and
its reorganization, or density.
Despite the almost universal significance of fibrosis
(Wynn, 2008; Hewitson, 2009), there is a lack of effective
anti-fibrotic therapeutic strategies. A growing body of evidence suggests that relaxin may fulfil this need. This review
will focus on its anti-fibrotic effects and mechanism of action
in tissues of the circulatory, renal, hepatic and integumentary
systems. Moreover, the influence of sex on fibrosis progression will be discussed.
Relaxin
Relaxin is a member of a family of peptide hormones that is
structurally similar to insulin, but which diverged from insulin early in vertebrate evolution to form a distinct peptide
family based on their two-chain structures, receptor binding
motif and ability to bind and activate GPCRs (refer to pp.
5846–7 in Alexander et al., 2015b; Bathgate et al., 2006), as
opposed to the tyrosine kinase receptors that are activated
by insulin and the insulin-like growth factors. The relaxin
peptide family is encoded by seven genes in humans, which
include the relaxin genes RLN1, RLN2 and RLN3, as well as
the insulin-like peptide genes INSL3, INSL4, INSL5 and INSL6
(Bathgate et al., 2006). In most other species though, only five
of these genes exist, including RLN1 and RLN3, the species
equivalents of human RLN2 and RLN3 respectively. The product of the human RLN2 gene (H2 relaxin) and its species
equivalent gene (relaxin) represent the major stored and circulating forms of relaxin in their respective species and will
be the forms of relaxin primarily discussed in this review.
Recombinant human relaxin [synthetically or recombinantly
produced drug form of relaxin (rhRLX), now also referred to
as serelaxin] is the drug-based form of H2 relaxin, which will
also be discussed throughout the review.
Anti-fibrotic effects of relaxin in the
cardiovascular system
Fibrosis is a hallmark of almost all forms of cardiovascular disease and a key contributor to atrial, ventricular and vascular
stiffness, impaired cardiac contractility and heart failure
(Mandavia et al., 2013; Frieler and Mortensen, 2015; Ling
et al., 2016). A common fibrotic process develops from a
number of cardiac pathologies including ischaemic injury and
myocardial infarction; hypertrophic, dilated and restrictive cardiomyopathies; valvular heart diseases; hypertension; diabetes;
metabolic disorders; and cardiac arrhythmia, amongst others.
Continuous exogenous administration of rhRLX, human
gene-3 relaxin (H3 RLX) or mouse relaxin (mRLX) to profibrotic factor-stimulated cardiac fibroblasts in vitro (at
100 ng·mL 1 over 1–5 days) or various experimental models
of cardiovascular disease in vivo (at 0.1–0.5 mg·kg 1·day 1 over
2–4 weeks, where 0.5 mg·kg 1·day 1 produces circulating
levels of ~20–40 ng·mL 1) has effectively reduced the cardiac
fibrosis associated with each model investigated (Table 1).
The anti-fibrotic effects of rhRLX have consistently
been demonstrated in various preclinical models of
British Journal of Pharmacology (2017) 174 962–976
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C S Samuel et al.
Table 1
Various models of cardiovascular disease that have been used to demonstrate the anti-fibrotic effects of relaxin
Model studied
Type of relaxin used
Length of treatment (days)
Ang II-stimulated atrial fibroblasts (rat) (Samuel et al., 2004a)
rhRLX
3
Ang II-stimulated cardiac fibroblasts (rat) (Gu et al., 2012)
rhRLX
1
TGF-β1-stimulated atrial fibroblasts (rat) (Samuel et al., 2004a)
rhRLX
3
TGF-β1-stimulated ventricular fibroblasts (rat) (Samuel et al., 2004a)
rhRLX
3
TGF-β1-stimulated cardiac fibroblasts (mouse)
(Sassoli et al., 2013; Squecco et al., 2015)
rhRLX
1–5
PMA-stimulated cardiac fibroblasts (rat) (Wang et al., 2014)
rhRLX
3
High glucose-stimulated cardiac fibroblasts
(rat) (Wang et al., 2009; Su et al., 2014)
rhRLX
2
Age-related fibrosis (mouse) (Samuel et al., 2004a) (Samuel et al., 2007)a
rhRLX/mRLXa
14/120a
β2-adrenoreceptor-induced cardiomyopathy (mouse)
#
(Samuel et al., 2004a; Bathgate et al., 2008) (Hossain et al., 2011a)c
(Chan et al., 2012; Samuel et al., 2014)
Ad-mRLXb/H3 RLXc/rhRLX
14
Myocardial infarction (pig)(Formigli et al., 2007)d,
(rat) (Bonacchi et al., 2009), (mouse) (Samuel et al., 2011)
hRLXd/rhRLX
7–30
Hypertension (rat) (Lekgabe et al., 2005; Gu et al., 2012)
rhRLX
14
Type 1 diabetes (rat) (Samuel et al., 2008)
rhRLX
14
Atrial fibrillation (rat) (Parikh et al., 2013; Henry et al., 2016)
rhRLX
14
a
Synthetically produced mRLX.
b
adenovirus-mediated mRLX (Ad-mRLX).
c
H3 RLX was used.
d
mouse skeletal myoblasts were engineered to produce relaxin based on the hRLX sequence.
cardiovascular disease and heart failure in vivo, regardless of
aetiology, including models of ageing (Samuel et al., 2007;
Samuel et al., 2004a), fibrotic cardiomyopathy (Chan et al.,
2012; Samuel et al., 2014), myocardial infarction (Formigli
et al., 2007; Bonacchi et al., 2009; Samuel et al., 2011),
hypertension (Lekgabe et al., 2005; Gu et al., 2012), type 1
diabetes (Samuel et al., 2008) and atrial fibrillation (Parikh
et al., 2013; Henry et al., 2016) (Table 1). Furthermore,
rhRLX (0.5 mg·kg 1·day 1) was found to have improved
anti-fibrotic efficacy over the clinically used ACE inhibitor,
enalapril (48 mg·kg 1·day 1), in an experimental model of fibrotic cardiomyopathy, but also augmented the anti-fibrotic
efficacy of enalapril when both treatments were administered
10 days after the onset of injury (Samuel et al., 2014).
