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Altered Expression of RXFP1 Receptor Contributes to the Inefficacy of Relaxin-Based Anti-Fibrotic Treatments in Systemic Sclerosis C

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Altered Expression of RXFP1 Receptor Contributes to the Inefficacy of Relaxin-Based Anti-Fibrotic Treatments in Systemic Sclerosis C Altered expression of RXFP1 receptor contributes to the inefficacy of relaxin-based anti-fibrotic treatments in systemic sclerosis C. Corallo1, A.M. Pinto2, A. Renieri2, S. Cheleschi3, A. Fioravanti3, M. Cutolo4, S. Soldano4, R. Nuti1, N. Giordano1 1Scleroderma Unit, Department of ABSTRACT DcSSc-affected fibroblasts only, but not Medicine, Surgery and Neurosciences, Objective. Relaxin is a potent anti-fi- in LcSSc-unaffected and healthy ones. University of Siena; brotic hormone that has been tested to Conclusion. The absence/altered ex- 2Medical Genetics, Department of Medical ameliorate fibrosis in systemic sclerosis pression of relaxin receptor RXFP1 in Biotechnologies, University of Siena; 3Rheumatology Unit, Department of (SSc), but with controversial results. the affected fibroblasts of SSc patients Medicine, Surgery and Neurosciences, The aim of the study is to sequence re- could explain the inefficacy of relaxin- University of Siena; laxin receptor gene RXFP1 and to as- based anti-fibrotic treatments in the 4Research Laboratory and Academic sess its mRNA expression and protein disease. Division of Clinical Rheumatology, levels in the skin of SSc patients and Department of Internal Medicine, healthy subjects. Introduction University of Genova, Italy. Methods. Fibroblasts were isolated Systemic sclerosis (scleroderma, SSc) Claudio Corallo, PhD from unaffected/affected skin samples of is a rare, heterogeneous disease with a Anna Maria Pinto, MD, PhD (n=16) limited-cutaneous-SSc-(LcSSc) high associated mortality, characterised Alessandra Renieri, MD, PhD Sara Cheleschi, PhD and from affected ones of (n=4) diffuse- by progressive fibrosis of the skin and Antonella Fioravanti, MD cutaneous-SSc-(DcSSc) patients. Fibro- internal organs such as lungs, heart, Maurizio Cutolo, MD blasts from healthy subjects were used kidneys and gastrointestinal tract, cou- Stefano Soldano, PhD as controls. Sequencing of exonic target pled to widespread macro- and micro- Ranuccio Nuti, MD regions of interest for RXFP1 gene was vascular alterations (1). SSc occurs in Nicola Giordano, MD performed, coupled with mRNA tran- susceptible individuals as defined by Please address correspondence to: script variant analysis. RXFP1 mRNA genetic studies and it is stimulated by Dr Claudio Corallo, and protein levels were assessed by initialing events, although these initi- Scleroderma Unit, quantitative-real-time-PCR-(qRT-PCR) Dipartimento di Scienze Mediche, ating factors are poorly understood at Chirurgiche e Neuroscienze, and by immunocytochemistry-(ICC). present (2). The main abnormalities of Università di Siena, Alpha-smooth-muscle-actin-(α-SMA) the disease are related to the connective viale Bracci, synthesis induced by transforming- tissue, in which the excessive produc- 53100 Siena, Italy. growth-factor-beta-1-(TGF-β1) stimu- tion of collagen and other extracellu- E-mail: [email protected] lation was investigated in all fibro- lar matrix proteins are responsible of Received on January 12, 2019; accepted blasts with and without pre-treatment a progressive and irreversible fibrosis in revised form on April 29, 2019. with serelaxin (a recombinant form of (3). There is no resolutive cure for SSc, Clin Exp Rheumatol 2019; 37 (Suppl. 119): human relaxin-2 targeting the receptor although the existing therapeutic strate- S69-S75. RXFP1). gies are able to keep the symptoms un- © Copyright CLINICAL AND Results. Sequencing of RXFP1 gene der control (4). In fact, vascular altera- EXPERIMENTAL RHEUMATOLOGY 2019. showed no relevant mutations in all tions are well managed through the use fibroblast populations. The analysis of of prostacyclin analogues in secondary Key words: systemic sclerosis, mRNA transcripts revealed the pres- Raynaud Phenomenon (RP) (5), and fibrosis, relaxin, RXFP1 receptor, ence of 13 different mRNA isoforms of through the use of endothelin receptor sequencing, serelaxin RXFP1 (7 coding and 6 non-coding) antagonists (ERA) or phosphodiester- upregulated in LcSSc/DcSSc-affected ase type 5 inhibitors (PDE-5i) for pul- samples and not in LcSSc-unaffected monary arterial hypertension (6, 7). Au- and in healthy ones. On the contrary, toimmune response is also kept under ICC demonstrated the absence of control through the immunosuppres- RXFP1 in LcSSc/DcSSc-affected fibro- sive drugs (8). Only fibrosis remains blasts and the presence in LcSSc-un- untreated, since there are no treatments affected and in healthy ones. To prove able to strongly interfere with the de- these findings, serelaxin pre-incubation velopment of this process (9). was unable to counteract TGF-β1- In order