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Improved Chemical Synthesis and Demonstration of the Relaxin Receptor Binding Affinity and Biological Activity of Mouse Relaxin †
Abstract
The primary stored and circulating form of relaxin in humans, human gene-2 (H2) relaxin, has potent antifibrotic properties with rapidly occurring efficacy. However, when administered to experimental models of fibrosis, H2 relaxin can only be applied over short-term (2-4 week) periods, due to rodents mounting an antibody response to the exogenous human relaxin, resulting in delayed clearance and, hence, increased and variable circulating levels. To overcome this problem, the current study investigated the therapeutic potential of mouse relaxin over long-term exposure in vivo. Mouse relaxin is unique among the known relaxins in that it possesses an extra residue within the C-terminal region of its A-chain. To enable a detailed assessment of its receptor interaction and biological properties, it was chemically synthesized in good overall yield by the separate preparation of each of its A- and B-chains followed by regioselective formation of each of the intramolecular and two intermolecular disulfide bonds. Murine relaxin was shown to bind with high affinity to the human, mouse, and rat RXFP1 (primary relaxin) receptor but with a slightly lower affinity to that of H2 relaxin. When administered to relaxin-deficient mice (which undergo an age-dependent progression of organ fibrosis) over a 4 month treatment period, mouse relaxin was able to significantly inhibit the progression of collagen accumulation in several organs including the lung, kidney, testis, and skin (all p < 0.05 vs untreated group), consistent with the actions of H2 relaxin. These combined data demonstrate that mouse relaxin can effectively inhibit collagen deposition and accumulation (fibrosis) over long-term treatment periods.
... The synthesis was performed analogous to literature with slight modifications. [3] Solutions of Cys(pyridin-2-ylthio)-Phe-Cys(Acm) (287 mg, 521 µmol, 1.1 equiv) in water (80 mL) and Cys(Acm)-Phe-Cys (6) (210 mg, 473 µmol, 1.0 equiv) in water (120 mL) were adjusted to pH 8.5 with NH4OH and subsequently combined. After 16 h of S10 stirring the reaction was stopped by addition of AcOH (2 mL). ...
... The synthesis was performed analogous to literature with slight modifications. [3] A 20 mM Iodine solution in HOAc (18 mL) was dropped to a solution of Cys(Acm)-Phe-Cys-S-S-Cys-Phe-Cys(Acm) (5) (320 mg, 363 µmol, 1 equiv) in HOAc (290 mL) and ...
... (calc. 761.15262).S8The synthesis was performed according to literature with some modifications.[3] Cys- ...
Small peptides are involved in countless biological processes. Hence selective binding motifs for peptides can be powerful tools for labeling or inhibition. Finding those binding motifs, especially in water which competes for intermolecular H-bonds, poses an enormous challenge. A dynamic combinatorial library can be a powerful method to overcome this issue. We previously reported artificial receptors emerging form a dynamic combinatorial library of peptide building blocks. In this study we aimed to broaden this scope towards recognition of small peptides. Employing CXC peptide building blocks, we found that cyclic dimers of oxidized CFC bind to the aromatic peptides FF and YY ( K ≈ 229–702 M ⁻¹ ), while AA binds significantly weaker ( K ≈ 65–71 M ⁻¹ ).
... This was carried out as previously described [21,22]. For canine relaxin bearing C-terminal acid on its two chains, synthesis of the chains was carried out using appropriately Fmoc-amino acid-preloaded hydroxymethylphenoxyacetyl-derivatised PEG-polystyrene. ...
... The chemical synthesis of a heterodimeric two-chain, three disulfide linked peptide remains a formidable undertaking and has been the subject of many attempts to both optimize and simplify, most recently by way of one-pot processes [27,28]. Our approach of using efficient Fmoc-solid phase synthesis of suitably S-protected A-and B-chains followed by stepwise formation of the three disulfides has proven to be highly effective and robust with many assemblies of insulin-like peptides successfully accomplished [21,22,[29][30][31][32][33]. In order to acquire sufficient quantities of canine relaxin to undertake detailed structural and biological studies, we employed our approach (Figure 2A) and subsequently obtained highly purified peptide (Figures 2B and C) in overall yield of more than 6% relative to the starting crude B-chain. ...
... Importantly, the activity decrease matched the decreased affinity and is likely the result of species differences in the ligand receptor pairing. Notably, mouse relaxin has a lower affinity for, and activity at, human RXFP1 than its native mouse RXFP1 receptor [22]. The peptide was then assessed for secondary structural conformation using CD spectroscopy (Figure 4). ...
Background: The chemical synthesis of multi-disulfide bonded heterodimeric peptides such as insulin has long been of significant scientific and commercial interest as well as a major challenge. The development of improved protocols which includes regioselective disulfide bond formation has greatly advanced the capacity to prepare and study insulin-like peptides including canine relaxin, an important regulator of parturition and indicator of canine maternal health. Methods: Separate, efficient solid phase synthesis of the two constituent chains (24 residue A and 35 residue B) was followed by stepwise formation of each of the three disulfide bonds, one intra within the A-chain and two interchain, by oxidation, thiolysis and iodolysis respectively. Results: Synthetic canine relaxin having a total of 59 residues was prepared in good overall yield and shown by several criteria to be highly purified. The peptide was shown to be less potent than human relaxin (H2 relaxin) in binding to and activating the human relaxin receptor, RXFP1, in transfected cells. A circular dichrosim spectroscopic analysis showed that the canine relaxin also possessed significantly less secondary structure compared to H2 relaxin which may account for its reduced activity. Conclusions: The synthetic protocols developed in our laboratory enabled the successful preparation of the complex, small insulin-like protein, canine relaxin. This will, in turn, allow a detailed study of both the tertiary conformation of this peptide and its role in canine reproduction.
... Continuous exogenous administration of rhRLX, human gene-3 relaxin (H3 RLX) or mouse relaxin (mRLX) to pro- fibrotic factor-stimulated cardiac fibroblasts in vitro (at 100 ng·mL À1 over 1-5 days) or various experimental models of cardiovascular disease in vivo (at 0.1-0.5 mg·kg À1 ·day À1 over 2-4 weeks, where 0.5 mg·kg À1 ·day À1 produces circulating levels of ~20-40 ng·mL À1 ) has effectively reduced the cardiac fibrosis associated with each model investigated ( Table 1). 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 À1 ·day À1 ) was found to have improved anti-fibrotic efficacy over the clinically used ACE inhibitor, enalapril (48 mg·kg À1 ·day À1 ), in an experimental model of fi- brotic 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). ...
... Furthermore, rhRLX (0.5 mg·kg À1 ·day À1 ) was found to have improved anti-fibrotic efficacy over the clinically used ACE inhibitor, enalapril (48 mg·kg À1 ·day À1 ), in an experimental model of fi- brotic 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 demon- strated similar anti-fibrotic efficacy to that of hRLX in murine models of cardiomyopathy induced by transgenic overexpression of β 2 -adrenoceptors. All forms of relaxin stud- ied 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). ...
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 fibrosis-related 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, anti-hypertrophic, 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.
... A second independent production of RXFP1 knockout mice reported a complete absence of a male phenotype, with homozygous mutant males being indistinguishable from wild types and with no impairment of fertility (Kamat et al., 2004). The early phenotype of the RLN knockout mouse has been well characterized by Samuel et al. (2003Samuel et al. ( , 2007. These mice were generated on a 129sv genetic background and largely from the F2 and F3 generations. ...
... In addition, testes exhibited marked fibrosis, particularly in older animals, assessed both by Masson trichrome staining and by increased hydroxyproline content. This effect could be reversed by chronic relaxin infusion (Samuel et al., 2007). Significantly, the incidence of apoptotic cells in the seminiferous compartment was elevated as measured by Bax and caspase-9 staining (Samuel et al., 2003). ...
The human genome project has identified, besides ovarian relaxin (RLN), six other relaxin-like molecules (RLN3, H1-RLN, INSL3-6), most of which appear to be expressed in the testis and/or male reproductive system, together with four different G-protein-coupled receptors responsive to one or other of these peptides. Earlier work on relaxin in the male assumed the simplistic hypothesis of only a single relaxin-like entity. This review systematically examines the expression and physiology of relaxin-like molecules in the male reproductive system in order to reappraise the importance of this hormone system for male reproductive function. Although there are important species differences, only INSL3 and INSL6 appear to be generally expressed at a moderately high level within the testis, whereas ovarian RLN is consistently a major secretory product of the prostate epithelium. However, all members of this relaxin-like family appear to be expressed also at a low level in different organs of the male reproductive system, suggesting possible autocrine/paracrine effects. The four receptors (RXFP1-4) for these peptides are also expressed to differing levels in both somatic and seminiferous compartments of the testis and in the prostate, supporting relevant functions for most members of this interesting peptide family. Recent studies of relaxin family peptides in prostate pathology highlight their functional importance in the clinical context as potential causative, diagnostic and therapeutic agents and warrant more specific and detailed studies of their roles also in regard to male fertility and other aspects of male reproductive function.
... The chemical ligand provides ideal chelating templates for the complexation of various metal ions due to the ability of their structures to contain nitrogen and sulfur atoms. The chemical functional ligands are useful for diverse potentiality both in medicinal chemistry and drug delivery [34][35][36]. Another promising Schiff base group has strong potential to be used as a chelating polydentate ligand that can be coordinated with copper ions to former stable complexes. ...
The chemical ligand-based conjugate adsorbent was prepared for selective and sensitive toxic lead (Pb(II)) ions monitoring and removal from contaminated water to save water quality and human health. Both in the monitoring and removal operations, the solution pH played a vital role by the conjugate adsorbent. The data clarified that pH 3.50 was suitable for simultaneous monitoring and removal of Pb(II) ions. In the monitoring, a significant color was formed upon the addition of Pb(II) ion even in the presence of ultra-trace level. Then the solution pH, reaction time, foreign ion, and initial concentration effect were systematically measured. The conjugate adsorbent exhibited an extremely low detection limit (0.35 µg/L) to comply with the material’s applicability in real sample onsite monitoring. In the reaction time effect, color optimization and high removal efficiency was achieved by increasing the reaction time. However, the diverse foreign ions were not adversely affected in the monitoring and removal to clarify the conjugate adsorbent as sensitive and selective towards the Pb(II) ions. The conjugate adsorbent was shown highly ordered structure and was able to open high functionality for Pb(II) ion monitoring and removal. The adsorption data also revealed that the present conjugate adsorbent exhibited adsorption capacity (172.87 mg/g) and was well fitted by the Langmuir adsorption isotherm with monolayer coverage. The adsorbed Pb(II) ion was desorbed using 0.30 M HCl and then simultaneously regenerated into the initial form for several cycles use after rinsing with water. Then it is expected that the optimum protocol effectively captured the Pb(II) in a sustainable and environmentally friendly manner for the production of clean water to safeguard public health and environmental remediation.
... For comparison, we synthesized native H2 relaxin using the previous approach, [31] which resorted to stepwise disulfide bond formation due to the impracticality of the direct oxidative folding. [32] First, the B chain incorporating Cys-(Acm) at B23 and a pseudoproline dipeptide at B26 Ser -B27 Thr was made through Fmoc SPPS. ...
Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next‐generation drugs. Separate synthesis of multiple peptide fragments and tedious chain‐to‐chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre‐made diaminodiacid bridge at A‐B chain terminal disulfide can be easily and rapidly synthesized by a single‐shot automated solid‐phase synthesis and expedient one‐step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.
... rhRLN has been shown to have full bioactivity with even slightly lower EC50 compared to rat relaxin in a mouse RXFP1 receptor bioassay (Scott et al., 2004), having high homology in those domains responsible for receptor binding (Bathgate et al., 2006). Thus, altogether relaxin-treated mice received a total of 50 ng rhRLN in 30 µl vehicle per uterus, equivalent to ∼270 nM which, accounting for degradation and dilution, should still be more than sufficient to activate murine RXFP1 receptors (EC50 ca 1 nM; Samuel et al., 2007). Although mouse seminal relaxin concentration is not known, data from other species suggest a seminal concentration of ∼0.2-2 nM (Ivell et al., 2011). ...