Likewise, both H3 RLX (Hossain et al., 2011a) and mRLX
(Samuel et al., 2007; Bathgate et al., 2008) have also demonstrated similar anti-fibrotic efficacy to that of hRLX in
murine models of cardiomyopathy induced by transgenic
overexpression of β2-adrenoceptors. All forms of relaxin studied to date mediate their anti-fibrotic actions by inhibiting
the effects of various pro-fibrotic factors (discussed below)
rather than by directly regulating collagen or other ECM
proteins per se, and rhRLX demonstrated greater anti-fibrotic
efficacy than enalapril due to its greater ability to inhibit both
TGF-β1 expression and its signal transduction at the level of
intracellular Smad2 phosphorylation (Samuel et al., 2014).
However, rhRLX did not affect chronic pressure overloadinduced cardiac hypertrophy or fibrosis that was associated
with biochemical wall stress rather than elevated TGF-β1
levels (Xu et al., 2008).
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Relevant mechanisms of the anti-fibrotic
actions of relaxin
Several studies conducted at the in vitro level have offered more
in-depth insights into the mechanisms and signal transduction
pathways associated with relaxin’s anti-fibrotic actions in the diseased myocardium, primarily involving rhRLX. rhRLX (and H3
RLX; Hossain et al., 2011a) acting via the relaxin family peptide
receptor 1 (RXFP1 receptor) specifically inhibits cardiac fibroblast
proliferation and differentiation into activated myofibroblasts
and, hence, myofibroblast-mediated aberrant collagen deposition (the basis of fibrosis) when cells are stimulated with either
angiotensin II (Samuel et al., 2004a; Gu et al., 2012), TGF-β1
(Samuel et al., 2004a; Sassoli et al., 2013; Squecco et al., 2015),
phorbol 12-myristate 13-acetate (PMA; Wang et al., 2014) or
high glucose (Wang et al., 2009; Su et al., 2014). The ability of
rhRLX to inhibit TGF-β1-mediated cardiac fibrosis progression
was shown to involve activation of the Notch-1 and NO pathways, and down-regulation of Smad2 (Samuel et al., 2014)
and/or Smad3 (Sassoli et al., 2013) phosphorylation. This
collectively results in the rhRLX-induced inhibition of the
pro-fibrotic actions of TGF-β1 on myofibroblast differentiation
and aberrant ECM/collagen deposition.
Furthermore, by suppressing the TGF-β1/Smad2 and/or
TGF-β1/Smad3 axes, which themselves promote fibrosis by
inhibiting the actions of matrix-degrading MMPs and
promoting tissue inhibitor of metalloproteinase (TIMP)
activity (which are natural inhibitors of MMPs) (Spinale
and Villarreal, 2014), rhRLX releases and promotes
various MMPs (including MMP-1 and its rodent orthologue
MMP-13, MMP-2 and MMP-9) (Lekgabe et al., 2005; Formigli
Anti-fibrotic actions of relaxin
et al., 2007; Parikh et al., 2013; Samuel et al., 2004a, 2008,
2011, 2014; Sarwar et al., 2015a,b) and/or decreases both cardiac TIMP-1 (Samuel et al., 2008) and TIMP-2 (Sassoli et al.,
2013) activity, which would probably result in the degradation of existing collagen fibres. Hence, relaxin mediates its
anti-fibrotic effects in the heart by suppressing aberrant profibrotic cytokine-induced ECM/collagen deposition, while
also being able to promote MMP-induced ECM/collagen
breakdown (Figure 1).
rhRLX may also indirectly inhibit cardiac fibrosis through its
anti-inflammatory (Nistri et al., 2003; Perna et al., 2005; 2008),
antioxidant (Perna et al., 2005), anti-hypertrophic (Dschietzig
et al., 2005; Moore et al., 2007; Parikh et al., 2013) and antiapoptotic (Moore et al., 2007; Bonacchi et al., 2009; Samuel
et al., 2011; Zhang et al., 2015) actions, while being able to
promote tissue repair via its angiogenic (Formigli et al., 2007;
Samuel et al., 2011; Segal et al., 2012), vasodilatory (Conrad
and Shroff, 2011; McGuane et al., 2011a,b) and wound healing
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(Mu et al., 2010) properties. However, the effects of rhRLX have
largely been shown to be independent of blood pressure regulation (Du et al., 2010; Samuel et al., 2008, 2014). These combined
effects of rhRLX, along with its direct anti-fibrotic actions, have
resulted in its ability to improve cardiac output and ventricular
performance (Perna et al., 2005; Teichman et al., 2008), while
being able to reduce cardiac contractility and ventricular
stiffness (Samuel et al., 2008; Du et al., 2010) as well as atrial
(Parikh et al., 2013) and ventricular (Nistri et al., 2008)
arrhythmias.
Anti-fibrotic actions of relaxin in the
kidney
The close association between cardiovascular pathology and
renal dysfunction is well documented and significant.
Patients with conventional risk factors for cardiovascular
Figure 1
Summary of the mechanisms of relaxin’s anti-fibrotic actions that are mediated through RXFP1 receptors and RXFP1–AT2 receptor heterodimers.
Relaxin specifically ameliorates the effects of pro-fibrotic stimuli such as TGF-β1 and Ang II, the former by inhibiting Smad2 (pSmad2) and/or
Smad3 (pSmad3) phosphorylation, which is dependent at least in part on the pERK1/2, NO and Notch-1 pathways. This causes decreased expression and deposition of interstitial (types I, III and V) and basement membrane (type IV) collagens and reduced activity of TIMP-1 and TIMP-2, accompanied by increased expression and activity of various MMPs, including MMP-1/13, MMP-2 and/or MMP-9. The end result is a decrease in the
rate of collagen deposition and increased collagen degradation, allowing clearance of the fibrotic scar.
British Journal of Pharmacology (2017) 174 962–976
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C S Samuel et al.
disease also suffer renal dysfunction. The pathology of the
heart and kidney is therefore inexorably linked (Hewitson
et al., 2015).