to treat SSc-related fibrosis, Competing interests: none declared. driven upregulation of α-SMA in LcSSc/ relaxin (RLX), a dimeric peptide hor- Clinical and Experimental Rheumatology 2019 S-69 Relaxin inefficacy in scleroderma / C. Corallo et al. mone belonging to the family of insu- Based on clinical and in vitro findings, solution (2.4 U/ml; Sigma-Aldrich) for lin-like peptides (10), has been used the aim of the study is to investigate the 30 minutes. Fibroblasts obtained were in the past to ameliorate the fibrotic reasons of the alteration of RLX recep- passaged twice and cultured at a density process in SSc (11). In fact, besides tor in the affected skin of SSc patients of 1x106 cells per flask in Dulbecco’s functional association with reproduc- by sequencing the exonic target regions Modified Eagle Medium (DMEM; tion, RLX plays other physiological of interest for gene RXFP1 to evaluate Sigma-Aldrich) supplemented with roles, including the inhibition of col- the presence of possible mutations in penicillin (100 U/ml; Sigma-Aldrich), lagen biosynthesis and/or the stimula- fibroblasts derived from SSc patients streptomycin (100 μg/ml; Sigma-Al- tion of collagen breakdown (12). How- and healthy subjects. As further con- drich), amphotericin B (0.25 μg/ml; ever, one of the major mechanisms firmation, the potential of serelaxin (a Sigma-Aldrich), glutamine (2 mM; of the RLX anti-fibrotic effect is the recombinant form of human H2-RLX) Sigma-Aldrich), and 10 % fetal bovine antagonism of transforming-growth- to reduce fibrotic conditions was tested serum (FBS, Sigma-Aldrich), followed factor-beta (TGF-β) signalling (13). In in vitro in SSc affected fibroblasts and by incubation at 37°C in an atmosphere particular, in vitro studies showed that in healthy fibroblasts stimulated with of 5% CO2 and 95% air until confluence RLX binding to its receptor (RXFP1) TGF-β. (1 week) in 75-cm2 flasks (BD Cos- results in the activation of the pro- tar, Cambridge, MA, USA). Viability tein kinase ERK1, with downstream Materials and methods was estimated by trypan blue staining activation of endothelial nitric oxide Patients enrollment, skin biopsy, (Sigma-Aldrich). Fibroblasts were used synthase (eNOS) and increased nitric fibroblast isolation and culture at third passage (P3) for all the in vitro oxide production (14). This in turn ac- Patients (n=16) affected by limited- experiments. tivates soluble guanylyl cyclase and cutaneous-SSc (LcSSc) and (n=4) by cGMP production (14). This pathway diffuse-cutaneous-SSc (DcSSc) in ac- mRNA isolation and quantitative has been shown to inhibit the phospho- cordance with the description of LeRoy real-time polymerase chain reaction rylation of Smad2/3, resulting in de- et al. (21) and who fulfilled the 2013 Fibroblasts were collected in TRIzol creased TGF-β signalling (14). There American College of Rheumatology/ reagent (Sigma-Aldrich). Total RNA are three different isoforms of RLX European League Against Rheumatism was extracted following the manu- (H1, H2 and H3) in humans (15). Sci- diagnostic criteria for SSc (22) were facturer’s instructions. The total RNA entists agreed on the fact that H2-RLX enrolled into the study. We performed content of the samples was quantified is the isoform responsible for reducing skin biopsies by using a 3-mm punch by measuring the absorbance at 260 organ fibrosis (16). These evidences on (mid-forearm) affected skin graded nm using an Ultrospec 2000 spectro- justified the use of human H2-RLX in as 2 according to the modified Rodnan photometer (Amersham Pharmacia SSc. In fact, preclinical animal models skin score (mRSS) (23). Unaffected ar- Biotech, Piscataway, NJ, USA). The showed encouraging results: H2-RLX eas (upper-arm) of skin from the same RNA was then reverse-transcribed us- treatment, or H2-RLX delivered by patients with LcSSc and from healthy ing a random hexamer MultiScribe adenovirus, effectively reduced car- subjects (n=10) were also taken. The enzyme (Applied Biosystems, Foster diac fibrosis induced by β-adrenergic unaffected skin was defined by clinical City, CA, USA). Quantitative real-time stimulation in rodents (17), while RLX palpation (graded as 0 according to the polymerase chain reactions (qRT-PCR) knockout mice developed pulmonary mRSS) and by histological examina- were run in the StepOne Real-Time fibrosis that was reversed after RLX tion that excluded SSc-related lesions. PCR System (Applied Biosystems) treatment (18). On the contrary, despite All patients and healthy subjects gave using TaqMan chemistry (Invitrogen, animal models were promising, clinical their fully informed, voluntary, written Carlsbad, CA, USA). Two microliters trial outcomes were not so convincing: consent according to the principles of of complementary DNA in a final vol- although a phase II trial by RLX sub- the Declaration of Helsinki and in com- ume of 20 μl were amplified using the cutaneous infusion was encouraging,
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