The hormone relaxin is important in female reproduction for embryo implantation, cardiovascular function, and during labor and lactation. Relaxin is also synthesized in males by organs of the male tract. We hypothesized that relaxin might be one component of seminal plasma responsible for eliciting the female cytokine response induced in the uterus at mating. When recombinant relaxin was injected into the uterus of wild-type (Rln+/+) mice at estrus, it evoked the production of Cxcl1 mRNA and its secreted protein product CXCL1 in four of eight animals. Mating experiments were then conducted using mice with a null mutation in the relaxin gene (Rln−/− mice). qRT-PCR analysis of mRNA expression in wild-type females showed diminished uterine expression of several cytokine and chemokine genes in the absence of male relaxin. Similar differences were also noted comparing Rln−/− and Rln+/+ females mated to wild-type males. Quantification of uterine luminal fluid cytokine content confirmed that male relaxin provokes the production of CXCL10 and CSF3 in Rln+/+ females. Differences were also seen comparing Rln−/− and Rln+/+ females mated with Rln−/− males for CXCL1, CSF3, and CCL5, implying that endogenous relaxin in females might prime the uterus to respond appropriately to seminal fluid at coitus. Finally, pan-leukocyte CD45 mRNA was increased in wild-type matings compared to other combinations, implying that male and female relaxin may trigger leukocyte expansion in the uterus. We conclude that male and/or female relaxin may be important in activating the uterine cytokine/chemokine network required to initiate maternal immune adaptation to pregnancy.
... RLN acts also as an antagonist for fibrosis, and promotes wound-healing, neo-angiogenesis, and vasodilation. 19,35,36 It also affects both contraction and relaxation of ligaments around the pelvis for the maintenance of pregnancy and parturition. 18,37 RLN has been shown to promote matrix remodeling by increasing cell proliferation, reducing α-SMA expression, and decreasing collagen synthesis in renal fibroblasts. ...
Purpose
The aim of this study was to investigate the anti-fibrotic effect of relaxin in subsynovial fibroblasts activated by transforming growth factor beta (TGF-β).
Materials and Methods
To test the anti-fibrotic effect of an adenovirus-relaxin construct (Ad-RLN) on subsynovial fibroblasts in vitro, cells from subsynovial connective tissue of patients with carpal tunnel syndrome were activated with TGF-β1 and exposed to Ad-RLN (as a therapeutic gene) or adenovirus-lacZ construct (as a marker gene) for four hours. Subsynovial fibroblast cultures without adenoviral exposure served as controls.
Results
We observed induction of gene expressions of collagen I, III and IV, as well as the abatement of alpha-smooth muscle actin (a-SMA) synthesis, Smad2 phosphorylation, and fibronectin at the protein level, in comparison to controls. In addition, protein expressions of matrix metalloproteinase (MMP) I was significantly induced, whereas the protein expressions of tissue inhibitor of metalloproteinases (TIMP) I and IV were reduced due to relaxin expression.
Conclusion
RLN prevents excessive synthesis of extracellular matrix by reducing the expressions of its components, such as fibronectin, a-SMA, and phosphorylated Smad2, by increasing the expression of MMPs; and by decreasing the expression of TIMPs.
... Decades after its discovery [12], relaxin was and still is considered as a hormone of pregnancy and parturition despite its detection in various males species, including pigs. At present, numerous studies in rodents have brought strong evidences indicating the crucial but reversible roles of relaxin on male reproduction [33][34][35]. Here, we show that (i) boar reproductive tract and sex glands express relaxin receptors, (ii) boar testes rather than prostate and seminal plasma accumulate higher levels of relaxin, (iii) relaxin protein is present in the vicinity of spermatozoa upon the onset of spermatogenesis, (iv) spermatozoa possess relaxin and its receptors, suggesting autocrine and/or paracrine actions during their epididymal and post-epididymal lifespan. ...
Relaxin levels in seminal plasma have been associated with positive effects on sperm motility and quality, and thus having potential roles in male fertility. However, the origin of seminal relaxin, within the male reproductive tract, and the moment of its release in the vicinity of spermatozoa remain unclear. Here, we assessed the longitudinal distribution of relaxin and its receptors RXFP1 and RXFP2 in the reproductive tract, sex accessory glands, and spermatozoa of adult boars.
Spermatozoa were harvested from three fertile boars and reproductive tract (testes and epididymis) and sex accessory gland (prostate and seminal vesicles) tissues were collected post-mortem from each boar. Epididymis ducts were sectioned into caput, corpus, and cauda regions, and spermatozoa were mechanically collected. All samples were subjected to immunofluorescence and/or western immunoblotting for relaxin, RXFP1, and RXFP2 detection. Immunolabeled-spermatozoa were submitted to flow cytometry analyses and data were statistically analyzed with ANOVA.
Both receptors were detected in all tissues, with a predominance of mature and immature isoforms of RXFP1 and RXFP2, respectively. Relaxin signals were found in the testes, with Leydig cells displaying the highest intensity compared to other testicular cells. The testicular immunofluorescence intensity of relaxin was greater than that of other tissues. Epithelial basal cells exhibited the highest relaxin immunofluorescence intensity within the epididymis and the vas deferens. The luminal immunoreactivity to relaxin was detected in the seminiferous tubule, epididymis, and vas deferens ducts. Epididymal and ejaculated spermatozoa were immunopositive to relaxin, RXFP1, and RXFP2, and epididymal corpus-derived spermatozoa had the highest immunoreactivities across epididymal sections. Both vas deferens-collected and ejaculated spermatozoa displayed comparable, but lowest immunofluorescence signals among groups. The entire sperm length was immunopositive to both relaxin and receptors, with relaxin signal being robust in the acrosome area and RXFP2, homogeneously distributed than RXFP1 on the head of ejaculated spermatozoa.
Immunolocalization indicates that relaxin-receptor complexes may have important roles in boar reproduction and that spermatozoa are already exposed to relaxin upon their production. The findings suggest autocrine and/or paracrine actions of relaxin on spermatozoa, either before or after ejaculation, which have possible roles on the fertilizing potential of spermatozoa.
... Despite these disappointing findings there are many studies in animals that show a role for relaxin in controlling collagen turnover. In relaxin knockout mice, there is a progressive increase in tissue fibrosis with age in male mice that is prevented (Samuel et al., 2007) or reversed in lung (Samuel et al., 2003b), kidney (Samuel et al., 2004b), and heart (Samuel et al., 2004a) by the administration of relaxin. ...
Relaxin, insulin-like peptide 3 (INSL3), relaxin-3, and INSL5 are the cognate ligands for the relaxin family peptide (RXFP) receptors 1-4, respectively. RXFP1 activates pleiotropic signaling pathways including the signalosome protein complex that facilitates high-sensitivity signaling; coupling to Gαs, Gαi, and Gαo proteins; interaction with glucocorticoid receptors; and the formation of hetero-oligomers with distinctive pharmacological properties. In addition to relaxin-related ligands, RXFP1 is activated by Clq-tumor necrosis factor-related protein 8 and by small-molecular-weight agonists, such as ML290 [2-isopropoxy-N-(2-(3-(trifluoromethylsulfonyl)phenylcarbamoyl)phenyl)benzamide], that act allosterically. RXFP2 activates only the Gαs- and Gαo-coupled pathways. Relaxin-3 is primarily a neuropeptide, and its cognate receptor RXFP3 is a target for the treatment of depression, anxiety, and autism. A variety of peptide agonists, antagonists, biased agonists, and an allosteric modulator target RXFP3. Both RXFP3 and the related RXFP4 couple to Gαi/Gαo proteins. INSL5 has the properties of an incretin; it is secreted from the gut and is orexigenic. The expression of RXFP4 in gut, adipose tissue, and β-islets together with compromised glucose tolerance in INSL5 or RXFP4 knockout mice suggests a metabolic role. This review focuses on the many advances in our understanding of RXFP receptors in the last 5 years, their signal transduction mechanisms, the development of novel compounds that target RXFP1-4, the challenges facing the field, and current prospects for new therapeutics.
Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.
... Native recombinant DNA-derived human relaxin-2 used as control was obtained from Corthera Inc. (San Francisco, CA, a subsidiary of Novartis AG, Basel, Switzerland). Synthetic europium-labelled H2 relaxin, unlabelled H2, and human INSL3 were prepared in-house as previously described [16,17]. ...
Human (H2) relaxin is a two-chain peptide member of the insulin superfamily and possesses potent pleiotropic roles including regulation of connective tissue remodeling and systemic and renal vasodilation. These effects are mediated through interaction with its cognate G-protein-coupled receptor, RXFP1. H2 relaxin recently passed Phase III clinical trials for the treatment of congestive heart failure. However, its in vivo half-life is short due to its susceptibility to proteolytic degradation and renal clearance. To increase its residence time, a covalent dimer of H2 relaxin was designed and assembled through solid phase synthesis of the two chains, including a judiciously monoalkyne sited B-chain, followed by their combination through regioselective disulfide bond formation. Use of a bisazido PEG 7 linker and " click " chemistry afforded a dimeric H2 relaxin with its active site structurally unhindered. The resulting peptide possessed a similar secondary structure to the native monomeric H2 relaxin and bound to and activated RXFP1 equally well. It had fewer propensities to activate RXFP2, the receptor for the related insulin-like peptide 3. In human serum, the dimer had a modestly increased half-life compared to the monomeric H2 relaxin suggesting that additional oligomerization may be a viable strategy for producing longer acting variants of H2 relaxin.
... The strategy used for the synthesis of bombyxin-IV was shown to work well for synthesis of H2 relaxin although the overall yield was modest, being about 10% relative to 50 Synthetic biomolecules the starting crude B-chain intermediate ( Figure 3II). It also allowed the preparation of relaxin-2 from other species including horse and mouse [19,20], and led to the first-ever acquisition of human relaxin-3 which was previously shown to be refractive to production via the random chain refolding approach [2,21]. Unlike with H2 relaxin, careful placement of the thiol protecting groups was also required in order to minimize the detrimental effect of the final iodine oxidation step used for disulfide linking of the Cys(Acm) residues. ...
The relaxin subfamily of peptides within the human insulin superfamily consists of seven members including relaxin-2 and relaxin-3. The former is a pleiotropic hormone that is a vasodilator and cardiac stimulant in the cardiovascular system and an antifibrotic agent whereas the latter is primarily a neuropeptide involved in stress and metabolic control. Both possess the unique three-disulfide heterodimeric peptide structure of insulin. Consequently, the synthesis, both chemical and biological, of relaxin-2 and relaxin-3 has long represented a special challenge to further understanding their structural and functional relationships. This review highlights past and recent developments in the use of chemical and recombinant DNA methods of synthesis of these peptides and current resulting knowledge of their biology.
... Three orthogonal cysteine S-protecting groups (Trt, tBu and Acm) were used that allowed the sequential formation of three disulfide bonds. Solid phase synthesis of the separate, selectively S-protected A-and B-chains followed by their purification and subsequent stepwise formation of each of the three disulphide bonds via oxidation, thioloysis, and iodolysis [28][29][30] led to the successful preparation of H3 peptides. Their analytical RP-HPLC profiles and MALDI TOF MS spectrometric analysis (H3 acid theoretical, MH+5501.52 found, MH+5500.63; ...
Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains' C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors.
... The results suggest that human relaxin may be effective against hepatic fibrosis at lower doses than previously reported. Consistent with this concept, a recent study described the effect of murine relaxin, which would not trigger the antibody response, on spontaneous fibrosis in the relaxin-null mice (35). In that study, murine relaxin at 100 lg/kg/day for an extended period was effective in reducing fibrosis in a number of tissues. ...
Hepatic fibrosis is characterized by excess collagen deposition, decreased extracellular matrix degradation and activation of the hepatic stellate cells. The hormone relaxin has shown promise in the treatment of fibrosis in a number of tissues, but the effect of relaxin on established hepatic fibrosis is unknown. The aim of this study was to determine the effect of relaxin on an in vivo model after establishing hepatic fibrosis METHODS: Male mice were made fibrotic by carbon tetrachloride treatment for 4 weeks, followed by treatment with two doses of relaxin (25 or 75 μg/kg/day) or vehicle for 4 weeks, with continued administration of carbon tetrachloride.