Early studies showed that continuous 2–4 week infusion
of rhRLX with osmotic mini-pumps ameliorated progressive
fibrosis in several experimental rodent models of fibrosis
including
bromoethylamine-induced
renal
papillary
necrosis (Garber et al., 2001), experimental anti-glomerular
basement membrane nephritis (McDonald et al., 2003) and
reduction of renal mass by either surgical ablation or
infarction (Garber et al., 2003). The last two models are
particularly insightful because they showed a decrease in hypertension in the infarction model, but no change in blood
pressure in the normotensive ablation model, suggesting that
structural–functional effects can be independent of changes
in blood pressure (Garber et al., 2003). Also key is the
observation that systemic administration of rhRLX
concurrently limits both cardiac and renal fibrosis in the
spontaneously hypertensive rat, indicating that rhRLX may
simultaneously ameliorate similar pathologies in multiple
organs (Lekgabe et al., 2005). Treatment of acute renal injury
with rhRLX also limited loss of structure and function after
ischaemia–reperfusion (Yoshida et al., 2013) and cisplatin
exposure (Yoshida et al., 2014).
However, rhRLX may not be universally renoprotective,
as it did not prevent diabetic renal complications in mouse
models of type I diabetes (Wong et al., 2013; Dschietzig
et al., 2015), when TGF-β1 was not elevated in either model.
Importantly, in a phase II scleroderma trial (Khanna et al.,
2009), cessation of rhRLX after 24 weeks of administration resulted in a significant decline in renal function, as estimated
by creatinine clearance (Khanna et al., 2009). It is unclear if
this was related to scleroderma-specific factors.
Establishing that pretreatment with rhRLX can slow progression is an important proof of principle, but the reality is
that most renal patients present clinically with established fibrosis. It is those studies in established renal disease that will
provide the greatest clinical insight.
Relevant pleiotropic actions of relaxin
The pleiotropic properties of rhRLX can also indirectly limit
renal fibrosis and promote wound healing by stimulating
NO production (Mookerjee et al., 2009; Sasser et al., 2011;
Chow et al., 2012; Chow et al., 2014), improving the local environment through maintaining glomerular filtration rate
(Conrad, 2004), preventing parenchymal cell loss through
apoptosis (Yoshida et al., 2013) and enhancing cell survival
through maintaining vascular supply via angiogenesis
(Unemori et al., 2000; Hewitson et al., 2010). Its recognized
direct cellular actions in the kidney include a reduction in
inflammation (Yoshida et al., 2014), oxidative stress (Sasser
et al., 2011) and ECM synthesis (fibrogenesis) and contraction
(Masterson et al., 2004). There is also circumstantial evidence
to suggest that a rhRLX-mediated increase in activity of
MMP-2, MMP-9 or MMP-1 (and its analogue MMP-13 in the
rodent) may directly limit renal scarring by increasing
collagen degradation (Figure 1). Nevertheless, the spatial
and temporal context of these collagenases are important as
degradation of collagen IV can limit fibrosis by removing excess collagen and, in other circumstances, exacerbate
injury through destruction of basement membranes. An
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British Journal of Pharmacology (2017) 174 962–976
MMP-induced vasodilatation occurs through ET1–32 production (Jeyabalan et al., 2003), which again may improve
wound repair and the environment in which the kidney
functions.
rhRLX may also indirectly reduce fibrosis by augmenting
repair with mesenchymal stem cells (MSCs). A combination
of rhRLX and bone marrow-derived MSCs attenuated kidney
fibrosis after 7 days of unilateral ureteric obstruction
(Huuskes et al., 2015). This was accompanied by reduced
tissue damage and enhanced MMP-2 activity compared with
either treatment alone, while rhRLX also directly increased
MSC proliferation and migration (Huuskes et al., 2015).
Signal transduction pathways of the
anti-fibrotic actions of rhRLX
Extensive cell culture studies using rat renal myofibroblasts
have provided a mechanistic insight into rhRLX’s anti-fibrotic
actions. rhRLX inhibits myofibroblast differentiation and collagen synthesis and promotes MMP expression/activity by
myofibroblasts through binding to its cognate receptor, RXFP1
(Mookerjee et al., 2009). Recent studies also suggest that heterodimer formation between the RXFP1 receptor and other GPCRs,
in particular angiotensin receptors, may be necessary (Chow
et al., 2014). Regardless, subsequent signal transduction through
a neuronal NOS–NO–cGMP pathway appears to be central to
the anti-fibrotic actions of rhRLX in renal myofibroblasts. More
specifically, rhRLX inhibits phosphorylation of Smad2 in both
human (Heeg et al., 2005) and rat renal (myo)fibroblasts
(Mookerjee et al., 2009). These effects are only partially dosedependent, with robust effects seen at physiological concentrations (Mookerjee et al., 2009). Importantly, the anti-fibrotic
actions of rhRLX may well be contingent on TGF-β1, which
may explain the lack of efficacy in models without aberrant
TGF-β1 expression (Wong et al., 2013; Dschietzig et al., 2015).
The influence of sex on the anti-fibrotic
actions of relaxin
There are sex differences in the incidence, prevalence and
progression of cardiovascular and renal disease, with males
being at greater risk than females prior to menopause (Silbiger
and Neugarten, 2008; Barrett-Connor, 2013). For example, it
has been demonstrated that cardiac fibrosis associated with
left ventricular hypertrophy due to aortic stenosis is greater
in men compared with women (Petrov et al., 2014). These
data suggest that there are sex differences in the adaptation
to pressure overload. Indeed, this greater cardiac fibrosis was
associated with higher cardiac tissue levels of TGF-β1, Smad2
phosphorylation, and higher fibrosis related gene expression
of collagens I and III, MMP-2 and MMP-9 in men than
women (Petrov et al., 2010; Petrov et al., 2014). Similarly, in
chronic kidney disease, tissue damage is greater in males than
females, and this has been associated with enhanced TGF-β1
activation and reduced NO bioavailability (Silbiger and
Neugarten, 2008). It is notable that these pathways are responsive to the actions of relaxin. Therefore, relaxin may
act as a protective buffer against cardiovascular and renal disease in pre-menopausal women.