Relaxin significantly decreased total hepatic collagen and smooth muscle actin content at both doses, and suppressed collagen I expression at the higher dose. Relaxin increased the expression of the matrix metalloproteinases MMP13 and MMP3, decreased the expression of MMP2 and tissue inhibitor of metalloproteinase 2 (TIMP2) and increased the overall level of collagen-degrading activity. Relaxin decreased TGFβ-induced Smad2 nuclear localization in mouse hepatic stellate cells.
The results suggest that relaxin reduced collagen deposition and HSC activation in established hepatic fibrosis despite the presence of continued hepatic insult. This reduced fibrosis was associated with increased expression of the fibrillar collagen-degrading enzyme MMP13, decreased expression of TIMP2, and enhanced collagen-degrading activity, and impaired TGFβ signalling, consistent with relaxin's effects on activated fibroblastic cells. The results suggest that relaxin may be an effective treatment for the treatment of established hepatic fibrosis.
... These effects were likely mediated through the interaction of RLX with its specific receptor RXFP1, which is fairly expressed by both NIH/3T3 fibroblasts and cardiac stromal cells and is known to play a major role in the modulation of RLX-induced tissue remodeling [16,44,45]. ...
The hormone relaxin (RLX) is produced by the heart and has beneficial actions on the cardiovascular system. We previously demonstrated that RLX stimulates mouse neonatal cardiomyocyte growth, suggesting its involvement in endogenous mechanisms of myocardial histogenesis and regeneration. In the present study, we extended the experimentation by evaluating the effects of RLX on primary cultures of neonatal cardiac stromal cells. RLX inhibited TGF-β1-induced fibroblast-myofibroblast transition, as judged by its ability to down-regulate α-smooth muscle actin and type I collagen expression. We also found that the hormone up-regulated metalloprotease (MMP)-2 and MMP-9 expression and downregulated the tissue inhibitor of metalloproteinases (TIMP)-2 in TGF-β1-stimulated cells. Interestingly, the effects of RLX on cardiac fibroblasts involved the activation of Notch-1 pathway. Indeed, Notch-1 expression was significantly decreased in TGF-β1-stimulatedfibroblasts as compared to the unstimulated controls; this reduction was prevented by the addition of RLX to TGF-β1-stimulated cells. Moreover, pharmacological inhibition of endogenous Notch-1 signaling by N-3,5-difluorophenyl acetyl-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT), a γ-secretase specific inhibitor, as well as the silencing of Notch-1 ligand, Jagged-1, potentiated TGF-β1-induced myofibroblast differentiation and abrogated the inhibitory effects of RLX. Interestingly, RLX and Notch-1 exerted their inhibitory effects by interfering with TGF-β1 signaling, since the addition of RLX to TGF-β1-stimulated cells caused a significant decrease in Smad3 phosphorylation, a typical downstream event of TGF-β1 receptor activation, while the treatment with a prevented this effect. These data suggest that Notch signaling can down-regulate TGF-β1/Smad3-induced fibroblast-myofibroblast transition and that RLX could exert its well known anti-fibrotic action through the up-regulation of this pathway. In conclusion, the results of the present study beside supporting the role of RLX in the field of cardiac fibrosis, provide novel experimental evidence on the molecular mechanisms underlying its effects.
... Samuel et al. [27] reported that relaxin injection increases collagen metabolism as a result of its antifibrotic properties in vivo. Samuel et al. [28] also reported that the administration of synthetic (JNK inhibitor), or SP600125 (p38 inhibitor) for 1 hr before relaxin challenge. The induction of MMP-1 mRNA by relaxin was suppressed by the MAPK inhibitors (Tukey test, * * p < 0.01 indicates a significant difference from the corresponding control at the respective time point, † indicates p < 0.05, † † p < 0.01 indicates a significant difference from relaxin (A)). ...
The relapse of teeth that have moved during orthodontic treatment is a major clinical issue with respect to the goals of successful treatment. Relaxin has an influence on many physiologic processes, such as collagen turnover. In this study, we determined the effects of relaxin on the relapse and remodeling of periodontal tissue after experimental tooth movement in rats, and we explored the molecular mechanism underlying these processes. To induce experimental tooth movement in rats, 10 g of orthodontic force was applied to the molars. After 14 days, the spring was removed, and then animals began receiving relaxin at a dose of 500 ng/ml for 1 week. The results were evaluated by micro-computed tomography and immunofluorescence staining. In addition, the effects of matrix metalloproteinase (MMP)-1 and MMP-8 production were investigated in human periodontal ligament (hPDL) cells in vitro. The expression of MMP-1 and MMP-8 was analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Furthermore, we demonstrated the signaling pathways involved in relaxin-regulated MMPs expression. The relapse distances and percentages were significantly decreased in the experimental group compared with the controls in vivo. A double-immunofluorescence analysis for Col-I/MMP-1 and Col-I/MMP-8 detected the expression of relaxin in the PDL. Relaxin significantly increased the MMP-1 and MMP-8 expression in a time-dependent manner in hPDL cells in vitro. Furthermore, a p38 inhibitor (SB203580) significantly inhibited the MMP-1 and MMP-8 expression. Our results indicated that relaxin modulates the collagen metabolism, and this hormone may therefore be useful to prevent orthodontic relapse following orthodontic treatment.
A relaxin‐like gonad‐stimulating peptide (RGP), Aso‐RGP, featuring six cysteine residues, was identified in the Crown‐of‐Thorns Starfish (COTS, Acanthaster cf. solaris) and initially produced through recombinant yeast expression. This method yielded a single‐chain peptide with an uncleaved C‐peptide (His Tag) and suboptimal purity. Our objective was to chemically synthesize Aso‐RGP in its mature form, comprising two chains (A and B) and three disulfide bridges, omitting the C‐peptide. Furthermore, we aimed to synthesize a newly identified relaxin‐like peptide, Aso‐RLP2, from COTS, which had not been previously synthesized. This paper reports the first total chemical synthesis of Aso‐RGP and Aso‐RLP2. Aso‐RGP synthesis proceeded without major issues, whereas the A‐chain of Aso‐RLP2, in its reduced and unfolded state with two free thiols, presented considerable challenges. These were initially marked by “messy” RP‐HPLC profiles, typically indicative of synthesis failure. Surprisingly, oxidizing the A‐chain significantly improved the RP‐HPLC profile, revealing the main issue was not synthesis failure but the peptide's aggregation tendency, which initially obscured analysis. This discovery highlights the critical need to account for aggregation in peptide synthesis and analysis. Ultimately, our efforts led to the successful synthesis of both peptides with purities exceeding 95 %.
The novel organic compound of ammonium (4-chlro-2-mercaptophenyl)carbamodithioate (ACMPC) was prepared and then subsequently grafted onto the mesoporous silica by direct approach for the preparation of composite adsorbent (CPA). The lanthanide (Ln(III)) intra-series separation behavior was measured using CPA and then the samarium (Sm(III)) ion was selected according to the high adsorption ability by CPA for effective separation, adsorption, and recovery in the solid-liquid approach. The mesoporous silica and CPA were characterized systematically for the evaluation of affecting experimental criterion. The solution acidity played an important factor as the alkaline pH area was avoided due to the hydroxide precipitation probability and pH 5.0 was selected for selective and effective Sm(III) ion separation and recovery. The data clarified that CPA exhibited high kinetic performances with high adsorption ability. The Sm(III) adsorption was highly fitted with the Langmuir adsorption isotherm model with monolayer coverage and the maximum adsorption capacity was determined as 155.13 mg/g. In addition, the diverse foreign ions were not reduced the Sm(III) ion adsorption significantly, and the CPA has approximately no adsorption capacity for other ions at this pH. The Sm(III) ion was strongly coordinated with the ACMPC and the expected highly stable complexation mechanism with the soft donor of N- and S- atoms. The elution of Sm(III) ions from the saturated CPA was desorbed successfully with 0.30 M HNO3. The regenerated CPA that remained maintained the high selectivity to Sm(III) ions and exhibited almost the same adsorption capacity as that of the original CPA. Therefore, the proposed CPA offered a cost-effective material and may be considered a viable alternative for effectively Sm(III) ion separation and recovery from waste samples as potential materials.
The prevalence of dyes in aquatic environments raises severe concerns on a global scale. Methyl orange (MO) is a typical anionic organic dye, which is widely used in industrial wastewater such as textile and paper making. Then the treatment of water contaminated with dyes is an important aspect. The recent decade has witnessed adsorption technology emerging as an advanced dye wastewater treatment with great potential and a grand blueprint, in which the specific surface area and active sites of the adsorbent are considered to be the two most important characteristics largely impacting the adsorption performance. In this study, chitosan-treated nanocomposite was prepared as an effective adsorbent for the removal of MO from contaminated water. The dye removal parameter was performed according to the solution acidity, reaction time, initial concentration, competing for ion affinity, maximum adsorption capacity, and reuse with potential use. The solution pH played a key role in MO dye removal and a suitable pH of 7.0 was selected according to high adsorption ability. The adsorption results were highly fitted with the Langmuir adsorption isotherm model and the maximum adsorption was 172.17 mg/g. The results revealed that introducing chitosan could improve the adsorption capacity and rate effectively even though sacrificing part of specific surface areas of the cotton, indicating that active sites might play a dominant role during the MO adsorption. In addition, the fabricated nanocomposite adsorbent was recycled rapidly by the eluent and regenerated simultaneously, which exhibited the advantages of easy operation as a potentially cost-effective material. The chitosan-based nanocomposite displayed high reusability based on the elution and simultaneous regeneration ability. Therefore, as a cheap green nanocomposite adsorbent with high adsorption performance for MO, chitosan-based fibrous nanocomposite adsorbent is expected to become one of the best candidate materials for future industrial wastewater treatment.
The hybrid donor chemical ligand of 5-tert-butyl-2-hydroxybenzaldehyde thiosemicarbazone (THTB) was prepared and then embedded onto inorganic porous silica as hybrid conjugate materials (HCM). The Europium (Eu (III)) ion was selected from the lanthanides (Ln(III)) series for green and robust adsorption and recovery based on the adsorption, complexation, and selectivity tendency from the standpoint of the pH-dependent factor. The chemical compound of THTB consisted of ON N-, and S-donor atoms and was able to make stable complexation with Ln(III) ions in optimum conditions due to the open functionality of the HCM. A surface complexation with a good complexation fitting to the experimentally collected data was used to describe the adsorption mechanism. The Eu(III) ion adsorption performance was measured with batch equilibrium methods. The affecting experimental protocols including solution pH, contact time, initial Eu(III) ion concentration, foreign ions effect, and recovery were carried out and evaluated consistently. The Eu(III) ion adsorption by the HCM was at pH 5.0 and this pH was selected to avoid the precipitation problem to ensure the adsorption mechanism. The co-existing several metal ions were not interfered with Eu(III) ion adsorption by the HCM due to the high affinity between Eu(III) ion and the functional groups of HCM. The bonding mechanism suggested that ON N-, and S-donor atoms of THTB were strongly coordinated to Eu(III) with 2:1 ratio complexation. The Langmuir adsorption isotherm model was plotted due to the HCM morphology and applied to validate the adsorption isotherms according to the homogeneous ordered frameworks. The Eu(III) ion adsorption capacity by the HCM was 176.31 mg/g as expected because of the high surface area of the HCM. The adsorbed Eu(III) ion was completely eluted from HCM with the eluent of 0.20 M HNO 3 and simultaneously regenerated into its initial form without significant deterioration. This study could be of great applicative utility for Eu(III) ions from waste aqueous solutions as green technology.
Commercially available insulins are manufactured by recombinant methods for the treatment of diabetes. Long-acting insulin drugs (e.g., detemir and degludec) are obtained by fatty acid conjugation at LysB29 ε-amine of insulin via acid-amide coupling. There are three amine groups in insulin, and they all react with fatty acids in alkaline conditions. Due to the lack of selectivity, such conjugation reactions produce non-desired byproducts. We designed and chemically synthesized a novel thiol-insulin scaffold (CysB29-insulin II), by replacing the LysB29 residue in insulin with the CysB29 residue. Then, we conjugated a fatty acid moiety (palmitic acid, C16) to CysB29-insulin II by a highly efficient and selective thiol-maleimide conjugation reaction. We obtained the target peptide (palmitoyl-insulin) rapidly within 5 min without significant byproducts. The palmitoyl-insulin is shown to be structurally similar to insulin and biologically active both in vitro and in vivo. Importantly, unlike native insulin, palmitoyl-insulin is slow and long-acting.
The organic ligand of 3–(((5–ethoxybenzenethiol)imino)methyl)–salicylic acid (EBMS) was synthesized and then immobilized onto the mesoporous silica for the fabrication of facial composite adsorbent (FCA). The FCA was successfully utilized for the heavy lanthanide of Thulium(Tm(III)) separation, adsorption, and recovery in the solid-liquid approach. The FCA characterizations and affecting experimental parameters were assessed systematically. The Tm(III) ion was selected due to the high adsorption ability of the FCA according to the intra-series separation behaviors. The solution pH played a key role based on hydroxide formation in the basic pH region and a slightly acidic pH (3.50) was selected based on the high adsorption ability. The FCA was shown high kinetic performance and diverse competing metal ions did not interfere with Tm(III) adsorption. The Tm(III) adsorption was well-fitted with the Langmuir adsorption isotherm model with monolayer coverage and the maximum adsorption capacity was determined as 168.57 mg/g. The Tm(III) ion was strongly coordinated with the EBMS and the expected bond distance between Tm-N was shorter than the other bond length of Tm-S atoms in the complexation mechanism. The adsorbed Tm(III) ion was completely desorbed from the FCA with the suitable eluent of 0.25 M HNO3 and then simultaneously regenerated the FCA into the initial form without significant deterioration in the original functionality. The highest selectivity and maximum adsorption capacity of FCA to Tm(III) ions indicated that the proposed EBMS ligand-based composite adsorbent high potentiality to separate and recover the Tm(III) ions from waste samples effectively.
Dye wastewater has a severe influence on the natural water environment. Using materials functioned by adsorption to remove dyes has received much attention due it’s to its high decolorization or removal efficiency. The study aims to complement chitosan-treated cotton composite material for toxic dye removal from contaminated water to save human health. The work identifies and analyses the removal of reactive dye (Remazol Brilliant Red F3B (RR)) and proffers its high adsorption ability with chitosan-cotton composite. The dye removal parameter was performed based on the solution pH, contact time, initial concentration, competing for ion affinity, maximum adsorption capacity, and reuses with potential use. The solution pH was shown important parameter to RR dye uptake and a suitable pH of 7.0 was selected according to high adsorption ability. The competing ions were not adversely affected in the dye adsorption as expected of a stable bonding mechanism. The adsorption results were highly fitted with the Langmuir adsorption isotherm model and the maximum adsorption was 169.33 mg/g. The chitosan-cotton composite displayed high reusability based on the elution and simultaneous regeneration ability. The adsorbed RR dye was eluted using ethanol and reused for the next operation after washing with water without significant deterioration.
The functional organic ligand of 4-tert-Octyl-4-((phenyl)diazenyl)phenol (TPDP) was immobilized directly onto the mesoporous silica for the fabrication of composite adsorbent to detect and remove the toxic copper (Cu(II)) ions from contaminated water. The mesoporous silica and the composite adsorbent were characterized systematically using different instrumentations. Upon addition of a trace amount of Cu(II) with composite adsorbent, a significant color was formed to visualize the Cu(II) ion detection at optimum experimental protocol. The pH played a key factor in the detection and removal operation and the optimum pH was 4.0 for this study. The effect of pH, color optimization, contact time, competing ions, and concentration was assessed systematically both in the detection and removal operations. The limit detection by the composite adsorbent to Cu(II) ion was 0.28 µg/L. The diverse metal ions did not interfere during the Cu(II) ion detection and removal by the composite adsorbent and complied with the high sensitivity for onsite uses as potential materials. The proposed adsorbent also exhibited high adsorption capacity and was well-fitted in the Langmuir adsorption isotherms in monolayer coverage and the maximum adsorption capacity was as high as 184.73 mg/g. The Cu(II) ion was eluted from the composite adsorbent using 0.15 M HCl and then simultaneously regenerated into the initial stage without losing its major functionality for the next use operation. However, the adsorption efficiency was slightly decreased after several cycles of use according to the data. Then it is estimated that the fabricated ligand-based composite adsorbent in the real waste sample treatment for detection and removal of Cu(II) ions as a low-cost material without using highly sophisticated instrumentations.
Chemical synthesis of insulin superfamily proteins (ISPs) has recently been widely studied to develop next‐generation drugs. Separate synthesis of multiple peptide fragments and tedious chain‐to‐chain folding are usually encountered in these studies, limiting accessibility to ISP derivatives. Here we report the finding that insulin superfamily proteins (e.g. H2 relaxin, insulin itself, and H3 relaxin) incorporating a pre‐made diaminodiacid bridge at A‐B chain terminal disulfide can be easily and rapidly synthesized by a single‐shot automated solid‐phase synthesis and expedient one‐step folding. Our new H2 relaxin analogues exhibit almost identical structures and activities when compared to their natural counterparts. This new synthetic strategy will expediate production of new ISP analogues for pharmaceutical studies.
The Layered double hydroxides (LDHs) were used for the toxic diverse metal ions accumulation owing to their functional sites and complexation capabilities. Their intercalation with ligands like sodium hexametaphosphate (SHMP) can enhance the active sites for heavy metal adsorption. Here, the Mg/Al-LDHs were inscribed with SHMP using the co-precipitation method to detoxify wastewater by removing lead (Pb²⁺) and cadmium (Cd²⁺). Mg/Al LDH-SHMP composite adsorbent was investigated using several instrumentations such as XRD, FT-IR, and FE-SEM-EDX for identifying the functional and morphological properties which are accountable for lead and cadmium cations adsorption. The adsorption was studied in a batch method to investigate the adsorption performance by optimizing pH, adsorbent dose, time of contact, concentration, and adsorption temperature. The adsorption isotherm, thermodynamics, kinetics of the adsorption progress, and mechanism were thoroughly investigated. The pseudo-second-order model gave the highest outcomes in terms of fitting the kinetic data, with the greatest correlation coefficient for both cations. The trend of isotherm was adjacent to the Langmuir model with the highest removal amount of 45.66 and 24.34 mgg⁻¹ for Pb²⁺ and Cd²⁺, respectively. The thermodynamic investigation exhibited that the adsorption reaction was spontaneous endothermic, and random in character. The capacity for absorption improved as a result of the higher temperature. Moreover, Pb²⁺ and Cd²⁺ accumulation onto the Mg/Al LDH-SHMP surface was plausible with surface complexations, isomorphic substitution, precipitation, and heavy metal chelation. The Mg/Al LDH-SHMP composite could be an ideal material for Pb²⁺ and Cd²⁺ accumulation from the wastewater for its high adsorption capacity and fast adsorption kinetics.
Amino Acids, Peptides and Proteins comprises a comprehensive review of significant developments at this biology/chemistry interface. Each volume of this Specialist Periodical Report opens with an overview of amino acids and their applications.
In keeping with the preceeding volumes in the series, this volume presents contributions from across the globe addressing the hot topics in the field. Disulfide-containing peptides and proteins are investigated by NMR, and mass spectrometry is used to determine inter-peptide distant constraints. Further chapters review the latest literature on antimicrbial peptides, modifications by Cytochrome P450 and the relaxin-family neuropeptides. Self-assembly and the moleculatr recognition of designed peptides are also discussed, and the latest in peptide and protein-based pharmaceuticals are reviewed. Volume editor Max Ryadnov also contributes a chapter on biofunctional peptide design.
As the published literature in the field continues to grow, researchers in academia and industry will find this comprehensive review of the current research and thought an essential first point of reference.
The formation of disulfide bonds is probably the most influential modification of peptides and proteins. An elaborate set of cellular machinery exists to catalyze and guide this process. In recent years, significant developments have been made in both our understanding of the in vivo situation and the in vitro manipulation of disulfide bonds. This is the first monograph to provide a comprehensive overview of this exciting and rapidly developing area. It offers in-depth insights into the mechanisms of in vivo and in vitro oxidative folding of proteins as well as mono- and multiple-stranded peptides. Procedures applied for laboratory and industrial purposes are also discussed by top experts in the field. The book describes the enzymes involved in the correct oxidative folding of cysteine-containing proteins in prokaryotes and eukaryotes. It then goes on to discuss the mimicking of these enzymes for successful in vitro folding of proteins (including synthetic replicates) and to deal with important issues concerning cysteine-rich peptides. The ability of natural bioactive peptides to fold correctly, and in high yields, to form defined structural motifs using cysteine sequence patterns is still puzzling. With this in mind, synthetic procedures for establishing native cysteine frameworks are discussed using selected examples, such as the potential of selenocysteines. The biotechnological and pharmaceutical relevance of proteins, peptides, their variants and synthetic replicates is continuously increasing. Consequently, this book is invaluable for peptide and protein chemists involved in related research and production.
Purpose
The aim of this study was to investigate the effects of relaxin (RLN) expression on fibrosis inhibition in synovial fibroblasts.
Materials and methods
Tissue cells from patients with knee osteoarthritis and >30° flexion contractures were utilised. Synovial fibroblasts were activated by TGF-β1 (two nanograms per millilitre) and then exposed to Ad-RLN as a therapeutic gene, adenovirus-lacZ construct as a marker gene, and SB505124 as an inhibitor for TGF-β1 signal for 48 h. The mRNA expression levels of collagens and MMPs were analysed by reverse transcription-polymerase chain reaction. Also, fibronectin, phosphorylation of Smad2 and ERK1/2, alpha smooth muscle actin, TIMP-1, TIMP-2, MMP-1 and MMP-13 levels were estimated using western blotting, and the total collagen synthesis was assayed.
Results
Ad-RLN-transduced synovial fibroblasts demonstrated 17%, 13%, and 48% reduction in collagen I, III and IV mRNA expression levels, respectively, and a 40% decrease in MMP-3, MMP-8, 20% decrease in MMP-9, MMP-13 mRNA expression, compared to non-Ad-RLN-transduced cells. In protein expression, Ad-RLN-transduced synovial fibroblasts demonstrated 46% increase in MMP-1, 5% decrease in MMP-2, 51% increase in MMP-9, and 22% increase in MMP-13, compared to non-Ad-RLN-transduced cells. Ad-RLN-transduced synovial fibroblasts showed a 25% decrease in TIMP-1 and 65% decrease in TIMP-2 protein expression at 48h, compared to non-Ad-RLN-transduced cells. Ad-RLN-transduced synovial fibroblasts demonstrated a 45% inhibition of fibronectin in protein expression level and 38% decrease in total collagen synthesis at 48h, compared to non-Ad-RLN-transduced cells.
Conclusion
Relaxin expression exerted anti-fibrogenic effects on synovial fibroblasts from patients with knee osteoarthritis and flexion contractures. Therefore, relaxin could be an alternative therapeutic agent during the initial stage of osteoarthritis with flexion contracture by exerting its anti-fibrogenic effects.
The insulin superfamily of peptides is ubiquitous within vertebrates and invertebrates and is characterized by the presence of a set of three disulfide bonds in a unique disposition. With the exception of insulin-like growth factors I and II, which are single chain peptides, the remaining 8 members of the human insulin superfamily are two-chain peptides containing one intramolecular and two intermolecular disulfide bridges. These structural features have long made the chemical synthesis of the peptides a considerable challenge, in particular, including their correct disulfide bond pairing and formation. However, they have also afforded the opportunity to develop modern solid phase synthesis methods for the preparation of such peptides that incorporate novel or improved chemical methods for the controlled introduction of both disulfide bonds and their surrogates, both during and after peptide chain assembly. In turn, this has enabled a detailed probing of the structure and function relationship of this small but complex superfamily of peptides.
This report presents an entirely chemical, general strategy for the synthesis of relaxin-2 and insulin-like peptide 5. Historically, these two peptides have represented two of the more synthetically challenging members of the insulin superfamily. The key synthetic steps involve two sequential oxime ligations to covalently link the individual A-chain and B-chain, followed by disulfide bond formation under aqueous, redox conditions. This is followed by two chemical reactions that employ diketopiperazine cyclization-mediated cleavage and ester hydrolysis to liberate the connecting peptide and the heterodimeric product. This approach avoids the conventional iodine-mediated disulfide bond formation and enzyme-assisted proteolysis to generate biologically active two-chain peptides. This novel synthetic strategy is ideally suited for peptides such as relaxin and insulin-like peptide 5 as they possess methionine and tryptophan that are labile under strong oxidative conditions. Additionally, these peptides possess multiple arginine and lysine residues that preclude the use of trypsin-like enzymes to obtain biologically active hormones. This synthetic methodology is conceivably applicable to other two-chain peptides that contain multiple disulfide bonds. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.