Anti-fibrotic actions of relaxin
The literature is unclear as to whether serum relaxin levels
are greater in women than in men. In the main, data suggest
that relaxin levels are greater in females during the luteal
phase of the oestrous cycle and pregnancy (Bani, 2008). A recent study (Wolf et al., 2013) detected no difference in serum
relaxin levels between men and women. However, given that
the subjects were athletes, it is possible that ovulation and
relaxin secretion was suppressed in these females. Thus,
higher circulating levels of relaxin may confer cardio-renal
protection during the reproductive years in women.
Surprisingly few studies have examined whether relaxin
has sexually dimorphic effects in ameliorating cardio-renal
disease. Studies examining the response to rhRLX infusion
observed no difference in the blood pressure, cardiac output,
total peripheral resistance or renal function (Danielson et al.,
2000; Conrad et al., 2004) between male and female rats. In
other studies, the age-related cardiac and renal fibrosis observed in relaxin knockout (RLN1-KO) mice was exacerbated
in male but not female mice (Du et al., 2003; Samuel et al.,
2004b), which was later found to be associated with testosterone and elevated androgen receptor levels in RLN1-KO mice
being causative of the problem (Hewitson et al., 2012). These
data suggest that the effects of relaxin may be of greater importance in males than in females. However, an alternative
explanation put forward was that females have additional
protective mechanisms that compensate and mask the loss
of relaxin (Du et al., 2003; Metra et al., 2013). Interestingly
though, while neither ovariectomy or oestrogen replacement
therapy affected the cardiac and renal fibrosis measured in female RLN1-KO mice (Lekgabe et al., 2006), ovariectomized
RLN1-KO mice had exacerbated lung myofibroblast burden
and subepithelial collagen levels (fibrosis), which were both
diminished by oestrogen replacement therapy, suggesting
that the potential compensation offered by oestrogen at least
may be organ specific. In the Relaxin in Acute Heart Failure
trial, subgroup analysis of the response to acute administration of rhRLX did not detect a sex difference in outcomes
(Metra et al., 2013). However, systemic adenoviral delivery
of mRLX attenuated left ventricular fibrosis in male and female β2-adrenoceptor transgenic mice (Bathgate et al.,
2008), suggesting that rhRLX may be equally effective for
the treatment of cardiovascular and renal disease in both
sexes. However, more detailed studies examining the response to rhRLX administration in models of disease in both
males and females are required to validate this. It would also
be important to conduct studies in both young and aged
animals, as cardiovascular and renal disease occur predominantly in post-menopausal women, when loss of ovarian
hormones may reduce receptor and/or second messenger signalling pathways.
There are also intriguing data to suggest that relaxin may
have an enhanced action in females. As detailed above,
rhRLX’s NO-promoting and TGF-β1 inhibitory actions were
found to involve an interaction with the angiotensin type 2
receptor (AT2 receptor), through heterodimers formed between the RXFP1 and the AT2 receptors (Chow et al., 2012;
2014) (Figure 1). Moreover, we have demonstrated that the
renal and vascular effects of AT2 receptor stimulation are enhanced in females in association with increased AT2 receptor
expression (Sampson et al., 2008; Hilliard et al., 2011, 2013;
Brown et al., 2012). It is therefore possible that signalling
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via RXFP1–AT2 receptor heterodimers may contribute to the
cardio-renal protection observed in young females and that
this mechanism may be down-regulated post-menopause
since the enhanced role for the AT2 receptors in females is lost
with ageing and reproductive senescence (Mirabito et al.,
2014a,b). This possibility warrants further investigation, as
restoration of AT2 receptor expression in older women, or
up-regulation in males, may represent a therapeutic target
worth pursuing.
In contrast, it is also possible that a deficit in relaxin may
play a role in the progression of renal disease in females. In
general, females are protected from renal disease except in
the case of diabetic nephropathy, in which progression and
severity of renal damage is greater in females (DiamondStanic et al., 2012; Barrett-Connor, 2013). Evidence suggests
that this is due to a reduction in the oestrogen to testosterone
ratio in females in response to the diabetic environment and
a similar change in hormonal balance also occurs postmenopause, a time when the risk of cardio-renal disease starts
to increase in women (Maric, 2009; Diamond-Stanic et al.,
2012). Given the association between relaxin secretion and
oestrogen status (Lippert et al., 1996; Seeger et al., 2000),
relaxin may be altered in diabetic patients. However, rhRLX
infusion in type I diabetic models has not been shown to
improve renal fibrosis, for the reasons outlined above (Wong
et al., 2013; Dschietzig et al., 2015). By contrast, in a high-fat
diet model of insulin resistance, rhRLX infusion reversed collagen accumulation in the heart (Bonner et al., 2013). As these
studies were all conducted in males, in the future, it will be
important to examine the effect of rhRLX replacement
therapy for diabetic nephropathy in females, in which an
aged-related reduction in relaxin might be associated with
sex hormone imbalance. Thus, several lines of evidence suggest that relaxin may explain the protection from cardiovascular and renal disease enjoyed by pre-menopausal women,
while its lack may contribute to the exacerbation of disease
in females.
Anti-fibrotic effects of relaxin in the
lung
Fibrosis in the lung is seen histopathologically in asthma,
chronic obstructive pulmonary disease, pulmonary fibrosis
(including idiopathic pulmonary fibrosis), bronchopulmonary
dysplasia and pulmonary hypertension. Its significance in these
diseases is that it is associated with progression, severity of disease and resistance to treatment, and contributes to the functional end points of dyspnoea, airway hyperresponsiveness
(AHR) and forced vital capacity (FVC) and forced expiratory volume in 1 s (FEV1).