A new synthetic route to human relaxin-2 has been established through a sequential disulfide bond formation process in the absence of iodine. It is enabled by a combination of cysteine protection with penicillin G acylase-labile Phacm and a newly identified thiol activator bis(5-(2-methoxyethoxy)-2-pyrimidinyl disulfide. The long-standing challenges in relaxin B-chain assembly and its poor solubility have been solved by the insertion of two isoacyl dipeptide segments. The overall yield was 25% from the B chain and 5.8% from the B-chain starting resin.
Human insulin-like peptide-6 (INSL-6) belongs to the insulin superfamily and shares the distinctive disulfide bond configuration of human insulin. In this report we present the first chemical synthesis of INSL-6 utilizing fluorenylmethyloxycarbonyl-based (Fmoc) solid-phase peptide chemistry and regioselective disulfide bond construction protocols. Due to the presence of an oxidation-sensitive tryptophan residue, two new orthogonal synthetic methodologies were developed. The first method involved the identification of an additive to suppress the oxidation of tryptophan during iodine-mediated S-acetamidomethyl (Acm) deprotection and the second utilized iodine-free, sulfoxide-directed disulfide bond formation. The methodologies presented here offer an efficient synthetic route to INSL-6 and will further improve synthetic access to other multiple-disulfide-containing peptides with oxidation-sensitive residues.
The synthesis of insulin has inspired fundamental advances in the art of peptide science while simultaneously revealing the structure-function relationship of this centrally important metabolic hormone. This review highlights milestones in the chemical synthesis of insulin that can be divided into two separate approaches: (i) disulfide bond formation driven by protein folding and (ii) chemical reactivity-directed sequential disulfide bond formation. Common to the two approaches are the persistent challenges presented by the hydrophobic nature of the individual A-chain and B-chain and the need for selective disulfide formation under mildly oxidative conditions. The extension and elaboration of these synthetic approaches have been ongoing within the broader insulin superfamily. These structurally similar peptides include the insulin-like growth factors and also the related peptides such as relaxin that signal through G-protein-coupled receptors. After a half-century of advances in insulin chemistry, we have reached a point where synthesis is no longer limiting structural and biological investigation within this family of peptide hormones. The future will increasingly focus on the refinement of structure to meet medicinal purposes that have long been pursued, such as the development of a glucose-sensitive insulin. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.
The highly conserved ectodomain of M2 protein (M2e) of influenza virus A is one of the most studied vaccine targets for induction of cross-strain protection. It has been reported that the native form of M2 is a homo-tetramer linked by disulfide bonds and that antibodies reactive to this form correlate with M2-mediated protection. Although vaccine constructs mimicking the quaternary structure of M2 have been described, the chemical syntheses of disulfide-linked dimeric and tetrameric forms of M2e have not yet been reported. By exploiting orthogonal chemistry during the syntheses, we successfully assembled dimeric and tetrameric forms of M2e but found that the antibodies that they elicited had protection properties similar to those of the monomeric form. There was, therefore no advantage mimicking the native conformation to improve vaccine efficacy. Nevertheless, the synthetic strategies have the potential for use in preparation of other dimeric or tetrameric constructs.
Leucine-rich repeat-containing G-protein coupled Receptor 7 (LGR7) is a member of the insulin-relaxin receptor superfamily ....
An iodine-free synthetic route to insulin analogues has been established via a directed disulfide bond formation strategy. This method is completely compatible with oxidation-sensitive residues. The key step is constructing the third disulfide bond via a novel procedure involving phenylacetylaminomethyl group (Phacm), immobilized Penicillin G Acylase, and Ellman's reagent. We expect that this method could be broadly utilized for synthesizing insulin-like and other cysteine-rich peptides, in particular, where oxidation-sensitive residues are present in the sequence.
Over the past few decades, research on the peptide hormone, relaxin, has significantly improved our understanding of its biological actions under physiological and diseased conditions. This has facilitated the conducting of clinical trials to explore the use of serelaxin (human recombinant relaxin). Acute heart failure (AHF) is a very difficult to treat clinical entity, with limited success so far in developing new drugs to combat it. A recent phase-III RELAX-AHF trial using serelaxin therapy given during hospitalization revealed acute (ameliorated dyspnea) and chronic (improved 180-day survival) effects. Although these findings support a substantial improvement by serelaxin therapy over currently available therapies for AHF, they also raise key questions and stimulate new hypotheses. To facilitate the development of serelaxin as a new drug for heart disease, joint efforts of clinicians, research scientists and pharmacological industries are necessary to study these questions and hypotheses. In this review, after providing a brief summary of clinical findings and the pathophysiology of AHF, we present a working hypothesis of the mechanisms responsible for the observed efficacy of serelaxin in AHF patients. The existing clinical and preclinical data supporting our hypotheses are summarized and discussed. The development of serelaxin as a drug provides an excellent example of the bilateral nature of translational research.
Dupuytren's disease is a fibroproliferative connective tissue disorder characterized by contracture of the palmer fascia of the hand. Relaxin (RLN) is a multifunctional factor which contributes to the remodeling of the pelvic ligament by inhibiting fibrosis and inflammatory activities. The aim of this study was to investigate the effect of the RLN gene on the inhibition of fibrosis in myofibroblastic cells. Myofibroblast cells with adenovirus LacZ (Ad-LacZ) as a marker gene or adenovirus relaxin (Ad-RLN) as therapeutic gene showed transgene expressions in beta-galactosidase assay and Western blot analysis. Myofibroblastic cells with Ad-RLN demonstrated a 22% and 48% reduction in collagen I and III mRNA expressions respectively, a 50% decrease in MMP-1, 70% decrease in MMP-2, 80% decrease in MMP-9, and a 15% reduction in MMP-13 protein expression compared with cultures with viral control and saline control. In addition, myofibroblastic cells with Ad-RLN showed a 40% decrease in TIMP 1 and a 15% increase in TIMP 3 protein expression at 48 h compared to cultures with viral control and saline control. Also, myofibroblastic cell with Ad-RLN demonstrated a 74% inhibition of fibronectin and a 52% decrease in total collagen synthesis at 48 h compared with cultures with viral control and saline control. In conclusion, the RLN gene render antifibrogenic effect on myofibroblastic cells from Dupuytren's nodule via direct inhibition of collagen synthesis not through collagenolytic pathway such as MMP-1, -13, TIMP 1, and 3. Therefore relaxin can be an alternative therapeutic strategy in initial stage of Dupuytren's disease by its antifibrogenic effect. © 2013 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.
Enzyme-labile protecting groups have emerged as a green alternative to conventional protecting groups. These groups introduce a further orthogonal dimension and eco-friendliness into protection schemes for the synthesis of complex polyfunctional organic molecules. S-Phacm, a Cys-protecting group, can be easily removed by the action of a covalently immobilized PGA enzyme under very mild conditions. Herein, the versatility and reliability of an eco-friendly combination of the immobilized PGA enzyme and the S-Phacm protecting group has been evaluated for the synthesis of diverse Cys-containing peptides.
We determined the precise role of relaxin family peptide (RXFP) receptors-1 and -2 in the regulation of MMP-9 and -13 by relaxin, and delineated the signaling cascade that contributes to relaxin's modulation of MMP-9 in fibrocartilaginous cells. Relaxin treatment of cells in which RXFP1 was silenced resulted in diminished induction of MMP-9 and -13 by relaxin, whereas overexpression of RXFP1 potentiated the relaxin-induced expression of these proteinases. Suppression or overexpression of RXFP2 resulted in no changes in the relaxin-induced MMP-9 and -13. Studies using chemical inhibitors and siRNAs to signaling molecules showed that PI3K, Akt, ERK and PKC-ζ and the transcription factors Elk-1, c-fos and, to a lesser extent, NF-κB are involved in relaxin's induction of MMP-9. Our findings provide the first characterization of signaling cascade involved in the regulation of any MMP by relaxin and offer mechanistic insights on how relaxin likely mediates extracellular matrix turnover.
The S-acetamidomethyl (Acm) protecting group is widely used in the chemical synthesis of peptides that contain one or more
disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the
sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly
as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated
with the iodination step. We report a simple method for simultaneous post-cysteine (Acm) group removal quenching of iodination
and isolation. Use of large volumes of diethyl ether for direct precipitation action of the oxidized peptide from the 90 or
95% aqueous acetic acid solution affords nearly quantitative recovery of largely iodine-free peptide ready for direct purification.
It was successfully applied to the synthesis of various peptides including human insulin-like peptide 3 analogues. Although
recovery yields were comparable to the traditionally used ascorbic acid quenching method, this new approach offers significant
advantages such as more simple utility, minimal side reactions, and greater cost effectiveness.
We herein describe the first synthesis of the native antimicrobial protein HBD-1 making use of an orthogonal thiol protection strategy and a novel dicarba analogue thereof. The robust hydrocarbon linkage was installed by replacement of one disulfide bond using on-resin ring closing metathesis. The unprecedented 59-membered C-terminal cysteine macrocyclic fragment thus formed then engages in native chemical ligation allowing convergent access to this unique synthetic protein analogue.
In the equine industry, pregnancy loss during the third trimester constitutes a large percentage of fetal and neonatal mortality and represents a major financial loss and time investment for the breeder. Early identification of placental insufficiency would, in some cases, make it possible to sustain the pregnancy through medical intervention. Recent work suggests that relaxin is a valuable clinical tool for diagnosing placental insufficiency and monitoring treatment efficacy in mares. Relaxin is a polypeptide member of the insulin superfamily that consists of a two-chain structure and three disulfide bonds in a disposition identical to that of insulin. It is typically produced in the ovary during pregnancy and has primary roles in maintaining mammalian pregnancy and facilitating the delivery of the young via remodelling of the reproductive tract. The placenta is the primary source of relaxin in the mare during pregnancy. Its primary structure has been determined and shown to be the smallest of the known mammalian relaxins. It consists of a 20 residue A-chain and a 28-residue B-chain. To undertake detailed biophysical and biological characterization of the peptide, its chemical synthesis was undertaken using regioselective disulfide formation methods. The synthetic equine relaxin showed typical α-helical structure under physiological conditions. The peptide was found to bind to the relaxin receptor, LGR7, in vitro, and its binding affinity was found to be higher than that of the “gold standard”, porcine relaxin, and similar to that of the human relaxin-2 (H2 relaxin).
In earlier studies we identified in a human genomic library a gene (human relaxin gene H1) coding for a relaxin-related peptide. We now have evidence that the human genome possesses an additional relaxin-related gene (designated human relaxin gene H2) which appears to be selectively expressed in the ovary during pregnancy. Nucleotide sequence analysis revealed striking differences in the predicted structures of relaxin encoded by these two genes. Chemical synthesis of biologically active relaxin based on the sequence obtained from ovarian cDNA clones confirmed that the expressed gene (H2) encodes an authentic human relaxin. The expressed gene appears to be transcribed into two different sized mRNAs and preliminary evidence suggests that the mRNA transcripts possess different 3'-untranslated regions. There was no evidence for the expression of human relaxin gene H1 in the ovary and so far it is unclear whether gene H1 is expressed in another tissue or whether it represents a pseudogene. From the sequence data presented here it will now be possible to construct oligonucleotide probes and raise antibodies against synthetic peptides which could then be used to identify sites of relaxin biosynthesis and specifically quantitate the expression from either the H1 or H2 relaxin genes.
Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis.
We have identified a novel human relaxin gene, designated H3 relaxin, and an equivalent relaxin gene in the mouse from the Celera Genomics data base. Both genes encode a putative prohormone
sequence incorporating the classic two-chain, three cysteine-bonded structure of the relaxin/insulin family and, importantly,
contain the RXXXRXX(I/V) motif in the B-chain that is essential for relaxin receptor binding. A peptide derived from the likely proteolytic processing
of the H3 relaxin prohormone sequence was synthesized and found to possess relaxin activity in bioassays utilizing the human
monocytic cell line, THP-1, that expresses the relaxin receptor. The expression of this novel relaxin gene was studied in
mouse tissues using RT-PCR, where transcripts were identified with a pattern of expression distinct from that of the previously
characterized mouse relaxin. The highest levels of expression were found in the brain, whereas significant expression was
also observed in the spleen, thymus, lung, and ovary. Northern blotting demonstrated an ∼1.2-kb transcript present in mouse
brain poly(A) RNA but not in other tissues. These data, together with the localization of transcripts in the pars ventromedialis
of the dorsal tegmental nucleus of C57BLK6J mouse brain by in situhybridization histochemistry, suggest a new role for relaxin in neuropeptide signaling processes. Together, these studies
describe a third member of the human relaxin family and its equivalent in the mouse.
Relaxin is a hormone important for the growth and remodeling of reproductive and other tissues during pregnancy. Although binding sites for relaxin are widely distributed, the nature of its receptor has been elusive. Here, we demonstrate that two orphan heterotrimeric guanine nucleotide binding protein (G protein)-coupled receptors, LGR7 and LGR8, are capable of mediating the action of relaxin through an adenosine 3',5'-monophosphate (cAMP)-dependent pathway distinct from that of the structurally related insulin and insulin-like growth factor family ligand. Treatment of antepartum mice with the soluble ligand-binding region of LGR7 caused parturition delay. The wide and divergent distribution of the two relaxin receptors implicates their roles in reproductive, brain, renal, cardiovascular, and other functions.
Relaxin (RLX) is a peptide hormone with known antifibrotic properties. However, its significance in the lung and its role as a therapeutic agent against diseases characterized by pulmonary fibrosis are yet to be established. In this study, we examined age-related structural and functional changes in the lung of relaxin-deficient mice. Lung tissues of male and female RLX knockout (-/-) and RLX wild-type (+/+) mice at various ages were analyzed for changes in collagen expression and content. We demonstrate an age-related progression of lung fibrosis in RLX -/- mice with significantly increased tissue wet weight, collagen content and concentration, alveolar congestion, and bronchiole epithelium thickening. The increased fibrosis was associated with significantly altered peak expiratory flow and lung recoil (lung function) in RLX -/- mice. Treatment of RLX -/- mice with relaxin in early and developed stages of fibrosis resulted in the reversal of collagen deposition. Organ bath studies showed that precontracted lung strips relaxed in the presence of relaxin. Together, these data indicate that relaxin may provide a means to regulate excessive collagen deposition in diseased states characterized by pulmonary fibrosis.
Relaxin family peptide 1 (RXFP1) receptor (LGR7) and RXFP2 receptor (LGR8) were recently identified as the receptor targets for H2 relaxin and insulin-like peptide 3 (INSL3), respectively. In this study, we define the pharmacology of these two receptors by using a number of receptor chimeras and relaxin family peptides. We have identified two binding sites on these receptors: one primary, high-affinity site within the ectodomain and a secondary, lower affinity site within the transmembrane region. The primary site was found to dictate receptor binding characteristics, although the lower affinity site also exerts some influence and modulates ligand affinity for the primary site in a manner dependent upon the peptide in question. Not all relaxin peptides were able to bind to the RXFP2 receptor, indicating that the relaxin-RXFP2 receptor interaction is species-specific. INSL3 was found to exhibit characteristics of a partial agonist at the RXFP2 and chimeric RXFP1/2 receptors, with low maximal cAMP responses but high potency in coupling to this pathway. cAMP accumulation studies also revealed that the binding sites couple to cAMP signaling pathways with differing efficiency: the high-affinity site signals with high efficiency, whereas the lower affinity site signals with little to no efficiency. Comparisons between RXFP1, RXFP2, the chimeric receptors, and the truncated receptors revealed that the interaction between receptor sites is critical for optimal ligand binding and signal transduction and that the ectodomain is essential for signaling. Evidence obtained in this study supports a two-stage binding model of receptor activation: binding to the primary site allows a conformational change and interaction with the low-affinity transmembrane site.
The hormone relaxin has been shown to cause coronary vasodilation and to prevent ischemia/reperfusion-induced cardiac injury in rodents. This study provides evidence that relaxin, used as an adjunctive drug to coronary reperfusion, reduces the functional, biochemical, and histopathological signs of myocardial injury in an in vivo swine model of heart ischemia/reperfusion, currently used to test cardiotropic drugs for myocardial infarction. Human recombinant relaxin, given at reperfusion at doses of 1.25, 2.5, and 5 microg/kg b.wt. after a 30-min ischemia, caused a dose-related reduction of key markers of myocardial damage (serum myoglobin, CK-MB, troponin T) and cardiomyocyte apoptosis (caspase 3, TUNEL assay), as well as of cardiomyocyte contractile dysfunction (myofibril hypercontraction). Compared with the controls, relaxin also increased the uptake of the viability tracer 201Thallium and improved ventricular performance (cardiac index). Relaxin likely acts by reducing oxygen free radical-induced myocardial injury (malondialdehyde, tissue calcium overload) and inflammatory leukocyte recruitment (myeloperoxidase). The present findings show that human relaxin, given as a drug to counteract reperfusion-induced cardiac injury, affords a clear-cut protection to the heart of swine with induced myocardial infarction. The findings also provide background to future clinical trials with relaxin as adjunctive therapy to catheter-based coronary angioplasty in patients with acute myocardial infarction.
Fibrosis (progressive scarring) is a leading cause of organ failure worldwide and causes loss of organ function when normal tissue is replaced with excess connective tissue. Several organs are prone to this process regardless of etiology. The pleiotropic hormone, relaxin, is emerging as a novel antifibrotic therapy. Relaxin has been shown to limit collagen production and reorganization, while stimulating increased collagen degradation. It not only prevents fibrogenesis, but also reduces established scarring. This review summarizes (1) the levels at which relaxin inhibits collagen production and existing collagen overexpression in induced models of fibrosis, and (2) the collagen-related phenotypes of relaxin- and LGR7-deficient mice. Recent studies on relaxin-deficient mice have established relaxin as an important, naturally occurring regulator of collagen turnover and provide new insights into the therapeutic potential of relaxin.
Relaxin-3 is a member of the human relaxin peptide family, the gene for which, RLN3, is predominantly expressed in the brain. Mapping studies in the rodent indicate a highly developed network of RLN3, RLN1, and relaxin receptor-expressing cells in the brain, suggesting that relaxin peptides have important functional roles in the central nervous system. A regioselective disulfide-bond synthesis protocol was developed and used for the chemical synthesis of human (H3) relaxin-3. The selectively S-protected A and B chains were combined by stepwise formation of each of the three insulin-like disulfides via aeration, thioloysis, and iodolysis. Judicious positioning of the three sets of S-protecting groups was crucial for acquisition of synthetic H3 relaxin in a good overall yield. The activity of the peptide was tested against relaxin family peptide receptors. Although the highest activity was demonstrated on the human relaxin-3 receptor (GPCR135), the peptide also showed high activity on relaxin receptors (LGR7) from various species and variable activity on the INSL3 receptor (LGR8). Recombinant mouse prorelaxin-3 demonstrated similar activity to H3 relaxin, suggesting that the presence of the C peptide did not influence the conformation of the active site. H3 relaxin was also able to activate native LGR7 receptors. It stimulated increased MMP-2 expression in LGR7-expressing rat ventricular fibroblasts in a dose-dependent manner and, following infusion into the lateral ventricle of the brain, stimulated water drinking in rats, activating LGR7 receptors located in the subfornical organ. Thus, H3 relaxin is able to interact with the relaxin receptor LGR7 both in vitro and in vivo.
Although the hormone relaxin was discovered 80 years ago, only in the past 5 years have the receptors for relaxin and three other receptors that respond to related peptides been identified with all four receptors being G-protein-coupled receptors. In this review it is suggested that the receptors for relaxin (LGR7) and those for the related peptides insulin-like peptide 3 (LGR8), relaxin-3 (GPCR135), and insulin-like peptide 5 (LGPCR142) be named the relaxin family peptide receptors 1 through 4 (RXFP1-4). RXFP1 and RXFP2 are leucine-rich repeat-containing G-protein-coupled receptors with complex binding characteristics involving both the large ectodomain and the transmembrane loops. RXFP1 activates adenylate cyclase, protein kinase A, protein kinase C, phosphatidylinositol 3-kinase, and extracellular signaling regulated kinase (Erk1/2) and also interacts with nitric oxide signaling. RXFP2 activates adenylate cyclase in recombinant systems, but physiological responses are sensitive to pertussis toxin. RXFP3 and RXFP4 resemble more conventional peptide liganded receptors and both inhibit adenylate cyclase, and in addition RXFP3 activates Erk1/2 signaling. Physiological studies and examination of the phenotypes of transgenic mice have established that relaxin has roles as a reproductive hormone involved in uterine relaxation (some species), reproductive tissue growth, and collagen remodeling but also in the cardiovascular and renal systems and in the brain. The connective tissue remodeling properties of relaxin acting at RXFP1 receptors have potential for the development of agents effective for the treatment of cardiac and renal fibrosis, asthma, and scleroderma and for orthodontic remodelling. Agents acting at RXFP2 receptors may be useful for the treatment of cryptorchidism and infertility, whereas antagonists may be used as contraceptives. The brain distribution of RXFP3 receptors suggests that actions at these receptors have the potential for the development of antianxiety and antiobesity drugs.
Insulin-like peptide 3 (INSL3), a member of the relaxin peptide family, is produced in testicular Leydig cells and ovarian
thecal cells. Gene knock-out experiments have identified a key biological role in initiating testes descent during fetal development.
Additionally, INSL3 has an important function in mediating male and female germ cell function. These actions are elicited
via its recently identified receptor, LGR8, a member of the leucine-rich repeat-containing G-protein-coupled receptor family.
To identify the structural features that are responsible for the interaction of INSL3 with its receptor, its solution structure
was determined by NMR spectroscopy together with in vitro assays of a series of B-chain alanine-substituted analogs. Synthetic human INSL3 was found to adopt a characteristic relaxin/insulin-like
fold in solution but is a highly dynamic molecule. The four termini of this two-chain peptide are disordered, and additional
conformational exchange is evident in the molecular core. Alanine-substituted analogs were used to identify the key residues
of INSL3 that are responsible for the interaction with the ectodomain of LGR8. These include ArgB16 and ValB19, with HisB12 and ArgB20 playing a secondary role, as evident from the synergistic effect on the activity in double and triple mutants involving these
residues. Together, these amino acids combine with the previously identified critical residue, TrpB27, to form the receptor binding surface. The current results provide clear direction for the design of novel specific agonists
and antagonists of this receptor.
This chapter discusses physiology and molecular biology of the relaxin peptide family. Relaxin-a member of a family of peptide hormones-includes seven members in humans-three relaxin peptides (H1, H2, and H3) and insulin-like peptides (INSL) 3, 4, 5, and 6. All these genes evolved from an ancestral relaxin-3 gene in (lower) vertebrates. The reproductive hormone relaxin is the product of one gene in most species, RLN1. Relaxin-3 is likely an important neuropeptide but has no known roles in reproduction. In contrast, relaxin has vital physiological roles during pregnancy, although its actions vary between species. Hence, relaxin has essential actions on the cervix, pubic symphysis, vagina, uterus, and mammary apparatus during pregnancy. It is also a mediator of the important cardiovascular changes that occur during pregnancy in many species. Relaxin plays a crucial role in implantation, especially in primates. In addition, relaxin has nonreproductive actions in wound healing, cardiac protection, and as an antifibrotic agent. INSL3 is essential for testis descent by promoting gubernacular development in the fetus. It also plays an important role in female and male germ cell maturation and survival, respectively.
A method, based on that of Stegemann but with a simplified procedure, has been developed for the spectrophotometric determination of hydroxyproline. Radical improvements have been achieved in the stability of the reagents and of the final color. The variation of the color yield with the conditions at various stages in the analytical procedure has been minimized, and been made independent of hydroxyproline concentration over a wider range than hitherto reported. In addition, a procedure has been developed which involves no thermostat bath, as the color development is carried out overnight at room temperature instead of in an accurately timed period at 60° C.