In a murine model of chronic allergic airways disease
(AAD), which mimics several features of human asthma, systemic treatment with 0.5 mg·mL 1 rhRLX via mini-osmotic
pumps was able to reduce total lung collagen and
peribronchial ECM deposition to that seen in control mice
(Royce et al., 2009). Furthermore, rhRLX treatment reversed
epithelial thickening and significantly improved AHR but
did not influence airway inflammation or goblet cell metaplasia. rhRLX therapy was also attempted in the most widely
used preclinical model of parenchymal pulmonary fibrosis
British Journal of Pharmacology (2017) 174 962–976
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C S Samuel et al.
induced by bleomycin administration. In this model, mice
are typically given a single or double dose of bleomycin –
originally used for the treatment of cancers where fibrotic
side effects in the lung were identified (Della Latta et al.,
2015). Furthermore, rhRLX administration significantly reversed established parenchymal fibrosis as well myofibroblast
contractility (Unemori et al., 1996; Pini et al., 2010; Huang
et al., 2011).
Given the fact that relaxin is a hormone and the RXFP1 receptor appears to be expressed widely throughout the body, it
is important to limit off target effects. This is achievable for
lung diseases as the organ is available for inhaled delivery.
However, there are a number of issues with regard to lung delivery that must be addressed. In a murine AAD model of
asthma, a similar reversal of established airway fibrosis was
achieved with daily intranasal administration of rhRLX over
2 weeks (Royce et al., 2014a) as that observed in earlier studies
in response to continuous systemic administration (Royce
et al., 2009). Intranasal administration to mice involves simply allowing the mouse to inhale a bolus of rhRLX
micropipetted to the nares. In humans, the lung is orders of
magnitude larger, and hence, the challenge is for drugs inhaled at the nose or mouth to reach the small airways
(<2 mm), which are of particular pathological significance
(Thien, 2013), and the alveolar sacs. As such inhaled medications need to be delivered as dry powder or nebulized to
achieve a small enough particle size to reach the terminal
parts of the respiratory tree, rhRLX has yet to meet this, but
promising new nebulization and delivery technologies are
under development that enable a larger range of drugs to be
nebulized without conformational changes or loss of bioactivity (Cortez-Jugo et al., 2015).
It is well-documented that rhRLX has a strong antifibrotic effect especially against established fibrosis, particularly with its ability to down-regulate TGF-β1-mediated collagen production (Royce et al., 2014a; Unemori et al., 1996) and
up-regulate gelatinases (MMP-2 and MMP-9) to help digest
aberrant collagen accumulation in the lung and airway wall
(Royce et al., 2009; Chow et al., 2012) (Figure 1). However, if
rhRLX is to have potential as a treatment for fibrotic lung diseases, it is important that it is able to complement existing
therapies, in particular inhaled and oral corticosteroids.
rhRLX has been combined with methylprednisolone to treat
established fibrosis in an experimental model of chronic
AAD and it was found that combination therapy with the corticosteroid and rhRLX more effectively reduced subepithelial
collagen thickness compared with either therapy alone
(Royce et al., 2013). However, corticosteroids are not without
their limitations, such as steroid resistance in some patients,
side effects that limit optimal dosing in very young children
and, from the point of view of fibrosis, the fact that blocking
inflammation does not eliminate fibrosis, as is clear in the
adult asthma population where airway remodelling remains
rife despite corticosteroids being in widespread use for decades. This reflects the fact that airway remodelling and fibrosis are often not due to chronic inflammation alone but arise
from other aetiologies such as epithelial damage and genetic
susceptibility (Holgate et al., 2006).
MSCs are another therapeutic approach used experimentally in combination with rhRLX for lung disease treatment
(Royce et al., 2015). Used alone, bone marrow-derived MSCs
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British Journal of Pharmacology (2017) 174 962–976
have been shown to have potential for treating a range of fibrotic lung diseases (Royce et al., 2014b) and have been used
successfully to treat AAD (Ogulur et al., 2014) and
bleomycin-induced (Ortiz et al., 2003; Moodley et al., 2009)
disease models. Stem cell therapies, in general, are thought
to heal either by engraftment into the host tissue or by
secretion of exosomes and proteins that have paracrine and
immunomodulatory effects in diseased tissue. In many
inflammatory lung diseases, there are areas of epithelial
damage where engraftment of MSCs would conceivably be
of benefit, but it seems more plausible that secretions of MSCs
explain most of the beneficial effects seen in mouse models
(Ge et al., 2013). It was hypothesized that combining rhRLX
with MSCs would augment the therapeutic efficacy of the
latter, by improving the micro-environment via removal of
aberrant ECM accumulation and fibrotic stimuli (Formigli
et al., 2007; Huuskes et al., 2015). In a chronic AAD model,
this combination treatment further reversed disease-induced
airway inflammation and airway/lung fibrosis compared with
either treatment alone and further increased MMP-2 and
MMP-9 levels (Royce et al., 2015). Another avenue for this
method is for the overexpression of relaxin by MSCs, which
would provide both a novel delivery method of rhRLX and
the likely positive effects of rhRLX in the immediate microenvironment of the MSC. This protein overexpression has already been used with other MSC treatments including overexpression of the CXCR4 receptor to enhance homing
(Yang et al., 2015) and with murine C2C12 myoblasts overexpressing relaxin to reduce fibrosis and promote angiogenesis
in ischaemically damaged organs (Formigli et al., 2007).
Much progress has been made on rhRLX in the acute heart
failure trials towards being a realistic drug treatment for
human disease. In addition, it has a good safety profile, the
ability to reverse aberrant collagen (without effecting basal
ECM required for normal structure) and other advantages
compared with other anti-fibrotic drugs both currently used
and under development. Given these factors and the
outstanding need for novel therapies to treat lung diseases
such as asthma (estimated 235 million sufferers 2004) and
chronic obstructive pulmonary disease (64 million sufferers)
(World Health Organization Global Burden of Disease),
relaxin has great potential as a future therapy for fibrosis
associated with various lung diseases.
Anti-fibrotic effects of relaxin in the
liver
Hepatic fibrosis results from the accumulation of fibrillar collagens and other ECM proteins in response to chronic injury
from a variety of causes, including alcohol overuse, viral
hepatitis and nonalcoholic steatohepatitis (Trautwein et al.,
2015). The liver cells responsible for the production of the
majority of the collagen in hepatic fibrosis are the hepatic
stellate cells (HSCs), specialized cells that reside in the
perisinusoidal regions (Friedman, 2008; Puche et al., 2013).
HSCs are unusual in that, in the normal (quiescent)
state, they function as lipid storing cells and are not
fibroblastic in nature. With liver injury, inflammatory
cytokines such as TGF-β1 and PDGF stimulate HSCs, causing
their transdifferentiation to an activated myofibroblastic
Anti-fibrotic actions of relaxin
phenotype, characterized by proliferation, contractility and
high expression of α-smooth muscle actin (α-SMA), and fibrillar collagens. At the same time, HSCs secrete TIMP-1 and
TIMP-2 (Friedman, 2008; Puche et al., 2013), resulting in a
shift in the balance between matrix production and degradation and a net increase in collagen accumulation. When the
cause of liver injury is removed, the number of activated
HSCs is decreased, through either apoptosis or reversion to
the quiescent phenotype, allowing clearance of the excess
collagen and a return to the normal liver architecture and
functioning. With chronic injury, however, the collagen accumulation causes disruption of liver architecture and can
eventually lead to cirrhosis. For this reason, HSCs have been
a prime target in the development of new treatments for advanced hepatic fibrosis and cirrhosis (Trautwein et al., 2015).
The first study of relaxin’s effects in the liver was in rats
treated for 4 days with porcine relaxin (Bani et al., 2001).
Relaxin caused dilation of the sinusoids and changes in
contractile, actin-rich cells that were probably either
myofibroblasts or activated HSCs. The HSCs were confirmed
as a target of relaxin in studies using culture-activated
rat HSCs, where rhRLX caused a decreased collagen
deposition and synthesis, accompanied by decreased secretion of TIMP-1 and TIMP-2, but without a change in the gene
expression of MMPs or α-SMA (Williams et al., 2001). In another study, porcine relaxin treatment of culture-activated
rat HSCs also caused decreased collagen synthesis and deposition, as well as decreased secretion of type I collagen, TIMP-1
and TIMP-2 (Bennett et al., 2003). Furthermore, relaxin decreased the level of α-SMA protein, increased the degradation
of type I collagen and increased the expression of MMP-13,
the major rodent fibrillar collagenase. Similar results were observed using primary human HSCs (Fallowfield et al., 2014).
Taken together, these studies provided evidence that human
or porcine relaxin can inhibit the pro-fibrotic properties of
activated HSC and promote conditions that favour collagen
degradation (Figure 1).
HSCs are the major liver cells that express RXFP1. Early
studies using PCR detected RXFP1 expression in human liver
(Hsu et al., 2002; Hsu et al., 2003), but not female mouse liver
(Scott et al., 2006). It was later revealed that quiescent rat
HSCs express very low levels of the RXFP1 receptor, which increased markedly during transdifferentiation in culture, and
the RXFP1 receptor was detected in fibrotic mouse liver and
cirrhotic human liver (Bennett et al., 2005, 2007). These findings were later confirmed in primary rat and human HSCs
and in rat and human hepatic fibrosis (Fallowfield et al.,
2014). The signalling pathways activated by relaxin in HSCs
are consistent with those in fibroblasts from other tissues
and include cAMP, cGMP, NO and Akt (Bennett et al., 2007;
Fallowfield et al., 2014). Furthermore, recent findings suggest
that rhRLX activation of RXFP1 receptors can result in activation of the anti-fibrotic transcription factor PPARγ, through a
mechanism involving cAMP, PKA, p38-MAPK and PPARγ
coactivator protein 1α (PGC1α; Singh and Bennett, 2010;
Singh et al., 2015).
Several studies have been conducted to assess the efficacy of
relaxin in the treatment of hepatic fibrosis. The first was a
prevention study using CCl4-induced hepatic fibrosis in rats,
with concomitant treatment with rhRLX (~0.5 mg·kg 1·day 1)
for 4 weeks (Williams et al., 2001), in which rhRLX reduced
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the overall liver collagen content as assessed by hydroxyproline
content. Later studies focused on more clinically relevant
models of established hepatic fibrosis in mice, using 4 weeks of
CCl4 treatment to induce fibrosis, followed by rhRLX treatment
via osmotic pumps with continued CCl4 administration.
In one study using short-term treatment with porcine relaxin
(0.5 mg·kg 1·day 1), relaxin had a small effect on fibrosis after
1 week, but little effect after 2 weeks (Bennett et al., 2009). A later
study of 4 week treatment with rhRLX (25 or 75 μg·kg 1·day 1)
showed significant reductions in the levels of liver collagen and
α-SMA and reduced expression of types I and III collagen, α-SMA
and TIMP-2, increased expression of MMP-3 and MMP-13 and
increased collagen degrading activity (Bennett et al., 2014).
Finally, another study showed that with short-term (72 h) administration of 0.5 mg·kg 1·day 1 rhRLX into rats made fibrotic
with 8 weeks of CCl4, rhRLX reduced the amount of α-SMA protein, but not the amount of liver collagen, and decreased the
gene expression of type I collagen, α-SMA, MMP-3 and TGF-β
(Fallowfield et al., 2014). Furthermore, in both CCl4 and bile
duct ligation models, rhRLX reduced portal pressure and increased the hepatic oxygen supply, consistent with other studies
using perfused liver models (Boehnert et al., 2008; Fallowfield
et al., 2014).
Taken together, the studies conducted thus far show
some positive effects of relaxin on hepatic fibrosis and
haemodynamics, at least in part by reducing the activated
phenotype of HSCs and/or promoting matrix degradation.
However, there are some differences in the various experimental models used thus far, and further studies are needed
to determine the optimal conditions necessary for effective
relaxin treatment of hepatic fibrosis. Nevertheless, these collective preclinical results led to a phase II trial of recombinant
human relaxin on haemodynamics in patients with
compensated cirrhosis and portal hypertension, which was
completed in early 2015 (clinicaltrials.gov identifier
NCT01640964). The results of this trial, when released, may
clarify the potential role of relaxin in the treatment of human
liver disease.