We study zero-sum stochastic games in which players do not observe the actions of the opponent. Rather, they observe a stochastic signal that may depend on the state, and on the pair of actions chosen by the players. We assume each player observes the state and his own action. In a companion paper we proposed a candidate for the max-min value, we proved that player 2 can defend this value, and that player 1 can guarantee it in the class of absorbing games. In the present paper we prove that player 1 can guarantee this quantity in general stochastic games. An analogous result holds for the min-max value.
Relaxin, a peptide hormone responsible for the widening of the birth canal in mammals, has been purified from the ovaries of pregnant hogs. The amino acid sequences of its constituent A and B chains were determined, and the positions of the disulfide cross-links were established. Relaxin was shown to be identical to insulin with respect to its disulfide bond distribution, but significant homology was lacking in other positions. These findings suggest that relaxin and insulin were derived from a common ancestral gene. Since the intrauterine mode of propagation is synonymous with the development of mammals, the genetic distance between insulin and relaxin should therefore permit an estimate of the earliest possible time of commitment of one evolutionary branch to the development of mammals. This event was estimated to have occurred about 5 X 10(8) years ago.
Relaxin is a peptide hormone synthesized in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its major biological effect is to remodel the mammalian reproductive tract to facilitate the birth process. Determination of the structure of human relaxin is thus a first step in opening up the possibility of clinical intervention in cases of difficult labour. However, the limited availability of human ovaries during pregnancy has prevented both direct amino acid sequence determination and isolation of cDNA clones obtained from relaxin producing tissue. Our approach has therefore been to screen directly for a human relaxin gene using an homologous porcine relaxin cDNA probe. We report here the successful identification of a genomic clone from which the structure of the entire coding region of a human preprorelaxin gene has been determined. Synthesis of biologically active relaxin has shown that the novel gene structure described herein codes for an authentic human relaxin. We believe this is the first successful synthesis of a biologically active hormone whose structure was predicted solely from the structure of a genomic clone.
According to a recently published cDNA sequence, mouse relaxin has an extra amino acid in the C-terminal end of the A chain and thus an interchain loop consisting of 25 amino acids instead of the usual 24-membered ring. Because of the restrictive disulfide link arrangement the extra residue can be expected to cause a loop out in the C-terminal alpha-helix. We have chemically synthesized authentic mouse relaxin as well as an analog without the additional A chain residue and found that the native hormone, although active, was inferior to its insulin-like analog. This result is in harmony with our previous study which suggests that the surface of relaxin represented by the C-terminal helix of the A chain is positioned opposite to the surface that contains the receptor interaction site and therefore is less sensitive to modifications.
Relaxin is a polypeptide hormone that has a variety of physiological effects both on remodelling of collagen and on uterine contractility. These are most apparent during pregnancy. The sequences of relaxin cDNAs derived from ovaries of late-pregnant random-bred Swiss mice have been established. Multiple subclones obtained from three independent polymerase chain reaction experiments were found to encode relaxins which were identical except at position 11 in the A chain (Ile or Val). All mouse relaxin cDNAs expressed in the ovary during pregnancy had an extra tyrosine inserted prior to the final A chain cysteine residue, a result confirmed by direct sequencing of relaxin peptides. Whilst this tyrosine insertion must have local effects on the folding of the A chain, structure-activity studies will clarify whether it perturbs functional interaction with the relaxin receptor. We have shown that there is a single relaxin gene in the mouse genome, and that expression during pregnancy occurs in the ovary but is not detectable in the placenta, uterus or fetus.
We have used gene targeting to generate relaxin (rlx)-deficient mice. The majority (15 of 17) of homozygous (rlx-/-) mice are fertile and produce normal litters. However their mammary development is deficient; pups are unable to suckle and die within 24 h of birth unless cross-fostered to a wild-type (rlx+/+) foster mother. The nipples of rlx-/- animals do not enlarge significantly during pregnancy, and their histology retains the appearance of the virgin state. Breast parenchyma is somewhat underdeveloped at term even though milk is produced. Mammary ducts become grossly dilated in these animals. Heterozygous (rlx+/-) mice lactate normally. The interpubic ligament does not relax during pregnancy in rlx-/- mice. Plasma osmolality during late gestation was significantly higher (P < 0.001) in rlx-/- mice than in wild-type controls.
A linear peptide containing two reduced cysteine residues can be rapidly converted to its oxidized cyclic form containing an intramolecular disulfide bond by adding an excess of 2,2'-bispyridyl disulfide (2,2'-dipyridyl disulfide or 2,2'-dithiodipyridine) to conventional buffer solutions. The reactants and products are easily separated by reverse-phase chromatography. This reaction will find wide application in forming intramolecular disulfide bonds because of its selectivity for free sulfhydryl groups, quickness, safety, and applicability under acidic conditions.
Relaxin, a hormone of the insulin-growth factor family, promotes collagen remodeling. In rodent models of pulmonary and dermal fibrosis, relaxin reduced interstitial fibrosis. To study relaxin's effect in renal disease, we used the experimental bromoethylamine (BEA) model that leads to severe renal interstitial fibrosis, a decrease in glomerular filtration rate, and albuminuria at one month.
Rats were injected with BEA one week prior to implantation of an osmotic pump delivering relaxin (2 microg/hour) or vehicle continuously for 28 days.
BEA caused a significant decrease in creatinine clearance, which was partially prevented by relaxin. In the relaxin-treated BEA rats, serum creatinine was normal, and albumin excretion was slightly decreased. By morphometric measurement, relaxin administration was associated with a significant decrease in interstitial fibrosis at the corticomedullary junction. This was accompanied by a decrease in the number of ED-1 positive cells (an index of macrophage infiltration) and in the intensity of immunohistochemical staining for transforming growth factor-beta. This antifibrotic effect of relaxin did not appear to be mediated by systemic hemodynamic changes since the mean arterial pressure was not significantly different among the groups.
Relaxin may have a useful application in decreasing interstitial fibrosis and thereby slowing the progression of renal disease.
Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis.
To determine the effects of relaxin on activated HSC.
Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively.
Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression.
These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.
To investigate cardiac phenotypes in mice deficient in the peptide hormone relaxin by gene targeting.
Echocardiography and cardiac catheterization were performed on male and female relaxin deficient (Rlx(-/-)) mice as well as heterozygous (Rlx(+/-)) and wildtype (Rlx(+/+)) littermates aged between 8 and 24 months. Collagen expression and content in the heart were analysed by real-time PCR, hydroxyproline assay and histology.
Heart rate, blood pressures, left ventricular (LV) dimensions, fractional shortening and maximal and minimal dP/dt did not differ significantly between the three genotypes of either gender at any age. However, 8-10-month-old Rlx(-/-) males exhibited a greater transmitral flow velocity (A-wave) at the late LV diastolic phase. Male Rlx(-/-) mice aged between 12 and 24 months had significantly higher LV end-diastolic pressures, a 30% increase in atrial weight and 10-30% increases in lung and liver weights. Male mice also showed an age-dependent increase (P<0.01) in LV collagen content that was more pronounced in Rlx(-/-) than control littermates (P<0.01). Procollagen type-1 expression was also significantly higher in the LV of Rlx(-/-) males compared with either Rlx(+/-) or Rlx(+/+) males at 6, 9 and 12 months of age. Age-matched female Rlx(-/-) mice did not display any of these cardiac phenotypes seen in Rlx(-/-) males.
Male Rlx(-/-) mice had impeded LV diastolic filling and increased atrial weights, most likely due to an increase in ventricular collagen content and chamber stiffness. These phenotypes in the Rlx(-/-) males were not observed in Rlx(-/-) females, indicating the importance of other gender-related factors in cardiovascular function.
Male mice deficient in relaxin showed retarded growth and marked deficiencies in the reproductive tract within 1 month of age. At 3 months of age, male reproductive organ weight (including the testis, epididymis, prostate, and seminal vesicle) from relaxin null (RLX-/-) mice were significantly (p < 0.05) smaller than those of wild-type (RLX+/+) male mice. Histologic examination of RLX-/- mouse tissues demonstrated decreased sperm maturation (testis), increased collagen, and decreased epithelial proliferation in the prostate compared with tissues obtained from RLX+/+ animals. The degree of sperm maturation in the testes of sexually mature RLX-/- mice (3 months) resembled that of immature (1 month) RLX+/+ mice and correlated with a decrease in fertility in RLX-/- mice. The marked differences in the extracellular matrix of the testis and prostate in RLX-/- males also correlated with an increase in the rate of cell apoptosis. Relaxin and LGR7 (relaxin receptor) mRNA expression was demonstrated in the prostate gland and testis of the normal mouse. Data from this study demonstrate that relaxin is an important factor in the development and function of the male reproductive tract in mice and has an essential role in the growth of the prostate and maintenance of male fertility. Relaxin may mediate its effects on growth and development by serving as an antiapoptotic factor.
Relaxin has vital physiological roles in pregnant rats, mice, and pigs. Relaxin promotes growth and softening of the cervix, thus facilitating rapid delivery of live young. Relaxin also promotes development of the mammary apparatus, thus enabling normal lactational performance. The actions of relaxin on the mammary apparatus vary among species. Whereas relaxin is required for development of the mammary nipples in rats and mice, it is essential for prepartum development of glandular parenchyma in pregnant pigs. During pregnancy relaxin also inhibits uterine contractility and promotes the osmoregulatory changes of pregnancy in rats. Recent studies with male and nonpregnant female rodents revealed diverse therapeutic actions of relaxin on nonreproductive tissues that have clinical implications. Relaxin has been reported to reduce fibrosis in the kidney, heart, lung, and liver and to promote wound healing. Also, probably through its vasodilatory actions, relaxin protects the heart from ischemia-induced injury. Finally, relaxin counteracts allergic reactions. Knowledge of the diverse physiological and therapeutic actions of relaxin, coupled with the recent identification of relaxin receptors, opens numerous avenues of investigation that will likely sustain a high level of research interest in relaxin for the foreseeable future.
Relaxin (RLX) is a peptide hormone that stimulates the breakdown of collagen in preparation for parturition and when administered to various models of induced fibrosis. However, its significance in the aging kidney is yet to be established. In this study, we compared structural and functional changes in the kidney of aging relaxin-1 (RLX-/-) deficient mice and normal (RLX+/+) mice.
The kidney cortex and medulla of male and female RLX+/+ and RLX-/- mice at various ages were analyzed for collagen content, concentration, and types. Histologic analysis, reverse transcription-polymerase chain reaction (RT-PCR) of relaxin and relaxin receptor mRNA expression, receptor autoradiography, glomerular isolation/analysis, and serum/urine analysis were also employed. Relaxin treatment of RLX-/- mice was used to confirm the antifibrotic effects of the peptide.
We demonstrate an age-related progression of renal fibrosis in male, but not female, RLX-/- mice with significantly (P < 0.05) increased tissue dry weight, collagen (type I) content and concentration. The increased collagen expression in the kidney was associated with increased glomerular matrix and to a lesser extent, interstitial fibrosis in RLX-/- mice, which also had significantly increased serum creatinine (P < 0.05) and urinary protein (P < 0.05). Treatment of RLX-/- mice with relaxin in established stages of renal fibrosis resulted in the reversal of collagen deposition.
This study supports the concept that relaxin may provide a means to regulate excessive collagen deposition during kidney development and in diseased states characterized by renal fibrosis.
Cardiac fibrosis is a key component of heart disease and involves the proliferation and differentiation of matrix-producing fibroblasts. The effects of an antifibrotic peptide hormone, relaxin, in inhibiting this process were investigated. We used rat atrial and ventricular fibroblasts, which respond to profibrotic stimuli and express the relaxin receptor (LGR7), in addition to two in vivo models of cardiac fibrosis. Cardiac fibroblasts, when plated at low density or stimulated with TGF-beta or angiotensin II (Ang II), accelerated fibroblast differentiation into myofibroblasts, as demonstrated by significantly increased alpha-smooth muscle actin expression, collagen synthesis, and collagen deposition (by up to 95% with TGF-beta and 40% with Ang II; all P < 0.05). Fibroblast proliferation was significantly increased by 10(-8) m and 10(-7) m Ang II (63-75%; P < 0.01) or 0.1-1 microg/ml IGF-I (27-40%; P < 0.05). Relaxin alone had no marked effect on these parameters, but it significantly inhibited Ang II- and IGF-I-mediated fibroblast proliferation (by 15-50%) and Ang II- and TGF-beta-mediated fibroblast differentiation, as detected by decreased expression of alpha-smooth muscle actin (by 65-88%) and collagen (by 60-80%). Relaxin also increased matrix metalloproteinase-2 expression in the presence of TGF-beta (P < 0.01) and Ang II (P < 0.05). Furthermore, relaxin decreased collagen overexpression when administered to two models of established fibrotic cardiomyopathy, one due to relaxin deficiency (by 40%; P < 0.05) and the other to cardiac-restricted overexpression of beta2-adrenergic receptors (by 58%; P < 0.01). These coherent findings indicate that relaxin regulates fibroblast proliferation, differentiation, and collagen deposition and may have therapeutic potential in diseased states characterized by cardiac fibrosis.