Anti-fibrotic effects of relaxin on the
integumentary system and connective
tissues
Research involving relaxin’s effect on clinical skin pathology
has a notable past, with scleroderma as the most frequently
studied pathology. Scleroderma is a spectrum disorder and
includes a milder, limited form as well as a ‘diffuse’ form
and involves fibrosis of internal organs. In its limited form,
common manifestations in the skin are limited to the extremities and include skin tightness and ulcerations, thickening,
altered vascularity and sensitivity of the hands and digits
(Raynaud’s syndrome), difficulty in swallowing (dysphagia)
and localized dilation of small blood vessels (CREST syndrome). In diffuse scleroderma, major organ fibrosis occurs
along with widespread skin involvement. Early reports indicated that oestrogen priming followed by 20–40 mg doses of
porcine relaxin (then known as ‘releasin’) partially resolved
skin tightness and Raynaud’s symptoms, prompted healing
of skin ulcerations that were refractory to corticosteroid
British Journal of Pharmacology (2017) 174 962–976
969
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C S Samuel et al.
treatment and, in limited cases, relieved dysphagia (Casten
and Boucek, 1958; Ismay, 1958; Evans, 1959; Rivelis et al.
1965). No improvements were reported in heart, lung or kidney function. Therapeutic effectiveness often required weeks
of therapy (3–5 weeks; Casten and Boucek, 1958) with therapy continuing for protracted periods (up to 30 months),
with discontinuation often exacerbating symptoms. Reports
of complications were rare, mainly focused on injection site
soreness. Contrary to these reports, others observed treatment failure in four patients treated with 80 mg of releasin
for 3–4 weeks (Jefferis and Dixon, 1962). These reports suggested that treatment efficacy was most often observed with
continued therapy and that response variability was present
in the outcomes.
More recently, clinical safety trials were conducted to establish tolerability of rhRLX to treat patients with systemic
disease of short (<5 year) duration. A 24 week randomized
control trial showed both safety and preliminary measures
of efficacy for 25 μg·kg 1·day 1 subcutaneous rhRLX infusion; a higher dose (100 μg·kg 1·day 1), however, failed to
show an advantage over the placebo (Seibold et al., 2000). A
14% improvement over placebo in ratings of standardized
scoring of skin thickness was observed in this trial. A subsequent FDA phase III efficacy randomized clinical trial involving over 200 patients failed to show efficacy over placebo and
evidenced seven instances of serious adverse events involving
renal crisis (Khanna et al., 2009).
The failure of this phase III trial forced a reassessment of
relaxin therapeutics for treating skin-associated fibrosis, most
especially in the context of targeting tissue competence to respond to relaxin. Certainly, in vitro studies have lent support
for therapeutic trials (Unemori and Amento, 1990). rhRLX
(1–100 ng·mL 1) dose-dependently reduced type I collagen
production from most (but not all) of a small sample of biopsies derived from patients with scleroderma (Unemori et al.,
1992), and these effects were synergistically enhanced by
co-administration of IFN-γ (100 U·mL 1). Furthermore,
rhRLX significantly improved the cosmetic appearance and
histology of porcine excisional wounds when administered
at 130 μg·kg 1·day 1 for 6 weeks (Stewart, 2009). Currently,
studies are underway to determine the effect of relaxin treatment in animal models of dermal wounding, and the expression of relaxin receptors in biopsy samples from patients with
Dupuytren’s disease (Cooney, unpublished data).
That fibroblasts are likely targets for relaxin therapy is also
supported from other clinically relevant anatomical sites,
suggesting a systemic-wide tropism. Treatment with rhRLX
(up to 100 ng·mL 1) of primary cultures of human fibroblasts
obtained from vaginal wall biopsies resulted in reduced TGF-β
1 translation (Wen et al., 2008). Fibroblasts comprising knee
ligaments and thumb express relaxin receptors (Dragoo
et al., 2003; Galey et al., 2003; Lubahn et al., 2006). An in situ
study of female lower shank tendons showed an inverse relationship between serum relaxin levels and patellar tendon
stiffness (Pearson, et al., 2011). These data strongly suggest
that fibroblasts located in connective tissue are competent
to respond to rhRLX.
However, the scope of relaxin’s apparent cellular interaction within the integument extends beyond resident
fibroblasts. Several studies have shown that keratinocytes
derived from normal skin express RXFP1 receptors (Cooney
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British Journal of Pharmacology (2017) 174 962–976
et al., 2010; Giordano et al., 2012). Relaxin effects have also
been isolated to a substituent of the ECM, fibrillin-2, in the
form of down-regulated gene transcription and translation
in human dermal fibroblasts in response to rhRLX dosing at
30 ng·mL 1 (Samuel et al., 2003b). Cell lines derived from
the eye epithelium and surrounding sites (human cornea,
conjunctiva, sebaceous and lacrimal glands) express both
RXFP1 and RXFP2 receptors and respond to dosing with
INSL3 and rhRLX (Hampel et al., 2012; Hampel et al., 2013).
Blood vessel endothelium and endothelial precursors have
been shown to be rhRLX targets. The administration of
rhRLX 0.1 mg·kg 1·day 1, s.c., to rats enhanced angiogenesis
in wound chambers, increased VEGF and bFGF signalling and
production and ultimately determined that effects were likely
to be mediated by monocytes infiltrating hypoxic tissue areas
(Unemori et al., 2000). Importantly, effects were confined to
the site of wounding and were not seen in alveolar
pulmonary macrophages. Others showed increases in the
transcription and translation of mediators of angiogenesis
and vasculogenesis in genetically diabetic mice dosed with
25 μg·day 1 of porcine relaxin with consequent improvement in wound healing time (Bitto et al., 2013) and that
rhRLX signalled through RXFP1 receptors to induce
mobilization of bone marrow-derived endothelial progenitors (Segal et al., 2012). Overall, there are an abundance of
histological sites in the integument that express relaxin
receptors and respond to dosing in vitro.