Thirst motivates animals to seek fluid and drink it. It is regulated by the central nervous system and arises from neural and chemical signals from the periphery interacting in the brain to stimulate a drive to drink. Our research has focussed on the lamina terminalis and the manner in which osmotic and hormonal stimuli from the circulation are detected by neurons in this region and how that information is integrated with other neural signals to generate thirst. Our studies of osmoregulatory drinking in the sheep and rat have produced evidence that osmoreceptors for thirst exist in the dorsal cap of the organum vasculosum of the lamina terminalis (OVLT) and in the periphery of the subfornical organ, and possibly also in the median preoptic nucleus. In the rat, the hormones angiotensin II and relaxin act on neurons in the periphery of the subfornical organ to stimulate drinking. Studies of human thirst using functional magnetic resonance imaging (fMRI) techniques show that systemic hypertonicity activates the lamina terminalis and the anterior cingulate cortex, but the neural circuitry that connects sensors in the lamina terminalis to cortical regions subserving thirst remains to be determined. Regarding pathophysiological influences on thirst mechanisms, both excessive (polydipsia) and inadequate (hypodisia) water intake may have dire consequences. One of the most common primary polydipsias is that observed in some cases of schizophrenia. The neural mechanisms causing the excessive water intake in this disorder are unknown, so too are the factors that result in impaired thirst and inadequate fluid intake in some elderly humans.
Relaxin is well known for its actions on collagen remodeling. To improve our understanding of the physiologic role(s) of relaxin, the relaxin gene-knockout (RLX-KO) mouse was established by our group and subsequently phenotyped. Pregnant RLX-KO mice underwent inadequate development of the pubic symphysis as well as the mammary glands and nipples compared to wild-type mice, thus preventing lactation. Later studies showed that these deficiencies were associated with increased collagen, primarily in the nipple and vagina. Analysis of male RLX-KO mice also demonstrated inadequate reproductive tract development. The testis, epididymis, and prostate of RLX-KO mice showed delayed tissue maturation and growth associated with increased collagen deposition. In nonreproductive tissues, an age-related increase in interstitial collagen (fibrosis) was also detected in the lung, heart, and kidneys of RLX-KO mice and was associated with organ dysfunction. From 6-9 months of age and onwards, all organs of RLX-KO mice, particularly male mice, underwent progressive increases in tissue weight and collagen content (all P < .05) compared with wild-type animals. The increased fibrosis contributed to bronchiole epithelium thickening and alveolar congestion (lung), atrial hypertrophy and increased ventricular chamber stiffness (heart) in addition to glomerulosclerosis (kidney). Treatment of RLX-KO mice with recombinant human relaxin in early and developed stages of fibrosis caused the reversal of collagen deposition in the lung, heart, and kidneys. Together, these findings suggest that relaxin is a naturally occurring inhibitor of collagen deposition during normal development, aging, and pregnancy and can be used to prevent the progression of fibrosis.
Human LGR8, initially discovered as a low-affinity relaxin receptor, has now been characterized as the INSL3 receptor. To investigate LGR8 function in the rat, an LGR8 ortholog was identified in the rat genome, and the full-length sequence was cloned and expressed. Rat LGR8 bound INSL3 with high affinity, clearly demonstrating that it is the rat INSL3 receptor. Interestingly, native rat relaxin did not activate rat LGR8, indicating that relaxin is not an endogenous ligand for rat LGR8. LGR8 mRNA expression was demonstrated in the gubernaculum at the time of testis descent and in the testis associated with germ cells.
Rodent models have been used for many years to probe the actions of relaxin. Identification of the orthologs of human leucine-rich repeat-containing g-protein-coupled receptor 7 (LGR7), the relaxin receptor, in mouse and rat will enable characterization of the response of LGR7 to relaxin in these species. Partial LGR7 homologous sequences from mouse and rat were discovered in the Celera and NCBI gene databases, amplified, cloned, and sequenced. At the protein level, mouse and rat LGR7 are 85.2% and 85.7% identical to human LGR7. Mouse and rat LGR7 were able to bind to and be activated by relaxin ligands.
The antifibrotic effects of the peptide hormone relaxin on cardiac and renal fibrosis were studied in 9- to 10-month-old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY). Rats (n=8 to 9 per group) were allocated into 3 groups: WKY controls, vehicle-treated SHR (SHR-V), and relaxin-treated SHR (SHR-R). Relaxin (0.5 mg/kg per day) was administered via subcutaneously implanted osmotic mini-pumps over 2 weeks before hearts and kidneys were harvested for analysis. Collagen content was analyzed by hydroxyproline assay, gel electrophoresis, and quantitative histology. Zymography was used to determine matrix metalloproteinase (MMP) expression and Western blotting to determine proliferating cell nuclear antigen (PCNA) expression and alpha-smooth muscle actin (alpha-SMA)/myofibroblast expression, whereas cardiac hypertrophy was assessed by myocyte size and real-time polymerase chain reaction of associated genes. The left ventricular (LV) myocardium of SHR-V contained increased collagen levels (by 25+/-1%, P<0.01 using biochemical analysis and 3-fold; P<0.01 using quantitative histology), enhanced expression of PCNA (by 70+/-8%; P<0.01), alpha-SMA (by 32+/-2%; P<0.05), and the collagen-degrading enzyme MMP-9 (by 70+/-6%; P<0.05) versus respective levels measured in WKY controls. The kidneys of SHR-V also contained increased collagen (25+/-2%, P<0.05 using biochemical analysis and 2.4-fold; P<0.01 using quantitative histology). Relaxin treatment significantly normalized collagen content in the LV (P<0.01) and kidney (P<0.05), completely inhibited cell proliferation (P<0.01) and fibroblast differentiation (P<0.05) in the LV, and increased MMP-2 expression (by 25+/-1%; P<0.05) without affecting MMP-9 in the LV compared with that measured in SHR-V. Thus, relaxin is a potent antifibrotic hormone with a rapid-occurring efficacy that may have therapeutic potential for hypertensive disease.
The omnipresent 6-kDa polypeptide relaxin (RLX) is emerging as a multifunctional endocrine and paracrine factor in a broad range of target tissues including cardiovascular tissues. To explore the pathophysiological roles of RLX in ischemic cardiovascular diseases, we studied the changes in RLX mRNA level in the myocardium and the effect of RLX supplements in rats with isoproterenol (ISO)-induced myocardial injury. In ISO-treated rats, RLX levels in myocardia and plasma increased 3.7- and 6.9-fold, respectively (P<0.01), the mRNA level increased significantly in myocardia compared with controls. Co-administration of RLX (0.2 and 2.0 microg/kg/d) and ISO increased left-ventricular pressure development and decreased left ventricular end-diastolic pressure (LVDEP) (all P<0.01). Malondialdehyde content in myocardia and lactate dehydrogenase and creatine phosphokinase activities in plasma in RLX-treated rats decreased markedly compared with that in ISO-treated alone rats (P<0.01 or P<0.05). In the high-dose RLX group, fibroblastic hyperplasia was relieved in myocardia, hydroxyproline level was lower, by 33% (P<0.05), and endothelin content in plasma was lower, by 31% (P<0.01) than in the ISO-alone group. Compared with control group, any indexes in sham rats treated with high-dose RLX were unaltered (all P>0.05). These results showed an up-regulation of myocardial RLX during ISO-induced myocardial ischemia injury and the protective effect of RLX on ISO-induced cardiac inhibition and fibrosis, which suggests that RLX could be an endogenous cardioprotective factor in ischemic heart diseases.
The hormone relaxin, known for its action on the female reproductive tract, is also able to act on organs and systems different from the reproductive ones, including the blood vessels, the heart and the brain. Relaxin causes vasodilation in several organs stimulating the biosynthetic pathway of nitric oxide (NO), a potent vasodilator. Relaxin also has a cardioprotective action: it reduces the inflammatory activation of neutrophils and their adhesion to the endothelium, and protects against myocardial injury caused by ischemia and reperfusion (I-R) in experimental animal models of myocardial infarction. Its mechanisms of action chiefly depend on the hormone's vasodilatory and anti-inflammatory properties. Recently, an additional form of relaxin has been discovered in the brain, where it has been postulated to act locally as a neurotransmitter. Relaxin, acting mainly on circumventricular organs, stimulates water drinking and vasopressin release and appears to be involved in the regulation of behavioural processes. Based on its properties on the cardiovascular system, it is possible to hypothesise that relaxin could regulate the vascular tone in the central nervous system and, going a step further, could protect the brain from IR-induced damage, possibly by an NO-mediated mechanism. This latter possibility is supported by the observation that relaxin is able to up regulate the endogenous production of NO in several target cells, as NO, at appropriate levels, is known to be involved in the protection against neural pathophysiological processes such as I-R-induced injury.
Relaxin is a peptide hormone with anti-fibrotic properties. To investigate the long-term effects of relaxin deficiency on the ageing skin, we compared structural changes in the skin of ageing relaxin-deficient (RLX-/-) and normal (RLX+/+) mice, by biochemical, histological, and magnetic resonance imaging analyses. Skin biopsies from RLX+/+ and RLX-/- mice were obtained at different ages and analyzed for changes in collagen expression and distribution. We demonstrated an age-related progression of dermal fibrosis and thickening in male and female RLX-/- mice, associated with marked increases in types I and III collagen. The increased collagen was observed primarily in the dermis of RLX-/- mice by 1 mo of age, and eventually superseded the hypodermal layer. Additionally, fibroblasts from the dermis of RLX-/- mice were shown to produce increased collagen in vitro. Recombinant human gene-2 (H2) relaxin treatment of RLX-/- mice resulted in the complete reversal of dermal fibrosis, when applied to the early onset of disease, but was ineffective when applied to more established stages of dermal scarring. These combined findings demonstrate that relaxin provides a means to regulate excessive collagen deposition in disease states characterized by dermal fibrosis and with our previously published work demonstrate the relaxin-null mouse as a model of progressive scleroderma.
Although originally characterised as a reproductive hormone, relaxin has emerged as a multi-functional endocrine and paracrine factor that plays a number of important roles in several organs, including the normal and diseased cardiovascular system. The recent discovery of the H3/relaxin-3 gene, and the elusive receptors for relaxin (Relaxin family peptide receptor; RXFP1) and relaxin-3 (RXFP3/RXFP4) have led to the re-classification of a distinct relaxin peptide/receptor family. Additionally, the identification of relaxin and RXFP1 mRNA and/or relaxin binding sites in the heart and blood vessels has confirmed that the cardiovascular system is a target for relaxin peptides. While evidence for the production of relaxins within the cardiovascular system is limited, several studies have established that the relaxin genes are upregulated in the diseased human and rodent heart where they likely act as cardioprotective agents. The ability of relaxin to protect the heart is most likely mediated via its antifibrotic, anti-hypertrophic, anti-inflammatory and vasodilatory actions, but it may also directly stimulate myocardial regeneration and repair. This review describes relaxin and its primary receptor (RXFP1) in relation to the roles and effects of relaxin in the normal and pathological cardiovascular system. It is becoming increasingly clear that relaxin has a number of diverse physiological and pathological roles in the cardiovascular system that may have important therapeutic and clinical implications.
Experimental relaxation of the pubic ligament of the guinea pig
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1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide
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HBTU, N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-methylmethanaminium hexafluorophosphate N-oxide;
hydro-chloric acid; HEK, human embryonic kidney; LGR7, leucine-rich repeat-containing G-protein coupled receptor 7; LV, left ventricle
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HCl, hydro-chloric acid; HEK, human embryonic kidney; LGR7, leucine-rich repeat-containing G-protein coupled receptor 7; LV, left ventricle;
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