Limitations of using rhRLX as a therapeutic
and challenges to consider for future studies
Whilst these collective findings highlight the therapeutic
potential of rhRLX as an anti-fibrotic, there are a number of
recognized barriers to clinical translation that are noteworthy. Firstly, the clinical efficacy of various anti-fibrotic agents
is difficult to evaluate in general due to the lack of reliable biomarkers that could indicate when fibrosis has developed to
the point when these agents should be appropriately administered, the lack of reliable non-invasive end points that could
demonstrate if they have regressed disease progression, and
the slow pathogenesis of fibrosis in humans (compared to
that in animals), which can take decades to develop.
Secondly, rhRLX is a peptide-based drug with a short in vivo
half-life (~10 min; Nair et al., 2015). Although this has been
overcome at the experimental level by administering rhRLX
to various animal models via subcutaneously implanted
osmotic mini-pumps and at the clinical level via
microinfusion pumps (Erikson and Unemori, 2001), weekly
injections (McGorray et al., 2012) or intravenous administration (Weiss et al., 2009), the relatively short duration of
rhRLX treatment may have contributed to the failed clinical
trials to date that have attempted to demonstrate its antifibrotic efficacy in patients with varying conditions, particularly given that fibrosis is a chronic condition that is likely
to require long-term treatment to diminish its progression.
Thirdly, the requirement for longer term rhRLX treatment
will only add to its expense to produce. While small
molecules that activate RXFP1 receptors (Xiao et al., 2013)
and single-chain derivatives of rhRLX (Hossain et al., 2011b;
Hossain et al., 2016) have recently been developed and will
be cheaper to manufacture, further work is required to
Anti-fibrotic actions of relaxin
determine their anti-fibrotic efficacy when chronically administered over long periods. The latter might be difficult to
test in rodent models though which have been found to
mount antibody responses to rhRLX, which in turn have
caused increased and variable circulating levels of rhRLX beyond 10 days post-administration (personal communication;
Dr. Elaine Unemori; Corthera Inc. and Novartis AG, San
Mateo, CA, USA). Consistent with this, mice administered
0.5 mg·kg 1·day 1 rhRLX undergo a doubling of circulating
human relaxin levels after 14 days of administration compared with that after 5 days (Samuel et al., 2003a), which
may lead to higher concentrations of the drug producing
lower physiological responses, given its well-demonstrated
bell-shaped dose–response effects (Unemori et al., 1996;
Danielson and Conrad, 2003; Teerlink et al., 2009).
Furthermore, the importance of understanding tissue
competence to respond to rhRLX along with signalling pathways is paramount to ensuring both efficacy and safety of
rhRLX in human clinical trials. Recently, Giordano et al.
(2012 showed that skin biopsies from patients with limited
systemic scleroderma showed weak or no staining for RXFP1
receptors. In line with this, only 30% of Dupuytren’s nodules
stain positive for the RXFP1 receptor (Cooney, unpublished
observation). These findings beg the question of how receptor expression changes over the course of disease. Restoration
of calcium signalling via NO is impaired in the endothelium
of spontaneously hypertensive rats and cannot be remediated
by rhRLX, unlike that of Wistar Kyoto control rats (Nistri
et al., 2015), suggesting differential responsiveness based on
phenotype or pathological state. Indeed, as in the disease
state, alterations in the ECM environment of cell culture
matter, especially in the context of myofibroblasts. Material
properties of the ECM affect mechanical signal transduction
in myofibroblasts, which, in turn, alters gene transcription
(Vi et al., 2010). These data show that culturing myofibroblasts on tissue culture plastic or even type I collagen
substrates significantly alters the feedback loop between cells
and ECM and underscores the importance of recognizing the
distinction between the pathological environment versus
one that fosters cell growth and proliferation.
Conclusions
rhRLX continues to hold promise as a therapeutic to treat several fibrotic conditions associated with up-regulated TGF-β1
and myofibroblast burden. In addition to directly downregulating intracellular Smad activity to suppress TGF-β1
signal transduction and the pro-fibrotic actions of TGF-β1
on myofibroblast differentiation and aberrant ECM
synthesis, it is able to regulate matrix degradation by altering
the MMP-to-TIMP balance (summarized in Figure 1).
Additionally, it may also indirectly inhibit fibrosis progression via its other organ-protective actions, including its
anti-inflammatory, anti-hypertrophic, anti-apoptotic, angiogenic and vasodilatory effects, and can also promote wound
healing and tissue function through these combined actions.
Although a number of high-quality randomized trials to date
have failed to demonstrate the clinical efficacy of its matrix
remodelling actions in human subjects (which have been
well characterized in various experimental models), it is
BJP
possible that the positive effects of rhRLX on blood pressure
and renal function that have been observed in various trials
may be associated with its ability to inhibit fibrosis progression in affected organs. A significant challenge for future trials is to identify patients who may benefit from rhRLX
therapy, identify appropriate end points for a peptide-based
therapeutic, which requires continuous administration, consider the timing and length of treatment as well as appropriate route of administration and ensure tissue penetrability
beyond the direct site of administration. Application of gene
sequencing to human biopsy material (to elucidate whether
perturbations involving single nucleotide polymorphisms
or deletions/additions that impair RXFP1 receptor functioning affect therapeutic responses to rhRLX), and the development of small molecule mimetics of relaxin that have
improved pharmacokinetics and biodistribution properties
compared with native H2 relaxin, will undoubtedly aid this
effort. Cell culture models that better mimic in situ tissue
conditions will also provide more relevant information.
Overall, this field of relaxin investigation remains vibrant
and full of potential.
Acknowledgements
This article was supported in part by National Health &
Medical Research Council (NHMRC) of Australia Senior
Research Fellowships to C.S.S. (GNT1041766) and K.M.D.
(GNT1041844) and NHMRC Project Grants to T.D.H. and C.
S.S. (GNT1078694) and to K.M.D., C.S.S and T.D.H.
(GNT1101552) and by US Department of Veterans Affairs
Merit Review (BX000849) and National Institutes of Health
(AA015509) Grants to R.G.B.
Conflict of interest
The authors declare no conflicts of interest.
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